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WO1999066059A1 - Nouveaux vecteurs de fragmentation et leurs utilisations - Google Patents

Nouveaux vecteurs de fragmentation et leurs utilisations Download PDF

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WO1999066059A1
WO1999066059A1 PCT/EP1999/004106 EP9904106W WO9966059A1 WO 1999066059 A1 WO1999066059 A1 WO 1999066059A1 EP 9904106 W EP9904106 W EP 9904106W WO 9966059 A1 WO9966059 A1 WO 9966059A1
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fragmentation
vector
anyone
vector according
aforegoing
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PCT/EP1999/004106
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Jurgen Del-Favero
Christine Van Broeckhoven
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Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/20—Pseudochromosomes, minichrosomosomes
    • C12N2800/206—Pseudochromosomes, minichrosomosomes of yeast origin, e.g. YAC, 2u

Definitions

  • Novel fragmentation vectors and uses thereof are novel fragmentation vectors and uses thereof.
  • the present invention relates to the development of new vectors for fragmentation of yeast artificial chromosomes.
  • YAC fragmentation is based on homologous recombination of a sequence on the YAC with a target sequence present on the fragmentation vector.
  • fragmentation can be performed from the centric as well as the acentric YAC side.
  • Vectors containing a selectable marker and a telomere can be used for acentromeric fragmentation, while the addition of a centromere to the vector, allows centromeric fragmentation. In either case deletion derivatives are generated with different nutrient requirements than their parent YAC.
  • YAC fragmentation with either repetitive or unique sequences as target for homologous recombination has a number of applications for analysis, modification and selection of cloned sequences in YACs.
  • YAC fragmentation with a repetitive target sequence is useful for the construction of detailed fragmentation panels, representing deletion derivatives covering the complete parent YAC.
  • a repetitive target sequence such as Alu
  • Such a panel can be used for the construction of restriction maps of YACs (Cook and Tomilinson, 1996) and has additional application in delineating markers and genes within YACs.
  • fragmentation with repetitive sequences allows removal of chimaeric parts of a YAC or can be employed in the creation of minimal YACs for the production of transgenic cell lines or animals, by removing the non-essential DNA from a YAC of interest. Targeting with specific sequences also has useful applications.
  • Fragmentation by cDNA or specific exons of a gene allows determining the orientation and/or the estimation of the physical length of a gene (Del-Favero et al., 1999). Furthermore, it is possible to construct minigenes with intact promoter regions and spliced exons, without prior knowledge of the promoter.
  • the present invention provides a fragmentation vector for producing deletion fragments of yeast artificial chromosomes, said vector comprising at least one telomere, at least one selectable marker and at least one repetitive element allowing for homologous recombination between said vector and said yeast artificial chromosome, whereby said repetitive element comprises at least 3 repetitions, preferably at least 7 repetitions of a certain triplet and whereby homologous recombination can occur with a frequency of at least about 1 %, preferably at least 5%.
  • the repetitive sequence does not need to be a triplet. It may as well be a doublet or longer (tetra, penta....) sequence which is repeated.
  • the invention is also applicable to sequences which have no or hardly any repetition at all, such as unique sequences of at least 9 bp, preferably at least 12 bp, more preferably at least 27 bp, which are much shorter target sequences than thought possible until the present invention.
  • the invention provides a fragmentation vector for producing deletion fragments of yeast artificial chromosomes, said vector comprising at least one telomere, at least one selectable marker and at least one sequence element identical to a sequence occurring in said yeast artificial chromosome allowing for homologous recombination between said vector and said yeast artificial chromosome, whereby said element has a length of between 9 and about 250 bp, preferentially between 9 and 150 bp and whereby homologous recombination can occur with a frequency of at least about 1%, preferably at least 5%.
  • Fragmentation vectors known in the art include non-centromeric as well as centromeric fragmentation vectors.
  • the present invention is of course applicable for both kinds.
  • the fragmented YACs need to have different nutrition requirements (preferred) or resistance than the parent YAC.
  • the invention in a preferred embodiment provides a fragmentation vector whereby said selectable marker upon homologous recombination generates a deletion fragment having a different nutrition requirement than its parent yeast artificial chromosome.
  • the vectors according to the invention comprise a SP6 and T7 sequence enabling the direct sequencing of both ends of isolated fragmented YAC ends.
  • the vectors according to the invention may be produced in a circular form, but for homologous recombination to occur they generally need to be linear.
  • the invention thus comprises both situations, so that in one embodiment the invention provides a fragmentation vector according to the invention which is linear and which comprises a telomere at one of its ends and recombination element at its other end.
  • the invention provides a fragmentation vector according to the invention which is circular and which comprises said element and said telomere separated by a restriction site unique for said fragmentation vector.
  • a typical vector according to the invention is presented in Figure 1.
  • PACs/BACs were chosen as anchor clones for constructing human "ready to sequence" contigs.
  • PACs/BACs are advantageous over YACs since PACs/BACs are easier to manipulate and are much more stable as compared to YACs.
  • PACs/BACs contigs are still under construction it is possibly a limiting step in the HSP. This is mainly due to underrepresentation and uneven spacing of the available markers. Therefore, PAC/BAC library construction results in clusters of PAC/BAC clones separated by uncovered gaps. In a classical approach these gaps are filled by end sequencing of the PACs/BACs flanking the gaps, followed by primer design. These end probes are then used to re-screen a PAC/BAC library. This process is iterated until all gaps are covered. Although straightforward, it is a time consuming process, with an average cycle time between two weeks and 1 month.
  • Generating more markers in a PAC/BAC independent manner can shorten this process.
  • several strategies can be followed.
  • One such strategy is to use plasmid libraries from flow sorted human chromosomes. Random sequencing of several thousand of these clones will generate enough new markers to construct complete PAC/BAC from an entire chromosome.
  • not all chromosomes can be sorted as a single chromosome resulting in extra, time consuming, and verification steps to isolate these chromosome specific clones.
  • the clones are generated ad random, no positional information is available, hampering the efficient construction of PAC/BAC contigs.
  • This method is applicable on every chromosome or part of a chromosome or specific region of a chromosome for which a YAC contig map is available. Based on the accessible YAC information it is crucial to select these clones that assemble into the best minimal tiling path (MTP) with respect to chimerism and stability.
  • MTP minimal tiling path
  • the proposed acentric fragmentation vectors to use in this method are based on the pDV1 vectors.
  • As target site for homologous recombination a 100 bp Alu repeat fragment is used enabling a fragmentation efficiency of at least 80% (Del- Favero et al., 1999).
  • a high throughput approach is preferred and is reflected in the use of 96-well microtiter plates for growth (MacMurray et al., 1991 ) and transformation (Smith et al., 1995) of the selected YAC clones. Resulting transformants will be plated on 132 mm petri-dishes containing growth media (supplemented with 5- fluoro-orotic acid) which will only the growth of fragmented YAC clones.
  • YAC end rescue includes DNA preparation of the fragmented YAC DNA, restriction digestion of the isolated DNA and circularization of the digested DNA by ligation. All these steps are performed in a 96 well format.
  • Transformation of the ligation products into E. coli cells is achieved on a one by one basis and resulting colonies are plated on selective medium. 1 colony of each transformation mix is chosen ad random for sequencing.
  • Plasmid template DNA is prepared in 96 well plates and subsequently sequenced using high-throughput sequencing equipment. The resulting sequences are processed to remove contaminating and repetitive sequences. Also, databank searches are performed to exclude all known sequences. Next, all newly generated sequences are subjected to primer design software. The generated primers are used to PCR amplify the new chromosome specific markers.
  • the basic vector pDVO (for details see figure 8), was constructed by inserting a 4.5 kb EcoRI/Sall fragment of the fragmentation vector pBCL8.1 carrying LYS2 and TEL (Lewis et al., 1992), into the plasmid vector pGEM3zf(-) digested with EcoRI/Sall. Next an End Rescue Site (ERS) was ligated in the EcoRI site.
  • the ERS was designed to contain the recognition sequences of four restriction enzymes: EcoRI, BamHI, Kpnl and Clal.
  • two complementary oligonucleotides 5' TTCGGATCCGGTACCATCGAT 3' SEQ. ID.
  • a mixture of a (CAG) 7 /(CTG) 7 and (CAG) 10 /(CTG) 10 adapter or a (CCG) 10 /(CGG) 10 adapter sequence was blunt end ligated in the Pstl site, after filling in this site with dNTPs and Klenow DNA polymerase, of the pDV1 basic vector.
  • the ligation mixture was transformed in DH5 ⁇ -cells and the obtained colonies were transferred to Hybond N+ membranes, treated by standard methods and hybridized with a ⁇ - 32 P labeled (CAG) 10 or (CCG) 10 oligonucleotide as a probe.
  • the orientation of the adapter relative to the vector telomere was determined by sequencing different selected clones using the SP6 universal primer ( Figure 1 and 2).
  • the resulting fragmentation vector were: the pDVCAG vector with the adapter sequence in a 5' (CAG) 7 3' orientation relative to TEL; the pDVCTG vector has the adapter sequences oriented in a 5' (CTG) 10 3' position relative to TEL; the pDVCCG vector oriented in the 5'(CCG) 10 3' position relative to the telomere and the pDVCGG vector containing the adapter sequence in a 5'(CGG) 10 3' relative to the telomere ( Figure 1 ).
  • YAC clones were grown in AHC medium (6.7 g/l of Yeast Nitrogen Base without amino acids, 10 g/l Casein hydrolysate acid and 55 mg/l adenine hemi- sulphate).
  • a chemical yeast transformation protocol with alkali cations was used in all fragmentation experiments (Gietz et al., 1992). Before transformation fragmentation vectors were linearized by digestion with Sail and two ⁇ g of linearized DNA was used per transformation. Transformants were selected on minimally supplemented Synthetic Drop-out (SD) media (Sherman, 1991) lacking lysine (SDLys " ). After culturing for 3 days at 30°C transformants were replica plated.
  • SD Synthetic Drop-out
  • Transformants of the acentric fragmentation were replica plated to SDIys " trp ' ura ' and SDIystrp ura * media. Transformants only growing on SDIys " trp ura + have the correct phenotype corresponding to the replacement of URA3 by LYS2. Transformants of the centric fragmentation were replica plated to SDIyslrp ura " and SDIys " trp + ura " media. Colonies only growing on SDIys " trp + ura " correspond to the replacement of TRP1 by LYS2.
  • High molecular weight (HMW) yeast DNA, embedded in low melting point agarose plugs was prepared as described by Southern et al. (1987). PFGE analysis was carried out using the CHEF Mapper XA apparatus (BioRad). Conditions for optimal separations were as determined by the embedded algorithm. YACs were visualized on an UV transilluminator after ethidium bromide staining. Their size was estimated using as length markers the Lambda concatemere ladder (Boehringer Mannheim) and the chromosomes of yeast strain AB1380. YACs were also visualized after Southern blotting and hybridization with radiolabeled LYS2 or Alu probes.
  • Hybridizations were incubated overnight in 7% SDS, 0.5 M Na 2 HP0 4 /NaH 2 P0 4 pH7.2, 1 mM EDTA at 65°C and subsequently washed at 65°C in 2 ⁇ SSC (1 ⁇ SSC is 0.15 M sodiumchloride and 0.015 M sodium citrate), 1 % SDS for 15 min. 1 ⁇ SSC, 1 % SDS for 15 min. and O. ⁇ xSSC, 1 % SDS for 30 min., followed by an overnight exposure to a Kodak X-ray film.
  • HMW yeast DNA of fragmented YACs was isolated in agarose plugs and YAC end sequences were obtained by end rescuing.
  • 20 ⁇ l of a agarose plug was equilibrated with 1 ⁇ TE (10 mM Tris-HCI pH8, 1 mM EDTA) and agarose was removed by treatment with gelase as described by the manufacturer (Epicentre Technologies).
  • the DNA was digested with one of the 4 restriction enzymes of the ERS in the presence of 2 ⁇ OPA buffer (1 ⁇ OPA is10 mM Tris- acetate pH7.5, 10 mM magnesium acetate, 50 mM potassium acetate) in a total volume of 50 ⁇ l for 3 hours.
  • SCA7 is the causative gene for autosomal dominant cerebellar ataxia with retinal degeneration if the normal (CAG) 10 repeat present in the first exon is expanded above 38 repeats (Del- Favero et al., 1998).
  • YAC clone 965a3 was separately fragmented with pDVCAG and pDVCTG and transformation efficiencies were determined after LYS2 selection. A 2-fold higher transformation efficiency was obtained with pDVCTG (360 colonies/ ⁇ g vector DNA). Replica plating on selective medium identified transformants that had integrated LYS2 into the YAC insert by recombination. A recombination efficiency of 10% was obtained with pDVCTG versus 2% with pDVCAG.
  • SPG4 is one of the loci for dominant spastic paraplegia's (SPG), a clinical and genetically heterogeneous group of neurodegenerative disorders, mainly characterized by spasticity of the lower limbs.
  • SPG spastic paraplegia's
  • Clinical and genetical analysis of Belgian pedigrees confirmed previous data localizing the SPG4 gene between the flanking markers D2S400 and D2S376, separated by a genetic distance of 4 cM (De Jonghe et al., 1996). Furthermore, within certain families, anticipation was observed leading to the possible involvement of triplet repeats in the etiology of the disease.
  • YAC clones 937d3 (1800 kb); 931 e1 (1700 kb); 802a5 (300 kb) and 895c12 (1500 kb) spanning the SPG4 region (De Jonghe et al., 1996), were fragmented separately with pDVCAG and pDVCTG .
  • YAC clones 802a5 and 895c12 yielded transformation efficiencies of 100-200 colonies per ⁇ g vector DNA and upon replica plating yielded recombination efficiencies of 2% independently of the vector used suggesting the absence of CAG/CTG sequences in these YACs. Repetition of the experiment yielded the same results and therefore both YACs were not further analyzed.
  • yeast DNA was prepared from 24 individual fragmented YACs of 931 e1 obtained with pDVCAG and 22 individual fragmented YACs of 937d3 obtained with pDVCTG, which were analyzed by PFGE, southern blotting and hybridization with LYS2.
  • YAC 931 e1 resulted in 2 sets of equally sized fragmented YACs containing respectively 18 clones of 1600 kb (B36) and 2 of 1500 kb (B35), while YAC 937d3 contained 4 sets of respectively 4 clones of 1600 kb (C43); 10 of 900 kb (C44); 2 of 350 kb (C42) and 2 of 50 kb (C45) (Table 1 and Fig. 4).
  • the resulting plasmids were analyzed by restriction digestion with EcoRI and Sphl and showed an identical digestion pattern for each fragmented YAC clone from each of the 6 fragmentation sets. Subsequent sequence analysis of most clones showed the presence of identical sequences in each set, confirming the restriction digestion data and consequently proving that each set represents products of a specific recombination event.
  • a BLASTN similarity search (Altschul et al., 1990) with the sequence of each fragmentation set against GenBank division's dbEST and dbSTS, showed only BLASTN hits for the sequences of C44, C43, B36 and B35 (Table 1 ).
  • Sets C44 and B36 were respectively identical to the 3' and 5' sequences flanking the (CAG) 9 repeat contained within the human marker UT2172 (GenBank Ace. N° L18017).
  • C43 matched perfectly with a human macronuclear mRNA sequence (GenBank Ace. N°: L37700)(30).
  • Set B35 showed identity to marker WI-9543 and to expressed sequence tags (ESTs) located in UniGene cluster Hs.27287.
  • CAG/CTG repeat content and the opposite flanking sequence were determined for each fragmentation set using the Genome walker kit (Clontech).
  • the number of repeat units was determined in both human and YAC DNA by PCR amplification with flanking primer sets (Table 1) followed by sequence analysis. No differences were observed in the number of repeat units between human and YAC DNA.
  • B35 GenBank Ace N° AF154411
  • C42 GenBank Ace N° AF154410
  • C45 GenBank Ace N° AF154408
  • C43 GenBank Ace N° AF154409 contained an imperfect CAG repeat sequence: cacCAGcacCAGCAG (Table 1).
  • CAG/CTG repeats To verify the chromosomal localization of the CAG/CTG repeats, we PCR amplified DNA from a monochromosomal mapping panel (Athwal et al., 1985). This analysis showed that all CAG/CTG repeats mapped to chromosome 2 except C43, which is located on chromosome 18. Subsequently, the polymorphic character of the CAG repeats was determined in 30 control individuals by a fluorescence-based PCR test allowing analysis of CAG repeat fragments on an ABI automated sequencer equipped with GENESCAN software for size determination of the PCR fragments. The size differences between the polymorphic fragments were interpreted in terms of number of triplet repeats.
  • yeast DNA was prepared for 24 individual fragmented YACs from each of the 4 experiments and was analyzed by PFGE, southern blotting and hybridization with LYS2. Fragmentation products from YAC 931 e1 with pDVCGG showed the presence of 7 equally sized fragmented YACs, while fragmentation products of pDVCCG contained 3 sets of respectively 10, 4 and 2 similar fragmentation products. For YAC 937d3 3 sets containing 13, 7 and 2 equally sized fragmented YACs were obtained with pDVCGG, while 7, 4 and 3 equal sized fragmented YACs were found with pDVCCG (Table 2).
  • Table 1 Summary of the CAG/CTG fragmentation data obtained with YAC clones 931 e1 and 937d3 from within the SPG4 locus.
  • Table 2 Summary of the preliminary CGG/CCG fragmentation data obtained with YAC cones 931 e1 and 937d3 from within the SPG4 locus.
  • Figure 1 Schematic representation of the new fragmentation vectors, linearized with Sal I, between target sequence (TS) and telomere (TEL) sequences.
  • Figure 3 Pulsed Field Gel analysis of the fragmentation products of the SCA7 YAC.
  • the pulsed field gel was run under the following conditions: 6V/cm; 14°C for 28 h with a pulse time from 30s to 2m30s. Subsequently the gel was blotted and hybridized with the Lys2 probe.
  • the 840 kb band which is present in every lane represents the endogenous Lys2 gene, located on yeast chromosome II of the yeast strain.
  • the left panel [(CAG) 7 fragmentation vector] shows only fragmentation products in lanes 3, 4, 9 and 12, indicating the absence of a major CAG repeat.
  • the right panel [(CTG) 10 fragmentation vector] shows more fragmentation products and more important six YACs of the same length, indicating an identical recombination site (Lanes 2, 3, 4, 6, 7 and 11).
  • ⁇ represents the ⁇ concatemere size marker (Boehringer Mannheim).
  • FIG. 4A represents the analyzed fragmentation products of YAC clone 937d5 with the (CTG) 10 fragmentation vector.
  • Figure 4B shows the analyzed fragmentation products of YAC clone 931 e1 with the (CAG) 7 fragmentation vector.
  • Figure 5 Nucleotide sequences of the obtained CAG repeats. (SEQ. ID. NO's: 3-7) For each triplet repeat the complete sequence is shown. The underlined sequences represent the obtained sequence after end rescue of the fragmented YACs. The recombinatory CAG repeat is indicated in bold. The double underlined sequence in sequence C represents the part of the sequence that was identical to the macronuclear mRNA sequence present in the database.
  • FIG. 6 YAC contig map spanning the SPG4 candidate region.
  • Horizontal lines represent YAC clones: thick lines represent parental YACs, thin lines represent fragmented YACs. Black circles represent positive STS/STR hits. Open squares represent the absence of a positive STS/STR hit.
  • the SPG4 candidate regions based on informative recombinants are indicated: SPG4 candidate region between markers D2S400 and D2S367 (De Jonghe et al. 1996).
  • Figure 7 Nucleotide sequences of the obtained CGG/CCG repeats.(SEQ.ID.NO's: 8-12)
  • the YAC was fragmented twice in a 15 kb interval indicating the sensitivity of the CGG/CCG method to recombine with C-G rich regions.
  • Bases 4820-4961 Yeast telomere sequence
  • Bases 4967-4972 Pstl restriction site. Function: cloning site for Target sequence (e.g. triplet repeat).
  • Bases 4973-4978 Sphl restriction site. Function: isolation of cloned fragmented end in combination with the restriction site used to generate the plasmid.

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Abstract

La présente invention concerne de nouveaux vecteurs de fragmentation des chromosomes artificiels de levure. Ces nouveaux vecteurs, qui se caractérisent par l'utilisation d'une courte séquence cible, sont particulièrement utiles pour isoler les régions flanquantes de triplets répétés relativement courts qui pourraient jouer un rôle dans l'expression aberrante des gènes humains.
PCT/EP1999/004106 1998-06-12 1999-06-11 Nouveaux vecteurs de fragmentation et leurs utilisations WO1999066059A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995005847A1 (fr) * 1993-08-20 1995-03-02 University Of Medicine & Dentistry Of New Jersey Fonction du facteur accessoire de l'interferon gamma et de son recepteur

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995005847A1 (fr) * 1993-08-20 1995-03-02 University Of Medicine & Dentistry Of New Jersey Fonction du facteur accessoire de l'interferon gamma et de son recepteur

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
COOK GP AND TOMLINSON I M: "High resolution restriction mapping of YACs using chromosome fragmentation", NUCLEIC ACIDS RESEARCH, vol. 24, no. 8, 15 April 1996 (1996-04-15), OXFORD GB, pages 1585 - 1586, XP002084325 *
DEL-FAVERO J ET AL: "YAC fragmentation with repetitive and single-copy sequences: detailed physical mapping of the presenilin 1 gene on chromosome 14", GENE: AN INTERNATIONAL JOURNAL ON GENES AND GENOMES, vol. 229, no. 1-2, 18 March 1999 (1999-03-18), pages 193-201, XP004161174, ISSN: 0378-1119 *
HAMER L ET AL.: "Isolation of yeast artificial chromosomes free of endogenous yeast chromosomes: Construction of alternate hosts with defined karyotypic alterations", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA., vol. 92, December 1995 (1995-12-01), WASHINGTON US, pages 11706 - 11710, XP002084324 *
HEARD E ET AL.: "Creation of a deletion series of mouse YACs covering a 500 kb region around Xist", NUCLEIC ACIDS RESEARCH, vol. 22, no. 10, 25 May 1994 (1994-05-25), OXFORD GB, pages 1830 - 1837, XP002084323 *
HEUS J J ET AL.: "Centromeric and noncentromeric ADE2-selectable fragmentation vectors for yeast artificial chromosomes in AB1380", GENOME RESEARCH., vol. 7, no. 6, June 1997 (1997-06-01), COLD SPRING HARBOR LABORATORY PRESS US, pages 657 - 660, XP002084577 *
LEWIS BC ET AL.: "Creation of a yeast artificial chromosome fragmentation vector based on lysine-2", GENETIC ANALYSIS: TECHNIQUES AND APPLICATIONS, vol. 9, no. 3, June 1992 (1992-06-01), pages 86 - 90, XP002117240 *

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Fisher et al. Construction of two YAC contigs in human Xp11. 23–p11. 22, one encompassing the loci OATL1, GATA, TFE3, and SYP, the other linking DXS255 to DXS146
Street et al. Physical mapping of potassium channel gene clusters on mouse chromosomes three and six
Hogan Chapter 4—Principles and techniques of molecular biology

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