Patent Application: US-73434496-A

Abstract:
dna fragments and methods for obtaining them are disclosed which when put into mammalian cells together with a dominant marker gene are able to form functional centromeres . the sequences can be used to generate probes for these centromeres . cell lines containing the functional centromeres are also provided . methods are taught for isolating mammalian centromeric dna as well as for producing cell lines carrying an excess of mammalian centromeres linked to a dominant selectable marker gene .

Description:
it is the discovery of the present invention that a segment of human dna can be isolated and introduced into mouse cells and results in a functional centromere . the functional centromeres containing dna of the present invention are preferably linked to a dominant selectable marker . this can be a resistance marker , such as the aminoglycoside - 3 &# 39 ; phosphotransferase - ii which provides resistance to g418 ( sigma ). other such markers known in the art may be used . the method of isolating centromeric dna of the present invention can be applied to any higher eukaryote , especially mammals . preferably a human cell line will be employed . metaphase chromosomes are isolated according to techniques known in the art . the chromosomes are then fragmented . endonuclease digestion and mechanical shearing can be used to fragment the chromosomes . desirably the majority of the fragments will be in the size range of less than 1 μm and some chromosomes will remain unbroken . unbroken chromosomes can be readily removed from the preparation by centrifugation at about 1 , 500 g for about 10 minutes . a human serum containing anti - centromere autoantibodies can be employed in the method of the invention . this is available from patients with crest syndrome . alternative sources of antibody may be used , such as monoclonal or animal derived polyclonal sera containing anti - centromere antibodies . the antibodies are incubated with the preparation of chromosome fragments under conditions where antibody - antigen complexes form and are stable . it is convenient if the antibodies are bound to a solid support . preferably a support such as protein - a sepharose cl4b ( pharmacia ) is used to facilitate separation of bound from unbound chromosomal fragments . however other methods to accomplish this goal can be used , as are known in the art , without employing an antibody bound to a solid support . the dna fragments comprising centromere dna are liberated from the antibodies and centromeric proteins by a deproteinization treatment . ultimately the dna is purified from all proteins , by degrading the proteins and extracting them from the chromosome fragment preparation . any such treatment known in the art may be used including but not limited to proteases and organic solvents such as proteinase k and phenol . the centromeric dna fragments can be used for any purpose or application known in the art . for example , they can be labelled and used as probes ; they can be ligated to vectors to clone or all part of the sequences ; and they can be used to purify centromeric proteins by attachment to a solid support . in one particular embodiment of the invention the centromeric dna fragments are used to probe a library of genomic dna from humans or other mammals for clones which hybridize . hybridizing clones can be analyzed for their ability to perform functions which centromeric dna possesses . one such function is to bind to centromeric proteins . another such function is to form a structure in cells which can be cytologically detected using appropriate immunostaining with anti - centromere antibodies which particularly stain centromeres . according to another method of the present invention a cell carrying an excess of mammalian centromeres is formed . the cell may be human or other mammalian . the centromere may comprise dna isolated from the same or a different mammalian species as the cell . the method involves cotransfection of a cell with two dna molecules : one is a dna carrying centromeric dna ; the other is a dna carrying a dominant selectable marker . preferably these two dna molecules contain sequences which allow concatamer formation , for example phage dnas such as λ phage . the first dna molecule may be isolated from a library of genomic dna using , for example , as a probe the centromeric fragments taught above . alternatively the first dna molecule may result from cloning the centromeric fragments taught above into a phage , for example λ , after manipulations to create fragments of the appropriate sizes and termini . the second dna molecule is readily within the reach of those of skill in the art , for example aλ phage carrying a drug resistance marker . it is believed to be desirable to employ λ phage dna because it concatemerizes , however the absolute necessity of this has not been determined . further , even if this property is necessary , other viral dnas or dna constructs may be able to supply this function . such other means of achieving concatemerization are also contemplated within this method . after cotransfection , cells are selected which express the dominant selectable marker , for example by growth in amounts of g418 which are cytotoxic for the cells without the marker . this selected population of cells is further screened to detect cells with an excess of mammalian centromeres . this screening can be done by standard cytogenetic techniques , as well as by immunostaining with anti - centromere antibodies . desirably the lambda , marker , and centromeric dna ( from the λ clone ) will all be localized at the site of the extra centromere . this can be determined by in situ hybridization studies , which are well known in the art . one cell line made by the methods described above is ec3 / 7 which has been deposited at the european collection of animal cell cultures , porton down , u . k . under accession no . 90051001 , under the conditions of the budapest treaty . the sequence of the dna insert in the lambda phage which was used to make the ec3 / 7 cell line , ( referred to as cm8 ) was determined by standard techniques and is shown in fig1 . the sequence does not correspond to any in dna sequence banks . the present invention also contemplates nucleic acid probes , preferably of at least 10 nucleotides , which hybridize to a dna molecule having the sequence shown in fig1 . one such molecule is cm8 , the lambda phage clone from which the sequence was derived . probes can be radiolabeled , biotin labeled or even unlabeled , for example , depending on the use for which they are desired . the following examples do not limit the invention to the particular embodiments described , but are presented to particularly describe certain ways in which the invention may be practiced . human colon carcinoma cell line ( colo 320 ) was grown as a suspension in rpmi medium supplemented with 10 % foetal calf serum ( fcs ). metaphase chromosomes of colo 320 cells were isolated by our standard method ( hadlaczky , et al . ( 1982 ), chromosomes , 86 : 643 - 659 ). isolated metaphase chromosomes were resuspended in 1 ml of buffer ( 105 mm nacl , 50 mm tris - hcl ph 7 . 5 , 10 mm mgcl 2 , 5 mm 2 - mercaptoethanol ) at a concentration of 1 mg / ml dna and digested with 500 u ecori restriction endonuclease for 1 h . the suspension was diluted with 4 ml of ipp buffer ( 500 mm nacl , 10 mm tris - hcl , 0 . 5 % np - 40 , ph 8 . 0 ) and sonicated for 5 × 50 s with an mse 5 - 70 sonicator . this treatment resulted in a suspension containing chromosome fragments and a few (& lt ; 1 %) unbroken small chromosomes . the suspension was centrifuged at 1500 g for 10 min to remove unbroken chromosome fragments . the supernatant contained only small (& lt ; -- 1 μm ) chromosome fragments as judged by light microscopy . two hundred fifty mg of protein - a sepharose cl4b ( pharmacia ) was swollen in ipp buffer and incubated with 500 μl human anti - centromere serum lu851 ( hadlaczky , et al . ( 1989 ), chromosoma , 97 : 282 - 288 ) diluted 20 - fold with ipp buffer . suspension of sonicated chromosome fragments ( 5 ml ) was mixed with anti - centromere sepharose ( 1 ml ) and incubated at room temperature for 2 h with gentle rolling . after 3 subsequent washes with 25 ml ipp buffer the sepharose was centrifuged at 200 g for 10 min . isolation of dna from the immunoprecipitate was carried out by proteinase - k treatment ( merck , 100 μg / ml ) in 10 mm tris - hcl , 2 . 5 mm edta , ph 8 . 0 containing 1 % sds , at 50 ° c . overnight , followed by repeated phenol extractions and precipitation with isopropanol . all general dna manipulations were done according to ( maniatis , et al . ( 1982 ) molecular cloning -- a laboratory manual ( cold spring harbor laboratory , cold spring harbor , n . y .). results of electrophoresis of immunoprecipitated and supernatant dna are shown in fig2 . the bulk of dna from chromosome fragments which did not bind to the anti - centromere sepharose ( supernatant ) ranged from several hundred base pairs to 5 kb ( fig2 lanes a and b ), while dna from chromosome fragments which bound to the anti - centromere sepharose contained an additional population of high molecular weight ( 9 - 20 kb ) fragments ( fig2 lanes c and d ). this distribution of fragments sizes is consistent with the notion that the centromeric dna is in the structurally most stable region of mammalian chromosomes ( hadlaczky , et al . ( 1981 ), chromosoma , 81 : 557 - 567 ), thus rendering this dna relatively resistant to enzymatic digestion and mechanical shearing . this example demonstrates the use of the high molecular weight immunoprecipitated dna as a hybridization probe to screen a genomic dna library . the high molecular weight dna was isolated from the agarose gel described in example 1 , by electroelution , labelled with 32 p - datp by random oligonucleotide priming ( feinberg , et al . ( 1983 ), anal . biochem ., 132 : 6 - 13 ) and used as a probe for screening a λ charon 4a human genomic library ( maniatis , et al . ( 1978 ), cell , 15 : 687 - 701 ). a hybridizing clone ( cm8 ) was obtained which contains a 14 kb human dna insert . the restriction map of this insert for some restriction endonucleases is shown in fig3 . southern hybridization of parts of the 14 kb insert to human lymphocytic genomic dna indicates that the 14 kb insert represents a continuous piece of dna in the genome and is not the ligation product of a number of fragments . this example demonstrates that the copy number of the 14 kb insert of clone cm8 is consistent with it being present on each chromosome in the human genome . southern blotting experiments were performed in which a single copy dna probe ( xv2c ) ( estivill , et al . ( 1987 ), nature , 326 : 840 - 845 ) and the central xhoi - ecori fragment of the cm8 insert ( fig2 ) simultaneously hybridized with serial dilutions of human peripheral lymphocyte dna . the probes were labelled by random oligonucleotide priming ( feinberg , et al . ( 1983 ), anal . biochem ., 132 : 6 - 13 ). by comparing the signal of the cm8 probe to the known single copy probe , the copy number of cm8 was estimated to be 16 - 32 per haploid genome . this example shows the use of the cm8 dna as a probe to human metaphase chromosomes . radioactive in situ hybridization with 3 h - thymidine labelled cm8 dna to human ( colo 320 ) metaphase chromosomes was performed according to the method of pinkel , et al . ( 1986 ), proc . natl . acad . sci . u . s . a ., 83 : 2934 - 2938 . a preferential centromeric localization of silver grains was observed ( fig4 ). in non - radioactive in situ hybridization according to the method of ( graham , et al . ( 1973 ), virology , 52 : 456 - 467 ), using biotin - labelled subfragments or the whole cm8 insert it was not possible to detect a positive hybridization signal by our standard method . furthermore , using a hybridization method which is suitable for single copy gene detection with a biotin - labelled probe ( lawrence , et al . ( 1988 ), cell , 52 : 51 - 61 ), apart from the typical r - band like alu hybridization pattern ( korenberg , et al . ( 1988 ), cell , 53 : 391 - 400 ), no specific hybridization signal was detected on any of the chromosomes with the whole 14 kb cm8 insert . possible explanations for this negative result are that these sequences are virtually inaccessible to the hybridization probe , due to their compact packing in the midst of the centromere structure , and that the biotin system is less sensitive than the radioactive one . the sequence of the human genomic insert of λ cm8 was determined using the dideoxy method ( sanger , et al . 91980 ), j . mol . biol ., 143 : 161 - 178 ; biggin , et al . ( 1983 ), proc . natl . acad . sci . u . s . a ., 80 : 3963 - 3965 ). see fig1 . the sequence of the 13 , 863 bp human cm8 clone was compared with a complete nucleic acid data bank ( microgenie , beckman ) and showed no homology to any known sequence . however , a 300 bp alu repeat deficient in the flanking direct repeat sequences was found in the 2 . 5 kb ecori - xhoi fragment ( fig3 ), which explains the alu type in situ hybridization pattern . this example demonstrates the use of the cm8 dna to form centromeres in mammalian cells . in order to detect any in vivo centromere function of the cm8 dna , it was introduced with the selectable aph - ii gene into mouse lmtk - fibroblast cells . the mouse fibroblast cells were maintained as a monolayer in f12 medium supplemented with 10 % fcs . the calcium phosphate method ( harper , et al . ( 1981 ), chromosoma , 83 : 431 - 439 ) was used to transfect the cells with 20 μg λ cm8 and 20 μg λ gtwesneo dna per petri dish ( 80 mm ). a 2 minute glycerol shock was used . the λgt wesneo was made by cloning the pag60 plasmid ( colbere - garapin , et al . ( 1981 ), j . mol . biol ., 150 : 1 - 14 ) into a λ gtwes ( leder , et al . ( 1977 ), science , 196 : 175 - 177 ) bacteriophage vector . the whole λ cm8 and λ gt wesneo constructions were used for transfections for two reasons . first , to separate the marker gene from the cm8 sequences , in order to avoid inactivating the aph - ii gene , a process which may occur during centromere formation . second , λ dna is capable of forming long tandem arrays of dna molecules by concatamerization . concatamerization was postulated as being necessary to form centromeres since , in s . pombe 4 to 15 copies of conserved sequence motifs form centromeres ( chikashige , et al . ( 1989 ), cell , 57 : 739 - 751 ). considering these two facts a multiplication of the putative centromeric dna by concatamerization might increase the chance of centromere formation . transformed cells were selected on growth medium containing 400 μg / ml g418 ( genticin , sigma ). individual g418 resistant clones were analyzed . the presence of human sequences in the transformed clones was monitored using southern blots probed with subfragments of the cm8 insert . screening for excess centromeres was achieved by indirect immunofluorescence using human anti - centromere serum lu851 ( hadlaczky , et al . ( 1989 ), chromosoma , 97 : 282 - 288 ). the chromosomal localization of &# 34 ; foreign &# 34 ; dna sequences was determined by in situ hybridization with biotin labelled probes . eight transformed clones have been analyzed . all of the clones contained human dna sequences integrated into mouse chromosomes . however , only two clones ( ec5 / 6 and ec3 / 7 ) showed the regular presence of dicentric chromosomes . individual cells of clone ec5 / 6 carrying di -, tri -, and multicentromeric chromosomes exhibited extreme instability . in more than 60 % of the cells of this cell line the chromosomal localization of the integrated dna sequences varied from cell to cell . due to this instability , clone ec5 / 6 was deemed to be unsuitable . however , cells of clone ec3 / 7 were stable , carrying either a dicentric ( 85 %) or a minichromosome ( 10 %). centromeres of dicentric chromosomes and minichromosomes were indistinguishable from the normal mouse centromeres by immunostaining with anti - centromere antibodies ( fig5 a and b ). this example shows that the newly introduced dna in the ec3 / 7 cell line contributes to centromere formation . in situ hybridization with biotin labelled cm8 , aph - ii gene , and λ phage dna were carried out . chromosomes were counterstained with propidium iodide ( pinkel , et al . ( 1986 ), proc . natl . acad . sci . u . s . a ., 83 : 2934 - 2938 ) for in situ hybridization experiments while in indirect immunofluorescence with dna binding dye , hoechst 33258 used . all observations and microphotography were made by using an olympus ahbs vanox microscope . forte 400 professional black and white , and fujicolor 400 super hg colour film were used for photographs . without exception these three probes hybridized onto the same spots : either on the distal centromere of the dicentric chromosome ( fig5 c ) or on the centromere of the minichromosome ( fig5 d ). in less than 5 % of the ec3 / 7 cells an alternative localization of the hybridization signal was found . these included cells with more than one integration site , cells without a detectable signal , or cells where the hybridization was found on chromosomes other than that identified as the dicentric chromosome . in less than 0 . 5 % of the cells a tandem array of the hybridization signal was observed on the dicentric chromosomes ( fig6 a - c ), suggesting that the additional centromere was capable of autonomous &# 34 ; duplication .&# 34 ; at least some of these duplicated centromeres appeared to be functional . this was indicated by the existence of a minichromosome with double centromeres . both centromeres of this minichromosome showed positive immunostaining with anti - centromere antibodies ( fig7 a ). minichromosomes carrying double centromeres might be breakage products of multicentromeric chromosomes . indirect immunofluorescence of mouse metaphase cells was performed as described by hadlaczky , et al . ( 1989 ), chromosoma , 97 : 282 - 288 . when indirect immunofluorescence and in situ hybridization were performed on the same metaphases , mitotic cells were resuspended in a glycine - hexylene glycol buffer ( hadlaczky , et al . ( 1989 ), chromosoma , 97 : 282 - 288 ), swollen at 37 ° c . for 10 min followed by cytocentrifugation and fixation with cold (- 20 ° c .) methanol . after the standard immunostaining ( hadlaczky , et al . ( 1989 ), chromosoma , 97 : 282 - 288 ) metaphases were photographed , then coverslips were washed off with phosphate buffered saline and slides were fixed in ice - cold methanol - acetic acid , air - dried and used for in situ hybridization . to demonstrate the integration of the human cm8 clone sequence and the aph - ii gene in the centromere region , immunostaining of centromeres with anti - centromere antibodies followed by in situ hybridization with cm8 and aph - ii probes was carried out on the same metaphase plates of ec3 / 7 cells . the in situ hybridization signals with both biotin - labelled cm8 and aph - ii probes showed a colocalization with the immunostained centromeric region of the chromosomes carrying additional centromeres ( fig7 ). forty - six independent subclones derived from a single cell were isolated and analyzed . each of the subclones carried the dicentric chromosome . the percentage of minichromosome - containing cells varied between 2 % and 30 % in different subclones . we were unable to isolate a subclone which carried the additional centromere exclusively in a minichromosome . this result suggested that the minichromosomes were unstable and they can be regarded as the products of regular breakages of the dicentric chromosomes . a preliminary analysis by immunostaining of ec3 / 7 cells ( 103 metaphases ) cultured for 46 days in non - elective medium showed that 80 . 6 % of the cells contained either a dicentric ( 60 . 2 %) or a minichromosome ( 20 . 4 %). subsequent in situ hybridization with biotin labelled probes proved the presence of the &# 34 ; foreign &# 34 ;, dna in the additional centromere . these results indicate that no serious loss or inactivation of the additional centromeres had occurred during this period of culture under non - selective conditions . this example shows that the cm8 insert concatamerized to form the functioning centromere of cell line ec3 / 7 . dna of the ec3 / 7 cell line and human lymphocyte dna were digested with restriction endonucleases and probed with subfragments of the cm8 insert in a southern hybridization experiment . comparing the intensity of the hybridization signal with ec3 / 7 dna to that with the human dna , the minimum number of integrated human sequences in the additional centromere was estimated to be ≧ 30 . the copy number of cm8 in human lymphocytic dna was determined as described above in example 3 . __________________________________________________________________________sequence listing ( 1 ) general information :( iii ) number of sequences : 1 ( 2 ) information for seq id no : 1 :( i ) sequence characteristics :( a ) length : 13875 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : genomic dna ( iii ) hypothetical : no ( iv ) anti - sense : no ( v ) fragment type :( vi ) original source :( xi ) sequence description : seq id no : 1 : 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