Patent Application: US-14982702-A

Abstract:
the fra16d fragile site is shown to be located within a gene encoding a protein termed for . the fragile site is the location of breakpoints of a variety of chromosomal rearrangements and other mutations associated with tumour cell lines . the for protein is shown to be expressed as a number of splice variants . the coding region of the gene encoding for protein has been dna sequenced as has the fra16d fragile sites . protein interactive ww domains have been identified as has an oxidoreductase domain . this invention provides for certain diagnostic and potential therapeutic benefits .

Description:
nine dna probes , ach202 ( d16s14 ), c311f2 , c302a6 ( d16s1075 ), c301f10 ( d16s373 ), 16 - 87 ( d16s181 ), c306d2 , 16 - 08 ( d16s162 ), c307a12 and cri - 0119 ( d16s50 ) which had been physically mapped into the 16q23 region ( 30 ) were chosen for fluorescence in situ hybridisation ( fish ) against fra16d expressing chromosomes . four of these markers mapped within the same somatic cell hybrid breakpoint interval defined by the cell lines cy113 ( p ) and cy121 ( 30 ). one of these , c306d2 mapped proximal to fra16d by fish while the others , c307a12 , cri - 0119 and 16 - 08 mapped distal to fra16d . these probes were therefore used as starting points to isolate a contig of cloned dna spanning fra16d . in the los alamos national laboratory database ( www - ls . lanl . gov ) an sts sequence from c306d2 was found within the ceph yacs my903d9 , my912d2 and my933h2 while an sts in c307a12 was found in my891f3 and my972d3 . these yacs were obtained from ceph and the prepared dna subjected to pst i digestion , southern blotted and probed with 16 - 08 , 16 - 87 , cri - 0119 , c306d2 and c307a12 in succession in order to confirm their content . in addition a search of the whitehead institute database ( www - genome . wi . mit . edu ) revealed that the two sets of yacs were joined into a contig by the yacs my801b6 , my845d9 and my944d8 . each of these yacs was used as template dna to assess sts content ( d16s518 , afma336yg9 , wi2755 , stsg - 10102 and d16s3029 ) and subjected to fish to assess position with respect to fra16d ( fig1 b ). additional probes were generated from the yac 801b6 by subcloning pst i digests of yac dna and screening with total human dna as probe . these subclones were digested with hinc ii to identify and isolate non - repetitive dna fragments as probes . this generated markers h13m , h22s , h23m , h29m and h40m . genome system inc . bac library filters were screened with the probes d16s518 , afma336yg9 , wi - 2755 , stsg - 10102 , h22s , h29m and d16s3029 and nine bac clones including 379c2 , 325m3 and 353b15 were identified . an additional sts , named 2as , was established by ‘ bubble ’ pcr from the end - fragment of bac 353b15 and was isolated as described by gecz et al ( 31 ). briefly , the bac dna was digested with alu i and ligated to the annealed bubble linkers . the final pcr was carried out with a combination of not i - a bubble primer and sp6 - promoter primer as described except an annealing temperature of 55 ° c . was used . these stss and hybridisation probes were used to establish restriction maps of the yac my801b6 and the bacs ( fig2 a ). the yac my801b6 and the bac 325m3 were used as dna templates for establishing lambda subclone libraries in lgem 11 or lgem 12 vectors ( promega ) according to the supplier &# 39 ; s protocol . my801b6 and 325m3 appeared to have intact human dna inserts , based on comparative pulsed field gel mapping of the yacs and bacs across the region ( data not shown ). fra 16d - expressing metaphases were obtained from peripheral blood lymphocytes by standard methods . briefly , cultures were grown for 72 hours in eagle &# 39 ; s minimal essential minimal medium , minus folic acid , supplemented with 5 % fetal calf serum . induction of fra16d was with 0 . 5 um aphidicolin ( dissolved in 70 % ethanol ) added 24 hours before harvest ( 32 ). dna clones were nick - translated with biotin - 14 - datp , pre - associated with 6 ug / ul total human dna , hybridised at 20 ng / ul to metaphase preparations , and detected with one or two amplification steps using biotinylated anti - avidin and avidin - fitc as previously described ( 33 ). hybridisation signal was visualised using an olympus ax70 microscope fitted with single pass filters for dapi ( for chromosome identification ), propidium iodide ( as counterstain ) and fitc . fra16d - expressing chromosomes were scored for signal only when the width of the fragile site gap was greater than the width of one chromatid , so that signal was unambiguously proximal or distal to the gap ( fig3 ). only fluorescent dots which touched chromatin were scored as signal — the few fluorescent dots which lay within the fragile site gap but did not touch proximal or distal segments were therefore not scored as signal since there was a possibility that they comprised non - specific background . lambda clones which gave very poor fish results ( high non - specific hybridisation to other chromosomes ) were not able to be scored with respect to the fragile site . this is likely to be due to the large amount of repetitive dna within these particular clones — see below . the tumour cell lines lovo , ht29 , kato iii , sw480 , ags , mda - mb - 436 and ls180 were purchased from the american type culture collection . lovo and ags cells were grown in hams f12 medium with 2 mm l - glutamine , 10 % fetal calf serum in 5 % co 2 , kato iii cells were grown in rpmi1640 medium with 2 mm l - glutamine , 20 % fetal calf serum in 5 % co 2 , ht29 cells were grown in mccoy &# 39 ; s 5a medium with 1 . 5 mm l - glutamine , 10 % fetal calf serum in 5 % co 2 , ls180 cells were grown in eagle &# 39 ; s minimal essential medium with 2 mm lglutamine and earle &# 39 ; s salts and non - essential amino acids , 10 % fetal calf serum in 5 % co 2 , sw480 cells were grown in leibovitz &# 39 ; s l15 medium with 2 mm l - glutamine and 10 % fetal calf serum , mda - mb - 436 cells were grown in leibovitz &# 39 ; s l 15 with 16 mg / ml glutathione and 0 . 026 units / ml insulin . pcrs for the detection of individual sequence tagged sites from across the fra16d region were duplexed ( 34 ) with control pcrs from the dystrophin gene on the x chromosome ( dmd pm or dmd49 , ref 35 ) or the aprt gene on chromosome 16 ( 33 ). this allowed verification that the pcr reaction was working in the absence of a fra16d region pcr product ( fig4 ). suitable pcr primers for alu29 , 17sp6 , alu20 , 178poly , 5 . 1a6 , rd69 , im7 were used or for 504ca , forward 5 ′- aacacagctcttatcacatcc - 3 ′ ( seq id no 6 ), reverse 5 ′- tggctgtamgtcagaactg - 3 ′ ( seq id no 7 ); while others were as given in database accessions , d16s518 ( genbank z24645 ), afma336yg9 ( gdb 1222843 ), wi2755 ( genbank g03520 ), stsg - 10102 ( genbank z23147 ), d16s3029 ( gdb 605884 ), wi - 17074 ( g22903 ), im9 ( genbank r05832 ), d16s3096 ( genbank ), d16s516 ( gdb 200080 ). pcrs for genbank aa368108 ( forward 5 ′- taatcctcagcctctagaatgcct - 3 ′ ( seq id no 8 ), reverse 5 ′- gtatgatgattttcagggagaaac - 3 ′) ( seq id no 9 ) and genbank aa398024 ( forward 5 ′- tgtcctcaactgattcttacaaac - 3 ( seq id no 10 ), reverse 5 ′- tcaatgggttaggcacagacc - 3 ′ ( seq id no 11 )) were derived from partial sequence analysis of bac353b15 . control pcrs for fra3b deletions were d3s1234 ( gdb 186387 ), d3s1300 ( gdb 188420 ) and d3s1841 ( gdb 254090 ). a contig of yac clones was established in the 16q23 . 2 region between markers c306d2 and c307a12 which were found by fish to map proximal and distal to fra16d , respectively ( fig1 b ). the individual yacs from this contig were also used as hybridisation probes to further localise the fragile site . these experiments identified the yac 801b6 as spanning fra16d , and therefore this yac was used as a source of dna for subcloning the region to provide shorter dna fragments for further refinement of the fragile site position . in addition , bac clones were identified from the region to provide redundancy of cloned human dna in an effort to avoid potential problems of instability of human dna in yacs , as has previously been noted for other fragile site regions , including fraxa ( 37 ), fra 1 ob ( 38 and o . handt , pers . comm .) and a chinese hamster aphidicolin inducible fragile site region ( 39 ). a pulsed - field gel restriction map of yac 801b6 was constructed by using hincii restriction fragment subclones of the yac for use as hybridisation probes ( h13m , h22s , h23m , h29m and h40m ) ( fig2 a ). the position of the bacs ( 379c2 , 325m3 and 353b15 ) with respect to the yac restriction map was determined by both the restriction mapping of the bacs and the positioning of common markers by pcr or hybridisation ( fig2 a ). the sts ( d16s518 , afma336yg9 , wi2755 , stsg - 10102 and d16s3029 ) content of the yacs and bacs was also determined to assist in map construction . subclone libraries of dna from yac 801b6 and bac 325m3 were generated using the lambda vectors igem12 and igem11 ( promega ), respectively and assembled into a contig by end - fragment hybridisation and restriction mapping . the integrity of the yac restriction map was verified by comparison with that of the bacs , 325m3 and 353b 15 . for the region between the bacs the integrity was verified by the use of long range pcr using human chromosomal dna as template . ( data not shown ). there have been difficulties in determining the precise localisation of common chromosomal fragile sites using fish ( refs fra3b ( 13 , 40 , 41 , 42 ), fra7g ( 18 , 19 ) and fra7h ( 43 ). the fish data have been interpreted as due to the fragile sites being spread out over long dna sequences ( eg 100 &# 39 ; s of kb ) or that there are multiple fragile sites at a single locus . an alternative explanation is that the dna in the immediate vicinity of the fragile site is not tightly ‘ packaged ’ into chromatin . we therefore chose to score only those chromosomes where the width of the gap or break at the fra16d fragile site was greater than that of one chromatid ( fig3 ). this approach was intended to reduce the possibility that the ‘ unpackaged fragile site dna ’ might be looping back over the distant side of the fragile site and therefore give a false ‘ spanning ’ signal — particularly for probes that are very close to or within the fragile site region . in addition , while the use of pre - reassociation in the hybridisation process dramatically improved the signal to noise ratio , it did render repeat rich regions poor hybridisation probes . this was particularly evident in the fra16d region where there is an abundance of dna repeat sequences of various kinds . the results of the fish experiments are plotted in fig4 . the closest clearly proximal probe to fra16d is 11 - 44 while the closest unequivocally distal probe is 1433 . these probes map at a distance of ˜ 200 kb apart . however , this 200 kb region includes consistent scatter of distal signal around 11 - 38 and 11 - 27 and the poor hybridisation between 1181 and 1511 ( due to repetitive dna content ). therefore this 200 kb defined by fish analysis is likely to be the maximum sequence required to define fra16d rather than provide any evidence that the fragile site is spread over such a distance . the fra3b fragile site — fhit gene intron 4 region is a frequent site of deletion in various types of cancer ( 8 ). homozygous fra3b deletions have been detected in various human adenocarcinoma cell lines including ( gastric ) ags , kato iii ; ( breast ) mda - mb - 436 ; ( colon ) lovo , ht29 , sw480 and ls180 ( 8 ). since these deletions are somatic events that presumably occur as a result of exposure of these cells to certain environmental factors ( 11 ), we chose to analyse tumour cell lines which exhibit fra3b deletions for the presence of homozygous deletion at the fra16d locus . stss that were either mapped to the fra16d region ( fig1 ) or generated from partial sequence analysis through the region ( data not shown ) were used to screen for homozygous deletion in various tumour cell line dnas . the stss were duplexed with a pcr from the dystrophin locus , as an internal control . the results for the analysis of one of the fra16d region markers , stsg - 10102 is shown in fig4 . of the seven tumour cell lines tested , the stomach tumour cell line ags was found to be homozygously deleted at stsg - 10102 and a series of contiguous markers through the region , ( table 1 ) thus suggesting the presence of minimal deletions spanning the fra16d region in each chromosome 16 present in the ags cell line . the maximal extent of heterozygous deletion in the ags tumour cell line in the fra16d region was determined by genotyping polymorphic markers . the markers d16s518 and d16s3029 both gave two alleles indicating proximal and distal outer limits to the deletion of either chromosome 16 in ags cells ( fig2 a ). the markers afma336yg9 and 504ca were uninformative and therefore did not aid in delineating the limits of heterozygous deletion . open reading frames of 372 ( for i ), 423 ( for ii ), 198 ( for iii ) and 45 ( for iv ) amino acids were obtained for the respective mrna sequences ( fig7 ). identical n - termini , unique c - termini . the region in which the chromosomal fragile site fra16d is located has recently been shown to be associated with two types of chromosomal instability in cancer . in multiple myeloma , translocation of ig loci into the 16q23 region causes the dysregulation of the c - maf proto - oncogene on the affected allele . while these breakpoints are spread over at least 500 kb they bracket both the c - maf gene and the fra16d fragile site ( 1 and fig1 ). the dysregulated expression results in elevated c - maf mrna levels , which is thought to contribute to neoplasia . these translocations were not identified by conventional cytogenetic analysis . their detected frequency in multiple myeloma cell lines suggests an incidence of ˜ 25 %. using representational difference analysis to identify differences between the genomes of normal and tumour cells , the fra 16d region has also been shown to be the site of homozygous deletion in three different types ( lung , ovary and colon ) of adenocarcinoma ( 29 ). the commonly deleted region includes fra16d , with the minimal deletion in colon tumour cell line corresponding almost exactly to the ˜ 200 kb region shown by our fish studies to span the fra16d fragile site . if common aphidicolin fragile sites confer susceptibility to mutagen induced dna instability in cancer then tumour cell lines which have been shown to have such instability at one fragile site are likely to exhibit instability at another fragile site . by analysing tumour cell lines with known fra3b deletions , we have found that the ags cell line derived from a stomach cancer exhibits homozygous deletion spanning fra16d . heterozygosity of the flanking markers d16s518 and d16s3029 indicates that the chromosome 16 deletions are confined to the immediate vicinity of fra16d . taken together these deletion data confirm the hypothesis that fra16d is associated with specific chromosomal instability in cancer . given that the observed deletions are homozygous they are therefore likely to represent the loss of a negative function ( eg tumour suppressor ) rather than the gain of a tumour promoting function . if the analogy with the fra3b locus holds then a gene either spanning or , at least partially , within the fra16d commonly deleted region may contribute to neoplasia as a consequence of quantitative and / or qualitative effects of the deletion . alternatively , the proximity of the fra16d deletions to the c - maf gene suggests that they have the potential to affect c - maf expression . the fra3b fragile site is associated with a region of ‘ late ’ replication ( 48 ) as are the ‘ rare ’ fragile sites fraxa and fraxe ( 49 , 50 ). assuming that replication timing is affected by proximity to fragile site loci and , given the coupling of replication with transcription , the deletion of the fra16d region may lead to an alteration in the timing , with respect to the cell cycle , of the expression of genes in the area — including c - maf . abbreviations bac , bacterial artificial chromosome ; dapi , 4 ′, 6 - diamindino - 2 - phenylindole ; fish , fluorescence in situ hybridisation ; fitc , fluorescein isothiocyanate ; loh , loss of heterozygosity ; fhit , fragile histidine triad ; fra , fragile site locus ; pcr , polymerase chain reaction ; sts , sequenced tagged site ; yac , yeast artificial chromosome cell lines ags , hct116 , hs578bst , hs578t , ls180 , mda - mb453 and t47d are from the department of cytogenetics and molecular genetics , wch collection and were originally obtained from the american type culture collection or the european collection of cell cultures . ags and ls180 cells were grown as described in example 1 . hs578bst cells were grown in opti - mem with l - glutamine , 0 . 0 mg / ml epidermal growth factor , 0 . 5 mg / ml hydrocortisone , 8 % fetal calf serum in 5 % co 2 . t47d , mda - mb - 453 and hs578t cells were grown in rpmi 1640 with l - glutamine , 10 % fetal calf serum in 5 % co 2 . c ) restriction fragments of l clones ( for sequencing between bac 325m3 and bac 353b 15 ). for dna sonication and cloning we modified the protocol from the sanger centre ( http :// www . sanger . ac . uk / teams / team53 / sonication . shtml ): 1 mg of each bac - dna were sonicated in 300 ml h 2 o and 8 ml 10 × mung bean buffer ( 500 mm naac , 300 mm nacl , 10 mm znso 4 ph 5 . 0 ) on ice for 20 seconds using the ultrasonic inc . heat systems sonicator w - 225 ( 50 % duty , 3 . 5 power ). after reducing the volume to 80 ul , blunt ends were created with adding 40 u of mung bean nucleases ( biolabs ) and incubating the mixture at 30 ° c . for 25 minutes . the products were size fractioned on a 1 % agarose gel and fragments ranging from 0 . 7 - 2 kb were extracted with the qiaquick gel extraction kit ( qiagen ). 1500 ng of sonicated dna ( used in 500 ng aliquots ) were ligated into puc18 - sma vector ( pharmacia ) at 16 ° c . overnight and transformed into sure cells ( electroporation - competent , stratagene ). 600 and 1500 clones of the sonication libraries of bac 325m3 and 353b 15 , respectively , were gridded on 96well plates and sequenced in one direction using the m13 - forward primer . sequences were assembled into contigs using the staden package ( mrc ) on an unix computer and edited in lasergene ( macintosh ). for a selected number of clones additional sequences with the m13 - reverse primer were retrieved and assembled . additional sequencing primers were designed and pcr - products sequenced to close gaps between contigs . 10 mg of each bac dna was mixed with 200 ml 10 × tm buffer ( 500 mm tris - hcl , ph 7 . 5 , 150 mm mgcl 2 ), 1 ml sterile glycerol and h 2 0 added to 2 ml . the mixture was pipetted into an ipi - nebulizer and nebulized at 10 psi for 45 seconds . the nebulized dna was then precipitated , end - repaired , size - fractioned and cloned as described for the sonicated dna . 300 and 500 nebulized clones of bac 325m3 and 353b 15 , respectively , were sequenced as described above and included in the assemblies . subclones for sequencing of bac 353b15 were picked randomly , whereas bac 325m3 subclones were selected after hybridisation of specific l - clones of the tile path , made from the bac 325m3 . c ) subcloning of restriction fragments of 1 clones between 1 - 32 and 1 - 191 was done in puc19 - vector . clones were sequenced with m13 - forward and m13 - reverse primers as well as with sequence - specific primers . in some cases subclones derived from specific restriction fragments were also subject to sonication , shotgun cloning and sequencing . sequencing was performed with the abi big dye terminator kit from perkin elmer . in cases where sequencing with the big dye terminator kit failed , drhodamine terminator kit was used , as recommended for gt - rich or homopolymeric regions by the abi - dna sequencing guide . probes for hybridisation on multiple tissue northern blots from clontech were : a ) exon 7 ( 186 bp ), positions 690 through 876 of af227526 b ) part of exon 9a ( 779 bp ), positions 1182 through 1961 of af227527 c ) exon 3 - 6a ( 366 bp ), positions 291 through 657 of af227528 d ) part of exon 1a ( 163 bp ), positions 298 through 461 of af227529 . rna was extracted from 1 × 10 7 cells for each of the cell lines using the rneasy mini kit from qiagen : the cells were disrupted by addition of 600 ul lysis buffer rlt ( supplied with the kit ). the lysed cells were homogenised by passing 5 - 10 times through a 21g ( 0 . 8 × 38 mm ) needle attached to a 5 ml syringe . 600 ul of 70 % ethanol were added and the samples were applied to rneasy mini spin columns . purification and elution of the samples were carried out according to the kit &# 39 ; s manual . 35 - 98 ug of total rna were obtained . reverse transcription was carried out in a 40 ul reaction volume using 12 - 33 ug of total rna from cell lines ags , hct116 , mda . mb . 453 , ls180 , t47d , hs578t and hs578bst , respectively , according to the product sheet of gibco brl superscript rnase h - reverse transcriptase kit except for the addition of 20 u rnase inhibitor ( rnasin , promega ) to the mixture . aliquots of 100 ng of cdna were amplified in pcr reactions using various cdna - primer combinations under standard pcr conditions ( 10 cycles of 94 ° c . for 30 sec , 60 ° c . for 30 sec , 72 ° c . for 30 sec , then 25 cycles of 94 ° c . for 30 sec , 55 ° c . for 30 sec , 72 ° c . for 30 sec ). a ) hhcma - f ( atcttggcctgcaggaacatggca ) ( seq id no 12 ) and wb85 - f ( ttattctgca cttttctggcggag ) ( seq id no 13 ), foriii specific c ) wb85 - e12 ( ttactacgccaatcacaccgagga ) ( seq id no 15 ) and wb85 - a ( tgaattagctccagtgaccacaac ) ( seq id no 16 ), common in fori , for ii and for iii complete 5 ′- ends of transcripts fori , forii and foriii were determined by 5 ′ race experiments including first strand cdna synthesis , purification , tdt tailing of the cdna , pcr of dc - tailed cdna and nested amplification according to the instruction manual of gibcobrl . 1 ug of total rna of cell lines hs578bst ( normal ) and t47d ( tumour ) were taken as templates . first strand cdna synthesis was conducted with the following specific gsp 1primers : fori ( coxido - r , 5 ′- ttatttcagcactcagctcaaagtcac - 3 ′), ( seq id no 17 ) forii ( hhcma - b , 5 ′- agcaaagagacctatgcctagccca - 3 ′), ( seq id no 18 ) foriii ( wb85 - f , 5 ′- ttattctgcacttttctggcggag - 3 ′). ( seq id no 13 ) fori and forii ( coxido - 32 , 5 ′- atatctgtaaatcgatgggactctg - 3 ′), ( seq id no 19 ) foriii ( wb85 - a , 5 ′- tgaattagctccagtgaccacaac - 3 ′). ( seq id no 16 ) nested amplification was done with 5 ul of a 1 : 100 dilution of gsp2 - pcr products and the gsp3 - primers : and foriii ( wb85 - e , 5 ′- tcctcggtgtgattggcgtagtaa - 3 ′) ( seq id no 21 ) in combination with the auap - primer ( gibcobrl ) ( seq id no 21 ). pcr - products were extracted with qiaquick - kit from agarose - gels after electrophoresis and sequenced directly with gsp3 - primers and the primer tj96 - c : the 3 ′ race system for rapid amplification of cdna ends ( gibco brl ) was used to determine the alternatively spliced 3 ′- ends of transcripts encoding fori . 3 mg of total rna of the normal fibroblast cell line sf4635 and the tumour cell lines ags and hct116 were taken as templates for first strand synthesis . instead of the adapter primer ( ap ) supplied with the kit , the following variant of this primer was used : this allowed a nested pcr approach in the subsequent pcr reactions . the target cdna was amplified with a primer overlapping the fori exon 8 / exon 9 boundary ( 5 ′- accaagtccatggtttcagactg - 3 ′) and a race - nested primer ( 5 ′- cgtcgactagtacgtacagt - 3 ′). a second round of amplification was performed with exon 9 specific primer # 9327 ( 5 ′- actgcctggtagaaggaggtcacttct - 3 ′) and the abridged universal amplification primer ( auap , 5 ′ ggccacgcgtcgactagtac - 3 ′) supplied with the 3 ′- race kit . 1 ml of first round pcr product was used for the nested pcr reaction . bands were cut out from agarose gels , purified with gene elute gel purification kit ( sigma ) and directly sequenced with primer # 9327 . chromosomal dna sequences corresponding to the alternative exons 10 , 10a and 10b were identified by blast searches of sequence databases . exon 10 was located in genbank ac009141 , exon 10a in genbank af179633 and exon 10b in genbank af009145 ( see fig6 and 10 ). the preliminary cdna sequence of the for iv transcript is incomplete at its 5 ′ end at this stage . the sequence determined so far derives from overlapping est - clones qf42f03 × ( ai149681 ) and tm79cll . xl ( ai570665 ). the latter was sequenced additionally with the internal primer tj96 - c ( 5 ′- ggaggcagctcgtcctcactg - 3 ′) ( seq id no 22 ). deletions in cell lines ags and hct116 were determined in duplex - sts - pcr reactions as described in example 1 . all primers are listed from 5 ′→ 3 ′ in table 1 . four regions of homozygous deletion ( referred to ashzd i - hzd iv ) were detected in the ags cell line . the proximal breakpoint for hzd i in ags was narrowed down to 654 base pairs between stss 16d - 15 / 16d - 36 (+) and 16d - 1 / 16d - 60 (−); the distal breakpoint of hzd i of 3962 base pairs is between sts 16d - 70 (−) and 16d - 47 (+). the proximal breakpoint for hzd ii in ags was narrowed down to 3030 base pairs between stss 16d - 57 (+) and 16d - 67 (−); the distal breakpoint of hzd ii of 1720 base pairs is between sts 16d - 68 (−) and 16d - 54 (+). the proximal breakpoint for hzd iii in ags was narrowed down to 209 base pairs between stss 16d - 51 (+) and 16d - 55 (−); the distal breakpoint of hzd iii of 5690 base pairs is between sts 16d - 202 (−) and 16d - 69 (+). the proximal breakpoint for hzd iv in ags was narrowed down to 5179 base pairs between stss 16d - 30 / 16d - 44 (+) and eta 1 (−); the distal breakpoint of hzd iv of ˜ 1500 base pairs is between sts im7 (−) and 410s1 a (+). two regions of homozygous deletion ( referred to ashzd i and hzd ii ) were detected in the hct116 cell line . the proximal breakpoint for hzd i in hct116 was narrowed down to 1835 base pairs between stss 16d - 19 (+) and 16d - 61 (−); the distal breakpoint of hzd i of 1549 base pairs is between sts 16d - 62 (−) and qz19h11 (+). the proximal breakpoint for hzd ii in hct116 was narrowed down to 422 base pairs between stss 16d - 63 (+) and 16d - 30 (−); the distal breakpoint of hzd ii of 1513 base pairs is between sts 16d - 66 (−) and 801a (+). for determining the presence of exon 9 of for i ( 51 bp ) in the ags cell line a duplex pcr with genomic primers from the dystrophin gene ( dmd ) as described in example 1 was carried out with primers 8040 / 8041 ( table 1 ). the dna sequence spanning fra 16d was determined by a combination of approaches . firstly , a tile path of lambda subclones of yac my801b6 and bac 325m3 was restriction mapped with restriction endonucleases ecori , hindiii , bamhi and saci in order to provide a reference framework with which to anchor the dna sequence . secondly , either whole bac dna preparations of bac325m3 or bac353b 15 or specific restriction fragments from the lambda subclone tile path were used as feedstock dna for construction of random insert plasmid libraries . sequences from the region between bac325m3 and bac353b15 ( l subclone tile path 132 to 1191 ) were subjected to long range pcr and restriction digest analysis in order to verify the integrity of this sequence . sequenced subclones were also ordered by hybridisation with individual lambda subclones from the minimal tile path . the dna sequences were therefore assembled in a directed rather than random manner . this approach greatly assisted in the assembly of those regions that were rich in dna repeats . the 270 kb contiguous sequence , with an average 4 - fold sequence coverage , spanning fra16d has been deposited in genbank ( accession number af217490 ) ( fig6 ). pcr analysis of sequence tags across the fra16d region was used to refine the location of deletion breakpoints in the ags and hct116 tumour cell lines ( fig6 ). both cell lines showed two distinct regions of homozygous deletion indicating a minimum of three deletion events on the two chromosome 16s in each cell line . four regions of the fra16d spanning sequence were particularly difficult to determine because of their composition ( as evident by dna polymerase pausing in sequencing ). each of these sequences coincided with breakpoint regions in hct 116 or ags tumour cell lines ( fig6 ). the unstable regions consisted of : 1 ) a polya homopolymer region at 144 to 145 kb of dna sequence af217490 ; 2 ) an imperfect ct - repeat of 320 base pairs at position 177 - 178 kb ; 3 ) an 8 kb region at position 191 - 199 kb encompassing a poly a homopolymer region followed by an at - repeat ; a polyt homopolymer repeat and two inverted ( hairpin - forming ) repeats and 4 ) a tg repeat followed by a homopolymer region ( poly t ) at 212 - 213 kb . this fourth sequence is located within a common breakpoint region for the ags and hct116 cell lines at 211 . 7 - 219 . 9 kb of af217490 . pcr across each of the breakpoint regions in ags and hct116 cell lines using primers from positive flanking stss failed to produce products suggesting that additional cryptic instability ( e . g . inversions or amplifications ) may also be present . the locations of three previously identified multiple myeloma breakpoints ( 1 ) was determined by either scanning of partial database sequences ( for anbl 6 ( 5 ′, 3 ′) and jjn3 ) or by pcr of stss on the tile path of lambda subclones spanning fra16d ( for mm . 1 ). scanning of the 270 kb sequence spanning fra16d by blast homology searches revealed a paucity of est homologies . the exceptions were consecutive exons corresponding to sequences from the est qg88f04 .× 1 ( fig6 ). these exons therefore locate fra16d within a 260 kb intron . blast searches with the qg88f04 .× 1 est sequence revealed considerable overlap with clusters of ests the longest available sequence of which was hhcma56 ( u13395 ). ests qg88f04 and hhcma56 clearly have distinct 3 ′ end sequences and were therefore referred to as transcript i and transcript ii . another cluster of ests ( transcript iii ) was found to share 5 ′ but not 3 ′ end sequences with transcripts i and ii . a fourth cluster of ests ( transcript iv ) was found to share sequence homology , however this overlap is between the 5 ′ most sequences of transcripts i - iii and the 3 ′ end of the est cluster suggesting that it may represent an overlapping gene rather than another alternatively spliced transcript . 5 ′ race experiments using mrna from normal ( hs578bst ) and tumour ( t47d ) cells were utilised to extend and confirm the sequences of the clusters of genbank est sequences of transcripts i - iv and to determine the organisation of the alternatively spliced mrnas which they represent . transcripts i , ii and iii were found to have a common 5 ′ end indicating a common promoter . the exons shared and utilised in the alternatively spliced mrnas were identified in bac sequences af217491 , af217492 , ac009044 , ac009280 and ac009129 ( fig6 ). the confinement of distribution of est sequences amongst exons confirmed that the different transcripts were due to alternate splicing . transcripts i - iii share common initiation methionine with an adjacent 5 ′ kozak translation initiation sequence and an upstream in - phase termination codon . the open reading frames code for proteins of 41 . 2kd , 46 . 7kd and 21 . 5kd respectively . each of these open reading frames shares homology with the oxido - reductase family of proteins and therefore the gene has been named for ( fragile site fra 16d oxido - reductase ) with the alternative spliced transcripts i - iii referred to as fori , forii and foriii respectively . northern blot analysis with various for exon probes identified the 2 . 3 kb forii transcript as the predominant and ubiquitously expressed mrna with fori and forii mrnas showing a similar pattern of expression . a dna probe spanning the 5 ′ exons detected additional rnas with a different tissue specific pattern . a cluster of ests ( fig6 ) with homology limited to exon 1 of the for gene was found from a blast search of the databases . this suggests that these transcripts ( referred to as foriv ) might arise from a different promoter and may well constitute a different gene , the 3 ′ end of which overlaps with the 5 ′ end of for ( fig6 ). the 3 ′ end sequences of these ests contain a very short open reading frame ( 4 . 1kd ) which is truncated with respect to that seen in the for transcripts . the complete fori - foriii mrna and partial related transcript sequence ( for iv ) were determined from 5 ′ race and rt - pcr products and deposited in genbank ( af227526 , af227527 , af227528 , af227529 ). rt - pcr and 5 ′- race were used to detect the various for transcripts in normal and tumour cells . striking differences between the presence / absence of for i and for iii transcripts was noted for the ‘ normal ’ fibroblast - like cell line hs578bst and various tumour cell lines ( fig4 ). 5 ′- race and rt - pcr products for transcript specific pcrs were sequenced to confirm the identity of the respective products . the sequence of the aberrant rt - pcr product from mda - mb - 453 cell line generated using a foriii specific primer contains a retroviral element ( herv - h ) 5 ′ of exons 5 and 6a of for ( genbank af239665 ). in addition , one est ( qz23c04 .× 1 ) identified in database blast searches contains exons 1 , 2 and 3 of for spliced at the 3 ′ end to another retroviral element ltr13 . homozygous deletion of fori exon 9 detected in ags tumour cells suggests that the gain of fori transcript will not be a common event in tumour cells . similarly , the loss of foriii transcript is not common to all tumour cells as foriii specific rt - pcr products were readily detected in both ags and hct116 cells ( fig1 ). the alternative spliced mrnas transcribed from the gene each show homology to the oxido - reductase superfamily of proteins . the open reading frames of the alternatively spliced for gene mrn as i - iii have a common n - terminus which contains a ww domain ( fig1 ). the ww domain is truncated in foriv open reading frame , however since this mrna appears to originate from a distinct promoter it may well be that an upstream reading frame is utilised in this mrna . the open reading frame from the for iii transcript retains the ww domain however it is truncated for approximately half the length of the oxido - reductase homology ( fig1 ). given the proposed role of the fhit gene in mediating the biological consequences of fra3b associated dna instability in cancer cells we sought to identify the closest gene to 20 fra16d which might mediate the biological effects of fra16d associated dna instability in cancer . sequence analysis of the fra16d spanning dna sequence revealed the for gene as the sole transcript in the immediate vicinity of the minimal region of homozygous deletion in cancer cells . alternative exons of this gene were found to flank both the fra16d fragile site and the tumour cell deleted regions — the alternative exon 9 being deleted in the ags cell line . no additional authentic transcripts from within the for gene intron were evident . differential expression of alternative spliced and aberrant for transcripts in normal and tumour cells rt - pcr and 5 ′- race gave differing patterns of for transcript expression in various normal and tumour cell lines . it will be of interest to determine whether there are differences in the ratio of for transcripts which are consistent with the biological characteristics of various cell types e . g . neoplastic state or metastatic potential . it is unlikely that the presence of for i transcripts will be a common property of tumour cells since at least the ags cell line is homozygously deleted for the fori exon 9 . additional aberrant for transcripts , including sequences fused to retroviral ltrs , were detected in tumour cells . it may well be that the ratio of the various for transcripts is perturbed by dna instability in the region and that it is the resultant alteration in relative abundance of the various for encoded proteins which mediates the biological consequences of dna instability at fra16d . for example the homozygous deletion in ags cells deletes exon 9 of the for i transcript and may have an effect on the stability of the for iii transcript , however this deletion is unlikely to have any direct effect on the forii transcript which terminates well outside the homozygously deleted region . the for encoded proteins show sequence homology to the oxido - reductase family of proteins and contain a ww domain . other members of this family of proteins include the yes proto - oncogene associated proteins and nedd - ubiquitin ligases . the open reading frame from the foriii transcript retains the ww domain however it is truncated for approximately half the length of the oxido - reductase / ubiquitin - ligase homology ( fig1 ). the foriii protein is therefore likely to be able to bind proteins that recognise the common fori and forii ww domain but not able to perform the enzymatic function encoded by the fori and forii proteins ( possibly ubiquitination ). such characteristics make the foriii protein a likely competitor of fori and / or for ii . since ubiquitination facilitates the process of specific protein turnover foriii could therefore act to prolong the half - life of its substrate by competing with fori and / or forii . influencing this ratio may have therapeutic benefits . thus the provision of reduced foriii production by perhaps use of antisense to foriii transcript may stabilise the balance . alternatively over expression of fori and / or forii could tip the balance the other way . ww domains are regions of protein - protein interaction that bind polyproline - rich motifs ( py domains ) in specific partner proteins . specificity in this interaction is determined by differences in particular amino acid in the various ww domains . proteins known to bind to 30 ww domains include the yes proto - oncogene product and p53 binding protein - 2 ( pirozzi et al ., ( 1997 ) j . biol . chem 272 , 14611 - 14616 ). alteration in the relative levels of the for encoded proteins as a consequence of fra16d associated instability is therefore likely to influence the biological function of the py - motif containing - protein ( s ) which is ( are ) the normal binding partner that the for proteins share through their ww domain . the majority of deletions in the 16q23 . 2 region are heterozygous with the homozygous deletions being confined and limited in number . cells which still have the capacity to produce forii protein ( from a normal chromosome 16 for allele ) might have an elevated level of foriii ( through fra16d associated deletion of the other chromosome 16 allele ) and therefore have a selective “ heterozygote ” advantage . the finding of aberrant for related transcripts spliced to retroviral rna sequences in tumour cells that do not necessarily exhibit fra16d homozygous deletion ( e . g . mda - mb - 453 , fig1 ) suggests that dysfunction of the pathway involving the for ww domain could be a common event in neoplasia perhaps through other forms of fra16d related dna instability such as dna insertion or translocation . three out of five previously mapped multiple myeloma translocations ( 21 ) map within the for gene suggesting that dna instability at the fra16d locus and aberrant expression of the for gene may have a variety of roles to play in various forms of cancer . for the purposes of working the invention a large number of references to pertinent methodologies are set forth in the following us patent documents :— u . s . pat . no . 5 , 981 , 218 to rio et al , u . s . pat . no . 5 , 928 , 884 to croce et al , u . s . pat . no . 5 , 945 , 522 to cohen et al , and u . s . pat . no . 5 , 837 , 492 to tavtigian et al . these documents are incorporated herein entirely specifically for purposes of permitting working of the invention . for the purposes of this specification the word “ comprising ” means “ including but not limited to ”, and the word “ comprises ” has a corresponding meaning . reference in this specification to a document is not to be taken as an admission that the disclosure therein constitutes common general knowledge in australia . 3 . hecht & amp ; sutherland ( 1984 ) cancer genet . cytogenet . 12 , 179 - 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