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:
FIELD OF THE INVENTION  
         [0001]    This invention relates to the field of cancers and in particular to nucleotide sequences of the fragile site FRA16D, of the FOR gene and amino acid sequences of its encoded proteins, as well as derivatives and analogs thereof and agents capable of binding thereto, and uses of these, such as in diagnosis and therapy.  
         BACKGROUND OF THE INVENTION  
         [0002]    Cancers are a significant factor in mortality and morbidity, with onset rates of forms of cancer being quite high in all places of the world. Early detection greatly improves the chances of remission and considerably reduces the chance of the cancer metastasizing. The treatment of early stage cancers is also much more benign so that there are less severe residual effects resulting from the treatment. Accordingly early detection of cancers is a high priority in management of the diseases. Similarly treatment of various cancers are of mixed outcome and it is desirable to provide for alternative treatments at least for certain forms of cancers.  
           [0003]    Cancers are of many different types and severity, however the uncontrolled proliferation of cancers cells is invariably associated with damaged DNA of one form or another. Some types of cancer are familial in the sense that there is an increased risk of contracting cancer, but the hereditary characteristics in most cancers are not simple and there is only usually a few fold increased risk among family members as compared to the general population. The DNA damage in most cancers are associated with somatic mutations the acquisition of which is thought to be associated with exposure to certain environmental factors.  
           [0004]    A very large number of genes have been identified as being associated with the onset of cancer and this reflects the complexity of the regulation of normal cellular proliferation. These genes can be categorised into three groups the first of which includes the so called oncogenes or protooncogenes which are often associated with positive control elements, enhancing cellular proliferation in the normal cellular cycle. Certain mutations in these positive control elements trigger uncontrolled proliferation. A second group are the so called tumour suppressor genes, which are genes that normally suppress proliferation, and inactivation or reduction in activity of these leads to abnormal proliferation. These tend to act in a recessive fashion. A third group are the so-called mutator genes which are normally responsible for maintaining genome integrity during the proliferative cycle, and if these are defective then the general mutation rate increases and the consequent chance of providing for a transforming mutation increases.  
           [0005]    One mapping technique to locate the site of chromosomal lesion in a cancer cell is known as the loss of heterozygosity (LOH) technique. Eukaryotes have two copies of each chromosome, apart from the sex chromosomes, and as a result cancers that result from mutations in a tumour suppressor generally require two mutations. Sometimes one mutation will be inherited, and a second mutation is required to trigger the cancer leading to loss of function of both copies of the gene in the individual. Quite often these secondary mutations will be deletions and their location can be detected by checking the presence of highly polymorphic genetic markers from the tumour tissue and from another site such as blood. The markers that are heterozygous in normal tissue and have become homozygous in the cancer tissue can give an indication of the lesion concerned.  
           [0006]    The LOH technique is however quite difficult to routinely perform and interpret reliably, this is particularly so because any tumour sample usually is also contaminated by non-tumour tissue, and it is at times difficult to distinguish a result because of a decreased relative intensity, and quantitative amplification techniques will often need to be employed. Another limitation relates to the availability of a suitably dense array of markers which generally leads to the detection only of larger deletions. A single tumour may have LOH in many distinct regions, but LOH will only be detected in those regions that have been tested. The LOH technique is thus unsuited to diagnostic purposes.  
           [0007]    The use of these LOH studies have identified a number of sites some of which correspond to regions of the chromosome termed fragile sites.  
           [0008]    Fragile sites appear as breaks, gaps or decondensations on metaphase chromosomes. These non-random breaks appear in defined locations on human chomosomes under appropriate conditions.  
           [0009]    There are two distinct forms of chromosomal anomaly referred to as fragile sites (Sutherland et al., 1998)). The ‘rare’ form is polymorphic in the population and is accounted for by the expansion of repeat DNA sequences beyond a copy number limit. The ‘common’ form is present at many loci in all individuals. Despite determination of the complete sequence analysis of the common fragile site, FRA3B (Boldog et al., 1996; Inoue et al., 1997; Mimori et al., 1999) and the partial sequence analysis of the common fragile sites, FRA7G and FRA7H (Huang et al., 1998a,b; Mishmar et al., 1998) the molecular basis for common fragile sites is not yet understood.  
           [0010]    Fragile sites are also distinguished by the culture conditions required for their induction. Common fragile sites are (mainly) induced by aphidicolin, whereas the rare fragile sites are induced by either high or low concentrations of folate or the AT-rich binding chemicals such as distamycin A or by bromodeoxyuridine. The role of chromosomal fragile sites in human genetic disease was thought to be restricted to fragile X syndrome caused by the FRAXA fragile site, however a mild form of mental retardation has been associated with FRAXE and the FRA11B fragile site appears to predispose to 11q breakage leading to some cases of Jacobsen syndrome.  
           [0011]    Fragile sites have been proposed to have a determining role in cancer associated chromosomal instability. There are in excess of 100 fragile sites in the human genome of which the fragile site FRA11B is located within the CBL2 proto-oncogene (Jones et al., 1994, 1995) and the FRA3B, FRA7G and FRA16D sites have been located within or adjacent to regions of instability in cancer cells (Ohta et al., 1996; Sozzi et al., 1996; Engelman et al., 1998; Huang et al., 1998a,b; Chen et al., 1996; Latil et al., 1997).  
           [0012]    Recent detailed molecular analysis of fragile site loci has demonstrated that the common fragile site FRA3B is located within a region subject to localised deletion and that this deletion is frequently observed in certain forms of cancer (Ohta et al., 1996; Sozza et al., 1996). FRA3B lies proximal to the major region of LOH on chromosome 3p previously shown to be responsible for deletion of the VHL tumour suppressor (Gnarra et al., 1994). The cancer-associated FRA3B deletions can result in inactivation of a gene (FHIT—Fragile Histidine Triad) which spans the fragile site (Croce et al U.S. Pat. No. 5,928,884). The FHIT gene product has been shown to have a role in tumour growth (Siprashvilli et al., 1997) but quite what the significance or nature of that role is subject of active research at the present.  
           [0013]    Another common fragile site FRA 7G has also been shown to be located within an about 1 Mb region of frequent deletion in breast and prostate cancer (18,19) as well as squamous cell carcinomas of the head and neck, renal cell carcinomas, ovarian adenocarcinomas and colon carcinomas (20). The human caveolin-1 and -2 genes are located within the same commonly deleted region as FRA 7G. Caveolin-1 has been shown to have a role in the anchorage dependent inhibition of growth in NIH 3T3 cells (21). The caveolins are therefore candidates for the tumour suppressor gene presumed to be located in the FRA 7G region (20).  
           [0014]    Another common fragile site which is aphidicolin inducible is the FRA16D site. FRA16D has been localised at 16q23.2 within a large overlapping region of chromosomal instability in breast and prostate cancer as defined by loss-of-heterozygosity (24,25). One study has found that a significant proportion (77%) of breast cancers carries a deletion at 16q23.2, including the marker D16S518 in the immediate vicinity of FRA16D (24).  
           [0015]    There has been no characterisation of a nucleic acid or protein associated with the FRA16D site and the physical location of FRA 16D has not yet been determined. Such a characterisation is desirable to enable potentially early diagnosis and assessment of risk as well as potentially providing for a therapeutic treatment.  
         SUMMARY OF THE INVENTION  
         [0016]    The inventors have produced a detailed physical map of the FRA16D region which provides markers to identify a relationship between this fragile site and DNA instability in neoplasia and which, further, may allow better diagnosis of cancers associated with the region. This analysis reveals the existence of an intimate relationship between the location of FRA16D and homozygous deletions in various tumours, culminating in the coincidence of two tumour cell DNA breakpoints with the most likely position of the fragile site.  
           [0017]    The inventors have also characterised the nucleic acid associated with FRA16D especially by nucleic acid sequencing. Analysis of the DNA sequence and EST sequences associated with the region has identified a number of introns and exons which are found to exist in at least four different splice variants of what will be termed protein FOR. RNA analysis has also been conducted and thus far at least four species of mRNA associated with the region have been detected.  
           [0018]    In a first aspect the invention could be said to reside in a method of detecting genetic variations of a 16q23.2 target in the 16q23.2 region of the chromosome, said method comprising the steps of contacting target nucleic acid with one or more oligonucleotides suitable for use as hybridisation probe or PCR priming specific for binding the 16q23.2 specific target, and ascertaining the binding of said oligonucleotide.  
           [0019]    It will be understood from the specification that the 16q23.2 specific target might be selected to be within the group comprising the FOR gene, the FRA16D site, or mRNA encoding FOR protein or two or more of these collectively. The target may include chromosomal rearrangements and mutations thereof and the rearrangements or mutations may, in one form, be cancer associated. The variations may include markers in the region such as set forth in this specification including in FIGS. 1, 2 and  6 .  
           [0020]    The 16q23.2 target within the FOR gene might be selected from one or more of the group comprising exons 1A, 1, 2, 3, 4, 5, 6, 6A, 7, 8, 9, 9A, 10, 10A, 10B or exons located between two adjacent exons or control elements in other adjacent regions that effect an altered expression of the FOR gene. Such adjacent regions may have a promoter, enhancer elements or other regulatory elements. The target may be any one of the splice variants currently identified as FOR I, FOR II, FOR III or FOR IV or it might include other combinations of two or more of the exons.  
           [0021]    It is noted in particular that breakpoints of three out of five 16q23.2 translocations associated with multiple myeloma map within the alternate splice of this FOR intron, that is, between exons 8 and 9A, and in one form a preferred target is the intron between exons 8 and 9A or a portion thereof.  
           [0022]    In some circumstances the method might be used to detect any rearrangements in a larger target area. Thus it might be desired to use a plurality of oligonucleotides which might be selected to bind to a range of target binding sites within the 16q23.2 specific target to detect for a range of changes. This might be used for example to detect for chromosomal rearrangements such as deletions within the FRA16D site or beyond that in the broader 16q23.2 region. The plurality of oligonucleotides or a plurality of specific binding sites of the 16q23.2 target are preferably spacially separated so that binding of each of the plurality of oligonucleotides or binding to the plurality of specific binding sites can be separately ascertained. The spacial separation might, for example, be conveniently provided as an array on a solid support, for example in a form that is common referred to as a gene chip (see for example patent specifications U.S. Pat. No. 5,288,514 and U.S. Pat. No. 5,593,839). Instead of a plurality of oligonucleotides it may be desired that the target be probed by a single oligonucleotide.  
           [0023]    Alternatively the target area might be small, thus for example the method might be used to ascertain the presence or absence of a particular mutation or allelic variation in the 16q23.2 target. Thus for example a target of the 6A, 1A, 9 or 10 or 9A exon will distinguish between FOR I, FOR IV, FOR II and FOR III transcription variants. These may also be used to quantify differences in expression of the splice variants FORII and FORI on the one hand and FORII on the other. It might be expected that because the FORIII only has the WW domains in contrast to FOR II and FOR I a significant biological effect may result from variations in the balance of expression of these different variations of FOR, such variations may give an indication of individuals who are at risk of contracting a form of tumour. A small target area might also be adequate for use with gross chromosomal rearrangements in so far as this might be used to determine the presence or absence of junctions of known chromosomal rearrangements, or alternatively the binding or non binding of one or more of a plurality of oligonucleotides. The target area might also be selected to allow for assessment of the presence or absence of cancer associated point mutations or small DNA rearrangements, using suitably selected oligonucleotides.  
           [0024]    The base sequence of the oligonucleotide chosen will depend upon several factors known in the art. Primarily the sequence of the oligonucleotide will be determined by its capacity to bind to the target nucleic acid sequence. The nature of the sequence will depend to some extent on the stringency of the hybridisation required, and whether or not it is desired for one oligonucleotide to detect variation in sequence or not. If variation in one nucleotide is required the stringency of the hybridisation will be high. The length of the oligonucleotide will also be determined by the stringency of the reaction required.  
           [0025]    The binding might be by in situ hybridisation of a chromosomal spread, or other suitable spacial arrangement of the target region such as for example on a so called gene chip. Such hybridisation methods will generally provide for an oligonucleotide and be capable of binding the target over a span of at least 15 nucleotides. In the case of hybridisation techniques the oligonucleotides will generally carry a label which can be detected by known measuring methods, especially when bound to the 16q23.2 target. Such labels might include radiolabels such as  32 P or a fluorescent marker.  
           [0026]    The method might require a preamplification step whereby the target nucleic acid is amplified, to make it easier to ascertain the binding or non binding of the nucleic acid to the target site.  
           [0027]    On the other hand the oligonucleotide might be suitable for amplification of a segment of the target nucleic acid such as by PCR, in which case the size of the target may be somewhat different. With this variation two oligonucleotides might be selected, to provide for amplification of at least part of the target nucleic acid, at least one of the oligonucleotides is required to bind in the target.  
           [0028]    The target nucleic acid might be presented in any one of a number of physical forms. Nucleic acid from an individual might be isolated and perhaps digested by a restriction enzyme and spread out such as by electrophoresis on an agarose or polyacrylamide gel, so that binding of the oligonucleotide can be effected whilst the target nucleic acid is supported by the gel or this might be supported on other solid medium such as a gene chip or a metaphase chromosomal spread. Alternatively the oligonucleotide or oligonucleotides might be fixed, and the target nucleic acid might either be diminished in size, or not, and then binding of fragmented targets to the fixed oligonucleotide determined.  
           [0029]    The target nucleic acid might be in the form of chromosomal DNA, or might be cDNA or mRNA.  
           [0030]    This method might also be used to detect other variants, homologs or analogs of the FRA16D site, FOR gene, or other nucleic acid sequences disclosed in this specification. Thus it might be, for example desirable to determine analagous gene in livestock, domestic, laboratory or sporting animals. Alternatively one might wish to determine another analogous protein that plays a similar role in humans.  
           [0031]    In a second aspect the invention relates to a method of detecting the number of alleles for one or more markers in the 16q23.2 target, and this may be a means of perhaps providing a measure of the loss of heterozygosity in an individual. This aspect of the invention therefore relates to locating a deletion that overlaps with the FRA16D region. The method might be achieved by providing a first set of one or more oligonucleotides and a second set of one or more oligonucleotides the first set of oligonucleotide being specific for a first variant of the target nucleic acid, the second set of oligonucleotides being specific for a second variant of the target nucleic acid, the first and second set of oligonucleotides being labelled so as to be capable of being distinguished, and the method comprising the steps of comparing the proportion of binding of the first and second set of oligonucleotides. A method of this sort is set forth in U.S. Pat. No. 5,928,870 to Lapidus et al, which for purposes of practicing the invention is incorporated herein by reference.  
           [0032]    It will be understood that the above method is useful in categorising the risk of contracting certain types of cancer associated with the FRA16D fragile site or other portion of the 16q23.2 region.  
           [0033]    In a third aspect the invention could be said to reside in a method of determining the level of expression of the FOR gene or any one or more exon thereof, by determining the level of mRNA expression using a probe specific for the FOR gene or exon thereof. This might be used to determine the dysregulation of FOR expression. It will be understood that it may be desired to also determine the level of expression of variants of the gene or exons including rearrangements and mutants including those associated with cancers. This is likely to give a prognosis in relation to at least certain cancers that are currently contracted or perhaps an indication of the risk of contracting one or more types of cancer.  
           [0034]    In a fourth aspect the invention could be said to reside in an isolated nucleic acid molecule selected from the group comprising  
           [0035]    a) any one or more of the nucleic acids sequences disclosed in the figures hereto or parts thereof  
           [0036]    b) FRA16D site  
           [0037]    c) FOR gene, or exons thereof  
           [0038]    d) mRNA of the FOR gene  
           [0039]    e) cDNA of the FOR gene  
           [0040]    f) variants of the above including, chromosomal rearrangements and mutations of sequences set out in a) to e) including those variants associated with cancers  
           [0041]    g) nucleic acid sequence capable of hybridising specifically to any sequence of a to e above or its complement, and especially those capable of doing so under stringent conditions.  
           [0042]    The nucleic acid molecule might include a mosaic from within the above molecules such as a combination of two or more of the group comprising the following, exon 1A, 1, 2, 3, 4, 5, 6, 6A, 7, 8, 9, 9A, 10, 10A, 10B or introns located between two adjacent exons or control elements in other adjacent regions that effect an altered expression of FOR, and it will be understood that such a mosaic includes a molecule encoding cDNA of variants of the FOR protein, whether a wild type allele, a mutated version, or otherwise rearranged. It will thus be understood that the invention includes antisense molecules to any regions of control that might be contemplated above. Such antisense molecules may be used to vary the expression of such protein as are produced by the FOR gene or perhaps adjacent genes such as the c-MAF gene. One may also wish to reduce the expression of one of the splice variants of FOR to provide treatment of a given condition, thus for example it might be desired to have antisense specifically to FOR III if FOR III is overexpressed in the condition.  
           [0043]    It will be understood that such nucleic acids include portions of nucleic acids that are suitable for use as primers or probes.  
           [0044]    The invention may also be said to include nucleic acids encoding a tumour associated gene from a human or animal capable of hybridizing with any nucleic acid of the fourth aspect of the invention.  
           [0045]    In a fifth aspect the invention could be said to reside in a recombinant vector including one or more nucleic acid sequences as set out above, and preferably operably linked to a control element such as might include a functional promoter. The recombinant vector might be used as an expression vector to produce or overproduce FOR protein or variants thereof, or perhaps overproduce nucleic acids associated with the FOR gene such as an antisense molecule. Suitable vectors are generally available commercially or may be constructed as described elsewhere or as is known in the art.  
           [0046]    In a sixth aspect the invention could be said to reside in an isolated protein molecule, the protein molecule being selected from the group comprising the following:  
           [0047]    a) a FOR protein, or  
           [0048]    b) a mutant or variant FOR protein which might optionally be associated with a cancer  
           [0049]    In a seventh aspect the invention could be said to reside in a polypeptide produced by any two or more exons selected from the group comprising 1A, 1, 2, 3, 4, 5, 6, 6A, 7, 8, 9, 9A, 10, 10A, 10B joined, said exons being either complete exons or partial, and may be variants.  
           [0050]    The invention might also encompass a purified cancer associated protein including a string of amino acids unique to a FOR protein and more particularly as set out in FIG. 9, preferably said amino acid string being at least 10 amino acids long and exhibiting at least 70% amino acid homology more preferably at least 90% homology.  
           [0051]    The protein may have an oxidoreductase domain and/or one or more WW domains or may have a role in DNA replication of chromosomal division.  
           [0052]    In another form the purified cancer associated protein includes an amino acid string with an amino acid sequence homology of greater than 70% but more preferably greater than 90% with an amino acid string selected from the group comprising:  
                               TGANSGIGFETAKSFALHGAHVILACR,   (SEQ ID No 1)                   LHVLVCNAATFALPWSLTKDGLETTFQVNHLGHFYLVQLLQDVL,   (SEQ ID No 2)               YNRSKLCNILFSNELHRRLSPRGVTSNAVHPG   (SEQ ID No 3)          
 
           [0053]    In another form the purified cancer associated protein includes a WW domain having an amino acid string of 10 amino acid or greater or preferably 20 amino acids or greated with an amino acid sequence homology of greater than 70% but preferably greater than 90% with an amino sequence selected from the group comprising the region 16 to 49 or 57 to 90 of the FOR gene (as graphically illustrated in FIG. 10A), being the amino acid strings  
                               DELPPGWEERTTKDGWVYYANHTEEKTQWEHPKT   (SEQ ID No 4)                   and               GDLPYGWEQETDENGQVFFVDHINKRTTYLDPRL   (SEQ ID No 5)          
 
           [0054]    In another form the purified cancer associated protein includes at least one oxidoreductase domain having an amino acid string of 10 amino acid or greater or preferably 20 amino acids or greater with an amino acid sequence homology of greater than 70% but preferably greater than 90% with an amino sequence selected from the group comprising the region 130 to 156 or 204 to 247 or 293 to 324 of the FOR gene (as graphically illustrated in FIG. 10A).  
           [0055]    In an eighth aspect the invention includes an agent capable of selectively binding a FOR protein or fragment or variant thereof. Such agents may be particularly useful in diagnostic methods. Such an agent may also be used to bind a protein containing a string of amino acids unique to FOR or variant thereof and in particular such variants that are currently known to be associated with one or more forms of cancer. The agent may selectively bind to the variant FOR as compared to an FOR protein not associated with cancer. Such an agent might be an agonist or an antagonist of FOR function. It might therefore be desired to provide for a number of agents each capable of selectively binding to a separate one of a number of variants of FOR so that it is possible to distinguish between variants. Thus for example it might be desired to target the C terminus of respectively FOR I, FOR II, FOR III and FOR IV to distinguish between these four proposed forms. The invention therefore also encompasses a method of detecting variants of the FOR protein. Measuring the relative levels of these four and other forms of FOR protein is likely to give an indication of regulatory perturbations which may be associated with certain cancers.  
           [0056]    The nature of the agents can vary depending on their intended use. Thus for a diagnostic method an antibody or fragment thereof, such as an Fab fragment, of a recombined molecule carrying the variable region of an antibody recognising the desired portion of the FOR may be adequate. The antibody might be polyclonal however preferably the antibody is a monoclonal antibody prepared by known techniques.  
           [0057]    Alternatively small molecules capable of binding the desired portion of the FOR protein may be used, such small molecules might include peptides, proteins, nucleic acids or sugars or other organic molecules. These can be isolated by screening using known techniques from libraries of suitable compounds. Such small molecules can then be tested for antagonist or agonist properties to potentially provide a therapeutical agent which have the potential to be used in the treatment of cancers. These agents would be administered by clinicians in an appropriate manner.  
           [0058]    Also useful therapeutically might be the provision of an isolated protein of the seventh aspect of the invention, particularly those forms that mimic the action of a wild type FOR, and perhaps simply the purified FOR. It is anticipated that the FOR protein in at least one of its forms is a tumour suppressor, that is, its absence increases the risk of aberrant cell division leading to a cancer. Accordingly one form of therapy may include the administration of such a protein to an individual who is considered at risk, particularly if they are found to have an altered FOR protein. Such administration would be in conformity with normal practices in a suitable excipient. It may also be the case that the aberrant FOR protein actively enhances tumourigenesis and accordingly it might be appropriate to administer an antagonist of the aberrant variant at the same time. Alternatively the administration of the antagonist on its own may be of therapeutic benefit. Thus for example FORIII is anticipated to be a competitor of FORII and/or FORI, and thus expression of FORIII at higher or lower levels relative to FORII and/or FORI is likely to have a therapeutic effect.  
           [0059]    Another form of treatment which is becoming increasingly contemplated is to provide for a method of gene therapy and one method of undertaking cell therapy is to provide for certain progenitor cells which include incorporated therein a vector capable of producing an appropriate form of FOR protein. Accordingly in a ninth aspect the invention could be said to reside in a recombinant host cell having stably inserted therein DNA of any one of the forms of DNA contemplated in the third aspect of the invention. In preference the DNA is capable of producing a tumour suppressing form of FOR, and most conveniently this will be a wild-type form of FOR, which may simply be a cDNA molecule or the FOR gene. Alternatively however it may also be desired to have a host cell which has a DNA sequence capable of producing an antisense molecule in the case where a tumour promoting form of the FOR molecule is produced by the individual to be treated, the antisense capable of reducing the level of expression of the FOR molecule.  
           [0060]    Methods of gene therapy are not limited to cases where the appropriate nucleic acid is delivered in a host cell, but also includes the administration of the nucleic acid specifically to the site of interest.  
           [0061]    The recombinant host cell may not necessarily be used for therapeutic purposes, it may also be used for over-expression of the protein, or a nucleic acid associated with FOR, or the 16q23.2 region, and may therefore be bacterial, yeast, plant, animal, preferably mammalian or human. Additionally the invention contemplates the provision of a transgenic non-human animal carrying recombinantly altered or overexpressing 16q23.2 DNA, preferably FRA16D or FOR gene, or other DNA of the fourth form of this invention. The recombinant DNA might be incorporated into the chromosome of the host, alternatively the host cell may carry said recombinant DNA in a self replicating element such as a plasmid.  
           [0062]    The agents of the eighth aspect may be used for ascertaining the level of expression of FOR, variants or exons thereof, to determine whether there is an altered level of expression. Thus a western blot using a labelled agent may be used for the purpose using known techniques.  
           [0063]    This is another means of measuring dysregulation of expression. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0064]    [0064]FIG. 1: Positional cloning of FRA16D and location of loss of heterozygosity and translocation in cancer.  
         [0065]    A. The locations of loss-of-heterozygosity regions in breast and prostate cancer and the approximate location of the FRA16D fragile site are indicated with respect to genetic markers (downward arrows) in the 16q23.2 region. Markers in the vicinity of FRA16D are shaded. The approximate location as determined by Chesi et al. (1) of multiple myeloma breakpoints and the c-MAF gene (bar) are also shown by upward black arrows. Not to scale.  
         [0066]    B. Map of the contig of YAC subclones across the FRA16D region with respect to genetic markers and FRA16D. Open boxes indicate those YACs which map by fluorescence in situ hybridisation proximal to FRA16D, grey boxes are those which span FRA16D and black boxes indicate those YACs which map distal to FRA16D. Not to scale.  
         [0067]    [0067]FIG. 2: Positional cloning of FRA16D and the extent of heterozygous and homozygous deletion in the AGS tumour cell line.  
         [0068]    A. Pulsed-Field gel map of ˜1 Mb of the ‘Right Hand Side’ (RHS) of YAC My801B6 and the location of BACs, genetic and STS markers (key markers are boxed). Restriction sites between Afma336yg9 and WI2755 are shown in B. The AGS stomach cancer cell line homozygous deletion is indicated—shaded circles denote the presence and open circles the absence of PCR products for the STS markers. Maximal region of heterozygous deletion in AGS cell line is indicated by polymorphic D16S518 and D16S3029 PCR products, indicated as A and B alleles. The two AGS cell line chromosome 16s are indicated by shaded bars.  
         [0069]    B. Restriction map of the critical FRA16D region (Afma336yg9 to D1653029) showing the location of key members of the lambda subclone tile path used for FISH in FIG. 3. Clones designated l-n are from 325M3; others are from 801B6. Open boxes represent those subclones found to map proximal (on the basis that &gt;85% of their FISH signals were proximal to FRA16D), grey boxes those which appear to span the fragile site (less than 85% on one side or other of FRA16D) and black boxes those which are distal to the fragile site (on the basis that &gt;85% of their FISH signals were distal to FRA16D). l clones which gave high background on FISH were not scored. These and other l clones for which FISH data were not obtained are included as thin boxes. STS localisation of the AGS homozygous breakpoints are indicated by the presence (shaded circles) and absence (open circles) of PCR products.  
         [0070]    [0070]FIG. 3: Fluorescence in situ hybridisation (FISH) of lambda subclones against FRA16D expressing chromosomes.  
         [0071]    Each panel contains two FRA16D expressing partial metaphases, with and without FISH signal merged. In each case the width of the gap or break at the fragile site is greater than the width of the chromatid. (a) 1504 showing signal proximal to FRA16D; (b) 1181 showing signal proximal and distal to FRA16D; (c) 1191 (upper) and 18 (lower) showing signal distal to FRA16D. Images of metaphase preparations were captured by a cooled CCD camera using the ChromoScan image collection and enhancement system (Applied Imaging International Ltd.). FISH signals and the DAPI banding pattern were merged for figure preparation.  
         [0072]    [0072]FIG. 4: Fluorescence in situ hybridisation mapping of the lambda subclone tile path across FRA16D.  
         [0073]    The individual lambda clones were scored against chromosomes where the FRA16D gap or break was greater than the chromatid width. Each increment represents a single FISH signal. n=number of chromosomes scored. Scores were plotted as proximal (p) and distal (d) with respect to FRA16D. Maximum location for FRA 16Ds indicated by arrows. Location of BAC clones 325M3 and 353B15 is also shown. The boxed lambda contig subclones indicate those for which FISH signal results with respect to the FRA16D fragile site were obtained—open boxes, had &gt;85% signal proximal to FRA16D; grey boxes, spanning (&lt;85% signal on one side or other of FRA16D) and black boxes, had &gt;85% signal distal to FRA16D. While this figure is not to scale the location of the lambda clones can be determined from their position in FIG. 2. Thin boxed lambda clones are those for which FISH data was not obtained.  
         [0074]    [0074]FIG. 5: Duplex PCR deletion detection at the FRA16D locus in tumour cell lines.  
         [0075]    PCR products from the duplex of STSG-10102 and dystrophin DMD Pm were subjected to agarose gel electrophoresis and ethidium bromide staining. Template DNAs were seven tumour cell lines and blood bank and no DNA controls. Markers are HpalI digested pUC19. The position of the STSG-10102 and DMD Pm PCR products are indicated by large grey-filled arrows while the primer dimer PCR artefact is indicated by a small white arrow.  
         [0076]    [0076]FIG. 6: A. Extent of loss of heterozygosity regions in breast (25) and prostate cancer (24) in relation to the cytogenetic position of the FRA16D fragile site as determined by fluorescence in situ hybridisation of a tile path of subclones as show in FIG. 4.  
         [0077]    B. Map of YACs which span FRA16D region showing approximate location of multiple myeloma breakpoints (MM.1, ANBL6, JJN3) determined by Chesi et al., (1). Location of homozygously deleted regions in AGS and HCT 116 tumour cell lines as determined by STS content. The locations of various partial BAC sequences (as evident by STS content) are indicated. Striped boxes=determined sequence accession numbers.  
         [0078]    C. The location of the FRA16D spanning DNA sequence and the respective exons of the alternative spliced FOR gene transcripts (numbered black boxes). Clusters of ESTs sequences representative of each of the alternative mRNA 3′ ends are given.  
         [0079]    [0079]FIG. 7: A. Northern blots of RNA from various human tissues. Expected FOR mRNAs (I-IV) are indicated for the respective DNA probes which span various exons of the FOR gene. H, heart, Br, brain; Pl, placenta; Lu, lung; Li, liver; sM, skeletal muscle; K, kidney; P, pancreas. Arrows indicate FOR mRNAs (FOR I approx. 1.3 kb, FOR II approx 2.2 kb, FOR III approx 0.74 kb)  
         [0080]    B. Northern blots of RNA from various human tissues, spleen, thymus, prostate, testis, ovary, small intestine, colon, peripheral blood leukocytes.  
         [0081]    Probes (I, II and III) and (I and II) are as indicated in FIG. 6. FOR I, FOR II and FOR III mRNAs are indicated. Additional transcripts hybridizing to the FOR probes are indicated by grey arrows.  
         [0082]    [0082]FIG. 8 A. Is a composite DNA sequence of the predicted FOR I transcript (SEQ ID No 28) constructed by conjoining overlapping EST, RT-PCR and 5′ RACE DNA sequences.  
         [0083]    B. Is a composite DNA sequence of the predicted FOR II transcript (SEQ ID No 29) constructed by conjoining overlapping EST, RT-PCR and 5′ RACE DNA sequences.  
         [0084]    C. Is a composite DNA sequence of the predicted FOR III transcript (SEQ ID No 30) constructed by conjoining overlapping EST, RT-PCR and 5′ RACE DNA sequences.  
         [0085]    D. Is a composite DNA sequence of the predicted FOR IV transcript (SEQ ID No 31) constructed by conjoining overlapping EST and RT-PCR DNA sequences.  
         [0086]    [0086]FIG. 9 are composite amino acid sequences predicted for the sequences for FOR I (SEQ ID No 32), FOR II (SEQ ID No 33), FOR III (SEQ ID No 34) and FOR IV (SEQ ID No 35) as shown in FIG. 8, unique sequences are underlined.  
         [0087]    [0087]FIG. 10 A. Is a diagrammatic representation of the four FOR amino acid sequences showing the locations of the alternate splice sites, the position of the exons, the three predicted oxido reductase domains, and the predicted WW domains. The sequence numbers refer to the amino acid sequence.  
         [0088]    B. Is an alignment of the sequences WW domains (SEQ ID No 4 and SEQ ID No 5) with each other and with the WW domain consensus sequence.  
         [0089]    [0089]FIG. 11 sets out DNA sequences for each of the exons identified for the FOR protein as well as a small amount of flanking intron sequence. The exon sequences are in uppercase, while the intron sequence is in lower case. Some nucleotide sequences are in bold, splice donor (GT) and acceptor (AG) sites, polyadenylation signals (AATAA) and initiation Methionine (ATG). For exons 1 and 1A an upstream in phase termination codon is in italics and confirms the correct open reading frame in these mRNAs.  
         [0090]    [0090]FIG. 12 is about 270 kb of DNA sequence that overlaps and defines within it the FRA16D fragile site (SEQ ID No 53), which is shown to reside between exons 8 and 9, this sequence has been deposited in the GenBank database and has been assigned accession number AF217490 as indicated in FIG. 6.  
         [0091]    [0091]FIG. 13 is DNA sequence deposited with GenBank database and identifed by accession number AF217492 as indicated in FIG. 6, and which encompasses exon 7 (SEQ ID No 52).  
         [0092]    [0092]FIG. 14 is DNA sequence deposited with GenBank database and identifed by accession number AF217491 as indicated in FIG. 6, and which encompasses exon 6 (SEQ ID No 51).  
         [0093]    [0093]FIG. 15 shows FOR transcripts in normal and tumour cells. Products that were subjected to sequence analysis are indicated by arrowheads.  
         [0094]    A RT-PCR were either ‘specific’ for the FOR III transcript or ‘general’ being able to detect FOR I-III mRNAs.  
         [0095]    B 5′RACE specific for the FOR I, FOR II and FOR III transcripts in ‘normal’ HS578BST cells and T47D tumour cells. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
     EXAMPLE 1  
     Mapping of the FRA 16D Fragile Site  
       [0096]    Materials and Methods  
         [0097]    Isolation of DNA Probes and YACs in the FRA16D Region  
         [0098]    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 (FIG. 1B).  
         [0099]    Additional Probes, STSs and BACs from the FRA16D Region  
         [0100]    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 (FIG. 2A).  
         [0101]    Subcloning and Contig Assembly  
         [0102]    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).  
         [0103]    Fluorescence In Situ Hybridisation  
         [0104]    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 (FIG. 3). 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.  
         [0105]    Tumour Cell Lines  
         [0106]    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.  
         [0107]    PCR Detection of Homozygous Deletion in Tumour Cell DNAs  
         [0108]    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 (FIG. 4). 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).  
         [0109]    Results  
         [0110]    Positional Cloning of FRA 16D  
         [0111]    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 (FIG. 1B). 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).  
         [0112]    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) (FIG. 2A). 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 (FIG. 2A). The STS (D16S518, Afma336yg9, WI2755, STSG-10102 and D16S3029) content of the YACs and BACs was also determined to assist in map construction.  
         [0113]    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).  
         [0114]    Localisation of FRA16D by Fluorescence In Situ Hybridisation (FISH)  
         [0115]    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 (FIG. 3). 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.  
         [0116]    The results of the FISH experiments are plotted in FIG. 4. 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.  
         [0117]    Detection of Homozygous Deletion in Tumour Cell Lines  
         [0118]    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.  
         [0119]    STSs that were either mapped to the FRA16D region (FIG. 1) 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 FIG. 4. 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.  
         [0120]    Detection of Heterozygous Deletion in AGS Tumour Cell Line DNA  
         [0121]    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 (FIG. 2A). The markers Afma336yg9 and 504CA were uninformative and therefore did not aid in delineating the limits of heterozygous deletion.  
         [0122]    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 (FIG. 7). Identical N-termini, unique C-termini.  
         [0123]    WW domains were identified by ProfileScan searches (at http://www.expasy.ch/prosite/).  
         [0124]    Discussion  
         [0125]    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 FIG. 1). 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%.  
         [0126]    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.  
         [0127]    Taken together these deletion data confirm the hypothesis that FRA16D is associated with specific chromosomal instability in cancer.  
         [0128]    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  
       EXAMPLE 2  
     DNA Sequencing of the FRA16D Fragile Site and the FOR Gene  
       [0129]    Materials and Methods:  
         [0130]    Cell Lines  
         [0131]    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 .  
         [0132]    Large Scale Sequencing of FRA16D  
         [0133]    Sequencing of the 270 kb region spanning FRA16D consisted of  
         [0134]    a) Sonication libraries and  
         [0135]    b) Nebulization libraries of BAC clones 325M3 and 353B 15 and  
         [0136]    c) Restriction fragments of l clones (for sequencing between BAC 325M3 and BAC 353B 15).  
         [0137]    a) Construction of Sonication Libraries:  
         [0138]    For DNA sonication and cloning we modified the protocol from the Sanger Centre (http://www.sanger.ac.uk/Teams/Team53/sonication.shtml):  
         [0139]    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.  
         [0140]    b) Construction of Nebulization Libraries:  
         [0141]    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.  
         [0142]    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.  
         [0143]    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.  
         [0144]    The final sequence was analysed using:  
         [0145]    BLAST (http://www.ncbi.nlm.nih.gov/BLAST),  
         [0146]    REPEATMASKER (http://ftp.genome.washington.edu/cgi-bin/RepeatMasker), and  
         [0147]    GENSCAN (http://CCR-081.mit.edu/GENSCAN.html).  
         [0148]    Northern Blot Hybridisation  
         [0149]    Probes for hybridisation on multiple tissue northern blots from Clontech were:  
         [0150]    a) exon 7 (186 bp), positions 690 through 876 of AF227526  
         [0151]    b) part of exon 9A (779 bp), positions 1182 through 1961 of AF227527  
         [0152]    c) exon 3-6A (366 bp), positions 291 through 657 of AF227528  
         [0153]    d) part of exon 1A (163 bp), positions 298 through 461 of AF227529.  
         [0154]    RNA Extraction  
         [0155]    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.  
         [0156]    RT-PCR  
         [0157]    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.  
         [0158]    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).  
         [0159]    Primers (5′-3′) used in RT-PCR were:  
         [0160]    a) HHCMA-F (ATCTTGGCCTGCAGGAACATGGCA) (SEQ ID No 12)and wb85-F (TTATTCTGCA CTTTTCTGGCGGAG) (SEQ ID No 13), FORIII specific  
         [0161]    b) FOR-ex3 (GAACAAGAAACTGATGAGAACGGA) (SEQ ID No 14)and wb85-F, FORIII specific  
         [0162]    c) wb85-E12 (TTACTACGCCAATCACACCGAGGA) (SEQ ID No 15)and wb85-A (TGAATTAGCTCCAGTGACCACAAC) (SEQ ID No 16), common in FORI, FOR II and FOR III  
         [0163]    5′ RACE  
         [0164]    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)          
 
         [0165]    PCRs of the dC-tailed cDNA were carried out with the GSP2-primers:  
                                       FORI and FORII   (coxido-32,   5′-ATATCTGTAAATCGATGGGACTCTG-3′),   (SEQ ID No 19)                   FORIII   (wb85-A,   5′-TGAATTAGCTCCAGTGACCACAAC-3′).   (SEQ ID No 16)          
 
         [0166]    Nested amplification was done with 5 ul of a 1:100 dilution of GSP2-PCR products and the GSP3-primers:  
         [0167]    FORI and FORII (coxido-21,5′-ACATGAAGAGGCACATTCTTGGCCT-3′) (SEQ ID No 20)  
         [0168]    and FORIII (wb85-E, 5′-TCCTCGGTGTGATTGGCGTAGTAA-3′) (SEQ ID No 21) in combination with the AUAP-primer (GibcoBRL) (SEQ ID No 21).  
         [0169]    PCR-products were extracted with Qiaquick-Kit from agarose-gels after electrophoresis and sequenced directly with GSP3-primers and the primer tj96-C:  
         [0170]    5′-GGAGGCAGCTCGTCCTCACTG-3′ (SEQ ID No 22).  
         [0171]    3′ RACE  
         [0172]    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:  
         [0173]    RACE-AP/VAR (5′-GGCCACGCGTCGACTAGTACGTACAGT {TTT } 5 T-3′).  
         [0174]    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.  
         [0175]    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 FIGS. 6 and 10).  
         [0176]    cDNA Sequence of FOR IV (AF227529)  
         [0177]    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).  
         [0178]    Determination of Breakpoints in Cell Lines AGS and HCT116  
         [0179]    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.  
         [0180]    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 (+).  
         [0181]    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 (+).  
         [0182]    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).  
         [0183]    Results  
         [0184]    DNA Sequence Spanning FRA16D  
         [0185]    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) (FIG. 6).  
         [0186]    Relationship Between Deletion and Translocation Breakpoints and FRA16D  
         [0187]    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 (FIG. 6). 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 (FIG. 6). 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.  
         [0188]    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).  
         [0189]    Alternatively Spliced FOR Gene Spans Fragile Site FRA16D  
         [0190]    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 (FIG. 6). 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.  
         [0191]    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 (FIG. 6). 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.  
         [0192]    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 (FIG. 6) 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 (FIG. 6). 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).  
         [0193]    FOR mRNA in Normal and Tumour Cells  
         [0194]    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 (FIG. 4). 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 (FIG. 15).  
         [0195]    FOR Encoded Proteins  
         [0196]    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 (FIG. 10).  
         [0197]    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 (FIG. 10).  
         [0198]    Discussion  
         [0199]    Identification of the FOR Gene Spanning FRA16D  
         [0200]    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.  
         [0201]    Differential Expression of Alternative Spliced and Aberrant FOR Transcripts in Normal and Tumour Cells  
         [0202]    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.  
         [0203]    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.  
         [0204]    Possible Function of FOR and Role in Neoplasia  
         [0205]    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.  
         [0206]    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 (FIG. 10). 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.  
         [0207]    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.  
         [0208]    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, FIG. 15) 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.  
         [0209]    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.  
         [0210]    For the purposes of this specification the word “comprising” means “including but not limited to”, and the word “comprises” has a corresponding meaning.  
         [0211]    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.  
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         1 
         
           
             53  
           
           
             1  
             27  
             PRT  
             Homo sapien  
             
               Cancer associated protein  
             
           
            1 

Thr Gly Ala Asn Ser Gly Ile Gly Phe Glu Thr Ala Lys Ser Phe 
  1               5                  10                  15 

Ala Leu His Gly Ala His Val Ile Leu Ala Cys Arg 
                       20                  25 

 
           
             2  
             44  
             PRT  
             Homo sapien  
             
               Cancer associated protein  
             
           
            2 

Leu His Val Leu Val Cys Asn Ala Ala Thr Phe Ala Leu Pro Trp 
  1               5                  10                  15 

Ser Leu Thr Lys Asp Gly Leu Glu Thr Thr Phe Gln Val Asn His 
                 20                  25                  30 

Leu Gly His Phe Tyr Leu Val Gln Leu Leu Gln Asp Val Leu 
                 35                  40 

 
           
             3  
             32  
             PRT  
             Homo sapien  
             
               Cancer associated protein  
             
           
            3 

Tyr Asn Arg Ser Lys Leu Cys Asn Ile Leu Phe Ser Asn Glu Leu 
  1               5                  10                  15 

His Arg Arg Leu Ser Pro Arg Gly Val Thr Ser Asn Ala Val His 
                 20                  25                  30 

Pro Gly 

 
           
             4  
             34  
             PRT  
             Homo sapien  
             
               Cancer associated protein  
             
           
            4 

Asp Glu Leu Pro Pro Gly Trp Glu Glu Arg Thr Thr Lys Asp Gly 
  1               5                  10                  15 

Trp Val Tyr Tyr Ala Asn His Thr Glu Glu Lys Thr Gln Trp Glu 
                 20                  25                  30 

His Pro Lys Thr 

 
           
             5  
             34  
             PRT  
             Homo sapien  
             
               Cancer associated protein  
             
           
            5 

Gly Asp Leu Pro Tyr Gly Trp Glu Gln Glu Thr Asp Glu Asn Gly 
  1               5                  10                  15 

Gln Val Phe Phe Val Asp His Ile Asn Lys Arg Thr Thr Tyr Leu 
                 20                  25                  30 

Asp Pro Arg Leu 

 
           
             6  
             21  
             DNA  
             Artificial Sequence  
             
               PCR Primer for 504CA (forward)  
             
           
            6 

aacacagctc ttatcacatc c                                               21 

 
           
             7  
             19  
             DNA  
             Artificial Sequence  
             
               PCR Primer for 504CA (reverse)  
             
           
            7 

tggctgtamg tcagaactg                                                  19 

 
           
             8  
             24  
             DNA  
             Artificial Sequence  
             
               PCR Primer for GenBank AA368108 (forward)  
             
           
            8 

taatcctcag cctctagaat gcct                                            24 

 
           
             9  
             24  
             DNA  
             Artificial Sequence  
             
               PCR Primer for GenBank AA368108 (reverse)  
             
           
            9 

gtatgatgat tttcagggag aaac                                            24 

 
           
             10  
             24  
             DNA  
             Artificial Sequence  
             
               PCR Primer for GenBank AA398024 (forward)  
             
           
            10 

tgtcctcaac tgattcttac aaac                                            24 

 
           
             11  
             21  
             DNA  
             Artificial Sequence  
             
               PCR Primer for GenBank AA398024 (reverse)  
             
           
            11 

tcaatgggtt aggcacagac c                                               21 

 
           
             12  
             24  
             DNA  
             Artificial Sequence  
             
               RT-PCR Primer HHCMA-F, FORIII specific  
             
           
            12 

atcttggcct gcaggaacat ggca                                            24 

 
           
             13  
             24  
             DNA  
             Artificial Sequence  
             
               RT-PCR Primer wb85-F, FORIII specific  
             
           
            13 

ttattctgca cttttctggc ggag                                            24 

 
           
             14  
             24  
             DNA  
             Artificial Sequence  
             
               RT-PCR Primer FOR-ex3  
             
           
            14 

gaacaagaaa ctgatgagaa cgga                                            24 

 
           
             15  
             24  
             DNA  
             Artificial Sequence  
             
               RT-PCR Primer wb85-E12  
             
           
            15 

ttactacgcc aatcacaccg agga                                            24 

 
           
             16  
             24  
             DNA  
             Artificial Sequence  
             
               RT-PCR Primer wb85-A  
             
           
            16 

tgaattagct ccagtgacca caac                                            24 

 
           
             17  
             27  
             DNA  
             Artificial Sequence  
             
               5′RACE Primer coxido-R  
             
           
            17 

ttatttcagc actcagctca aagtcac                                         27 

 
           
             18  
             25  
             DNA  
             Artificial Sequence  
             
               5′RACE Primer HHCMA-B  
             
           
            18 

agcaaagaga cctatgccta gccca                                           25 

 
           
             19  
             25  
             DNA  
             Artificial Sequence  
             
               5′RACE Primer coxido-32  
             
           
            19 

atatctgtaa atcgatggga ctctg                                           25 

 
           
             20  
             25  
             DNA  
             Artificial Sequence  
             
               5′RACE Primer coxido-21  
             
           
            20 

acatgaagag gcacattctt ggcct                                           25 

 
           
             21  
             24  
             DNA  
             Artificial Sequence  
             
               5′RACE Primer wb85-E  
             
           
            21 

tcctcggtgt gattggcgta gtaa                                            24 

 
           
             22  
             21  
             DNA  
             Artificial Sequence  
             
               Direct Sequencing Primer tj96-C  
             
           
            22 

ggaggcagct cgtcctcact g                                               21 

 
           
             23  
             53  
             DNA  
             Artificial Sequence  
             
               3′RACE Primer RACE-AP/VAR  
             
           
            23 

ggccacgcgt cgactagtac gtacagtttt tttttttttt tttttttttt ttt            53 

 
           
             24  
             23  
             DNA  
             Artificial Sequence  
             
               PCR Primer overlapping FORI exon 8/exon 9 
                         boundary  
             
           
            24 

accaagtcca tggtttcaga ctg                                             23 

 
           
             25  
             20  
             DNA  
             Artificial Sequence  
             
               3′RACE Primer RACE-NESTED  
             
           
            25 

cgtcgactag tacgtacagt                                                 20 

 
           
             26  
             27  
             DNA  
             Artificial Sequence  
             
               Exon 9 Specific Primer #9327  
             
           
            26 

actgcctggt agaaggaggt cacttct                                         27 

 
           
             27  
             20  
             DNA  
             Artificial Sequence  
             
               3′RACE Primer AUAP  
             
           
            27 

ggccacgcgt cgactagtac                                                 20 

 
           
             28  
             1225  
             DNA  
             Homo sapien  
             
               FOR I mRNA  
             
           
            28 

ggtctcgttt ggagcgggag tgagttcctg agcgagtgga cccggcagcg ggcgataggg     60 

gggccaggtg cctccacagt yagccatggc agcgctgcgc tacgcggggc tggacgacac    120 

ggacagtgag gacgagctgc ctccgggctg ggaggagaga accaccaagg acggctgggt    180 

ttactacgcc aatcacaccg aggagaagac tcagtgggaa catccaaaaa ctggaaaaag    240 

aaaacgagtg gcaggagatt tgccatacgg atgggaacaa gaaactgatg agaacggaca    300 

agtgtttttt gttgaccata taaataaaag aaccacctac ttggacccaa gactggcgtt    360 

tactgtggat gataatccga ccaagccaac cacccggcaa agatacgacg gcagcaccac    420 

tgccatggaa attctccagg gccgggattt cactggcaaa gtggttgtgg tcactggagc    480 

taattcagga atagggttcg aaaccgccaa gtcttttgcc ctccatggtg cacatgtgat    540 

cttggcctgc aggaacatgg caagggcgag tgaagcagtg tcacgcattt tagaagaatg    600 

gcataaagcc aaggtagaaa caatgaccct ggacctcgct ctcctccgta gcgtgcagca    660 

ttttgctgaa gcattcaagg ccaagaatgt gcctcttcat gtgcttgtgt gcaacgcagc    720 

aacttttgct ctaccctgga gtctcaccaa agatggcctg gagaccacct ttcaagtgaa    780 

tcatctgggg cacttctacc ttgtccagct cctccaggat gttttgtgcc gctcagctcc    840 

tgcccgtgtc attgtggtct cctcagagtc ccatcgattt acagatatta acgactcctt    900 

gggaaaactg gacttcagtc gcctctctcc aacaaaaaac gactattggg cgatgctggc    960 

ttataacagg tccaagctct gcaacatcct cttctccaac gagctgcacc gtcgcctctc   1020 

cccacgcggg gtcacgtcga acgcagtgca tcctggaaat atgatgtact ccaacattca   1080 

tcgcagctgg tgggtgtaca cactgctgtt taccttggcg aggcctttca ccaagtccat   1140 

ggtttcagac tgcctggtag aaggaggtca cttctgattg tcagtgactt tgagctgagt   1200 

gctgaaataa aatgataaac aagtc                                         1225 

 
           
             29  
             2219  
             DNA  
             Homo sapien  
             
               FOR II mRNA  
             
           
            29 

tcgggccccg acgcgcgcgg gtctcgtttg gagcgggagt gagttcctga gcgagtggac     60 

yccggcagcgg gcgatagggg ggccaggtgc ctccacagty agccatggca gcgctgcgct   120 

acgcggggct ggacgacacg gacagtgagg acgagctgcc tccgggctgg gaggagagaa    180 

ccaccaagga cggctgggtt tactacgcca atcacaccga ggagaagact cagtgggaac    240 

atccaaaaac tggaaaaaga aaacgagtgg caggagattt gccatacgga tgggaacaag    300 

aaactgatga gaacggacaa gtgttttttg ttgaccatat aaataaaaga accacctact    360 

tggacccaag actggcgttt actgtggatg ataatccgac caagccaacc acccggcaaa    420 

gatacgacgg cagcaccact gccatggaaa ttctccaggg ccgggatttc actggcaaag    480 

tggttgtggt cactggagct aattcaggaa tagggttcga aaccgccaag tcttttgccc    540 

tccatggtgc acatgtgatc ttggcctgca ggaacatggc aagggcgagt gaagcagtgt    600 

cacgcatttt agaagaatgg cataaagcca aggtagaaac aatgaccctg gacctcgctc    660 

tgctccgtag cgtgcagcat tttgctgaag cattcaaggc caagaatgtg cctcttcatg    720 

tgcttgtgtg caacgcagca acttttgctc taccctggag tctcaccaaa gatggcctgg    780 

agaccacctt tcaagtgaat catctggggc acttctacct tgtccagctc ctccaggatg    840 

ttttgtgccg ctcagctcct gcccgtgtca ttgtggtctc ctcagagtcc catcgattta    900 

cagatattaa cgactccttg ggaaaactgg acttcagtcg cctctctgca acaaaaaacg    960 

actattgggc gatgctggct tataacaggt ccaagctctg caacatcctc ttctccaacg   1020 

agctgcaccg tcgcctctcc ccacgcgggg tcacgtcgaa cgcagtgcat cctggaaata   1080 

tgatgtactc caacattcat cgcagctggt gggtgtacac actgctgttt accttggcga   1140 

ggcctttcac caagtccatg caacagggag ctgccaccac cgtgtactgt gctgctgtcc   1200 

cagaactgga gggtctggga gggatgtact tcaacaactg ctgccgctgc atgccctcac   1260 

cagaagctca gagcgaagag acggcccgga ccctgtgggc gctcagcgag aggctgatcc   1320 

aagaacggct tggcagccag tccggctaag tggagctcag agcggatggg cacacacacc   1380 

cgccctgtgt gtgtcccctc acgcaagtgc cagggctggg ccccttccaa atgtccctcc   1440 

aacacagatc cgcaagagta aaggaaataa gagcagtcac aacagagtga aaaatcttaa   1500 

gtaccaatgg gaagcaggga attcctgggg taaagtatca cttttctggg gctgggctag   1560 

gcataggtct ctttgctttc tggtggtggc ctgtttgaaa gtaaaaacct gcttggtgtg   1620 

taggttccgt atctccctgg agaagcacca gcaattctct ttcttttact gttatagaat   1680 

agcctgaggt cccctcgtcc catccagcta ccaccacggc caccactgca gccgggggct   1740 

ggccttctcc tacttaggga agaaaaagca agtgttcact gctccttgct gcattgatcc   1800 

aggagataat tgtttcattc atcctgacca agactgagcc agcttagcaa ctgctgggga   1860 

gacaaatctc agaaccttgt cccagccagt gaggatgaca gtgacaccca gagggagtag   1920 

aatacgcaga actaccaggt ggcaaagtac ttgtcataga ctcctttgct aatgctatgc   1980 

aaaaaattct ttagagatta taacaaattt ttcaaatcat tccttagata ccttgaaagg   2040 

caggaaggga agcgtatata cttaagaata cacaggatat tttggggggc agagaataaa   2100 

acgttagtta atccctttgt ctgtcaatca cagtctcagt tctcttgctt tcacattgta   2160 

cttaaacctc ctgctgtgcc tcgcatccta cgcttaataa aagaacatgc ttgaatatc    2219 

 
           
             30  
             711  
             DNA  
             Homo sapien  
             
               FOR III mRNA  
             
           
            30 

tgccccgacg cgcgcgggtc tcgtttggag cgggagtgag ttcctgagcg agtggacccg     60 

gcagcgggcg ataggggggc caggtgcctc cacagtyagc catggcagcg ctgcgctacg    120 

cggggctgga cgacacggac agtgaggacg agctgcctcc gggctgggag gagagaacca    180 

ccaaggacgg ctgggtttac tacgccaatc acaccgagga gaagactcag tgggaacatc    240 

caaaaactgg aaaaagaaaa cgagtggcag gagatttgcc atacggatgg gaacaagaaa    300 

ctgatgagaa cggacaagtg ttttttgttg accatataaa taaaagaacc acctacttgg    360 

acccaagact ggcgtttact gtggatgata atccgaccaa gccaaccacc cggcaaagat    420 

acgacggcag caccactgcc atggaaattc tccagggccg ggatttcact ggcaaagtgg    480 

ttgtggtcac tggagctaat tcaggaatag ggttcgaaac cgccaagtct tttgccctcc    540 

atggtgcaca tgtgatcttg gcctgcagga acatggcaag ggcgagtgaa gcagtgtcac    600 

gcattttaga agaatggaaa acaaaatacc accctccgcc agaaaagtgc agaataaaaa    660 

ttttccacta gcaaaagaag gaaaaaataa aagatcttga atagtctcat c             711 

 
           
             31  
             524  
             DNA  
             Homo sapien  
             
               FOR IV mRNA  
             
           
            31 

tcgggccccg acgcgcgcgg gtctcgtttg gagcgggagt gagttcctga gcgagtggac     60 

ccggcagcgg gcgatagggg ggccaggtgc ctccacagtc agccatggca gcgctgcgct    120 

acgcggggct ggacgacacg gacagtgagg acgagctgcc tccgggctgg gaggagagaa    180 

ccaccaagga cggctgggtt tactacgcca agtaaggggg ccgcagtggg gccgcggacg    240 

cacctgggac cctgcacagc ccacggacgc cacctgcgcg gggaggacgc gcactccagc    300 

gcagcgcgtg cggtgcaaag tgaaagtaac tgttaaggag cttcagggaa aagggtccag    360 

ggttcccagt aggggccggc ccccttggtg ggcctcgggt ccagcggggg tcacctggtg    420 

gcttcccggc gcgccctctg ctgttcagga tgcagcactg cgcggcgcgg cgagggcaaa    480 

gcggcctcat ccccgccaaa aaataaagat gttttaaaaa gcgc                     524 

 
           
             32  
             363  
             PRT  
             Homo sapien  
             
               FOR I mRNA open reading frame  
             
           
            32 

Met Ala Ala Leu Arg Tyr Ala Gly Leu Asp Asp Thr Asp Ser Glu 
  1               5                  10                  15 

Asp Glu Leu Pro Pro Gly Trp Glu Glu Arg Thr Thr Lys Asp Gly 
                 20                  25                  30 

Trp Val Tyr Tyr Ala Asn His Thr Glu Glu Lys Thr Gln Trp Glu 
                 35                  40                  45 

His Pro Lys Thr Gly Lys Arg Lys Arg Val Ala Gly Asp Leu Pro 
                 50                  55                  60 

Tyr Gly Trp Glu Gln Glu Thr Asp Glu Asn Gly Gln Val Phe Phe 
                 65                  70                  75 

Val Asp His Ile Asn Lys Arg Thr Thr Tyr Leu Asp Pro Arg Leu 
                 80                  85                  90 

Ala Phe Thr Val Asp Asp Asn Pro Thr Lys Pro Thr Thr Arg Gln 
                 95                 100                 105 

Arg Tyr Asp Gly Ser Thr Thr Ala Met Glu Ile Leu Gln Gly Arg 
                110                 115                 120 

Asp Phe Thr Gly Lys Val Val Val Val Thr Gly Ala Asn Ser Gly 
                125                 130                 135 

Ile Gly Phe Glu Thr Ala Lys Ser Phe Ala Leu His Gly Ala His 
                140                 145                 150 

Val Ile Leu Ala Cys Arg Asn Met Ala Arg Ala Ser Glu Ala Val 
                155                 160                 165 

Ser Arg Ile Leu Glu Glu Trp His Lys Ala Lys Val Glu Thr Met 
                170                 175                 180 

Thr Leu Asp Leu Ala Leu Leu Arg Ser Val Gln His Phe Ala Glu 
                185                 190                 195 

Ala Phe Lys Ala Lys Asn Val Pro Leu His Val Leu Val Cys Asn 
                200                 205                 210 

Ala Ala Thr Phe Ala Leu Pro Trp Ser Leu Thr Lys Asp Gly Leu 
                215                 220                 225 

Glu Thr Thr Phe Gln Val Asn His Leu Gly His Phe Tyr Leu Val 
                230                 235                 240 

Gln Leu Leu Gln Asp Val Leu Cys Arg Ser Ala Pro Ala Arg Val 
                245                 250                 255 

Ile Val Val Ser Ser Glu Ser His Arg Phe Thr Asp Ile Asn Asp 
                260                 265                 270 

Ser Leu Gly Lys Leu Asp Phe Ser Arg Leu Ser Pro Thr Lys Asn 
                275                 280                 285 

Asp Tyr Trp Ala Met Leu Ala Tyr Asn Arg Ser Lys Leu Cys Asn 
                290                 295                 300 

Ile Leu Phe Ser Asn Glu Leu His Arg Arg Leu Ser Pro Arg Gly 
                305                 310                 315 

Val Thr Ser Asn Ala Val His Pro Gly Asn Met Met Tyr Ser Asn 
                320                 325                 330 

Ile His Arg Ser Trp Trp Val Tyr Thr Leu Leu Phe Thr Leu Ala 
                335                 340                 345 

Arg Pro Phe Thr Lys Ser Met Val Ser Asp Cys Leu Val Glu Gly 
                350                 355                 360 

Gly His Phe 

 
           
             33  
             414  
             PRT  
             Homo sapien  
             
               FOR II mRNA open reading frame  
             
           
            33 

Met Ala Ala Leu Arg Tyr Ala Gly Leu Asp Asp Thr Asp Ser Glu 
  1               5                  10                  15 

Asp Glu Leu Pro Pro Gly Trp Glu Glu Arg Thr Thr Lys Asp Gly 
                 20                  25                  30 

Trp Val Tyr Tyr Ala Asn His Thr Glu Glu Lys Thr Gln Trp Glu 
                 35                  40                  45 

His Pro Lys Thr Gly Lys Arg Lys Arg Val Ala Gly Asp Leu Pro 
                 50                  55                  60 

Tyr Gly Trp Glu Gln Glu Thr Asp Glu Asn Gly Gln Val Phe Phe 
                 65                  70                  75 

Val Asp His Ile Asn Lys Arg Thr Thr Tyr Leu Asp Pro Arg Leu 
                 80                  85                  90 

Ala Phe Thr Val Asp Asp Asn Pro Thr Lys Pro Thr Thr Arg Gln 
                 95                 100                 105 

Arg Tyr Asp Gly Ser Thr Thr Ala Met Glu Ile Leu Gln Gly Arg 
                110                 115                 120 

Asp Phe Thr Gly Lys Val Val Val Val Thr Gly Ala Asn Ser Gly 
                125                 130                 135 

Ile Gly Phe Glu Thr Ala Lys Ser Phe Ala Leu His Gly Ala His 
                140                 145                 150 

Val Ile Leu Ala Cys Arg Asn Met Ala Arg Ala Ser Glu Ala Val 
                155                 160                 165 

Ser Arg Ile Leu Glu Glu Trp His Lys Ala Lys Val Glu Thr Met 
                170                 175                 180 

Thr Leu Asp Leu Ala Leu Leu Arg Ser Val Gln His Phe Ala Glu 
                185                 190                 195 

Ala Phe Lys Ala Lys Asn Val Pro Leu His Val Leu Val Cys Asn 
                200                 205                 210 

Ala Ala Thr Phe Ala Leu Pro Trp Ser Leu Thr Lys Asp Gly Leu 
                215                 220                 225 

Glu Thr Thr Phe Gln Val Asn His Leu Gly His Phe Tyr Leu Val 
                230                 235                 240 

Gln Leu Leu Gln Asp Val Leu Cys Arg Ser Ala Pro Ala Arg Val 
                245                 250                 255 

Ile Val Val Ser Ser Glu Ser His Arg Phe Thr Asp Ile Asn Asp 
                260                 265                 270 

Ser Leu Gly Lys Leu Asp Phe Ser Arg Leu Ser Ala Thr Lys Asn 
                275                 280                 285 

Asp Tyr Trp Ala Met Leu Ala Tyr Asn Arg Ser Lys Leu Cys Asn 
                290                 295                 300 

Ile Leu Phe Ser Asn Glu Leu His Arg Arg Leu Ser Pro Arg Gly 
                305                 310                 315 

Val Thr Ser Asn Ala Val His Pro Gly Asn Met Met Tyr Ser Asn 
                320                 325                 330 

Ile His Arg Ser Trp Trp Val Tyr Thr Leu Leu Phe Thr Leu Ala 
                335                 340                 345 

Arg Pro Phe Thr Lys Ser Met Gln Gln Gly Ala Ala Thr Thr Val 
                350                 355                 360 

Tyr Cys Ala Ala Val Pro Glu Leu Glu Gly Leu Gly Gly Met Tyr 
                365                 370                 375 

Phe Asn Asn Cys Cys Arg Cys Met Pro Ser Pro Glu Ala Gln Ser 
                380                 385                 390 

Glu Glu Thr Ala Arg Thr Leu Trp Ala Leu Ser Glu Arg Leu Ile 
                395                 400                 405 

Gln Glu Arg Leu Gly Ser Gln Ser Gly 
                410 

 
           
             34  
             189  
             PRT  
             Homo sapien  
             
               FOR III mRNA open reading frame  
             
           
            34 

Met Ala Ala Leu Arg Tyr Ala Gly Leu Asp Asp Thr Asp Ser Glu 
  1               5                  10                  15 

Asp Glu Leu Pro Pro Gly Trp Glu Glu Arg Thr Thr Lys Asp Gly 
                 20                  25                  30 

Trp Val Tyr Tyr Ala Asn His Thr Glu Glu Lys Thr Gln Trp Glu 
                 35                  40                  45 

His Pro Lys Thr Gly Lys Arg Lys Arg Val Ala Gly Asp Leu Pro 
                 50                  55                  60 

Tyr Gly Trp Glu Gln Glu Thr Asp Glu Asn Gly Gln Val Phe Phe 
                 65                  70                  75 

Val Asp His Ile Asn Lys Arg Thr Thr Tyr Leu Asp Pro Arg Leu 
                 80                  85                  90 

Ala Phe Thr Val Asp Asp Asn Pro Thr Lys Pro Thr Thr Arg Gln 
                 95                 100                 105 

Arg Tyr Asp Gly Ser Thr Thr Ala Met Glu Ile Leu Gln Gly Arg 
                110                 115                 120 

Asp Phe Thr Gly Lys Val Val Val Val Thr Gly Ala Asn Ser Gly 
                125                 130                 135 

Ile Gly Phe Glu Thr Ala Lys Ser Phe Ala Leu His Gly Ala His 
                140                 145                 150 

Val Ile Leu Ala Cys Arg Asn Met Ala Arg Ala Ser Glu Ala Val 
                155                 160                 165 

Ser Arg Ile Leu Glu Glu Trp Lys Thr Lys Tyr His Pro Pro Pro 
                170                 175                 180 

Glu Lys Cys Arg Ile Lys Ile Phe His 
                185 

 
           
             35  
             36  
             PRT  
             Homo sapien  
             
               FOR IV mRNA open reading frame  
             
           
            35 

Met Ala Ala Leu Arg Tyr Ala Gly Leu Asp Asp Thr Asp Ser Glu 
  1               5                  10                  15 

Asp Glu Leu Pro Pro Gly Trp Glu Glu Arg Thr Thr Lys Asp Gly 
                 20                  25                  30 

Trp Val Tyr Tyr Ala Lys 
                 35 

 
           
             36  
             999  
             DNA  
             Homo sapien  
             
               FOR Exon 1 and flanking introns  
             
           
            36 

ccaggccctg cccctttgac gccggccgtc gcgatattgc ggagactgga tttcagcttc     60 

gtggtcggcg gagcggcccc tggagggcgc agtgcgcagg cgtgagcggt cgggccccga    120 

cgcgcgcggg tctcgtttgg agcgggagtg agttcctgag cgagtggacc cggcagcggg    180 

cgataggggg gccaggtgcc tccacagtca gccatggcag cgctgcgcta cgcggggctg    240 

gacgacacgg acagtgagga cgagctgccc cgggctggga ggagagaacc accaaggacg    300 

gctgggttta ctacgccaag taagggggcc gcagtggggc cgcggacgca cctgggaccc    360 

tgcacagccc acggacgcca cctgcgcggg gaggacgcgc actccagcgc agcgcgtgcg    420 

gtgcaaagtg aaagtaactg ttaaggagct tcagggaaaa gggtccaggg ttcccagtag    480 

gggccggccc ccttggtggg cctcgggtcc agcgggggtc acctggtggc ttcccggcgc    540 

gccctctgct gttcaggatg cagcactgcg cggcgcggcg agggcaaagc ggcctcatcc    600 

ccgccaaaaa ataaagatgt tttaaaaagc gcacatgctc agctccctcc tgcaggctct    660 

gggttgcagg gataggagtt ttgttgtgtt ttgttttgtt ttgtccagac cggtattgct    720 

cagtcatcca ggctggagtg caggggtgcc atcatagccc actgtagcct ctacctactg    780 

ggttcagaca atcctctcat ctcagcctct tgagtggctg ggactacaag cgtgcactac    840 

tatgcccgac taatttttta agtatttgta gagactaggg tcgcaccatg ttgcccaggc    900 

tggtctcgaa ctactgggct caagcaggcc acctgcctca gcctccagaa ttgagattac    960 

aggcgtgagc cactgcgcct agccaggagt attttttag                           999 

 
           
             37  
             1000  
             DNA  
             Homo sapien  
             
               FOR Exon 1A  
             
           
            37 

ccaggccctg cccctttgac gccggccgtc gcgatattgc ggagactgga tttcagcttc     60 

gtggtcggcg gagcggcccc tggagggcgc agtgcgcagg cgtgagcggt cgggccccga    120 

cgcgcgcggg tctcgtttgg agcgggagtg agttcctgag cgagtggacc cggcagcggg    180 

cgataggggg gccaggtgcc tccacagtca gccatggcag cgctgcgcta cgcggggctg    240 

gacgacacgg acagtgagga cgagctgcct ccgggctggg aggagagaac caccaaggac    300 

ggctgggttt actacgccaa gtaagggggc cgcagtgggg ccgcggacgc acctgggacc    360 

ctgcacagcc cacggacgcc acctgcgcgg ggaggacgcg cactccagcg cagcgcgtgc    420 

ggtgcaaagt gaaagtaact gttaaggagc ttcagggaaa agggtccagg gttcccagta    480 

ggggccggcc cccttggtgg gcctcgggtc cagcgggggt cacctggtgg cttcccggcg    540 

cgccctctgc tgttcaggat gcagcactgc gcggcgcggc gagggcaaag cggcctcatc    600 

cccgccaaaa aataaagatg ttttaaaaag cgcacatgct cagctccctc ctgcaggctc    660 

tgggttgcag ggataggagt tttgttgtgt tttgttttgt tttgtccaga ccggtattgc    720 

tcagtcatcc aggctggagt gcaggggtgc catcatagcc cactgtagcc tctacctact    780 

gggttcagac aatcctctca tctcagcctc ttgagtggct gggactacaa gcgtgcacta    840 

ctatgcccga ctaatttttt aagtatttgt agagactagg gtcgcaccat gttgcccagg    900 

ctggtctcga actactgggc tcaagcaggc cacctgcctc agcctccaga attgagatta    960 

caggcgtgag ccactgcgcc tagccaggag tattttttag                         1000 

 
           
             38  
             1000  
             DNA  
             Homo sapien  
             
               FOR Exon 2  
             
           
            38 

ctacaggcac gtgccactgt ccccagctaa ttttgtattt ttggtagaga cagggtttca     60 

ccatgttggc caggatggtc tccatctcct gaccttgtga tctgctcccc tagggctccc    120 

aaagtgctgg gattacaggt gtgagccacc gctcccgacg gcctctagat attttgagtg    180 

attcagcaaa cctcctaaag ttgaccccgt agctggggtc acagtcctct ttctccttct    240 

tccccctact tccttcttat atctggctat ctgggagaga aaaaatttaa tacaattgat    300 

tactttttag aagagttaat ttttacttat tactgtggat tttttgtttt ttaacagtca    360 

caccgaggag aagactcagt gggaacatcc aaaaactgga aaaagaaaac gagtggcagg    420 

aggtttgtat gttgttgtct aaggatcttg gatggaagca ttaagtagat gaggaaatgt    480 

cactggcaga gaggtgacag gttattgtgt gtttagggag ggctctctgg tagagaacca    540 

gactcccact ctgaggagct tatgggattt ggcagaagaa gatgatgaga tcatagggtg    600 

gagtgaggat gatttcttac tggtcttaaa gtaccaaaat ggccagatgt ggtggctcat    660 

gcctgtaatc ccagcacttc gggaggccga ggcggatgga tcacctgagg tcaggagttt    720 

gagactagcc tggccaacat gttgaaagcg catctcttct aaaaatacaa aaattagctg    780 

ggcatggttg catgcgcctg taattccagc tactttggag gctgaggcac gagaagtgct    840 

caaacccagg agctggaggt tgcagtgagc caagatcaca ccactgcgct ccagcctcgg    900 

tgacagagtg agatgctgtc tgaaaaatag cgacaacaac aacaacaaaa accagaaaat    960 

aattgaggcc tttgattcac agtttatgat gttttcctat                         1000 

 
           
             39  
             1000  
             DNA  
             Homo sapien  
             
               FOR Exon 3  
             
           
            39 

acaagaggca aaaatgtgga gcccagggtg ggatcagggg ccttctgcag ctgtcgcagt     60 

tggaacatgt gacgaaagcc agttgatgtg acaactgctg ggtgggaggg acaggcttgg    120 

gggcggggct gggagggctc cttcccttcc tgacccaggg atggtcttta cttctccctg    180 

gcacctgtag acctgtcttt cttgtgtttc agatttgcca tacggatggg aacaagaaac    240 

tgatgagaac ggacaagtgt tttttgttga gtaagtgtct gcaaagaaac cactctcagc    300 

tgttttgctt tttaatagga atttttaatt ataaaagtaa tacatagtaa ctgtagaaaa    360 

atacaaagta taacagtgtg tatctgtaat cttagcagaa gtgactactt ttaacatcgc    420 

tggaactttt aacatcgcta gatttattct tctattttcc ccgcacacgc atccttaaaa    480 

aaaaaaagta gggaggccag gcatggtggc tcatgcctgt aatcccagca ctttgggagg    540 

tcgaagtggg tggatcctga ggtcaagaga tcgagaccat tctggccaac atggtgaaac    600 

tccgtctcta ctaaaaatat aaaaatgagc tgggtgtggt ggcacgtgcc tgtggtccct    660 

gctactcggg agggtaaggc gggagaatca cttgaaccca ggaggcggaa gttgcagtga    720 

gctgagatcg cgccactgca ctccagcctg ggtgacagaa tgagactcct tctcaaaaaa    780 

aaaaaaaaaa aaaaaagagg gaatgcactg tgtggactgt ttagtaacct gctttttcca    840 

tgtaatatat tatgagcatt ttcttgtatc catatctatt tttcaaaaag atgctattta    900 

gcagctctag agttattatt ttgtacagat atactataat ttatcaaatt gcctattgga    960 

cattaatgct aaattcctgt tatagagaac ctataattat                         1000 

 
           
             40  
             1000  
             DNA  
             Homo sapien  
             
               FOR Exon 4  
             
           
            40 

cttctctacg tctttaggcc cttgcacagg gctaagagat ttaattgaaa tctactgaaa     60 

agagtggctg acagatctta tagctacatt tacatgaatt acataaaagc ccaaaacctt    120 

ctcaagaagc cttttttgag atctaaggat acatggcaat agttattgta tgttacagcg    180 

tatgttatag gcagttctga aggaaaggat tcgatgacta tctttttttg gcacaaatgt    240 

gatccttctg gaggccagaa gatagattca gtgggcccca gttctttcag gtttaaggaa    300 

taagcatttt ggtctatgaa aaatggggtt ttcctaaagt ataagattgt cttatattta    360 

taaatgcctg tgttcattgc tgtgggttca ctgctttctc ttttgggcag ccatataaat    420 

aaaagaacca cctacttgga cccaagactg gcgtttactg tggatgataa tccgaccaag    480 

ccaaccaccc ggcaaagata cgacggcagc accactgcca tggaaattct ccagggccgg    540 

gatttcactg gcaaagtggt tgtggtcact ggagctaatt caggaatagg taggctcttc    600 

acttagttat ttatctttgg gactgctata atgagatcca cttagatcta gctataatgg    660 

aattttgttt agtggttctc tgatttaaac atgactttta tccttttcag atatcgtttc    720 

attaacatca ctacctcttt ttaaatccta atgttgtcat ggaagcctgt gtaggggctg    780 

accttgaagt ctctgaaagc tgaacactca gcaaaagact gtggctattt tggtattcag    840 

ggatgagaga caacaggctc cgtctaagag tttttgacct ggtctgcatg gtgtatgggc    900 

atattccaat tacctgggtt attcaaacca aaattgattg gtaatagaat acggggaaca    960 

acaaggtatt atgtctttgg aacaaaggat actacaggtt                         1000 

 
           
             41  
             1000  
             DNA  
             Homo sapien  
             
               FOR Exon 5  
             
           
            41 

gtttttgcag atcttggccc ccaaaattct tagtcatagt gcccttctga tgccttcaaa     60 

cagatatggg tgtgtatgca cgtgtgttat tttgtccatc ttttctaatt gttctcagta    120 

ggaaatgggg ctaagaaacc taggtaacac tggtagaagc agaggtgctg gcgacactta    180 

ccatagagta gagcataaag tccttatcgt gggcacagga tctggtcatt attgatggtt    240 

aaatggctgg acatgccgtg aactcttcta tgtcccagtc ttcatctttg aaatcactgc    300 

atccctagga caagttttct caccatcaca gtgttgacag tccgggccag atggttctat    360 

gttgtcaggg gctgccctgt tcatggtaag atgttttgta atgcctggtg tttacccact    420 

agatgccagt aggactctac cccacaactg tggccactga aaatgtctcc agacatttgc    480 

ttctgtcccc tggggatcaa aatcacccct ggttgagaac ttggggtaat ttaagtggtg    540 

ctccggtaaa ggccattcaa catgactcac tgtgttgatg ttatgttttc taacattgac    600 

tttcctttaa accatagggt tcgaaaccgc caagtctttt gccctccatg gtgcacatgt    660 

gatcttggcc tgcaggaaca tggcaagggc gagtgaagca gtgtcacgca ttttagaaga    720 

atgggtaagt gcttgactgt tgttgttttt tttaattgtc aaatacacat gccgggctaa    780 

ccatatggag atttcagtgg agtgtgtaaa gtttattgct cttggaatca tgtctttatt    840 

tttaaagtca tcttcacttt gtttgtctta tgaatgaaag catgagcaag tccattatta    900 

ttcacgcatt tgtttgtagt tcattgtcag tgtcatctgc tttattagtc aggggtttga    960 

gaatgtttct tcctttaact catttattaa gaaaaataaa                         1000 

 
           
             42  
             1000  
             DNA  
             Homo sapien  
             
               FOR Exon 6  
             
           
            42 

gcttttgaaa ttaagatatt gtccattgca tctttccatg atcgaggcgt tgtcgtgcat     60 

tcccctcctt acagcgtttt aggatgcaga ttgagaggag ataacgttag cccgaagcgc    120 

ctttgcagcc tcttttgcgc ggctgaaaca cagcacaacc cacccccccg ccccaccccc    180 

cagcctgtag gtttagcaga atcccagcct cacatcctcc ccgaacttgg cagtaaaagc    240 

cctgttcttc cattcattcc gatgatttat attctctctg ggcgtcttat attaaacagg    300 

ggaattccga catgttccat aacacattta ctgtaacttg ataccatgaa ctacacttgc    360 

tgttatttat catttctttt tattttctct cattgcagca taaagccaag gtagaaacaa    420 

tgaccctgga cctcgctctg ctccgtagcg tgcagcattt tgctgaagca ttcaaggcca    480 

agaatgtgtg agtgttccag tggagggtta tagatcataa tttcttgcta ttgtaatatc    540 

tttatcagat gaacacaatt gggagaatgc aaggctgttg tgttgtcttg gcgtccaaac    600 

aggaggctca tttatattgg ccctgttaag gtgaaccgta ttttcttgac tcacagtcac    660 

cttcattatg agatgtgtca tcaatctaat aacagcttcc cacataccaa aagagaagac    720 

actattaaag cactagtaaa agtggctaat aaaagcttgg caatagtaag atgcatcctg    780 

attataagat tttttgtagt gcatttcaga atggagtaag agtatattta aattgcattc    840 

aggaacaagt aaactcaatt atccaatatg gcagggaggt tgacaatcca agcacccaaa    900 

aaacctctag tttctaaagc cttcgatgat ttgatgtggt acatggatgt ggttccaaaa    960 

aacatggact cacattcctt ttatttattt tttttcatcc                         1000 

 
           
             43  
             1000  
             DNA  
             Homo sapien  
             
               FOR Exon 6A  
             
           
            43 

acatttgttt ctctccagga tctcattggc ggtttgctgg tattatattg ccatttatat     60 

tggaaaggca ggcagtgcga aaactgtagt ctattaagag tttgtgagtg tgtgtatgtg    120 

tatacagtga aataatatct agtgtaatgt gaagtgacag tcaaaattac agcttttctc    180 

ttgccgacaa gggtacttct ctgccaagta tagtaatatt ttggtttttt tgttcaaggt    240 

tgccagcaat tctggcttac tctcttgaga gccatattag tgtgtggaag gaaggagctt    300 

cctgtcaggg gaaatactat tattttgata tatatttgtt ggtgccaaac tgcaattaaa    360 

taggaggtgt tggaagaagg ttttatttaa nccagcttga atttgttaat taattagcta    420 

aaacttattt ttggtttgct tcaagaaaca gttctatgtt gttttcataa tttaatttta    480 

cacaactgtc ttttgtttgt atcttacaga aaacaaaata ccaccctccg ccagaaaagt    540 

gcagaataaa aattttccac tagcaaaaga aggaaaaaat aaaagatctt gaatagtctc    600 

atcaattaca tcttcttttg tgggtatttc ctggtctttt catgttttga cttctatctc    660 

ggtgatctga cagacatgta cgatttgcaa caacatctat tatatgattt aatttatacc    720 

tttatcagtt tgcagttatg ctttacgact cttcaggtga ctaaaganaa aagagggtgt    780 

tttaaagtgt gtgtgtttgt gtgtgcgtgt acgtgtatac atgtgttttt ctgtgtacat    840 

aagtgtgtgt gttttaaatt gtttgaaaaa cactagntcc atctctattg tattatctga    900 

gggatgctag ctcgttactt gggatagtaa caagtttctt ggtgacaact cctncctccc    960 

attgtcaagg aagactgagg atgtcaccag agttcgctgt                         1000 

 
           
             44  
             1000  
             DNA  
             Homo sapien  
             
               FOR Exon 7  
             
           
            44 

gtggttgaac ctgagttcca actagggtga caactcttcg cagattgcct gccgctgtcc     60 

tggttttcac actgaaagtt ccgtatccta ataagcctct catcctcagc aacggaggac    120 

agttgcccac tcaaagcctt gtgacattct agggtatcct atttctacat ggtgggaatc    180 

tagaagacgg agaaagaatt tctcattccc gaaggagcat ggattatcct tggttgtagt    240 

gtttatgttc cacatcacgt ggattcccga aggagcatgg attatccttg gttgtagtgg    300 

ttatgtccac atcacatggg atattttatt tttcaggcct cttcatgtgc ttgtgtgcaa    360 

cgcagcaact tttgctctac cctggagtct caccaaagat ggcctggaga ccacctttca    420 

agtgaatcat ctggggcact tctaccttgt ccagctcctc caggatgttt tgtgccgctc    480 

agctcctgcc cgtgtcattg tggtctcctc agagtcccat cggtgggttt gaattgcata    540 

tttgttcact tatccccttt ctcataccag ctaatattcc cccaaggctc tcattctgaa    600 

aataattttc attagtcctg cttgagacat gtgggtggac tcagcttggc tcacttaatt    660 

tttccaggtc ttttttgttc gcctgcgatt gtgggggact gtttagaagg actttctaga    720 

gcaaggaaga ttgcctttac gactatactt caagctcctc attgattttc gcttacagat    780 

ggaataataa cttcatgaaa aactcaatgg catgaaccta ttattggatt tgtaattcaa    840 

caacttcaac atcttaccaa gaagaatgtg cagttattct agcaggagaa acaatgcaat    900 

tagagcctgc gagatgaaat caaattgttt tataatgaga aattagggaa ttcgaggcag    960 

acattagctg tgtaattgtg gaaagggaag aactgtagtt                         1000 

 
           
             45  
             1000  
             DNA  
             Homo sapien  
             
               FOR Exon 8  
             
           
            45 

tttttgtatt tttagtagag atggcatttt gccatgttgg ccaggctggt cttgaactcc     60 

cgacctcagg tgatccactc gtctaagact cccaaagtgc tcggattaca gatgtgagcc    120 

actgcaccca gcattcctta gatttccaat aaaaataaaa agctgtgtgg gaagtcagaa    180 

cttggttgct tcatgtcata tttcctattt ttaagattta cagatattaa cgactccttg    240 

ggaaaactgg acttcagtcg cctctctcca acaaaaaacg actattgggc gatgctggct    300 

tataacaggt ccaagctctg caacatcctc ttctccaacg agctgcaccg tcgcctctcc    360 

ccacgcgggg tcacgtcgaa cgcagtgcat cctggaaata tgatgtactc caacattcat    420 

cgcagctggt gggtgtacac actgctgttt accttggcga ggcctttcac caagtccatg    480 

gtaagagaac agcttctggc gccgcaaaca ccttgggtcc tagagaaacc tgcacacttg    540 

tgtctccacc tttttacctc ttgcgggcat gagtctggtc tcagtaataa cattgtccag    600 

cccatcataa agggctcttg aacacatttt catcaacttt aggttaagtc tgtttgggta    660 

aatgcgtctt ggagggctgg gtagaagatg tgggtttcag tatcatgtta agtatggctg    720 

aaagtcctta tggaaatggt gatttttttg tttgtttggt tttgtttttt ttggggtttt    780 

ttattcagaa actttgaaaa tctattttgt tgaatggagc acttgaaaac tgctgttttg    840 

tgtcagtagg taaacaacaa acattggtga ctactgaatt ttcagcagat gtgattcctt    900 

tgtttcacag aaaaactgga tcttttgttc taaatttttt tcttctaatg ggtataatcc    960 

tctgttggag agtcctttga tagctaggag tgtgttttct                         1000 

 
           
             46  
             1000  
             DNA  
             Homo sapien  
             
               FOR Exon 9  
             
           
            46 

tagaccccct ttgataatct ccactaagca gacatactcg atacatcttc actaatgagt     60 

tctgacttca taaaaagtat taatgacttc tttttgaaag taagagtgct ttgaatacca    120 

gtcgttattg ctttagaagt tcataaaagc aaaagcacag tatttccccc agtgtttgtg    180 

cgataagaga atagaatgta ggtcccagcg ccttagaatt ttaagctatg ccttctcttg    240 

gtttgtgaat ttccaggttt cagactgcct ggtagaagga ggtcacttct gattgtcagt    300 

gactttggtg agttcttacc ttgtaaaaga tttacaatta tttcattttc aacatagctt    360 

tatcttatga caaaggtgac agaaaggaaa tctcctaagt tggcctacag ggtgctttag    420 

aaaacatctg gctgggcatg gtggttcaca cctgtaatct ccacactttg ggaggctgaa    480 

gtaggctgaa gtgggaggat ggttagagcc taggagttcg agaccagtct gggcaacaac    540 

gtgagatcct gtctctacaa aaaataaaaa aaattatctg ggtatagtgg tgtgcacctg    600 

aagtcccagc taactgggag tctgaggcaa ggaaattgtt tgagcctagg aggttgagag    660 

tgcagtgagc cgtgttgctg ccactgtact ccagcctggg caacaggaca agaccgtgtc    720 

tccaaaagga aaaaaataat aaagcactgt ctctctctac ccttgcagta tccctgtagg    780 

agagagttac tattagctcc cagtttatag gtgagtgata tggtttggat gtgtccccac    840 

ccaaatctca acttgaattg tatctgccag aattcccaca tgttgtggga gggacccagg    900 

gggaggtaat tgaatcatgg ggcccagcct ttcccatgct attctcataa tagtgaataa    960 

gtctcatgag atctgatggg tgtatcagga gtttccgctt                         1000 

 
           
             47  
             1500  
             DNA  
             Homo sapien  
             
               FOR Exon 9A  
             
           
            47 

aagtcgaggg gttggatgaa tttgttcttg cacgttcaga aggataccat ctttttctct     60 

gtgtggaaga ggcgcctgcc acgacgtatt gatatgtgta tttattttcc aagcctgtcc    120 

ctgtatgagg tgtcaaaagt tacaccagct ttacaagcgg agtttatgaa ctcgtttttc    180 

caggatagtc acattatact ttttacagtc atgtgctttc agcccagtac cctttgctat    240 

gccaagatcc agctgaaact gaaccaggtg ggggaggcct gctaatgccc aggcagtcga    300 

aatgacgcca tctcatcact ccttctctta aaattttttt ttgtctttct tcttggattt    360 

ccagcaacag ggagctgcca ccaccgtgta ctgtgctgct gtcccagaac tggagggtct    420 

gggagggatg tacttcaaca actgctgccg ctgcatgccc tcaccagaag ctcagagcga    480 

agagacggcc cggaccctgt gggcgctcag cgagaggctg atccaagaac ggcttggcag    540 

ccagtccggc taagtggagc tcagagcgga tgggcacaca cacccgccct gtgtgtgtcc    600 

cctcacgcaa gtgccagggc tgggcccctt ccaaatgtcc ctccaacaca gatccgcaag    660 

agtaaaggaa ataagagcat tcacaacaga gtgaaaaatc ttaagtacca atgggaagca    720 

gggaattcct ggggtaaagt atcacttttc tggggctggg ctaggcatag gtctctttgc    780 

tttctggtgg tggcctgttt gaaagtaaaa acctggttgg cgtgtaggtt ccgtatctcc    840 

ctggagaagc accagcaatt ctctttcttt tactgttata gaatagcctg aggtcccctc    900 

gtcccatcca gctaccacca cggccaccac tgcagccagg ggctggcctt ctcctactta    960 

gggaagaaaa agcaagtgtt cactgctcct tgctgcattg atccaggaga taattgtttc   1020 

yattcatcctg accaagactg agccagctta gcaactgctg gggagacaaa tctcagaacc  1080 

ttgtcccagc cagtgaggat gacagtgaca cccagaggga gtagaatacg cagaactacc   1140 

aggtggcaaa gtacttgtca tagactcctt tgctaatgct atacaaaaaa ttctttagag   1200 

attataacaa atttttcaaa tcattcctta gataccttga aaggcaggaa gggaagcgta   1260 

tatacttaag aatacacagg atattttggg gggcagagaa taaaacgtta gttaatccct   1320 

ttgtctgtca atcacagtct cagttctctt gctttcacat tgtacttaaa cctcctgctg   1380 

tgcctcgcat cctacgctta ataaaagaac atgcttgaat atcatcacct gaagtttgta   1440 

ttgtttcttt aaatgtttgt ttcagtttgt ttttgttttt cattttttag aaaagaaatc   1500 

 
           
             48  
             595  
             DNA  
             Homo sapien  
             
               FOR Exon 10  
             
           
            48 

ctttttctca agtgttgaaa taaattaatg gtgtggccat ggttctcgta cttcaggtcc     60 

ctgtgagccc ctggggtcct acacaacttg gggtaatgct atggtcacct gcatcccctc    120 

ctctccagcc ctcacctggt tttctcttct ccttccaaga aaaaacaccc ataccattct    180 

taacatctct ggaagctggg ggtcaggaga gggagatcca taaagttctt gtcttccccg    240 

actcaagaag cttaagggta cattcactca ctcatgaatt gactggttta ttcattcact    300 

ggttgattaa ttcattcatt cactggttaa ttaattaatt catgtcatcc tttggatgtc    360 

ttgcagagct gagtgctgaa ataaaatgat aaacaagtca aaaacaaaaa ggcctctgac    420 

ttaacaggac ttccgtgatg cgggggaaga agacaatagg caagtaagca agtaaatata    480 

caagataaat agaaactgtg atgagggagt taaagaaatt aaactggtcc ttgtggaagc    540 

aacttcgggg gaggaggaaa gacagggaag accattttaa ggaggtgatg tttga         595 

 
           
             49  
             959  
             DNA  
             Homo sapien  
             
               FOR Exon 10A  
             
           
            49 

aacagaaaaa catgccatca tctttaattt cctgcatcag ctaagcattt atttcatgta     60 

ugtcctcacac tagattgagt ggttccatat taatatccta cgtcaaagat gagtaaattg   120 

uagatgcgtag gttctaattt gcccagtgtc gtggtgtgtg atcttgacca ctagcctaaa   180 

uctgctatttt atgtccaccc atcaacctct acggctgccc ctcatttgaa cacacaaaat   240 

uatagttgtgc ggcttggtgg agccatctaa attccgttag gccatttgcc aatgctgcta   300 

uttaggggcga aatgtcatgc gcttgatcta aatgtactta ggagaattct caggacctga   360 

utgaattatta ttcggaattt tatggcctca caggttgcag gcttcatacc aactgcagct   420 

uaatgagctat gggccccgag aaacactgag gacacacggc gttctgcaca cagagtgggc   480 

utgtttctgtc tgttctcccc ctgcaccctt ctcagatgca atctcaagtc ataggagaac   540 

uttgtgcaaat gtttctcctg gatggtttcc tttagagcat gtgtcctata acttgaaatg   600 

ugttgtctgag cagaatgttt ttagaagtta gattttttta ggggggaaac aggaaccaaa   660 

ugcaaagccaa tagagatctt gaaaaaaaaa aaaaaaagaa aaaaccaccg tggtattcta   720 

uggaagaaaaa agcatttttc gaatgaaaac tttttattat atttgatata ttctgcttcc   780 

ucttccctagt atgtattaat gagatgaaat cacttcttaa ttttcaggtt aatattaaag   840 

uttgaagccca tccctctacc ctgaggactc tgccagcctc tggcagtatt cctttccaac   900 

uttccacttgc cccaaatagg tagaagttag cctttatttt tggtgtcatg tcttctttc    959 

 
           
             50  
             921  
             DNA  
             Homo sapien  
             
               FOR Exon 10B  
             
           
            50 

tttctccccg acatgcccct gccaacctcc ccccatcatt ccagctggga gaggccatca     60 

aatgtagctt ggagagctat agaaaccttg agctctgagc ccaaggagca agttggagtc    120 

ctgcctccag ctgtgtagct cagataacag ctggagcaga gaacatgctg ttctctctgc    180 

tagaatatct gacccaattc tgactagtaa agagagttaa tagtttgtac ctcaagtcat    240 

tcttgtccct gtgtgaagga agagaagcag tcatttccct ccacctccaa cacacactcc    300 

gtccccactc tgtctctctt ggctgttttt ctcttcattg ttgttaaact cacgttcatt    360 

tctcttgtag tcagggaaat gaagagccac tttcaacaat tctgaagaag aaatatggga    420 

tgttttgctt cgggatggag gctggagcag gagtttctgg agtctgccag tacccagttt    480 

gaatcccagc tctgcctttt atcagctggg tgttgggcaa gttagctgac ttttgtgagt    540 

tttctcatca ttaaaatgag aacactgtta ttggtctttc gggattgttt tgagaaatga    600 

gatatcgaga catgcctggc acaaggcctt aattcttctt catggtcaag aaatggcaga    660 

ttttccccct tccattccca cccttgcaca tagtaggttc tcagcaagta tttgtagatg    720 

taatcgacca gcagagatca tttgtaccct taacacccac agagagtcac agatgctttc    780 

actgaaggag ggtgtcccaa gactcaatgg cagggaataa aaatgccaag tcatgtaagt    840 

attccacaaa gttagagggg aggagtaagt atctcttatt cgtgcatctt tatggtatga    900 

ccaagggctc atgatttgta a                                              921 

 
           
             51  
             15004  
             DNA  
             Homo sapien  
             
               DNA region encompassing exon 6  
             
           
            51 

gatgggcgtt tattatgaga tacacgaaga cagagaccag aggttcccca agtagcccct     60 

ggacctgcag tgtcagcatc acctggtagc ttgttacaca taaaaattct tgggctttat    120 

tccacacata ataaatcaaa agcttgtggg gtggggcctg cagtctgagg tttatttttt    180 

cgaaaactgt aagttccagg gtacatgtga aaaatgtgca agtttgaaaa ataggtaatc    240 

atctgccacg gtggtcatgc tgcaaagatc aactcatcac ctggatatga agcccagcgt    300 

ccagtacctg ttgttacaga tgctctaccc accacatgca agccccagtg tgtgttatcc    360 

gcctccaccg tgtgtcaatg tgttctcatc atgcagctcc catttataaa cgagagcata    420 

cagtgtttgg ttttcagttt ctgcataagt tcgcttagga taacggcttc cagctccatc    480 

catgtccctg cagaggacat gatctctttc ctttttatga ttatataata ttccatggta    540 

tatatgtacc atattttctt catcctatca tttatgagca tttggagtga ttccgtgtcc    600 

ttgctattgt gaaataccat ttgacccagc aatcccatta ctgggtatgt aaccaaagga    660 

atagaaatga ttgtattata aaggtacatg cacatgtatg ttgattgcag cagttttcac    720 

agtagaaaag acatggaatt cacccaatcc gaattttaac aagtcttcca tgtgattctg    780 

agacagactc atgttcgtgt ggttctggct ctgcagaact gaggtgggcc ccaaaacttg    840 

cgtttctaaa aggtactagc tgagcatgat gactcacatt tgtaatccca ccactttgtg    900 

aggccgaggc gggtggatcg cttgaggcca ggagtttgag accagccttg ccaacatggt    960 

gaaactccat ctctactaaa aatataaaaa tttgccgggt gtagtggtgc acacctgtag   1020 

tcccagctat ttggaaggct ggggcaggag aatcgcttga acctgggagg cagatgttgc   1080 

agtgagctga gatcaggcca ctgcactcca acctgggtga cagagccaga gtctgtctca   1140 

aaaaacgaaa aacaaaaaaa accaaaagac taaatgtata aaaggtaccc aggtgatgtt   1200 

gatgctactt gtcttggcca atgaaatgaa aagcctacag gccaggcatg gtggctcatg   1260 

cctgtgatcc acaactttgg gaggctgagg caggcagatc acctgaagtc aagaattcaa   1320 

aaccagcctg accaacatgg tgaaacccca tccctactaa aaatacaaaa ataacaccaa   1380 

aaaaaaaaaa aaaccacaac aaattagcca ggtgtggtga tgtggcctat agtcccagct   1440 

atttgggagg ctgaggcagg agaatctctt gaatccagga ggtgaacgtt gcagtgagcc   1500 

aagatctcac cactgcactc tagcctgaac ttcagagaga gactatgtct caaaaaaaaa   1560 

aaaaaaaaaa agcctacagt tgacaggcag ataacagagg gagcaaagga gtcttgtgga   1620 

agacaatagg gagtgttgag gacctgtcaa acaacacagt ccccatctgt actttgggag   1680 

ggaaggactt cttaattcca gctgcctgtt gccatggggg aacatagacc cagaactgcc   1740 

atcctttcta atttttttaa aaattaaaag gctaaaagtt ttggcttata aaaatggtat   1800 

ttcctgagtt tttacaatta tagtattaag atattcctag gtttttgttt gttttcgaga   1860 

cgaagtttcg ctctcatttc ccaggatgga gtgcagtggt gcaatctcct ccgcctccca   1920 

gtttaagcca ttctcctgcc tcagcctcct gagtagctgg gattacaggc gcccgccacc   1980 

atgcccggct aattttttgt attttttagt acagacgggg tttcagcatg ttggccaggc   2040 

tggtctcaaa ctcctgacct cacgtgatcc acccgccttg gcctcctaaa gggttgggat   2100 

tacaggtgtc agctagtgca cctgacccta ggataatctt aattttaagt aactggtaac   2160 

tcataaattt ttaaacactc tgcagttcaa agaaaacatg tttgtggtag gtagccagtt   2220 

tataacctgt gtaagactgt gttattagca aatccctcaa tgtgttgacc aagagtgggt   2280 

aggaatcctg cagacagaat cttttgaagg tggtttgttt ttttattcct ttataagtac   2340 

agttgtagat ttacaaagta attgagtata tagtacataa ggtcttgtgt acccaccgat   2400 

cgcaattgag catatagtac atgctgtcct gtgtacccac tgatccctgg tcctgcccct   2460 

ggtatgaggg tattaacatc ttgcattagt gtggcacatt tgctacaatt aataaaccaa   2520 

tatcgataca ttattaacta gtctatagtt tacatgaggg gctgctgctt gtgttgtaca   2580 

ttctataggt ttgttcaatg cataatgaca ggtgtctgtc atttcattat ggagtgaaac   2640 

cattccaatt tcatacagcg tagtttcacg cctctaaaag tcccctgaaa gtgttattaa   2700 

aagtactatc tggccatgtc ctcatttccc cagcaaagtc cttctgagaa tttaggcgct   2760 

ttaggctaga ggatctcagt gctcagaaaa gcggtattgc attgtgattt tgatttggat   2820 

tttcttgatg gcttaggata ttgaacatct cttcatatga ttattggatg gatgattttt   2880 

ttcagcggtt atcaggtcac acctgtaatc atgcccacct cattttctta cagtgcagtg   2940 

tgtgctttta ggcacatacc aagagcttac agagatcctt ctggttcaga ggaaagattc   3000 

acatccccaa caaacactgc tccccgatta caatatagag aggctaaatt atagtccata   3060 

tcacttttcc tttaaatccc tttacttttt tgtgctacaa gagtttcaga ataataattc   3120 

tgcagaaaaa agtactgatt gtccagcagt tgtgtcaggt agaaaactgg gatgcccgac   3180 

tgcattggtt tagccagaac tgtggccatg aaaccttgcc tggcccccag ggatggggtg   3240 

aggggggata tcatatacgc tttaggtggg gcagaggggt ggtctgcccc tgtgcaggca   3300 

ggaatgttca ggaagcaggc tgcctctgtt tctactctaa atatttaaca ttcacggatg   3360 

gcatggatcc tgactaagcg gagcagatgc tggctctgta ttgcacatag ctcctgaata   3420 

tcagccctgc ttggctatgc atgtgttttt tgttagtttt tgagacagag tctcggtctc   3480 

ttgcccaggc tggaatgcag tggcacgatg ttggctcact gcaaaccctg cctcccaggt   3540 

tcaagcgatt ctcctctccc agcctcccga gtctctggga ttacagacgt gcatcaccag   3600 

gcccagctaa tttttatatt tttactcgag acgtagtttc gccatgttgg ccaggctggt   3660 

ctcaaactcg tgaccttagg tgatatgcgc ccctcagctt cctaaagtgc tgggattaga   3720 

ggcatgagcc accagacatg acctgtgttt atattgttaa atcaccattg tcaatcttga   3780 

aaggagactt tgttttgatg tatgtaagat tgagttttta ctcatctcta catagtttcc   3840 

tcagattgct tttttttttt tttgagaccg agtctagctc tgtcacccag gctggagtgc   3900 

agtggcacaa tctttgctca ctgcaacctc tgcctcccat gtttcagtga ttctcttgct   3960 

tcagattccc gtgtacaggc atgccaccac acctggctaa tttttgtatt tttagtagag   4020 

atggcatttt gccatgttgg ccaggctggt cttgaactcc cgacctcagg tgatccactc   4080 

gtctaagact cccaaagtgc tcggattaca gatgtgagcc actgcaccca gcattcctta   4140 

gatttccaat aaaaataaaa agctgtgtgg gaagtcagaa cttggttgct tcatgtcata   4200 

tttcctattt ttaagattta cagatattaa cgactccttg ggaaaactgg acttcagtcg   4260 

cctctctcca acaaaaaacg actattgggc gatgctggct tataacaggt ccaagctctg   4320 

caacatcctc ttctccaacg agctgcaccg tcgcctctcc ccacgcgggg tcacgtcgaa   4380 

cgcagtgcat cctggaaata tgatgtactc caacattcat cgcagctggt gggtgtacac   4440 

actgctgttt accttggcga ggcctttcac caagtccatg gtaagagaac agcttctggc   4500 

gccgcaaaca ccttgggtcc tagagaaacc tgcacacttg tgtctccacc tttttacctc   4560 

ttgcgggcat gagtctggtc tcagtaataa cattgtccag cccatcataa agggctcttg   4620 

aacacatttt catcaacttt aggttaagtc tgtttgggta aatgcgtctt ggagggctgg   4680 

gtagaagatg tgggtttcag tatcatgtta agtatggctg aaagtcctta tggaaatggt   4740 

gatttttttg tttgtttggt tttgtttttt ttggggtttt ttattcagaa actttgaaaa   4800 

tctattttgt tgaatggagc acttgaaaac tgctgttttg tgtcagtagg taaacaacaa   4860 

acattggtga ctactgaatt ttcagcagat gtgattcctt tgtttcacag aaaaactgga   4920 

tcttttgttc taaatttttt tcttctaatg ggtataatcc tctgttggag agtcctttga   4980 

tagctaggag tgtgttttct tctttacttt cccaaagaca attttggatg aatcatggta   5040 

ctgtggttac atttggaagt gtttacaaag gtgataagat gtttttatag tttggtgttc   5100 

atttatcgac catattaaga accttcttct atgaaatggt tttagtagga agtattttga   5160 

atgaagaaag cgtattttcc acaatatatt tggtagatta tttttcaaaa ccaaaggact   5220 

tcaaaaatgt cttcctatta gtggacacca gtattccagt tacccaaact taaaatctac   5280 

ttgacagaaa tcaccctttt cccaaacaca tttgcctttt agcgatatca caggccttca   5340 

cagttggacc taggatatta ataaaacaaa gcaaaacaac aacaaaagaa gaactattgc   5400 

aggcctatta tcctccagct gatctcacga ccttggaaac tgtctgctgc cttctcttac   5460 

actcattccc acctgccacc cttaaccttc gtatttgggg atgacttttt tttttttttt   5520 

tttttttttt tttttgagac ggagtctcgc tctgtcgccc aggctggagt gcagtggcgg   5580 

gatctcggct cactgcaagc tccgcctccc gggttcacgc cattctcctg cctcagcctc   5640 

ccaagtagct gggactacag gcgcccacca ctatgcccgg ctaatttttt gtatttttag   5700 

tagagacggg gtttcaccgt tttagccggg atggtctcaa tctcctgacc tcgtgatccg   5760 

cccgcctcgg cctcccaaag tgctgggatt acaggcgtga gccaccgcgc ccggcctggg   5820 

gatgactttt aaaaccacat caaaagtctt ttgaagtgct taggaggagg taagtaagta   5880 

tatcttttat tctaatcacc caagtggttt ttaggcagag taaacagaca tattccaaga   5940 

gtcgaatact taggatcagt ggcagtgaag tgaaaggacc tacggagaat ggtgttccat   6000 

gccgcagggt gggattgatc tctaggaagt aaatgaatga aacttacctg taggtggatc   6060 

acagggtttt gaaatctccc catcactttg ctcccgatga ctgcataagc gcagcctctt   6120 

catgaaagtt tctttgacca tggttatgcc tcatactttg ttgtgtcctt cagtttacat   6180 

gaaaagaagg aatatggatc tgaactgggt gccagtgcca tttaaatgag gcatttagga   6240 

atggggagag gagagaggtg gattcttcct tattttctgc tggcccagtg ccttccgctt   6300 

taacaagccc agtcaaccat catttgtaat ctttgggtct tgagtcatct cacttgtatt   6360 

tgatcattga tgtgttttaa ggagctcggt tcagtggcat cagagagttc gttttctcct   6420 

tcttccataa ggtccctaat cagaggaacg ggatgggaaa gctttagaga tgagtctgtg   6480 

actatggaat ttggtgaggg gtgatataac catcctgcca tttgttgtag actcgaatgg   6540 

atgaggagtc attgatagaa aatagtggaa actgttattc tgtggcctga tcccattgct   6600 

tagggactct gtgccagggc atgtgggaaa agaaggaagg caagttctgc taaaatataa   6660 

agtcagggta aacattttta tttattggag aagagttgat tattatctaa tatttaatat   6720 

aatatgctta ataaagtcag acttgaaagt tggctggttg gacctaacgg gagtatctct   6780 

aaaaaatacc agcaaattaa gttggatcgg tgactgtatc atgatatcgg agtgttagct   6840 

gttttgggag tacgtgtgct caggacttgg gctgagtgga aaaccagagt atggggttag   6900 

aaatggaggc agagggacta ccatgtcggg gaggatgcac aatgaattgg acgaaggttg   6960 

caactcagtg agcaccgtac ctgtagtgtg tcttagtaat agcagatgcc caacctccat   7020 

gcctaactcc tttgcctacc ccaccagtgt gcagcagcct ctgccgcacc ctactgcagt   7080 

ctctcctatt tccctggcag gagagctcag agactacttg tgaacaggcc tcaatgccta   7140 

tagcaaggca aggcttggga gaaacaaatg tcctctggga gcagccttgg gtaaaagatt   7200 

aactgcgcac tgagctttag atccctgagt gagaaagctg tgggaaaatg agtgtgtagc   7260 

tcatgagagg gtttggagaa aagtggtgac atcgcaaaaa tcccaggttt tgttgaagtt   7320 

gtctgtggag gggcactcat agaaagggca ctggacgtga gcatttccct tgtagttgag   7380 

atgggatttg acatgacttc ctggtggtgg caagacaaga aagtggcaac ctgggacagt   7440 

ggacctcaca gctctcctgc taatggtgct gatggctaag ctcagctctt acccttgacc   7500 

actggaacaa cttagacatg tcaccagctt tctgagcctg tctcattacg atgaaaattg   7560 

tgatacaggt tagtgtaagg accgacaata atgcatataa catcaataat gatggatata   7620 

gaatttctgc agttattata atctggctct ccagtatggt agtcgtgggc cacatgtggc   7680 

tattgagttc taaaaatgtg gctaatagaa ctgaggaagt gaattttcaa tttgattcca   7740 

tgtttgttaa ttcgaactga aaaacaccac gtgtgccaag tggctgccat actggagagc   7800 

gtggtcaaat gacatgccta gctcagtgcc tttcgcatgg gaggtatgca gtacttcctg   7860 

ccacgatggc tgttactctt gtcgttgtga gcagtgcctt ggtcccgatg atgattctcc   7920 

aaaatgtaat ctctgctgac agaacaagag tctgcaaatt gggagtctgg aaagggaaga   7980 

gaagggcttt gtccctggcc ccaaagactc agggagaggt ttactggcca ggtagaaagg   8040 

gcgtccaggt gaagggaccc tggttcagtg atctccaacc tgcacgtctc atttgtgagg   8100 

aagccgtagg gtgtggactt ttcttgtttc tgaccttgag tgattagaaa atagcagctt   8160 

ttatatgtca caaatggact ttagataagc atgaagatga cgaagactgt ggaatatgta   8220 

gctaagagtc ttactaaaat cctctctagt gtatttatat atttaagcgt ttggtagtac   8280 

ttttttagcc atcagcgttc tctatattag gttgatacag aagttattga ggtttttgcc   8340 

attgaaagta atacgtcact tcatgcagag gtggaattgg cgctctgagc ttgcttgtca   8400 

aattcacagg cattcacttt tctttcataa acatttagga ctctgtattt aggagaggaa   8460 

tcactgtgaa tgtagacctc gggttggtca ttttcctagt aagatagacc agactgactg   8520 

gaaaagtcac cctcttgggc tatcctgcgt atgtggctcg gcattgcttc cgtcaggata   8580 

atgcatcagt ttcttggaca aagggtgcat ttctgctatt tgaatgcaga catatttttg   8640 

gatataagcc acgcagttgc atcccatttc ttccgtaggt aaaggctggg tttctaattg   8700 

tgattgagaa gcttagcata gatgcaatgt ccctatcacc agatggctag tgtgctctgc   8760 

cttgtctgcc ttttacctta gagagggtgc ttccttctga cacgggtatt gctggagtac   8820 

acattctgtt gcatgaggtc aggggagcaa gaaatacaac ccagacttgc gctcggaagc   8880 

cctgccttca tttcttctct gtagctggct cccttaagta ttaaacagca gtattctaat   8940 

atcaacgtcc cttttttatt tgctgtgact tgctgtgact acggtgattt tagttacagt   9000 

aattaggtca ctcttcatgg agtcgggcta ttttaagtgc ctctttaagg aatattgttg   9060 

cagatcagta attttacaat tggatgttct tgtgcaactt tattaaatgt gatctgtctt   9120 

ttaaatgttg cccttttaca gttacacatt tctaattgtt gctgtaaatg cattatcact   9180 

ttgaaattaa ttaatttttc cattttggct gcctgcaata taataatcac taatgagaaa   9240 

ctcacaggaa tcaattctca gcacagcggc ccctactggt aaagccatgt ctctgcctcc   9300 

ctcttcttct tcagatccag agaactggac aagtgtattg gggcgtcctg ggagggaagt   9360 

gtttgtttct tcctcccttc aatttatggt gaaataaaaa tcacagataa acttggaaag   9420 

tgagttattt ttaagtgtcg ttctaaaaaa gatgcaatat acttcaaata gctgcttcta   9480 

aaatatgtta aaggaaacta acatttgctg agtactttct aataatgaag tcacatcata   9540 

cgtgcattac atgtttattt tcttgactct agagcattct tttatgcagt taaattactc   9600 

taaaatgttt tgcacctgca cgcattaatg tgtattgttt ggtaagaact ttcgagttgg   9660 

tgaaaaaatg tattgtattt tagtatctat atttatatat acatattagt ctatgggttt   9720 

taattcacaa aaccaggtgt tgtatctggt gcacataagg gattttcaag gctaatcttg   9780 

ggtttttgca gttgtaacca tgtgtgatga ctttagaatc ctaccacact tccaaataga   9840 

gatgcagatt gaccattttc ccagatacca cgatgcttta tgtatcagca tatattttca   9900 

ttaatttctc ataatttctg ttagaattaa taattccaag ttctacaatc ttagaattat   9960 

tatctttatt ttgtaattga aaagcacaga aacctcatgc agcgtgttca agaaaatgat  10020 

ataccaggct tgtaaacttt cacttgctaa ttgcagctgc atttgctttg tccaatgcac  10080 

ggtaatgccc ctggcatcca tatctagtta tgcagttatg cttccttttt aaaatgtttg  10140 

acagcacctt cactgaaatg aatgctgcca ccaccgccaa ccccaccttt tttttttcca  10200 

cataagcttt catttctggt aaatacatcc ttgccagagt tcccaggtcc aagccggtcc  10260 

tttgtggacc agtatgcaga tttgaagttg agcactgtca cttgcctctt aggagtgttt  10320 

tgtgcaacca catatttgtg ttggcagata acagtagaaa gaatatcatg tgttagggta  10380 

gcaagagctt agcagatgtt ttagggaact gcattttcaa atcattttac attaaaaaat  10440 

ggcttacgtt tatggaactt ctgcgacgat cgggttacgt ttcctgtgcc cgtgtaaagc  10500 

cagtcatcgt gctgagtttt aacggatgtg cagctaccct gctaatggat gggccggcaa  10560 

tgctggtcct gttgcaaagt tataaattat aactagaaaa agcacaagat gtcattcttc  10620 

taaccttcgt gaaagccaaa cggggtaatt ctgcccttga ataacccgct gcagggtgat  10680 

ttatagttgt acaatttaaa acgtaatgaa ggagataata gatgttgatt tgttcctgaa  10740 

attagtattt tagaaaaata cttctccccc tttccatctt taaatacctg cagctgacta  10800 

cactatagat actatcaagg gcaaagcccc ctgtttgaat gtgtattggt acattggtgt  10860 

atcacataca aggcagaggg tgtataaaac aggctttctg ttgcgagggg gaaatatggt  10920 

tttcaaagtg gaaatttcat tgtattcttc atgaatctaa acattttgag actcctgagt  10980 

gaggtttgaa ttttctggtt gcctttctct tgccagtcca gatatgttag aaaactggac  11040 

tctcaggacc cctctgtggt tggaccacac tggcgctttc ctgcagttgt ctgtcaacag  11100 

aaggacctgg tagctggctt gtcctcttac accactgagg ccaccttctt gcaggaagaa  11160 

acatacttct tcctgccctg gattaacatt accattggtt ttagtagtct tacgtcatta  11220 

tgtccaaagc ttttaagttt acagtgcttg cttagtccag tgaccaatgg atgtaagtaa  11280 

gttggaaaag ggcaaaggat ttagaatcat gagggttgta ttcaaactgc agcctctact  11340 

cccttttagt tacctaattt tgaaatcatt tgacatttct gaggttaatt ttctacatca  11400 

gtttagctgc tttaggccgc aagttattaa cacccactta aaggtggctg aaataataga  11460 

ggttttgctt cccatgtatc cagagttgct gaggcagcaa gggttgtgtt tggctactat  11520 

atgagtagct cctttgtgat aatctttact tttccctcat gcttacaaga tggcttctgt  11580 

ggtgccagcc attgcatcct atttgataag gtctgaaggc acagatggga gggatgaggc  11640 

tcctcattgc atgccttttt cctttatcaa gagacagagc attctgcttc agaactgtcc  11700 

tcctgagttg acttcccgtc agatcccact ggttgggact gagtcatctg tttgtatcct  11760 

agttataaag gagactagaa aagtgcataa ctggcttttt gagcctctag agtggcaggc  11820 

gaactctgat tttgaggaaa aaggataggg taatggtgtt gacacaggca ctgttgaata  11880 

cgtatgtaca tattttaata gttttctcat taatcaaatg gaaatgctat cgattttata  11940 

ggatcgttgt gagaagaagt gagaattgaa atgaaaggca tctagtcttc cccggtcccc  12000 

atccacttga cccaccatac ctcaagccac ttctggctcc atgctctttc tttccttagg  12060 

gttttgaaac atgttcttca gcctagaata ctcctcctcc actcttaatt tctacccatc  12120 

ctgcaaaact cagtctgaac agcacacgtc aaggaagcag ttttggtctc tcagattata  12180 

tgaagctctc caatatgttc tttcagaaca gccagtgcct agaatactgc ctgactcgta  12240 

gtagcactcc ttatgtgttg aatgagtaaa tgaagagtcc ctggaatttc ttcttcataa  12300 

cattgaacac aactgcagtt tgaaaacata atttctgtag tttttttttt ttcttttttt  12360 

ttgagacgga gtgtctctcc gttgcccgga ttggagtgta gtggcacaat cccggctcac  12420 

tgcaacctcc gcgcctcccc agttcaagca attctcctgc ctcagcctcc caagtagctg  12480 

ggattacaag cactggccac caaacccagc taatttatat atttttagta gaagcggggt  12540 

ttcaacatgt tggccaggct ggtctcaaac tcctgacctc aggtgatcca cctgccttgg  12600 

ccttccaaag tgtggtggga ttccaggcat gagccactgt gcctggcctg tagtttcttt  12660 

ttcttttttc ttttcttttc ttttttttga gatgaagtct tgctctgtca cctaagctgg  12720 

aatgcagtgg tgcaatcttg gctactgcag cctccgcctc caagttcaag cgattctcct  12780 

gtgtcagcct cctgagtagc tgaaatacag gtgtgcacca ccacgcacgg ctgatgtttg  12840 

tatttttagt agagacggga tttcaacatg ttggccaagc tcgtctcaaa cctgcctcag  12900 

cctcccaaag tgctgggatt acagatgtga gccactgcac ccagcctgta gtttcttatt  12960 

taatgtctgt ccccccttct cttgacagtg gtgtttttgc cttcacacta ctgtatccct  13020 

ggtccatacc gtaaatgttc tgtaaatgtt tgtaaatgaa tacatgaaag atgaccaggt  13080 

gctgaatcag tgagagtcct taatgtactt cttatcattt atccctctga ggtaggcaag  13140 

ggattatagt ttagaatgtg taaatacgta aaagataagg caaggagacc tgctgagagt  13200 

cacacagcaa gcccccaagg tcccccaggc tcatggtcaa atgacatgta agggacgtat  13260 

ccagcggtgt gggtatgttt ggagggaatg aatgtaagga gagagaggtg aggcacagga  13320 

gagacaggat gagctttaga ggtagacagc tctcgggtcc agccctagct ctctcccatt  13380 

ctaactcttg ggcaaggttc aaaacctgat taagattcat gcataaaact gagatgttag  13440 

aactacctca tgggcttgtc ttgagaatta aagacgttat gaacttaagg cctctacagt  13500 

gtgctagatg cttaagaaat gttaactact attgttattg gcttgcagta gaaattaaag  13560 

attatgaatg ttcagcattc agaatatact agatactcaa taaccattat tgctgtcatt  13620 

attaacatta ttttccccag ctttactgag gtatgagtga caaaaatttc atatatgcgc  13680 

atgagtgtgt gtgtgtgtgt gtgtgtgtgt gtgtggctgg gcatggtggc tcttgcctat  13740 

aatcctaaca gttgaggagg ccaagctggg aggatgactt gagcctggga gttggaggct  13800 

gcagtgagct atgatggtgc ccctgcactc catcctgggt gacatagtga gaccctatct  13860 

ctaaaggaaa aaaaaaatta tatgtattcg tggtgtgaaa tgtgatgttt tgatattcat  13920 

atgcattgtg aaatgactac cacaatcaag ctaattaaca catcggtcaa cttacatagc  13980 

tatcattttt tgtgtgtggc aagaacattg aaagtctagt ctcagcaatt ttcaagtata  14040 

taatacgtta ttattattac tatcgttaac tgtattcacc atgctgtaca atagatctcc  14100 

agaatgtatt cattctgtct aactgaaact taggatcctt tgaccactat ctccccattt  14160 

atcaccccct cccacttccc cactaccagc ccctcctggg aactgccctc tactctctgc  14220 

ttctgtgagt ttgactgctt taaattccac acaaaaagtg agatcataga gtatttgtct  14280 

ttctgcgcct ggcttatttt acgataaatt gattgcttct cttcccattg tttatagata  14340 

cccattctcg taatgatggc actgtgtctg tcatagaagg cctaattctc cctgttaaca  14400 

ttgaacataa aaacctcata tgggccaggc acagtggctc atacctgaaa tcccagcact  14460 

ttgggaggct gaggcggatg aatcacctga ggttgggagt tcgagaccag cctggtcaac  14520 

attgcgaaat tctgtctcta ctaaaaagat acaaaaaaat tggctggctg gtcatggtgg  14580 

ctcatgcctg taatcccagc actttgggag gccgagccgg gcgaatcacg aggtcaggag  14640 

attgagacca tcctagctaa catggtgaaa ccctgtctct accaaaaata caaaaaaatt  14700 

agccaggcgt ggtggtgggg cgcggtcgtg ggtgcctgta gtcccagcta ctcagaggct  14760 

aaggcaggag aatggcgtga acccgggagg tggaggttgc agtgagccaa gatcgcacca  14820 

ctgcactcca gcctgcgtga cagagcgaga ctccatctca aaaaaaaaaa aaaaaaaaaa  14880 

aggctgtgtg tggtgactcg tgtctattgt cccagctact tgggaggctg agacatgaaa  14940 

atcacatgaa cctgggaggt ggaggttgca gtgagctgac atcgcatcac tgcactccag  15000 

cctg                                                               15004 

 
           
             52  
             4559  
             DNA  
             Homo sapien  
             
               DNA sequence encompassing exon 7  
             
           
            52 

ttttagttga catgtcatta tggattcttg ggcataaacg tttatatgaa ttttgaatat     60 

taggaaataa tcttggaagc tatattagtt ttctaaagct attataacaa attaccacag    120 

atggagtgcc tcaaaacaac agaattttac tctcttacag tttcagagac caaacatctg    180 

aaatcaagtg gtttgcagcg ttggctcctt ctggaggctg gaaggagcat ctgttccatg    240 

cctttctcct agcttcccat gactgccagc cacccttgga atttcttggc ttttatctta    300 

gtcagtctaa tctctgcctc tgtctttcca tctccttctc tgtgggtgtt ccttctcccc    360 

ttctgttttg taaggatagg cctgtcattg gatttaaggc ccaccttaat ccaagatgat    420 

ctcattttaa gatgtgggac ttaatcacat ttgcgaagac cctttttcca aataaggtcg    480 

cattcacaga tccctaccat tcaactcact atggaagcaa tgggatggga ggtaccattc    540 

aactcacaga ttcctaccat tcaacccact ataaagcagt ggcatatgaa aaggatggga    600 

ggttgctatg ggaatggtct gccccagatg caggtaaaat ggggaagcat tttatataga    660 

atggaaaaac aacagtaaag ctggctaaaa cttgatctgc tttttacagt catgtgctga    720 

caattctaaa cattatcaat gataaaatac ttctccctca tgatattaac tgtgcaattt    780 

ttgctgcccc tgctacatac atgctttatt gcttggaaac aggattactt ggtgaaataa    840 

tgtgaatctt tcaattctca tgatacatgt tttcaactta atatccaggg tgcaggaata    900 

tatattccca ccaactttat acaagataat acaaatataa tagtatgaat aaatatgcta    960 

atttgataga aaatatttta atatttacta ctagtgtagt tggacgtgtt aaatattgca   1020 

atggttattt gtaatctacc tttataaaat ttctaatgtc tgccactgtt tctgttagac   1080 

tcttaagaag atacgtgttt gtctcataga tttgtaagaa ctctttatat actaaaaata   1140 

tggggcttct atatgattgc atatattcgt cacttttgtt ccactgtttt agagagcaga   1200 

agattttaat ttctataaag atttttatgt ttccctttat tgcatgccaa tgtttgtttt   1260 

ttaaaaactt gtacgtctta ggctgcgatg tgaaaagtgt cttttttttg atttggaaac   1320 

agagtcttgc tctgatgcct gtagtggtat gatcatagct cattgcagcc cttgaactcc   1380 

tgggctcaag taatcctcct atctcagcct ccgaagtagc tgggactaca gccacacacc   1440 

accacacctg gctcactttc gggtggttgt tgttgagatg gggtcttgct gtgttgctca   1500 

ggctggtttt gaatactggg ctcaagtgat tatcctgcct tagcctccca aagtgctgga   1560 

atttcagagg tgagccactg tgcccagcct cctgtttttt ttacatgtat atatatatat   1620 

atatatatat atatatatat atatatatat atatacacac acacacacac atatatatat   1680 

acacacacac atatatatat acacacacat atatatacac atatatacac atatatatac   1740 

acacatatat atacacacac acatatatat acacacacat atatatacac acacatatat   1800 

atatatacat atacacacac acacacacac acacacacac acacacacac atatattttt   1860 

tttttctttt agatggagtg tctctctgtt gcccaggctg gggtgcagtg acatgatctc   1920 

tgctcactac aacctctgcc tcctgggttc aagtgattct cctgccttag cctcccaagt   1980 

agctgggatt acaggcgtgt gccaccacgc tgggctaact tttgtatttt tagtagagat   2040 

gggggtttca ccatgttggt caggctggtc ttgaacttct gacctcgtga tttgcctgcc   2100 

tcagcctccc aaagtgctga gattacaggc gtgagccgcc atgcctgccc ttatttttat   2160 

atatcttcca taacctttct atctctttct ttctctttct ctgtttctgt gtgtgtctgt   2220 

gtctgataat aaaatacatt aaattttatt ttataacata aatgttttat aaggtcggta   2280 

gatttgttta tgattttaat ggctgcatag tattctatca tatggatata ttctaaatgt   2340 

aataattcct caatggtata tttagtttac tttatatttg ctgctttagt ttgtcacaaa   2400 

cactgtggat agccttgtat atatttattt tgttgtatag ttcttattgg tacagtatga   2460 

tttttcacaa gtatacaatt actttctgag aatttagctt tttgattcac gtgaaagcct   2520 

tacttgtatt gttaacaatt ttgttacgtt tgtgaggcct aggcagaagg attgcttgag   2580 

accaggaact caagaccacc ttgaccacct tgggcaacat agggggagcc catctctaca   2640 

aaaataaaaa ataagaaata aaatagccat gcatggtggt gtgcccctgt agtcccagtt   2700 

actcgggagg ctaaggtgga aggatcgtct gagcctggga gtttgagact gcagtgagtt   2760 

atgaccacac cactgcactc cagcctggat gacagagaaa gatctcgtca aaaaaaaaaa   2820 

aaaaattaaa tatatacaaa ataagggaga atactatgac aggcttccac ttacctatct   2880 

ttaggcttct ttataaaatg ttcatttaat ctgatctcta taagaaattc tgtatcctta   2940 

aacaacatgt gcccttctct ttacacagag cgattacccc cttgctatgt tttattaaca   3000 

gctagcaagt gctgggtcct gtgtcagttg ttttgttttt tctttttctt tgtttttctt   3060 

ttcttttctt ttcttttttt tttttgtttt tttttttttt ggagatagag tcttgctctg   3120 

ttggtcaggc tggagtgcag tggcatgatc ttagctcact gcaacctctg cctcctgggt   3180 

tcaagtgatt ctccggcctc agcctcctga gtaactggga ttacagacat ataccaccac   3240 

acctggctaa ttttttttgt gttttgacag agatggggtt tcaccatgtt ggtcaggctg   3300 

gtctcaaact cctgacctaa aatggtccac ctgccttggc ctcccaaagt gctgggatta   3360 

caggcgtgag cccccgcgcc tggccctgtg tcagcctttt taacagcata atttttcctt   3420 

tattagtcat aactacttta agggctattt tgatttacag acaaagtgag acttggatga   3480 

attaagaaag aattaatcag gtggttgaac ctgagttcca actagggtga caactcttcg   3540 

cagattgcct gccgctgtcc tggttttcac actgaaagtt ccgtatccta ataagcctct   3600 

catcctcagc aacggaggac agttgcccac tcaaagcctt gtgacattct agggtatcct   3660 

atttctacat ggtgggaatc tagaagacgg agaaagaatt tctcattccc gaaggagcat   3720 

ggattatcct tggttgtagt gtttatgttc cacatcacgt ggattcccga aggagcatgg   3780 

attatccttg gttgtagtgg ttatgtccac atcacatggg atattttatt tttcaggcct   3840 

cttcatgtgc ttgtgtgcaa cgcagcaact tttgctctac cctggagtct caccaaagat   3900 

ggcctggaga ccacctttca agtgaatcat ctggggcact tctaccttgt ccagctcctc   3960 

caggatgttt tgtgccgctc agctcctgcc cgtgtcattg tggtctcctc agagtcccat   4020 

cggtgggttt gaattgcata tttgttcact tatccccttt ctcataccag ctaatattcc   4080 

cccaaggctc tcattctgaa aataattttc attagtcctg cttgagacat gtgggtggac   4140 

tcagcttggc tcacttaatt tttccaggtc ttttttgttc gcctgcgatt gtgggggact   4200 

gtttagaagg actttctaga gcaaggaaga ttgcctttac gactatactt caagctcctc   4260 

attgattttc gcttacagat ggaataataa cttcatgaaa aactcaatgg catgaaccta   4320 

ttattggatt tgtaattcaa caacttcaac atcttaccaa gaagaatgtg cagttattct   4380 

agcaggagaa acaatgcaat tagagcctgc gagatgaaat caaattgttt tataatgaga   4440 

aattagggaa ttcgaggcag acattagctg tgtaattgtg gaaagggaag aactgtagtt   4500 

agagcatatt agaaatctgg ccgtgcctct tttggttaaa atttcaatta aaacatcag    4559 

 
           
             53  
             26040  
             DNA  
             Homo sapien  
             
               DNA sequence containing the FRA16D fragile site  
             
           
            53 

gcttttgaaa ttaagatatt gtccattgca tctttccatg atcgaggcgt tgtcgtgcat     60 

tcccctcctt acagcgtttt aggatgcaga ttgagaggag ataacgttag cccgaagcgc    120 

ctttgcagcc tcttttgcgc ggctgaaaca cagcacaacc cacccccccg ccccaccccc    180 

cagcctgtag gtttagcaga atcccagcct cacatcctcc ccgaacttgg cagtaaaagc    240 

cctgttcttc cattcattcc gatgatttat attctctctg ggcgtcttat attaaacagg    300 

ggaattccga catgttccat aacacattta ctgtaacttg ataccatgaa ctacacttgc    360 

tgttatttat catttctttt tattttctct cattgcagca taaagccaag gtagaaacaa    420 

tgaccctgga cctcgctctg ctccgtagcg tgcagcattt tgctgaagca ttcaaggcca    480 

agaatgtgtg agtgttccag tggagggtta tagatcataa tttcttgcta ttgtaatatc    540 

tttatcagat gaacacaatt gggagaatgc aaggctgttg tgttgtcttg gcgtccaaac    600 

aggaggctca tttatattgg ccctgttaag gtgaaccgta ttttcttgac tcacagtcac    660 

cttcattatg agatgtgtca tcaatctaat aacagcttcc cacataccaa aagagaagac    720 

actattaaag cactagtaaa agtggctaat aaaagcttgg caatagtaag atgcatcctg    780 

attataagat tttttgtagt gcatttcaga atggagtaag agtatattta aattgcattc    840 

aggaacaagt aaactcaatt atccaatatg gcagggaggt tgacaatcca agcacccaaa    900 

aaacctctag tttctaaagc cttcgatgat ttgatgtggt acatggatgt ggttccaaaa    960 

aacatggact cacattcctt ttatttattt tttttcatcc ttttcagtct ttcaaaattc   1020 

cagttggaga aagccttagt tagggcctag catattttga tcctatcata tgctagcatc   1080 

cctttctaac agagaaggtt gtaggagaaa gggagagaag cggaaggggg tggggagaca   1140 

gagagacaga caggaggcct caaaccctga aacactgagc taaggaaagt gatcatggca   1200 

agctacacta attacaatac tttgtttcca agtgtttatt tttactcata tttagggcag   1260 

gcaatcctgg tttctcgttg aacatagagg tttgaatttc attaataaat aacttcattt   1320 

attttttttc agtgacttga ttcaaacatg aggattaagt taataatagc acaggttgtg   1380 

cgaaggataa gataattaca caagaggcac cagaaccact gaatgtggag agctctcata   1440 

aatgacaagc tgcctttggg ttaggctctg ttgggaacat tagttctgca gtgttgcaag   1500 

cagatgaagg atgtgaggga agggatctta aaccagatat tcaaatggcc ctgtggggag   1560 

ctgacaccac actgctgtct agtgtccaat tctccttgca tggctgtgtc acccaggttg   1620 

gaacgtagtg cacaatctcg ccttaatgca acgtcccact gcgggcccaa gtgattttcc   1680 

tgtcccagcc tcctgagtag ctgggactac agatgcctgc caccatggcc tgctaatttt   1740 

tgtattttta gtagacatag ggtttcacca tattggtcag actggtctca aactcctggc   1800 

ctcaggtgat ccacccgcct cagcctctga aaatgctggg attacacaca tgaaccactg   1860 

cgcccagccg ctctacttta ttagatttaa aaagtttgct ctcagctggg tgcagtggct   1920 

tatgcctgta atcccagcag tttgggaggc caaggcgggg agggtcatga ggtcaagaga   1980 

tcaagaccat cctggccaac attgtgaaac cccatctcta ctaaagatac aaaaaattag   2040 

ctgggtgtag tggtgcacac ctctagtcct agctacttgg gaggctgagg caggaaaatc   2100 

gcttgaacct gggaggcaga ggttgcagtg agccaagatg gcaccactgc actccagcct   2160 

ggcgacagat tgagactccg tctcaaaaaa aaaaaaaaaa aaaaaaaaag tttgtactca   2220 

gcctggcgtg gtggctcacg cctgtaatcc cagcactttg tgaggccgag gtgggcggat   2280 

cacctgaagt caggagttcg agaccagcct gggcaacaag gtaaaacccc gtctctacta   2340 

aaaatgcaaa attaactggg cggggaggca catgcctgaa atcccaacta cttgggaggc   2400 

tgaggcagga gaatggcttg aacccgggag gtggaggttg cagtgagctg agactgaacc   2460 

agtgcactcc agcccgggtg acaagagcaa aactctgtct caaaaaaaaa aaaaaaaaat   2520 

tgactcaggc tcttgctgga gtatgtcagt gtcccaagtc attgaggtct acattagagt   2580 

cagtcttagt gagagttgca acatcgccta agcgcagacc ccaagctggc ttgtaggaac   2640 

agtgagataa catcccccag ccagccagat ctgagagagc cctcctgtgt gtgtcctttg   2700 

cagcgcttgg ctgttagtag cttccaccct ttgccagaac tatcaagagg cacctcaaca   2760 

ggctgcaaca ctagcattca agacatttgt tcgctgattt gcttcgtcat tcatttatcc   2820 

accggttcct tcacttcaca cacatttcag gagcacctct aaagccccag acattcttct   2880 

gggctctgag aagagagtgg tggccctgga agccacagta accgcacaca agctgtcacc   2940 

tcagttgtca gctattatgg atggctgact cctcaaactc atctgtctgt ggttgtttgg   3000 

gagggaatga taatgacaag aacagcagcg gagtcagcag ctcatttact gagggcttat   3060 

gggccatacg tggaaaatat tttgcatgta cgttttgatt caattctcaa agcagtcctt   3120 

gggagtagat gctattaata tctctactat acagataagg aaagtgaggc tagagaggat   3180 

atataattta tccaaggcat gtggaatgtg ggtagagggg ccgatttagc ctcttcacgg   3240 

ctccaacttg accaccgtac tatactgtgt ttcattttag tttatttttt atttattttt   3300 

tgagacaggg tctcgctgtg ttgcccaggc tgaagtgcag tggtgcaatc tctgttcatt   3360 

gcaacctctg gccttgggtt caagcgattc tcatgcctca tcctccagag tagctgggat   3420 

tacaggtgca ggttaccaca cacagctaat ttttgtattt tctgtagaga tgaagtttca   3480 

ccatgttggc caggctagtc aactcctgac ctcaagtgat ctctccacct tggcctccca   3540 

aactgctgga attacaggcg tgagccaccc tgcccgaccc tttcatttta aatgtgatag   3600 

aaacagactc agagaatgtg agtgactttt cgaagggtca ctagcaagtt ggtgacgggc   3660 

atggggtttc actccagtct gcctcttgtt cttttcctca tagcatgtga gatctcacaa   3720 

ttcactgccg ctgaataact ttgtccagtt cctctgtcag aatgtgttgg aagtgagcag   3780 

ctttgctgaa gagagccaac tgttacataa tagggatcat cttccacgtc ttaactggac   3840 

cattagtttt taaccattaa gtagttggga aatcatcaag ttcaagtgct ttttgtaatc   3900 

accaagctca aaactttgga ggaaactctc acccattttg ccctaatccc agattttagc   3960 

acaggacttt ggaattttag aaagtacttc taggttgaga aacaagggtg attttccata   4020 

tagagacctc tgtagcacaa ccctctttgt atgtgtccca tgagagtaca ggattttaca   4080 

tggcttagag aaatactgtg gtggtgaaaa tatgcataaa aaagcgagag attttacttt   4140 

tgaaatgctg ccaaagtgaa atctcttttg gataggcaga tgacagttta gtacttgcag   4200 

gtctttcgat tctgacaact gtcagaagtg aatttaagtg ctggtgacta ggctgccaga   4260 

gaaacacatg taactctatt cttatcaccc cccatcttca gacattaaaa ttttcttgtg   4320 

tgtaaaacat aatgacaaat gaaagaaagg ctgccagttc agaaccaatc tcttttcacg   4380 

ctgttgcatt gaggagagga aaaaatccct taaatttgaa ttccatgtgg atgctcttat   4440 

ttaatatgcc gtttcaaagc ctgtatgcga tcaagccaga gtattatcag gctctgagtt   4500 

cctgtgtgta ttcccttggt ccatgctcca tctcaagtgt ttctccacac tgggataggc   4560 

agcatattgt gtagtcttag agcagaaaag gtagaaacag attcttagta caaacactgg   4620 

tgagaaaagc agcatattgt actgtggaac cacccgccgt ctgccttctg tgggagatgg   4680 

ggatcagcct cttcctggat ctcttctttt tattcgggtg ctgggaggtg aggctgcaaa   4740 

ggaggctgtt gtagagggag ccctcctaac catgattatg taaacaatag ctgctaggca   4800 

acttgttgtt tgttttatgc aaggcagtgt gctaagcact tacatacatg tattctgtct   4860 

gtagtttgtt ataagagtcc tgcacactta accatgatta tccctacttt gcaaattaca   4920 

aggttgcagt gcagagagcc taagacactt tctcaacttg agcaagtagg agatccagaa   4980 

tttgaaccca aatctctctg aatccaaaac gactttttgt tactttgcac acttcagtca   5040 

taggaatttc agtgtggatt tctgtattgg ttatgtattg ccgagtaaga aatgactctg   5100 

gccaggcacg gtggctcaca catgtaatcc cagcacctgg ggaggccgca gtgggtggat   5160 

tgcttgaggc caggagtttg agaacagcct ggccaacatg gcaaaacccc gtctgtacta   5220 

aaaatacaaa aattagccgg gcatggtgat gcatacctga agtcccagct actcaggagg   5280 

ctgaggcatg agaattgctt gagctctggg gtgggcagag gttgcagtga gccgagatca   5340 

tgcctggggg acagtgagac tctgtctcaa acaaattact ccaaaattta gtggctaaaa   5400 

aacaacataa aacaagaaac atttctgttg tgcctcctct tttcctccct ccctctttct   5460 

catagttaat ttgtttgaat caggattcaa ataaggtcga tgcattttta atctataggt   5520 

tttgtctcct tttttttttt ttccttaaaa aaatttcttt taatgcaaca gatttaaggg   5580 

accagggtgt tctctagagc aggggtccat gacctctagg ccacaaacca atagcggagc   5640 

cgtggcctgt tataaattgg gtggcatggc agggagtgag tggtgggcaa gcataggaag   5700 

ctgcgtctgt gcttagagcc actctctgtc actctcatta gtgcctcagg tcctccccct   5760 

gtcagatcag caacagcatt agtctcatag gaccgtgatc cctattgtga gccacacatg   5820 

caagggatct aggctgcatg ctccttacga aaatctaatg cctgatgatc tgtcactgcc   5880 

tcccatcacc ttcaagatgg gattgtctag ttctaggaaa acaagctcag ggctcccacc   5940 

gattgtacat tatggtgagt tggataacta tttcattata tattacaacg taataataat   6000 

agaaataaag tgcacaataa atgtcatgtg cttgaatcgt cccaaagcca cccccactcc   6060 

tttgagtcta tggaaaaatt gtcttccaga aaaccacttg ctggtgccaa aaaaatttgg   6120 

aaacctctgc tctagagatt tctgcagtct gtactttggt gattgcatct ctgtggtgtc   6180 

ttttaacata ctcttctgtc tcctgagttt cctgtgaaag ggtagataac tcaagagact   6240 

caatcaactt cgtgtttaat gttttcgtaa gaatacttca gaggcagtgg tgtaatcgtc   6300 

caagagtaca cacaggcctg atcgccaaag ccagttttct tcaggcccag cacgtcctga   6360 

cctcctcact cctctgccca ctctctgttt actcactccc attttcattc attgattctg   6420 

ttattctcca ctaagttttc tgggtggtca gcccaggaag cctcagcctg cttccagata   6480 

gttctcctca gagttagcgg tagcagcggt cgtcggccag tgtgtctcgg gtcaaattat   6540 

tttgggcatt actggaaagt tccaaaccaa agcctgtggt aagttccacc caaactggct   6600 

tgtgagggat atttttgttt aaactgcatt cctggagagg agccagactt cctactgcct   6660 

ggcagcagtg gtgtccatag gagtatgaag agctgggacc ctttctaatc actcagacca   6720 

aatattgggg ttttctgaaa tggactgaga aataacagta tgtttttatg aggctttgca   6780 

cattttcctt cctagcagta gctgcttagt ctactgaaaa gtgcatattt tgaacagggc   6840 

ctagaagagt taacagctcc tagagagagg tgctctgtaa tactttttct tcttcaaaaa   6900 

atggtttatg gctgggcgca atggttcatg cctataatcc cagcgctttt gggaggccaa   6960 

ggtgagagga ttgcttaagc ccagaaacgt gagaccagcc tgggcaacgt agtgagacgc   7020 

tgtctctgta ttacaaaatt ttttaaaaaa cggtttgtag gtccttaagt ccctgataaa   7080 

atagagaact gaattgcaat cctggaactt aaaaagttgg tgacgacacc tgagatattt   7140 

attacttaga ttgcagttac tgggtcagct tgtataatac tgaccaaggg tttttgattc   7200 

ttcctggaat tgataggaaa ttcatattaa aataattacc caagtccaaa catttttaga   7260 

actgcatttt tgatcatgga tttttatgtc tcttctgaac tttctgtcac cggtataatt   7320 

taaagaaatt atacttaagc tttgtctcac ttagaagata atatagaaca gtggtgtttt   7380 

tttaattaaa aaaaaagtta aaataacggt tttgtatcct tgctttactt cttaaacata   7440 

tgggaggaaa aaaaatcttt aacaagttta tttattttca ttttctgcta aattactttc   7500 

agaacttgaa tctactaatc ccagatataa tattcttgga ttcatattcc aaattttgct   7560 

gtctcaaatc catctaggga agtgggtggg ctataaatta taaataaatt ccaaattttg   7620 

tgggatgaat taccctgaag accaacgtgt aaattacata ttaatctttc tttttctccc   7680 

tagctcggtt ttaagaataa tgttttagcc aacatatctg cattactctt ggctcaatat   7740 

gagaaatcca tttttggttt gcctaacaga agatcatgtt gctttgcttc tctacacagt   7800 

atgaaaaccc aaagaaaaga aaaacagagg cagttttttg ctctaatgaa tgctctaaat   7860 

ctagctctta attatgattt tttaaggaaa attttgaaaa gtctacaagt taaatttttt   7920 

tttctatccc atacattttc catcctaagg cattgaaaaa gcacactgtg aaatacttag   7980 

tgtatctaga aacatcaggg aagaatgctt ccctcctaag caaaattttg ccttctgaaa   8040 

ctttttcagc attcagtctt tttatataat acttagaaaa atatttctga aatagatcat   8100 

acactctctt cccaaaaaca tcaaagtatg accgtaaagg gcagaggtag gtaaacttct   8160 

tgtaaggggc cagagagtga atatttgaga attttcagac tattaagatc tctgttgcaa   8220 

ctgcttgctt ttgccgtggt agcctgaaag cagccgtaga tagtatggaa atggatgatc   8280 

atggcagtgt tgcaataaaa ctttacaaaa cagacaatgg gccagattgg ccaggggcca   8340 

tagtatgcta cccctgggca acaacctgta tgccctggag tagtgtaaag aacgtgggtg   8400 

ttgggggtca actgacgctt ccagctctac cacttactgg ctgtgtggct ttgggcaaac   8460 

tactgaaaat ctctcagcgt cactttccaa gtgtgtgtaa tgtgtatttt cacagtgctt   8520 

tgcaggttgt tgattattga aaatagccat aatgcatgaa attaccagac acatctcact   8580 

ttatggagcc tggggctatt ggtaatatgc atttctttct catcttgatc gtaaaatgat   8640 

cttagaaagg tttctgagaa tatatagagt ttaagacagc aataagacaa ctaattaatt   8700 

aaacaggaaa aggggatgtt gtgctcagag aggaagtgtg ggtctcataa gggctttcac   8760 

aatcgtttga gaggacacgt gtgatgtctc atgcctgtta tcccagcact ttgggaggcc   8820 

aaggcaggca ggttgcttga gttccggagt ttgagaccag cctcggtaat ttggcaaaac   8880 

cttgtctcta caaaaattac agaaattagt tgggtgtggt ggtgcacacc tgtagtccca   8940 

gctgcttggg aggctgacga gtaaggatca cttgagccag catggtggag gctgccatga   9000 

tcatgctact gcactccagc ctgggcaaca gagccagaac ctgtcttgta aagaaaagga   9060 

aaaagagaga gaagggcaga aagaaagaag ggaaggaaga aaggaaaatt gggcccagga   9120 

atgatcttta caatgcctga caaccaagag aagaagggaa atgagcttca cattgcctgc   9180 

aagctctaag gtgacaagag ccaagagaaa ttattgttac tgtagtgatg ttccactgag   9240 

gatcataaag tactttatta ctctactgag tatggttatt ggatatgtgt tcttcttttt   9300 

ctttttcttt atcttttttg ctattctttt gttattcttg atttatgctg atggaaagcc   9360 

atggacccaa ggatgcttca cagttttctt taggagtaaa tgcttagatt ccatgttctt   9420 

tgacatgagc tatgtctgtt cctctcgagt ggaagcatcc ttttcagatg agttgccaga   9480 

aaagcagcca gctctggata agtgaggtac agcagaacac actgcaaata ctaggaatcc   9540 

ttaagtacag tggaacccca aagcactcta cctgctttct ttctcacctc cttaaaaact   9600 

ttttttgccc tcacctcatc atttattcag cagtcacaac agtgccaaga acttggctag   9660 

agattggaaa taaagcttat gccttctctc atatctcctg gaccttattt ctttcttaca   9720 

agaattgtga tgcttaacca gtttttttga taaccttttt ataaatgcca acccttccaa   9780 

aaaacctgcc cccctggtgg agagaagaat tattacatca attaggggtc acttagcatg   9840 

acatttgtcg gaaaaaaaaa agttagtgag cctttttgcc atattaaaag tcatcactgc   9900 

caagacataa atgaaaatgt gttcgaatta accacaccaa tgttcacaaa ataaacattt   9960 

ttgatttccc aacagaatcc taggtttaac tatcactatc atctttcatg aaatcaaagt  10020 

catatatgta aattgaacac aactttccct tccatagaga gtaaaaacca cgctttggag  10080 

ggtagataca attaccccag ggttgtcttt tcccactcct cacaatccca ccagtgcaca  10140 

tgcaaggtga tgtccttcct ttagctatag caaataatgt taattattgt tggtgttaaa  10200 

taatgattat gtaaagcact agactagaca ttcgtcggca aagtttttct gcaaagaatc  10260 

agatagtaaa tatttttgct cttatcagcc agacagtctc tgcggcaacc attcaagcat  10320 

tgttgaatac attgttgagt gttacatgag actattgtaa tataaaagta gcctgggaca  10380 

acacataaat aactgggtgt ggctgtgtcc taataaaact ttatttacaa agaacaggaa  10440 

gtggcttgga tttggtatct ggcctggcag ctgtggttta ctactcctta gacggtggcc  10500 

cagagaccct ttaaatgaaa ttcattttac tagcaccctt tttcatcatg agaaatatat  10560 

tctgtttttc ttagaaaatg ggttatgtta ggtctggtca aggtaaataa gtgttgagag  10620 

tcgatgttgt gtgcatagtt aatttcaatt ctttgaagaa gctcccccat gatatttcac  10680 

ggctgagaga agaggaaaga gtttaagtgg aacagtgtgc tttgctgagc tttggaaata  10740 

ttaccatata gggaagcagg tcaataagac aactaagtgc tgtttcaata acgaagatac  10800 

tgaagcgcta attggagtat ggaaccatat aatgatgata ataattgcta atatttatca  10860 

gctatttatc atgtgtcatg tacagctaag cacttacata ccatcccatt ttatccttat  10920 

aatgactcta agagtgggtt aaaaaatggc agaagaatgg cagtgtttaa tggttcagcc  10980 

tggtgcaatg attaaccaca ttttacagac taagaaatta agaacctcaa ccaagttcat  11040 

gctagctggt acgcagtaag gctagaactt cgtccaaaat ctcttcttct gttgagctca  11100 

gcttgcatag tagcttggag aatcagaaag atctgtctca tttgggaaat ataccatgta  11160 

aaaaacattg tttctaaagg agatttgtcc catgagtaaa atagatgatg gacagccact  11220 

gcctagtggg acaattagaa aggtcagttc aaggttggag gagatgcttc tttcagccaa  11280 

ttttcctttt tctcaggatc acctcaggtg atccgcccac ctcaacctcc caaactgctg  11340 

ggattatagg cgtgagccac cgctcctggc ctttcagcca attttctatc accaaaggga  11400 

aatcgttttg ctggaatatg tggtaaagga ggttaaagtc aaaagaaatt ctcgtcccgc  11460 

tcagttaagg tactcagact attttccaac caatcaaaag gggtgctgct tcatggagtt  11520 

cgtttaagct aaagcggcag ctgttgactg tcatttgcat catctttaaa catttactgt  11580 

gaatgtcact gtccatttcc actttctttc tacttgtctt caaattgatg cttatcaagt  11640 

agacagaaga agaccaaggg gtcgttttgc tatttatacc tccaaattga tggcgtgatc  11700 

actctcaagt gcaaacccag ccctgacact gtcctgtttg gcaatgtcct gctatctgac  11760 

ctgcaaatag ctacacttcc tgctgtggcc cacccaggcc tctgggatct gatcccttct  11820 

cccatgtcac ctgtggccat gtcttcccca acccaggctt ctctgctccg ctgcttatgt  11880 

tctggatcct gactctgagc ctttgcttgt gtggctcctc tcccacttgt ccatcatcat  11940 

gtcagttatg taagcataga ttccaaagtg acaatgaggg tagttaggat ggcaagagga  12000 

gaaaaaccaa gctggattca tggattgctt gtgtcacgtc ctctacagag cctcccttct  12060 

acctgctctt acccgggcac cacagttgat gagttatttt ttggaccatc agcaacacac  12120 

ccaatcattg tacatggaat acttcgggga tgcttttttc tatattgatt ttacttcatt  12180 

aaactgagct ccagaggagc agaaactttg gctaatacat cttggtctta gcttgtaata  12240 

tctgtgctac aacttattaa gatggtgacg ctggcaaatt ccttaatctt tctaattctt  12300 

aatttcatca gcaaagatgg gaaaggatac taacaccact ctgggttgtt gagaggattc  12360 

aataactgaa tatttataaa gcgtagtacc tgatacttaa taaaaaagtg aattttaacc  12420 

tgtgtcatca ttgtcatcgt ctttatcatc ctttgcaatt attacattta ctgccttcta  12480 

gtacaaggaa ggggatgggt ggctggctgg ctaggtggat agaaggatgg aagaggtaag  12540 

tacaaggaag gggatgagtg gctggctggc tagatggata gaaagatgga acaggtaagt  12600 

ataaggaagg ggataggtgg ctggctggct agatggatgg aaggggtgga aggggatgca  12660 

agaggtaagt acaaggaagg ggatgggtgg ctggctggct agatggatag aaggttggaa  12720 

gacaggaagg atggaacgga agaagaggga agaacaaaaa tggaaacaca tagtactgtt  12780 

aggtgaactg aacttctaag gtgccgattc tcagtgatag aatcttgagt tgatacctcc  12840 

ttgggtggca tggagcctat acctttgtag atcttgggaa acaacttcta aaagaatcat  12900 

agttgtatgt aatcgtaagt acaacaataa ttattttagg cacataaccc aaaggttttc  12960 

ttacaaggaa tctatgaacc ttaagagtga gggcttctgt taagagtaag ggcttccctg  13020 

gagtggacat ggatcatggg actgagccag cttggcattg ttgggttgaa cagggagcga  13080 

cacctctcag cccagtctat caagcctgct ctttgacctg cagtgagacc acccacgcag  13140 

acatcaatgc agcaaatccc cggcgtcagg gtttcaacat ttggttactc tcagagaact  13200 

ctcgatttat ataagacttg gaaaaagggt ttgagtttct gtggtttaca attatatttc  13260 

ccaacttggc ccatgaatcc agcttggttt ttctcctctt agcatcctaa ccaccctcat  13320 

tgtcactttg gaatctacac ttacataatt gacatgttga gaagctgtgc taaaaccaca  13380 

ctgaaatcac attttaataa catgggaacc atcttttccc agtaaattgt tgagaagcta  13440 

attcttgtca gcctaaaaac ttgaatatac atttgaataa atcagcggtg ctacaccgtg  13500 

gcagcctgct gaaaatcccc aaggaagatt atatttttag ttgagctact tgtcactgca  13560 

ctgtgttttt aatattgtga gtcctttctt gtcttcattt ttgaagaatc tattgcatac  13620 

cttgtcattc agaaaaacat aaacgggacc tctcaattag cggtaaagtt cgtcagttta  13680 

acttttaagc ttaaactcct gttatagttc ggtcacttac tcgtcatcaa aaagatattt  13740 

gagctgatat tatgcaataa tttataacca aaaacaggag gaaaaggtct ctgtttgtct  13800 

cagaagtaca agttatgcat aaggtgacaa attacacagc tttgggaaat gggtcttaat  13860 

ggaatgcacc aggctaatag aaaagcagtg cctcgatttc ccacctcaga tctgaaaact  13920 

cctgagagac tgacagggcg gttccccagc ttggctgtgt gaggaatcag ctgggaatcg  13980 

aacactgcag tctggctgtc acccccagga ttctaatgta attgttctgc attgtggatc  14040 

gatggatgga tggatgaatg gctggacaga aggaagactt ggaaaaaatc tttgagtttc  14100 

tgtggtttac aattttattt cccaacttgg cccatgaatc cagcttggtt tttctcctct  14160 

taccatccta aggagggagg gagggaggaa aagggggaag gaagcaggta gaaagggaac  14220 

agggaaggga ggactggagg aaggaaagca gggatcattc tggagtgtac actgggcatt  14280 

tgattttaaa gcaaatccaa gagatcatta aatttgtatc aatgaataca ctgttacccc  14340 

tcaatcaaca ctacaattca tatatcagaa tagcactttg ttacttgttt ctgagatagg  14400 

gctggctctg tcacccagac tggtgtgcag tggcacgatc acagctcact atggcctcag  14460 

ccaccagggt tcaagtgatc cttccactca gcctcctgag tagttgggac tacatgcctg  14520 

caccatcatg cctggctaat ttttattttt gtagagacga ggtctccttg tgttgtccag  14580 

gctggtctca aacttctgga atcaagtgat cctcctgccc ctgcctccca aaatactggg  14640 

attacaggca tgagccatgt ccagctgcct tgtttttttc tttgaagaga atgatagacc  14700 

ttccatagga aaaatgttaa atatgtgtgt gcagaaataa agatttcaaa tatttttgca  14760 

agatattttt tctaatacca cttttttcta cattttccat aatttagtga agatagtaaa  14820 

ttaacaaagt ggaaaagact gaatatttta agaaaagcca agtttaaaaa ttttgaacct  14880 

aatatttctt aaagtagcta aaattcagat attgagaata aattcaactt gacatggcaa  14940 

aattctaata ggctgaaata atgttttggt ctagaactat gtagctttgt gtagcccatc  15000 

aattgtctaa aaaaagagta acctattttg atgaaactcg ctgtatcttg taacctgtat  15060 

cctgtcttgg tattgtggga gtatatatga tttaggggaa agagtctgga gagaccttaa  15120 

gtctgcttta gggaaagggt gaggaacccc actgaaggct acttaacgca ttttgagaat  15180 

gtcagtaaag atttctcaga gcccagggat ttttttttta attgagacat aatttacata  15240 

caatacaatt ccttttttct tttttttgag acggagtctt gctctgtcgc ccaggctgga  15300 

gtgtgatggt gtgatcttgg ctcactgcaa cctctgcctc ctgggttcaa gtgattctcc  15360 

tgcctcagcc tactgagtag ctgggattac aggtgtgcgc caccacgctt ggctaatttt  15420 

tgtattttta gtagacacgg ggtttcacca tgttggttaa gctggtctca aactcctgac  15480 

ctctagatcc gcctgcctct gtaatcccaa agtgctggga ttacaggcgt gagcccctat  15540 

gcccagccaa aattcattat tttaagctgt acaattgagt gatttttatt acgttcacaa  15600 

agttacgtta ccattaccac ttttgaattg cagaacaatt tcatcactcc aaaaagaaat  15660 

ctcataacaa ttagcagtta ttttccatcg ctacttcctc cagtcccaag gcaatcagca  15720 

gtctgctttc tatctctacc tgtttgccta ttctggacat ttcatatgag tggagttaga  15780 

taatacatgg ccttttgtga ctggctttca cttagcataa tgttctagag gttcatttat  15840 

gttattgcaa aaatcagtac ttcatttctt tttatggctg aaaaaaattc cattatgtag  15900 

atgtgccata tttgtttatc tggttgtcag tcggatattt ctgttgttcc tactttttgg  15960 

ctactgtaaa agatgctgct atgaacatta ttgtgattat tataccttat ttgtaaacat  16020 

catgggtggg gggttgcagt aaacatgttg gaaagtaggg ttggaggtcc gtagaaattg  16080 

ggggcttcag cacttccccc aagctcaaca ccaaccccct ttctgagccc ctcttgaagg  16140 

agagttccct gggacgtgcc tggtattggt acaatcagtc aggaagcatt tttcctgggg  16200 

agaaacttac aagtccacga tcaaagccaa caagagacaa ggtgttacat gactcatttt  16260 

cggtttaaga agtgacaggc tgattctaag ttgggttcaa ttattttgtt aaagcgtttt  16320 

gcttatttga cttctcctga cctcggaaat aattctaacc aatcagtgct ggctcccatt  16380 

ggccctgggg tctggttgct ttacagctgg tgacaggggg accactccac taccacatgt  16440 

gaattaatcc tcaactccag agccaagtgc cattctccag caaggttgta tttcttcatt  16500 

agctattccc agggcccaga aagtcccaga ggatgtcaga gtacattaat ttttatcata  16560 

acatggaatc tttcaggtct gaatggcagc acacggctgt caggggcttc tgaactctat  16620 

tacagctcca tatatctcta ggcaaaacag aggaaagagt cgtcattggc aagggagatg  16680 

tacaaaatgc atgagatgtt ttattttttg agtgacttga ccacgtgctt aagcacattc  16740 

cccaaacaat ttttttctta ttgtttgtaa gttgtaagtt gtaaattcac ctctgccacc  16800 

acctattaaa gcccactccc tgcattaaaa ctgtataaag tgtatttaaa taaactctct  16860 

ttgcatgatg tgaatgaaat cgtcatctgg tacttaaaac tattctataa agttattaaa  16920 

aaattaatgt tcccttccca tgatttttct gcagaattta tgcatccatg atactgcaga  16980 

agttcataaa taatggcttg tattgctgct ttagtattgc tttatgccta cgaaatataa  17040 

tgttaatttg tagcaatgct aatgtgtttt caggaaggct ctttgtttat tgcctttatt  17100 

ttccccactt accaagtggg taaaatgctt tgagggttgc attttatgta ttcaggaggc  17160 

ccaggtatta ttttaataga agcactattg acaaatacca gtcatccccc ctgtgccagg  17220 

ccctggatga ggcactgctt cgcatggggg ctccccagat tgtcccacaa ggaaagcata  17280 

gtcaaagaca aagttttcag ttgtaagagt aaatgtgttc tgcctaggca ttgtcaagta  17340 

atttactgcc agctctagcc cttcactcaa gtttcctgga tacttttgac ttcttagcca  17400 

tggatgtgtt tgaaggctgc atggaccttc acttacttgc actgcaggtc agcctaattg  17460 

catgagctct gtggaccaca gagcagggtt ttccaaagtt caccaagaca aatattgtat  17520 

tatcttaaca tatattcatt ttttaaaact gaaaatcaga agagcaactc cacctagcag  17580 

aagtcttttg caaagggcga ggcgaggcta aaaagtatag aagagttcgt ttccagtgca  17640 

attttataaa cacagatggt ccttaaatta agcaaatggt acctaaatga ctgtgttgtg  17700 

gataatggta acagagggag ggactcgggg gttttttaaa aagtactgat tgtatgcagt  17760 

gttttaaaca gataactgtg atcttagtgt gatgaaagat gctgggagat ttcaccagtg  17820 

gtatcttatt atttttcggg gattttgtaa ttcaacaaaa ttctgttgta tgccaagcat  17880 

aaccctaggt gtgagagcac aaggtgactt cagataccac ctctatcctt caggggtttg  17940 

gggcccatta ttatgactta atccattttg ggcgtgagaa gctgagggtc acagaaagaa  18000 

ccaattccct ctttaaataa tgccacccca accctcctca tctgccaggt ctttcccttc  18060 

ttctatttgt atgaataata gtcactttct cttgtggagt tcgctaaatt ctactttggc  18120 

ctatcaaatt tctttcatat cacaactaaa tttcttaagg acgggactat ggttcatttg  18180 

tcagacgaac aaatgggaat ttgccaagag acacttgggt taatttacgt cttttccatc  18240 

caagggcact atgttgaagt gaggctagta ggtcatgagt gtggttgaag ttactttttc  18300 

ttactttccc gaccagcccc catccttact gcacttaaag ttgattgtcc attttattaa  18360 

atgtccccag gaagccagaa cacagggcag taaagtgctg aatgcaaagg gcaggagaaa  18420 

aatggaaaca accagaactg taacaccaag gaatgagacc tgcatgtcag atatcatgcc  18480 

cattgcacta agtgccattg gggcacaatt atcaaatgga tgcattttcc ctagaaaacc  18540 

atcttggaga gcatgtggat gtacttctat tttacatttc cccctattta caatcaatga  18600 

gattgagatt ttgttgctgg gactgctgat gatgggatgg gaaaatataa tcaaggtaat  18660 

ggacatgagg caaaaattta aggaaatgac aaaaacaaga gtatttccat tttcagttaa  18720 

gtgtatgtac tgatgttctg gaattcacta taagaagttg caaatggtgc atgaaatgaa  18780 

aaattcctgg tggtctccag gggacacagc cggtgctgtg ctccactctg ggtaactgtt  18840 

ttggattatt ttctctattc caactgaaat aaaaaaaaat taattaaatg tggctaggtt  18900 

atcttgacag cagaatccat tcccagttaa ttattatttt aatacttgat ggtgtctgtc  18960 

aaattgtcga catgtgacgg tcctttcaaa tttaaaggaa tagctgatgg tcactggcca  19020 

cccaagctga tactgatttt atatgttgat gtttctcatt ttatttgctc tttccttgaa  19080 

tatttattca ggacattctc taccagacat attgagtaag ggcaacagaa acaatacata  19140 

agtatcttat aaatgtggaa aacaatgtat atgtgttttt tatctctcaa tgattggtgg  19200 

gtaccatatc cccaaagtag aatgagcatt tgagaaaaca ggaaatatcc tcttttaggc  19260 

accatctctg tcaaggctga tgctgggctt tttatatatt ttctctaatt cttgtggctg  19320 

tcaaacaagg tgggcattat cattcccttt ataggggaca cagctgtggc tcagaggggt  19380 

ttattcactt tcctgagggc cacacacata atgagaggca gacacaggtg acgaagtgag  19440 

ttttccctgt cacgccatct tatctgtcac atacctctct gacatgctaa aattgcacta  19500 

aacaaaagaa ttctcttatg cacatatcat gcaaaagata ttctttaact ggggatcatg  19560 

tttctcattc catcaataga atgactaaca ttttctgagg gtgtctcacg tgaaagtaaa  19620 

tcgctcatgt ttgttctttt taaaagatgc ccttcgtatt gtgtatcttg cagtcttgct  19680 

ttctcaaact taagccaact atatcgtcat ttttgcaaaa tcactgcgtc agtttactat  19740 

tatttaatgt ttattgctac caattttaag aaatccttta taggactatt tgtgaaattg  19800 

attttgtgag gatgatgata taatttccat tacattacag catataaata taaatatata  19860 

tatatatata tatatatata tatatatata tatatatata tatattttat tatttttttt  19920 

tgagacggag tcttgctctg tcaccaggct ggagtgcagt ggtgcaatct cggctcactg  19980 

taacctccgc cgcccgggtt caagcgattc ccctgcctta gcctcctgag tagctgggac  20040 

tacaggcatg tgctaccaca cccagctaat tttttgtatt ttagtagaga tggtttcacc  20100 

atgttggcga ggatggtctc agtctcctga cctcgtgatc cgcctgcctt ggccttccaa  20160 

agtgctggga tatacatttt tttttttttt ttgagagatg gagtgtcact ctgttgccga  20220 

ggctggagtg tagtggcgca atctcggctc actgcaacct cctcctccgg ggttcaagcg  20280 

attctcctgc ctcagcctcc ccagtagctg ggattacagt cgtgtgccac cacgcctggt  20340 

tagtttttgt atttttagta gagatgggtt tcactgtgtt ggctaaggtg gtctcaaact  20400 

catgacctca agtgatccgc ccgcttcagc ctcccaaagt gctgggatta caggcgtcag  20460 

ccactgtgcc cggccggata gaaataattt ttataaactc cttggatgct acctaaaatc  20520 

atcttgtttt gctagtggca catgctgcat tttgggcagc tgtggccttg gtggattgct  20580 

gaagtagatt tgaccttacc tggactgagg cagctgttga agggaattgc tgtgttcagt  20640 

gtatactgcc atccatgatt tcatgaaacc agctctagct atttaagcag gggtcaaact  20700 

tagaattcta cattattttt ttcccttttc tgggaggaaa gacagttgaa caccagcaaa  20760 

gactaagaaa tttcttagaa gactgtgggt ccttgggccc tttctattga atttcagagt  20820 

atttccaaat actatgaagt cttgcagctt agttgagaaa tgccccagat ggtgtgacat  20880 

tctgcttcca ggagggattg gaaagtattt ccttttacat aacattccac tcagctcatt  20940 

cctttgctgt gtctgaaatt gaatccccca aagccacaat tatcttaaca ttcagaagag  21000 

tgtttattta atctgcaaaa tcttgcctca cttttgggga gcatgttaac aatttcactt  21060 

acaaatcttc tgtgtaactc aaccccatgg tggtgtctac tgctgctcct agactcttta  21120 

aagcaccttt ctcatctcag gtttgaaatg atatgtctca ttcttgggtt ccttgagtcg  21180 

taatgggttt gtcttgtctc cacagcataa atgactcttt cttgatcaac tagaaccaca  21240 

tcaacttctt ccctccagct tcagtgatat attgtgaaac atggctattc aacgtcctgt  21300 

agaccaaatg ccataagaaa aatagcattg attcaaacgt atccatccag atacctaaaa  21360 

aagttttact tcttaccaca tcttgagtct gggcaaacac gcacttccta tggacattga  21420 

ttactgtcta ctgtagagat aacatttgca catacagatt atggcacatg gtagaaagtg  21480 

ttaagtaatg taggaatgga catatcccaa gcaaaattgg aagccaagtc ccctgtccct  21540 

gctcaagttg gtatgactgg tgtatggtgc cttaatgggt acttaaagtc caggtgagag  21600 

tggcaggagg cagccaaatg cctaggtaga taggagccgg tccctgttga aaccccactt  21660 

ccaagttgaa gacagtttaa agactgaaag ccaagctaca agttaaatcc tcggaccaga  21720 

ttgagaactt gtcttcttac ttggtgcact cttctgattg atccccacct ttcacctatt  21780 

ttacatattc ctgcccttcc ctaactggtt tcctatgctg tcatgcccac ctttgagtgt  21840 

tgccttcact ttaaccttct gtgcatgctc acaaagtaat tagcatgtac cctccattct  21900 

gagttaatat aaggccccag acccagccac atggggcaac tttactgcct tcaggtaggg  21960 

gaaccacccc caccacattc cctctccact gagagttttc cttttagtta ataaattcgg  22020 

ctccactcac tctccattgt ctgcatgcct aattcttcct ggttgtgaga caagcagttg  22080 

gacctagctg agctaaggag cagaaagact gtatcacagg gaacttgtgt aacagcttga  22140 

tctcctgtcc tacgtagcta tctattggta agaagttgaa ggaacttgtg tcattccgtt  22200 

gtgcctgtcg tcttgacctt gtaaaaggtc ttgggtaagc atgcaagaag ttttgaagag  22260 

ggagatacag ctaatttgca gataaagagc aagggaagaa ttcctggaga aaggaagagt  22320 

ttcctgagtc acctttggga ggtaggaagg gtttgacatg taagctgggc atctgggaac  22380 

gagtgaggga ttctgtgagc cccatctcag tggaccactc aaggaaggtg ggtaagccct  22440 

gggtaataag tgtgtaagca gggaacagaa agtactgtga tttaaaatat gttaattttt  22500 

ctactgtaca gatgagagcc agcttggaga tgggctgtag ctcaagcatc ttacctacct  22560 

ctgatttctt aatgccacgt tataaggctg ctgcttatag ctcttgaagt cactccaaaa  22620 

acagatgagt gagaccctgt tgctaaagtc ccactgggtg tagattattc acagatgtat  22680 

acacagtggc tcactccagg taggatgtga tcagtgcttt tagaaataca gaaagtccta  22740 

ttggtttaaa aaaaattttt ttttgtaatg aattgagttt taaagctagc actgtacaat  22800 

aaaagggtga atttcactat gaattatgac aaacacagtc atagagctgc catcatcact  22860 

gtcaagatac agaacagtgc catcaccccc caaatgtcct ctgtgcctta ctgtagtcat  22920 

acctgctcct gacacctagc ccctgggaac cattgatctt ttttctgtcc ctagttgttt  22980 

gtcttctctg caatgttaca taaacagaaa tattctgggt gtcgcttttg gagtttggat  23040 

tccttcccat agcataatgc atgcagtatt tgtctgtttc tgctaaatta cccccaagac  23100 

ctagcgactt aaaacagcaa acatacacta tctcactctg ttagttttcc attgctgttg  23160 

taataaatta ccacaaactt atgtgcttaa aacccaaagc tattatctga cagctctgta  23220 

ggttaaagat ttgtcatggt tctcattggg ctaaaattaa ggtgtcggta tggctctgct  23280 

gctttctaga gactccaggg agaatatttt tccttgcctt ttccagcttc taaaggtcac  23340 

ccacaacatc ttcaaaacca gcactgttgc atctctgacc ttagccctgt agtcccattt  23400 

ccctctagcc acaatcggga aaggatctca ggactgttgt gatgacactg tgcttaccta  23460 

gattatctag catgagctcc ctgtctcaag gtgatagagt ttgcatgttt ttctcctgca  23520 

catgccatgt tgaaacataa tccctagtgt tggcggtggg tgtgctggga ggtatttgga  23580 

tcatgggggt ggaaccctca tgcatgactt acggccatcc ctttggtgat aagtgagttc  23640 

acatgatatc tggcaccttc cttcctctgt tgctcttgcc ctcaccatgt tagctgccta  23700 

ctcccctttt gccttccgcc atgactgtaa gcctcctgag gcctcaccgg aagccaagca  23760 

catgcttctt gtagagtctg cagaatcgga gccaattaaa tctcttttca ttataaatta  23820 

tccagcctca gttacttctt tattgcagtg caagaatagc ttaacacaca aggtcttacc  23880 

cttgatcacg gtctgcagca tgtcttttac catgtaaggt aatatgttca gtggctgtgg  23940 

gggttaggat gtggacttct ttgggggact tttatttttc ccagttacta tttttgtgac  24000 

tcaggaattt agggacagtt tggctggttg tttctggctc agggtctttc ttgggctgca  24060 

atcaagatgt cagctggggg ctgggcatgg tggctcactc ctgttatcac agcactttgg  24120 

gaggtcgagg tgagtggacc atttgaggtt aggagtttga gaccaacctg gccaacatgg  24180 

tgaaacgcca tactaaaagt acaaaaatta gttgggcatg gtggcacatg catgtaatcc  24240 

cagttagttg gggggctaag gctggagaac agcttcaaaa caggaggcgg aggttgtggt  24300 

caactgagat cacaccactg cactccagcc tgggtgaaag agcaaggccc cacctcaaaa  24360 

aaaaaaaaaa aaaaaaaaag ttagctgggg ccaaggtcat ctcaagtctt gactatgaaa  24420 

ggacctaaca tcatgcatgg ctgttggcag aggtcacttc ctcatgagca ttaaactgaa  24480 

agctataatt cctgactatt gaccagaggc gaacctcagt tccctgccat gtgggcctct  24540 

tcatggggca gttgataaca cagcagttag cttccattgg attgagtaag caagagagca  24600 

agaacaggag tgatacagaa gccagcatct ttttgtaaac caatctcaga agaattgtcc  24660 

atgatttttt ctatattctg ttagttagaa acaaattcct aggtctcacc cacccttgag  24720 

gtgaggggat tacacaaaag tatgaaaacc aggaagcagg gagcattggg agtcatttgg  24780 

accctgccta ggacagtgcg tttgagatcc atgttattct atccattagt agtagtttgc  24840 

ttctttttaa tgttgagtaa tatgccattg tgtgtttata tattgctaat ggattttaaa  24900 

gagggctaat caacgtgttg attagaggga aattttcttc agtgaaataa tatttgagca  24960 

aaaccttgaa gaacaattag gaatttgaca gagggaatgg cacgaataaa gacccagact  25020 

taatcaagtg aggggcgtac tcatccatgg ggcagtggat gattcacctg cctggagctt  25080 

aaggagaagg gggtgtagtt gtggctgttg ctcagaagga cctgaatgtc aggatgcatt  25140 

tgtgtttaat ttagcagaca tttaggaacc attaaaagtt tttgagaatg ggtaggtaag  25200 

attagaatag tattttaata agtttaacta ggctttaacc agtattcata aaaagtcaag  25260 

tgggagaatg tagaggtgac aagaccaggt gtgggctatt gcagtgacta cacctaaaag  25320 

gaacagagct agaaacatgg agtgacatga agtcattgtc ttaggttggt gatcctagca  25380 

gctgagcctg ggatggggat tcttgttcct atgatcgatt aggggactgt tcccagagga  25440 

gagcggagag ggaagcaggc tttggtttct actggaaatc tgtttccgcc cgattccatg  25500 

gggagctctg gagaaggaat tgtatcaccg agttggtcct ccttgtcaac agagcatgtg  25560 

tgtgggggtg atgacttccc agatgtgagg gtgccctcca gcaatggaca gttatctgga  25620 

ggagttcatg ctccagaggt agtgcctacc tctgtggaga agtaggggat ggcacggtat  25680 

tttaggggtt cctaatgagc ctagagatac tgacattgac cagtcatgga gactaaagga  25740 

gaagatggga tagcacattg ttttcctaag tatttgtaat caggtgactg gtttgcaaat  25800 

atcagccatt acagaaataa agggtatcag gacggggagg agatgtgagg agagaagatg  25860 

aagagtttag atttaggcat gttgaccctg aatttgtggg aaggtatcca ggaagataag  25920 

actgacaggt gggaatgaga ttctggaact tggaaagaat atagtggcta gagctaaggg  25980 

tttgaagtct tcatggcgat tttgttcgga gtcatggatt tggaagaaac tgctgcaggg  26040