Summary Human DNA can he cloned as yeast artificial chromo- somes (YACs), each of which contains several hundred kilobases of human DNA. This DNA can be manipulated in the yeast host using homologous recombination and yeast selectable markers. In relatively few steps it is possible to make virtually any change in the cloned human DNA from single base pair changes to deletions and insertions. In order to study the function of the cloned DNA and the effects of the changes made in the yeast, the human DNA must be transferred back into mammalian cells. Recent experiments indicate that large genes can be transferred from the yeast host to mammalian cells in tissue culture and that the genes are transferred intact and are expressed. Using the same methods it may soon be possible to transfer YAC DNA into the mouse germ line so that the expression and function of genes cloned in YACs can be studied in developing and adult mammalian animals.

Introduction Burke et al. developed a method for cloning large fragments of human DNA into yeast artificial chromo- somes (YACs)('). Large fragments of human DSA are ligatcd in vitru to two separate vector fragments, one containing a yeast ceiitromere, selectable marker and telomere, the other a second yeast selectable marker and telomere. On transformation into yeast (Saccharo- myces cerevisiac), thc recombinant DNA is maintained as an extra yeast chromosome which can be seen as an

additional band when the yeast chromosomes are separated by pulsed field gel electrophoresis and visualized hith ethidium bromide and UV light. Several libraries of yeast strains containing human DNA have been made. including three recent libraries with an average size of human insert greater than 350 kb(2-4). A number of YACs containing human genes have been isolated and characterized. In gcneral the YACs seem to be unrearranged copies of the human DNA, thou h

and others which contain non-contiguous pieces of human DNA'6). Furthermore, large contigs of overlap- ping YACs spanning several megabases of DNA have been assembled attesting to the power and fidelity of this cloning

Once in the yeast host. the YAC DNA can be manipulated by the very powerful combination of homologous recombination and yeast selectable markers. The next stage of developing this technology is to introduce the YAC DNA back into mammalian cells for functional analysis. Transfer into cells in tissue culture will be adequate for analysis of genes which are expressed in these cells. However, to study develop- mentally important genes. for example, it will be necessary to re-introduce YAC DNA into animals.

Manipulation of DNA in yeast is a well developed field. Using homologous recombination with specifi- cally tailored constructs, one can generate alterations ranging from single base pair changes to deletions, replacements or insertions of any size. The methods as applied to yeast DNA in general have recently been reviewed(9). Some examples of specific modifications which will be useful for engineering YACs, for subsequent analysis in mammalian cells, are illustrated in Fig. 1. Subtle mutations can be made by sequentially introducing and then removing a yeast selectable marker (Fig. lA)('"-''); this could be uscd to alter transcription factor binding sites in the promoter region, or amino acids in the coding region. New DNA can be inserted to make null mutations or substitute a

for the original coding region reporter (Fig. IB) . Thc YAC can be deleted from either end by recombination between the repetitive Alu sequences in the YAC and constructs containing AIu, telomere

there are YACs which rearrange at a high frequency f 5 )

(IFne

TEL

TEL TEL

TRPl CEN

TEL

D.

TEL

TRPl - CEN Human gene

URA3

Fig. 1. Diagram showing some of the ways that inairimalian DNA can be altered following cloning in a YAC. Two YACs are shown with the pubitions of the yeast selectable markers (TRPl URA31, centromere (CEN), ielomeres (TEL) and the human genes under investigation. Homologous recombination is indicatcd by X. A. Introduction of a subtle mutation: a selectable marker (H) is inserted and thcii replaced by the desired mutation (x). B. Replacement of the human gene with a reporter gene (3) and yeast selcctable marker (H). C. Removal of the righthand end of the YAC. D. Joining of the two YACs to give one larger YAC containing the entire human gene.

and, if necessary. centromcrc scquences (Fig. lC)(”); this could be used to locate and delimit the extent of a gene. In addition. larger YACs spanning a required region can be generated by splicing togethcr YACs in vivo using meiotic recombination (Fig. 1D) as has been described for the cystic fibrosis gene(@ and the BCL2 proto-oncogene(’‘).

There are a variety of methods of transferring DNA to mammalian cclls, but. with the exception oi microinjection. the efficiency is not great enough to allow identification of transformed cells in the absence of a selectable marker. In some cases the YAC itself will contain a gene for which one can select, for example, the h oxanthine phosphoribosyltransferase (HPRT) gene”? and the phosphoribosylglycinamide formyl- transferase (GART) gene(“), both of which can be selected in inutant mammalian cell lines. For YACs that do not contain a selectable marker. it is possible to introduce one by homologous recombination in the yeast host. The selectable gene can be introduced into the known vector sequence of the YAC(”). into a defined internal sequence(18), or into a repetitive internal elcmen t W

This article does not focus on the structural analysis of YACs or their manipulation in the yeast host but rather discusses the various different methods of transferring DNA from yeast into mammalian cells in

tissue culturc. These techniques should be applicable for generation of transgenic or chimaeric mice.

Fusion of Yeast Spheroplasts with Mammalian Cells It is possible to fuse yeast spheroplasts with mammalian cells using polyethylene glycol such that a plasmid in the yeast is stably transferred to the mammalian cells(20). As the yeast DNA is never extractcd, the DNA is not shcared, and this method should be suitable for transferring hundreds of kilobases of DNA intact. Indeed, fusion of Sc~iizosaccliarom)?c~s pombe sphero- plasts with mammalian cells allows transfer of an entire S. pombe chromosome(21). Initial experiments to transfer human DNA from yeast to mammalian cells used YACs carrying the bacterial aminoglycoside phosphotransferase (neo) gene linked to mammalian control elements. which confers resistance to G418 on mammalian cells. This work showed that cell fusion can be used to transfer YAC DNA to mammalian cell^(^*.'^.^^). The YAC DNA is often transferred intact and becomes integrated into the inammalian genomic DNA. Finally, when YACs containing selectable genes of mammalian origin are uscd, these larger (40-50 kb) genes are transferred intact and are c x p r e s s ~ d ( ~ ~ - ~ ~ ) . Fig. 2B shows an analysis of a cell line generated by fusion of yeast carrying a YAC containing the human

A. yGART2

s s s S I.’ I I 50 70 110 300 70

I

kb

300 -

110 - 70 - 50 -

Total Left Right human end end

Probe: DNA (e) 0 )

B. yGART2 integrated in hamster genome

S s s s s S J u l I I - I

260 70 110 300 370 -- r

kb 370 - 300 - 260 -

110 - 70 -

Total Left Right human end end

Probe: DNA (*)

Fig. 2. Analysis of a YAC containing the human CART gene (yGART2) and a ccll line made by fuhion of the yGART2-containing yeast with Chinese hamster cells. A. Map of yCART2. The positions of the Sfil restriction endonuclease sites are shown above thc line with the sizes of the fraginciits indicated in kb below. Total yeast DNA was cut with S$I. size fractionated by pulsed ficld gel electrophorebis, transferred to a filter and hybridized sequentially with total human DNA. a left-vector fragment, and a right-vector fragment, as indicated. Thc filter was incompletely strippcd between probing with total human DNA and the right-vector fragment. so that residual signals are visible in the lane probed with the right-end fragment. B. Map of yGART2 (thick line) integrated into the hamster genome (thin lines). Total DNA from the fusion cell line was treated as dcscrihed in A:

GART gene (yGART2), with Chinese hamster ovary (CHO) cells defective in the GART gene. GART gene expression was used to select cells which had taken up YAC DNA. The YAC, as maintained in the yeast host is 600kb. On digestion with the restriction enzyme S'I, five fragments are generated which can be seen on transfer of the DNA to a filter and hybridization with total human DNA (Fig. 2A). The left end of the YAC is located in a 50 kb S'I fragment, whereas thc right end i s in a 70kb SfiI fragment which comigrates with the internal 70 kb SjI fragment (Fig. 2A). In this fusion cell line, a single copy of the YAC is integrated into the hamster genome as shown in Fig. 2B. The 300kb, 110 kb and 70 kb internal Sfil fragments of the YAC are present in the fusion cell line DNA. In addition the left end of the YAC is now located in a 260 kb fragment, and the right end in a 370kb SfiI fragment (Fig. 2B). This indicates that the YX4C has integrated, essentially intact, into the hamster genome and a human gene on the YAC is expressed. The position of integration of the human DNA in YACs integrated into a rodent chromosome has also been visualized by in situ hybridization(18).

However, not all fusion cell lines contain an unrearranged copy of the YAC and some cell lines contain DNA from more than one copy of parts of the YAC. For the CHO ccll line, four out of seven fusion cell lines contained all the S'I fragments of the YAC and both vector ends. Two of these contained no other human DNA whereas the other two contained parts of a second copy of the YAC("). In contrast, when fusion cell lines were generated from a YAC containing the human HPRT gene (yHPRT) and HPRT-negative mouse (L A-9) cells, only one of ten fusion cell lines contained any vector DNA, even though all the cell lines contained most of the 680 kb YAC as determined by the Alu-containing fragments. When a selectable marker has been inserted into one of the vector arms of the YAC, it is important that the entire YAC with the vector sequences is transferred. In the case of yHPRT and L A-9 cells, reliance on a selectable marker on one of the vector arms would have led to a much reduced frequency of fusion products that could meet the selection.

In addition to taking up YAC DNA, the fusion cell lines contain a variable amount of yeast genomic DNA(15319). Fig. 3 shows analysis of a fusion cell line generated from yHPRT and mouse L A-9 cells. The fusion cell line contains the entire unrcarranged human HPRT gene (Fig. 3A) and most or all of the Alu- containing Hind111 fragments present in the YAC (Fig. 3B). In addition, many of the Tyl-containing fragments in the yeast genome are also present in the fusion cell line (Fig. 3C); Ty-1 is a yeast repetitive element of which about 30 copies are widely dispersed in the yeast genome. For many experiments the yeast DNA is irrelevant, but its prehence is indicative of major alterations in the structure of the recipient genome.

A.

12- 12-

8-

15 -

7 -

4-

HPRT cDNA

4 -

Alu TYl Fig. 3. Analysis of a YAC containing the human HPRT gene (yHPRT) and a cell line inadc by fusion of thc yHPKT containing yeast with mouse cells. DNA from the yeast strain or the fusion cell line (indicated above each lane) was cut with HiridITT, size fractionatcd on an agarose gel. transferred to a filter and liybridized with various DNA probes. A. Hybridization with the human HPRT cDNA. The yeast DNA migrates faster than the mammalian DNA because thew is only 0.5 % as much DNA clectrophoresing in this lane. B. hybridization with a human Alu sequence. C. hybridization with a yeast rcpctitive Ty1 clcniciit. Sizes are showii in kb.

In experiments reported to date with mouse and Chinese hamster cell lines, YACF have been transferred by spheroplast fusion at fre uencies between 1 and 50 colonies per lo6 ~ells( '~.I ' .~' q, 19,22) . Th' IS frequency is high enough to determine whether a particular YAC contains a functional gene or can transfer a specific phenotype to mammalian cells. but is probably not high enough to allow screening a large population of YACs for rare clones that contain a selectable gene. In addition, some recipient cell lines are very resistant to fusion and uptake of yeast DNA: for instance, fusion lines are obtained from the human cancer cell line HuTu 80 (ATCC HTB 40) at a frequency less than 1 "/, of that observed when L A-9 cells are uscd in parallel experiments (C.H., unpublished observation).

Transfer of Purified Yeast DNA Fusion between mammalian cells and yeast sphero- plasts does not always work and has the drawback of introducing yeast DNA into the mammalian cells along with the YAC. A variety of othcr methods are available

for efficiently transferring purified DNL4 into mam- malian cells including calcium phosphate coprecipi- tation, lipofection and electroporation. With some mammalian cell lines, thousands of transformants can be generated with a microgi-am of DNA, so it is feasible to transform with total high-molecular-weight yeast DNA and then select for those transformants that have taken up thc selectable marker located on the YAC. For this purpose, DNA containing intact yeast chromo- somes, which does not inhibit transformation, can be made by sucrose density centrifugation(23). Calcium phosphate precipitation has been used to transfer two 40-50 kb YACs containing the glucose-6-phosphate dehydrogenase and ribosomal RNA genes(17). Although the YAC DNA is transferred intact, no analysis of the amount of yeast DNA cotransferred was made. However, as calcium phosphate precipitation normally transfers about 100 kb of concatemerized DNA to individual cells(24). these transfectants would be expected to contain yeast DNA in addition to the YAC DNA. Lipofection has been used to transfer a YAC containing the GART gene to CHO cells(’‘), and in this case yeast DNA was transferred with the YAC. Electroporation generally leads to integration of a single copy of the input DNA(”) and would be expected to lead to transfer of the YAC DNA without other yeast DNA. Indeed, a cell linc containing the GAKT gene has been obtained by electroporation, and there is no yeast Tyl repetitive DNA in this cell line (A.G.: unpublished results).

The disadvantage of these methods is that the YAC DNA may be sheared during DNA preparation and transformation. However, as many mammalian genes span less than 50kb of genomic DNA, these genes could be transferred intact if the selectable markcr was located in the vicinity of the gene. In many cases it will be convenient to work with a YAC in the l0Okb size range as this will be more easily manipulated than larger YACs but still large enough to contain sizable functional genes. The efficiency of these methods is often no greater than fusion, as a YAC only comprises from 0.5 to 5 % of the yeast DNA.

Transfer of Gel-Purified YAC DNA Cloning of human DNA in YACs is fundamentally different from cloning in bacterial plasmids or viruses in that the YAC can only be separated from other yeast chromosomes by size. Although methods for isolating high-molecular-weight YAC DNA from gels have been p ~ b l i s h e d ( ” * ~ . ~ ~ ) , none of these methods give particu- larly pure or concentrated DNA. Phenol extraction of DNA from low melting agarose gels, or electroelution by standard methods, give modest amounts of purified DNA ranging in size from SO to 200 kb. If precipitated with ethanol, this DNA can be prepared at about 10 ng p - ’ and used for electroporation, lipofection or microinjection.

One advantage of calcium phosphate precipitation as

a method of transferring DNA to mammalian cells i s that any one cell integrates a large amount of DNA. and a selectable marker can be supplied by co-transform- ation(”). In many instances, YACs are used to clonc a large region of DNA, possibly spanning several megabases, in pursuit of a particular gene that has only been regionally localized. Since most mammalian genes appear to span less than 5Okb, calcium phosphate- mediated transformation might provide a suitable technique with which to search for the gene of interest using a functional assay. Up to 200ng of YAC DNA ranging from 50-100 kb in size could be isolated from one pulsed-field gel and used to transform mammalian cells, while providing the selectable marker by co- transformation.

Methods of DNA transfer which utilize purified DNA should be greatly aided by the use of YACs which can be amplified in the yeast host. One system for amplifyin YACs 10- to 20-fold has recently been described?”), and it should be possible to modify existing YACs so that they can be amplified in a similar fashion.

Transgenic and Chimaeric Mice Transgenic mice are typically made by microinjection of DNA into one of the pronuclei of a fertilized oocyte, followed by implantation in pseudopregnant females for development to term(29). The size of the DNA which can be transferred will probably be limited due to the problems associated with preparation of purified, concentrated YAC DNA, as well as the vulnerability of the DNA to shearing during passage through the microinjection needle.

DNA can also be introduced into the germ line of mice by altering mouse embryonic-stem (ES) cells cultured zn vitro and then reintroducing these cells into a blastocyst-stage embryo(30). In some cases, the genetically altered ES cells give rise to germ line cells and hence genetically altered mice in the next generation. Electroporation and microinjection(”) can be used to introduce new DNA into cultured ES cells and both of these methods are amenable to transfer of purified YAC DNA. In addition, fusion of ES cells with yeast spheroplasts might give altered ES cells which can participatc in the germ line of chimaeric mice. However, when fusion is carried out with other mouse cell lines, yeast genomic DNA is transferred as well as the YAC DNA. This extra yeast DNA, and other genomic rearrangements that take place after fusion, might affect formation of chimaeric mice and incorpor- ation of these ES cells into the germ line.

Perspectives Cloning of DNA into YACs has already changed the face of human gene cloning due to the larger size of the DNA fragments and the greater tolerance of yeast than E. coli for complex eukaryotic This has meant that large intervals of DNA defined by genetic

linkage(@ and chromosomal deletions and translo- c a t i o n ~ ( ~ ~ ) can now be spanned in relatively few steps and without getting stuck by unclonable pieces of DNA. Some genes associated with mammalian diseases have been isolated in YACs by such walking stratcgies. When these diseases have cellular phenotypes, it should be possible to confirm that a particular YAC contains the relevant wild-type genc by transfer into mutant mammalian cells and observation of compleinentation.

Using E. coli based vectors, cloning has been restricted to approximately 50 kb fragments for cos- mids, and, more recently 100kb fragments using P1 based vectors(34). However, human gencs and gcne clusters range in size up to several 100 kb and functional units such as centromeres may be even larger. Thus it has often been impossible to clone entire genes as a single piece of DNA. In some instances minigene constructs are adequate for in vivo tests of genc expression and function. However, even when whole genes have been cloned and transferred to mammalian cells or transgcnic mice, the cxprcssion of the gcnes has often been disappointingly low or incorrectly con- trolled(3s). YACs are now being made with an average size of over 300 kb and these can be joined together by in vivo recombination in yeast to generate yet larger clones. These YACs may contain the entire functional mammalian gene including long-range controlling elements which allow position-independent expression of the gene. When DNA from such YACs is transferred to mammalian cells the gene of interest will be in its normal context and may be expressed and controlled in the same way as the endogenous gene. Methods of this type may facilitate the identification and analysis of long range regulatory elements such as the human b-globin locus controlling regions which act as enhancers(”), the Dmsuphila specialized chroinatiii structures (scs) surrounding the hsp70 genes which act as domain boundaries(37), and the chicken A elcment located at the 5‘ end of the chicken lysozyme locus which is a scaffold attachment site and imparts high levels of position-independent expression to the gene(”).

Fine manipulation of small cloned genes has recently been facilitated by the introduction of the polymerase chain reaction. However, such manipulation remains a formidable task for genes spanning even tens of kb. Subtle mutations can be made by homologous recombi- nation in mammalian cells, but this procedure either requires transformation by micr~in jec t ion(~~) or two rounds of homologous re~ombination(~’), both of which are technically difficult. These mutations can easily be introduced into genes cloned in YACs(“) which can then be transferred to mammalian cells. The effects of the alterations can be studied in the presence of the wild-type gene or in a background with a null allele of the gene.

The transfer of intact YACs spanning over 600 kb of human DNA to cells in tissuc culture is a step towards long-range functional analysis of mammalian genes.

Similar transfer into the germ line of mice would have even more striking implications.

Acknowledgements We thank Dr. M. V. Olson and Dr. D. Gottlieb for critical reading of the manuscript. Clare Huxley is a Lucille P. Markey Visiting Fellow and this work was supported in part by a grant from the Lucille P. Markey Charitable Trust. Andreas Gnirke is a Fellow from the Deutsche Forschungsgemeinschaft.

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ZIEMIN, S . , ?:AILLOU-hIiLLkR. P.. LICHIER. P.. EVANS. G . A, . KERXY, J. H. ,

40 B4RTOY. M. c . . HOEKSTRA, &I. F. AND EWERSON, B. M. (1990). Site- of a complex gene locus. !$ucl.

Department of Genetics, Washington University Medical School, 4566 Scott Ave., St. Louis. MO 63110, USA.

THE INTERNATIONAL FEDERATION OF SOCIETIES FOR HISTOCHEMISTRY AND CYTOCHEMISTRY ANNOUNCES THE

9th International Congress of Histochemistry and Cytochemistry

MAASTRICHT, THE NETHERLANDS

August 30-September 5, 1992 Plenary Lecture topics:

In sitzi hybridization; Intravital microscopy; Cytochemistry at the EM level; Enzyme histochemistry; Immunocytochemistry; Intracellular Transport; Nerve-immune system interactions; Peroxisomes.

Symposia will highlight the impact of histo- and cytochemistry in the areas of Developmental biology and agcing; Cell growth and differentiation; Neurosciences; Plant Cell Biology ;

Diagnostic Pathology; Toxicology: Receptors and Ligands: Tntracellular calcium.

Workshops will focus on iiew mnth in: Tissue processing tcchniques; Flow and image cytometry: Morphometry: Autoradiography ;

Confocal microscopy.

Organizing Secretariat: Prof. Dr. F. C. S. Ramaekers, Department of Molecular Cell Biology; University of Limburg; P.O. Box 616;

6200 MD MAASTRICHT, The Netherlands; Tel. 31-43-888642; Fax. 31-43-437640.