Isolating Vector Insert Junctions from Yeast Arttflctal Chromosomes
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Downloaded from genome.cshlp.org on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press Isolating Vector_insert Junctions from Yeast Arttflctal Chromosomes Gary A. Silverman Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02115 USA The physical mapping of complex ge- of the set. (13) By analyzing partial or the basis of STS generation by random, nomes can be achieved by assembling complete restriction fragment patterns single-end, clone-limited (isolation of an contigs of yeast artificial chromosomes (fingerprints) of individual h phage or STS from one end of a clone), and dou- (YACs). In the early stages of map con- cosmid clones, contigs spanning exten- ble-end, clone-limited strategies (isola- struction, randomly generated markers sive portions of the Escherichia coli, (14) C. tion of STSs from both ends of a clone). can be used to screen YAC libraries, order elegans, (is) and Saccharomyces cerevi- Clearly, the generation of STSs via the the clones, and assemble the contigs. siae (13'16) genomes have been con- double-end, clone-limited strategy, espe- However, as the maps increase in size, structed. Contig construction using cially in the latter stages of map con- this process becomes less efficient, as YACs has proved more arduous. The fin- struction, is the most efficient method randomly generated markers are less gerprinting of YACs by restriction map- for completing contig assembly. (24) In likely to add new clones to the ends of ping, although feasible,(17'18) is con- practical terms, however, the benefits of the contigs. This problem can be over- founded by the large genomic insert, contig construction by a directed ap- come by a more directed approach such repetitive sequences, multiple YACs in a proach can be offset if methods for iso- as the generation of markers from the single cell, and chimeric fragments. As lating STSs from the ends of the clones ends of YAC inserts. The purpose of this an alternative to fingerprinting meth- are inefficient or labor intensive relative report is to consider the practical aspects ods, Green and Olson proposed the con- to those used for generating random STSs. of the methods used to isolate vector- cept of sequence tagged site (STS)-con- The need to develop methods that insert iunctions from YAC clones. Al- tent mapping. ~19) STSs are small, single- easily and reliably yield terminal DNA though many techniques are available, copy fragments in the genome that can sequences from the genomic insert was the high throughput and success rate of be recovered or detected by unique PCR apparent early in the evolution of YAC PCR-based strategies may prove to be the primers. (2~ Conceivably, YAC contigs cloning. This was prompted by the sim- most efficient means for closing the gaps spanning entire chromosomes could be ple notion that the termini from YAC in- between contigs and completing the assembled by using STS-specific PCR as- serts will maximize a chromosomal walk physical maps of complex genomes. says to screen libraries and detect over- by detecting clones in the library with The advent of yeast artificial chromo- lapping clones. This concept was sup- the least amount of overlap. The purpose some (YAC) cloning systems has facili- ported by the use of 16 STSs to align 30 of this report is to review the utility of tated greatly the mapping, cloning, and YACs into a 1.5-Mb contig spanning the the techniques used to isolate YAC vec- functional analysis of complex genomes cystic fibrosis gene region. (19) On a larger tor-insert junctions. The plethora of (as reviewed). (1-3) YACs spanning the eu- scale, the generation of additional STSs available techniques suggests that no chromatic segments of the human y(4) should permit complete mapping of an preferred method has emerged. How- and 21 (s) and extensive portions of the entire genome. In the early stages of map ever, PCR-based techniques, rather than Caenorhabditis elegans (6) and Drosophila construction, the random generation of conventional plasmid or k phage sub- melanogaste/7) genomes attest to their STSs may prove to be the most efficient cloning, have experienced the most importance in genome mapping. The method to initiate contig assembly.~z1-23) widespread use (Table 1). All of the tech- positional cloning of disease-associated However, if the probability of isolating a niques provide the means to isolate STSs genes such as the neurofibromatosis random STS is predicted by the Poisson that can be used to build contigs and fill type 1, ;8) adenomatous polyposis coli distribution, then the efficiency of map gaps. In addition, these end fragments (APC), ;9'~~ and the Huntington's disease construction will diminish greatly as the can be used to assess the extent of over- genes (~1,12) also relied on cloning and contigs expand to cover 60-80% of the lap with other clones, determine telom- mapping with YACs. genome. ~z4) Specifically, calculations eric-centromeric orientation of YACs, The physical map of a genomic re- based on the Poisson distribution predict increase marker density for pulsed-field gion can be deduced by the assembly of that 60% of the total effort would be re- gel electrophoresis (PFGE) mapping, contiguous clones (contigs). Contigs are quired to complete the final 13% of the generate new restriction-fragment length a collection of cloned DNA segments map if a random approach is used exclu- polymorphisms, and identify chimeric with individual members that share sively. Palazzolo et al. (z4) present com- YAC clones. overlap with at least one other member puter simulations of contig building on The techniques described in this re- 3:141-1509 by Cold Spring Harbor Laboratory Press ISSN 1054-9803/93 $5.00 PCR Methods and Applications 141 Downloaded from genome.cshlp.org on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press TABLE 1 Methods for Isolating YAC gene, is subcloned into a plasmid vector. _URA3 side) are linearized by endonu- Vector-insert Junctions The appropriate clone is isolated by clease digestion and are used to trans- URA3 complementation of E. coli con- form yeast via a lithium acetate proce- Plasmid rescue taining the pyrF mutation. (26) dure. Transformants are selected for Plasmid rescue after vector arm Newer YAC vectors such as pYAC- lysine prototrophy, pICL and pLUS inte- modifications, in vivo neo, (27) pJS97/98, (28) and the pGS966- grate by homologous recombination Conventional subcloning k phage 999 series (29) permit plasmid rescue from into the ampicillin resistance gene of the plasmids both sides of the cloning site. They con- LA and a segment of pBR322-related se- Genomic sequencing tain ColE1 origins, antibiotic resistance quence in the RA, respectively. Integra- PCR genes, and several common restriction tion site-specific PCR assays can be used Alu-vector sites in both vector arms. As an added to confirm that plCL and pLUS are in- anchor-vector feature, some of these vectors contain T3 serted appropriately into the vector inverse or T7 bacteriophage RNA polymerase arms. By linearizing the plasmids before promoters that permit riboprobe synthe- transformation, 70--100% of the lysine sis from either side of the cloning site. prototrophs contain integrating vectors port have been successful in isolating The widely available Washington Uni- at the appropriate pYAC4 site. As ex- vector-insert junctions from the left or versity, (3~ CEPH, (31) ICI, (32) and ICRF (3~) pected, transformation with nonlinear- right arms of the pYAC series of vec- human genomic YAC libraries were con- ized plasmids dramatically reduces the tors. (zs) For reference, the left arms (LA) structed using the pYAC4 vector. To fa- percentage of Lys + cells with correct in- of pYAC vectors contain the CEN4, ARS1, cilitate plasmid rescue directly from sertions. Plasmid rescue of the YAC vec- and TRP1 sequences, whereas the right these clones, Hermanson et al. (34) devel- tor-insert junctions is achieved by di- arm (RA) contains the URA3 gene. With oped a set of integrating plasmids to ret- gesting miniprep DNA of the Lys § some modifications, these methods will rofit the pYAC4 vector arms with ele- transformants with a restriction enzyme prove useful in isolating terminal frag- ments present in the newer vectors: dual that cleaves in the polylinker, ligating ments from DNA propagated in other ColE1 origins of replication, antibiotic the restriction fragments, transforming cloning systems (e.g., Pls, BACs, PACs, resistance genes, and clusters of restric- E. coli and selecting for ampicillin (plCL) and cosmids). tion sites (Fig. 1). These plasmids, pICL or kanamycin resistance (pLUS) (Fig. 1). (integrating into CEN side with LYS2) Over 50 end fragments, up to 20 kb in and pLUS (LYS2 integration into the size, were rescued using this technique. PLASMID RESCUE The isolation of a YAC vector-insert junc- tion by plasmid rescue was described by Burke et al. (2s) YAC DNA is digested with a restriction enzyme that generates a te- lomere-free junction fragment. The frag- ments are self-ligated, and the resultant plasmids are used to transform E. coli. The advantages of plasmid rescue are its technical simplicity and the ability to obtain fragments as large as 20 kb. These fragments can be used as fluorescence in situ hybridization (FISH) probes, and their larger size also increases the likeli- hood of obtaining unique sequence in- formation. Unfortunately, the original design of the pYAC series of vectors placed a ColE1 origin of replication and ampicillin resistance gene only on the LA. Furthermore, the limited number of available restriction sites in the vector arm (XhoI, NdeI) limits the usefulness of this technique to isolating those ge- nomic inserts that have either a XhoI- SalI or NdeI rare-cutting restriction site within -20 kb of the LA cloning site.