commentary - Journal of Cell Science

COMMENTARY Telomere cloning and mammalian chromosome analysis

WILLIAM R . A. BROWN, MELANIE J. DOBSON and P H I L I P MacKINNON Biocl~etnistryDepnrtnrent, Oxford University, South P a t h Road, Oxford OX1 3QU, Englotld

Summary Although eucaryotic chromosomes vary in size over five orders of magnitude and are constituents of diverse genetic systems the fundamental features of their telomeres appear to be almost completely conserved. This can be exploited to enable molecular cloning of human telomeres in yeast and suggests that many of the ideas that will arise from studies of telomeres in the experimentally tractable ciliates and yeasts will hold true of mammalian telomeres. T h e particular value of cloned mammalian telomeres is that they contribute reagents for mapping mammalian chromosomes and that they provide one set of elements for the construction of artificial mammalian chromosomes.

Introduction Human telomeric D N A has recently been cloned in yeast artificial chromosomes (YACs) (Cross et al. 1989; Brown, 1989; Cheng et al. 1989; Reithman et al. 1989). T h e success of these cloning experiments depended upon heterologous function of the human telomeres in yeast cells. Such an approach is potentially applicable to the telomeres of a wide variety of other organisms. T h e purpose of this review is to consider the background to these experiments, to compare them with other approaches to telomere cloning and to describe some of the opportunities that cloned telomeres present for the structural and functional analysis of the chromosomes of humans and other mammals.

Telomere biology Is conserved in evolution Telomeres are the protein-DNA complexes at the natural ends of chromosomes. They were originally identified by cytologrcal studies that showed that whereas the natural ends of chromosomes are stable, chromosome ends generated by X-irradiation are fusagenic (Muller, 1938). Subsequent cytologcal work has revealed that telomeres have the additional properties of associating Journal of Cell h e n c e 95, 521-526 (1993) Pnnted In Great Br~tmn0T h e Company of Biolog~stsLlmted 1990

both with one another and with the nuclear membrane (Dancis and Holmquist, 1979; for a review of telonlere molecular biology: Blackburn and Szostak, 1984). Telomeres also have unique biochen~icalproperties. Complete replication of a linear double-stranded chromosomal D N A molecule poses the eucaryotic cell a particular problem. T h e root of the difficulty lies in the machinery of template-dependent DNA replication. In all examples hitherto analysed D N A replication proceeds in a 5' to 3' direction and is primed by a short polynucleotide. This primer is subsequently enzymically degraded to reveal a gap that is usually filled by D N A synthesis initiated 5' of the gap. Gaps at the 5' ends of double-stranded D N A molecules cannot be filled, and thus complete replication of telomeric D N A by this mechanism acting alone is in~possible. Many ideas as to the nature of the additional mechanism have been presented (reviewed by Blackburn and Szostak, 1984) but experimental analysis has revealed an enzymically novel telomere replication machinery that is likely to be widely conserved in both the plant and animal kingdoms. Discovery of this machinery has largely been the result of studies of telomere replication in the holotrichous ciliate Tetrahjmma themophila. T h e macronucleus of cells of this organism contains approximately 200 copies of a 21 kilo base pair (kb) 1'mear palindromic molecule that includes two copies of the ribosomal RNA genes (Gall, 1974). Abundance, uniformity and small size made these n~olecules a good source of telomeric DNA. Sequence analysis demonstrated that this telomere consists of 40-60 repeats of the sequence T T G G G G oriented 5' to 3' toward the chromosome end (Blackburn and Gall, 1978). T h e terminal 12-16 bases of the array are unpaired (Henderson and Blackburn, 1989) and the remainder are associated with the complen~entarysequence CCCCAA (Blackburn and Gall, 1978). T h e C-rich strand is extensively nicked (Blackburn and Gall, 1978). I t seems probable that both strands of this sequence form novel but as yet poorly defined secondary structures that involve a variety of non-Watson-Crick base pairs (Henderson et al. 1987; Lyamichev et al. 1989; Sundquist and Klug, 1989; Williamson et a[. 1989).

Table 1. Telomeric simple sequence DNA Species Protozoa Holotrichous ciliate Tetrahymena thermophila Glaucoma chatloni Hypotrichous ciliate Oxytricha fallax Stylonicha pustulata Euplotes crassus Paramecium tetraurelia Haemoflagellate Trypanosoma brucei Slime moulds Physamm polycephalum Didymium iris Dictyostelium discoideum Fungi Saccharomyces cerevisiae Schizosaccharomyces pombe Sporozoa Plasmodium berghei Plants Arabidopsis thalhana Mammals Homo sapiens

Sequence of G-rich strand telomenc repeat unit

TTGGGG* TTGGGG TTTTGGGG TTTTGGGG TTTTGGGG TTGGGG,TTTGGG

Reference

Blackburn and Gall (1978) Katzenet al. (1981) P\utz etal. (1982) Klobutcher et al. (1981) Klobutcheref al. (1981) Baroinefa/. (1987)

TTAGGG

Blackburn and Challoner (1984) Van der Ploeg et al. (1984)

TTAGGG TTAGGG

F o r n e y s al. (1987) Forney et al. (1987) Emery and Werner (1981)

TG,.j

TG,_3 T1 -2 ACAd_ i Co_ i G i _£

TTCAGGG, TTTAGGG

Shampayef al. (1984) Walmsley et al. (1984) Sugawara and Szostak (1986) Ponzi etal. (1985)

TTTAGGG

Richards and Ausubel (1988)

TTAGGG TTGGGG*

Moyzis^a/. (1988) Allshire et al. (1989)

• T h e T T G G G G repeated sequence is a minor component of the human telomeric sequence array.

Telomeric DNA has now been identified in many other organisms (Table 1). The abundance and small size of the chromosomes in the macronuclei of other ciliated protozoa made direct identification of the telomeric DNA also possible in these species. In many of the other organisms listed in Table 1, however, the telomeric DNA was identified after selective cloning or, in the case of human telomeric DNA, by a probe isolated during a random cloning exercise. In all organisms that have been studied the telomeric DNA is tandemly repeated and of low complexity. One strand is G-rich and oriented 5' to 3' towards the chromosome end. The observation of evolutionarily conserved features of both the primary and secondary (Henderson et al. 1987) structures of the telomeric terminal array suggested that the mechanism of telomere replication might also be evolutionarily conserved. Support for this idea and an indication that telomeric sequence replication involves non-DNA-template-mediated sequence addition came from the observation that telomeric DNA from Tetrahymena (Szostak and Blackburn, 1982) or from the hypotrichous ciliate Oxytricha nova (Pluta et al. 1984) would function in the yeast Saccharomyces cerevisiae. Linear DNA molecules that include an origin of DNA replication and a selectable marker gene but lack two telomeres are unstable as episomes; if they are introduced into yeast by transformation they are either lost from the cell or they integrate into the yeast genome. However, if ligated to telomeric DNA from either of the ciliates, then they are maintained extrachromosomally. Analysis of 522

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such molecules after their replication in yeast revealed that the telomeric DNA, initially derived from the ciliate, had been modified by the addition of several hundred extra base pairs of yeast telomeric DNA (Szostak and Blackburn, 1982; Pluta et al. 1984; Shampaye^a/. 1984). Studies of the haemoflagellate Trypanosoma brucei provided additional evidence for the importance of sequence addition in telomere replication. The expressed variant surface antigen gene of the blood form of this parasite lies close to a telomere, and probes from this gene can be used to analyse telomere length. During multiplication of the parasite in blood the telomeres grow at a rate of about 10 base pairs every cell division (Bernards et al. 1983). Telomere growth does not of course continue indefinitely, and periodically the chromosome end shortens. The basic mechanism of telomeric DNA sequence addition became clear with the demonstration of a telomere terminal transferase enzyme in Tetrahymena thermophila (Grieder and Blackburn, 1985). A similar activity has subsequently been demonstrated in Euplotes (Shippen-Lentz and Blackburn, 1989), Oxytricha (Zahler and Prescott, 1988) and human fibroblasts (Morin, 1989). These enzymes synthesise autologous G-rich strand repeats onto an oligonucleotide substrate with a sequence of the G-rich strand of either autologous or heterologous telomeric DNA. They are ribonucleoproteins. In Tetrahymena where the RNA component has been sequenced it has been shown to include a sequence CAACCCCAA complementary to one and a half copies of the G-rich strand telomeric repeat (Grieder and Black-

burn, 1989). The evolutionary conservation of the enzymic activity and of one component of its substraterecognition specificity strongly suggests that it plays a central role in the life of the cell. Cloning telomeres Three approaches have been used to clone selectively telomeric DNA. The most powerful relies upon heterologous telomere function in the yeast S. cerevisiae. Linear DNA with a centromere, an origin of replication and a single telomere is unstable as an episome in yeast but the stability of such a molecule is greatly increased by the addition of a second telomere. Linear episomes of this type are often called yeast artificial chromosomes (YACs). Complementation of this sort had previously been used in cloning of a yeast (5. cerevisiae) telomere (Szostak and Blackburn, 1982) but since the essential elements of telomere function are conserved between yeast and humans it has been possible to apply this strategy (Fig. 1) to the cloning of human telomeres (Cross et al. 1989; Brown, 1989; Cheng et al. 1989; Reithman et al. 1989). The YACs isolated by this strategy were confirmed as originating from telomeres by hybridisation to a previously mapped proterminal DNA sequence probe (Brown, 1989), by the ability of subcloned DNA sequences to detect human genomic DNA fragments that are sensitive to the exonuclease BaB\ (Cross et al. 1989; Cheng et al. 1989) or less precisely by hybridization in situ (Reithman et al. 1989). The proterminal regions of the chromosomes of many species are rich in repeated sequences distinct from the terminal simple sequence array (reviewed by Blackburn and Szostak, 1984). These have been called telomere-associated repeats; human telomeres contain such repeats (Cheng et al. 1989; Brown, MacKinnon and Dobson, unpublished observations) and this has prevented chromosome assignment of many of the rather short clones obtained so far. In the experiments of Reithman and colleagues (1989), however, the capacity of YACs to clone long stretches of DNA was exploited (Burke et al. 1987) and one of the telomeric clones was able to be assigned to the long arm of human chromosome seven. In any molecular cloning experiment it is of interest to establish if there has been sequence rearrangement during propagation of the sequence in the novel biological host. The length of the TTAGGG array in human telomeres varies between 2 and 20 kb depending on the cell type. In those telomeric YACs where it has been examined, the TTAGGG array is less than 1.5 kb in length and it thus appears that the yeast has abbreviated the terminal simple sequence array (Brown, 1989). The mechanism behind this rearrangement is undefined. A second modification to have been observed is the expected addition of yeast telomeric DNA sequences onto the human telomere (Dobson, MacKinnon and Brown, unpublished). It is as yet unclear what effect if any these modifications will have upon the functional potential of the cloned DNA in mammalian cells. There is no evidence, despite extensive restriction site and sequence analysis, for any other rearrangement. This is

encouraging, given the large number of repeated sequences that have been detected in the clones, and presumably reflects the greater stability of such sequences in yeast as opposed to bacteria. A second approach has been used for the molecular cloning of telomeres from Tiypanosoma bmcei (Van der Ploeg et al. 1984), from Plasniodium bei-ghei (Ponzi et al. 1985) and from the plant Arabidopsis thalliana (Richards and Ausubel, 1988). In this strategy high molecular weight DNA is first digested with Bal3l, flush ended by the action of the Klenow fragment of Eschericliia coli DNA polymerase I and then ligated to plasmid vector sequences. This procedure tags telomeric sequences, although any randomly broken end is potentially clonable. The DNA is then restricted with an enzyme that does not cut in the plasmid sequences and circularized by ligation at low DNA concentrations. The resulting population of plasmids is enriched in telomere-derived sequences as compared to what would be obtained in a random cloning exercise. These may be identified, after introduction into bacteria, either by colony hybridisation using a sequence probe recognizing the terminal array or, where this is unknown, by screening the plasmids directly for the presence of sequences that detect Bal3lsensitive, telomeric DNA. The third strategy for telomere cloning also used E. coli as a host but in this approach the high molecular weight DNA was linker tailed after Bal3l and Klenow treatment, restricted and then cloned into a bacteriophage vector. Telomeric clones were identified using a previously identified proterminal sequence probe. This approach was used for the cloning of telomeres from Paramecium (Baroin et al. 1987) and relied upon the preexistence of the proterminal sequence probes for its selectivity. In light of the YACs' ability to clone large fragments and to select directly for heterologous telomeres it seems unlikely that these E. co//-based telomere-cloning strategies will be widely used in the future. Structural and functional studies with cloned telomeres Molecular clones of human telomeric DNA provide reagents for asking structural and possibly functional questions about human chromosomes. We may consider these individually. What sequence elements are present in the protetvtinal regions of human chromosomes? Preliminary evidence suggests that there are repeated sequence elements common to the proterminal regions of different chromosomes (Cheng et al. 1989; Brown, Dobson and MacKinnon unpublished). We should like to know how many different types of telomere-associated repeat exist in the human genome, what is the chromosomal distribution of such repeats and how extensive is the homology that exists between different telomeres. In Drosophila melanogaster (Liitzelschwab et al. 1986) and the yeast Saccharomyces cerevisiae (Charron et al. Telomere cloning and cluvmosome analysis

523

URA3

Q

ARS1. TRP1

BamHl

Notl

i ^

Restrict with BamHl and enrich for telomeric DNA on silver caesium sulphate gradient

Restrict with BamHl and Notl Dephosphorylate

I BamHl I

Notl

I TEL

BamHl

URA3

I

BamHl

ARS177?«|CEN4

I BamHl

I

BamHl I

f Ligate; introduce into yeast by spheroplast transformation Screen for human telomeric YACS by colony hybridisation with probe specific for TTAGGG array

TEL

URA3

ARS1TRP1 CEN4

TEL

URA3

ARS1TRP1 CEN4

Fig. 1. Telomere cloning in yeast artificial chromosomes. The diagram outlines the central steps used in the telomere cloning experiments described by Brown (1989). The plasmid pTV2 contains two marker genes (URA3 and TRP1) capable of being selected in yeast, a centromere (CEN4) for accurate chromosome segregation at mitosis, an origin of DNA replication in yeast (ARS1) and a single telomere, TEL, derived from the ribosomal DNA of Tetrahymena. Restriction with BamHl and Notl releases a large DNA fragment, which contains these functional elements and also a smaller stuffer fragment that does not enter into the cloning and is not illustrated. The larger fragment includes only a single telomere and is incapable of functioning as an artificial linear chromosome. This deficiency may be complemented by ligation of the fragment to human telomeric DNA. Human DNA is illustrated at the top right by wavy lines and the telomeric sequences by open boxes. The DNA is first cut with Saw HI to generate clonable ends and then enriched for telomeric sequences by fractionation on a caesium sulphate gradient in the presence of silver ions. The partially purified telomeric DNA is then ligated to the vector fragment; the mixture is then introduced into yeast and chaemeric chromosomes identified by hybridisation with a probe for the human telomeric DNA.

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1989), genes have been identified close to telomeres. We should like to know what proterminal genes exist in the human genome and to understand how their chromosomal position affects their mutability and other aspects of their genetics. \Wiat is the recombination behaviour of human proterminal DNA? There is some evidence to suggest that the frequency of recombination events increases particularly in male meiosis near human telomeres (Donis-Keller et al. 1987). Is this true of all telomeres and in both sexes? How does the sequence organisation of a telomere reflect its recombination behaviour? Is there a correlation between minisatellite sequence density (Royle et al. 1988) and recombination? Are there features unique to the long-range sequence organisation around telomeres? Long-range restriction site maps have been constructed around the ends of the short arms of the human sex chromosomes (Brown, 1988; Petit et al. 1988), around the tip of the short arm of chromosome 4 (Bucan et al. 1990) and around the alpha globin complex at the tip of the short arm of chromosome 16 (Fischel-Ghodsian et al. 1987). All of these maps include an unexpectedly high density of CpG islands (Bird, 1986). Is this feature common to all telomeric regions? Hybridisation in situ indicates that human telomeres are folded back into the metaphase chromosome (Moyzis et al. 1988). What sequence elements are responsible for this particular configuration of the metaphase chromatin? Can cloned human telomeres function in mammalian cells? Experimental analysis of the relationship between the sequence organisation and function of mammalian chromosomes requires the construction of artificial mammalian chromosomes. As in the case of yeast this will require an origin of DNA replication, a centromere, telomeres and possibly other functional sequences that remain to be identified. It is now appropriate to start to try and build such an artificial chromosome. Two approaches now seem feasible. In a so-called bottom-up approach one may proceed by analogy with the S. cerevisiae and combine the cloned telomeres with other sequences whose sequence identity may suggest functional potential: viral replication origins or satellite DNA sequences, for example. An alternative strategy is to target telomeric DNA directly into mammalian chromosomes by homologous recombination. Introduction of telomeres in this way into yeast chromosomes (Vollrath et al. 1988) causes sequence-specific chromosome breakage. It may be possible to use this approach to engineer a small fully functional chromosome that would be capable of being replicated and manipulated in yeast, and of being structurally completely analysed.

tution. What proteins beside the telomerase comprise the telomere, what is their function and how do they interact with the telomeric DNA? In Oxytricha a protein heterodimer containing two subunits of 55 000Mr and 43 000il'/r has been identified that binds non-covalently to the (T 4 G 4 ) 2 telomere tail (Gottschling and Zakian, 1986; Raghuraman and Cech, 1989). This interaction is distinctive in that it is stable in 2 M NaCl. Telomeric DNA sequences and the purified protein can be reconstituted into a complex with many of the properties of the telomere purified from macronuclei. In light of the striking evolutionary conservation of many aspects of telomere biochemistry it seems probable that similar proteins will be found in mammalian cells. Telomeres, meiosis and the nuclear membrane Telomeres are often seen to be associated with the nuclear membrane (see, for example, Moens, 1973; Dancis and Holmquist, 1979). The molecular basis of the association is undetermined while its functional significance is a matter of speculation. Chromosome synapsis is initiated near to the telomeres during the zygotene stage of meiotic prophase. It is tempting to interpret this as suggesting the existence of synapsis promoting sequences in the proterminal region of the chromosomes. Another explanation does away with this notion but instead suggests that that synapsis is initiated near the telomeres because they are tethered at the nuclear membrane. According to this idea chromosome synapsis occurs when two homologues are aligned in phase over a critical distance and that synapsis would occur near the telomeres, because it is at these points that the chromosomes are in register. For this idea to be valid the telomeres need to be mobile in the plane of the nuclear membrane. Chromosome synapsis is obviously central in meiosis but may be important in aspects of the regulation of gene expression. Telomere tethering and lateral diffusion may be a device to ensure that it can occur. We thank Mark Jobling for comments on a draft manuscript. References ALLSHIRE, R. C , DEMPSTER, M. AND HASTIE, N. D. (1989). Human

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