Abstract:
The present invention comprises a method method to identify tumor suppressor genes by detecting genes in a mouse retroviral insertion mutagenesis model which expression is inhibited by methylation of the viral insertion or the VIS-flanking gene. This is preferably accomplished by first randomly cutting the mouse genomic DNA, immunoprecipitating the methylated DNA and amplifying the VIS-flanking DNA by inverse PCR, optionally followed by cloning and sequencing of the amplicons. 
     Next to the already known tumor suppressor genes Smad1 and Mad1-like, several putative tumor suppressor genes have been found. The tumor suppressing properties of these genes, as indicated in Table 3 also form part of the present invention. Further use of these genes and/or its substrates or downstream products, for diagnosis and therapy of cancer, preferably AML is envisaged.

Description:
[0001]    The invention is related to the field of cancer, more specifically to the field of leukaemia and to the detection of genes playing a role in the development of said cancer. 
         [0002]    Retroviral integration mutagenesis is considered a powerful tool to identify cancer genes in mice (Suzuki, T., et al, 2002, Nat. Genet. 32:166-174; Erkeland, S. J. et al., 2004, J. Virol. 78:1971-1980; Joosten, M. et al., 2002, Oncogene 21:7247-7255; Mikkers, H. et al., 200, Nat. Genet. 32:153-159; Neil, J. C. and Cameron, E. R., 2002, Cancer Cell 2:253-255; Akagi, K. et al., 2004, Nucleic Acids Res. 32: D523-527). Identification of genes generally takes place by amplification of the genomic sequences flanking the virus integration site (VIS), whereby VIS-flanking genes common to independent tumors (i.e. common VIS genes) are considered bona fide disease genes. However, VIS genes not yet found common often also belong to gene classes associated with cancer and may qualify as disease genes. Further, genes located more distantly from the VIS may also be involved in disease, but the likelihood of this happening and the influence of the distance between the gene and the VIS is unknown. Recently, it has been established that the genes, detected in this mouse model, have clinical relevance for human cancers (Erkeland, S. J. et al., 2006, Cancer Res. 66:622-626). 
         [0003]    It is generally assumed that expression of VIS flanking genes is most frequently increased due to the transcription enhancing activities of the viral LTR. Thus, in that case it would only be possible to find genes that play an active role in the forming or maintenance of the tumor. It would be desirable to search for (common) VIS-flanking genes, that are effective in the above indicated mouse retroviral integration mutagenesis models, of which the expression is decreased by the viral insertion, since these genes would likely act normally as tumor suppressor genes. With the current models, it is very difficult to discriminate between genes that are overexpressed and genes of which the expression is inhibited. 
         [0004]    Thus, there is need for a method using retroviral integration mutagenesis, which allows for the detection of genes inhibited because of the viral insertion. 
         [0005]    The inventors now have discovered that such genes can be identified by investigating the methylation pattern which in some instances occurs during retroviral integration. As is well known, one of the defence mechanisms of cells against viral attack is methylation of the viral DNA, thereby marking said DNA as ‘foreign’, whereafter the methylated DNA is silenced by endogenous silencing mechanisms. The methylation takes place at the so-called CpG islands in the LTR of the virus, through mechanisms which are well known in the art. In this way expression of the viral DNA and the DNA of the VIS-flanking genes is prohibited. It has further appeared that this methylation is able to spread over the VIS-flanking genes, which thus results in further inactivation (inhibition of expression) of the VIS-flanking genes. 
         [0006]    One embodiment of the present invention is a method to identify tumor suppressor genes by detecting genes in a mouse retroviral insertion mutagenesis model which expression is inhibited by methylation of the viral insertion or the VIS-flanking gene. This is preferably accomplished by first randomly cutting the mouse genomic DNA, immunoprecipitating the methylated DNA and amplifying the VIS-flanking DNA by inverse PCR, optionally followed by cloning and sequencing of the amplicons. 
         [0007]    Next to the already known tumor suppressor genes Smad1 and Mad1-like, several putative tumor suppressor genes have been found. The tumor suppressing properties of these genes, as indicated in Table 3 also form part of the present invention. 
     
    
     
       LEGENDS TO THE FIGURES 
         [0008]      FIG. 1 . Taqman strategy for detection of methylated CpG in integrated LTR&#39;s of MuLV. These LTR&#39;s are known to possess 516 CpG&#39;s. Analysis is focused on CpG&#39;s 161-337, which are core CpG&#39;s known to be target for methylation. Two rounds of PCR are performed on bisulphite-treated genomic DNA. The first regular PCR is done with methylation insensitive primers to amplify the region containing CpG&#39;s 161-337. The second (Taqman PCR) round is performed with nested primers within this region in which the reverse (RV) primer is either methylation sensitive (M1) or methylation insensitive (M1u). Signals are quantified by Taqman light cycler. Probe and primer compositions are given in text. Delta Ct values calculated by subtracting Ct values obtained with RV primer M1u from Ct values obtained with RV primer M1 provide a quantitative measure of the methylation status of LTRs in a given tumor sample. 
           [0009]      FIG. 2 . Results of methylation detection experiments (Taqman) in leukaemia samples from mice infected with the Graffi 1.4 murine leukaemia virus. To generate a reference line for the Taqman assay, mixing experiments with methylated LTR-containing plasmid (341) and nonmethylated LTR-containing plasmid (340) were performed and delta Ct values calculated as described with  FIG. 1  (upper Table). These cloned LTR sequences are derived from bisulphite-treated genomic DNA from a Graffi-1.4-induced tumor. PCR-amplified LTR sequences from this tumor were cloned into TA vector and sequenced to detect methylation status. This showed that the assay is linear between delta Ct values 0 and 8.00 (Graph). Based on these values, 5 categories of methylation, (&lt;5; 5-12.5; 12.5-25; 25-50; and 50-100) were defined (lower Table). 
           [0010]      FIG. 3 . Results of the agarose gel with the amplicons from the inverse PCR after MeDIP enrichment for methylated DNA. Tumor cell samples from different leukemic mice (99-12, 99-49, etc), derived from liver (Li) spleen (Spl) or bone marrow (BM) were analyzed. Bands with sizes greater than the viral LTR sequence only (marked by line) represent fragments that consist in part of LTR sequence and in part of flanking genomic sequences. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0011]    In the research that led to the present invention, a number of genomic regions were identified to be involved in tumor development by proviral tagging. Proviral tagging (Berns. 1988. Arch Viro. 1.102:1-18; Kim et al. 2003. J Virol. 77:2056-62) is a method that uses a retrovirus to infect normal vertebrate cells. After infection, the virus integrates into the genome thereby disrupting the local organization of the genome. This integration affects the expression or function of genes, depending on the integration site of the virus, which may for instance be in a coding region, a regulatory region or a region nearby a gene. If a cellular gene involved in tumor development is affected, the cell will acquire a selective advantage to develop into a tumor as compared to cells in which no genes involved in tumor development are affected. As a result, all cells within the tumor originating from the cell affected in a gene involved in tumor development will carry the same proviral integration. Through analysis of the region nearby the retroviral integration site, the affected gene can be identified. 
         [0012]    Mouse retroviral insertion mutagenesis models are known for several types of cancer. For acute myeloid leukaemia (AML) the Graffi 1.4 (Gr-1.4), BXH2 and AKxD murine leukaemia virus (MuLV) models have been proven useful for finding genes involved in the development, maintenance and spread of leukaemia. 
         [0013]    Acute myeloid leukemia (AML) is the most frequent form of acute leukemia in adults and is one of the most aggressive forms of leukemia, which is acutely life threatening unless treated with different kinds of chemotherapy. Depending on the AML subtype determined by various clinical parameters, including age, and laboratory findings, for instance cytogenetic features, allogeneic stem cell transplantation might follow the remission induction by chemotherapy. The 5 years overall and disease free survival rate of adult AML is currently in the order of 35-40%. There is a strong need for a more precise diagnosis of AML, which allows for better distinction between the prognostic subtypes and for new therapeutic strategies for the large contingent of patients that can not be cured to date. The currently available laboratory techniques allow for a prognostic classification, but this is still far from optimal. Still, most patients cannot satisfactorily be risk-stratified and still a majority of patients are not cured by currently available treatment modalities. 
         [0014]    The pathogenesis of leukemia is complex. Before becoming clinically overt, leukemic cells have acquired multiple defects in regulatory genes that control normal blood cell production. In human leukemia, until now only few of these genes have been identified, mainly by virtue of the fact that these genes were located in critical chromosomal regions involved in specific chromosome translocations found in human AML. Studies in mice, particularly those involving retroviral tagging, have yielded only relatively small numbers of retroviral insertions and target genes per study, but have nonetheless made clear that there are at least a few hundred genes that can be involved in the pathogenesis of murine leukemia. There is a strong conservation between the mouse and human hematopoietic systems, as is for instance evident from the fact that the biological properties of the hematopoietic progenitor cells and the regulators (hematopoietic growth factors) are largely similar. Therefore, it is not surprising, that is recently has been established (Erkeland, S. J. et al., 2006) that these genes have human, clinical relevance. 
         [0015]    Also for other cancers such models exist, e.g. mice infected with murine mammalian tumor virus (MMTV) as a model for breast cancer and mice infected with e.g., Moloney virus or Cas-Br-M virus for B and T cell lymphoma&#39;s. 
         [0016]    Because MuLV preferentially, albeit not exclusively, integrate into the 5′ promoter region of genes, it is generally assumed that expression of VIS-flanking genes is most frequently increased due to the transcription enhancing activities of the viral LTR. However, CpG islands in the viral LTR are a potential target for de novo methylation, which could form the initiating event to silencing the (expression of the) viral insert and the VIS-flanking genes. 
         [0017]    In mammalian cells, approximately 3.5 to 5% of the cytosine residues in genomic DNA are present as 5-methylcytosine (Ehrlich et al., 1982, Nucl. Acids Res. 10:2709-2721). This modification of cytosine takes place after DNA replication and is catalyzed by DNA methyltransferase using S-adenosyl-methionine as the methyl donor. Approximately 70% to 80% of 5-methylcytosine residues are found in the CpG sequence (Bird, 1986, Nature 321:209-213). This sequence, when found at high frequency in the genome, is referred to as CpG islands. Unmethylated CpG islands are associated with housekeeping genes, while the islands of many tissue-specific genes are methylated, except in the tissue where they are expressed (Yevin and Razin, 1993, in DNA Methylation: Molecular Biology and Biological Significance. Birkhauer Verlag, Basel, p. 523-568). This methylation of DNA has been proposed to play an important role in the control of expression of different genes in eukaryotic cells during embryonic development. Consistent with this hypothesis, inhibition of DNA methylation has been found to induce differentiation in mammalian cells (Jones and Taylor, 1980, Cell 20:85-93). 
         [0018]    Methylation of DNA in the regulatory region of a gene can inhibit transcription of the gene. This is probably caused by intrusion of the 5-methylcytosine into the major groove of the DNA helix, which interferes with the binding of transcription factors. 
         [0019]    Existence of methylation has been shown in the present mouse model by a methylation sensitive Q-PCR ( FIG. 1 ). However, other strategies for demonstrating methylation, such as MeDIP and methylation sensitive restriction enzyme digestion, may be employed. By Q-PCR, it was found that LTR methylation in the applied model occurs with variable frequencies, ranging from &lt;5% to 50-100% (See Table 2). However, these estimations are currently provisional and need to be verified by other methods. 
         [0020]    Since tumors developed in these cases, where the proviral insertion (and possibly a part of the flanking genes) were methylated and thus the expression of these genes was inhibited, this means that knock-out of these genes apparently is a trigger for the development or maintenance of the tumor. Thus, it is envisaged, that these genes, which are subject to transcription and translation in a normal, wild-type cell, would then act as tumor suppressors. 
         [0021]    As is exemplified in the Experimental part, it is possible to retrieve the identity of the VIS-flanking genes from samples of the tumors. In the present invention, this is accomplished by digesting the genomic DNA with a restriction enzyme, enrichment of methylated DNA fragments by immunoprecipitation and applying an inverse PCR on these fragments. The amplified fragments are then subjected to gel electrophoresis, which yields several bands, which can be sequenced and from which the identity of the genes can be retrieved. 
         [0022]    However, the invention is not limited to the above-applied method. Any method known in the art which enables isolation of VIS-flanking genes surrounding a methylated viral insert would be feasible to detect potential tumor suppressor genes. 
         [0023]    There are several ways whereby the identified genes can be assayed for their tumor suppressor function. Firstly, growth factor dependent cell lines are available that faithfully recapitulate normal myeloid cell proliferation, survival and differentiation in response to exogenous stimuli, such as granulocyte colony-stimulating factor (G-CSF). Based on the cellular features of AML cells, it is a reasonable assumption that reduced expression of tumor suppressor genes in this model will have negative effects on the induction of myeloid differentiation and stress-induced (e.g., by growth factor deprivation) apoptosis induction, or positive effects on pro-survival and proliferation signaling pathways. A murine interleukin3-dependent cell-line engineered to express the human G-CSF receptor is particularly suitable for these studies (De Koning et al, Blood 91: 1924, 1998). Genes of interest (single or multiple) can be knocked-down in these cells using siRNA or shRNA approaches and changes in cell proliferation, survival and differentiation and expression of genes and activation of signaling pathways involved herein can be taken as functional endpoints. This analysis can be extended to primary bone marrow stem cells and progenitor cells using in vitro and in vivo approaches in mice. For the latter, hematopoietic stem cells transduced with siRNA or shRNA can be transplanted into irradiated recipient mice, which can be monitored for defects in blood cell production and possible development of leukemia. These experiments may also be performed in (genetically modified) mouse strains that are already predisposed to tumor development due to other genetic abnormalities. In addition, genetic approaches may be taken to knock out genes in mouse embryonic stem cells to generate gene deficient mouse strains and to cross these mice with relevant tumor-prone strains to study cooperativity of gene defects in tumor development. 
         [0024]    Thus, an embodiment of the present invention are the tumor suppressor genes, that were found in the VIS-flanking genes of the methylated samples. These genes are listed in Table 3. The person skilled in the art will recognise that some of the genes found are already known as tumor suppressor genes (Smad1 and Mad1-like), but the largest part of the listed genes are unknown to play a role in suppression of tumors. Ideally, a tumor suppressor gene is found in more than one sample, which confirms its importance in tumor suppression. Expression of the genes of interest will be analyzed in clinical AML, by employing gene array-based expression profiling (Valk et al, N Engl. J Med 2004 Apr. 15; 350(16):1617-28), to determine their relevance for human disease and to establish their potential prognostic value, along the lines similar to those described in the study by Erkeland et al (Erkeland, S. J. et al., 2006, Cancer Res. 66:622-626). 
         [0025]    The genes from Table 3, and optionally further identified by the above described expression profiling may be used to develop diagnostic tools to further risk-stratify cancer, in particular AML. As is shown in WO 2005/080601 genetic expression information, alongside with clinical parameters, can be used to classify AML, and, on basis of said classification, predictions can be made about responsiveness to a particular therapy. It is envisaged that the genes of the present invention will be a further aid for such a classification and determination of susceptibility to therapy. 
         [0026]    The genes from Table 3 may potentially also form the starting point for the design of therapeutic strategies. One such a strategy can be to increase expression of the gene in vivo, e.g. by enhancing the activity of the promoter and/or by genetic therapies using (viral) vectors coding for the gene. Another strategy aimed at restoring activities of critical downstream substrates of these genes is envisaged. Now the tumor suppressor genes of the invention are known, a person skilled in the art can easily detect downstream gene products and/or substrates. Depending on the nature of such products and/or substrates therapy will consist of administration of these products and/or substrates to restore natural levels, or closing down pathways that would deplete the produced amounts by e.g. siRNA treatment. 
         [0027]    Experimental Part 
       1. Protocols 
       [0028]    I. PCR to Amplify LTR Sequences after Bisulphite Treatment
 
Take 2 ul of DNA from tumor samples and treat with bisulphite as described in protocol of DNA EZ methylation kit D5002 (ZymoResearch/BaseClear)
 
       Use 1 ul for PCR: 
       [0029]      
         [0000]    
       
         
               
               
             
           
               
                 1 ul template 
                 5′ 94° C. 
               
               
                   
               
               
                 1 ul bsLTRrv1 
                 10 cycles 
               
               
                   
               
               
                 1 ul bsLTRfw2 
                 30″ 94 C. 
               
               
                   
               
               
                 6 ul NTP&#39;s (1.25 mM/NTP) 
                 30″ 50 C. 
               
               
                   
               
               
                 5 ul buffer 
                 1′ 72 C. 
               
               
                   
               
               
                 0.25 ul Taq 
                 7′ 72 C. 
               
               
                   
               
               
                 storage at 4 C. 
               
               
                   
               
               
                 bsLTRrv1: CCCAAAATAAACAATCAATCAATC 
               
               
                   
               
               
                 bsLTRfw2: GAGAATAGGGAAGTTTAGATTAA 
               
             
          
         
       
     
       II. Quantification of Methylated LTR by Quantitative PCR (Taqman) 
       [0030]      
         [0000]    
       
         
               
               
             
           
               
                   
                 2 μl DNA (from PCR I) 
               
               
                   
                 0.25 μl dNTP&#39;s (10 mM) 
               
               
                   
                   
               
               
                   
                 0.25 μl probe: bsLTR M1 
               
               
                   
                 (5′-AAACGCGCGAACAAAAACGAAAAACGAACTA-3′) 
               
               
                   
                 or 
               
               
                   
                   
               
               
                   
                 UM2 (5 pmol/μl) 
               
               
                   
                 (AAACCATATCTAAAAACCATCTATTCTTACCCCC) 
               
               
                   
                   
               
               
                   
                 2.5 μl buffer A 
               
               
                   
                 0.125 μl Ampli Taq Gold 
               
               
                   
                   
               
               
                   
                 1 μl bsLTRtqm Fw2 (10 pmol/μl) 
               
               
                   
                 (GGTTAAATAGGATATTTGTGGTGAGTAG) 
               
               
                   
                   
               
               
                   
                 1 μl bsLTRtqm Rv1 (10 pmol/μl) 
               
               
                   
                 (AATTCTTAAACCTCTTTTATAAAACTC) 
               
               
                   
                   
               
               
                   
                 5 μl MgCl2 (25 mM) 
               
               
                   
                 12.875 μl MQ (end volume 25 μl) 
               
             
          
         
       
     
       Cycling Protocol: 
       [0031]      
         [0000]    
       
         
               
               
             
           
               
                   
               
             
             
               
                  1x 
                 10 min 95° C. 
               
               
                 45x 
                 15 sec 95° C. 
               
               
                   
                 30 sec 58° C. 
               
               
                   
                 30 sec 60° C. 
               
               
                   
               
             
          
         
       
     
       III. MeDIP on Methylated CpGs 
     Reagents 
       [0032]    Proteinase K (10 mg/ml)
 
Mbo1 enzyme (GATC) &amp; Neb 3 buffer; MboI R0147L, Biolabs
 
α-Methylcytidine antibody (1 μg/μl) BI-MECY-0500, Eurogentech, Maastricht
 
Pre-immune serum IgG (12 μl/μl, diluted to 1 μg/μl), Mouse IgG technical grade from serum, Sigma, Zwijndrecht
 
Ip buffer (should be cold!): PBS solution
 
       0.05% Triton-X-100 
       [0033]    100% ethanol 
       3M NaAc, ph 5.5 
     Phenol/chloroform 
       [0034]    Glycogen 20 μg/μl Roche 901393 (optional)
 
Protein G-Sepharose beads
 
       Protocol 
     Day 1 
     Digestion of Genomic DNA 
       [0035]    Take 10 microgram genomic DNA and digest o/n with 50 units of Mbo1 (10 μl) in total of 100 μl (Neb buffer 3) 
       Day 2 
     Antibody Incubation 
       [0036]    Take 2×40 μl of digestion product and denaturise DNA for 10′ at 95° (also for enzyme inactivation)
 
Keep 4 μl as 10% input control, add 200 μl IP-buffer and put on at 4° on a roller until prot K will be added
 
Put denaturised samples directly on ice
 
Add 20 μg antibody (20 μl) and add total volume up to 500 μl with IP-buffer (1 sample with α-methylcytidine and 1 with mouse pre-immune serum IgG)
 
Incubate samples for 2 hr at 4° on a roller
 
Incubation with Dynabeads
 
         [0037]    Wash 60 μl of Dynabeads (M-280 Sheep anti mouse IgG 112.01, Dynal Biotech) per tumor sample; 3× 
         [0000]    Add 1000 μl IP-buffer to pooled beads and place in magnet for 2 minutes, remove supernatant, at the last step: resuspend beads thoroughly in 110 μl IP-buffer per tumor sample
 
Add 50 μl of beads to the + and − sample of each tumor and incubate for 2 hr at 4° on a roller
 
       Washing Beads 
       [0038]    Wash samples 3× with 700 μl IP-buffer, finally resuspend beads in 200 μl IP-buffer
 
Add 200 μl IP-buffer to the 10% input sample
 
       Elution of DNA 
       [0039]    Add 2 μl proteinase K (=20 μg) to the input, + and − samples and incubate or for 3 hrs at 50°
 
Discard beads and keep the supernatant
 
       DNA Recovery 
       [0040]    Add 200 μl phenol/chloroform and spin down (spin 5′ at 13 k rpm)
 
Collect supernatant
 
       Add 500 μl 100% EtOH 
       [0041]    Add 20 μl 3M NaAc pH 5.5 and optional 0.5 μl (10 μg) glycogen
 
Incubate o/n at −20° C. to precipitate DNA (or at −80° C. until sample is frozen)
 
       Day 3 
     Spin 30′ 13 k, 4° C. 
       [0042]    Decant supernatant
 
Add 500 μl ice cold 70% EtOH
 
       Spin 10′ 13 k, 4° C. 
     Dilute DNA in 20 μl MQ 
       [0043]    PCR after MeDIP 
       Samples 
     Use 1 μl of the MeDIPped DNA for PCR 
       [0044]    PCR on 3 different samples:
 
Input control (should always be positive)
 
IgG control (controls for the amount of aspecific binding)
 
A-methylcytidine sample (positive if DNA was methylated)
 
       Sequences 
       [0045]      
         [0000]    
       
         
               
             
           
               
                 Amplification of 3 different sequences 
               
               
                 H19: positive control, H19 ICR1 fw 
               
               
                 (ACATTCACACGAGCATCCAGG) × 
               
               
                 H19 ICR1 rv (GCTCTTTAGGTTTGGCGCAAT) 125 bp 
               
               
                   
               
               
                 LTR: L2N (Msp1) (ATCTGTGGTGAGCAGTTTCGG) × 
               
               
                 L3N (AGAGGCTTTATTAGGAACGGG) 287 bp 
               
             
          
         
       
     
       Tm: 58° 
     Elongation: 30″ 
       [0046]    Expected result: 
         [0000]                                                                α-           Primer   Input   IgG   methylcytidine                           H19   +   −   +           LTR   +   −   + (if methylated)                       − (if not                       methylated)                        
III. Inverse PCR after MeDIP
 
         [0047]    Take 8 μl MeDIPped DNA 
         [0048]    Add 2 μl dilution buffer, and add up to 10 μl with MQ-H 2 O 
         [0049]    Add 10μ T4 DNA ligation buffer 
         [0050]    Add 1 μl T4 DNA ligase 
         [0051]    Leave at RT for 15′ 
         [0052]    Heat inactivate T4 DNA ligase at 65° for 15′ 
         [0053]    Take 2 μl for PCR in total of 50 μl (L5×L6) 
         [0054]    Take 2 μl of this dilution and perform nested PCR (L5N×L6N) 
       PCR Program: INVPCR1 (60°) and INVPCR2 (56°) 
       [0055]    10′ 94°
 
30 cycles
 
30″ 94°
 
       30″ 60° (L5×L6) or 56° (L5N×L6N) 
       [0056]    3′ 72°
 
End cycles
 
5′ 72°
 
4° storage
 
       Primers: 
       [0057]      
         [0000]    
       
         
               
               
             
           
               
                   
                 L5:  CAACCTGGAAACATCTGATGG 
               
               
                   
                   
               
               
                   
                 L6:  CCCAAGAACCCTTACTCGGC 
               
               
                   
                   
               
               
                   
                 L5N: CTTGAAACTGCTGAGGGTTA 
               
               
                   
                   
               
               
                   
                 L6N: AGTCCTCCGATAGACTGTGTC 
               
             
          
         
       
     
       I. Quantification of Methylated LTR by Quantitative PCR (Taqman) 
       [0058]    To establish whether integrated proviral sequences, specifically CpG islands in the long terminal repeat (LTR) sequences of Graffi 1.4 murine leukaemia virus (Gr-1.4 MuLV) were methylated in Gr-1.4 MuLV-induced tumors, and to what extent, a quantitative method involving methylation specific PCR, based on Taqman technology, was developed. ( FIG. 1 ). Methylation specific PCR (MSP) is a well established technique in genome research (Derks et al, Cell Oncol. 2004; 26 (5-6):291-9). To establish linearity of this assay, an experiment was performed with plasmid DNA&#39;s containing sequences derived either from the unmethylated LTR (plasmid 340) or the methylated LTR (plasmid 341). Based on this, a reference line was generated and methylation status categories defined ( FIG. 2 ). Next it was established that genomic DNA samples from normal somatic tissues (bone marrow, liver, spleen) do not give a specific signal in this assay, in line with the fact that these normal tissues are not expected not contain (methylated) Graffi 1.4 LTR sequences (Table 1). We then screened all Graffi 1.4-induced tumors (n=81). Distinct methylation categories were defined: high (n=7), medium-high (n=15), medium (n=12), low (n=20) and very low to none (n=27) (high and medium high samples shown in Table 2). 
         [0000]    
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Ct values in normal tissue samples. Ct value &lt;30 for 
               
               
                 M1u and &lt;34 for M1 indicate that no methylated 
               
               
                 LTRs are present. 
               
             
          
           
               
                   
                 Tissue 
                 Ct M1u 
                 Ct M1 
               
               
                   
                   
               
               
                   
                 normal bone marrow 
                 31.8 
                 35.4 
               
               
                   
                 Normal liver 
                 31.4 
                 37.9 
               
               
                   
                 Normal spleen 
                 31.8 
                 40.8 
               
               
                   
                 MQ (nested PCR) 
                 30.3 
                 33.9 
               
               
                   
                 MQ (Taqman) 
                 Not determined 
                 Not determined 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Methylation status of high and 
               
               
                 medium high methylated samples. 
               
             
          
           
               
                   
                   
                   
                 % 
               
               
                   
                   
                 delta 
                 methylation 
               
               
                 sample 
                 organ 
                 Ct 
                 mean 
               
               
                   
               
               
                 99-12 
                 lymph node 
                 1.6 
                 100-50  
               
               
                 99-23 
                 bone marrow 
                 2.1 
                 100-50  
               
               
                 99-49 
                 bone marrow 
                 2.2 
                 100-50  
               
               
                 99-5  
                 liver 
                 2.3 
                 100-50  
               
               
                 99-20 
                 spleen 
                 2.4 
                 100-50  
               
               
                 99-10 
                 liver 
                 2.5 
                 100-50  
               
               
                 99-44 
                 bone marrow 
                 2.6 
                 100-50  
               
               
                 99-55 
                 spleen 
                 3.1 
                 50-25 
               
               
                 00-10 
                 liver 
                 3.2 
                 50-25 
               
               
                 99-29 
                 bone marrow 
                 3.4 
                 50-25 
               
               
                 99-16 
                 spleen 
                 3.5 
                 50-25 
               
               
                 99-34 
                 spleen 
                 3.7 
                 50-25 
               
               
                 99-33 
                 bone marrow 
                 3.7 
                 50-25 
               
               
                 00-14 
                 liver 
                 3.7 
                 50-25 
               
               
                 99-36 
                 bone marrow 
                 3.9 
                 50-25 
               
               
                 00-9  
                 liver 
                 4.0 
                 50-25 
               
               
                 00-17 
                 liver 
                 4.1 
                 50-25 
               
               
                 00-22 
                 liver 
                 4.2 
                 50-25 
               
               
                 99-19 
                 spleen 
                 4.3 
                 50-25 
               
               
                 99-48 
                 liver 
                 4.4 
                 50-25 
               
               
                 00-4  
                 spleen 
                 4.5 
                 50-25 
               
               
                 99-18 
                 spleen 
                 4.5 
                 50-25 
               
               
                   
               
             
          
         
       
     
       II. MeDIP on Methylated CpGs 
       [0059]    The genomic DNA was digested with Mbo1. The fragmented DNA was enriched for methylated DNA by immunoprecipitation with MeDIP (incubation with antibodies directed against 5-methyl-cytosine, α-5MC). Primers L2N and L3N were generated to detect methylated LTR after MeDIP. Primers were also generated for the methylation imprinted gene H19, serving as positive control on the MeDIP procedure. Enrichment of LTRs after MeDIP with α5-mC was found in 25/34 samples tested thus far. Positive signals were found in all methylation categories, with generally the highest signal in the high to medium high methylation categories and lower signals in the low to very low categories. As expected, MeDIP on normal hematopoietic tissues was negative for LTR, but positive for the methylation imprinted gene H19. 
         [0000]    III. Inverse PCR after MeDIP and Identification of Flanking Genomic Regions 
         [0060]    MeDIP/iPCR was performed on the positively responding samples (all high and medium high methylation samples, except 99-10, 99-33, 99-34 and 00-17, and samples 00-18 (spleen), 99-3 (liver), 99-47 (liver), 00-19 (bone marrow), 99-56 (spleen), 99-7 (liver) and 99-58 (spleen) from the middle methylation samples and samples 00-5 (spleen) and 99-45 (bone marrow from the low methylation samples). This resulted in 1 to 7 bands per tumor sample ( FIG. 3 , results of medium and low methylation samples not shown). Bands were isolated and subjected to nucleotide sequencing to identify flanking sequences. Genes located within a distance of 500 Kb were identified (Table 3). These gene products include known suppressor genes such as Smad1 and Mad1-like, as well as a number of genes with as yet poorly characterized roles in cancer. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Genes located within a distance of 500 Kb of a methylated VIS 
               
             
          
           
               
                 tumor 
                   
                   
                   
                 gene 
                 protein 
               
               
                 sample 
                 gene 
                 distance 
                 human homologue 
                 annotation 
                 function 
               
               
                   
               
               
                 99-16 
                 A kinase anchor 
                  44 kb 3′ 
                 protein A kinase 
                 NM_018747 
                 regulates PKA 
               
               
                 band 1 
                 protein 7 
                   
                 anchor protein 7 
                   
                 distribution, 
               
               
                   
                   
                   
                 isoform gamma 
                   
                 probably to 
               
               
                   
                   
                   
                   
                   
                 cytoplasm 
               
               
                   
                 arginase 1, liver 
                 200 kb 5′ 
                 arginase-1 
                 NM_007482 
                 liverenzyme, 
               
               
                   
                   
                   
                   
                   
                 ureumcyclus 
               
               
                   
                 cofactor required for 
                 210 kb 3′ 
                 idem 
                 NM_027347 
                 co-activator of 
               
               
                   
                 Sp1 trancriptional 
                   
                   
                   
                 transcription by 
               
               
                   
                 activation subunit 3 
                   
                   
                   
                 Sp1 
               
               
                   
                 erytrocyte protein 4.1- 
                 280 kb 3′ 
                 band 4.1 like protien 2 
                   
                 x 
               
               
                   
                 like 
               
               
                   
                 ectonucleotide 
                 300 kb 5′ 
                 idem 
                 NM_134005 
                 hydrolysis of 
               
               
                   
                 pyrophosphatase/phosphodiesterase 3 
                   
                   
                   
                 extracellular 
               
               
                   
                   
                   
                   
                   
                 nucleotides 
               
               
                   
                 ectonucleotide 
                 400 kb 5′ 
                 idem 
                 NM_008813 
                 hydrolysis of 
               
               
                   
                 pyrophosphatase/phosphodiesterase 1 
                   
                   
                   
                 extracellular 
               
               
                   
                   
                   
                   
                   
                 nucleotides 
               
               
                 99-19 
                 cyclin D3 
                 intron 1 
                 G1/S-specific cyclin 
                 NM_007632 
                 G1 to S-phase 
               
               
                 band 1 
                   
                   
                 D3 
                   
                 transmission, 
               
               
                   
                   
                   
                   
                   
                 phosphorylation 
               
               
                   
                   
                   
                   
                   
                 of rb, 
               
               
                   
                 taube nuss 
                  3.3 kb 5′ 
                 idem 
                 NM_022015 
                 required for 
               
               
                   
                   
                   
                   
                   
                 basal and 
               
               
                   
                   
                   
                   
                   
                 activator- 
               
               
                   
                   
                   
                   
                   
                 dependent 
               
               
                   
                   
                   
                   
                   
                 transcription, 
               
               
                   
                   
                   
                   
                   
                 TATA-binding 
               
               
                   
                   
                   
                   
                   
                 protein initiation 
               
               
                   
                   
                   
                   
                   
                 factor 
               
               
                   
                 unknown seq 
                  36 kb 5′ 
                 x 
                 x 
                 x 
               
               
                   
                 Riken cDNa 
                  68 kb 3′ 
                 x 
                 x 
                 x 
               
               
                   
                 1700001C19 
               
               
                   
                 bystin 
                  92 kb 5′ 
                 idem 
                   
                 bystin is found in 
               
               
                   
                   
                   
                   
                   
                 the placenta 
               
               
                   
                   
                   
                   
                   
                 from the sixth-tenth 
               
               
                   
                   
                   
                   
                   
                 week of 
               
               
                   
                   
                   
                   
                   
                 pregnancy 
               
               
                   
                 guanylate cyclase 
                 105 kb 5′ 
                 Guanylyl cyclase 
                 NM_008189 
                 retinal 
               
               
                   
                 activator 1a (retina) 
                   
                 activating protein 1 
               
               
                   
                   
                   
                 (GCAP 1) 
               
               
                   
                 Trf (TATA binding 
                 110 kb 5′ 
                 Ubiquitin specific 
                 NM_020048 
                 de-ubiquitination 
               
               
                   
                 protein-related factor)- 
                   
                 protease homolog 49 
               
               
                   
                 proximal protein 
               
               
                   
                 homolog ( Drosophila ) 
               
               
                   
                 ubiquitin specific 
                 125 kb 5′ 
                 Ubiquitin carboxyl- 
                 NM_198421 
                 de-ubiquitination 
               
               
                   
                 peptidase 49 
                   
                 terminal hydrolase 49 
               
               
                   
                 guanylate cyclase 
                 110 kb 3′ 
                 Guanylyl cyclase 
                 NM_146079 
                 retinal 
               
               
                   
                 activator 1B 
                   
                 activating protein 2 
               
               
                   
                   
                   
                 (GCAP 2) 
               
               
                   
                 mitochondrial 
                 125 kb 3′ 
                 Mitochondrial 28S 
                 NM_183086 
                 x 
               
               
                   
                 ribosomal protein S10 
                   
                 ribosomal protein S10 
               
               
                   
                 transcriptional 
                 150 kb 3′ 
                 idem 
                 NM_172622 
                 basal cell cycle 
               
               
                   
                 regulating factor 1 
                   
                   
                   
                 regulatory 
               
               
                   
                   
                   
                   
                   
                 protein 
               
               
                   
                   
                   
                   
                   
                 interacting with 
               
               
                   
                   
                   
                   
                   
                 Sp1 to activate 
               
               
                   
                   
                   
                   
                   
                 the p21 and p27 
               
               
                   
                   
                   
                   
                   
                 gene promoters 
               
               
                   
                 fibroblast growth factor 
                 190 kb 5′ 
                 idem 
                 NM_144939 
                 FRS3 negatively 
               
               
                   
                 receptor substrate 3 
                   
                   
                   
                 regulates ERK2 
               
               
                   
                   
                   
                   
                   
                 signaling 
               
               
                   
                   
                   
                   
                   
                 activated via 
               
               
                   
                   
                   
                   
                   
                 EGF stimulation 
               
               
                   
                   
                   
                   
                   
                 through direct 
               
               
                   
                   
                   
                   
                   
                 binding to ERK2 
               
               
                   
                 progastricsin 
                 225 kb 5′ 
                 Gastricsin precursor 
                 NM_025973 
               
               
                   
                 (pepsinogen C) 
               
               
                   
                 transcription factor EB 
                 280 kb 5′ 
                 idem 
                 NM_011549 
                 TFE3 and TFEB 
               
               
                   
                   
                   
                   
                   
                 regulate E- 
               
               
                   
                   
                   
                   
                   
                 cadherin and 
               
               
                   
                   
                   
                   
                   
                 WT1 expression 
               
               
                   
                 forkhead box P4 
                 375 kb 3′ 
                 forkhead box protein 
                 NM_028767 
                 members of the 
               
               
                   
                   
                   
                 P4 
                   
                 forkhead box 
               
               
                   
                   
                   
                   
                   
                 gene family, 
               
               
                   
                   
                   
                   
                   
                 including 
               
               
                   
                   
                   
                   
                   
                 members of 
               
               
                   
                   
                   
                   
                   
                 subfamily P, 
               
               
                   
                   
                   
                   
                   
                 have roles in 
               
               
                   
                   
                   
                   
                   
                 mammalian 
               
               
                   
                   
                   
                   
                   
                 oncogenesis 
               
               
                 99-36 
                 DNA primase, p58 
                 intron 7 
                 DNA primase large 
                 NM_008922 
                 synthesizes 
               
               
                 band 2 
                 subunit 
                   
                 subunit 
                   
                 small RNA 
               
               
                   
                   
                   
                   
                   
                 primers for the 
               
               
                   
                   
                   
                   
                   
                 Okazaki 
               
               
                   
                   
                   
                   
                   
                 fragments made 
               
               
                   
                   
                   
                   
                   
                 during 
               
               
                   
                   
                   
                   
                   
                 discontinuous 
               
               
                   
                   
                   
                   
                   
                 DNA replication 
               
               
                   
                 RIKEN 1700001G17 
                  95 kb 5′ 
                 x 
                 x 
                 x 
               
               
                   
                 gene 
               
               
                   
                 Rab23, member of 
                 150 kb 5′ 
                 RAS related protien 
                 NM_008999 
                 GTPase 
               
               
                   
                 RAS proto-oncogene 
                   
                 Rab23 
                   
                 mediated signal 
               
               
                   
                 family 
                   
                   
                   
                 transduction and 
               
               
                   
                   
                   
                   
                   
                 intracellular 
               
               
                   
                   
                   
                   
                   
                 protein 
               
               
                   
                   
                   
                   
                   
                 transportation 
               
               
                   
                 Bcl2-associated 
                 175 kb 3′ 
                 BAG-family molecular 
                 NM_145392 
                 The BAG 
               
               
                   
                 athanogene 2 
                   
                 chaperone regulator 2 
                   
                 domains of 
               
               
                   
                   
                   
                   
                   
                 BAG1, BAG2, 
               
               
                   
                   
                   
                   
                   
                 and BAG3 
               
               
                   
                   
                   
                   
                   
                 interact 
               
               
                   
                   
                   
                   
                   
                 specifically with 
               
               
                   
                   
                   
                   
                   
                 the Hsc70 
               
               
                   
                   
                   
                   
                   
                 ATPase domain 
               
               
                   
                   
                   
                   
                   
                 in vitro and in 
               
               
                   
                   
                   
                   
                   
                 mammalian 
               
               
                   
                   
                   
                   
                   
                 cells. All 3 
               
               
                   
                   
                   
                   
                   
                 proteins bind 
               
               
                   
                   
                   
                   
                   
                 with high affinity 
               
               
                   
                   
                   
                   
                   
                 to the ATPase 
               
               
                   
                   
                   
                   
                   
                 domain of Hsc70 
               
               
                   
                   
                   
                   
                   
                 and inhibit its 
               
               
                   
                   
                   
                   
                   
                 chaperone 
               
               
                   
                   
                   
                   
                   
                 activity in a Hip- 
               
               
                   
                   
                   
                   
                   
                 repressible 
               
               
                   
                   
                   
                   
                   
                 manner 
               
               
                   
                 zinc finger protein 451 
                 190 kb 3′ 
                 zinc finger protein 
                 NM_133817 
               
               
                   
                   
                   
                 451 
               
               
                   
                 dystonin 
                 340 kb 5′ 
                 Bullous pemphigoid 
                 NM_010081, 
               
               
                   
                   
                   
                 antigen 1 isoforms 
                 NM_133833, 
               
               
                   
                   
                   
                 1/2/3/4/5/8 
                 NM_134448 
               
               
                 99-36 
                 lunatic fringe gene 
                 intron 1 
                 Beta-1,3-N- 
                 NM_008494 
                 embryonic 
               
               
                 band 4 
                 homolog 
                   
                 acetylglucosaminyltransferase 
                   
                 development 
               
               
                   
                   
                   
                 lunatic 
               
               
                   
                   
                   
                 fringe 
               
               
                   
                 12 days embryo 
                  5.5 kb 5′ 
                 x 
                 x 
                 x 
               
               
                   
                 eyeball cDNA, RIKEN 
               
               
                   
                 full-length enriched 
               
               
                   
                 library, 
               
               
                   
                 clone: D230015O06 
               
               
                   
                 tweety homologue 3 
                 10.5 kb 3′  
                 tweety 3 
                 NM_175274 
                 chloride channel 
               
               
                   
                   
                   
                   
                   
                 activity 
               
               
                   
                 galectin-related inter- 
                  45 kb 5′ 
                 PREDICTED: similar 
                 XM_132470 
               
               
                   
                 fiber protein 
                   
                 to galectin-related 
                 XP_132470 
               
               
                   
                   
                   
                 inter-fiber protein 
               
               
                   
                 carbohydrate 
                  85 kb 3′ 
                 idem 
                 NM_021528 
                 carbohydrate 
               
               
                   
                 sulfotransferase 12 
                   
                   
                   
                 metabolism 
               
               
                   
                 IQ motif containing E 
                  55 kb 3′ 
                 idem 
                 NM_028833 
               
               
                   
                 guanine nucleotide 
                 150 kb 3′ 
                 Guanine nucleotide- 
                 NM_010302 
                 Gα(12) 
               
               
                   
                 binding protein, alpha 
                   
                 binding protein, 
                   
                 stimulates cell 
               
               
                   
                 12 
                   
                 alpha-12 subunit 
                   
                 proliferation and 
               
               
                   
                   
                   
                   
                   
                 neoplastic 
               
               
                   
                   
                   
                   
                   
                 transformation of 
               
               
                   
                   
                   
                   
                   
                 NIH 3T3 cells by 
               
               
                   
                   
                   
                   
                   
                 attenuating 
               
               
                   
                   
                   
                   
                   
                 p38MAPK- 
               
               
                   
                   
                   
                   
                   
                 associated 
               
               
                   
                   
                   
                   
                   
                 apoptotic 
               
               
                   
                   
                   
                   
                   
                 responses, while 
               
               
                   
                   
                   
                   
                   
                 activating the 
               
               
                   
                   
                   
                   
                   
                 mitogenic 
               
               
                   
                   
                   
                   
                   
                 responses 
               
               
                   
                   
                   
                   
                   
                 through the 
               
               
                   
                   
                   
                   
                   
                 stimulation of 
               
               
                   
                   
                   
                   
                   
                 ERK- and JNK- 
               
               
                   
                   
                   
                   
                   
                 mediated 
               
               
                   
                   
                   
                   
                   
                 signaling 
               
               
                   
                   
                   
                   
                   
                 pathways, 
               
               
                   
                   
                   
                   
                   
                 results from 
               
               
                   
                   
                   
                   
                   
                 differential 
               
               
                   
                   
                   
                   
                   
                 proteome 
               
               
                   
                   
                   
                   
                   
                 analysis report a 
               
               
                   
                   
                   
                   
                   
                 role for SET in 
               
               
                   
                   
                   
                   
                   
                 Gα(12)-mediated 
               
               
                   
                   
                   
                   
                   
                 signaling 
               
               
                   
                   
                   
                   
                   
                 pathways and a 
               
               
                   
                   
                   
                   
                   
                 role for Gα(12) in 
               
               
                   
                   
                   
                   
                   
                 the regulation of 
               
               
                   
                   
                   
                   
                   
                 the leukemia- 
               
               
                   
                   
                   
                   
                   
                 associated SET- 
               
               
                   
                   
                   
                   
                   
                 protein 
               
               
                   
                   
                   
                   
                   
                 expression 
               
               
                   
                 caspase recruitment 
                 260 kb 3′ 
                 Caspase recruitment 
                 NM_175362 
                 genetic 
               
               
                   
                 domain family, member 
                   
                 domain protein 11 
                   
                 inactivation of 
               
               
                   
                 11 
                   
                   
                   
                 the MAGUK 
               
               
                   
                   
                   
                   
                   
                 family protein 
               
               
                   
                   
                   
                   
                   
                 CARD11/Carma 
               
               
                   
                   
                   
                   
                   
                 1/Bimp3 results 
               
               
                   
                   
                   
                   
                   
                 in a complete 
               
               
                   
                   
                   
                   
                   
                 block in T and B 
               
               
                   
                   
                   
                   
                   
                 cell immunity. 
               
               
                   
                   
                   
                   
                   
                 CARD11 is 
               
               
                   
                   
                   
                   
                   
                 essential for 
               
               
                   
                   
                   
                   
                   
                 antigen receptor- 
               
               
                   
                   
                   
                   
                   
                 and PKC- 
               
               
                   
                   
                   
                   
                   
                 mediated 
               
               
                   
                   
                   
                   
                   
                 proliferation and 
               
               
                   
                   
                   
                   
                   
                 cytokine 
               
               
                   
                   
                   
                   
                   
                 production in T 
               
               
                   
                   
                   
                   
                   
                 and B cells due 
               
               
                   
                   
                   
                   
                   
                 to a selective 
               
               
                   
                   
                   
                   
                   
                 defect in JNK 
               
               
                   
                   
                   
                   
                   
                 and NFκB 
               
               
                   
                   
                   
                   
                   
                 activation 
               
               
                   
                 eukaryotic translation 
                 170 kb 3′ 
                 Eukaryotic translation 
                 NM_133916 
                 results indicate 
               
               
                   
                 initiation factor 3, 
                   
                 initiation factor 3 
                   
                 that p116 plays 
               
               
                   
                 subunit 9 (eta) 
                   
                 subunit 9 (elF-3 eta) 
                   
                 an essential role 
               
               
                   
                   
                   
                   
                   
                 in the early 
               
               
                   
                   
                   
                   
                   
                 stages of mouse 
               
               
                   
                   
                   
                   
                   
                 development 
               
               
                   
                 sorting nexin 8 
                 220 kb 5′ 
                 idem 
                 NM_172277 
               
               
                   
                 FtsJ homolog 2 
                 280 kb 5′ 
                 Putative ribosomal 
                 NM_013393, 
                 FTSJ2 is a 
               
               
                   
                   
                   
                 RNA 
                 NM_177442 
                 nucleolar RNA 
               
               
                   
                   
                   
                 methyltransferase 2 
                   
                 methyltransferase 
               
               
                   
                   
                   
                   
                   
                 involved in 
               
               
                   
                   
                   
                   
                   
                 eukaryotic RNA 
               
               
                   
                   
                   
                   
                   
                 processing and 
               
               
                   
                   
                   
                   
                   
                 modification 
               
               
                   
                 nudix (nucleoside 
                 275 kb 3′ 
                 7,8-dihydro-8- 
                 NM_008637 
                 MTH1 protects 
               
               
                   
                 diphosphate linked 
                   
                 oxoguanine 
                   
                 cells from H2O2- 
               
               
                   
                 moiety X)-type motif 1 
                   
                 triphosphatase 
                   
                 induced cell 
               
               
                   
                   
                   
                   
                   
                 dysfunction and 
               
               
                   
                   
                   
                   
                   
                 death by 
               
               
                   
                   
                   
                   
                   
                 hydrolyzing 
               
               
                   
                   
                   
                   
                   
                 oxidized purine 
               
               
                   
                   
                   
                   
                   
                 nucleotides 
               
               
                   
                   
                   
                   
                   
                 including 8-oxo- 
               
               
                   
                   
                   
                   
                   
                 dGTP and 2-OH- 
               
               
                   
                   
                   
                   
                   
                 dATP 
               
               
                   
                 mitotic arrest deficient 
                 290 kb 5′ 
                 Mitotic spindle 
                 NM_010752 
                 1. MAD1 and 
               
               
                   
                 1-like 1 (Mad1-like) 
                   
                 assembly checkpoint 
                   
                 Proto-Oncogene 
               
               
                   
                   
                   
                 protein MAD1 
                   
                 Proteins c-myc 
               
               
                   
                   
                   
                   
                   
                 reciprocally 
               
               
                   
                   
                   
                   
                   
                 regulate 
               
               
                   
                   
                   
                   
                   
                 ribosomal DNA 
               
               
                   
                   
                   
                   
                   
                 transcription, 
               
               
                   
                   
                   
                   
                   
                 providing a 
               
               
                   
                   
                   
                   
                   
                 mechanism for 
               
               
                   
                   
                   
                   
                   
                 coordination of 
               
               
                   
                   
                   
                   
                   
                 ribosome 
               
               
                   
                   
                   
                   
                   
                 biogenesis and 
               
               
                   
                   
                   
                   
                   
                 cell growth 2. 
               
               
                   
                   
                   
                   
                   
                 Together these 
               
               
                   
                   
                   
                   
                   
                 data 
               
               
                   
                   
                   
                   
                   
                 demonstrate that 
               
               
                   
                   
                   
                   
                   
                 the MYC- 
               
               
                   
                   
                   
                   
                   
                 antagonist 
               
               
                   
                   
                   
                   
                   
                 MAD1 and 
               
               
                   
                   
                   
                   
                   
                 cyclin-dependent 
               
               
                   
                   
                   
                   
                   
                 kinase inhibitor 
               
               
                   
                   
                   
                   
                   
                 p27(Kip1) 
               
               
                   
                   
                   
                   
                   
                 cooperate to 
               
               
                   
                   
                   
                   
                   
                 regulate the self- 
               
               
                   
                   
                   
                   
                   
                 renewal and 
               
               
                   
                   
                   
                   
                   
                 differentiation of 
               
               
                   
                   
                   
                   
                   
                 HSCs in a 
               
               
                   
                   
                   
                   
                   
                 context- 
               
               
                   
                   
                   
                   
                   
                 dependent 
               
               
                   
                   
                   
                   
                   
                 manner. 3. Data 
               
               
                   
                   
                   
                   
                   
                 show that the 
               
               
                   
                   
                   
                   
                   
                 loss of Trrap 
               
               
                   
                   
                   
                   
                   
                 leads to 
               
               
                   
                   
                   
                   
                   
                 chromosome 
               
               
                   
                   
                   
                   
                   
                 missegregation, 
               
               
                   
                   
                   
                   
                   
                 mitotic exit 
               
               
                   
                   
                   
                   
                   
                 failure and 
               
               
                   
                   
                   
                   
                   
                 compromised 
               
               
                   
                   
                   
                   
                   
                 mitotic 
               
               
                   
                   
                   
                   
                   
                 checkpoints, 
               
               
                   
                   
                   
                   
                   
                 which are 
               
               
                   
                   
                   
                   
                   
                 caused by 
               
               
                   
                   
                   
                   
                   
                 defective Trrap- 
               
               
                   
                   
                   
                   
                   
                 mediated 
               
               
                   
                   
                   
                   
                   
                 transcription of 
               
               
                   
                   
                   
                   
                   
                 the mitotic 
               
               
                   
                   
                   
                   
                   
                 checkpoint 
               
               
                   
                   
                   
                   
                   
                 proteins Mad1 
               
               
                   
                   
                   
                   
                   
                 and Mad2. 
               
               
                 99-44 
                 Stearoyl-CoenzymeA 
                  95 kb 3′ 
                 Acyl-Coa desaturase 
                 NM_005063 
                 by globally 
               
               
                 band 1 
                 desaturase 1 
                   
                   
                   
                 regulating lipid 
               
               
                   
                   
                   
                   
                   
                 metabolism, 
               
               
                   
                   
                   
                   
                   
                 stearoyl-CoA 
               
               
                   
                   
                   
                   
                   
                 desaturase 
               
               
                   
                   
                   
                   
                   
                 activity 
               
               
                   
                   
                   
                   
                   
                 modulates cell 
               
               
                   
                   
                   
                   
                   
                 proliferation and 
               
               
                   
                   
                   
                   
                   
                 survival and 
               
               
                   
                   
                   
                   
                   
                 shows the role of 
               
               
                   
                   
                   
                   
                   
                 endogenously 
               
               
                   
                   
                   
                   
                   
                 synthesized 
               
               
                   
                   
                   
                   
                   
                 monounsaturated 
               
               
                   
                   
                   
                   
                   
                 fatty acids in 
               
               
                   
                   
                   
                   
                   
                 sustaining the 
               
               
                   
                   
                   
                   
                   
                 neoplastic 
               
               
                   
                   
                   
                   
                   
                 phenotype of 
               
               
                   
                   
                   
                   
                   
                 transformed cells 
               
               
                   
                 Stearoyl-CoenzymeA 
                 intron 4 
                 Acyl-Coa desaturase 
               
               
                   
                 desaturase 2 
               
               
                   
                 Stearoyl-CoenzymeA 
                  60 kb 3′ 
                 Acyl-Coa desaturase 
               
               
                   
                 desaturase 3 
               
               
                   
                 Stearoyl-CoenzymeA 
                  33 kb 5′ 
                 Acyl-Coa desaturase 
               
               
                   
                 desaturase 4 
               
               
                   
                 cDNA sequence 
                 110 kb 5′ 
                 Polycystic kidney 
                 NM_016112 
                 x 
               
               
                   
                 BC046386 
                   
                 disease 2-like 1 
               
               
                   
                   
                   
                 protein 
               
               
                   
                 biogenesis of 
                 150 kb 5′ 
                 biogenesis of 
                 XM_193940 
               
               
                   
                 lysosome-related 
                   
                 lysosome-related 
               
               
                   
                 organelles complex-1, 
                   
                 organelles complex- 
               
               
                   
                 subunit 2 
                   
                 1, subunit 2 isoform 1 
               
               
                   
                 CWF19-like 1, cell 
                 160 kb 5′ 
                 idem 
                 XM_129328 
               
               
                   
                 cycle control ( S. pombe ) 
                   
                   
                 XP_129328 
               
               
                   
                 wingless related MMTV 
                 190 kb 5′ 
                 Wnt-8b protein 
                 NM_011720 
               
               
                   
                 integration site 8b 
                   
                 precursor 
               
               
                   
                 gene model 341 
                 220 kb 3′ 
                   S. cerevisiae  SEC31- 
                 XM_140784 
                 WD40 domain 
               
               
                   
                   
                   
                 like 2 isoform a 
                 XP_140784 
               
               
                   
                 NADH dehydrogenase 
                 250 kb 3′ 
                 NADH-ubiquinone 
                 NM_026061 
               
               
                   
                 (ubiquinone) 1 beta 
                   
                 oxidoreductase ASHI 
               
               
                   
                 subcomplex 8 
                   
                 subunit, mitochondrial 
               
               
                   
                   
                   
                 precursor 
               
               
                   
                 hypoxia-inducible 
                 255 kb 5′ 
                 Hypoxia-inducible 
                 NM_176958 
               
               
                   
                 factor 1, alpha subunit 
                   
                 factor 1 alpha 
               
               
                   
                 inhibitor 
                   
                 inhibitor 
               
               
                   
                 paired box gene 2 
                 450 kb 5′ 
                 Paired box protein 
                 NM_011037 
               
               
                   
                   
                   
                 Pax-2 
               
               
                   
                 conserved helix-loop- 
                 190 kb 5′ 
                 Inhibitor of nuclear 
                 NM_007700 
                 New nuclear role 
               
               
                   
                 helix ubiquitous kinase 
                   
                 factor kappa-B kinase 
                   
                 of IKK-alpha in 
               
               
                   
                   
                   
                 alpha subunit 
                   
                 modifying 
               
               
                   
                   
                   
                   
                   
                 histone function 
               
               
                   
                   
                   
                   
                   
                 that is critical for 
               
               
                   
                   
                   
                   
                   
                 the activation of 
               
               
                   
                   
                   
                   
                   
                 NF-kappaB- 
               
               
                   
                   
                   
                   
                   
                 directed gene 
               
               
                   
                   
                   
                   
                   
                 expression 
               
               
                   
                 SPFH domain family, 
                 215 kb 5 
                 SPFH domain protein 
                 NM_145502 
               
               
                   
                 member 1 
                   
                 1 precursor 
               
               
                   
                 cytochrome P450, 
                 260 kb 5′ 
                 x 
                 NM_001001446 
               
               
                   
                 family 2, subfamily c, 
               
               
                   
                 polypeptide 44 
               
               
                   
                 carboxypeptidase N, 
                 310 kb 5′ 
                 Carboxypeptidase N 
                 NM_030703 
                 carboxypeptidase 
               
               
                   
                 polypeptide 1 
                   
                 catalytic chain 
                   
                 N regulates the 
               
               
                   
                   
                   
                 precursor 
                   
                 biologic activity 
               
               
                   
                   
                   
                   
                   
                 of SDF-1alpha 
               
               
                   
                   
                   
                   
                   
                 by reducing the 
               
               
                   
                   
                   
                   
                   
                 chemokine- 
               
               
                   
                   
                   
                   
                   
                 specific activity 
               
               
                   
                 dynamin binding 
                 390 kb 5′ 
                 idem 
                 NM_028029 
                 functions to bring 
               
               
                   
                 protein 
                   
                   
                   
                 together 
               
               
                   
                   
                   
                   
                   
                 dynamin with 
               
               
                   
                   
                   
                   
                   
                 actin regulatory 
               
               
                   
                   
                   
                   
                   
                 proteins 
               
               
                   
                 ATP-binding cassette, 
                 460 kb 3′ 
                 Canalicular 
                 NM_013806 
                 This protein is a 
               
               
                   
                 sub-family C 
                   
                 multispecific organic 
                   
                 member of the 
               
               
                   
                 (CFTR/MRP), member 2 
                   
                 anion transporter 1 
                   
                 MRP subfamily 
               
               
                   
                   
                   
                   
                   
                 which is involved 
               
               
                   
                   
                   
                   
                   
                 in multi-drug 
               
               
                   
                   
                   
                   
                   
                 resistance, 
               
               
                   
                   
                   
                   
                   
                 multispecific 
               
               
                   
                   
                   
                   
                   
                 organic anion 
               
               
                   
                   
                   
                   
                   
                 transporter 
               
               
                 99-48 
                 SMAD1 
                 exon 2 
                 idem 
                 NM_008539 
                 Smad1 has a 
               
               
                 band 1 
                   
                   
                   
                   
                 role in regulating 
               
               
                   
                   
                   
                   
                   
                 p38 MAPK, 
               
               
                   
                   
                   
                   
                   
                 Smad1, beta- 
               
               
                   
                   
                   
                   
                   
                 catenin and Tcf4 
               
               
                   
                   
                   
                   
                   
                 have roles in 
               
               
                   
                   
                   
                   
                   
                 controlling Myc 
               
               
                   
                   
                   
                   
                   
                 transcription, 
               
               
                   
                   
                   
                   
                   
                 Smad1 is an 
               
               
                   
                   
                   
                   
                   
                 effector of 
               
               
                   
                   
                   
                   
                   
                 signals provided 
               
               
                   
                   
                   
                   
                   
                 by the bone 
               
               
                   
                   
                   
                   
                   
                 morphogenetic 
               
               
                   
                   
                   
                   
                   
                 protein (BMP) 
               
               
                   
                   
                   
                   
                   
                 sub-group of 
               
               
                   
                   
                   
                   
                   
                 TGFbeta 
               
               
                   
                   
                   
                   
                   
                 molecules 
               
               
                   
                 methylmalonic aciduria 
                  75 kb 5′ 
                 Methylmalonic 
                 NM_133823 
               
               
                   
                 (cobalamin deficiency) 
                   
                 aciduria type A 
               
               
                   
                 type A 
                   
                 protein, mitochondrial 
               
               
                   
                   
                   
                 precursor 
               
               
                   
                 PREDICTED: 
                 125 kb 5′ 
                 x 
                 x 
                 x 
               
               
                   
                 hypothetical protein 
               
               
                   
                 LOC67687 
               
               
                   
                 OTU domain 
                 300 kb 5′ 
                 Putative HIV-1- 
                 XM_194424 
                 HIV-1 induced 
               
               
                   
                 containing 4 
                   
                 induced protein HIN-1 
                 XP_194424 
                 protein HIN-1 
               
               
                   
                 ATP-binding cassette, 
                 300 kb 3′ 
                 ATP-binding cassette 
                 NM_015751 
                 Alternatively 
               
               
                   
                 sub-family E (OABP), 
                   
                 sub-family E member 1 
                   
                 referred to as the 
               
               
                   
                 member 1 
                   
                   
                   
                 RNase L 
               
               
                   
                   
                   
                   
                   
                 inhibitor, this 
               
               
                   
                   
                   
                   
                   
                 protein functions 
               
               
                   
                   
                   
                   
                   
                 to block the 
               
               
                   
                   
                   
                   
                   
                 activity of 
               
               
                   
                   
                   
                   
                   
                 ribonuclease L. 
               
               
                   
                   
                   
                   
                   
                 Activation of 
               
               
                   
                   
                   
                   
                   
                 ribonuclease L 
               
               
                   
                   
                   
                   
                   
                 leads to 
               
               
                   
                   
                   
                   
                   
                 inhibition of 
               
               
                   
                   
                   
                   
                   
                 protein synthesis 
               
               
                   
                   
                   
                   
                   
                 in the 2- 
               
               
                   
                   
                   
                   
                   
                 5A/RNase L 
               
               
                   
                   
                   
                   
                   
                 system, the 
               
               
                   
                   
                   
                   
                   
                 central pathway 
               
               
                   
                   
                   
                   
                   
                 for viral 
               
               
                   
                   
                   
                   
                   
                 interferon action 
               
               
                   
                 anaphase promoting 
                 340 kb 5′ 
                 idem 
                 NM_026904 
               
               
                   
                 complex subunit 10 
               
               
                 99-56 
                 G-protein coupled 
                 exon 3 
                 G-protein coupled 
                 NM_173398 
                 ?? 
               
               
                 band 3 
                 receptor 171 
                   
                 receptor H963 
               
               
                   
                 purinergic receptor 
                 intron 1 
                 P2Y purinoceptor 14 
                 NM_001008497, 
               
               
                   
                 P2Y, G-protein 
                   
                 (P2Y14) 
                 NM_133200 
               
               
                   
                 coupled, 14 
               
               
                   
                 mediator of RNA 
                 intron 11 
                 x 
                 XM_887994 
               
               
                   
                 polymerase II 
               
               
                   
                 transcription, subunit 
               
               
                   
                 12 homolog (yeast)-like 
               
               
                   
                 G protein-coupled 
                  63 kb 3′ 
                 Probable G protein- 
                 NM_032399 
               
               
                   
                 receptor 87 
                   
                 coupled receptor 87 
               
               
                   
                 Usher syndrome 3A 
                 225 kb 5′ 
                 Usher syndrome type 
                 NM_153384, 
                 retinal and inner 
               
               
                   
                 homolog (human) 
                   
                 3 protein 
                 NM_153385, 
                 ear 
               
               
                   
                   
                   
                   
                 NP_700434, 
                 malformations 
               
               
                   
                   
                   
                   
                 NP_700435 
               
               
                   
                 15 days embryo head 
                 200 kb 3′ 
                 immunoglobulin 
                   
                 x 
               
               
                   
                 cDNA, RIKEN full- 
                   
                 superfamily, member 
               
               
                   
                 length enriched library, 
                   
                 10 
               
               
                   
                 clone: 4022435C0 
               
               
                   
                 purinergic receptor 
                 190 kb 3′ 
                 P2Y purinoceptor 13 
                 NM_028808 
               
               
                   
                 P2Y, G-protein coupled 
               
               
                   
                 13 
               
               
                   
                 purinergic receptor 
                 195 kb 3′ 
                 P2Y purinoceptor 12 
                 NM_027571 
               
               
                   
                 P2Y, G-protein coupled 
               
               
                   
                 12 
               
               
                   
                 seven in absentia 2 
                 430 kb 5′ 
                 x 
                 NM_009174 
                 Siah proteins 
               
               
                   
                   
                   
                   
                   
                 function as E3 
               
               
                   
                   
                   
                   
                   
                 ubiquitin ligase 
               
               
                   
                   
                   
                   
                   
                 enzymes to 
               
               
                   
                   
                   
                   
                   
                 target the 
               
               
                   
                   
                   
                   
                   
                 degradation of 
               
               
                   
                   
                   
                   
                   
                 diverse protein 
               
               
                   
                   
                   
                   
                   
                 substrates, an 
               
               
                   
                   
                   
                   
                   
                 expansion of 
               
               
                   
                   
                   
                   
                   
                 myeloid 
               
               
                   
                   
                   
                   
                   
                 progenitor cells 
               
               
                   
                   
                   
                   
                   
                 in the bone 
               
               
                   
                   
                   
                   
                   
                 marrow of Siah2 
               
               
                   
                   
                   
                   
                   
                 mutant mice 
               
               
                 99-56 
                 WAS protein family, 
                 intron 1 
                 Wiskott-Aldrich 
                 NM_153423 
                 WAVE2 acts as 
               
               
                 band 4 
                 member 2 
                   
                 syndrome protein 
                   
                 the primary 
               
               
                   
                   
                   
                 family member 2 
                   
                 effector 
               
               
                   
                   
                   
                   
                   
                 downstream of 
               
               
                   
                   
                   
                   
                   
                 Rac to achieve 
               
               
                   
                   
                   
                   
                   
                 invasion and 
               
               
                   
                   
                   
                   
                   
                 metastasis, 
               
               
                   
                   
                   
                   
                   
                 suggesting that 
               
               
                   
                   
                   
                   
                   
                 suppression of 
               
               
                   
                   
                   
                   
                   
                 WAVE2 activity 
               
               
                   
                   
                   
                   
                   
                 holds a promise 
               
               
                   
                   
                   
                   
                   
                 for preventing 
               
               
                   
                   
                   
                   
                   
                 cancer invasion 
               
               
                   
                   
                   
                   
                   
                 and metastasis, 
               
               
                   
                   
                   
                   
                   
                 WAVEs (WASP- 
               
               
                   
                   
                   
                   
                   
                 family verprolin- 
               
               
                   
                   
                   
                   
                   
                 homologous 
               
               
                   
                   
                   
                   
                   
                 proteins) 
               
               
                   
                   
                   
                   
                   
                 regulate the 
               
               
                   
                   
                   
                   
                   
                 actin 
               
               
                   
                   
                   
                   
                   
                 cytoskeleton 
               
               
                   
                   
                   
                   
                   
                 through 
               
               
                   
                   
                   
                   
                   
                 activation of 
               
               
                   
                   
                   
                   
                   
                 Arp2/3 complex 
               
               
                   
                 D164 sialomucin-like 2 
                  50 kb 5′ 
                 CD164 sialomucin- 
                 XM_131719, 
                 ulti- 
               
               
                   
                   
                   
                 like 2 
                 XM_900155, 
                 glycosylated 
               
               
                   
                   
                   
                   
                 XM_900160 
                 core 
               
               
                   
                   
                   
                   
                   
                 protein 
               
               
                   
                   
                   
                   
                   
                 24 
               
               
                   
                   
                   
                   
                   
                 (MGC- 
               
               
                   
                   
                   
                   
                   
                 24) 
               
               
                   
                 mitogen-activated 
                  65 kb 5′ 
                 idem 
                 NM_016693 
                 The encoded 
               
               
                   
                 protein kinase kinase 
                   
                   
                   
                 kinase was 
               
               
                   
                 kinase 6 
                   
                   
                   
                 identified by its 
               
               
                   
                   
                   
                   
                   
                 interaction with 
               
               
                   
                   
                   
                   
                   
                 MAP3K5/ASK, a 
               
               
                   
                   
                   
                   
                   
                 protein kinase 
               
               
                   
                   
                   
                   
                   
                 and an activator 
               
               
                   
                   
                   
                   
                   
                 of c-Jun kinase 
               
               
                   
                   
                   
                   
                   
                 (MAPK7/JNK) 
               
               
                   
                   
                   
                   
                   
                 and 
               
               
                   
                   
                   
                   
                   
                 MAPK14/p38 
               
               
                   
                   
                   
                   
                   
                 kinase, 
               
               
                   
                   
                   
                   
                   
                 apoptosis signal- 
               
               
                   
                   
                   
                   
                   
                 regulating kinase 2 
               
               
                   
                 AT hook, DNA binding 
                  90 kb 3′ 
                 idem 
                 NM_146155 
               
               
                   
                 motif, containing 1 
               
               
                   
                 solute carrier family 9 
                 200 kb 5′ 
                 Sodium/hydrogen 
                 NM_016981 
                 mice lacking 
               
               
                   
                 (sodium/hydrogen 
                   
                 exchanger 1 
                   
                 NHE1 
               
               
                   
                 exchanger), member 1 
                   
                 (Na(+)/H(+) 
                   
                 upregulate their 
               
               
                   
                   
                   
                 exchanger 1) 
                   
                 Na(+) channel 
               
               
                   
                   
                   
                   
                   
                 expression in the 
               
               
                   
                   
                   
                   
                   
                 hippocampal and 
               
               
                   
                   
                   
                   
                   
                 cortical regions 
               
               
                   
                   
                   
                   
                   
                 selectively; this 
               
               
                   
                   
                   
                   
                   
                 leads to an 
               
               
                   
                   
                   
                   
                   
                 increase in Na(+) 
               
               
                   
                   
                   
                   
                   
                 current density 
               
               
                   
                   
                   
                   
                   
                 and membrane 
               
               
                   
                   
                   
                   
                   
                 excitability 
               
               
                   
                 Gardner-Rasheed 
                 175 kb 3′ 
                 Proto-oncogene 
                 NM_010208 
                 Hck and Fgr 
               
               
                   
                 feline sarcoma viral 
                   
                 tyrosine-protein 
                   
                 function as 
               
               
                   
                 (Fgr) oncogene 
                   
                 kinase FGR 
                   
                 negative 
               
               
                   
                 homolog 
                   
                   
                   
                 regulators of 
               
               
                   
                   
                   
                   
                   
                 myeloid cell 
               
               
                   
                   
                   
                   
                   
                 chemokine 
               
               
                   
                   
                   
                   
                   
                 signaling by 
               
               
                   
                   
                   
                   
                   
                 maintaining the 
               
               
                   
                   
                   
                   
                   
                 tonic 
               
               
                   
                   
                   
                   
                   
                 phosphorylation 
               
               
                   
                   
                   
                   
                   
                 of PIR- 
               
               
                   
                 G-protein coupled 
                  75 kb 3′ 
                 Probable G-protein 
                 NM_008154 
                 Gpr3-defective 
               
               
                   
                 receptor 3 
                   
                 coupled receptor 
                   
                 mice may 
               
               
                   
                   
                   
                 GPR3 
                   
                 constitute a 
               
               
                   
                   
                   
                   
                   
                 relevant model 
               
               
                   
                   
                   
                   
                   
                 of premature 
               
               
                   
                   
                   
                   
                   
                 ovarian failure 
               
               
                   
                   
                   
                   
                   
                 due to early 
               
               
                   
                   
                   
                   
                   
                 oocyte aging 
               
               
                   
                 synaptotagmin-like 1 
                 100 kb 3′ 
                 synaptotagmin-like 
                 NM_031393 
                 SHD of Slp1/Jfc1 
               
               
                   
                   
                   
                 protein 1 
                   
                 specifically and 
               
               
                   
                   
                   
                   
                   
                 directly binds the 
               
               
                   
                   
                   
                   
                   
                 GTP-bound form 
               
               
                   
                   
                   
                   
                   
                 of Rab27A 
               
               
                   
                 WD and 
                 150 kb 3′ 
                 WD and 
                 NM_199306 
                 WD40 domain 
               
               
                   
                 tetratricopeptide 
                   
                 tetratricopeptide 
               
               
                   
                 repeats 1 
                   
                 repeats protein 1 
               
               
                   
                 nuclear distribution 
                 350 kb 3′ 
                 Nuclear migration 
                 NM_010948 
               
               
                   
                 gene C homolog 
                   
                 protein nudC 
               
               
                   
                 ( Aspergillus ) 
               
               
                   
                 nuclear receptor 
                 380 kb 5′ 
                 Nuclear receptor 0B2 
                 NM_011850 
                 SHP acts as a 
               
               
                   
                 subfamily 0, group B, 
                   
                 (Orphan nuclear 
                   
                 transcriptional 
               
               
                   
                 member 2 
                   
                 receptor SHP) 
                   
                 coregulator by 
               
               
                   
                   
                   
                   
                   
                 inhibiting the 
               
               
                   
                   
                   
                   
                   
                 activity of 
               
               
                   
                   
                   
                   
                   
                 various nuclear 
               
               
                   
                   
                   
                   
                   
                 receptors 
               
               
                   
                   
                   
                   
                   
                 (downstream 
               
               
                   
                   
                   
                   
                   
                 targets) via 
               
               
                   
                   
                   
                   
                   
                 occupation of the 
               
               
                   
                   
                   
                   
                   
                 coactivator- 
               
               
                   
                   
                   
                   
                   
                 binding surface 
               
               
                   
                   
                   
                   
                   
                 and active 
               
               
                   
                   
                   
                   
                   
                 repression 
               
               
                   
                 G patch domain 
                 400 kb 5′ 
                 G patch domain 
                 NM_172876 
               
               
                   
                 containing 3 
                   
                 containing protein 3 
               
               
                   
                 ATP binding domain 1 
                 410 kb 5′ 
                 idem 
                   
                 of the MDR/TAP 
               
               
                   
                 family, member B 
                   
                   
                   
                 subfamily are 
               
               
                   
                   
                   
                   
                   
                 involved in 
               
               
                   
                   
                   
                   
                   
                 multidrug 
               
               
                   
                   
                   
                   
                   
                 resistance 
               
               
                   
                 stratifin 
                 430 kb 3′ 
                 14-3-3 protein sigma 
                 NM_018754 
                 Stratifin was first 
               
               
                   
                   
                   
                   
                   
                 identified as an 
               
               
                   
                   
                   
                   
                   
                 epithelial cell 
               
               
                   
                   
                   
                   
                   
                 antigen 
               
               
                   
                   
                   
                   
                   
                 exclusively 
               
               
                   
                   
                   
                   
                   
                 expressed in 
               
               
                   
                   
                   
                   
                   
                 epithelia. the 
               
               
                   
                   
                   
                   
                   
                 functional role of 
               
               
                   
                   
                   
                   
                   
                 sfn in cell 
               
               
                   
                   
                   
                   
                   
                 proliferation and 
               
               
                   
                   
                   
                   
                   
                 apoptosis could 
               
               
                   
                   
                   
                   
                   
                 be relevant to 
               
               
                   
                   
                   
                   
                   
                 the regulation of 
               
               
                   
                   
                   
                   
                   
                 growth and 
               
               
                   
                   
                   
                   
                   
                 differentiation as 
               
               
                   
                   
                   
                   
                   
                 a tumor 
               
               
                   
                   
                   
                   
                   
                 suppressor 
               
               
                   
                   
                   
                   
                   
                 gene, stratifin 
               
               
                   
                   
                   
                   
                   
                 itself is subject to 
               
               
                   
                   
                   
                   
                   
                 regulation by 
               
               
                   
                   
                   
                   
                   
                 p53 upon DNA 
               
               
                   
                   
                   
                   
                   
                 damage and by 
               
               
                   
                   
                   
                   
                   
                 epigenetic 
               
               
                   
                   
                   
                   
                   
                 deregulation and 
               
               
                   
                   
                   
                   
                   
                 Gene silencing 
               
               
                   
                   
                   
                   
                   
                 of 14-3-3sigma 
               
               
                   
                   
                   
                   
                   
                 by CpG 
               
               
                   
                   
                   
                   
                   
                 methylation has 
               
               
                   
                   
                   
                   
                   
                 been found in 
               
               
                   
                   
                   
                   
                   
                 many human 
               
               
                   
                   
                   
                   
                   
                 cancer types 
               
               
                   
                 zinc finger, DHHC 
                 430 kb 3′ 
                 x 
                 NM_001017968 
               
               
                   
                 domain containing 18 
               
               
                   
                 phosphatidylinositol 
                 480 kb 3′ 
                 phosphatidylinositol 
                 NM_178698 
               
               
                   
                 glycan, class V 
                   
                 glycan class V 
               
               
                   
                 syntaxin 12 
                 280 kb 5′ 
                 idem 
                 NM_133887 
               
               
                   
                 protein phosphatase 1, 
                 325 kb 5′ 
                 Nuclear inhibitor of 
                 NM_146154 
                 NIPP1 has a role 
               
               
                   
                 regulatory (inhibitor) 
                   
                 protein phosphatase 1 
                   
                 in the nuclear 
               
               
                   
                 subunit 8 
                   
                   
                   
                 targeting and/or 
               
               
                   
                   
                   
                   
                   
                 retention of PP1 
               
               
                   
                 replication protein A2 
                 400 kb 3′ 
                 Replication protein A 
                 NM_011284 
                 Phosphorylation 
               
               
                   
                   
                   
                 32 kDa subunit 
                   
                 of the RPA2 
               
               
                   
                   
                   
                   
                   
                 subunit is 
               
               
                   
                   
                   
                   
                   
                 observed after 
               
               
                   
                   
                   
                   
                   
                 exposure of cells 
               
               
                   
                   
                   
                   
                   
                 to ionizing 
               
               
                   
                   
                   
                   
                   
                 radiation (IR) 
               
               
                   
                   
                   
                   
                   
                 and other DNA- 
               
               
                   
                   
                   
                   
                   
                 damaging 
               
               
                   
                   
                   
                   
                   
                 agents, which 
               
               
                   
                   
                   
                   
                   
                 implicates the 
               
               
                   
                   
                   
                   
                   
                 modified protein 
               
               
                   
                   
                   
                   
                   
                 in the regulation 
               
               
                   
                   
                   
                   
                   
                 of DNA 
               
               
                   
                   
                   
                   
                   
                 replication after 
               
               
                   
                   
                   
                   
                   
                 DNA damage or 
               
               
                   
                   
                   
                   
                   
                 in DNA rep 
               
               
                   
                 sphingomyelin 
                 410 kb 5′ 
                 Acid 
                 NM_133888 
               
               
                   
                 phosphodiesterase, 
                   
                 sphingomyelinase- 
               
               
                   
                 acid-like 3B 
                   
                 like 
               
               
                   
                   
                   
                 phosphodiesterase 
               
               
                   
                   
                   
                 3b precursor 
               
               
                   
                 X Kell blood group 
                 440 kb 5′ 
                 X Kell blood group 
                 NM_201368 
               
               
                   
                 precursor related family 
                   
                 precursor-related 
               
               
                   
                 member 8 homolog 
                   
                 family, member 8 
               
               
                   
                 eyes absent 3 homolog 
                 450 kb 3′ 
                 idem 
                 NM_010166, 
                 Experiments 
               
               
                   
                 ( Drosophila ) 
                   
                   
                 NM_210071, 
                 performed in 
               
               
                   
                   
                   
                   
                 NM_211356, 
                 cultured 
               
               
                   
                   
                   
                   
                 NM_211357 
                   Drosophila  cells 
               
               
                   
                   
                   
                   
                   
                 and in vitro 
               
               
                   
                   
                   
                   
                   
                 indicate that 
               
               
                   
                   
                   
                   
                   
                 Eyes absent has 
               
               
                   
                   
                   
                   
                   
                 intrinsic protein 
               
               
                   
                   
                   
                   
                   
                 tyrosine 
               
               
                   
                   
                   
                   
                   
                 phosphatase 
               
               
                   
                   
                   
                   
                   
                 activity and can 
               
               
                   
                   
                   
                   
                   
                 autocatalytically 
               
               
                   
                   
                   
                   
                   
                 dephosphorylate 
               
               
                   
                   
                   
                   
                   
                 itself 
               
               
                 99-58 
                 cleavage stimulation 
                  20 kb 3′ 
                 Cleavage stimulation 
                 NM_024199 
                 is involved in the 
               
               
                 band 1 
                 factor, 3′ pre-RNA, 
                   
                 factor, 50 kDa subunit 
                   
                 polyadenylation 
               
               
                   
                 subunit 1 
                   
                   
                   
                 and 3′end 
               
               
                   
                   
                   
                   
                   
                 cleavage of pre- 
               
               
                   
                   
                   
                   
                   
                 mRNAs 
               
               
                   
                 RIKEN cDNA 
                  23 kb 5′ 
                 x 
                 x 
                 x 
               
               
                   
                 F730031O20 gene 
               
               
                   
                 aurora kinase A 
                  30 kb 3′ 
                 Serine/threonine- 
                 NM_011497 
                 serine/threonine 
               
               
                   
                   
                   
                 protein kinase 6 
                   
                 mitotic kinase, 
               
               
                   
                   
                   
                   
                   
                 BRCA1 
               
               
                   
                   
                   
                   
                   
                 phosphorylation 
               
               
                   
                   
                   
                   
                   
                 by Aurora-A 
               
               
                   
                   
                   
                   
                   
                 plays a role in 
               
               
                   
                   
                   
                   
                   
                 G(2) to M 
               
               
                   
                   
                   
                   
                   
                 transition of cell 
               
               
                   
                   
                   
                   
                   
                 cycle, human 
               
               
                   
                   
                   
                   
                   
                 cancer cells 
               
               
                   
                   
                   
                   
                   
                 frequently exhibit 
               
               
                   
                   
                   
                   
                   
                 overexpression 
               
               
                   
                   
                   
                   
                   
                 of Aurora A 
               
               
                   
                   
                   
                   
                   
                 protein 
               
               
                   
                   
                   
                   
                   
                 regardless of the 
               
               
                   
                   
                   
                   
                   
                 cell cycle stage 
               
               
                   
                 RIKEN cDNA 
                  40 kb 5′ 
                 x 
                 x 
                 x 
               
               
                   
                 2410001C21 gene 
               
               
                   
                 (2410001C21Rik), 
               
               
                   
                 mRNA 
               
               
                   
                 RIKEN cDNA 
                  45 kb 5′ 
                 x 
                 x 
                 x 
               
               
                   
                 2010011I20 gene 
               
               
                   
                 (2010011I20Rik), 
               
               
                   
                 mRNA 
               
               
                   
                 Adult male spinal cord 
                  50 kb 3′ 
                 OTTHUMP00000031 
                 x 
                 x 
               
               
                   
                 cDNA, RIKEN full- 
                   
                 350 (Fragment) 
               
               
                   
                 length enriched library, 
               
               
                   
                 clone: A330041C17 
               
               
                   
                 PREDICTED: 
                  70 kb 5′ 
                 x 
                 x 
                 x 
               
               
                   
                 hypothetical protein 
               
               
                   
                 LOC76426 
               
               
                   
                 melanocortin 3 
                 200 kb 3′ 
                 idem 
                 NM_008561 
               
               
                   
                 receptor 
               
               
                   
                 transcription factor AP- 
                 150 kb 5′ 
                 Transcription factor 
                 NM_009335 
                 AP-2 gamma 
               
               
                   
                 2, gamma 
                   
                 Erf-1 
                   
                 seems to be 
               
               
                   
                   
                   
                   
                   
                 required in early 
               
               
                   
                   
                   
                   
                   
                 embryonic 
               
               
                   
                   
                   
                   
                   
                 development, 
               
               
                   
                   
                   
                   
                   
                 suggest a role of 
               
               
                   
                   
                   
                   
                   
                 AP-2 
               
               
                   
                   
                   
                   
                   
                 transcription 
               
               
                   
                   
                   
                   
                   
                 factors in the 
               
               
                   
                   
                   
                   
                   
                 maintenance of 
               
               
                   
                   
                   
                   
                   
                 a proliferative 
               
               
                   
                   
                   
                   
                   
                 and 
               
               
                   
                   
                   
                   
                   
                 undifferentiated 
               
               
                   
                   
                   
                   
                   
                 state of cells, 
               
               
                   
                   
                   
                   
                   
                 characteristics 
               
               
                   
                   
                   
                   
                   
                 not only 
               
               
                   
                   
                   
                   
                   
                 important during 
               
               
                   
                   
                   
                   
                   
                 embryonic 
               
               
                   
                   
                   
                   
                   
                 development but 
               
               
                   
                   
                   
                   
                   
                 also in 
               
               
                   
                   
                   
                   
                   
                 tumorigenesis 
               
               
                   
                 cerebellin 4 precursor 
                 350 kb 5′ 
                 cerebellin 4 precursor 
                 NM_175631 
                 neuromodulatory 
               
               
                   
                 protein 
                   
                   
                   
                 function 
               
               
                   
                 bone morphogenetic 
                 400 kb 3′ 
                 bone morphogenetic 
                 NM_007557 
                 BMP-7/OP-1, a 
               
               
                   
                 protein 7 
                   
                 protein 7 precursor 
                   
                 member of the 
               
               
                   
                   
                   
                   
                   
                 transforming 
               
               
                   
                   
                   
                   
                   
                 growth factor- 
               
               
                   
                   
                   
                   
                   
                 beta (TGF-beta) 
               
               
                   
                   
                   
                   
                   
                 family of 
               
               
                   
                   
                   
                   
                   
                 secreted growth 
               
               
                   
                   
                   
                   
                   
                 factors, is 
               
               
                   
                   
                   
                   
                   
                 expressed 
               
               
                   
                   
                   
                   
                   
                 during mouse 
               
               
                   
                   
                   
                   
                   
                 embryogenesis 
               
               
                   
                   
                   
                   
                   
                 in a pattern 
               
               
                   
                   
                   
                   
                   
                 suggesting 
               
               
                   
                   
                   
                   
                   
                 potential roles in 
               
               
                   
                   
                   
                   
                   
                 a variety of 
               
               
                   
                   
                   
                   
                   
                 inductive tissue 
               
               
                   
                   
                   
                   
                   
                 interactions 
               
               
                 00-10 
                 myosin 1H 
                  40 kb 5′ 
                 idem 
                 NM_146163 
                 ?? 
               
               
                 band 5 
               
               
                   
                 forkhead box N4 
                  40 kb 5′ 
                 forkhead box protein 
                 NM_148935 
                 expressed 
               
               
                   
                   
                   
                 N4 
                   
                 during neural 
               
               
                   
                   
                   
                   
                   
                 development in 
               
               
                   
                   
                   
                   
                   
                 the retina, the 
               
               
                   
                   
                   
                   
                   
                 ventral hindbrain 
               
               
                   
                   
                   
                   
                   
                 and spinal cord 
               
               
                   
                   
                   
                   
                   
                 and dorsal 
               
               
                   
                   
                   
                   
                   
                 midbrain 
               
               
                   
                 potassium channel 
                  45 kb 3′ 
                 idem 
                 NM_026145 
               
               
                   
                 tetramerisation domain 
               
               
                   
                 containing 10 
               
               
                   
                 acetyl-Coenzyme A 
                  65 kb 3′ 
                 acetyl-Coenzyme A 
                 NM_133904 
                 Acc2−/− mutant 
               
               
                   
                 carboxylase beta 
                   
                 carboxylase 2 
                   
                 mice have a 
               
               
                   
                   
                   
                   
                   
                 normal life span, 
               
               
                   
                   
                   
                   
                   
                 a higher fatty 
               
               
                   
                   
                   
                   
                   
                 acid oxidation 
               
               
                   
                   
                   
                   
                   
                 rate, and lower 
               
               
                   
                   
                   
                   
                   
                 amounts of fat 
               
               
                   
                 ubiquitin protein ligase 
                  83 kb 5′ 
                 ubiquitin protein 
                 NM_054093 
                 This gene 
               
               
                   
                 E3B 
                   
                 ligase E3 isoform B 
                   
                 encodes a 
               
               
                   
                   
                   
                   
                   
                 member of the 
               
               
                   
                   
                   
                   
                   
                 E3 ubiquitin- 
               
               
                   
                   
                   
                   
                   
                 conjugating 
               
               
                   
                   
                   
                   
                   
                 enzyme family. 
               
               
                   
                   
                   
                   
                   
                 The encoded 
               
               
                   
                   
                   
                   
                   
                 protein may 
               
               
                   
                   
                   
                   
                   
                 interact with 
               
               
                   
                   
                   
                   
                   
                 other proteins 
               
               
                   
                   
                   
                   
                   
                 and play a role in 
               
               
                   
                   
                   
                   
                   
                 stress response. 
               
               
                   
                 mevalonate kinase 
                 125 kb 5′ 
                 idem 
                 NM_023556 
                 Mevalonic 
               
               
                   
                   
                   
                   
                   
                 aciduria, with 
               
               
                   
                   
                   
                   
                   
                 psychomotor 
               
               
                   
                   
                   
                   
                   
                 retardation, 
               
               
                   
                   
                   
                   
                   
                 cerebellar ataxia, 
               
               
                   
                   
                   
                   
                   
                 recurrent fever, 
               
               
                   
                   
                   
                   
                   
                 and death in 
               
               
                   
                   
                   
                   
                   
                 early childhood, 
               
               
                   
                   
                   
                   
                   
                 and hyper- 
               
               
                   
                   
                   
                   
                   
                 immunoglobulin 
               
               
                   
                   
                   
                   
                   
                 D syndrome, 
               
               
                   
                   
                   
                   
                   
                 with recurrent 
               
               
                   
                   
                   
                   
                   
                 fever attacks 
               
               
                   
                   
                   
                   
                   
                 without 
               
               
                   
                   
                   
                   
                   
                 neurologic 
               
               
                   
                   
                   
                   
                   
                 symptoms, are 
               
               
                   
                   
                   
                   
                   
                 caused by 
               
               
                   
                   
                   
                   
                   
                 mevalonate 
               
               
                   
                   
                   
                   
                   
                 kinase deficiency 
               
               
                   
                 methylmalonic aciduria 
                 120 kb 3′ 
                 Cob(I)yrinic acid a,c- 
                 NM_029956 
               
               
                   
                 (cobalamin deficiency) 
                   
                 diamide 
               
               
                   
                 type B homolog 
                   
                 adenosyltransferase, 
               
               
                   
                 (human) 
                   
                 mitochondrial 
               
               
                   
                   
                   
                 precursor 
               
               
                   
                 uracil DNA glycosylase 
                 175 kb 3′ 
                 idem 
                 NM_011677 
                 Immunoglobulin 
               
               
                   
                   
                   
                   
                   
                 isotype switching 
               
               
                   
                   
                   
                   
                   
                 is inhibited and 
               
               
                   
                   
                   
                   
                   
                 somatic 
               
               
                   
                   
                   
                   
                   
                 hypermutation 
               
               
                   
                   
                   
                   
                   
                 perturbed in 
               
               
                   
                   
                   
                   
                   
                 mice deficient in 
               
               
                   
                   
                   
                   
                   
                 this enzyme 
               
               
                   
                 ubiquitin specific 
                 200 kb 3′ 
                 Ubiquitin carboxyl- 
                 XM_149655 
               
               
                   
                 peptidase 30 
                   
                 terminal hydrolase 30 
               
               
                   
                 transient receptor 
                 300 kb 3′ 
                 idem 
                 NM_022017 
                 Trpv4 gene in 
               
               
                   
                 potential cation 
                   
                   
                   
                 mice markedly 
               
               
                   
                 channel, subfamily V, 
                   
                   
                   
                 reduced the 
               
               
                   
                 member 4 
                   
                   
                   
                 sensitivity of the 
               
               
                   
                   
                   
                   
                   
                 tail to pressure 
               
               
                   
                   
                   
                   
                   
                 and acidic 
               
               
                   
                   
                   
                   
                   
                 nociception 
               
               
                   
                 glycolipid transfer 
                 350 kb 3′ 
                 idem 
                 NM_019821 
               
               
                   
                 protein 
               
               
                   
                 G protein-coupled 
                 400 kb 3′ 
                 G protein-coupled 
                 NM_019834 
                 GIT proteins are 
               
               
                   
                 receptor kinase- 
                   
                 receptor kinase- 
                   
                 GTPase- 
               
               
                   
                 interactor 2 
                   
                 interactor 2 
                   
                 activating 
               
               
                   
                   
                   
                   
                   
                 proteins (GAPs) 
               
               
                   
                   
                   
                   
                   
                 for ADP- 
               
               
                   
                   
                   
                   
                   
                 ribosylation 
               
               
                   
                   
                   
                   
                   
                 factor (ARF) 
               
               
                   
                   
                   
                   
                   
                 small GTP- 
               
               
                   
                   
                   
                   
                   
                 binding proteins, 
               
               
                   
                   
                   
                   
                   
                 and interact with 
               
               
                   
                   
                   
                   
                   
                 the PIX family of 
               
               
                   
                   
                   
                   
                   
                 Rac1/Cdc42 
               
               
                   
                   
                   
                   
                   
                 guanine 
               
               
                   
                   
                   
                   
                   
                 nucleotide 
               
               
                   
                   
                   
                   
                   
                 exchange 
               
               
                   
                   
                   
                   
                   
                 factors. GIT and 
               
               
                   
                   
                   
                   
                   
                 PIX transiently 
               
               
                   
                   
                   
                   
                   
                 localize p21- 
               
               
                   
                   
                   
                   
                   
                 activated protein 
               
               
                   
                   
                   
                   
                   
                 kinases (PAKs) 
               
               
                   
                   
                   
                   
                   
                 to remodeling 
               
               
                   
                   
                   
                   
                   
                 focal adhesions 
               
               
                   
                   
                   
                   
                   
                 through binding 
               
               
                   
                   
                   
                   
                   
                 to paxillin 
               
               
                   
                 ankyrin repeat domain 
                 460 kb 5′ 
                 Ankyrin repeat 
                 NM_026718 
               
               
                   
                 13a 
                   
                 domain protein 13 
               
               
                   
                 D-amino acid oxidase 1 
                 300 kb 3′ 
                 idem 
                 NM_010018 
               
               
                   
                 slingshot homolog 1 
                 325 kb 5′ 
                 slingshot homolog 1 
                 NM_198109 
                 Expression of a 
               
               
                   
                 ( Drosophila ) 
                   
                   
                   
                 phosphatase- 
               
               
                   
                   
                   
                   
                   
                 inactive SSH1 
               
               
                   
                   
                   
                   
                   
                 induces aberrant 
               
               
                   
                   
                   
                   
                   
                 accumulation of 
               
               
                   
                   
                   
                   
                   
                 F-actin and 
               
               
                   
                   
                   
                   
                   
                 phospho-cofilin 
               
               
                   
                   
                   
                   
                   
                 near the 
               
               
                   
                   
                   
                   
                   
                 midbody in the 
               
               
                   
                   
                   
                   
                   
                 final stage of 
               
               
                   
                   
                   
                   
                   
                 cytokinesis and 
               
               
                   
                   
                   
                   
                   
                 frequently leads 
               
               
                   
                   
                   
                   
                   
                 to the regression 
               
               
                   
                   
                   
                   
                   
                 of the cleavage 
               
               
                   
                   
                   
                   
                   
                 furrow and the 
               
               
                   
                   
                   
                   
                   
                 formation of 
               
               
                   
                   
                   
                   
                   
                 multinucleate 
               
               
                   
                   
                   
                   
                   
                 cells 
               
               
                   
                 coronin, actin binding 
                 400 kb 5′ 
                 Coronin-1C 
                 NM_011779 
                 This gene 
               
               
                   
                 protein 1C 
                   
                   
                   
                 encodes a 
               
               
                   
                   
                   
                   
                   
                 member of the 
               
               
                   
                   
                   
                   
                   
                 WD repeat 
               
               
                   
                   
                   
                   
                   
                 protein family. 
               
               
                   
                   
                   
                   
                   
                 WD repeats are 
               
               
                   
                   
                   
                   
                   
                 minimally 
               
               
                   
                   
                   
                   
                   
                 conserved 
               
               
                   
                   
                   
                   
                   
                 regions of 
               
               
                   
                   
                   
                   
                   
                 approximately 40 
               
               
                   
                   
                   
                   
                   
                 amino acids 
               
               
                   
                   
                   
                   
                   
                 typically 
               
               
                   
                   
                   
                   
                   
                 bracketed by gly- 
               
               
                   
                   
                   
                   
                   
                 his and trp-asp 
               
               
                   
                   
                   
                   
                   
                 (GH-WD), which 
               
               
                   
                   
                   
                   
                   
                 may facilitate 
               
               
                   
                   
                   
                   
                   
                 formation of 
               
               
                   
                   
                   
                   
                   
                 heterotrimeric or 
               
               
                   
                   
                   
                   
                   
                 multiprotein 
               
               
                   
                   
                   
                   
                   
                 complexes. 
               
               
                   
                   
                   
                   
                   
                 Members of this 
               
               
                   
                   
                   
                   
                   
                 family are 
               
               
                   
                   
                   
                   
                   
                 involved in a 
               
               
                   
                   
                   
                   
                   
                 variety of cellular 
               
               
                   
                   
                   
                   
                   
                 processes, 
               
               
                   
                   
                   
                   
                   
                 including cell 
               
               
                   
                   
                   
                   
                   
                 cycle 
               
               
                   
                   
                   
                   
                   
                 progression, 
               
               
                   
                   
                   
                   
                   
                 signal 
               
               
                   
                   
                   
                   
                   
                 transduction, 
               
               
                   
                   
                   
                   
                   
                 apoptosis, and 
               
               
                   
                   
                   
                   
                   
                 gene regulation, 
               
               
                   
                   
                   
                   
                   
                 Coronin 3 is 
               
               
                   
                   
                   
                   
                   
                 abundantly 
               
               
                   
                   
                   
                   
                   
                 expressed in the 
               
               
                   
                   
                   
                   
                   
                 adult CNS. All 
               
               
                   
                   
                   
                   
                   
                 murine brain 
               
               
                   
                   
                   
                   
                   
                 areas express 
               
               
                   
                   
                   
                   
                   
                 coronin 3 during 
               
               
                   
                   
                   
                   
                   
                 embryogenesis 
               
               
                   
                   
                   
                   
                   
                 and the first 
               
               
                   
                   
                   
                   
                   
                 postnatal stages 
               
               
                   
                 selectin, platelet (p- 
                 480 kb 5′ 
                 P-selectin 
                 NM_009151 
                 The 
               
               
                   
                 selectin) ligand 
                   
                 glycoprotein ligand 1 
                   
                 homozygous 
               
               
                   
                   
                   
                 precursor 
                   
                 PSGL-1-deficient 
               
               
                   
                   
                   
                   
                   
                 mouse was 
               
               
                   
                   
                   
                   
                   
                 viable and fertile. 
               
               
                   
                   
                   
                   
                   
                 The blood 
               
               
                   
                   
                   
                   
                   
                 neutrophil count 
               
               
                   
                   
                   
                   
                   
                 was modestly 
               
               
                   
                   
                   
                   
                   
                 elevated, In 
               
               
                   
                   
                   
                   
                   
                 contrast, 
               
               
                   
                   
                   
                   
                   
                 leukocyte rolling 
               
               
                   
                   
                   
                   
                   
                 2 h after tumor 
               
               
                   
                   
                   
                   
                   
                 necrosis factor 
               
               
                   
                   
                   
                   
                   
                 alpha stimulation 
               
               
                   
                   
                   
                   
                   
                 was only 
               
               
                   
                   
                   
                   
                   
                 modestly 
               
               
                   
                   
                   
                   
                   
                 reduced, but 
               
               
                   
                   
                   
                   
                   
                 blocking 
               
               
                   
                   
                   
                   
                   
                 antibodies to E- 
               
               
                   
                   
                   
                   
                   
                 selectin infused 
               
               
                   
                   
                   
                   
                   
                 into the PSGL-1- 
               
               
                   
                   
                   
                   
                   
                 deficient mouse 
               
               
                   
                   
                   
                   
                   
                 almost 
               
               
                   
                   
                   
                   
                   
                 completely 
               
               
                   
                   
                   
                   
                   
                 eliminated 
               
               
                   
                   
                   
                   
                   
                 leukocyte rolling 
               
               
                 00-10 
                 hypothetical protein 
                 240 kb 5′ 
                 x 
                 XM_135684 
                 x 
               
               
                 band 6 
                 LOC74236 
                   
                   
                 XP_135684 
               
               
                   
                 expressed sequence 
                 200 kb 3′ 
                 Melanoma-derived 
                 x 
                 x 
               
               
                   
                 AI987692 
                   
                 leucine zipper- 
               
               
                   
                   
                   
                 containing 
               
               
                   
                   
                   
                 extranuclear factor 
               
               
                   
                 RIKEN cDNA 
                 240 kb 3′ 
                 Melanoma-derived 
                 x 
                 x 
               
               
                   
                 9930109F21 gene 
                   
                 leucine zipper- 
               
               
                   
                 (9930109F21Rik), 
                   
                 containing 
               
               
                   
                 mRNA 
                   
                 extranuclear factor 
               
               
                   
                 0 day neonate thymus 
                 250 kb 3′ 
                 Melanoma-derived 
                 x 
                 x 
               
               
                   
                 cDNA, RIKEN full- 
                   
                 leucine zipper- 
               
               
                   
                 length enriched library, 
                   
                 containing 
               
               
                   
                 clone: A430110B17 
                   
                 extranuclear factor 
               
               
                   
                 Protein FAM49B 
                 300 kb 3′ 
                 Protein FAM49B (L1) 
                 NM_016623 
               
               
                   
                   
                   
                   
                 ( homo   
               
               
                   
                   
                   
                   
                   sapiens ) 
               
               
                   
                 development and 
                 500 kb 3′ 
                 130-kDa 
                 NM_010026 
                 SH3 domain 
               
               
                   
                 differentiation 
                   
                 phosphatidylinositol 
               
               
                   
                 enhancing 
                   
                 4,5-biphosphate- 
               
               
                   
                   
                   
                 dependent ARF1 
               
               
                   
                   
                   
                 GTPase-activating 
               
               
                   
                   
                   
                 protein