CA2086092A1 - Library screening method - Google Patents

Library screening method

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Publication number
CA2086092A1
CA2086092A1 CA002086092A CA2086092A CA2086092A1 CA 2086092 A1 CA2086092 A1 CA 2086092A1 CA 002086092 A CA002086092 A CA 002086092A CA 2086092 A CA2086092 A CA 2086092A CA 2086092 A1 CA2086092 A1 CA 2086092A1
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Prior art keywords
dna
targeting
yeast
dna fragment
gene
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CA002086092A
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French (fr)
Inventor
Douglas A. Treco
Allan M. Miller
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Shire Human Genetics Therapies Inc
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Individual
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/10Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034Isolating an individual clone by screening libraries
    • C12N15/1082Preparation or screening gene libraries by chromosomal integration of polynucleotide sequences, HR-, site-specific-recombination, transposons, viral vectors
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/87Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90Stable introduction of foreign DNA into chromosome
    • C12N15/902Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/905Stable introduction of foreign DNA into chromosome using homologous recombination in yeast
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

Abstract

Materials and methods for homologous-recombination screening of DNA libraries constructed in a eukaryotic host and methods for homologous-recombination chromosome walking for isolating overlapping DNA sequences for building an extended physical map of a chromosomal region.

Description

W092t01069 P~T/US91/04926
- 2~6~2 LIBRARY SCREENING METHOD
_______________________ Description Back~round of the Invent on A genomic DNA "library" is for~ed by digestin~
05 genomic DNA fro~ a particular organism with a suitable restriction enzyme, joining the genomic DNA
fragments to veceors and introducing the DNA frag-ment-containing vectors into a population of host cells. Complementary DNA (cDNA~ is DNA which has 10 been produced by an enzyme known as reverse transcriptase which can synthesize a complemen~ary strand of DNA (cDNA) using a mRNA strand as a template. A cDNA library is formed by digesting cDNA with a suitable restriction enzyme, joining the cDNA fragments to vectors and introducing the cDNA
fragment-containing vectors into a population of host cells.
In producing a DNA or cDNA library, the pieces of DNA have been fragmented into an unordered collection of thousands or millions of pieces. To isolate a host cell carrying a specific DNA sequence (i.e., a specific DNA clone), the entire library must be screened. Radioactively labeled or other-wise labeled nucleic acid probes are traditionally ~ 25 employed to screen a DNA or cDNA library. Nucleic -~ acid probes identify a specific DNA sequence by a .~ .
process of in vitro hybridization between comple-mentary DNA sequences in the probe and the DN~.
clone.

', .

W092/01069 PCT/US91/~4926 20~092 A specific DNA clone that has been identified - and isolated in this manner can contain DNA that is contiguous to the probe s~quence. A terminus of the DNA clone, therefore, can be used as a new probe to 05 rescreen the same or another DNA library to obtain a second DNA clone which has an overlapping sequence with the first DNA clone. sy obtaining a set of overlapping DNA clones, a physical map of a genomic region on a chromosome may be constructed. This process is called "chromosome walking" because each overlapping DNA clone which is isolated is one step further along the chromosome. Each DNA clone also can be studied to determine its physical relation-ship to a previously mapped genetic function and, thus, a series of overlapping DNA clones provides a physical map of a chromosome which is correlated to a map of genetic functions.
Chromosome walking is used, for example, to identify or localize a gene of interest, such as one thought to be causative of a disease or other condition This is done by using a DNA fragment which displays a restriction fragment length polymorphism (RFLP) shown to be genetically linked to (i.e., physically localized to the same ;~` 25 chromosome region as) a gene which causes or is associated with a disease, condition or phenotype, or a segment of DNA contiguous to such a RFLP, as an .- in vitro hybridization probe to screen a DNA library and pull out larger fragments of DNA in which all or part of the probe sequence is represented.
The usefulness of any DNA clone isolated in ~' this manner is that it includes DNA that is `:

`' .. - . ~ .. . . . . .

W O 92/01069 PC~r/US91/04926
3 2 ~ ~ S ~ ~ 2 contiguous to the RFLP sequence that is incrementally closer to the position of the - sou~ht-after gene than the original RFLP. To get a step closer, a labeled molecule corresponding to an 05 end o~ the newly isolated DNA clone is prepared and used to rescreen the library, with the goal being to isolate DNA clones that overlap with sequences found in the first DNA clone and that are incrementally closer to the gene of interest than either the starting probe or the first DNA clone isolated.
This procedure is repeated as needed. with the resulting DNA clones being used in gene~ic studies to assess whether they are more closely linked to the gene of interest. To walk over a distance of 10 million base pairs using presently-available chromosome walking techniques could require from 100 to 2,000 steps, depending on the DNA cloning vector ; system used. Any approach designed to decrease the work required to take a single walking step, or which would allow multiple walking projects to be carried out simultaneously would be a major advance.
The number of DNA clones which would be required to form a complete library of genomic DNA
is determined by the size of the genome and the DNA
clone capacity of the vector used to clone and propagate the segments of the genomic DNA.
Construction and screening of genomic DNA libraries of organisms with large genomes is labor intensive and time consuming. The development of vectors having a capacity for large DNA clones has helped to reduce ~he labor inv~lved in screening gen~mic ' : , . - .~

2~0~2 4 libraries. However, screening libraries remains time consuming, slow and labor intensive.

S___ary_of_the_I_ve_tion The present invention is a method of identi-05 fying and isolating a DNA fragment of interest (a target DNA fra~ment), from a DNA fragment library in a eukaryotic host cell, which is based on homologous recombination between the target DNA fragment and DNA present in a targeting vector introduced into the DNA fragment library. I~ further relates to targetin~ vectors and DNA fragment libraries con-structed in eukaryotic host cells as described herein.
The method of the present invention is used to screen a DNA fragment library constructed in a eukaryotic host cell in which ~enetic recombination (exchange of information between DNA present in a chromosome in the host cell and DNA introduced into the host cell) occurs by means of homologous recombination. In one embodiment, in which the eukaryotic host cell is yeast, genetic recombination occurs essentially exclusively by homologous recombination. DNA fragments in host cells are propa~ated in the form of artificial chromosomes which include, in addition to a DNA fragment insert, all of the DNA sequences necessary for the chromosome to participate in host cell replication ` and mitotic segregation in the same manner as . naturally-present host cell chromosomes. In general, the artificial chromosome is present in one copy or low-copy number in a host cell.
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2 ~ 2 The present method makes use of a targeting vector or vehicle which: 1) includes a DNA
sequence, referred to as targetin~ DNA, homologous to at least a portion of the target DNA fragment and 05 a selertable marker gene which is functional in host cells under appropriate conditions and 2) is non-replicating in the host cell. Preferably a double-strand break is made in the targeting DNA
present in the targeting vector, which generally is circular when purified from an E. coli host.
Alternatively, a gap can be introduced by making two cuts in the targeting DNA (e.g., with appropriately selected restriction enzyme(s)). The break or gap renders the vector linear, provides DNA ends which stimulate homologous recombination with host cell artificial chromosome sequences and increases the efficiency of stable transformation by homologous recombination.
The targeting vector is introduced into cells harboring the DNA fragment library, producing a ` mixed population of host cells, some of which contain the targeting vector and some of which do not. The resulting population of host cells is ; maintained under conditions appropriate for homologous recombination between DNA already present in the cell (i.e., prior to introduction of the targeting vector) and homologous sequences, such as those in the targeting vector. Subsequently, the population of cells is subj-ected to conditions appropriate for selection of host cells in which ' homologous recombination has occurred. Because the targeting vector is unable to replicate in the host : .
- - ~ : ...: .
: . -, 2 0 ~ 2 cell, stable transformati~n with the selectable marker gene can occur only throu~h homologous recombination. The selectable marker gene is replicated and, therefore, confers a stable 05 phenotype, only in host cells in which homologous recombination has occurred. Identification of such host cells--and, thus, of host cells containing the DNA fragment of interest--is carried out by ~ulturing the population of host cells under con-ditions (e.g,, culturing on appropriate media) inwhich only those host cells in which homologous recombination and stable transformation with sequences which are replicatableoccurred can survive. Growth of a transformed host cell is indicative of the presence of the target DNA
fragment. Host cells containing a target DNA
fragment are, as a resùlt, separated or isolated from host cells which do not contain the target DNA
fragment. The target DNA fragment can be removed from the host cell and sequenced or manipulated te.g., subcloned or mapped), using known techniques~
Alternatively, targeting DNA and a selectable marker gene for selection in yeast can be introduced into yeast host cells conta.ining the DNA fragment library by mating a yeast strain containing the targeting DNA and the selectable marker gene on a replicating yeast linear plasmid with the yeast host cells containing the library. In this embodiment, the two yeast strains must be of opposite mating types. ~omologous recombination occurs between the targeting linear plasmid and a library YAC having DNA homologous to targeting DNA, producing two .

WO92J010~9 PCT/US91/04926 linear molecules, each of which is a YAC. In one embodiment, the linear plasmid has negatively selectable markers flanking the targeting DNA
sequence. Each of the two recombination products 05 carries one of the two negatively selectable markers, making differential selection of the two reco~bination products possible. In another embodiment of the method in which mating of opposite yeast strain types is used, a yeast replicating plasmid is constructed in such a manner that the targeting DNA and a first selectable marker gene can be freed from the yeast replicon by recombination events and a second selectable marker gene, which is a negatively selectable marker gene, is used to select the replicon itself.
The replicating yeast plasmids described above can be introduced into host cells containing YACs by transformation.
In a preferred embodiment, the DNA fragment library is constructed in yeast, such as , Saccharomyces (S.) cerevisiae or Schizosaccharomyces (S.) ~ombe, ln which DNA fragments are present in yeast artificial chromosomes (YAC). Each yeast host cell contains one YAC or a few YACs, each present in one or few copies. A YAC includes, in addition to a DNA fragment, all of the DNA sequences required for chromosomes to replicate in yeast, segregate chromosomes to their progeny and stabilize chromosome ends. In this embodiment, the targeting ' 30 vector used is a bacterial plasmid or other vector which does not replicate in yeast and includes ~` targeting DNA and a selectable marker gene that .
.
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W O 92/01069 PC~r/~S91/04926 . , 0 ~ 2 functions in yeast. The targeting vector, which preferably has been linearized by introducing a double-strand break within the targeting DNA of the bacterial plasmid, is introduced into yeast cells.
05 The resulting mixed population of yeast cells is maintained under conditions appropria~e for homologous recombination to occur between targeting DNA and target DNA in the YAC. This is followed by selection of yeast cells stably transformed with the targeting DNA and selectable marker gene. Stable transformation of the yeast cells c.onfers on them a selectable phenotype, such as antibiotic resistance or nutrient prototrophy, such as amino acid prototrophy or nucleoside prototrophy. Growth of .~15 yeast cells under conditions compatible with survival only of stably transformed cells is indic-ative of the presence of the target DNA sequence.
Target DNA can be removed from the yeast cell and ~sequenced or manipulated, using known techniques.
.. 20 The present invention also relates to vectors useful in the present method. Vectors include ~ar8eting vectors, which are generally bacterial plasmids which do not replicate in yeast and include targeting DNA and a selectable marker gene functional in yeast. They may also include a bacterial origin of replication and a selectable ` marker gene for selection in bacteria. YAC arm vectors useful in the present method are also the subject of the present invention. These include a . 30 yeast selectable marker gene, a bacterial origin of replication, a bacterial selectable marker gene, a ~yeast telomere, and one or more cloning sites at : . , ' . -, WO92/~1069 PCT/US91/04926 - 2 ~

which tar~eting DNA is introduced or inserted into the vector. In addition, YAC arm vectors can include yeast centromere sequences and a yeast replication origin.
05 The pr0sent invention further relates to eukaryotic host cells, particularly yeast cells, constructed as described herein and useful for construction of YAC libraries from which a DNA
fragment of ineerest can be identified and isolated by the claimed method. In addition, the present invention relates to DNA fragment libraries, parti-cularly YAC libraries, constructed in such eukaryo-tic host cells.
The method, targeting vectors, YAC arm vectors and DNA fragment libraries of the present invention are useful for identifying and isolating a target DNA fragment, which can be genomic DNA or cDNA and can be an entire gene, gene portion or other DNA
sequence. The DNA in DNA fragment libraries screened by this method can be of any type, such as mammalian (particularly human), plant, insect, ; avian, fish, crustacean, molluscan, viral and protozoan. For example, they can be used to identi-fy and isolate a gene associated with a particular disease or condition, related genes within an organism's genome, and cDNA.
Further, as described herein, physically contiguous DNA sequences can be identified in a YAC
library in yeast cells (or other DNA iragment library) and used to construct a physical chromosome map. That is, the present method is useful for chromosome walking. In this embodiment, a first YAC

. .

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, WO92/0106g PCT/US91/04926 2 ~ 9 2 `-:

containing a target DNA fragment is isolated, using the claimed homologous recombination-based method described herein, and a terminus of the fragment is subcloned. In many instances, both termini will be 05 subcloned in order to determine the correct direc-tion for the walk to proceed. The terminus of the first target DNA fragment is then used as the targeting DNA present in the targeting vector, which is introduced into the YAC library. A second target YAC is isolated, which has as part of it the target DNA fragment, which partially overlaps the first target DNA in sequence. The second terminus is subcloned and used as ~he targeting DNA in a tar-geting ~ector introduced into the YAC library. This results in isolation of a third YAC containing R
. target DNA fragment, which partially overlaps the second target DNA fragment in sequence. This ~:~ process results in isolation of a series of YAC con-taining target DNA fragments which partially overlap .` 20 and can be repeated as many times as needed to `, construct the physical map sought. Chromosome walking can be carried out by the method of the present invention by using DNA which displays a restriction fra8ment length polymorphism (or RFLP) ` 25 as targeting DNA in the targeting vector to screen a YAC library. A terminus of target DNA isolated in ~ this manner is subcloned or isolated and the resul-- ting sequence used to isolate a contiguous DNA
fragment. This is repeated as often as needed to construct tbe physical map and, optimally, to reach W O 92/01069 PC~r/US91/04926 _1_12~g~ 2 a desired gene with which, for example, the RFLP is associated.
The method of the subject invention has nume~-ous advantages over other approaches tO screening 05 DNA libraries. F~r example, it is possible to screen a DNA fragment library many times simulta-neously. Libraries are stored as a pool of clones, thus eliminating the work to organize and screen a library that is distributed over many filter mem-branes. The time needed to screen a library isconsiderably le.ss than that needed with conventional methods.

Brief Descri~tion of the Drawin~s ____________ __________________ _ Figure 1 illustrates the identification of target DNA fragments in a YAC library by the homo-logous-recombination selection method of the present invention. The YAC includes telomeres (arrowheads), centromere/yeast origins of replication (filled circles), and a DNA fragment; in the case of clone #3, the DNA fragment contains within it a target DNA
fragment (solid rectangle~.
Figure 2 illustrates targeting (homologous reciprocal recombination) to generate a YAC that is marked for selection.
Figure 3 illustrates selection by homologous recombination of a DNA clone from a DNA YAC library using one-step gene disruption.
Figure 4 is a map of plasmid pl84DLARG, B:
BamHI; Sm: SmaI; P: PstI; ARG4: yeast ARG4 gene (arrow indicates direction of transcription); Cm:
chloramphenicol resistance gene; ORI (pACYC184):
Origin of replication from pACYC184; --~

,, ' .` ~ ~ - ' .

WO92/0106g PCT/US91/0~926 2~3~32 -hypothetical targeting sequence inserted into cloning site.
Figure 5 is a restriction enzyme and Southern blot analysis of clones selected by targeting with 05 human epsilon- and beta-globin sequences.
Figure 6 is DNA from eight colonies isolated by screening with fragment 8A. Lanes 1-4: clones 8A.1, 8A.2, 8A.3 and 8A.4; Lane 5: plasmid pl84-8A.
Lanes 6-7: clones 8A.5 and 8A.6; Lane 8: an example of DNA from an isolated colony which does not show the unit-length-linear band; Lane 9: clone 8A.11 1 microgram of total yeast DNA was loaded in lanes 1-4 and 5-9. 2 nanograms of plasmid pl84-8A
was loaded in lane 5. The electrophoreased DNA
~ 15 samples (all digested with KpnI) were transferred to ;, a nylon membrane and hybridized with a 32-P labeled ARG4 DNA probe. The arrow marks ~he position of the unit-length-linear band at 8.3 kb.
Figure 7 is DNA from each of the positive clones digested with XhoI and with either KpnI tfor those isolated by screening with fragment 8A) or ' AvaI (for those isolated by screening wlth fragment lOB). Samples were electrophoresed on a 1~ agarose gel, transferred to a nylon filter,- and hybridi~ed with 32p labeled pBR328 ~Boehringer Mannhei~
Biochemicals, Indianapolis, IN). Lanes 1-7: clones 8~.1, 8A.2, 8A.3, 8A.4, 8A.5, 8A.6, 8A.ll (all isolated by screening with fragment 8A); lanes 8-10:
clones lOB.6, lOB.29, lOB.41 (isolated by screening with fragment lOB).
Figure 8 shows analysis of YAC DNA for presence of unit-length-linear fragments hybridizing to an .
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WO92/01069 PCTtUSgl/04926 . . .
-13- 2 ~ 2 ARG4 DNA probe: Lane 1: EcoNI digest of plasmid pl84DLARG/PCRF 5, which contains the 852 base pair - PstI fragment from the human ADA locus clones into ~he PstI site of 184DLARG. 1 nanogram of digested 05 plasmid DNA was loaded; Lanes 2-3: empty (no samples loaded); Lanes 3-6: EcoNI digested YAC DNA
(approxima~ely 1 microgram) from candidate transformants 184ADA.B, 184ADA.C and 184ADA.D. The electrophoresed samples were transferred to a nylon mPmbrane and hybridized to a 32-P labeled fragment of ARG4 DNA. The arrow indicates the position of EcoNI linearized plasmid pl84DLARG/PCRF.5 (5.2 kb).
Figure 9 is a schematic representation of one embodiment of the present homologous recombination method, in which a YAC containing target DNA is identified using recombination with a linear yeast plasmid.
!

i Brief Description of ATCC De~osits ____________ _______________ _____ The following deposits have been made at the 20 American Type Culture Collection (June 28, 1990) under the accession numbers indicated. These deposits have been made under the terms of the Budapest Treaty.

1. Saccharomyces cerevisiae strain TD7-16d _________ ___ __________ , ATCC No. 74010.
2. Plasmid pl84DLARG, ATCC No. 40832.

Detailed Description of the Invention The present invention is based upon Applicant's discovery that the process of homologoos , " . . .

2 ~ 3 2 reco~bination which occurs in eukaryotic cells can be used for the purpose of screening DNA fragment libraries constructed in eukaryotic cells and identifying and isolating a DNA fragment of 05 interest, referred to as a target DNA fragment.
The present invention is a method of isolating a DNA fragment of interest, referred to as a target DNA fragment, from a DNA library constructed in a eukaryotic host in which genetic recombination occurs by homologous recombination. The target DNA
; fragment is generally present in a larger fragment containad in the eukaryotic host cell. The DNA used to construct the DNA libraries may be cDNA or genomic DNA which is of human or other origin. A
target DNA fragment is identified by the present method by introducing into the DNA fragment library a non-replicating targeting ~ehicle which contalns targeting DNA and an appropriate selectable marker gene and identifying eukaryotic host cells in which homologous recombination occurs between target DNA
and targeting DNA. Homologous recombination results in stable integration of targeting DNA and the selectable msrker gene into DNA in host cells, which are identified on the basis of a selectable phenotype conferred as a result of stable transfor-mation of host cells with the selectable marker gene. For example, they are identified on the basis of their ability to grow under conditions (e.g., in the presence of a drug or in the absence of an essential nutrient) incompatible with growth of host cells in which stable integration has not occurred.

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~: , The DNA library used in the present method is a population of eukaryotic host cells, such as yeast cells, containing a unit, such as an artificial chromosome, which includes a DNA fragment insert and 05 is rsplicated in the host cells. The DNA library is screened for DNA fragment insert(s), present in the arti~icial chromosome, all or a por~ion of which is a target DNA fragment, by introducing into the eukaryotic host cells a targeting vehicle, such as a bacterial plasmid, which is non-replicating in the eukaryotic host cells and includes a targeting DNA
sequence ~i.e., a DNA sequence homologous, at least in part, to the target DNA) and a selectable marker gene useful for selection in the host cell. Host cells containing the targeting vehicle are cultured under conditions appropriate for homologous recDmbi-nation between the targeting DNA sequence and target DNA to occur. Host cells stably transformed with the selectable marker are subsequently identified (i.e., by identifying host cells able to grow under conditions under which non-stably transformed cells cannot grow, and die).
In a specific embodiment of the present invention, which is exemplified by the Examples which follow, the DNA library is a population of yeast cells which contain artificial chromosomes carrying a DNA fragment insert and host cells containing target DNA are identified and isolated , from this YAC vector library.
A targeting vehicle, such as a bacterial plasmid, which is non-replicating in yeast is introduced into the population of host yeast cells ~ ,. . - - .

containing the DNA YAC library. The bacterial plasmid includes a targeting DNA sequence which is homologous, at least in part, to targe~ DNA of interes~ and a selectable marker gen~ that functions 05 in yeast. Preferably, the targeting plasmid is cut with a restriction endonuclease that introduces a double-strand break within the targeting DNA
sequence, thereby linearizing the bacterial plasmid and providing DNA ends which are recombinogenic, to s~imulate the process of homolo~ous recombination with the YAC sequences. The efficiency of homolo~ous recombination is, as a res~lt, increased.
Because the plasmid is non-replicating in yeast, stable transformation with the selectable marker can only proceed by homologous recombination.
The resulting host yeast cell population, which includes stably transformed host yeast cells (i.e., those in which the plasmid, including the selectable marker gene, has been stably integrated by homo-lo~ous recombination into DNA already present i~host cells prior to introduction of the targeting vehicle) and non-stably transformed host yeast cells, is cultured under conditions such that only stably transformed yeast cells are able to grow, In a correctly tar8eted event, the entire plasmid is stably incorporated in the host yeast cells by homo-logous recombination between the targeting DNA
sequence of the plasmid and homologous sequences (i.e., target DNA fragments) in the YAC. In other embodiments, such as when a linear targeting plasmid is used, it is not necessary, however, for the entir~ plasmid to become stably incorpora~ed, as . ~ .
, : ~

W092/01069 PCT/US9l/049~6 long as homologous recombination occurs to an extent sufficient to introduce a selectable marker gene into DNA already present in the host cells, such as in a YAC. Only those few h4st yeast cells which 05 contain a target DNA fragment(s) and have thereby undergone homologous recombination with ehe ~argeting plasmid are able to grow under the conditions used ~e.g,, in antibiotic-containing medium or medium lacking a nutrient essential to non-stably transformed cells), due tO the introduction of the yeast-selectable marker gene contained on the targeting plasmid. They are identified on the basis of.the selectable phenotype conferred by stable transformation of the selectable marker gene.
To prevent homologous recombination events between the plasmid-borne yeast-selectable marker gene and homologous sequences in the host yeast cells, it is preferable that host cell sequences homologous with targeting vector sequences have been deleted or almost entirely deleted from the genome of the host yeast strain before it is used for the YAC vector library. Alternatively, host cell sequences homologous with a yeast-selectable marker gene on the incoming targeting plasmid can be retained as a mutated, non-functional portion of the yeast chromosome. If this approach is used, ' however, more positive scores for homologous recom-bination will have to be screened to ensure that homologous recombination events which occur took place between the targeting DNA sequence on the ~ ,.... . .. . . .

WO 92/01069 PCI~/US91/04926 2 ~ 2 - 1 8 -bacterial plas~id and the target DNA sequence present on the YAC.
Figure 1 illustrates schematically the iso-lation of target DNA fragments from a YAC vector oS library by the method of the present invention. The targeting plasmid on the far left is introduced into a population of yeast cells (ovals), each of which contains a DNA YAC containing a different DNA
fragment. The plasmid includes a selectable marker gene for selection in yeast (diagonally lined section) and a targeting DNA fragment (solid section) in which a double strand break has been introduced. In this example, one host yeast cell (#3) contains a DNA fragment in a YAC that is homologous to a sequence carried on the targeting plasmid (solid sections on clone #3). Recombination between these two sequence occurs, resulting in the stable integration of the selectable marker carried on the plasmid into the yeast chromosome (YAC). The resulting population of cells is grown under con-; ditions appropriate for selection of host yeast cells stably transformed with the selectable marker gene. For example, they are plated on appropriate selective media, such as nutrient-deficient media.
Only those cells in which che selectable marker gene functions grow. Growth of.cells under these conditions is indicative of the presence of a target DNA fragment. Although YAC are exemplified herein, other yeast vectors, such as YCp vectors (YCp50, YCpl9) can be used to construct a DNA library.
' The general scheme for selection of a target D~A fragment from a DNA YAC library is shown in ' ` .

`:' . . . .
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:~ ': ' : ; - -. . .
, -19- 2 ~g ~3~ ~

Figure 2. Figure 2 illustrates the integration of a targeting plasmid (pl84DLARG) carrying a selectable marker (the yeast ARG4 gene; open box) and a segment of DNA that is homologous to a sequence in the DNA
YAC library (targeting DNA; sol$d arcs on plasmid).
05 The thin lines represent an insert of human or other (non-yeas~) DNA propagated as a yeast artificial chromosome (YAC). The solid black box is the target DNA fragment, a sequence of DNA which is a portion of YAC DNA present in a DNA clone, found in the library, that is homologous to the targeting sequence. The remaining portions of the DNA YAC are comprised of the YAC ~ector arms: the thick lines represent plasmid vector sequences for replication and selection in bacteria. The shaded boxes represent genetic ~arkers used for selection in yeast (yeast selectable markers URA3 and TRPl). The solid arrowheads and circle represent telomeres (TEL) and a centromere/ yeast replication ori~in tCEN/ARS), respectively. Figure 2a depices the targeting DNA (present in the targeting vector) aligning with the target DNA fragment in the YAC.
Figure 2b depicts the product of homologous recombination between the targeting DNA and tar8et DNA fragment. The targeting plasmid has been cut uniquely in the targeting DNA, at the site corres-ponding to the ~ertical arrow in the target sequence, ULL indicates the unit length linear restriction fragment that results from duplication of the target sequence (and the restriction site) on the YAC. As described in Example I, a ULL can be generated only if integration occurs into a DNA

:`' i -. :' : . .

WO 92/0106~ PCI /US91/04926 2 ~ 2 -20-sequence that contains the restriction enzyme site in question and contains sufficient homology surrounding that site to allow resyn~hesis (by repair) of the restriction enzyme site on the 05 targeting plasmid. Candidate clones that display a ULL are assumed to be homologous recombination events and are analyzed further.
In another embodiment of this method, a yeast-selectable marker gene on the incoming targeting DNA
molecule can be a bacterial gene engineered ~o be expressed in yeast and confer drug resistance, e.g., the CAT or neo genes from Tn9 and Tn903, or bacterial amino acid or nucleoside pro~otrophy genes (e.g., the E. coli argH, trpC, and pyrF genes).
In another embodiment of the method of the present invention, the targeting vector is a linear DNA fragment which includes a targeting DNA sequence homologous to a target DNA fragment to be identi~ied and/or isolated from the YAC library. In this embodiment, a selectable mar~er gene is inserted into the targeting DNA, producing a targeting DNA
sequence which includes two non-contiguous domains.
This embodiment is described in detail in Example II
and represented schematically in Figure 3. The targeting vector, which is a linear sequence which does not replicate in yeast, ls transformed into the pooled DNA YAC library, as described in Example I.
Homologous recombination occurs between the targeting DNA and the target DNA fra~ment.
In addition to the above-described embodiment, other approaches to introducing targe~ing DNA into host cells c ~n be used. For example, earg~ting DNA

` .

, ~: . : ., , : - : . .:
-2 ~ 9 ~

can be pres~nt on a replicating yeast linear plasmid (Murray, A.W. and Szostak, J.W., N_ture 305:189-193 - (1983)) in a yeast strain of mating type opposite to that of the host strain used for the library. The 05 linear plasmid has selectable markers flanking the targeting DNA sequence (i.e., one at each end of ehe tar~eting DNA); both markers are different fro~
those used in the construction of the YAC library and can be selected against (i.e., ne~atively selectable markers, such as LYS2, URA3 or ~YH2).
Homologous recombination between two linear molecules produces two linear molecules, each of which is a hybrid of the two parental molecules. In this embodiment, in which recombination occurs between the targeting linear plasmid and a library YAC, each of the two recombination products is a YAC
and each carries one of the two negatively selectable ~arkers, allowing for differential selection of the two recombination products.
The basis of this differential selection is illustratet in Figure 9. Filled circles, arrowheads, and open rectangles represent centromeric, telomeric, and marker gene sequences, respectively. The shaded boxes represent targeting ~5 or target sequences. URA3~ cells can be selected against (killed) by growth on ~edia containing the nucleoside analog 5-fluoro-orotic acid (5FOA), while LYS2 cells can be selected against by growth on media containing the amino acid analog alpha-amino-adipic acid (~aa). Molecule 1 is a - ..
- target YAC constructed in a vector system using ARG4 and TRPl as selectable markers (phenotype arg tFp .

:, - . . . . ~ : .

WO92/01069 PCT/~S91/04g26 2~g~2 5FOA ~aa ). Molecule 2 is a linear targeting plasmid in which the targetin8 sequence is flanked by ~RA3 and LYS2 (phenotype arg trp 5FOA ~aa ).
The phenotype of cells harboring molecules 1 and 2 05 in an unrecombined form is arg trp 5FOA ~aa .
Molec~les 3 and 4 are the produc~s of recombination between Molecules 1 and 2, resulting from a cross-over between the targeting and target sequence. The phenotype of Molecule 3 is arg trp 5FOA ~aa , and can be selected for by growth on 5FOA plates lacking arginine. The phenotype of Molecule 4 is arg trp 5FOA ~aaR, and can be selec~ed for by growth on ~aa plates lacking tryptophan. Thus, cells containing one or both non-recombinant molecules, as well as cells containing either of the recombinant products can be differentially selected (cells harboring only one or the other recombinant product arise by random loss events).
~ In such a scheme, the yeast cells harboring the targeting linear plasmids are msted to all members of the library and maintained under conditions favorable for spontaneous or induced homologous recomblnaeion (induced by, for example, meiosis or ultravlolet irradiation). Recombinant target YACs are selected by virtue of the unique phenotypes of the recombination products resulting from homologous recombination between the targeting sequence on the linear plasmid and YAC molecules harboring a suitable target sequence. Each of the two product YACs is truncated at the position of the target DNA
sequence, and the differential selection is used to - .- : : :

: .

WO 92/01069 PClr/US91/04926 isolate the two products separately, In order to isolate the two products of a single event, yeast cells harborings YACs and linear targeting plasmids are preferably plated or gridded out prior to 05 selection for recombinants. Selection is accomplished by replica plating onto the appropriate sel~cti~e plates.
In this embodiment, the relative orientation of the targeting sequence with respect to the two (negatively) selectable markers on the linear targetin~ plasmid is important. Recombination between a target YAC and only one of the two orientations of targe~ing linear plasmid will give _ rise to a stable recombinant (i.e,, a recombinant with one and only one centromere), YAC molecules with two centromeres show frequent breakage and unstable phenotypes and YAC molecules with no centromere are highly unstable by virtue of segreRation bias. In a preferred embodiment, linear tar8eting plas~ids are constructed with the targeting sequence present in both orientations and introduced into the library in separate matings.
As an alternative to mating to introduce linear targeting plasmids into the library, linear targeting plasmids can be introduced into host cells containing YACs by transformation, essentially as described in Example I.
ln another embodlment, a yeast replicating plasmid carrying a targeting sequence can be constructed in such a manner that the targeting DNA
and a selectable marker (SM1) can be freed from the yeast replicon by natural or induced recombination : .

- ~:, ,. , .; ~ ..................... . .

.~ ~ '. ' . :

WO92/0106g PCT/US91/04926 2 ~ 2 -2~

events, and such that the replicon itself can be selected against by virtue of a negatively selectable marker (SM2), such as URA3, LYS~ or CYH2.
Examples of inducible recombination systems which 05 can be engineered to function for this purpose are the fl~ mediated recomhiantion pathway of the yeast 2-micron plasmid and the cre- lox recombination _ _ _ _ _ _ _ system of bacteriopha~e Pl. The plasmid is introduced into a yeast strain of mating type opposite to that of the host strain used for the library. After mating to all members of the YAC
library, the targeting DNA sequence and selectable marker are released as a nonreplicating molecule and the selectable marker can only be stabilized by homologous recombination with a YAC harboring a suitable target DNA sequence. The targeted recombinants are selected by plating onto media which selects for SMl and against SM2.
As an alternative to mating to introduce the plasmid described in the preceding paragraph, the plasmid can be introduced by transformation, eseentially as described in Example I, followed by the induction step to free the targeting substrate . from the yeast replicon.

Identification and Isolation of a Target DNA
_ _ _ _ _ _ _ _ _ _ _ Fra~ment Using Homolo~ous Recombination The above-described embodiments of the present method are useful to identify and isolate any target DNA fragment, which can be an entire gene, a gene portion or other nucleotide sequence. For example, a gene of ~nterest, suob as a ~-globin gen- or `

: . ' , WO92/01069 PCT/U~91/0~926 -25- 2 ~ 2 adenosine deaminase gene, can be identified in a DNA
fragment library using the claimed method and, if desired, isolated from host cells by known methods.
Identification of target DNA fragments by the 05 present method is described in detail in Examples I, III and IV.

homolo~o_s-Reco__i_atio_ C__o_oso_e_Walki_~
~ he method of the present invention, by which a target DNA fragment is isolated from a DNA library, is useful for isolating physically-contiguous DNA
segments from a DNA YAC library in order to con-struct a physical chromosome map. That is, when used iteratively, each sime with targeting DNA
deri~ed from a YAC which overlaps with and extends beyond a previously identified region, it is a method for chromosome walking. In the present method of chromosome walking, a target DNA fragment present in a YAC iS isolated, as described above. A
terminus of this first target YAC fragment is subcloned into a plasmid vector. The terminus of the first DNA fragment is, thus, used as a second targeting DNA sequence, which is introduced into host yeast cells containing a DNA YAC library. The terminus of the first DNA fragment, which is contiguous to the first target DNA sequence, in turn becomes the second targeting DNA sequence. As used herein, the term contiguous includes sequences which are immediately adjacent to the first target sequence and those nearby or in proximity to the first target sequence (i.e., separated from the first target sequence by intervening nucleotide(s)).

.

., , ~ , .

W092/01069 PCT/US9ltO4926 ~ 2 -26-This second targeting DNA sequence should not have any homology with the first targeting DNA sequence, so that when it in turn is incorporated in a YAC at a point of homology with a second DNA clone, the 05 second DNA clone selected will have a different terminal DNA sequence. The ~er~inal subfragment from the second DNA clone is used to isolate the next (i.e,, the third) DNA clone. Each successive DNA clone is isolated by virtue of its homology with the termial subfragment of the previously isolated DNA clone. A series of overlapping clones is obtained by repeating this process; the process is repeated as needed to construct the physical map desired, The successive recovery of terminal DNA
fragments allows rescreening the same library or a second library for overlapping clones.
In one embodiment of the present invention, chromosome walking is carried out in order to ' determine the chromosomal location of a gene of ,~20 interest, such as a gene which causes a disease, by usin~ a DNA fragment displaying a RFLP genetically linked to the gene of interes~, or a DNA fragment contiguous with the RFLP, as targeting DNA in the targeting vector, A targeting vector, such as a bacterial plasmid, which includes the RFLP-displaying DNA or fragment contiguous to the RFLP-displaying DNA, as targeting DNA and a select-able marker gene, is introduced into a human DNA YAC
library. Homologous recombination between the targeting DNA and a target DNA fragment in the ;library results in the first step in walking to the gene of interest. A YAC containing the target DNA

2 ~ 2 fragment is identified in this way. One terminus or both termini of the target DNA fragment is used as targeting DNA in a targeting vector to rescreen the same library or screen a second library, as 05 described above. Also as described above, ~his is repeated, each time using a terminus of the tar~et DNA fragment isolated in the previous step as targeting DNA. This continues until the gene of interest is identified or the desired physical map is completed.

Host Cell Ty~es a_d CharacterLstics The method is described herein with particular reference to screening YAC DNA libraries constructed in yeast cells through ~he use of targeting DNA
sequences present in bacterial plasmids. It is to be understood, however, that this is merely for purposes of exemplification and that the present method can be carried out using other host cell types, provided that genetic recombination between vector-borne DNA and DNA already present in the host cell occurs by homologous recombination and that an appropriate targeting vector is available.
Appropriate eukaryotic host cells include those which normally (as they occur in nature) undergo genetic recombination essentially exclusively by homologous recombination ~e.g., Saccharomyces cerevisiae, Sch~zosaccharomyces pombe). As used herein, the term essentially exclusively means that homologous recombination occurs without significant levels of non-homologous recombination under the conditions used. Homologous-recombination selection ~- ,-, ~-., - ~
- : : ~ , ... ..

: , : . , WO92/010~9 PCT/US91/04926 2 ~ 28-of DNA clones could be utilized as a selection method in the cells of any organism in which 1) a suitable DNA cloning system exists and 2) the cells can be manipulated or induced, by gene~ic 05 engineering or genetic manipulation, to perform recombination which is predominantly based on DNA
sequence homology, or in which the targeting DNA can be treated in such a manner that it engages in ho~ologous-recombination as its preferred mode of recombination With these criteria met, one skilled in the recombinant DNA arts could perform homologous-recombination selectlon of DNA clones from a DNA library. Such organisms may include, but are not limited to, Schizosaccharomyces ~ombe, Droso~_il_ me1a_o~aster, Ho_o sa~ie_S, M_s muscu1us and S~o_o~tera fru~iperda.
Sacch_ro_yces ce_evisiae is a preferred host organism for ths selection of DNA clones using homo-logous-recombination because of its ability to route transforming DNA carrying double-strand breaks into a recombination pathway based virtually exclusively on DNA sequence homology.
Certain characteristics of host cells in which DNA fragment libraries are constructed should be considered and possibly modified to optimi~e use of such cells in the present method, such as by decreasing non-targeted svents and, thus, increasing the efficiency of the method. For example, as described below, it might be necessary to remove selectable markers present in the targeting vector from host yeast cells and to construc- targeting vectors in such a manner that they include no 2~g~

sequences homologous with those in the vector sequences used in the propagation of the DNA
library.
As described below, it has been determined that ~5 the selectable marker gene(s) chosen for the targeting vector should not normally be present in the host yeast genome or should be deleted from the normal chromosomal position(s) in the host yeast strain. Without this modification of the host strain, recombination even~s between the selectable marker and the yeast genome would occur at a higher rate. For near-complete (> 99%) coverage of the human genome, a DNA YAC library with an average frag~ent size of 300 kb would consist of approximately 50,000 members (Maniatis, T. et al., Molecular Cloning-A Laboratory Manual, pg 271, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1982). In order to isolate sequences that are represented only once in such a library, the ratio of targeted to non-targeted events should approXimate or exceed 50,000 to 1, for at this ratio one incorrect (non-targeted) clone will be isolated for every correct clone. In most cases, however, sequences in this library will be represented 3-5 times, and a ratio of 10,000-17,000 to 1 would be adequate. Non-targeted events result from ; recombination between the targeting plasmid and regions of homology in the yeast cell, and can be minimized by decreasing the extent of such homology.
As described in Example V, it was determined that deleting the chromosomal copies of selectable marker genes present on the vectors used is desirable - , , , ~ - - : , , -: : : ~: . , : , : ,, , 2 ~ 2 30 because it reduces the occurrence of non- targeted events. As described in Example V, these results indicate that the selection of a targeted clone (target DNA fragment) from a DNA YAC library is 05 feasible and particularly efficient in host yeast cells that carry no homology with selectable markers present on targetin~ vectors.
Non-targeted events might also occur as a result of homologous recombination between sequences on the targeting vector, such as the bacterial plasmid origin of replication or drug resistance marker, and homologous sequences on the YAC vector arms used to construct the DNA library. This homology can be minimized by constructing the targeting vector using a drug resistance marker that is not present in the YAC vector, and by using a bacterial plasmid origin of replication that is divergent from or non-homologous to the origin present on the YAC vector arms. The results described in Example V also place the frequency of non-homologous recombination at approximately 0.003 (l in 30,729), consistent with the invention described in this application. It was possible to select yeast cells carrying homology to the targeting vector even when only l in lO,000 of the cells transformed had such homology. In fact, at this dilution targeted events were isolated multiple (four) eimes, indicating that a clone represented once in a library of 40,000 clones could be isolated.

.:

.

WO 92/01069 PCr/US91104~26 , ~g~2 Tar~etin~_Vectors Tareeting vectors useful in the method described herein are also the subject of the present invention. Targeting vectors of the present 05 invention have two key characteristics: they are non-replicating in the host cell in which the DNA
fragment library is constructed and they include a DNA sequence, referred to as targeting DNA, whicn is homologous to a target DNA fragment which, for the purposes of the invention, is a DNA fragment comprising all or a portion of a desired clone to be identified in and isolated from the DNA library.
Targeting vectors will generally be bacterial plasmids of the YIp class, particularly in those cases in which yeast cell hosts are used. Vectors appropriate for other types of cell hosts can also be constructed using known techniques.
Sequences used as targeting DNA in the targeting vec~or can be entirely homologous to the target DNA fragment, although they need noe be.
They need be only sufficiently homologous that under the conditions used, genetic recombination between vector-borne DNA introduced into the cells and DNA
in YAC in the cells occurs by the host cell recombination pathway or process. Preferably~ a double strand break is introduced into a targeting DNA sequence. Alternatively, a double strand gap can be introduced. The free ends adjacent to the break or gap can be modified to prevent recircularization (e.g., by phosphatase treatment of the ends of the DNA or by creating non-complementary ends by using two different restr-iction enzymes~.

": . : ~ . -WO92/01069 PCT/US91/0~926 2 ~ 2 In addition to targeting DNA, targeting vectors include a selectable marker gene that functions in yeast, an origin of replication and a selectable marker that functions in bacteria (e.g., E. coll.).
05 The selectable marker gene is one which is func-tional (makes selection of transformed cells possi-ble) in the host cell type used for DNA fragment ; library construction. The choice of the yeast selectable marker gene can be made from among many various endogenous yeast ~ene loc i, e . ~ ., ARG4, LEU2, HIS3, HIS4, THRl, URA3, TRP1, LYS2, ADE2, ADE8, and MET2. Alternatively, the yeast selectable marker may be a marker gene that is not endogenous to the yeast genome, but is a foreign gene that confers a selectable phenotype, e.g., a bacterial gene engineered to be expressed in yeast and confer drug resistance on the yeast cells (such as the CAT
; or neo genes from transposons Tn9 and Tn903, respec-tively) or amino acid or nucleoside prototrophy (such as E. coli argH, trpC, or pyrF genes). Other , selectable marker genes useful for this purpose include genes which confer tolerance to metal ions (e.g., the CUPl gene, which confers resistance to copper ions), genes which confer an ability to progress through the cell cycle in cells with a mutant phenotype and genes which result in expres-sion of a cell surface marker.
The suitable selectable marker genes for selection in bacteria include the genes encoding : 30 resistance to the antibiotics, kanamycin, ampicill-in, tetracycline, spectinomycin, streptomycin, erythromycin, or any other marker, including genes : . .- . :, :
~: . ;. ::.. ~ . . :

WO 92101069 PCrIU~91/04926 2 ~ 2 encoding biosynthetic enzymes for which auxotrophic bacterial hosts exist.
Bacterial origins of replication may be derived from a variety of source, including pl5A (exempli-05 fied by the origin of plasmid pACYC184), ColE1,phage M13, phage fl, phage Lambda, or any other replicon that one trained in the art would recogni~e as providing an equivalent function.
vectors constructed and used to screen YAC DNA
libraries are described in detail in Example I and represented schematically in Fi~re 4. Targeting plasmid pl84DLARG contains a selectable marker functional in yeast (ARG4) and a bacterial origin of replication (derived from pACYC184) .
15 Targeting DNA molecules are not limited to molecules of the Yip class. The tar8eting DNA can be a fragment of DNA purified from a larger plasmid, with such a plasmid constructed in such a manner that the desired targeting sequence is interrupted by, among other sequences, a bacterial or yeast replicon. The plasmid is also constructed such that upon cleavage with a restriction enzyme that will release the replicon from the inner section of the targeting sequence, a yeast selectable marker ~5 remains covalently linked to the oueer two ends of the targeting sequence.
Alternatively, a selectable marker and a targeting sequence can be ligated together in vitro and ligation products consisting of one copy of the targeting sequence and one copy of the selectable marker (or multimers consisting of alternating targeting and selectable marker sequences in a uniform orientation~ are purified. These ligation : ~ . : :i: :: : : .. , . . , : , . ..

WO 9~/01069 PCI/US91/04926 2~6~2 products are circularized in vitro and cleaved with a restriction enzyme to introduce a double-strand break or gap in the targeting sequence and leaving the selectable marker intac~.
05 Finally, the two halves of a targeting sequence can be ligatsd to a selectable marker in a single three-way li~ation in vitro to generate a targeting molecule suitable for transformation.
The present invention is illustrated by the following Examples, which are not intended to be limiting in any way.

Methods Used Herein Unless otherwise noted, methods for plasmid purification, restriction enzyme di~estion of plasmid DNA and gel electrophoresis, use of DNA
modifying enzymes, ligation, transformation of bacteria, transformation of yeast by the lithium acetate method, preparation and Southern blot analysis of yeast DNA, tetrad analysis of yease, preparation of liquid and solid media for growth of E. coli and yeast, and all standard molecular bio-logical and microbiological techniques can be carried out essentially as described in Ausubel et a.l. ~Ausubel, F.M. et al., Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley-Interscience, New York, 1987).

EXAMPLE I- SELECTION BY HOMOLOGOUS-RECOMBINATION OF
__________ ________________________________________ A TARGETED DNA CLONE FROM A DNA YAC
LIBRARY
____ __ _ Plasmid pYAC4 (ATCC #67380) was used to con-struct ~ library of human genomic DNA. Human DNA

,` : ' . - . . .

,,f WO92/~1069 PCT/US91/04926 2 ~

was isolated from white blo~d cells (D. surke~ Ph.D.
Thesis, Washington Univ~rsity, st. Louis, MO., 1988), partially digested with EcoRI and ligated to pYAC4 arms digested with EcoRI and BamHI.
05 The ligation mixture was then used to transform yeast host strains, either MGD131-lOc or IV-16d, using the spheroplast method (Burgers, P.M.J. and Percival, K.J., Analytical_Biochemistry 163:391 397 (1987)). (The construction of host strains MGD131-lOc and IV-16d with the ~ppropriate marker deletions is described at the end of the Example.) Since the pYAC4 vector carries the yeast selectable markers TRPl and URA3, transformants can be selected for by growth on plates lacking tryptophan and uracil 11,625 YACs with an average size of 190 kb (0.73 human genome equivalents) are individually grown in the wells of 96-well microtiter plates; 0.1 ml was taken from each well and pooled in three subpools of approximately 4,000 clones each. For each subpool, an equal volume of 30~ glycerol was added and the subpool was aliquoted and frozen at -70~C.
For a library comprising 734 of one genome, and assuming equal representation of all clones, the probability that it contains any one specific human DNA sequence is just over 0.5. The probability that one of six different fragments of DNA is represented in the library is 1-(0.5) , or 0.98.
The construction of the targeting plasmid pl84DLARG is described below and illustrated in Figure 4. It carries the yeast ARG4 gene (Beacham, I.R. et al. (1984) Gene 29:271-279) as a selectable , .

WO92/01069 P~T/U~91/049~6 2 ~ ~3 ~ 36-marker, and its bacterial origin of replication i5 derived fro~ pACYC184 (Chang, A.C.Y. and Cohen, S.N.
(1978) Jour_al of Bacteriolo~y 134:1141 1156.), which shares only limited sequence homology to the 05 pBR322 origin used on pYAC4. The entire chromosomal copy (a 2.0 Kb HpaI DNA fragment) of ARG4 has been deleted in the library host strains IV-16d and MGD131-;0c The 2.2 kb BclI-ClaI fragment from pACYC184 (Chang, A.C.Y. and Cohen, S.N., (1978), Journ_1 of-sacteriolo~y 134:1141-1156.) containing the pl5A origin of replication and the chlora-mphenicol resistance gene was ligated to BamHI-AccI
digested pMLC28 (a derivative of pSDC12 carrying the pUC18 multiple cloning site; Levinson et al., J.
_~ Ge_., 2:507-517 (1984); plasmid pUC18 (ATCC ~37253) can substitute for pMLC28 in the construction of pl84DLARG described here). BamHI
and AccI cut this plasmid one time each, in the polylinker. The ligation mixture was digested with SacI and HindIII, which cut in the pMLC28 polylinker, and the digested DNA was created with T4 DNA polymerase to generate blunt ends. The DNA was ligated under dilute conditions to promote circularization, and the ligation mix was treated with the restriction enzyme AvaII (to linearize any parental molecules) prior to transformation into bacteria. One plasmid, pl84DL, carrying only the sequences contained within the larger of the two BclI-Clal fragments of pACYC184 and a permuted version of a portion of the pMLC28 polylinker was identified. Plasmid pHpa5 (provided by N. Schultes and J. Szostak; Department of Molecular Biology, ... .

~. :. ., ~ . :.

W092/01069 PCT/US9t/04926 37 2 ~ g ~ ~ ~ rJ

Massachusetts General Hospital, Boston, MA 02114) car~ies the ARG4 gene as a 2.0 Kb HpaI fragment inserted into the HincII site of pMCL12 (a deri~ative of pSDC12 carrying the pVC12 multiple 05 cloning site). Levinson et al., J. Mol. A~pl. Gen., 2:507-517 (1984). This plasmid was cut at the Ps~I
and SmaI sites flanking the ARG4 insert, and the ARG4 fragment was ligated to PstI-SmaI cut pl84DL.
A plasmid carrying a single copy of the ARG4 gene inserted in the orientation shown in Figure 4 was isolated and designated pl84DLARG. Figure 4 is a map of plasmid pl84DLARG.
Genomic fragments for tyrosine hydroxylase (chromosome 11), metallothionein II pseudogene (chromosome 4), anonymous DNA markers D16S3 and D16S37 (chromosome 16), and a 1.9 kb HindIII frag-ment located 5' of the epsilon globin gene (chromosome 11) were subcloned into pl84DLARG and used for selection of clones by recombination from a YAC library. With the exception of the tyrosine hydroxylase gene ~ragment, all of the fragments were blunt ended by treatment with T4 DNA polymerase and ligated to SmaI cut pl84DLARG. The tyrosine hydroxylase gene fragment was cloned into the BamHI
site of pl84DLARG. A 1.3 kb HpaI-BamHI fragment from the 5' end of the beta globin gene (chromosome 11) was blunt-end ligated to the same 2.2 kb BclI-ClaI fragment used to construct pl84DLARG. The beta- and epsilon-globin fragments are 1.3 and 1.9 kb fragments, respectively, from the human beta-hemoglobin locus on chromosome 11. The beta-globin fragment (ATCC ~39698) was subcloned . - ., .: , ~

' 1 : : .':~ . , : . .

wo 92/01069 PCl /US91/04926 .
~ 2 -38- .

from pHU5'be~a (Treco, D., et _1_, Mol._Cell._Biol_, 5:~029-2038, 1985), and includes sequences from positions 61,338 (HpaI site) through 62,631 (BamHI
si~e) in the Genbank HU~HBB sequence. This fragment 05 includes the 5' end of the human beta-globin gene.
The AvaII si~e at Genbank map position 62,447 was used to introduce a double-strand break for targeting, leaving 1.1 and 0.18 kb of homology on either side of the break. The 5' epsilon-globin probe (ATCC ~59157), is a ~indIII fragment and includes sequences centered approximately 15 kb 5 to the epsilon-globin gene (ATCC ~59157), from positions 3,266 through 5,172 in the Genbank HUMHBB
sequence. The ApaI sites at map positions 4,361 and
4,624 were used to create a 0.26 kb double-strand gap for targeting, leaving 1.1 and 0.5 kb of homology on either side of the gap.
Properties of the remaining four genomic DNA
fragments are as follows: tyrosine hydroxlase tchromosome 11; 2.3 kb BamHI fragment; ATCC ~59475;
double-strand break made with HindIlI, 0.6 kb from end); metallothionein pseudogene (chromosome 4; 2.8 kb HindIII-EcoRI fra~ment; ATCC #57117; double-strand break made with NdeI, 0.4 kb from end);
anonymous DNA marker D16S3 (chromosome 16; 1.5 kb HindIII fragment; ATCC #59447; double-strand break made with ApaI, 0.75 kb from end); D16S37 (chromosome 16; 2.3 kb HindIII fragment; ATCC
#59189; double-strand break made with ApaI, 0.95 kb from end).
Each targeting plasmid was linearized with a restriction en~yme that cuts within the human DNA

..
. .

WO92/0106~ PCT/US91/04926 (the targeting DNA) and 20 ~g of digested DNA was used to transform the pooled library. Equal volumes of the three library subpools were thawed, mixed and inoculated into CM -ura - trp ~edium containing 40 05 ~/ml each of kanamycin and ampicillin. This culture was ~rown overnight at 30C with vigorous shaking and harvested at a density of 1.86 x 10 cells/ml. The cells were transformed using the lithium acetate method (Ausubel, F.M. et al. Current Protocols in Molecular Biolo~y, Supplement 5, Green Publishing AssociaCes and Wiley- Incerscience, New York 1987). 20 ~g of plasmid cuc within the human DNA was used to transform 7 x 10 cells in a volume of 0.2 ml, and the entire transformation mix was spread onto the surface of eight selective plates (complete minimal media lacking uracil, tryptophan, and arginine) and incubated ae 30C for 3-7 days.
Transfor~ants were analyzed by restriction enzyme digestion and Southern hybridization analysis. DNA was prepared from each of the candidates and digested with the same enzyme used to linearize the targetin8 plasmid, The Southern blot was probed with 32p radiolabeled ARG4 DNA, Homo-logous integration events are identified by hybridi-zation to a single band of exactly the same size asthe linearized transforming DNA molecule [the "Unit Length Linear~ band (ULL); Figure 2]. A ULL can only be generated if integration occurs into a DNA
sequence that contains the restriction enzyme site in question, and contains enough homology surrounding that site to allow the re-synthesis (by repair) of the restriction enzyme site on the !

:, , ; "~ ' ' ~: -, ,.` ` ` ; ~'`
: ~
, WO9~/01069 PCT/USgl/04926 2~6~2 ~40-targeting plasmid. Candidates that display a ULL
are assumed to be homologous integration evenes and are subjected to further analysis. Unit length linears were seen for 6 of 21 epsilon-globin candi-05 dates analyzed and for 3 of 14 beta-globin candidates. No unit length linears were observed for the other targeting sequences used.
Figure 5 is a restriction enzyme and Southern blot analysis of clones selected by targeting with hu~an epsilon- and beta-globin sequences. In the left panel, DNA from nine clones selected as arg+
were digested with AvalI (the enzyme used to make the double-strand break in the beta-globin targeting sequence). In the right panel, DNA from nine clones selected as arg+ were digested with ApaI (the enzyme used to make the double-strand break in the epsilon-globin tar~eting sequence). The asterisks identify clones correctly selected by homologous recombi-nation. The lanes marked M were loaded with purified beta-globin targeting plasmid digested with AvaII (left panel), or purified epsilon-globin targeting plasmid digested with Apal (right panel).
The size of this marker fragment is identical to the size predicted for correctly targeted events. The arrowheads indicate the fragment size predicted for correctly targeted events, 5.6 kb in the left panel and 6.2 kb in the right panel. Hybridization was with P labeled ARG4 DNA.
Three each of the beta- and epsilon-globin positives were further analyzed by CHEF gel electro-phoresis (Chu, G., Vollrath, D., and David, R.W.
Science, 234:1582-1585 (1986)), and restriction ~ .

WO 92/01069 PCl /U~91/(~4926 enzyme and Southern hybridization analysis, probing with epsilon- or beta-globin DNA as appropriate.
This analysis demonstrated that all six YACs are identical and carry both beta- and epsilon-globin 05 DNA, as would be expected since these two genes lie only 40 kb apart on human chromosome 11. In all six YACs the ARG4 DNA has integrated onto a YAC of 190 kb and the pl84DLARG constructs have integrated as predicted into the homologous DNA within the globin 10 locus, Homologous recombination has been successfully used to isolate unique ~enes from a DNA YAC library.
The YACs isolated encompass the entire beta-~lobin locus from at least 16 kb 5~ to the epsilon gene down to the beta globin gene, along with about 130 kb of flanking DNA. In addition, a similar selection protocol performed with the same DNA YAC
library resulted in the isolation of YACs from ~he ~-globin locus after the library had been stored at -70C for over fourteen months. It is thus disclosed here, for the first time, that it is possible to isolate clones from a human DNA YAC
library by homologous-recombination selection.

Saccharomyces Cerevisiae Host Strain Construction T~e construction of strains of S. cerevisiae carrying chromosomal deletions of ARG4 (MGDl31-lOc) or ARG4 and TRP1 (IV-16d) can be carried out as follows:

, WO92/~1069 PCT/US91/04926 2 ~ 8 ~ 9 2 42-Deletion of_ARG4 The internal 2.0 kb HpaI fragment carrying the entire ;structural gene and regulatory ele~ents for the yeast argininosuccinate lyase gene (ARG4) is 05 deleted from a plasmid consistin~ of the 11 kb BamHI
fra~ment isolated from p(SP013)2 (Wang, H-T., et al., Molecular and Cellular Biolo~y, 7:1425-1435, 1987) inserted into the 8amHI site of pUC19 (ATCC
#37254j, by digestion with HpaI and relegation of the DNA under dilute conditions (1 ~g/ml). The rssulting plasmid is digested with BamHI and intro-duced into an S. cerevisiae strain carrying the wild-type alleles for ARG4, TRP1, URA3, and LEU2, and carrying any non-reverting his3 allele. The transformation is carried out in conjunction with any plasmid carrying yeast CEN and ARS elements, and the yeast HIS3 gene, using standard co-trans-formation conditions (Ausubel et al., 1989, Chapter 13). A useful plasmid for this purpose can readily be constructed by subcloning the 1.7 kb BamHI
fragment from pRB15 (ATCC #37062) into the BamHI
site of YCp50 (ATCC #37419). His Cells are screened for arginine auxotrophy by replica plating onto CM -arginine plates. His arg cells are grown in the absence of selection for HIS3, and single colonies are isolated and screened for histidine auxotrophs. DNA from his arg colonies is prepared and analyzed by restriction enzyme and Southern blot a~alysis to identify transformants carrying the ARG4 deletion (arg4Q). This protocol is used to generate strain MGD131-lOc used in Example I abo~e.

.
-. , -W092/0~069 PCT/US91~04926 Deletion of TRP1 _________________ In a yeast strain of opposite mating type as that used ~above, also carrying mu~ant alleles for LEU2 and URA3 (leu2 , ura2 ), an identical procedure 05 is carried out, but using a linear fragment of DNA
carryin~ a deletion of the yeast gene for N-(5'-phosphoribosyl)-anthranilate isomerase (TRPl). This is accomplished by subcloning the 8amHI-XhoI
fra8ment from pBR322-Sc4120 (Stinchcomb, D.T., et al., Journal of_Molec_ lar_ Biolo~y, 1~8:157 179, 1982) into BamHI-XhoI cut pGEM7, (Promega, Madison, Wisconsin) followed by deletion of the 1.2 kb EcoRi fragment containing TRPl and ARSl. The resulting plasmid, pK2, is digested with BamHI and XhoI and co-transformed with a HIS3-CEN-ARS plasmid, like that described above, selecting for histidine prototrophs, and following the strategy outlined above ~o identify cells carrying the TRP1 deletion ~trplQ). These cells are mated with cells carrying arg4~, and diploids heterozygous for the two deletions are isolated. This strain, TD7-16d, is sporulated, subjected to tetrad analysis, and spores with appropriate phenotypes are analyzed by restriction enzyme and Souehern blot analysis to identify a srrain with both the arg4a and trpl~
alleles. The genotype of TD7-16d is: a/~, arg4~/ARG4, LEU2/leu2-3,112, ura3-52/URA3, trpl-289/erpl~, ade2-lOl/ade2-lOl, cyhS/cyhr, (CYH2/cyh2), his3~1/his3~1.

, ~ : . : . -WO 92/01069 PCI/lJS91/04926 EXAMPLE_II SELECTION_BY_~OMOLOGOUS RECOMBINATION
OF A DN__CLONE_FROM___DNA_YAC_LIBRARY
USING ONE-STEP GENE DISRUPTION
______________________________ The method of one-step gene disruption 05 (Rothstein, R.J., Methods in EnzymoloF,y, ______________ ____ 101:202-211, Academic Press, New York, 1983) can be adapted for use in the selection of clones from DNA
libraries by homologous recombination. In this embodiment, a selectable marker is inserted into the targetin~ sequence. The targeting sequence, with the embedded selectable marker, is subsequently isolated as a single linear fragment (as diagrammed in Figure 3) and transformed into the pooled DNA YAC
library, as described in Example I. Correctly targeted clones arising as a result of homologous recombination between the targeting molecule and specific DNA clones within the library will carry a single copy of the targeting sequence that is disrupted by the presence of the selectable marker, and will migrate at a specific and predictable position after restriction enzyme digestion and Southern blot analysis, using either ARG4 or the targeting sequence as a radiolabeled probe. This is in contrast to the process described in Example I, in which the correctly targeted DNA clones have ~wo uninterrupted copies of th~ eargeting sequence flanking the selectable marker.
Figure 3 illustrates the selection by homo-logous recombination of a DNA clone from a DNA YAC
library using one-step gene disruption. The thin line represents an insert of DNA in the form of a yeast artificial chromosome (YAC). The solid box is the DNA fragment, a sequence of DNA constituting a ..
. : ~ ', ~' ' ' , WO92/~1069 P~T/US91/04926 ~45-portion of a DNA YAC clone found in the library that is homologous to the targeting sequence. In the diagram, the targeting sequence (solid boxes) has been modified by the insertion of the yeast ARG4 05 gene (open box). The remaining portions of the DNA
YAC are comprised of the YAC vector arms: the thick lines represent plasmid sequences for replication and selection in bacteria. The shaded boxes repre-sent genetic markers used for selection in yeast (yeast selectable markers URA3 and TRPl~. The solid arrowheads and circle represent teIomeres (TEL) and a centromere/ yeast replicaeion origin (CE~/ARS), respectively. Figure 3a depicts the targeting molecule aligning with the target sequence on the DNA YAC. Figure 3b depicts the product of homologous recombination between the targeting and target sequences, with the targeting sequence having replaced the target sequence.
As a specific ex.ample of this embodiment of the basic concept, the 1.9 kb 5' epsilon-globin fragment (see Example I) is subcloned into the HindIII site of pUClô (ATCC #37253). The resulting plasmid is digested with ApaI, dropping out a 0.26 kb ApaI
fra~rnent from the central portion of the 5' epsilon-globin insert. The 3' ApaI overhangs are made blunt with T4 DNA polymerase, and the resulting material is ligated to the purified ARG4 2.0 kb HpaI
fragment (Beacham, I.R., Ge_e, 29:271-179, 1984).
The resulting plasmid, with ARG4 disrupting the
5'epsilon-globin sequence, is digested with HindIII
and transformed into the DNA YAC library, as described in Example I. The specific example pre-sented results in the replacement of 0.26 kb of the ' , :: :
.:
., .

2 ~ 2 -46-S~ epsilon- globin DNA with th~ ARG4 sequence, since ApaI is not unique in the targeting sequence. For anzymes that are unique in the targeting sequence, however, the result will be a simple insertion.
5 EXAMPLE III: USE_OF_TERMINAL_FRAGMENTS_DERIVED_FROM
YE_ST_ARTIFICIA__CHROMOS_M__CLONES_F_R
THE ISOLATION OF CLONES KNOWN TO BE
___________________________________ PRESEN_ IN____EAS___RTIFICI_L
CHROMOSOME LIBRARY--A MODEL SYSTEM TO
_____________________________________ TEST_THE_FEASIBILITY_OF__IBRA_~' S REENING_BY_HOMOLOGOUS_RECOMBINA_ION
We used homologous recombination screening to extract a clone from the library that was known to exist within the library. Sinoe the vec~or arm containing the TRPl gene in YACs constructed with pYAC4 contains a plasmid replicon and a selectable marker (the beta-lactamase gene conferrin~
ampicillin resistance), the technique of "plasmid rescue" was used to isolate terminal fragments from two YACs constructed in the vector pYAC4. The restriction enzyme XhoI cleaves at a single site within the TRPl vector arm, at the junction between the telomere and pBR322 sequences. Complete digestion of YAC DNA with XhoI should produce a res~riction fragment devoid of telomeric sequences, containing a functional plasmid replicon and Amp marker, and har~borinz a segment of human DNA that was ad;acent to the vector arm in the original YAC
clone and extends to the terminal XhoI site in the human DNA insert.
A group of 161 YACs within the library were constructed using the host yeast strain MGD131-lOc .

(~enotype a leu2-3,112 ADE2 cyh2 his~l trpl-289 agr4~ ura3-52). Total 3NA from two clones in this group was digested with XhoI, ligated under dilute conditions to promote intramolecular 05 circularization, and transformed into E. coli (all steps carried out essentially as described in Ausubel et al., 1988 [above]. Plasmid DNA was isolated from ampicillin resistant colonies and subjected to restriction enzyme analysis. One human DNA fra~ment from each of the two rescued plasmids was subsequently blunt-ended by treatment with T4 polymerase and ligated into the SmaI site of pl84DLARG. The fragments, lOB and 8A, are 1 and 4 kb fragments, respectively, of human DNA lying adjacent to the TRPl vector arms in two differen~
YACs. The resultin~ constructs (plasmids pl84-lOB
and pl84-8A) were digested with a number of restriction enzymes which do not cleave pl84DLARG to identify an enzyme that would cut within the human DNA to promote targeting. 20 ~g of each construct was digested with the appropriate targeting enzyme and used for library s~reening, essentially as described in Example 1. Fragment 8A contains a single KpnI site lying 2.8 kb from one end and this enzyme was used to introduce a unique double strand break within the inserted sequence in pl84~8A.
Fragment lOB contains a single AvaII site lying 0.5 kb from one end and this enzyme was used to introduce a unique double strand break within the inserted sequence in pl84-lOB.
Eleven arg colonies resulting from screening with clone 8A were isolated and analyzed. Similar , .

2 ~ 2 to strain IV-16d (Example l and ATCC Accession No.
74010) strain M~Dl31-lOc carries a 2 kb deletion encompassing the entire ARG4 gene. However, the two strains differ with regard to their LEU2 ~enotype;
05 IV-l6d is leu and MGDl31-lOc has a leu phenotype.
Seven of the ll colonies displayed a leu phenotype, suggesting that they indeed represented independent isolates of the original YAC from which clone 8A was derived (a very strong possibility since strain MGDl31-lOc is the host for only 161 out of the 11,625 YACs (1.4~) in the libr~ry). Seventeen arg colonies resulting from screening with clone lOB
were isolated and analyzed. Three of the 17 colonies displayed the leu phenotype. The presence Of the leu ~arker strongly suggests that these clones represent isolates of the original YAC from which clone lOB was derived.
DNA was prepared from each of the seven leu colonies isolated by screening with clone 8A as well as one of the leu colonies. DNA was digested with the same enzyme used to linearize the transforming DNA molecule (KpnI~. A Southern blot of these digests were probed with 32-P labeled ARG4 DNA. As described in Example l, homologous integration events should reveal hybridization to a single fragment of e~actly the same size as the linearized transforming DNA molecule (referred to in Example l as a Unit Length Linear Fragment, or ULL). Of the eight clones analyzed, all seven in strain MGDl31-lOc (the leu colonies) represent homologous events, while the single leu+ transformant analyzed does not (Figure 6). Thus, seven out of eleven .' ~ ', J' ` ~, ' 49 2 ~ 9 2 candidate clones isolated were correcely targeted events. A similar analysis was performed on each of the three leu colonies isolated by screening with clone lOB. All ~hree clones displayed a ULL upon 05 Southern blot analysis, while 14 leu transformants did not.
To confirm that the three homologous events isolated by screening with clone lOB and the seven homologous events isolated by screening with clone 8A represent the independent isolates of tne same YACs, we have mapped the termini of the YACs in these ten clones. Figure 6 shows the result of this analysis. Three bands are evident in each lane, corresponding to the ULL, the left arm, and the right arm of the YAC. The bands migrate at identical positions in all seven YACs isolated with 8A, and at different, but identical positions in all three YACs isolated with lOB. These data show that the distance to the nearest KpnI site at each end of the seven 8A YACs is identical, while the three lOB
YACs display similar behavior for the positions of their terminal AvaII sites.

EXAMPLE IV: SCREENING OF A HUMAN YEAST ARTIFICIAL
CHROMOSOME LIBRARY BY HOMOLOGOUS
RECOMBINATION TO ISOLATE A YEAST
_______________ ________________ ARTIFICIAL CHROMOSOME CLONE DERIVED
FROM THE HUMAN ADENOSINE DEAMINASE
LOCUS
Synthetic oligonucleotides o6 and o7-2 were used in the polymerase chain reaction to amplify a l,~76 base pair fragment of the human ADA gene ```' '' . : :
- ' ~.

.. : , 2~6~99 correcponding to positions 34,243.-35,618 ~Genbank entry HUMADAG) from total human genomic DNA isolated from peripheral blood leukocytes. The amplified fra~ment was digested with PstI and the 852 base 05 pair subfragment corresponding to HUMADAG positions 34,349-35,201 was isolated and cloned into the ~s~I
site of plasmid pl84DLARG (Example 1) to genetic pl84DLARG/PCRF.5. One inser~ orientation was chosen (that with HUMADAG position 34,349 adjacent to the 3' end of the yeast ARG4 gene in pl84DLARG. The resultin~ plasmid was purified and 20 micrograms was linearized at the unique EcoNI site within the human ADA insert (corresponding to HUMADAG position 34,657) prior to transformation into the pooled YAC
library. Transfor~ation of the pooled YAC library was performed exactly as described in Example 1, with the exception being that the YAC library consisted of an additional 3,585 clones, for a total of 15,210 clones representing approximately 1.2 genome equivalents, ~ our arg transformants were isolated. Three of these are displayed in Figure 8 and all three displayed a unit-length linear fragment upon restriction enzyme digestion with EcoNI and Southern blot analysis. Analysis of the fourth arg transformant confirmed that i~ carries the same insert as YAC 184ADA.C and 184ADA.D. All four transformants harbor a similarly sized YAC of ca.
200 kb as judged by CHEF gel electrophoresis. The intensity of the ULL band in DNA prepared from YAC
184ADA.B and other data indicate that YAC 184ADA.B

.
,-WO92/01069 PCT/US91/n4926 2~3~2 has undergone multiple tande~ integrations of thetargeting plasmid.
Comparison of a representative YAC, YAC
184ADA.C, with human ~enomic DNA by restriction 05 enzyme and Southern hybridization analysis using multiple probes and restriction digests confir~ed that this YAC indeed conteins sequences from the human ADA locus.

OLIGONUCLEOTIDE o6 5' AGATCTGTTT GAGGCTGCTG TGAG

Bases numbered 1-24 corresponding to positions 34;243-34,266 in GENBANK Entry HUMADAG.

OLIGONUCLEOTIDE o7-2 lO 20 5' AGATCCGGCA ACTTGTAGTA CCCAGGATG

Bases numbered 7 29 corresponding to positions 35,618-35,596 in GENBANK Entry HUMADAG. Bases 1-6 correspond to one of the four possible recognition sequences for the restriction enzyme BstYI, added to facilitate cloning.

EXAMPLE_V: QUANTIFICATION OF EFFECT OF CHROMOSOMAL
DELETIONS OF HOMOLOGOUS SEQUENCES
PRESENT IN HOST CELL

W092/01069 PCT/US91/04~26 Orr-Weaver et al. (Proc. Natl. Acad. Sci. USA, Vol. 78, 10:6354-6358 (1981)) showed that a plasmid carrying the yeast LEU2 gene results in leu transformants at a frequency of 1.4-1.7 per ~g of 05 DNA when a double-strand break was made in the pBR322 portion of the plasmid. This is 1/lO of the frequency at which leu transformants arose when targeting was directed to the LEU2 gene by a double-strand break in LEU2 sequences (12-17 per ~g of DNA). Similarly, when a HIS3 containing plasmid was cut within pBR322 sequences, his transformants appeared at 1/50 of the ra~e observed when the same plasmid was cut within ~IS3. In both cases, the non-tar~eted prototrophs were demonstrated to be the results of recombination between the plasmid and the chromosomal leu2 and his3 mutant genes. Thus, screening a library for one clone out of 50,000 by homologous recombination without deletion of the chromosomal LE~2 gene would be expected to yield 5,000 leu+ transformants which arise through homolo-gous recombination with the yeast eenome when ~he targeting plasmid carries LEU2, even if a double-strand targeting break is made in another part of the plasmid. The results suggest, however, that dele~ing the chromosomal copies of LEU2 and HIS3 would eliminate virtually all of the nontargeted events.
The advantage of chromosomal deletions from host cells for the purposes of the method was quantified as follows: A plasmid carrying the yeast , ARG4 ("target") and URA3 ("marker") genes was transformed into a mixture of yeast cells after ~: ~ :: ': : : :: :, ;.

WO92/~1069 PCT/US91/049~fi ~33~2 making a double-strand break at the unique BclI si~e in the ARG4 sequence. All of the cells in the mixture had homology to URA3, bl~t only 1 in l,O00 or 1 in lO,OOO had homolo~y to ARG4 . This type of dilution experiment measures the relative frequencies of targeted and non-targeted events.
For example, using 1 ~g of DNA and a 1 to l,OO0 dilution, the isolation of 5 yeast colonies by homologous rerombination at ARG4 indicates that 5.000 cells were theoretically capable of a targeted event, but only 5/5,000 cells actually had the necessary homology at ARG4. The targeting frequency is therefore equivalent to 5,000 targeted events per ~g in an undiluted culture. If, in the same experiment, 5 colonies were isolated that were independent of homolo~y at A~G4 (recombination at URA3 or elsewhere, non-targeted events), the frequency of these non-targeted events is 5 per ~g, and the ratio of targeted to non-targeted events in this experiment would be 1,000 to 1.
For the 1 in 1,000 dilution, 78 targeted trans-formants were isolaeed (by recombination with ARG4;
equivalent to 78,000 targeted events) and 17 by recombination elsewhere (non-targeted events). At a dilution of 1 in 10,000, four targeted events (equivalent to 40,000 targeted events~ and seven non-targeted events were isolated. The ratio of targeted to non-targeted events is thus (78,000 +
40,000) divided by (17 + 7), or 4,917 to 1. This ratio would lead to approximately 10 incorrect events for every one correct event when screening a li~rary ~or a sequence present on 1 in SO,OO0 YACs, .
. ~- , - ' ' ' ' WO 92/OlOS9 PCl /US91/04926 ~6~2 which is several-fold too hi~h to be ~enerally acceptable, althou~h the use of URA3 as a targeting ~arker is clearly preferred over the use of the LEU2 or HIs3 markers previously used in tar~eting studies 05 (Orr-~eaver et al., 1981). 84% ~16 of 19 analyzed) of the non-targeted events where in fact due to recombination between the URA3 marker on the plasmid and the chromosomal ura3 locus. If there were no homology between the targeting plasmid and the chromosomal ura3 locus, then the non-targeted events resulting from homology at the ura3 locus are removed from the analysis and the ratio increases to 30,729 to 1. At this ratio, a sequence represented 3 times in 50,000 YACs would be correctly tar~eted 1.8 times for every one non-targeted event. This ratio would also result in the favorable ratio of one correct event for every 1.6 incorrect events when screening a library for a sequence present on only 1 in 50,000 YACs.
These results indicate that the selection of a targeted clone from a DNA YAC library is feasible and particularly efficient in host yease cells thae carry no homology with selectable markers present on targeting vectors.

'"' :!. : : : . ' .

2 ~ 2 PCT A~pll~ant's Guide - Volume I - Annex M3 ~ _ _ _ Intomltlionrtl Appilc~s~n Plo: PCT/
MICROORGAI'~ISMS
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In respect of those designations in which a European Patent is sought the Applicant hereby informs the European Patent Office under European Rule 28(4) that, until the publication of the mention of the grant of the European Patent or until the date on which the European Application has been refused or is withdrawn or is deemed to be withdrawn, the availability of the biological material deposi~ed with the American Type Culture Collection under Accession Nos. 74010 and 40832 shall be effected only by the issue of a sample to an expert nominated by the requester in accordance with European Rule 28(5) _ C. W~lUlll~nD 'r~ 0~ ~IICIt I~OIC~O~- ~RII IIIADO r (U ~r~o Inr~ ono nn nol hr r~ll d~lUon Utd 3~tttt) EP, AU, CA, JP and KR

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Claims (31)

1. A homologous-recombination method for identifying and isolating a target DNA fragment from a DNA fragment library constructed in eukaryotic host cells, comprising the steps of:
a) providing a DNA fragment library in a population of eukaryotic host cells in which genetic recombination between DNA
introduced into the eukaryotic host cells and DNA present in the eukaryotic host cells occurs by homologous recombination;
b) introducing into the population of host eukaryotic cells containing the DNA
fragment library a selectable marker gene for selection in the host cell and a targeting DNA sequence homologous in part to a target DNA fragment, the selectable marker gene and the targeting DNA sequence linked to one another, thereby producing a mixed population of eukaryotic host cells;
c) maintaining the mixed population of eukaryotic host cells under conditions appropriate for homologous recombination to occur, whereby eukaryotic host cells containing the target DNA fragment are stably transformed with the selectable marker gene and the targeting DNA sequence as a result of homogous recombination between the target DNA fragment and the targeting DNA sequence and stably transformed eukaryotic host cells with a selectable phenotype are produced; and d) culturing the product of step (c) under conditions appropriate for selection of stably transformed eukaryotic host cells with a selectable phenotype.
2. A method of Claim 1 wherein the eukaryotic host cells are yeast host cells, the targeting DNA
is present in a replicating linear vector contained within yeast cells which are of mating type opposite to the mating type of the yeast host cells and referred to as yeast cells containing the targeting DNA and the targeting DNA is introduced into yeast host cells by mating yeast host cells with yeast cells containing the targeting DNA.
3. A method of Claim 1 wherein the targeting DNA
vector is a linear DNA fragment, wherein the targeting DNA sequence is disrupted by the insertion wherein of the selectable marker gene for selection of the eukaryotic host cell.
4. A homologous-recombination method for identifying and isolating a target DNA fragment from a DNA fragment library constructed in eukaryotic host cells, comprising the steps of a) providing a DNA fragment library in a population of eukaryotic host cells in which genetic recombination between DNA
introduced into the eukaryotic host cells and DNA present in the eukaryotic host cells occurs by homologous recombination;
b) introducing into the population of host eukaryotic cells containing the DNA
fragment library a targeting DNA vector which is non-replicating in the eukaryotic host cells, the targeting DNA vector having a selectable marker gene for selection in the host cell and a targeting DNA sequence homologous in part to a target DNA fragment, thereby producing a mixed population of eukaryotic host cells;
c) maintaining the mixed population of eukaryotic host cells under conditions appropriate for homologous recombination to occur, whereby eukaryotic host cells containing the target DNA fragment are stably transformed with the selectable marker gene and the targeting DNA sequence present in the targeting DNA vector as a result of homologous recombination between the target DNA fragment and the targeting DNA sequence and stably transformed eukaryotic host cells with a selectable phenotype are produced; and d) culturing the product of step (c) under conditions appropriate for selection of stably transformed eukaryotic host cells with a selectable phenotype.
5. A method of Claim 4 wherein the targeting DNA
molecule is a targeting plasmid.
6. A method of Claim 5 wherein the targeting plasmid has a double-strand break introduced within the targeting DNA sequence.
7. A method of Claim 4 wherein the target DNA
fragment is cDNA.
8. A method of Claim 6 wherein the targeting plasmid is a YIp plasmid and DNA fragments are present in eukaryotic host cells in artificial chromosomes, the artificial chromosomes ad-ditionally comprising all of the DNA sequences necessary for the chromosome to participate in host cell replication and chromosome segregation.
9. A method of Claim 6 wherein the target DNA
fragment is: a mammalian DNA sequence; a human DNA sequence; a plant DNA sequence; a mammalian gene; a human gene; or a plant gene.
10. A method of Claim 8 wherein the selectable marker gene is selected from the group consisting of genes which confer a selectable phenotype on eukaryotic host cells and the selectable phenotype is: antibiotic resistance, nutrient prototrophy, tolerance to a metal ion, ability to progress through the cell cycle or expression of a cell surface marker.
11. A mammalian DNA sequence, a human DNA sequence, a plant DNA sequence, a mammalian gene, a human gene, or a plant gene isolated by the method of Claim 9.
12. A homologous-recombination method for identifying and isolating a target DNA fragment in a DNA fragment library constructed in yeast cells, comprising the steps of:
a) providing a DNA fragment library in a population of host yeast cells, wherein DNA fragments are present in yeast artificial chromosomes and a host yeast cell contains one yeast artificial chromosome or a low-copy number of yeast artificial chromosomes:
b) introducing into the yeast cells containing the DNA fragment library a selectable marker gene for selection in yeast linked to targeting DNA homologous in part to a target DNA fragment, thereby producing a mixed population of yeast cells;
c) maintaining the mixed population of yeast cells under conditions appropriate for homologous recombination between targeting DNA and target DNA fragments present in yeast artificial chromosomes, whereby yeast cells containing the target DNA
fragment are stably transformed with the selectable marker gene and the targeting DNA sequence as a result of homologous recombination between the target DNA
fragment and the targeting DNA sequence and stably transformed yeast cells with a selectable phenotype are produced; and d) culturing the product of step (c) under conditions appropriate for selection of yeast cells with a selectable phenotype.
13. A method of Claim 12 wherein the targeting DNA
molecule is a linear DNA fragment, wherein the targeting DNA sequence is disrupted by the insertion of the selectable marker for yeast into the targeting DNA.
14. A homologous-recombination method for identifying and isolating a target DNA fragment in a DNA fragment library constructed in yeast cells, comprising the steps of:
a) providing a DNA fragment library in a population of host yeast cells, wherein DNA fragments are present in yeast artificial chromosomes and a host yeast cell contains one yeast artificial chromosome or a low-copy number of yeast artificial chromosomes;
b) introducing into the yeast cells containing the DNA fragment library a targeting DNA vector which is non-replicating in yeast, the targeting DNA vector having a selectable marker gene for selection in yeast and targeting DNA
homologous in part to a target DNA
fragment, thereby producing a mixed population of yeast cells;

c) maintaining the mixed population of yeast cells under conditions appropriate for homologous recombination between targeting DNA and target DNA fragments present in yeast artificial chromosomes, whereby yeast cells containing the target DNA
fragment are stably transformed with the selectable marker gene and the targeting DNA sequence present in the targeting DNA
vector as a result of homologous recombination between the target DNA
fragment and the targeting DNA sequence and stably transformed yeast cells with a selectable phenotype are produced; and d) culturing the product of step (c) under conditions appropriate for selection of yeast cells with a selectable phenotype.
15. A method of Claim 14 wherein the targeting DNA
vector is a plasmid and the selectable pheno-type is: antibiotic resistance, nutrient prototrophy, tolerance to a metal ion, ability to progress through the cell cycle, or expression of a cell surface marker.
16. A method of Claim 15 wherein the targeting plasmid has a double-strand break introduced within the targeting DNA.
17. A method of Claim 16-wherein the target DNA
fragment is: a mammalian DNA sequence; a human DNA sequence; a plant DNA sequence; a mammalian gene; a human gene; or a plant gene.
18. A mammalian DNA sequence, a human DNA sequence, a plant DNA sequence, a mammalian gene, a human gene, or a plant gene isolated by the method of Claim 17.
19. A homologous-recombination method for identi-fying and isolating a target DNA fragment in a yeast artificial chromosome library, comprising the steps of:
a) providing a yeast artificial chromosome library in a population of host yeast cells, wherein DNA fragments are present in the yeast artificial chromosomes and one copy or a low-copy number of the yeast artificial chromosomes is present in host yeast cells;
b) introducing into the yeast artificial chromosome library of (a) a targeting plasmid which is a bacterial plasmid non-replicating in yeast, the targeting plasmid having a selectable marker gene for selection in yeast and targeting DNA
in which there is a double strand break;
c) maintaining the product of step (b) under conditions appropriate for homologous recombination between targeting DNA and target DNA fragments present in yeast artificial chromosomes, whereby homologous recombination between a target DNA
fragment and targeting DNA produces host yeast cells having a selectable phenotype;
and d) selecting host yeast cells having the selectable phenotype conferred in step (c).
20. A method of Claim 19 wherein the target DNA
fragment is: a mammalian DNA sequence; a human DNA sequence; a plant DNA sequence; a mammalian gene; a human gene; or a plant gene.
21. A mammalian DNA sequence, a human DNA sequence, a plane DNA sequence, a mammalian gene, a human gene or a plant gene isolated by the method of Claim 20.
22. A method of Claim 19 wherein the selectable phenotype is: antibiotic resistance, nutrient prototrophy, tolerance to a metal ion, ability to progress through the cell cycle or expression of a cell surface marker.
23. A method of Claim 14 further comprising isolating contiguous DNA segments from the DNA
fragment library, comprising the steps of:
a) subcloning a terminus of a target DNA
fragment obtained by the method of Claim 9 into a targeting DNA vector which is non-replicating in yeast, the targeting DNA vector having a selectable marker gene for selection in yeast, thereby producing a targeting DNA vector containing subcloned DNA which is the terminus of the previously isolated target DNA fragment;
b) introducing into the DNA fragment library provided the targeting vector produced in (a) as subsequent targeting DNA;
c) maintaining the product of (b) under conditions appropriate for homologous recombination between targeting DNA and target. DNA fragments in the DNA fragment library, thereby producing yeast cells which are stably transformed with the selectable marker gene and targeting DNA
as a result of homologous recombination between the target DNA fragment and the targeting DNA sequence and have a selectable phenotype;
d) culturing the product of step (c) under conditions appropriate for selection of yeast cells in which homologous recombi-nation has occurred;
e) selecting yeast cells, produced in (d), having a selectable phenotype;
f) subcloning a terminus of the target DNA
fragment present in yeast cells selected in (e) into a targeting DNA vector as in step (a); and g) repeating steps (b) through (e) as needed.
24. The method of Claim 23 wherein the targeting DNA vector is a bacterial plasmid having a double strand break in the targeting DNA
therein and the selectable phenotype is selected from the group consisting of:

antibiotic resistance, nutrient prototrophy, tolerance to a metal ion and impaired function of a gene essential for progression through the cell cycle.
25. Saccharomyces cerevisiae carrying a chromosomal deletion of at least one selectable marker gene.
26. Saccharomyces cerevisiae TD7-16d.
27. Saccharomyces cerevisiae IV-16d.
28. Saccharomyces cerevisiae MGD131-10.
29. Plasmid p184DLARG and functional equivalents thereof.
30. A plasmid non-replicating in yeast, comprising:
a) a yeast selectable marker gene;
b) a bacterial origin of replication;
c) a bacterial selectable marker gene; and d) a cloning site for insertion of targeting DNA.
31. A method of fragmenting human genomic DNA
suitable for incorporation in a recombinant-DNA
library which is to be used for mapping contiguous genomic DNA fragments, comprising:
digesting human genomic DNA with at least one restriction endonuclease selected from the group consisting of ApaI, NsiI and ScaI, thereby selecting against the occurrence of certain repetitive DNA sequences at the termini of the DNA fragments produced.
CA002086092A 1990-07-13 1991-07-12 Library screening method Abandoned CA2086092A1 (en)

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