T I G - - J u l y 1987, VoL 3, no. 7

Cloning in yeast: an appropriate scale for mammal,an genomes Howard Cooke Deparlment of Biolo~, Massachusetts l~titate of Technolo~, 77 Massachusetts Average, Cambridge, MA 02139, USA.

The ability to clone fragments of DNA in prokaryotic systems has caused an explosion in our under- standing of gene structure and flmc- tion. This technology has been adopted as a major tool by branches of biology as disparate as taxonomy and neurobiology. It has spawned a multi-million dollar industry and gen- erated the capacity to deal with major problems (AIDS springs to mind immediately) in a way and. with a speed which would have been impos- sible 15 years ago. But with current methods a gap between cytogenetic or genetic scales of analysis and the scale of molecular doning remains, especially when tb~ object of study is a human system. In current studies employing E. coil as a host organism, the size of the DNA fragment that can he cloned is limited by the biology of bacteriophage lambda.

Closing this gap is one reason for wishing to be able to clone large fragments of DNA 1'2. There are many examples of human diseases for which linked markers exist. In physical terms these markers may be separated from the gene by meffd- bases of DNA. Elaborate schemes have been devised to cover these distances by several 'jumping' steps ~'4 but this approach appears technically complex and fails to iso- late the DNA between take-off and landing points. Chromosome medi- ated gene transfer s "s capable of cloning DNA fragments that are megabases in length but the com- plexity of the host genome is a dis- advantage, as is the lack of control over the size of fragments trans- ferred. A further major problem with this approach is that rearrange- ments occL~r with relatively high frequency ¢.

The second reason for wanting a large scale cloning method is that some functional units are very large; for exm-aple, factor VIII covers 190 kbp or about 0. I% of the humm~ X chromosome 7, the Duchenne mus-

megahase or more s, and the dis- tances involved in immunogiobulin supergene family reanangements have so far been too great to be determined.

The third reason for cloning on this scale is to provide a starting point in the human genome sequencing pro- ject: an ordered set of clones seems logistically a simple place to begin. If fewer clones need to be ordered initially, fewer resources will be needed, the number of errors will

monitor be lower and they will be easier to detect.

Burke, Carle and Olsen 9 report an elegant approach to the cloning of large DNA fragments. By forsaking E. coli for Saccharomyces cerevisiae they have been able to exploit the availability of cloned telomeres m and centromeres ~1 to produce a 'yeast artificial chromosome' (YAC) that contains human DNA fragments hun- dreds of kilobases in size. A bacterial plasmid that carries two yeast telo- meres, a yeast centromere and autonomously replicating sequence (ARS), with selectable markers and a selectable cloning site, is used to generate two 'arms' (Fig. 1). One arm contains a teiomere and a select- able marker and the other contains another selectable marker m~d both a centromere and a telomere. Ligation of large fragments of DNA to these arms, followed by transformatio~ into yeast and selection gives rise to

EcoR1 cloning s,te

TEL ~ T E L BamH1 BamH1 Target DNA

/ /

BamH1 and EcoR1 d,gesl / Parl=al EcoR1 d,gest ~

T ¥ TRP ARS CEN

I ; " : ~ 1

URA \ / TEL TRP ARS CEN ~ ' L i g a l e URA TEL

Fig. 1. Construcgan of a yeast agi~ial chromosome c~rni~ system. A plasmid containing inverted repeats of tdomeric (TEL) s e q u e ~ , a centromere sequence (CEN4) and selectable marker~ (TRP I and URA3) provides the two vector'arms' after cleavage in a doningsite in the SUP4gene and,it the BamHlsites, which can be healed byyeast togive functional ~lomeres. After depbo~ho~,lalion the vector arms are l~ated to large DNA fro~nents. Transformation is used to introduce these constr~cts into yeast, where they are

cular dystrophy gene could span a maintained as s~thetic chromosomes. Modified from Ref. 9.

© ~9s7, ~ ~ . C.=b~d~ m6S- ~,_~OZ ®

onitor yeast containing synthetic telocen- tric chromosomes.

By pulsed field gel analysis 12'13 and hybridization, the authors are able to show that the fragments cloned are a faithful representation of the original genomic DNA at this level of resolution. They suggest that, since eukaryotic DNAs contain endogenous ARSs x4 and since yeast contains the usual eukaryotic spec- trum of repeated sequence organiza- tion, sequences which are unstable or difficult to clone in E. coli may be tolerated by S. cerevisiae because of similarities in the replication pro- cess. Probably only considerable use of the system will prove or disprove this point. Since these chromosomes are tagged by different DNA se- quences at each end they are a good substrate for mapping by partial digestion and end-labelling approaches. Plasmid rescue pro- vides a means of recovering terminal fragments which can then be used to isolate over'.apping clones. Both these approaches are facilitated by the low sequence complexity of the yeast genome. If walking on this scale becomes possible, physical linkage of genetic markers spaced a few centimorgans apart would be an order of magnitude less daunting than at present.

If these artificial chromosomes are open to the same recombinational processes as normal yeast chromo- somes then many possibilities arise.

Introduction of a plasmid, linearized in a target sequence and containing a genetic marker, into a library of yeast artificial chromosome clones would result in integration of the marker in clones containing the target se- quence. Selection for this marker would provide a powerful means of isolating clones that was not depen- dent on colony hybridization (in yeast this technique is difficult because of the cell wall and the low number of copies of DNA molecules per colony).

if a plasmid containing a target sequence and an inverted repeat of telomeric sequences were intro- duced, recombination with this plas- mid would break the artificial chromosome at the target site and so provide a physical mapping method which was not dependent on restric- tion endonucleases.

It seems unlikely that yeast will be able to express genes present on synthetic chromosomes containing mammalian DNA, since such se- quences will have large introns and inappropriate promoters. Whilst -.omplementation approaches are un- likely to be successful for these rea- sons, this is not a significant draw- back since for many human genetic diseases we do not know the pheno- type at the biochemical level. Where complementation could be useful, these artificial chromosomes could he tagged with a suitable marker and reintroduced into a mammalian cell

TIG--July 1987, Vol. 3, no. 7

by fusion. The attractiveness of cloning in

yeast will depend on a number of factors: how simply libraries can be generated, whether there are many regions of mammalian genomes that are unclonablc' in yeast, how large a DNA fragment can he introduced, the stability of ~ e artificial chromo- somes and whether they can be mani- pulated by the standard methods of yeast genetics. These questions re- main to be answered but the initial omens provided by Burke et aL look very promising indeed.

References I Gusella et al. (1983) Nature 306. 234-238 2 Beaudet, A. et al. (1986) Am. ]. Hum.

Genet. 39, 681-693 3 Poustka, A-M, et aL (1987) Nature 325,

353-355 4 Collins, F. et aL (1987)Science 235, 1046-

1049 5 McBride, O. W. and Ozer, H. L. (1973)

Proc. NatlAcad. Sci. USA 70, 1258--1262 6 Pritchard, C. A. and Gondfellow, P. N.

(1987) Genes Deo. 1, 172-178 7 Gitschier, J. etal. (1984)Nature 312, 326-

330 8 Monaco. A. P. et aL (1986)Na/ure 323,

646-650 9 Burke, D. T., Carle, G. T. and Olsen,

M. V. (1987)Sde~e 236, 806-813 I0 Szostak, S. W. and Blackburn. E.H.

(1982) Cell 29, 245-255 11 Clarke, L. and Carbon, J. (1984)Nature

287, 5O4-509 12 Schwartz, D. L. ar~d Cantor, C. (1984) Cell

37, 67-75 13 Carle, G. F. and Olsen, M . V . (1984)

Nucleic Acids Res. 12, 5647-5664 14 Williamson, D. H. (1985) Yeast 1, 1-14

j

Gene targeting in Dictyostelium: what do cells need myosin for? Rob Kay MR C Laboratory of Molecular Biology, Hills Road, Cambridge C B2 2QH, UK.

Gene targeting, in which an in-vitro manipulated gene is re-inserted into the genome of an organism by recom- bination with its chromosomal homo- Iogue, has proved to he a technique of great power in yeast biology. Starting only from a cloned gene, it allows the phenotype of a null muta- tion .to be disco-~-ered by a suitable gene disruption, and the phenotype of a modified gene by gene replace- ment. Gene targeting is not available as a routine technique in higher cells. Although many animal cells can be

transformed readily, the DNA is usu- ally inserted at random into their chromosomes and does not undergo homologous recombination. The resi- dent gene is therefore unaffected by the transformation and the experi- menter is reduced to learning about the function of a cloned gene by examining the effects of overpro- duction of the normal product or of production of a rare dominant product.

The first example of gene target- ing in the slime mould Die~yostelium

discoideum has just been reported and seems likely to herald a new era o[ gene manipulation in this organ- ism. Dictyostelium has a very differ- ent biology from yeast, allowing a new set of problems to be tackled. Unlike yeast, Didyostelium cells are motile, resembling leucocytes in their amoeboid and chemotactic movements. Although the nuclear envelope remains intact during mi- tosis, a typical eukaryotic spindle forms and the cells divide by binary fission. Also unlike yeast, Dictyoste- lium displays a phase of multicellular development in which the phenom- ena of cell differentiation and pattern- ing have many similarities with the same phenomena in the development of higher organisms.

De Lozanne and Spudich I report experiments in which the homo- logous recombination of a truncated myosin gene into its cellular homo- logue has been accomplished. The

~) 1987, EIse~er Pubbcauons, Ca~nbridge 0168 - 9525/87/$02_00