Patent Application: US-81104501-A

Abstract:
terminal differentiation is associated with repression in the expression of the p2p subset of hnrnp proteins . the 5173 base pair p2p cdna was cloned and characterized . the cdna contains a 4214 base pair open reading frame . probes to the p2p cdna detect a single 8 kb mrna in multiple murine tissues , in proliferating murine 3t3t cells but not in terminally differentiated 3t3t adipocytes . evidence that the p2p cdna can encode proteins with domains for hnrnp association was established by showing that the c130 monoclonal antibody , produced against a fusion protein derived from the p2p cdna , selectively detects native p2p hnrnp proteins . in addition , it was shown that p2p antisense oligonucleotides selectively repressed 30 - 40 kda p2p expression . since terminal differentiation is also associated with modulation in rb1 function , assays were performed which demonstrated that p2p cdna products interact with rb1 . evidence that the p2p cdna encodes a protein domain that binds rb1 was established using a gst - p2p fusion protein to selectively precipitate rb1 . data also show that this binding is competed by the adenovirus e1a protein , indicating that binding occurs through the “ pocket ” domain of rb1 . these results establish that the p2p cdna encodes protein domains involved in both hnrnp association and rb1 binding and complement recent reports that localize rb1 to sites of rna processing in the nucleus . the interaction of p2p cdna products and rb1 may therefore serve to modulate cell proliferation and / or other biological functions associated with tumor suppression by an rna processing mechanism .

Description:
the balb / c 3t3t mesenchymal stem cell line has been previously described in detail ( 14 ). growing monolayer cultures of these cells are maintained at 37 ° c . in 5 % co 2 in dulbecco &# 39 ; s modified eagle &# 39 ; s medium ( dme , sigma chemical , st . louis , mo .) supplemented with 10 % bovine calf serum ( bcs , hyclone , logan , utah ). to prepare quiescent undifferentiated 3t3t cells they were cultured in dme containing 0 . 5 % bcs for 3 to 4 days at low cell densities of 1 × 10 4 cells / cm 2 . in some studies , growing undifferentiated cells were treated with 50 mg / ml of p2p antisense or sense oligonucleotides . the human hematopoietic stem cell line k - 562 has also been well characterized ( 15 ) and these cells are grown as suspension cultures in rpmi 1640 supplemented with 10 % bcs . the procedures to induce 3t3t cells to undergo differentiation into adipocytes has previously been described ( 16 ). this process involves three steps : ( a ) predifferentiation growth arrest , ( b ) nonterminal differentiation and ( c ) terminal differentiation . these steps occur in a parasynchronous manner during a 3 - 10 day interval after low density , growing cells are cultured in heparinized dme containing 25 % human plasma ( hp ) on ethylene oxide treated petri dishes ( 16 ). it is possible to obtain highly enriched populations of cells at each of the differentiation states described above by using well documented modifications of these culture conditions and reagents ( 1 , 14 , 16 ). these methods were used to prepare cell populations for the current studies . cellular lysates were prepared as described by kaelin et al . ( 17 ). growing murine balb / c 3t3t , and human k562 cells were washed twice with ice - cold pbs , and lysed for 30 minutes at 4 ° c . in ice - cold ebc buffer ( 50 mm tris [ ph 8 . 0 ], 120 mm nacl , 0 . 5 % np - 40 , 200 mm sodium orthovanadate ) containing 10 mg / ml of the protease inhibitors aprotinin , leupeptin , and phenylmethylsulfonyl fluoride ( sigma ). the lysates were cleared of nuclei and debris by centrifugation at 14 , 000 × g for 15 minutes at 4 ° c . to clone p2p related sequences , approximately 1 × 10 6 plaques from an oligo ( dt ) random primed lgt11 murine 3t3 fibroblast cdna expression library ( clontech , palo alto , calif .) were screened using standard procedures ( 18 ) with monoclonal antibody ac88 or fa12 . the ac88 antibody , generated against hsp90 , cross - reacts with the p2p proteins and has previously been described ( 19 ). fa12 also recognizes p2ps and was prepared against core hnrnp proteins ( 20 ). briefly , e . coli strain y1090 was infected with recombinant λgt11 phage , plated on lb plates and incubated at 42 ° c . for 3 hours . subsequently , the plates were overlaid with nitrocellulose filters saturated in 10 mm isopropyl b - d - thiogalactoside ( iptg ) and incubated an additional 3 hours at 37 ° c . to induce the expression of the b - galactosidase fusion proteins . at the end of this period the nitrocellulose filters were air dried for 1 hour at room temperature and subsequently incubated in blocking buffer for 1 hour . antibody probing was performed at room temperature in the blocking buffer for 2 hours . the filters were washed three times in blocking buffer and probed with an alkaline phosphatase conjugated rabbit anti - mouse igg ( for ac88 ) or rabbit anti - mouse igm ( for fa12 ) and the filters developed as previously described ( 18 ). ac88 positive clones were further screened with monoclonal antibody fa12 . clones positive for both ac88 and fa12 were identified and isolated by multiple rounds of plaque purification . the resulting p2p cdnas were subcloned into the ecori site of the pgem3 vector ( promega , madison , wis .) and restriction endonuclease sites were mapped . various restriction endonuclease fragments of the p2p cdnas were also subcloned into the vectors additional 5 ′ p2p sequences were cloned using the rapid amplification of cdna ends ( 5 ′ race ) method ( 22 ). for race , gene - specific oligonucleotides were used to prime first strand cdna synthesis from murine 3t3t total rna using the cdna cycle kit ( invitrogen , san diego , calif .) and 5 ′- race was performed using a variety of different primer sets . amplified products were characterized by size analysis , cloned into the pcrii vector ( invitrogen ) and their dna sequences were determined . throughout this sequencing procedure periodic searches of the dna databases using the blast programs were performed for related sequences . as the sequencing of the 5 ′ end of the p2p cdna was being completed , one significant homology was discovered . a human cdna , designated rbq1 ( 23 ), was found to have extensive homology to a 5 ′ region of the murine p2p cdna . therefore , primers for the 5 ′ most region of rbq - 1 were also used in characterizing the p2p cdna using rt - pcr . total cellular rna was isolated from growing cells , quiescent undifferentiated cells , cells at the nonterminal differentiation state , and terminally differentiated cells . total cellular rna ( 20 mg ) from each sample was denatured and fractionated on 1 . 2 % formaldehyde - agarose gels and transferred by blotting to nitrocellulose filters . blots were prehybridized for at least two hours at 42 ° c . with 5 × denhardts solution , 5 × ssc , 500 formamide , 25 mm potassium phosphate , and 100 mg / ml denatured salmon sperm dna . hybridizations were carried out overnight at 42 ° c . in the same solution containing 10 % dextran sulfate and random - primed 32 p - labeled p2p cdna probes . after hybridization the filters were washed and autoradiographed with intensifying screens at − 70 ° c . tissue specific expression of the p2p mrna was determined using a mouse multiple tissue northern blot ( clontech ) according to the manufacturer &# 39 ; s protocol . p2p cdnas were removed from the pgem vectors using restriction endonuclease eco ri and ligated downstream from the bacteriophage t7 gene 10 promoter and translation initiation site the eco ri site of the pet5a , b and c vectors using standard procedures . bacterial clones were screened for the presence and orientation of the inserts by digestion with ecori and psti . individual clones containing the cdnas in all six possible reading frames were used for subsequent analysis . expression was achieved using the procedure described by studier et al ( 24 ). for each of the cdnas only one reading frame , which corresponded to the largest open reading frame , resulted in expression of a fusion protein antigenically related to p 2 ps . these fusion proteins were then used to produce an antip2p specific monoclonal antibody , c130 , at the university of tennessee memphis molecular resource center hybridoma laboratory . additional monoclonal antibodies , such as those designated c50 , c147 , and c164 were produced by injection of the transcript product of the p2p cdna into mice , in the same method as for production of the c130 antibody . polyclonal antibodies which bind to the p2p transcript are also conceived . the bacterial expression system consisting of the pet5 series of expression vectors , the bacteriophage ce6 and e . coli strain hms174 were gifts from dr . f . w . studier . p2p cdna sequences coding for p2p peptides were generated using rt - pcr and ligated into the pgex - kg vector to generate the following gst fusion proteins of specific cdna sequences given parenthetically : gst - p2p ( 1 - 332 ), gst - p2p ( 494 - 688 ), gst - p2p ( 753 - 909 ) and gst - p2p ( 918 - 1095 ). expression and purification of the gst - fusion proteins was performed as described ( 25 ). fresh overnight cultures of e . coli bl21 transformed with either pgex - kg or one of the above mentioned pgex - p2p recombinants were diluted 1 : 10 in lb medium containing 100 mg / ml ampicillin and incubated at 30 ° c . with shaking for one hour . fusion protein expression was induced by the addition of iptg to a final concentration of 0 . 1 mm and the cultures grown for an additional 3 hours ( 17 ). to recover the fusion proteins , the bacterial cultures were sedimented by centrifugation at 5000 × g for 5 minutes at 4 ° c . and resuspended in { fraction ( 1 / 10 )} volume of netn buffer ( 20 mm tris [ ph 8 . 0 ], 100 mm nacl , 1 mm edta , 0 . 5 % np - 40 ). the cells were lysed on ice by mild sonication and cellular debris was removed by centrifugation at 10 , 000 × g for 5 minutes at 4 ° c . glutathione - agarose beads were washed three times and resuspended ( 1 : 1 [ v / v ]) in netn ; bacterial supernatants were then mixed with the glutathione - agarose beads and rocked at 4 ° c . for one hour to allow the fusion proteins to bind . the beads were finally washed five times with netn buffer . for analysis of bound bacterial gst - or 6x - his proteins , the beads were boiled in 1 × sds sample buffer , analyzed by sds polyacrylamide gel electrophoresis and then the proteins were visualized by staining with coomassie blue . an e1a vector used to express the e1a protein as a 6x - his fusion protein was the gift of dr . margaret quilan , university of tennessee , memphis . expression and purification of the fusion protein was carried out using the his - bind kit following the manufacturer &# 39 ; s protocol ( novagen , madison , wis .). glutathione s - transferase ( gst ) - p2p fusion proteins were expressed and recovered on glutathione - sepharose beads as described above . whole - cell lysates of k562 cells ( 1 × 10 7 cells / sample ) were rocked with the beads for 1 hour at 4 ° c . and then washed five times with netn buffer . the beads were then boiled in 1 × sds loading buffer and the proteins separated on sds - polyacrylamide gels and transferred to nitrocellulose membranes . competition experiments were performed by adding an excess of the 6x - his e1a fusion protein to the cellular lysates prior to the addition of the gst - p2p fusion proteins . the rb1 protein was visualized by immunoblotting using anti - rb1 antibodies if8 or c15 ( santa cruz biotechnology , santa cruz , calif .). these antibodies were also used to immunoprecipitate native rb1 from the cellular lysates to serve as a positive control following the manufacturers &# 39 ; protocol . [ 0040 ] fig1 and 2 provide a summary of the characteristics of the p2p cdna that has been cloned . to clone the p2p cdna , a 3t3 cdna lgt11 library ( clontech ) was screened using the ac88 monoclonal antibody that detects both p2ps and hsp90 ( 7 , 19 ). ac88 positive clones were rescreened with the monoclonal antibody fa12 against core hnrnp proteins , which was previously shown to also react with the p2ps ( 7 , 20 ). two independent clones , designated clone a ( 1398 bp ) and clone b ( 1943 bp ), were found to be recognized by both antibodies . nucleotide sequencing of the cdnas showed that the 3 ′ most region of clone a and the 5 ′ most region of clone b were 100 % homologous over a 863 base pair region , suggesting that these were overlapping clones derived from a single rna species . the overlapping clones were joined through a unique hindiii restriction endonuclease site in the overlapping region to generate 2478 base pair cdna clone . this includes a 1658 base pair open reading frame and 820 base pair of 3 ′ untranslated sequence . additional screens of the cdna library using this cdna as the probe failed to give new clones with any additional 5 ′ cdna sequence . therefore , the cdna clone was extended towards the 5 ′ end using race ( rapid amplification of cdna ends ) methods whereby gene - specific oligonucleotides were used to prime first strand cdna synthesis from murine 3t3t total rna and 5 ′- race was performed . amplified products were cloned and their dna sequences were determined . this extended the 5 ′ sequence by 1015 base pairs and a gst - p2p fusion protein derived from this region was found to bind rb1 , i . e ., gst - p2p ( 753 - 909 ) [ fig1 ]. throughout the sequencing procedure , periodic searches of the dna databases using blast programs were also performed to search for related sequences especially those encoding rb1 binding domains . one significant homology was found with a human cdna , designated rbq1 ( 23 ), which was isolated by its rb1 binding characteristics . primers to the 5 ′ end of rbq1 were therefore used to further extend the p2p cdna sequence using rt - pcr methods to give a 5173 base pair p2p cdna . analysis of this cdna reveals a single long open reading frame extending from an atg codon at base 139 to a termination codon at base 4353 . the presence of two in - frame stop codons near the 5 ′ end of the cdna and several in - frame stop codons at the 3 ′ end of the cdna suggest that the cdna contains the entire coding region of the gene . this open reading frame has the potential to code for 1404 amino acids to generate a protein having a predicted molecular mass of 156 . 9 kda . the deduced amino acid sequence of the protein is shown in fig2 . this highly basic protein ( pi , 9 . 6 ) has multiple potential nuclear localization signals between amino acids 717 and 1323 which is in agreement with previous findings that p2ps represent a subset of nuclear hnrnp proteins ( 7 ). in addition , computer analysis of the sequence of the p2p cdna - derived open reading frame shows a unique cysteine - rich domain near the amino terminus ( amino acids 61 to 101 ) which closely resembles the consensus sequence of the “ ring ” class of zn ++ finger domains ( 26 ) and another domain near the amino terminus ( amino acids 79 to 97 ) that has been implicated in cell growth control , i . e ., the cell division sequence motif [ cdsm ] ( 27 ). p2p mrna expression in multiple tissues and repression by terminal adipocyte differentiation to establish the tissue distribution and the size of the p2p mrna , a mouse multiple tissue northern blot was probed with a p2p cdna probe . a single 8kb mrna was found in all tissues examined . very low , but detectable , levels of p2p mrna were found in kidney , brain , and spleen while moderate levels of p2p mrna were found in heart , lung , liver and skeletal muscle . the highest levels of p2p mrna expression were detected in testis ( fig3 a ) the use of probes to different 3 ′ and 5 ′ p2p cdna domains detected the same 8 kb rna by northern blotting ( data not shown ). to determine if terminal adipocyte differentiation has an effect on p2p mrna expression , total rna was isolated from rapidly growing 3t3t cells , quiescent serum - starved undifferentiated 3t3t cells , quiescent predifferentiated 3t3t cells , nonterminally differentiated 3t3t adipocytes , and terminally differentiated 3t3t adipocytes . then northern analysis was used to compare p2p mrna levels in cells at these states . fig3 b shows that the 8 kb p2p mrna is expressed in all specimens except those derived from cells at the terminal stage of adipocyte differentiation where its expression is markedly repressed . this result is in agreement with previous findings that p2p protein expression is repressed when murine 3t3t mesenchymal stem cells and normal human keratinocytes irreversibly lose their proliferative potential in association with terminal differentiation ( 7 ) or senescence ( 8 ). a monoclonal antibody produced against a p2p cdna - derived fusion protein reacts with native p2ps the carboxy - terminal portion of the p2p cdna orf ( base pairs 2695 to 4353 ) were subcloned into the pet5 series expression vector . in this system , the cdna was placed proximal to the bacteriophage t7 gene 10 translation initiation site such that individual plasmids were isolated containing the cdna in all six reading frames in phase with the gene 10 protein product . expression of the protein encoded in each reading frame was obtained by infecting e . coli strain hms174 harboring the recombinant plasmid with the bacteriophage ce6 as described above . this bacteriophage is a lambda - derived phage containing the gene for t7 rna polymerase . infected bacteria containing the recombinant pet5 vectors produce the t7 rna polymerase which in turn directs the expression of fusion proteins between the t7 gene 10 protein and the reading frame of the cdnas . only one reading frame which corresponds to the 3 ′ end of the large open reading frame [ fig1 ], resulted in expression of fusion proteins antigenically related to p2ps . the fusion protein was electroeluted from preparative gels and used to produce a p2p - specific monoclonal antibody at the university of tennessee , memphis , molecular resource center hybridoma laboratory . one hybridoma so generated was reactive against the purified fusion protein . the antibody , termed c130 , was therefore used to probe 3t3t nuclear and total cell extracts by western analysis . fig4 shows that the c130 monoclonal antibody specifically detects native p2p proteins in a manner similar to the pattern seen with ac88 . however , c130 and ac88 recognize separate epitopes because c130 detects only p2ps whereas ac88 shows cross - reactivity to heat shock protein 90 . these data support the conclusion that the cloned p2p cdna encode hnrnp - related p2p peptides . evidence that the p2p cdna encodes a rb1 binding peptide using a p2p - gst fusion protein because rb1 is required for muscle cell terminal differentiation ( 10 ) and data showing that p2p expression is modulated during terminal adipocyte differentiation state , studies were performed to determine if p2p cdna products might interact with rb1 . to accomplish this gst - p2p fusion proteins were periodically produced to different p2p cdna domains . cellular lysates were prepared from human k - 562 hematopoietic stem cells which contain abundant rb1 protein and these lysates were then precipitated with each of the four gst - p2p fusion proteins , i . e . gst - p2p ( 1 - 332 ), ( 484 - 688 ), ( 753 - 908 ) and ( 918 - 1095 ) as illustrated in fig1 . the lysates were also precipitated with gst protein alone as a negative control in these experiments . fig5 a demonstrates that one fusion protein , gst - p2p ( 753 - 909 ), specifically precipitates a protein that is detected by the anti - rb1 antibody if8 . fig5 a also shows that the gst - p2p ( 753 - 909 ) fusion protein preferentially binds the hypophosphorylated form of rb - 1 which is primarily expressed in the g 1 phase of the cell cycle thus suggesting a possible physiological role for the interaction of p2p cdna products and rb1 in the control of cell growth . most proteins that associate with the hypophosphorylated form of rb1 bind to a region of rb1 that has been termed the “ pocket ” domain ( 28 ). to determine if the interaction between rb1 and gst - p2p ( 753 - 909 ) occurs through the rb1 “ pocket ” domain , competition experiments were conducted using purified viral e1a protein . e1a is known to bind specifically to the rb1 pocket domain and to inhibit cellular proteins from binding to this region ( 29 ). fig5 b shows that the interaction between the gst - p2p ( 753 - 909 ) fusion protein and rb1 is blocked by the addition of purified e1a protein . this inhibition is specific for the e1a protein since the addition of another protein , dihydrofolate reductase , did not block the interaction of rb1 and the gst - p2p fusion protein ( data not shown ). therefore , gst - p2p ( 753 - 909 ) binds specifically to the hypophosphorylated form of rb1 and this interaction occurs through the rb1 “ pocket ” domain . p2p mrna and p2p protein is expressed in cells that have proliferative potential regardless of whether they are in a growing or quiescent state . conversely , the expression of p2p cdna products is repressed in cells that have lost their proliferative potential as a result of terminal differentiation or senescence . in contrast , transformed cells with malignant characteristics , especially sv40 transformed cells that lack the ability to terminally differentiate or senesce , lack the ability to repress p2p expression . it is conceived , therefore , that the proliferative potential of cancer cells , in general , may be blocked if p2p expression is repressed by the use of antisense oligonucleotide reagents that are targeted to bind to specific domains of the p2p mrna to block its translation . the p2p antisense oligonucleotide [ 5 ′ cagcaggagctgtgtt ′ 3 cdna ( 3424 - 3409 )] and a p2p sense oligonucleotide [ 5 ′ ctactaagccatcggc ′ 3 ( 3560 - 3575 )] have been prepared , isolated , and studied , as shown below in table i . the antisense oligonucleotides are prepared by jude labs ( memphis , tenn .) and biosynthesis ( louisville , tex .). these oligonucleotides ( 15 - 50 mg / ml ) were added to the culture media of growing 3t3t cells for various times up to 9 days and the effect of these treatments on p2p expression was determined by western blotting using the ac88 antibody to detect p2ps . additional data also suggests that p2p antisense oligonucleotide can repress cellular proliferation by greater than 50 %, whereas a p2p sense oligonucleotide has no effect . thus , p2p antisense reagents which bind to a domain of the open reading frame of p2p cdna can be used to repress p2p expression and cellular proliferation , which indicates that the repression of p2p expression may be able to repress the proliferative potential of normal , nontransformed cells , abnormal cells , and cancerous cells both in vitro and in vivo . the results of these studies establish the therapeutic value of p2p antisense reagents for the treatment of proliferative diseases , including cancer . 1 . scott , r . e ., hoerl , b . j ., wille , j . j ., jr ., florine , d . l ., krawisz , b . r . and hun , k . 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