WO2008113773A2 - Gene expression regulation technology and noncoding rnas for diagnosis and therapy - Google Patents

Gene expression regulation technology and noncoding rnas for diagnosis and therapy Download PDF

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WO2008113773A2
WO2008113773A2 PCT/EP2008/053132 EP2008053132W WO2008113773A2 WO 2008113773 A2 WO2008113773 A2 WO 2008113773A2 EP 2008053132 W EP2008053132 W EP 2008053132W WO 2008113773 A2 WO2008113773 A2 WO 2008113773A2
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seq
sequence
nucleic acid
acid molecule
promoter
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WO2008113773A3 (en
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Aldo Pagano
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Biorigen S.R.L.
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
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    • A01K2267/0312—Animal model for Alzheimer's disease
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2330/00—Production
    • C12N2330/10—Production naturally occurring

Definitions

  • the Antisense Technology induces translation inhibition of the target mRNA as a consequence of the formation of a long RNA:RNA duplex molecule that, in principle, inhibits mRNA translation.
  • This technology seemed initially very promising.
  • its efficacy is negatively affected by the fact that long RNA:RNA duplex molecules are often recognized as virus replicative intermediates by the cell, which subsequently activates an antiviral response (known as the Interferon Response) leading to the damage and/or the death of the host cell.
  • the authors present a novel gene expression downregulation technology aimed at minimizing or eliminating both the interferon response activation typical of the AS-based methods and the off-targeting limitation of the shRNA/siRNA/miRNAs gene silencing procedures.
  • the present technology maintains the positive features of AS-based methods such as the target specificity associated with long AS RNA sequences and the gene expression downregulation efficiency of shRNA/siRNA/miRNAs.
  • the authors described a set of noncoding snRNA-like transcription units with regulatory features. Two of them (called 21 A and 29A, respectively) were Alu/7SL-derived transcripts with an extragenic Pol III Type 3 promoter (4).
  • the AIu similar secondary structure of 21 A/29 A transcripts exhibit the two canonical loops (called AIu module) of the 7SL-derived molecules known to bind srp9 and srpl4 molecules and responsible for a transient translational blockade in primates (6).
  • AIu module the two canonical loops of the 7SL-derived molecules known to bind srp9 and srpl4 molecules and responsible for a transient translational blockade in primates (6).
  • this very long (>100 base pairs) target determinant coupled to the stabilization of the ribonucleoparticle by a specific and well tolerated secondary structure complex formation that escapes the antiviral response is referred to as TaD/ AM (Target Determinant/ AIu Module) gene silencing configuration.
  • TaD/ AM Target Determinant/ AIu Module
  • AD Alzheimer's Disease
  • Ib amyloid plaques in the brain
  • the most important approaches for its therapy are based on the disaggregation of amyloid plaques by different reagents such as specific antibodies or other specific molecules.
  • Other approaches attempt to block amyloid plaques formation (2b).
  • the very detailed studies performed until now on Alzheimer's Disease allowed to identify a series of proteins (amyloyd Beta peptide and its precursor, the Presenilins 1 and 2, Alpha and Beta Secretase and others) whose function and/or misfunction is associated with Alzheimer phenotype generation.
  • the authors propose in the present invemtion, a novel tool of analysis of AD together with an experimental plan for the identification of an efficacious therapeutic agent.
  • the noncoding fraction of the human genome includes a larger than expected number of ncRNA genes controlled by distal sequence element (DSE) and proximal sequence element (PSE) Pol Ill-readable promoters, thus likely to be transcribed by Pol III. They referred to them as co-genes since they could specifically co-act with a protein-coding pol II gene (3b).
  • DSE distal sequence element
  • PSE proximal sequence element
  • GPR51 is specifically expressed in the brain and it is part of the G protein-coupled receptor protein family whose misfunction has been associated to different neurodegenerative disorders (6b).
  • 38A maps in antisense configuration in the intron I of KCNIP4 (GC04M020407, 1 Ip 15) in a chromosomal region where alternative splicing events take place.
  • KCNIP4 interacts with one of the most studied components of AD pathways, the presenilin 2 protein.
  • 45 A lies in AS configuration in the intron I (in a region alternatively spliced) of APBB2 gene (NP 775098.2, 4pl4); whose protein product is associated to late onset AD manifestations.
  • the present model suggests that the expression modulation induced by 17A, 38A, 45 A and 51A might be specifically associated with AD and exerts a role in the onset of the pathological phenotype. Therefore 17A, 38A, 45A and 51A represent valuable targets for AD therapeutic agents. Results of specific experiments that demonstrate the action of 17 A, 38 A, 45 A and 51A in AD are reported and discussed below, together with their role as therapeutic molecular targets.
  • TaD/ AM a novel gene expression regulatory tool with advantageous features. They refer to this novel molecular configuration as TaD/ AM being composed by a long Target Determinant aimed to specifically silence the target gene expression fused with an AIu Module that confers a high stability to the whole silencer and that participates to the silencing mechanism by increasing significantly its efficiency.
  • TaD/AM shows several advantages: 1) This method allows the specific silencing of the desired target gene while minimizing the possibility of an undesired off-target phenomenon. This advantage is independent from the length of the Target Determinant of the sequence. 2) TaD/AM does not activate the antiviral response that is almost always triggered by the canonical antisense molecules.
  • TaD/AM allows the use of a Target Determinant sequence of any length (virtually between 15 and hundreds of basepairs). This peculiar characteristic is most likely the consequence of an AIu Module-driven specific ribonucleoparticle assembly.
  • TaD/AM configuration can be used both in vitro (by transfection/infection of in vitro cultured cells) and in vivo (by infecting mice with viral vectors harboring TaD/AM silencers or by the generation of TaD/AM-transgenic mice).
  • TaD/AM silencing procedure does not require a specific equipment and is of easy use with common instruments.
  • TaD/AM also comprises a pol III type 3 promoter selected from the group of:
  • the model proposed implies that the regulation of the alternative splicing events has to be ascribed to the specific expression modulation of four correspondent Pol III small RNAs whose transcription regulation is under the control of Pol Ill-specific promoters.
  • nucleic acid molecule comprising sequentially: a) a 7SL small-RNA derived sequence comprising at least the binding domain to srp9 and srpl4 proteins of the 7SL ribonucleocomplex b) a sequence identical or complementary to a target sequence; c) a pol III type 3 promoter.
  • the 7SL small-RNA derived sequence is an Alu-derived sequence. More preferably, the Alu-derived sequence is a 29A derived sequence. Preferably, the Alu- derived sequence is a 21 A derived sequence.
  • the sequence identical or complementary to a target sequence is of a length of at least 15 nucleotides. More preferably, it is of a length of at least 50 nucleotides.
  • the pol III type 3 promoter has a sequence comprised in the group of SEQ ID No.l to SEQ ID No.34.
  • an expression vector comprising the nucleic acid molecule as described above.
  • a further object of the invention is a host cell transformed with the expression vector as described above.
  • Another object of the invention is a non human transgenic animal bearing the nucleic acid molecule as described above.
  • nucleic acid molecule as described above, or of the vector as described above or of the host cell as described above to modulate the expression of the target sequence in vivo or in vitro.
  • the target sequence is involved in a pathological state.
  • a nucleic acid molecule comprised in at least one of the following sequence: Seq ID No. 68, Seq ID No. 69, Seq ID No. 70 and Seq ID No. 71 for the diagnosis of an age-related pathology.
  • the age-related pathology is a neurodegenerative disease. More preferably, the neurodegenerative disease is Alzheimer's disease.
  • nucleic acid molecule comprised in at least one of the following sequence: Seq ID No. 68, Seq ID No. 69, Seq ID No. 70 and Seq ID No. 71 for medical use.
  • Another object of the invention is a molecule able to be vehiculated into the CNS and to bind to the promoter activating region of a nucleic acid molecule comprised in at least one of the following sequence: Seq ID No. 68, Seq ID No. 69, Seq ID No. 70 and Seq ID No. 71.
  • the use of the transcribed region of the nucleic acid molecule comprised in at least one of the following sequence: Seq ID No.
  • the age-related pathology is a neurodegenerative disease. More preferably, the neurodegenerative disease is Alzheimer's disease.
  • FIG.l Promoter activity transfection assay.
  • a specific luciferase-silencing hairpin is transcribed by four PSE/DSE-dependent promoters (21A, U6, Hl, 29A).
  • a view of the silencing constructs enclosing the hairpin sequence is enclosed.
  • the promoter region encompasses the putative pol III Type 3 regulatory regions (PSE, DSE and TATA).
  • pMock is a negative control being a luciferase hairpin construct lacking the entire PSE/DSE- dependent promoter region, thus resulting transcriptionally inactive.
  • FIG. 2 TaD/AM Structure.
  • FIG.3 GFP-specific Antisense TaD/AM transfection in HeLa cells.
  • A Construct structures.
  • B Representative fluorescence microscope fields showing the silencing effects.
  • C Graphical quantitative determination of GFP silencing by different constructs.
  • FIG. 4 GFP-specific Sense TaD/AM transfection in HeLa cells.
  • FIG. 6 Luciferase gene expression silencing by TaD/AM in HeLA cells. Quantitative determination of luminescence emission after constructs transfection.
  • FIG. 7 Nucleic acid sequence phylogenetic tree of 2 Kbp of the whole collection of promoters. The Alzheimer's disease associated promoters are indicated by the arrows.
  • FIG. 8 KCNIP4 N-terminal fragment variants. In bold underlined is indicated the predicted signal peptide with IEA-GLED as cleavage site indicated by the arrow.
  • VARIANT-I Ace. N°: NM025221 (gene 1-2361; CDS 121-873); VARIANT-II Ace.
  • EF-hand calcium binding motif
  • a diverse superfamily of calcium sensors and calcium signal modulators most examples in this alignment model have 2 active canonical EF hands.
  • Ca2+ binding induces a conformational change in the EF-hand motif, leading to the activation or inactivation of target proteins.
  • Epsl5 homology domain found in proteins implicated in endocytosis, vesicle transport, and signal transduction.
  • the alignment contains a pair of EF-hand motifs, typically one of them is canonical and binds to Ca2+, while the other may not bind to Ca2+.
  • FIG. 9 Promoter activity transfection assay.
  • a specific luciferase-silencing hairpin is transcribed by three PSE/DSE-dependent promoter elements (pMock, pU6-ffl, p38A-ffl).
  • the promoter region encompasses the putative pol III type 3 regulatory regions (TATA box, PSE, and DSE).
  • pU6-ffl is the canonical pol III promoter used as positive control; pMock contains no promoter thus resulting transcriptionally inactive.
  • FIG. 10 Graphical representation of 38A expression in AD patients and controls. mRNA product of the housekeeping gene GAPDH (Glyceraldehyde 3 Phosphate Dehydrogenase) was measured and subsequently all the 38A expression values were normalized to their GAPDH counterparts.
  • FIG. 11 A) Dissociation curves. The merge of several analysis of the amplification product dissociation curves evidences that the two KCNIP4 splice variants [here referred to as Variant I (I) and Variant IV (IV)] are unambiguously distinguishable by two picks at different dissociation temperatures.
  • the splice variant I is characterized by a pick at 83.3 + 0.5 0 C while the alternative splice variant IV is dissociated at 80.8 0 C in the same conditions.
  • FIG. 12 In vitro transcription analysis of 17A and 38A.
  • a In vitro transcription of 17A (lanes 2, 7) or empty vector (lanes 1, 4).
  • Transcription products were either radiolabeled during synthesis, gel- fractionated and directly visualized (lanes 1, 2) or subjected to primer extension analysis (lanes 4-7; lane 5, no DNA during in vitro transcription; lane 6, no reverse transcriptase during primer extension). Radiolabeled RNA size markers were loaded on lane 3. The most abundant 17A-specif ⁇ c primer extension product is indicated by a red arrowhead. Shown in lanes 8-11 are the results of sequencing reactions primed with the same oligonucleotide utilized for primer extension, b: The in vitro transcription experiment was identical to the one reported in panel a, except that the specific template was the 38A transcription unit plus regulatory regions, and primer extension analysis was conducted using a 38 A- specific primer.
  • RNA size markers run in parallel are indicated on the left, c:
  • the in vitro transcription experiment was identical to the one reported in panel A, except that the specific templates were the human 7SK gene (lanes 1 and 10), promoter- less 7SK (lanes 3 and 9), the 7SK transcribed region fused with the 38A promoter region (lanes 2 and 8).
  • panels a-c are the sequences of 17A, 38A and hybrid 38A-7SK transcription units, respectively.
  • the PSE, TATA and terminator sequences are in red, the bases corresponding to the transcription start sites identified by primer extension are in blue, the transcribed regions are underlined.
  • the 38A-derived upstream sequence is in italic character.
  • a,b Real Time RT-PCR detection of KCNIP4 and GPR51 splice variants synthesis in SH-SY5Y (full bars) and SKNBE (striped bars) cell lines transiently transfected with either 17A or 38A expression plasmids.
  • FIG. 14 ncRNA expression and alternative splicing in AD cases, a: 17A and 38A expression in AD cases (orange columns) and non-AD control individuals (green columns) as determined by Real-Time RT-PCR.
  • AD cases 124 and 1024 have a familiar origin.
  • the amplification product dissociation curves unambiguously distinguish the RNAs of interest by peaks at specific temperatures (insets). The averaged results are also reported.
  • Asterisks indicate the concomitant highest expression of 17A and 38A in the same individuals keeping in line with a common deregulation of the members of the AP-cluster.
  • FIG. 15 Expression analysis of 38A a sequence variation in three different cell lines.
  • FIG. 16 ncRNA-induced perturbation of A ⁇ secretion a: Increased amyloid ⁇ secretion and perturbation of A ⁇ 42/A ⁇ 40 ratio in 17A/38A-overexpressing SHSY5Y cells.
  • X axis transfected plasmids.
  • Y axis quantitative determination of A ⁇ (pg/ml) secreted in the medium 48 hours after transfection as determined by sandwich ELISA (results were normalized to the pMock-transfected cell line).
  • FIG. 17 Schematic view of GPR51 (G protein-coupled receptor 51) locus.
  • Variant I is referred to as Variant a in AceView,http://www.ncbi.nlm.nih.gov/IEB/Research/Acembly and Variant II represents Variant b as referred to Aceview).
  • the alternatively spliced Variant II sequence is homologous to Pan troglodytes GPR51 (XP 520146.2) except for the additional 20 residues long predicted sequence stretch (underlined) most likely constituting a misread protein product. Both the variants encompass a predicted ANF receptor domain (receptor family ligand binding domain).
  • Variant I contains a 7tm 3 (7 transmembrane receptor, metabotropic glutamate domain) missed in Variant II.
  • GPR51 was reminescent of a possible involvement in AD generation due to the fact that: i) Alterations in signal transduction pathway of G-protein-coupled receptors (GPCRs) were found in the cerebrocortex and in the peripheral cultured tissues of AD patients (Leosco D. et AL, 2007) and ii) the membrane G protein-coupled receptors Kinases were significantly reduced in brain cortices of an early-onset AD transgenic model (Suo Z. et al., 2004).
  • FIG. 18 Schematic view of KCNIP4 locus.
  • Variant IV variant b in AceView
  • Variant I Variant a in Aceview
  • 38A maps in intron I of KCNIP4 gene (GC04M020407, I lpl5.31) in a region where alternative splicing events give rise to the distinct I and IV protein variants.
  • KCNIP4 regulates the electrophysiological properties of Kv Kb channel with a specific role of the splice variant IV in determining an altered kinetics of potassium channels (Holmqvist, M. H et al., 2002).
  • KCNIP4 is a Presenilin 1 and 2 protein interactor (Morohashi H. et al., 2002).
  • FIG. 19 Cerebral cortex samples.
  • FIG. 20 Speculative model of the AP cluster contribution to Alzheimer's disease.
  • EXAMPLE 1 TARGET DETERMINANT/ALU MODULE (TAD/AM) GENE EXPRESSION REGULATORY CONFIGURATION Materials and Methods
  • HeIa cells grown in DMEM supplemented with 10% FCS
  • the expression constructs containing the regions of interest cloned in the pTopo vectors were introduced into the cells using the Fugene 6 transfection reagent (Roche) according to the manufacturer's instructions.
  • a plasmid Expressing Luciferase (pGL3) and one expressing Renilla (pRL) were used as control of transfection efficiency (to which all the results were normalized).
  • RNAi-silencing assay 24, 48 and 72 hours after transfection cells were harvested and both firefly luciferase and Renilla luciferase activities were measured by Dual-Luciferase reporter assay system (Promega) according to manufacturer's protocol. RNAi-silencing assay
  • the hairpin sequence [targeting a firefly luciferase mRNA from a co-transfected expression plasmid (Promega)] is:5'GGAUUCCAUUCAGCGGAGCCACCUGAUGAAGCUUGAUCGGGUCUCGCUG AGUUGGAAUCCAUU-3' (SEQ ID No. 35).
  • Oligos used to subclone the novel Pol III Type 3 promoters within Not I/HinD III restriction sites were the following: HlFprom Not I: 5 '-ATGCGCGGCCGCATTTGCATGTCGCTATGTG-S ' (SEQ ID NO. 36)
  • Firefly (ffl) Luciferase as target to be silenced and with a pRL plasmid (Promega) expressing a Renilla Luciferase to which all the determinations were normalized. 24, 48 and 72 hours after transfection cells were harvested and firefly/Renilla luciferase activities were measured by Dual-Luciferase reporter assay system (Promega) according to the manufacturer's protocol.
  • the original plasmid constructs p21A and p29A were generated amplifying from a genomic DNA preparation the regions of interest; the PCR products were then subcloned into the pNEB193 vector.
  • the oligos used to generate p21A and p29A PCR fragments were the fo Ho wing :
  • the TaD/ AM constructs were generated by ligating together in the pSHAG vector the three elements of their construction (the 29A and or U6 promoter, the GFP sense Target Determinant, the GFP antisense Target Determinant, The Luciferase sense Target
  • the authors fused the two promoters of the Alu/7SL-derived transcription units (2 IA and 29A) with an hairpin able to down-regulate the expression of a co-transfected Luciferase plasmid (pGL3) by a mechanism of RNA interference.
  • pGL3 co-transfected Luciferase plasmid
  • luciferase hairpin construct lacking the entire promoter region was prepared (here referred to as pMock).
  • pMock As positive controls two similar constructs were prepared substituting the 21 A and/or 29 A promoters with that of two well known Pol III Type 3 transcription unit, Hl and U6 respectively.
  • the luciferase emission was 0.5 + 0.1 for the 21A-driven construct, 0.1 + 0.005 for the U6-driven sample, 0.2 + 0.01 for the Hl-driven construct and 0.1 + 0.02 for the 29A-driven construct (Fig. IA).
  • a AIu module expected to bind srp9 and srp 14 proteins (Fig. 2). All the constructs were then sequenced in order to assess the correct cloning procedures.
  • the authors analyzed the samples by a fluorescence microscope and counted several microscope fields for GFP positive cells evidencing that: a) the promoter without TaD/ AM shows a rate of positive cells (1.1 + 0.09) comparable to the unsilenced sample transfected with the GFP 47 plasmid.
  • results showed that: a) As previously demonstrated, the promoter alone does not induce variation in the number of GFP positive cells (1.1 + 0.2) when compared to the unsilenced sample; b) The construct enclosing the Target Determinant in Sense configuration butr lacking the AIu module inhibits with low efficiency the GFP expression (0.86 + 0.2); c) Strikingly, the GFP-sense TaD/ AM construct is the most efficient configuration for gene expression silencing (0.25 + 0.03). This could be explained by a conjugating and unexpected silencer effect of the sense configuration with the stability conferred by the AIu Module (Fig. 4). The constructs did not induce any other changes that could be associated with a possible antiviral response.
  • the authors prepared a Luciferase-specif ⁇ c Sense TAD/AM construct.
  • a 270 bp long luciferase fragment was subcloned in the GFP TaD/AM construct substituting the GFP sense sequence moiety.
  • the construct was co-transfected with a plasmid expressing luciferase (pGL3) and a plasmid expressing Renilla luminescent protein (pRL) in HeLa cell.
  • pRL Renilla luminescent protein
  • HeIa cells grown in DMEM supplemented with 10% FCS were grown in multiwell Petri dishes 16 hours before transfection.
  • a plasmid Expressing Luciferase (pGL3) and one expressing Renilla (pRL) were used as control of transfection efficiency (to which all the results were normalized).
  • the hairpin sequence [targeting a firefly luciferase mRNA from a co-transfected expression plasmid (Promega)] is: 5 ' GGAUUCCAUUCAGCGGAGCCACCUGAUGAAGCUUGAUCGGGUCUCGCUGA GUUGGAAUCCAUU-3' (SEQ ID No. 58).
  • Oligos used to subclone the novel Pol III Type III promoters within Not I/HinD III restriction sites were the following: 38AFprom Not I: 5 '-ATGCGCGGCCGCTTCACTAAGATCCAGTGC-S ' (SEQ ID NO. 59) 38ARprom HinDIII:5 '-GATCAAGCTTCATCAGGTGGCTCCCGCTGAATTGGAAT CCGATTCATGAAC ACAGAAT ATT-3' (SEQ ID No. 60).
  • the original plasmid construct p38A was generated amplifying from a genomic DNA preparation the regions of interest; the PCR products were then subcloned into the pNEB193 vector.
  • the oligos used to generate p38A PCR fragments were the following: 38A Forward: 5 '-AGCAATAGCAATCAGACCAG-S ' (SEQ ID NO. 61); 38A Reverse: 5 '-GTCCTGTTGGTACCCTTGT-S ' (SEQ ID NO. 62).
  • the insert obtained was then subcloned in pTOPO vector (Invitrogen) following manufacturer's instructions. Prior to transfection all the plasmids were sequenced by DNA Sequencing Kit (Applied Biosystems) following manufacturer's instructions.
  • RNA preparations of 8 post-mortem brain biopsis derived from Alzheimer's disease patients and 8 derived from healthy donors (no correlation with AD) were subjected to reverse transcription by Superscript II First Strand Synthesis Kit (Invitrogen) following manufacturer's instructions.
  • the cDNA obtained was measured by real-time quantitative RT-PCR using SYBR GREEN gene expression quantization, on PE ABI PRISM@ 7700 Sequence Detection System (Perkin Elmer).
  • the sequences of forward and reverse primers as designed by the Primer Express 1.5 software were KCNIP4 Sybr For (varl) 5'-ATGAAGCTCTTGCCCTGCTC-S', (SEQ ID NO.
  • Threshold cycle CT, which correlates inversely with the target mRNA levels, was measured as the cycle number at which the reporter fluorescent emission increases above a threshold level.
  • Glyceraldehyde 3 phosphate dehydrogenase (GAPDH) gene was examined by quantitative RT-PCR as described above.
  • the sequences for human GAPDH primers were 5'-GAAGGTGAAGGTCGGAGTC-S' (SEQ ID NO. 66), 5'- GAAGATGGTGATGGGATTTC-3' (SEQ ID No. 67).
  • Relative transcript levels were determined from the relative standard curve constructed from stock cDNA dilutions, and divided by the target quantity of the calibrator following manufacturer's instructions. Results Co-evolution of the four AD-associated Pol III promoter elements.
  • Table 1 Sequences of 2000 bp of 17A, 38A, 45A, 51A nucleic acid promoter sequences; the PSE elements are in bold, the TATA boxes are underlined. Promoters are followed by the transcript sequences indicated in italics. Transcript sequences are followed by 600 bp of downstream regions. 17A promoter (2000bp) Seq ID No. 68
  • a splice variant of KCNIP 4 gene contains a potential signal peptide region
  • 38A maps in the first intron of its putative gene target (KCNIP4, Uniprot/SWISSPROT Acc:Q6PIL6; from human gene ENSGOOOOO 185774, mapping at 4pl5.31) in antisense configuration and being, once expressed, most likely able to modulate its splicing mechanism
  • KCNIP4 putative gene target
  • KNIP4 does not contain a KDEL sequence motif in its C-terminal moiety (that would redirect the protein into the endoplasmic reticulum)
  • the authors fused its promoter to a luciferase-specific silencer hairpin; 48 hours after co-transfection of sk-NBE neuron-like cells with this construct together with a plasmid expressing luciferase (pGL3), the authors detected a significant decrease in luciferase activity (0.27 fold of the negative control emission, a DNA construct harboring the same hairpin without promoter, here referred to as pMock) thus demonstrating an efficient hairpin transcription driven by 38A Pol III promoter.
  • pMock plasmid expressing luciferase
  • KCNIP4 Variant IV is specifically expressed in Alzheimer's Disease patients.
  • EXAMPLE 3 RNA POLYMERASE III TRANSCRIPTS CONTRIBUTE TO ALZHEIMER'S DISEASE Material and methods
  • Transcription reactions were carried out in a final volume of 25 ⁇ l in the presence of 2 ⁇ g of template DNA and HeLa cell nuclear extract (100 ⁇ g) supplemented with 50 ng of recombinant human TBP.
  • the standard transcription mix contained: 5 mM creatine phosphate, 70 mM KCl , 5 mM MgCl 2 , 20 mM Tris/HCl pH 8, 1 mM DTT, 2 ⁇ g/ml ⁇ - amanitin, 0.5 mM CTP,ATP,GTP, 25 ⁇ M/ 10 ⁇ Ci UTP /[ ⁇ - 32 P]UTP, SUPERase IN (Ambion, 10 U), glycerol 10 % (v/v).
  • a double scale transcription reaction was performed as described, without including radiolabeled UTP.
  • the purified transcripts were resuspended in a final volume of 12 ⁇ l in the presence of 0.5 mM dNTPs and 1 pmole of specific 5 '-end-radio labeled probe.
  • the mixture was heated at at 65 0 C for 5 min and a mixture providing 50 mM Tris/HCl pH 8, 75 mM KCl, 3 mM MgCl 2 ,5 mM DTT, SUPERase IN (Ambion, 10 U) and 200 U Superscript III reverse transcriptase (Invitrogen) was added to a final volume of 20 ⁇ l.
  • the reactions were incubated for Ih at 60 0 C and subsequently for 15 min at 70 0 C to inactivate the enzyme.
  • the products were precipitated with ammonium acetate and gel- fractionated.
  • sequences of 17A forward and reverse primers were 5'-CCACCCTGCAACTGACACAT-S' (Seq ID No. 76) and 5'- GCAAAGGTGCTAATCTTGACTCTTG-3' (Seq ID No. 77).
  • sequences of 38A forward and reverse primers were 5'-CTATCAAAATTTCAAGGATATGCATCA-S' (Seq ID No. 78) and 5'-GATGCCTCAAGCTTTGTTTTGC-S' (Seq ID No. 79).
  • sequences of GPR51 (Vl) forward and reverse primers were 5'-TCCGTCACATCCATCATTGC-S' (Seq ID No.
  • KCNIP4 (V4) forward and reverse primers were 5'-TGGAACAGTTTGGGCTGATTG-S' (Seq ID No. 86) and 5'-CGGTGGCCATCTCCAGTT-S' (Seq ID No. 87).
  • G3PDH Glyceraldehyde 3 phosphate dehydrogenase
  • human G3PDH primers were 5'- GAAGGTGAAGGTCGGAGTC-3' (Seq ID No. 88) and 5'- GAAGATGGTGATGGGATTTC-3'(Seq ID No. 89) .
  • the sequences for human 5s rRNA primers were 5'-TACGGCCATACCACCCTGAA-S' (Seq ID No. 90) and 5'- GCGGTCTCCCATCCAAGTAC-3' (Seq ID No. 91).
  • the sequences for human 7SK RNA primers were 5'-AGGACCGGTCTTCGGTCAA-S' (Seq ID No. 92) and 5'- TCATTTGGATGTGTCTGCAGTCT-3' (Seq ID No. 93).
  • the sequences for human c- Myc primers were 5'-CGTCTCCACACATCAGCATAA-S' (Seq ID No.
  • AD Alzheimer's Disease
  • a ⁇ beta- amyloid
  • RNAs originate from transcription units mapping in the intronic regions of protein-coding genes of brain- specific proteins: Potassium Channel Interacting Protein, KCNIP4 5c 7c ; G Protein Coupled Receptor 51, GPR51 8c l lc ; Sortilin-Related Receptor 1, SORLl 12c l4c and Amyloid Peptide Beta Binding 2, APBB2 15c ' 16c .
  • KCNIP4 5c 7c G Protein Coupled Receptor 51, GPR51 8c l lc
  • Sortilin-Related Receptor 1 SORLl 12c l4c
  • Amyloid Peptide Beta Binding 2 APBB2 15c ' 16c .
  • the authors here show that two of these Pol III transcripts used as experimental models drive AD-specific alternative splicing events leading to the synthesis of distinct protein isoforms and influence A ⁇ secretion.
  • RNA Polymerase RNA Polymerase Ill-dependent non-coding RNAs
  • ncRNAs RNA Polymerase Ill-dependent non-coding RNAs
  • 17A SEQ. ID No. 68
  • 38A SEQ. ID No. 69
  • 45A SEQ. ID No. 70
  • 51A SEQ. ID No. 71
  • Pol III transcript-driven alternative splicing was also evidenced at protein level by immunofluorescence microscopy.
  • a polyclonal antibody raised against GPR51 N-terminal epitopes the authors observed a well-marked immuno fluorescent signal in pMock-transfected SHSY5Y cells (Fig. 13c); in contrast, a strongly decreased signal was detected in cells overexpressing 17A where the alternatively spliced GPR51 protein form (lacking the long N-terminal portion) is synthesized and not recognized by the same IgGs (FIG. 13d).
  • the same experiment was performed challenging 38A-transfected cells with a KCNIP4-specific antibody raised against the N-terminus of the protein.
  • AD Alzheimer's disease
  • the authors postulated a possible active role of the ncRNA cluster in this neurodegenerative disorder.
  • the authors measured by quantitative Real Time RT-PCR 17A and 38A transcript amounts in post-mortem cerebral cortex samples obtained from 18 AD patients and from 10 non-Alzheimer control individuals (Fig. 19).
  • AD cases exhibit a significantly increased expression level of 17A and 38A (13.3-fold and 10.5-fold on average, respectively) with respect to the non-AD control individuals suggesting the existence of a correlation between the expression of these ncRNAs and the AD phenotype (Fig. 14a).
  • the authors investigated the alternatively spliced forms of their corresponding mRNAs.
  • the authors measured in cortical extracts from AD and non-AD control individuals the relative amount of: 1) GPR51 splice variants 1 and 2 (the latter lacking a long N-terminal portion) 210 and 2) KCNIP4 splice variants I and IV (the latter harboring a predicted N-terminal signal peptide) 220 .
  • Table II 38A AD-associated genetic variations, 38A promoter sequence analysis of AD cases.
  • the three 38A novel alleles are indicated with ⁇ , ⁇ and ⁇ , and compared to the wild type promoter sequence.
  • the functional promoter elements (DSE, PSE and TATA box) are indicated in italics.
  • the base substitutions are indicated in red and bold whereas the point deletions are indicated by a star.
  • the underscored sequence corresponds to the transcribed region.
  • AAAATTATCTCT,4 TTTGCA GATGATATAATCCCTTT - ⁇ * G * G GAAAACCCTAAAGATTCCACAA AAAACTGACAGAATGAATTAATTCAGTAAACTTGCAGGATACAAAATCAACATACAAAAATCA GTAGCATTTTTATACACTAATAACAACATATCTGAAAAAGACGCTTTAAAATCCCATTTATGAA AGCATAAAAATAGTTAGAAATAAATTZ ⁇ CCA TAAA GGTGAAA ZATTTGTATACCGATAACZ4Z4 A4CCTTTGATAAAAAAAA ⁇ GTTGAAGAAGACACATATAAATAGAATAATATTCTGTGTTCATGAAT CAAAAAATTTAACAATGTTAAAATGTCTGTATTAACCAAAGCAATATACAAATTCAATGCAATT TCTATCAAAATTTCAAGGATATGCTTCACAGAAATAGAAAAAAAATTCTTGAAATTCATATGGA ACCACAGACACATAAAAACAGAAfAGGCAAAGGAACAATGAAAAAACAATGGAACCACAGACA
  • the authors fused the 38 Aa promoter to a luciferase silencer hairpin; 48 hours after co-transfection of this construct with a plasmid expressing luciferase in HeLa cells they detected a very strong decrease of luciferase activity in the samples promoted by the ⁇ genetic variant and a less pronounced luciferase inhibition in the sample transfected with a hairpin construct which expression was promoted by the wild type allele, thus, demonstrating an increased hairpin transcription driven by the novel altered 38A ⁇ Pol III promoters.
  • the experiment was repeated in parallele in three different cell lines providing the same result (Fig. 15).
  • Pol Ill-specific inhibition was confirmed by a decreased synthesis of Pol Ill-transcribed RNAs (5S rRNA, 7SK RNA, 17A RNA, 38A RNA) and a concomitantly unaffected expression of Pol II-dependent transcripts (cMyc, Glyceraldehyde Phosphate Dehydrogenase) (Fig. 16).
  • ncRNAs significantly up-regulates the synthesis of alternatively spliced transcript variants specifically in the brain of AD subjects; ii) causes a perturbation of the subcellular localization and/or abundance of the canonical splice variants; iii) increase the A ⁇ secretion and generate an unbalanced A ⁇ ratio, favoring the production of A ⁇ x-42 species.

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Abstract

A nucleic acid molecule comprising sequentially: a 7SL small-RNA derived sequence comprising at least the binding domain to srp9 and srp14 proteins of the 7SL ribonucleocomplex; a sequence identical or complementary to a target sequence; a pol III type III promoter; and derivatives thereof for medical and diagnostic use.

Description

GENE EXPRESSION REGULATION TECHNOLOGY AND NONCODING RNAS
FOR DIAGNOSIS AND THERAPY
BACKGROUND OF THE INVENTION Target Determinant/ AIu Module (TaD/ AM) gene expression regulatory configuration
At present methods of gene expression downregulation commonly used are based on two main mechanisms: the so called Antisense Technology (AS) and the gene silencing by short hairpins, small interfering and/or microRNAs (shRNAs, siRNAs and miRNAs, respectively). The Antisense Technology induces translation inhibition of the target mRNA as a consequence of the formation of a long RNA:RNA duplex molecule that, in principle, inhibits mRNA translation. This technology seemed initially very promising. However at the present state, its efficacy is negatively affected by the fact that long RNA:RNA duplex molecules are often recognized as virus replicative intermediates by the cell, which subsequently activates an antiviral response (known as the Interferon Response) leading to the damage and/or the death of the host cell. It is therefore clear that besides the advantage of a precise target selection offered by a long AS nucleic acid sequence, the AS technology has a main limitation due to the interferon response (1). In addition this technique is also limited by the empiric observation that often the AS-dependent silencing effect is only marginal; this considerably limits the usefulness of this approach. (2). On the other hand, the recent development of gene silencing and RNA interference procedures based on very short RNA molecules that are unable to activate the antiviral response seem, to the vast majority of the scientists, the proper solution to limit the interferon response. However, due to their very limited extension, miRNAs or siRNAs sequence can induce undesired effects of off-targeting that leads to an unspecific silencing procedure (3).
In the present invention the authors present a novel gene expression downregulation technology aimed at minimizing or eliminating both the interferon response activation typical of the AS-based methods and the off-targeting limitation of the shRNA/siRNA/miRNAs gene silencing procedures. In addition, the present technology maintains the positive features of AS-based methods such as the target specificity associated with long AS RNA sequences and the gene expression downregulation efficiency of shRNA/siRNA/miRNAs. In a recent work the authors described a set of noncoding snRNA-like transcription units with regulatory features. Two of them (called 21 A and 29A, respectively) were Alu/7SL-derived transcripts with an extragenic Pol III Type 3 promoter (4). This finding implies that upon transcription these molecules might be assembled in ribonucleoparticles together with two proteins of the 7SL ribonucleocomplex called srp9 and srpl4 (5). In the authors' recent experiments, 21 A is shown to be associatied with cell proliferation control by a mechanism of AS gene expression control acting at post-transcriptional level and most likely targeting multiple loci. Strikingly, besides the efficiency of gene expression downregulation showed by these Alu-related molecules, no interferon response was observed. Therefore, a peculiar and specific RNA secondary structure able to escape the antiviral response activation was hypothesized. In detail, the AIu similar secondary structure of 21 A/29 A transcripts exhibit the two canonical loops (called AIu module) of the 7SL-derived molecules known to bind srp9 and srpl4 molecules and responsible for a transient translational blockade in primates (6). The authors' experimental results pointed toward an AS action of 21 A due to a RNA:RNA duplex formed by more than 90 base pairs of the left hand of the transcript and the targets RNAs, thus ascribing to this sequence fragment the specificity of action. Considering the AS 21 A left hand action together with the 7SL similarity of the right hand potentially able to block translation and/or to stabilize the complex in order to escape the antiviral response activation, the authors hypothesized the possibility to use a novel nucleic acid ribonucleoparticle-forming configuration to generate a novel system for gene expression regulation. This configuration is built by fusion of a long (>200 basepairs) Target Determinant (TaD) sequence fragment, useful to specifically perturbate the target RNA expression, with the 191 bp long 21A AIu Module (right hand) forming the srp 9/14 ribonucleoparticle with translation inhibition features, and functioning as stabilizer for the interferon response evasion. In the present invention, this very long (>100 base pairs) target determinant coupled to the stabilization of the ribonucleoparticle by a specific and well tolerated secondary structure complex formation that escapes the antiviral response, is referred to as TaD/ AM (Target Determinant/ AIu Module) gene silencing configuration. Experimental results that evidence the advantage of this technology in gene expression control are reported below. Pol Ill-dependent noncoding RNAs in Alzheimer's disease
Alzheimer's Disease (AD) is a progressive brain disorder that gradually destroys the patient's memory and all related abilities such as learning, reasoning and other. It is a neurodegenerative disorder characterized by the presence of abundant amyloid plaques in the brain (Ib). At present, the most important approaches for its therapy are based on the disaggregation of amyloid plaques by different reagents such as specific antibodies or other specific molecules. Other approaches attempt to block amyloid plaques formation (2b). The very detailed studies performed until now on Alzheimer's Disease allowed to identify a series of proteins (amyloyd Beta peptide and its precursor, the Presenilins 1 and 2, Alpha and Beta Secretase and others) whose function and/or misfunction is associated with Alzheimer phenotype generation. However, despite a considerable experimental effort, the deep origin of AD remains unknown. In the present invention, the authors propose a novel model of AD that may provide an original novel way for the investigation of this disease, leading to the identification of novel molecules as targets for AD therapeutical approaches. In fact, based on experimental evidences, the authors suggest an uncommon origin of AD disease, that in their model, is associated with the overexpression of four specific small nuclear-like noncoding RNAs (ncRNA) transcribed by RNA Polymerase III (Pol III). Since the results of the present invention support the direct involvement of these four nucleic acid molecules (hereafter referred to as 38 A, 17 A, 45 A, 51A) in AD pathological phenotype generation, the authors propose in the present invemtion, a novel tool of analysis of AD together with an experimental plan for the identification of an efficacious therapeutic agent. In a recent study, the authors proposed that the noncoding fraction of the human genome includes a larger than expected number of ncRNA genes controlled by distal sequence element (DSE) and proximal sequence element (PSE) Pol Ill-readable promoters, thus likely to be transcribed by Pol III. They referred to them as co-genes since they could specifically co-act with a protein-coding pol II gene (3b). Given the very high sequence homology between pol III and pol II transcript pairs, and in the light of the results the authors have obtained in a previous study, they proposed that a large part of these novel elements may act as antisense inhibitors of protein translation and/or mRNA maturation, although some of them could play a role in gene-expression regulation with different mechanisms (3b). Therefore the authors not only isolated a collection of novel noncoding transcripts to be investigated for their potential regulatory action with respect to pol II target genes but also provided a collection of 33 novel PSE-dependent promoters useful for the identification of common regulatory regions specific for this type of promoters. Such finding is of particular relevance for the identification of specific molecules that can inhibit not only the function of the novel non coding transcripts but also the activity of the 33 novel PSE-dependant promoters subsequently abolishing or mediating the effect of such transcripts on the protein targets. In the context of a further detailed analysis of the Pol III collection mentioned above, the authors found that four small RNA elements (previously mentioned as 17A, 38A, 45 A, 51A) map in intronic portions of four protein genes whose biological roles might be directly or indirectly related to Alzheimer Disease. 17A maps in sense configuration in the intron 3 of GPR51 protein (Ref Seq protein NP 005449.59, 9q22-q31) in a region where alternative splicing events take place. GPR51 is specifically expressed in the brain and it is part of the G protein-coupled receptor protein family whose misfunction has been associated to different neurodegenerative disorders (6b). 38A maps in antisense configuration in the intron I of KCNIP4 (GC04M020407, 1 Ip 15) in a chromosomal region where alternative splicing events take place. KCNIP4 interacts with one of the most studied components of AD pathways, the presenilin 2 protein. 45 A lies in AS configuration in the intron I (in a region alternatively spliced) of APBB2 gene (NP 775098.2, 4pl4); whose protein product is associated to late onset AD manifestations. 51A maps in 1 Iq32.2 in the SORL 1 (other aliases: LRI l, LRP9, SORLA, SorLA-1, gp250, GenelD: 6653) intron 1 in sense configuration. This region is associated with alternative splicing events during SORLl mRNA maturation. SORLl has been recently associated to AD manifestations and is now being studied in the context of this pathology (4b).
Taken together this information suggests a potential involvement of 17A, 38A, 45A and 51A in AD development. Indeed their protein targets are directly and/or indirectly associated with AD onset. Speculating about the role of 17A, 38A, 45A and 5 IA, the authors hypothesized that after Pol Ill-driven expression they might regulate the splicing mechanism of the introns in which their are hosted, most likely driving the synthesis of alternatively spliced transcripts. This molecular mechanism is already known to be at the base of the synthesis of alternative proteins (also known as alternatively spliced variants) in other loci (5b). Thus, the present model suggests that the expression modulation induced by 17A, 38A, 45 A and 51A might be specifically associated with AD and exerts a role in the onset of the pathological phenotype. Therefore 17A, 38A, 45A and 51A represent valuable targets for AD therapeutic agents. Results of specific experiments that demonstrate the action of 17 A, 38 A, 45 A and 51A in AD are reported and discussed below, together with their role as therapeutic molecular targets.
DESCRIPTION OF THE INVENTION
In the present invention, the authors propose a novel gene expression regulatory tool with advantageous features. They refer to this novel molecular configuration as TaD/ AM being composed by a long Target Determinant aimed to specifically silence the target gene expression fused with an AIu Module that confers a high stability to the whole silencer and that participates to the silencing mechanism by increasing significantly its efficiency. When compared to siRNAs, shRNAs, miRNAs technologies, TaD/AM shows several advantages: 1) This method allows the specific silencing of the desired target gene while minimizing the possibility of an undesired off-target phenomenon. This advantage is independent from the length of the Target Determinant of the sequence. 2) TaD/AM does not activate the antiviral response that is almost always triggered by the canonical antisense molecules. Therefore, the TaD/AM method allows the use of a Target Determinant sequence of any length (virtually between 15 and hundreds of basepairs). This peculiar characteristic is most likely the consequence of an AIu Module-driven specific ribonucleoparticle assembly. 3) TaD/AM configuration can be used both in vitro (by transfection/infection of in vitro cultured cells) and in vivo (by infecting mice with viral vectors harboring TaD/AM silencers or by the generation of TaD/AM-transgenic mice). 4) TaD/AM silencing procedure does not require a specific equipment and is of easy use with common instruments. In the present invention TaD/AM also comprises a pol III type 3 promoter selected from the group of:
Human Genome Map 14qll.2 (784bp sequence) (SEQ ID No. 1) HA
ATTTGCATGTCGCTATGTGTTCTGGGAAATCACCATAAACGTGAAATGTCTTT GGATTTGGGAATCTTATAAGTTCTGTATGAGACCACTTTTTCCCATAGGGCGG AGGGAAGCTCATCAGTGGGGCCACGAGCTGAGTGCGTCCTGTCACTCCACTCC CATGTCCCTTGGGAAGGTCTGAGACTAGGGCCAGAGGCGGCCCTAACAGGGCT CTCCCTGAGCTTCGGGGAGGTGAGTTCCCAGAGAACGGGGCTCCGCGCGAGGT CAGACTGGGCAGGAGATGCCGTGGACCCCGCCCTTCGGGGAGGGGCCCGGCG GATGCCTCCTTTGCCGGAGCTTGGAACAGACTCACGGCCAGCGAAGTGAGTTC AATGGCTGAGGTGAGGTACCCCGCAGGGGACCTCATAACCCAATTCAGACTAC TCTCCTCCGCCCATTTTTGGAAAAAAAAAAAAAAAAAAAAAACAAAACGAAA CCGGGCCGGGCGCGGTGGTTCACGCCTATAATCCCAGCACTTTGGGAGGCCGA GGCGGGCGGATCACAAGGTCAGGAGGTCGAGACCATCCAGGCTAACACGGTG AAACCCCCCCCCATCTCTACTAAAAAAAAAAAATACAAAAAATTAGCCATTAG CCGGGCGTGGTGGCGGGCGCCTATAATCCCAGCTACTTGGGAGGCTGAAGCAG AATGGCGTGAACCCGGGAGGCGGAGCTTGCAGTGAGCCGAGATCGCGCCACT GCATTCCAGCCTGGGCGACAGAGCGAGTCTCAAAAAAAAAAAAACC
Human Genome Map 2p24.3 (3000 bp sequence) (SEQ ID No. 2) 12A
TGTATTTTAAATTATGATACATAATGACTATTTAACTTCCAAACAGAATTCACT CATTTACATTTATGAACATTCTGGGTATAATATCCAGAGGGAATTAAACCACTA TCTCAGAGAGATATCTGCATTCTGATGTTCACTGAAACATTATTCACAGTAGCC AAAATACAGAAACAACCTGTCTGTCAACGAATTAATGGATAAATAAAAGAGA TAAGGAATATATATATACACACACATACACACACAAGCACACACACACACATA CAATGGAAAATTATTCATCCTAAACGGAAATAAAATTCTGCTATTTACAAGAA GAAGAATGAAACTGGAGGACCTTCTGCTTAGAGAAATAAGTCAGACATAGAA AGACATATACTGCATGATCTGACTTGTATGTGGAATATAAAAAAGTAGAACTC ATGAAAATAGAGTAGAAGGGTGGTTACCAGAAGTTATGGGGTGGGAGAAATG GAGAGCTATTGGTCCAAGGATGCACACTTTGAATCATAAGGAATAAGTTCTGG AGACCTGATGTGCAGTAGGATGACTATAGTTAATAATCATGTATTATATGCTTG AAATTTGCTAAGAGAATAGATATTCAGTATTCTTACAACACACACAGACACAC ACACACACAGGTATGTCAGGTGATGGATATGTCAATTAGCTTGATTGTGGTGA TCATTTTTATAATATATACATATATCAAAATAGTATATTTCCAGTTTTTCACTTT TCTTTTAATTTTTATTATCATATATTTTACTATATAAAATATTTTTAACTAACAT GATGTCAGTCCAGCCTGACCAACATGGAGAAACCCCATCTCCACTAAAAATAC AAAATTAGCTGGGCATGGTGGTGCATGCCTGTAATCCCAGCTACTGGGGAGGC TGAGGCAGGAGAATCATTGGAACCTGGGAGGCGGAGTTTCTGGTGAGCTGAG ATCACACCATTGCACTCCAATCTGGGCAACAAAAGCAAAATTCTGCCAAAAAA AAAAAATTCTGGACAGAATTTTGCATAGAAAGCCCTTTTTCATCCCCAAATTAT AATAATAACATAGTACATTTTCTTTTTGTAATTCCAAGGGATCTATTTTTTGTTT ATTTTGACATATAGCTCTTAGGTTCTTTTGGCATTATTTAGTGTGTAAGAGTAA GTAAGGATATATTTTTATAGTTTTCCAAATAATAGCAAATATCCAGAAATAACT TATAAAACAGGTCATCCTTTCACCATAAATGTGAAATGCTACCTTTATCCTAT GTATTTGAATATATATACATATATATTCAAGTACATTCTCTCTATATATGTGTG TTTATAATATCATATATATACACACACATATGTGTGTGTGTGTGTGTGTGTGTG TGTTACCTCTTTCAATTCCATAGTGTTTTAAGTAATCTAATTTTGGCATACTGAA AATACTGATAAGAAAAATTCTTATTTTTTCTTTCAAAATTTCCTTTGCATTTAT AATACATTAATTTTCCAGATAATCTTTAGAATCAGCTTATCAAACTTTGTTAGA AGTGTATTTTATGTTAATCGAGACAATACTGGAGCTTGTAGAATAATTTCAAGA AGAAAGACTCATCTAACATATTTGAGTATTTTCATGCAAGGGCAGAGTATGTTT CTTCCTTTATTATTCTTTGCTCTCCTAAAGTAAAGATTTATAAGTGGTTTATAAT CCTTTTTTACTTTATATTAAGTTTATCTCTAGTTTTTTATAGTTTTTGCTATTATT ATGGCTATAATTGTCTTTAATTGCTATTTTTAATTGAATGTTAATGTGTTAAAG GAAAACCATTAATTTTTGTATACTGATTTGTGTCCTGTTAAGTTCATAAACTAG ATTACTACTTCTAAAAGTTTTATTTGATTGTTTTGACCTTTAGGTATAGAAAATC ACATTGCTTGCACCTTACTGCAAGTTCACAAATCCTTTCCATTACTTATACTTTG TAATTTTTTATCTGTATTTAAATTAAGTAGTCCAGTGCAGTCAATATTGAATAA ATGGTACTAGTTATAGCAGGCCAGTTTTACTTTTATTATGAATTCTTACAGTAT TATCAAAGACGTTCTTTATCAAGTGAGGCAGTTTTTCCTATTTGCAGTTTGTCA AGAGTACATTTTTAGTTTTACTATAAATTTGCGTTGAGTATTATCAAATGACTT CTTTCATATAGTGTCATTATCATGTATTTTCTCCTTTCACATTATCAAGTAGAGA ATTACATCAACAGTTGTCCTAATTCATATCATCCTGAACAAATTCTACTTAATC ATGGCACACTAAATATGTAATTTTTTATAATAGTTTGGAATCTGAATTTATATT TTTGAATAAAATTTGTCGATAGTTTTTATTTTTCTATATGTGATTAATTTGCATT TTTGGAACAATGATTTTGCCAGGTTCATAGACTGAGTGAGAAAGGTCCATTAA AACTAGATTCATGTATTATTTGCTGATTAATAAATTAAATAACTTAGAAGTTAT CTGTTAATAAATAGGTTACTAAAACTTGCCAGTGAAATTAGAGCTAAATTTTTA TTTTTGTGTCAAGTATTTGCCGAAATTCACTTTCTAAATTGTTATTGAACTATTT AAATTTTCTACCTATTCTTGAATCAAATTTTGTAATGTATATTTTTGTCAGTCTT ACATGTTGTTATAATTTTTAATATTATTTCATGCATTTGCATAAAATGTCACAA ATTTCTAAAATACGTTATGTTCCTGTAATTATGCTCATTATTTCATACTAACAAT TTTCATTCTAGTACTCTTTTTTCCCCTTTCATCAGACTTAACAAAAGAGTTGTCT ATTTTATGAATCTTTGCAAATAAGTAGCCCTTGATTTTTATTTTTAAGCCTATTT TTTATATGGAATTGTAATTGGAAGATTTAAAAAGTCAATATTGCACTGGAAAA TATAAAATAAAAATTAATGTTTAATTCTATGTGT
Human Genome Map 3pl2 (1921 bp sequence) (SEQ ID No. 3) 14A
TTCTCTTTTCTCCACATCCTCACCAACATGTTATTTTTTGTCTGTTTAATAATAG
CCATTCTAACTGATGTATGATTATATCTTATTTTGGTTTTAATTTGCATTTTTCT
GATTAGTAATGTTGAGCATTTTTAATATGCCTCTGGGCTATTTATATTTCTTCTT TTAAAAATGTCTATTCATGTTCTTTGCCGACTTTCTAATGGATGATGGAATGCT AAAGGCCCAGACTTAACCACTATGCAATATAGCCATGTAACAAAAGTGTACTT GTACCTCTTAAATTTATGCAAATAAAAACTCAAAAAAAAAAAAACAAAAAAA CCTAAGATGACTAAATGTCAGAAAACCAGGTTTTACATGCCACTTCATTTGCTG AAATACAACGTACACAGCCTGTTAAAATGAAGTCGTCTGCCCCCCAAAATATA TAATATTAATAAGGTCTCTACCTAGAATCACCAGTTTACAATAAATGCAGAGG ATAGATGCACATGTTAGAAAACACCATAAAGGTGAAATCACCCAAAGTCTAC TCGACAAATTATCCAACTTCTTCAACCATTAAATAGCATAAAAGTTAGGGGAG GGGAATCTGTTACAGAACAAAGGAGATAATAATATATCATGCAAACACAAAC CCATCTATATCTTGATTCAAATATAAATTGCAAAAAACGGCTTGAAATTACTAT AGAAATTTCAACAGAAACAAGGTCTTAGATAAACAGTCCCCAACTTTTTTGGT ACCAGGGACCAGTTTTGTGGGAGACAATTTGTCCACAGACAAAGGGTGGAGA GGTGGGGATGGCTTCAGGATGAAACTGTTCCACCTTAAATCATCAGGCATTAG TTAGATTCTCAGAAGGAGTACACAACACAAATCCCTCACATGTGCAGTTCACA ATAGAGTTCATGCTCCTACGAGAATCTAATGCTGCTGCCCATCTGACAGGAGG TAGAGCTCAGGCGGTAATGCTTGCTTGCCTGCCACTCACCTCTTGCTGTGTGGC TCCGTTCATAACAGGCCACAGACTGGAACCCATCTGCAGCCCCAGGGTTGGGG ACCCCTGTCCTAGATAACATTTAGTAAATACAGTTAATTCTTTTCAGTGCAATA ACTTTGTTGAGATTGTTTTTTAACTGGCTAGCCATATACAGAAAACAGAAACTG GACCCCTTCCTTACACCCTATACAAAAATTAACTAATAAAAAAACACTGACAT GTAAGACCTAAAACCATAAAAACCCTAGAAGAAAACCTGGGCCATACCATTCA GGACATAGGAAAGGACAAAGGCTTCATGACAAAAACACCAAAAGCAAAGGCA ACAAAAGCCAACATTGACAAATGAAATCTAATTAAACTAAAGAGATTCTGCAC AGCAAAAGAAACTATCATCAGAGTGAACAGGCAACCTACAGAATGGGAGATA ATTTTTGCCATATATTCTTCTGACAAAGGGCTAATCTTTGTCAGAATCTACAAG GAACTTAAAAAAATTTACAAGAAAAAAGCAACCCCATCAATAAGTGGGCAAA GGATGTGAACAGACACTTCTCAAAAGAAGACATTTATGCAGCCAACAAACAA ATGAAAAAAAGCTCATCATCACAGGTCATTAGAAAAATGCAAATCAAAACTAC AATGAGATACCATCTCACACCAGTTAGAACGGTGATCATTAAAAACTCAGGAA ACAACAGATGCTGGAGAGGACGTGGAGAAATAGGAACGCTTTTACACTGCTG GTGGGAGTGTACATTAGTACAACCATTTTGGAAGACAGTGTGGCAATTCCTCA AGCATCTAGAAACAGAAATACAATTTGACCCAGCCATCCCATTACTGGGTATA TACCCAAAGGATTATAAACCATTCTACTATAAAGGCACATGCACACGTATGTT TATTGTGG
Human Genome Map 9q22-9q31 (2521 bp sequence) (SEQ ID No. 4) 17A
TTTCAGCCTCCCTTCTACCCCACTCCAGGTACTTCTGCCTCTGTGGAATTCCTGC TGATTCTAAGCCATGATGAGCATGGCTACCCTACCCTCTGATCTTCCCTCCTAC CGTGCTGGGCTCCTGTAGGAGGGGATCCCTCTCTTCCTCCTCCACCAAATGTTG TCTCTTTTTGGAACCTTGTCTGAGCACTCTCCCCAGGTGGGATGAGTCACTTCC TCCCTTTGTTCCCAGGCCCCTTTGTTCCTGTTTCCCCTGAGAGGTCTCTGTCTTC TTCACCATGCTGGGAGTAACCTGAGGACAAGGTCAAGGCCGATGATGTCTATG AGCCCAAGAGAGGGTCTGGTGCGTAAAAGCTGTTTGAGAGAGTATGCAGAAG GAATGGACAAATGAAAATTAGAGACTGACTTACAACTGGGGAAACTTCTCGTT GACCCTTTCTGTTCCTAAAGAGAGTGTCACCGGATAGGGGTCAGGAGCCTGGG CTTTCAGTTGCAACAAGAAGACTTCTTTGCTGTGGGCTTTCTGAAAGACAGTTC CTCTCTCTGTGACTCTTCAAAACAGACATGACAATCATGTGTGCCCTGCTTGCC CCTGAGGCTGCGTTGAGAGATATAAAACCATCAGGAAAGTGCTCAGTGGCTGT GCACCTGCAGCCAGCACCTCTGGCCAGTGTTGGAGAGCAAGGAAGGGAAAGC CAAGGGAAGCCAATTCCTGGGAGCTTCTCCTGTCTGGGATGCCAAGGTGGAAA TGAACTTGAGACCCAGACCAAACTTGAGGCTCTTTCATAGTCAGGTAATTTGG GCACCCAGGGCATTGAGATCAGTCTGCCATTCACCCTGTGGCTAGCCACACCT ACCTTCAGCTTTTTGACACTGGTACAGGGATCGTTGGAGAAGCTCTCGGTGTCT GAAATCTCAATGTCCTCGCCATACAGAACTCCAGTCAGGTCATTCCGCACCTGT CAGCAAAGAGAAAGCAGAGGGTGGGTGTGCTGGGGACCACAGGAAGGGCCAG TTCCGAGGGGTCACCCTGGGGAAGTCAATTGGGCAAAGCGATTTTCTCTACCG ACAATGCAAAGTGAGTGGTTTTGTTTTACATTATTAACTAGACCGCCCCACAAA AACTTGAGGATCCCCCAGTCCCACCCTGCAACTGACACATGGATACAAGGAGG CCAGACAGGGAAGGGACTTTCCAAGATTGCCCAGGGAGTTCCTGCAAGAGTCA AGATTAGCACCTTTGCTGGTGTTTCTCCACCACATCACACTGTCTCCAAATCAG GCTATTCAATTGTGTCTTTGTTAATATTTTGCACTATTTATTTGCAACATTATTT CACTTTTATGGTGAGGAAATAGCTAAGATATTCAAAGACAATATAGAGTAAA GGAAAGAGGAAAGAAGTATGGAACCTGCCTATGATGTTACACGTAACTATGTG TCTACTGACACTCAGAATGAGGAATATCTATGGATGTGAAAAGCAAAGGGCTG CAAACTCCAGTGTTACCAGGACCAGCCAGTGTGTGAACTAGCCTGTGTGGAAG GATATTACAGAATGATAGGGCTGGGCGTGGGCTCACACCTGTAATCCCAGAGC TTTGGGAGGCCAAGACGGGGGGCTTGCTTGAGCCCAGGAGTTCAAGACTGCAG TGAGCCGTGATCACGCCACCGTACTCCAGCCTGGGTGACAGAACAAGACCTTG TCTCAACAGAACAAAACAAAACAAAAGACAGTAATAGTTGGTTGCTGAGTTAG AATGTGGGTCAAGGGTTACCAGACCTTCTGATTTTTGAGGGGAGAAATCAGAA ATTTAGACTTTTAAAATATAAAATCCCCTAATTTTTAAATGTTGATACTATTTTG AATTAAAAAAAAAAGAAAGTAAGGATCAAACCAAATAAACTTAAAGTCTGTA TCTGGCCTGTGGCCATGGCTGTGCACCCTCTGACATATAGCAATGGAAACTGG ATTTTGGGTTTTAGTAGCAAGAACTAGGCTGGGGTTAGGGGATCCAGCTTCCA GGTCCTGCTCTATCACTGACTTGCTTGTGACCTCAATCTCTCGTGTGATTCTTCC CTTCTCTGGGCCTCAGTTTCTTCCGCTTTAGGAGATGCTTAAAGCACTTCTTGTT CACACCCATTAGCATGGCTATTACCAAAAAGCAAAACCACAAGTGTTGGTGA AGATGTGGAGAAACTGGAACCCTTGTGTGTTGCTGGTGGAAATGTAAAATGCT GCCACTGCTGTGGAAAACAGTATAGCAGCCCCTCAAAAAAATAAATATAAAAT TACCACATGATCCAGCAATTCCACTTCTGGGCACGTACCCAAAAGAATTGGAA GCAGGGACTTGACAGACATTTTACACCCATGTTCATAAGAACATTTGTTCACTG CAGCTAAAAGGCAGAAGCAGCCCAAACGTTCACTGCTCGATGAATGGATAAAT GAATTGTGGTGTATACAGACAGTGAAATACTATTCAGCCTTCAAAAGGAATAA AATTCTGACACAT
Human Genome Map 3pl2.3-12.2 (2641 bp sequence) (SEQ ID No. 5) 19A
ACATATGATTTTTTCAAATTTTCTAAATAGTAATTATTTCCTAGCTCTGCCTTCT
GAAAAGTCCTAGAATTACAACAAGCTGGAAACAATGAACAAATGGAGCCTTC
AGACTGTAATCTCTAAATATGATTTTCCTTTTAGTGAAAAGATTTCTTTGGAGA AATAGTTGATTATAGATCTAGGTCAAGACATTTATGAGATGACCCTGGGACAT TTTATTTTTGTCAGAAAGCCTGGAAAGTATTAATGTGTCTACACAAAACAAAG GAACCAACTTAAAAGAGCAGTCACTGACCACAGCAGAGATAATTGAAGCATC AAACTGAATAAAAAATATAACTCATTCAAGCAGATGCAATTAGTTATCCTGAA AATGATAAATGCATGAATGTAATCAAACATTAATACTTGGTTTCCTGTGCAATT CCTTTTCAGGGTAATGAAAACTGATGAGTGAGAGTTAAATGAAGGACTCCAGA AAGAATGACAGTTACAATATAATGATTTGTGCCCCCCAAATAAAATAATTGAT CTACACAAAATACATCAGTATTAGGCAAAACTAGATGGTAAAAAAATTTCGAA GAAAAATAGATTACGGAGAACAAATCAGAACTCACTGATCAAACTTGATATGA CTAATTTATAGTTATTTGAGAAATCCACATGCTGTTTTCCATAGAGGTTGACTA TTTTACATTCCAACTAATAATGTATAAGGCATTCTCTTTTCTCCACATCCTCACC AACATGTTATTTTTTGTCTGTTTAATAATAGCCATTCTAACTGATGTATGATTAT ATCTTATTTTGGTTTTAATTTGCATTTTTCTGATTAGTAATGTTGAGCATTTTTA ATATGCCTCTGGGCTATTTATATTTCTTCTTTTAAAAATGTCTATTCATGTTCTT TGCCGACTTTCTAATGGATGATGGAATGCTAAAGGCCCAGACTTAACCACTAT GCAATATAGCCATGTAACAAAAGTGTACTTGTACCTCTTAAATTTATGCAAAT AAAAACTCAAAAAAAAAAAAACAAAAAAACCTAAGATGACTAAATGTCAGAA AACCAGGTTTTACATGCCACTTCATTTGCTGAAATACAACGTACACAGCCTGTT AAAATGAAGTCGTCTGCCCCCCAAAATATATAATATTAATAAGGTCTCTACCT AGAATCACCAGTTTACAATAAATGCAGAGGATAGATGCACATGTTAGAAAAC ACCATAAAGGTGAAATCACCCAAAGTCTACTCGACAAATTATCCAACTTCTTC AACCATTAAATAGCATAAAAGTTAGGGGAGGGGAATCTGTTACAGAACAAAG GAGATAATAATATATCATGCAAACACAAACCCATCTATATCTTGATTCAAATA TCAATTGCAAAAAACGGCTTGAAATTACTATAGAAATTTGAACAGAAACAAGG TCTTAGATAAACAGTCCCCAACTTTTTTGGTACCAGGGACCAGTTTTGTGGGA GACAATTTGTCCACAGACAAAGGGTGGAGAGGTGGGGATGGCTTCAGGATGA AACTGTTCCACCTTAAATCATCAGGCATTAGTTAGATTCTCAGAAGGAGTACA CAACACAAATCCCTCACATGTGCAGTTCACAATAGAGTTCATGCTCCTACGAG AATCTAATGCTGCTGCCCATCTGACAGGAGGTAGAGCTCAGGCGGTAATGCTT GCTTGCCTGCCACTCACCTCTTGCTGTGTGGCTCCGTTCATAACAGGCCACAGA CTGGAACCCATCTGCAGCCCCAGGGTTGGGGACCCCTGTCCTAGATAACATTT AGTAAATACAGTTAATTCTTTTCAGTGCAATAACTTTGTTGAGATTGTTTTTTA ACTGGCTAGCCATATACAGAAAACAGAAACTGGACCCCTTCCTTACACCCTAT ACAAAAATTAACTAATAAAAAAACACTGACATGTAAGACCTAAAACCATAAA AACCCTAGAAGAAAACCTGGGCCATACCATTCAGGACATAGGAAAGGACAAA GGCTTCATGACAAAAACACCAAAAGCAAAGGCAACAAAAGCCAACATTGACA AATGAAATCTAATTAAACTAAAGAGATTCTGCACAGCAAAAGAAACTATCATC AGAGTGAACAGGCAACCTACAGAATGGGAGATAATTTTTGCCATATATTCTTC TGACAAAGGGCTAATATCCAGAATCTACAAGGAACTTAAAAAAATTTACAAGA AAAAAGCAACCCCATCAATAAGTGGGCAAAGGATGTGAACAGACACTTCTCA AAAGAAGACATTTATGCAGCCAACAAACAAATGAAAAAAAGCTCATCATCAC AGGTCATTAGAAAAATGCAAATCAAAACTACAATGAGATACCATCTCACACCA GTTAGAACGGTGATCATTAAAAACTCAGGAAACAACAGATGCTGGAGAGGAC GTGGAGAAATAGGAACGCTTTTACACTGCTGGTGGGAGTGTACATTAGTACAA CCATTTTGGAAGACAGTGTGGCAATTCCTCAAGCATCTAGAAGCAGAAATACA ATTTGACCCAGCCATCCCATTACTGGGTATATACCCAAAGGATTATAAACCATT CTACTATAAAGGCACATGCACACGTATGTTTATTGTGG
Human Genome Map 14q22.1 (2341 bp sequence) (SEQ ID No. 6) 2OA
AGGCCTCAGTGTCCTAGACTAGCACAGAACAAGCAGATGAAACAAAGTTTATA TCAGAATGTCAACTGAAAAAGTATCATTTACCATAAATGGAAGATAATTGTAT TAAATTCTAGTTAGATGCTAAAACTTCAAGAACTTTTAGAGTCTGTACCTGCAT TCTGTTAAAAATATAGATTAAAAAATGCTAACATGTTAACACAAAGGACTTTC CAGAAAGACTTAAAGAAAAGTGAAAGGGGAATAACTGTCTTGCAATGTAATTC ATTGTCGTTTAAGACTGGGTCTATGGAACACCCTAAATCACCTGGTTCCATCAC GTTCTTTTTAACATGGAGATGGATAGTTTTTCCCCATACTCTATATATTGAGCA TTCTATAGTTCATGATTTTTCTGCATAGAGAATTGTTCAAGCCGGGGGTGCAGG CTCACCGACTGGATAGTGAATCAAGAAAATAGTGTGTTCATTAGTTCATCATTA CCCTGAGTTTCCAACAAGAATTTAGTACAGGAAAGTAGACAGCGGAGCTGGGA GCCATCTATTTGAAACTGTCTTAAGCAAAACTAAGAAACCGAGTAAGCTTGCT TTTGGTGTCTTTCATCCCTTCTTGTGTGCCCCCTAATTATTCACTCCCCAATGCC CAGACATTATGATGCCTTCTCCTGCTCAGAGACCTTTCTGGGAGGAAGACCTAC TCAGACCTGGTATTCCCTCATCCTAGGCTCTACCCTATTTTTCATCCAGCTGTTA AAGCTGAGTGACTAATTTCACACTTATGTACGAATGACCCATAACTGGCTTAAT GCTGTGACCATCTTGGGGGTATTCAAAGCTGATAAACACTTTTTTAAGTTATAT AATAATCAAAGAAGCTTATCTTTCTGCTTTATTTCAAATTTCACCCCACAGGCC TTACTTATTTTTAAGATCAATGATTTTGATGGGCCCCCCCTTCCCACTCTTAATT CAGGGTATTTCTGGCCCCATCCGGATCCAAACTCTAATGCTCATCTCTTCCATA CTGTCCTTTGCAGGTCATCGGTATTGCAAGAGTTGCATAAGGCCCAATTCAGTC TCTGCCCCAAAAGCTCAAGTCCAAACTTCAGAATCTGGGAGGACAAGGATTCA GGAAATTTTGTCAGAACTATGAGCTTTGAACTTTCACTTTTATGGTGAGGGTC ACATTTGGTCTGAATCAATTAATCCATTACCCGCCCCCCCCCCCCCCCCCACCA CCACCATGTGTGAATTCAAAATAATCAACTTGGGTTTATTATAAAAAACAAAA TATATTAATATAAGTATACTAAGATTTTTCTAGAAAACTTGGCCGGGCGCGGTG GCTCACGCCTGTAATCCCAGCACTTTGGGAGACCGAGGAGGGCGGATCACAAG GTCAGGAGATCGAGACCATCCTGGCTAACACGGTGAAACCCCATCTGTACTAA AAATACAAAAAATTAGCCGGGCGTGGTGGCGGGCGCCTGTAGTCCCAGCTACT CGGGAGGCTGAGGCGGGAGAATGGCGTGAACCCGGGAGGCGGAGCTTGCAGT GAGCCCAGATCGCGCCACTGCACTCTGCCTGGGTAACAGAGTGAGACCCTGTC TCAAAAAACAACAAACAAATAAACTTAGAAGAATATATGTGACTATTGGCCGG GCGCGGTGGCTCACGCCTGTAATCCCAGCCCTTTGGGAGGCCGAGGCGGGCAG ATCACGAGGTCAGGAGATAGAGACCATCCTGGCTAACATGGTGAAACCCTGTC TCTACTAAAAATAAAAAAATAAAAAATAAAAAATGCGAGGTGGCGGGCGCCT GTAGTCCCAGCTATTCAGGAGGCTGAAGCAGGAGAATGGCGTGAACCCGGGA GGCGGAGCTTGCAGTGAGCCGAGATCGCGCCACTGCACTCCAGCCTGGGCAAC AGAGTGAGACTCCGTCTCAAAAAAAAAAAAAAAAGAAGAAGAAGAAAGAAA AAGAAAAGAAAAAGGAAAAAGAAAACTTAATTCTGGCAATGGACTGTTTCTA AAATAATATATTAATACTACTTAATGAGGAAGAAAAAACCTCTGACATCCTAA AATGCCAAGTGTTTGCCTTTACCAAGGTTTAAGCACACATAAACACGCATATTC AAATACCACCCAAAGTGGAGGTGCAAAGATCAGCCTGTACCGCACAGTAACA CAGACTGGGTTGTTTTTTGTAAAGAAGGCAACTAGTCCAGTGAGTAATCCCTTC ATTTTCCACACACATACCCTTCTGTTTTCTCCCTCTCCTCCCCCCACACCCTCCA CTGCAGTTAAAACGTAATTCGAAGAAGCCTAAGGTAAAAGCCCCT
Human Genome Map 8q24.13 (2100bp sequence) (SEQ ID No. 7) 21A
GTGGACAGGGAAATCTTACCTTCCTGCCTCTCTATGTTCAGGCTGAGTGGGTCA
GAAGGAGAGTGTATTAGGTAAGAAAATTTATCAGTATTATTTAGTGAACACTG
GATTTATCCTTTTGCATTCTGGCTGTAGTACCCAACTTCCACATGGCAATGCAC CCTCACCTCAGCCCTCCGCCCACGTGGTCCCCTTGCTGAGCACTTTAATGAATG ACTGCATCTCATTTTCACAGCTATTTGATGCACCTGCTATTATTACTCTTATTAC CATTTTCCAGTGGGAAGCTGCTTCTTGGGCAGGGTGGATTTCCATCTGCGTCTC CTTTTCGGTGTTGAAAGCTGGTAAGTGAGGACACCAGGATTGGAACCTGGGTA GTCTGAGTCCAGAATCTCTATTTTCAAGTCTTCCTGCTCTCTGCTTCTGGCAAGT TTGATGTCCACTTTTGATCTTCACCTACATTCCAGCATAATAGCTACTTTTGGTT GTTTTCTCAGCAGCACAAGAGAAGTGTGGCGAGATTTTTAGGTGAGTCATCTA GAGAAGTTAATCTTATTTTGGGAATTCTACTGGCAGCTTCAGGTGGGGAAAAT TTTGTTATTTTCTATCCTCCTCTAGGTTCTAAAAGGGAAGAAAGATGGTGAGCG TAGAAAGATGTGACTGTATTCACTATTCACCCTTTGTCGGGTGGTGAGTAAGCA GCTTGCAAAAGCAATGAAGTTTGGAACAATCCAGAGAACCAAACTTTCAGCTG CCAGAGATGGCACCTGGTATCCTGGGTACATCTGCCTGTAGGGCCCAGAAAGA GCTGGAAGCCAAGTGCATGGATCAGGTCTGTAGGAAGGTGGGAGAGCCAGGA ATCGAGTGTCAGGGGGCATTTATTACCCATGGAAGCAGGTTTTTGTCAATTTTG TTCACTGCTGGATCACTAACACCTGGACTGGTGCCTGGCCAGGTGGTGGCTTCA TAATCATTTGTTGAGTGAATCAATGAATGAATGAATGAACAGCTGTAGCAGAT GCTAGCAGGGCTTCCTATTTCTTCCATCACCATAAAGGTGAAAGACATCATAA ACGGGAATTTAGACAATCCTCAGAAATTTTCAACTGCCATGTATCTTGACTTGA TGCTTCTAGTAGTTATATTTATTTGTAATTCAATCTTTCTTTTTAAATAGTTGAC C AAGTGTGGTGGCTC ACGTAGTCCCAGC4 CTTTGGGA GGCTGA GGCA GGA GGA T CA CTTGA GCCCA GGAA TTTGA GA CCA GCTTGGGC AA CA TA GTGA GA CCTCA TCTCTT AAAAAAAAAAATTAGCTGGGTGTGGTAGTGCACACCTGTGGTCCCAGCTACTT TAGAGGCTGAGGTAGAGGATTGCTTGAGCCTGGGAAGTTGGGGCTGTAGTGAG CTTTGATTGCATCACTGCACTCCAGCCTGGGTGACAGAGCAAGACCCTGTCTCT AAAAAATTAAATAAATAATAAAAAAATTAAAAAGTAACTCCCTTTTCTTTATT TTCAGGCTTCCTTCCCACCTGCTAATTCAAACACTTTACAACCAAAAATATCTT ACCTTGATCCTGTTTCTTTCTCTATAACCTCTCTATTTCTGTTTCTTTCAACCAA ATTTCTTAGGTCATCTATAATTTTGTTTCTACTTTTTCTATGCATGCCTCAATCC ATTGCCAACTCCTCAACCTGCCCCAAGTGCCCACAACTCCACCAAAAGTAATT CTAACATTTTACCAATCCAATACATCACAGTTTTTTATAAAAAACTTAAGAAAT ATACTTTAGTTGAATTTGAAAGAGTTGCCCACTTGTGTTAAATATTTCTTTCCTT GTGTCTGGGATATCATTTGATTCTGATTCTATTCCTAATTCTCTGACCACCCTTT CTTCGTAGATTTCTCTTCCTTTGTTCAGCCTTTCACATCCTTGGAGTTCCATCCT CTGGTGATTGTTTGTCCTGTTCCATACATTCTCCTTATATGAGCATTGTGTTTTA GCTTATGAATGGTCACATGACCTAGCCAGGCCAATCAGAGTCTTCCATGAGAC TTTTGTTTATTTATTTATTTATTAATTTATTTATTCTTCCACATGCCATC
Human Genome Map 6ql6-q21 (2100 bp sequence) (SEQ ID No. 8)
22A
GCACACCTGAGCAAGGGAGAGGAAAGGGTTCTTATTCCTGACACAGGTAGCCC CTACTGTTGTGTTGTTCCCCTGTTGGCTAGGGCTGGAACGCACAGTCAAAGCTA ATTCCGATTGGCTATTTTAAAGAGAGCAGGCGTAGGAGCCAGAATGGTGGGGC GAGTAGTTTGGCGGGAAGGTCAGTTACAGAACAGGTGACTCAGGATGACTCAG GTCAGAGCAGGTGACCAGGGGTGTCTCAGGATGGAGCAGGTGACCAGGGGTG ACACAGGATGGAGCAGGTGATAGAGGCTAGGAGGGGGTTGTTTACTGAAACT AGGGGCAAGGAGATGACGAGAACGAGAAAGTTAAACTTTAAAATGAAGAACA AAGAACAGGGGAGCTGAACATACTGATAGAACTCTTTCAAGTCTACTTAGGTA ACTATTTGTTTGTTTTTCTGCTTCTAAAATTTTGTTGAAATTTTCTCCTTTCTTAT TCTCATTGTTCTTGAGGTTTCGTGTATTTAAAAAATCTTCTTACTGTAATTGTCA TAGTTGAGTAGGGAGCAACGTTAGATTAATATATTCAATACTTCACTGTTACCT GGAATAAGAGCCCTCTCTTTAAACAAAATATTATGCAGAAATCTAATACAGGA AGCAAATAAAAACTAGAACTACTCTGGTTCAAATAGAGTGAAGACAGAGCAG ATCTTGTTCTTGTAATTGAAAGGAATATGATATAATAAGTATTGACAATATTTT CTTCTCACCAAATAAGTTTCTAATTCTATATATAAAGGAAATACTTTCAGAATA AAACGAATATATGAGTTTTATTTTTAAATCACAAAACGAAGTTCAAGAACATT TTTGAAACTGGGAAGATTCATATTTTAGTATCTGTCAAATGATGATAAATTCGG AAGCCAGTGTAATTTATACCCTAGGGGCTGAGGTCTAATTCAACATATTCCAGT TTCTATTTTCTAAAGCTAAAGAAACATGTGTTACAATGTAGATAGGGAATACTT TCTTAATGAACCATGCTGAACTGTAAGATTTTTAAGACTCCTTTTTAATGCATT ACATTACACTGTATCTTGTTTTCACATTTATGGTGAGGTTAATATAAAAGAGA CATTAAACAAATATATTTCTGCTCTTTACAAAGGATGATTATTGTTTTCTTACAT TTCAACTAAAAATTTCTATAATATTATACTTGCAAGAAGTATAACACTCTTAAT GAGCAATACAGTTAACCTTAAGGTTAACTTGCAAAATTTCATGTCTAATTTAGT ATCATTAACACATTGAAAAATCTCTCCTAAATTTCACTCATCTTGATCAAAATC CATGTTAAAGGTTTTGAAACTACACTTAATACATCTGCCTTATTTTATGCCCCC ACTTATACTACTAGTTATTAATGCACTTTGGACAGCTGGTTCCTCTGCCTTTTGA GGATTCTGTGGTGTAACTGATTGGTTCTCAGCTTTTTCCACTGCCCATTTGGGA TGCAACCCTTTCAAGTCTGCTTAGGTAACTATAATTTGTTCATGTGTTTTTCTAC TTCTAAAATTTGGCTGACATTTTCTCCTTATTCTTGTTGTTCTTGAGGTTTTATG CACTTAAAAAATCTTTCTACTGTAATTATCGTAGTTGAGTAGGGAGCAACATTA GATTCATGTATTCAATACTTCACTGTTACCTGGAATAAGAGCCTTTTTTAAGGG CTCTTACTGAAAAACACAATACACTTATGTTCTTCTATAATGTTTTAAGGAATT TTTTAACATTAATCTCCTGTCTCAGCCTTTAAGGCCATTAAATGACTTAAGATA GTTGCTGTGGCTCCAAACATTGTATCCACATTTCCACAAGAGGAAGTAAAAAA AGAAAAAAATGAGTCTTGCCCTTTCCTTGTAAAGACAATTTCCACAAGGTTCA CATTCCTCTGGTCAGAAATCAGTCATACACCCACGCCTAGCTGGAAGGTAGGT TGGGTAATGTGGACTTTAATTCAGACAGTATTGTGTCAGTAAAAGTCAGGGGT TCTATTATTAAAGAAGCAGTGTAAACAGACATGAGAAAACAAACTGTAGCCTC TGTGCTT
Human Genome Map Xq21.3 (2160 bp sequence) (SEQ ID No. 9)
23A
TAAATATAAATAAATCATCACTAGGTATGTTCTAATAAAATTTCAGAACACCA AAGATGAAGATAAATTTTAAAAGTAGCCAGAGGAAAAAGGTGAATTACCTTTA AAAGGTTACAGTTTAACAAGAAGTCTGACTTTTCAACTACAAAGATTTTCAGA ATAATATCTTCAGTATGCTGAATGAAGTTTTAAAAAGCAGCTGCCAAAAGAGT TTTAAACTGTGTAGGTATATTTCAAGACTAAAGGGAAATGAAAACATCTGTAG ATAATGAAATCTGTAATTACCAACTGAACTTAAGGATATGCTTCAAGTGGAAG AAAAGTTAATTGAAATGGAGGGTCTGAGATGTAAGACAGAATGAGAAATAAA TAAAATAGCAAATATATAAATAAATCTAGATGAACACTGACTGCAAAAAGCTA CAATAATGATAACATCTTTGGGCATTTAAAAAGATAATTAAAATAAGTAAGAG GAGTGACAAATTCTTTGGGAGGTGATTAAATTGAATGAAAGTATTCTAAGGTA CTTGCACAGTCACAAGGGGTGAAAAAAGGTTTTGTTTATAGTAAGACTTTGTC AAGTAGGCATATGTAATTTCCAGGTTACCCATAAAAAATTCAAAACAGAAAGT GAAATTTCCAAATTAGTAGAGAAAAAAGTGACATAATAAAAATTATTCAATTC AAAATGAGGCAAAAAAGAAGAGACAAAGAAACACGGGATATTTAGTACAAAT AAAAATCACAACATAATACAGTTCATTAAAACTCAAATTTGTTAGTAATTCCAT TGATTATATATGAATTATGTGTTCCATTTATGAAGCAAAATATCAGAAGAAAA ATCCACCCATATACTGTTTTAAAGAAACATTTAAGACATAAGAATACAGAAAG TTTGGCAGTAAATGAATCAAAAATGAAATAATGAGTCTACGTGAGGATAATCA ATGTTCACTGAAATATTAGGGTGAAATGCTGATGGAGAACTTTATAATAGATA TACCAGGTCTATAACACCTGAGCCTAATCAAATGTAACACCATAAAAGTGAA ATATCCAGACGCTATCCACCAGATTATAGGAAATGCAAAGCAAAAGAATATT AAAAGACATAAAGATGACTCAATCTTGCAATTCCAGAACATGGGAACTTCTAA AAGATAAATTGTTTTAATCAATATGTAGTAAAAAGGGAAAGGGAACTGTTATT GAATAAAAGTGACATCGTGACCAAATGTAATGTAATAACTTTGGACACTGCTT GAAGAAACCAACTATAAAAATTCATATTGAGTCAGTCAAGAACATGTTTATAT TGACTGGAATTTTATTACTTTAAGGATTAGTATTAATTTTTCAGTGTAGTAATG GATTGTAGTTATAGTAAAAAAAAAGTTCTTATTTTTGAAATTTACATTGAATTA TTGATGAATAAAATTATGTGATATTTGGAATTTTCTTTAACATAATTTTCATTAT TAATAATAAAATCATGAAAAGGAACAACTCTTGTTGAATGCACATTGGAACTC TGTTGAAGCAGGCATTTCTGACCTAGGGGGAAAAAAAACATAAAAGAGAAGA TTTTTATGTGATAAATACAGGTGGTTGCCAGGGGCTGCCGGGTGGGGAAAATG GGGAGATGTTAGTCAAATGGTACAAAGTTTCAGTTGTGCAGGATGAGTAAATA AGCTCTGGAGATCCAGTGTACAACATGATGACTATAGTTAATAATACTGTATT ATATACTTAAAATTTTCTGAGTAGGTTTGAAACGTTCTCGCCATACACACACAG AAAAGGGTAACTGTGAGGTGATGAATACGTATTCAAGCTAATCACGTAATTAG CTCGATTGTGGTATTTATTTCACAATGTATAAGTAAATTAATAAATCACATTGT ACTCAACTATATATATTTTTTGTCAATTATACCTCAATAAAGCTGGGGAAAATG TAAAAATAAATAAATAAATTACCGAAAAAACCCAAACATCCATAAATGAAAA TGATACCAAATCTGGCGCCACTTTTTACAATGGATGTAAAAGTCAAGAGTTAA AATCTTTAACATGCATGCTTACTATGTCGAAAGATCACGTACATGAAAACAAA CATACTTTATTGTGATTTTTTTGAATGTAAGCGATGAA
Human Genome Map 12q21 (2219 bp sequence) (SEQ ID No. 10) 24A
TATAATTATTAACTGAAGTCATAGTTTACATTAAGGCTTACACTTTGTGTTGAA TAGTTCTATGGATGAGGGAAGGGGCTAAAATGCATAATTTTATGCATTCACCA TTAAAATATCATGGGGAATAGTTTTACTGTCTTAAAAATTTCCTTCATTTCAAT TATTTGTTCTTCTCTCCACTCTCTAAAGCCCTGGAAACCACTTATCATTTTATTG TCTCTATATTTCTGTCTTTTTCAGAGCGTCATGTAGCTGGACTTATACAGCAAG TAGCCTCTTCAGATTGGCTTCTTTAACTTAGTAATATTCATGTAACATTGCTCCA TGTGTTTTCGTGGCTTAATAGGTCATTCCTTTTCATTACTGATCATTTTATTCTG TGCATGTACCACAATTTGTTCGTCTACTACTGAATGATGTCTTGATTGTTTCGGT TGTTGGTGATTATGAATAAACTTGCTATAAACATTTACTTGTGTGGATGTAAGT TTTCAACTTATTCAGATAATATTTAAAAGAGCAATTGCTGTATAGTATGGTAAG ATTATGTTTAGCCTTGTATGGAACTGCCAAAGTGGCTGTACCATTTTGTATTCC TACCAGCAATGAATGAAAACACCTGTTGATCTGCATCCTTACCACTATATGATA TTGTCATATTTCAGATTTTAATCCGTCTAATAGATGTGTAGTGGTAGATAGTTG CTTAATTTTCAATTCTCTTATGACATACAATGTTTAACATCTTTTTATATGTATA TTTGCTATCTGTATATCCTCTTTGGTGAGGTGTCTGTTCAGATCTTTTTCCCATT TTAAATTGGATTGTTTTCTTATTTTTGAGTTTTAAGTGTTCTTTTTATATTTTAAG TGCAAGCCCTTTATCAGATATGTATTTTGTGCATATTTTCCCACTCTGTGGCTTG TATTTTAATTCTCTTAATAATATCTTTGCAGAAGTTTTTAATTTGAACAAATTT CACTTTTATGGTGTGCTTAAGAAGTTGTATCTAAAAACACAAGGTCACCTATA TTTTCTCCTGTTACAGAAGTTTTAGACTGTGGGTTTTTTATTTAGCTCTATGATC CATTTTGAGCTAATTTTTGTGAACTGTGTAAAGTCTATGTCTGGATTCTTTTTTT TTCCAATGTAGATATCCAGTTGTTCCAGCATCACTTGTTGAAAAGATTATCTTT TCTACAGTGAATTGCATTTGTTTCTTTGTCTAAGATCAGTTTACTATATTTGTGT GGGTCTATTTCTAGGCTCTCTATTCTGTTCTATTGGTCTATGTGTTAATTCTTCC ATAACATGCTGTTTTGACTATTGCAGCTTTATAGTAAATTTTCCATTTGAATTG TGTCATATTCTTCTTTGTTCTTCTTCTTGTGTATTATGTTGCCTATTCTGAGTCTT TTTGTATTTTAATATAAACTTTCTTGTCAATTTGTTGATACACAGAAAATAACTT GCTTGGATTTTAATGGGAGTTGCAATGAATGTGGAAATTAAGTTGAGAAGAAT TGACATCTTAGCAATATAGAGTCTTTCCTGTCCCTATACATAGAATATCTATCT AGATCTTCTTTGATCTCCTTCATCAGACTTTTGTAGTTTTAGCCACATAGATCCT GTACATATTTTGTTTGATCTATACTTAAATATTTTATGTAATCAATTGACTTTTG TATATTAACTTTTTATCCTACAACCTTGCTATAACAGCTTATTAGTTTCAGGGA CTTTTTGCCAACTATGGGATTTTCTGCATATAAATCATGCAAAATATGCAATCA TGTCATCACCAAACAAATATAGTTCTATCTATGCCTTCCCAATATGTACACCCT TTATTTCTTTTTCTTGTCTTATTGCATTGGCCAGGCCTTCCAGTACAATGTTGAA AAGGAATGGTGAGATACAATATTCTTGCCTCTTTTTTCATTTTACGAGGAAAGC ATTCCTTTTAATAGTAGGCAGTCAGAATATAATATGTAATATTTTTAAAGGCAA TAAATAGACATCT AAGTGAGTTATTTT AAAATTGAGAGTTT AAAATCAAATAA AACTAAAAGAATTTAATAGTTTGCCTCTGTATCATGGAATGAAGGAGTTAACA AGAGCTGATGAGAGAATCTGCTATTTGTCACTATATCTTTTATTAGCATTTGAC TTTTAAAATATGTTACAATGAATATTTTAATAATTTTCTTCATAA
Human Genome Map 7q22 (2160 bp sequence) (SEQ ID No. 11) 27A
ACATCTGTCTGTTTTGTGTGGCCGTCACAAAATAATCAAAGACTAAGTAATTTA TAAAGAACAAAAATTTATTTCTCATAGTTAATGGAGGCTGAGAAGTCCAAGAC GAAAGTGCTGGCATCTCATGAGGGTCTTCTTCCTGTGTCCCCACAGGGCATAA GAGTGTAAGACTATGAACTCATTTCTGCAAGCCCTTTATACAATGATGTTAATT CATTCATGAGAGTGGGGACCTCGTGACCTAAACACCTCCCATTAGATTCTACCT CCCAACACTGTTGCACTGTGCATTGAGTTTCTAACACATAAATTTTGGGGGGCA CATTCAAACCATAACATGGAGTTTTCTGCATTGAGAAATGAAGGATCCATTTA ATACAGGGACCTCAAAATACAAAGAGAAAACTGACTGGCTGTATGGAGCTAG ACGAAGAGGAGTAAGAAACTACTATTTGCAAGGCTGTGTAATTCCAAGGACT GTTATTCTTGGATGCTATGATGTTTTTAAAGAACAAACTACTATACATTTGTAA GTTATTAAATTATTAATATTATTTGAGAATTTCAAATGGCTTAACTAATCAATG TGACATAGTGGGAAAATTGGGCCTTTAATTGAAGACACAATTTGCTGATTACC ACTTGGTAACTTAGCCCTTGCTTCTCTAACCCTTAGTTCATCTTGTAAAATGCAT TAGTTCTACTCCATAGGGGTATTGTGAGATTTAAACGCAGTGCAGCATATTAA GCACCCAGTGTAGTCCCTGATACATAGTGAAACATCAATAATAAATTGTTGCT ACTGGTAGAAATCCCTTGGCGTTTGGTAGATTTCCAATAAATACTAATTCTTCT AAAACTTTTAATGATTATGTAGATAGATATATGCCTAGATCTGGTAACAAATAT GCTATATCAATAGTCAAAACATTCTCTCTTAATTTTATTATGATATATATTGGA AATCTTAGTGTGGTTTTGATTATACTAACATAATTATGTGGCATTAGTATGCCA AATGTACTCACAGTTATGCCAAAATTACCTGCCCCAAATTACAGCTAATCCTTT CTTTGGTCCTAGGAGAGATACGCACACTAGGGATATCACCATAAAAGTGAAG AAACACTTTATTACTGGCTGGGCTTGTTTCTGAAATTCTAACACAGAGTTCTTA TAACATGGACTTTTCCTTGCCTCCTAGTTCAAGCTTGAGGGCTTACTGTGCTCT TGCAGGGAAAGATAAAAGAAAGTGTCAGAGTGAAAGAATGGTCAAATGTATG AACTCTTCTTTTATTTATTAATTTAAATACAGTGACTCTGTTCACTAGTAAACA CACCTAACCCCTGCCTTAGAGTCAGATTAACATCTTCTTTGAGGACAGCCCAAA GAAGAAAAATGCAAGGATGAAGCCTAGAGAGGTTTCCATCTCGTATACTTATA TTCCACTATCTTTGGTTCTTTCTTTCAACCATTAGACTTAAACCCAACTGTATAA TTAATCAAACATGTGGATATTTCCTTGGAGGAAGAAATAGAGAAGTGTCAGGG AAGTTCGACCGCCACCTAAGTGTGTCTGCTTTTTTAATGCTGCCTTATGGTCTA AAGAGATGGGTGAAAAGCAGAGTATTCATTTCAAGGCCATACTATATTATATG CCATCTATTCACTCCAGGCTGCTTGTTGTCAAGGAAGAATAAAAACCTTGATAT CAAAGAGAATTAAGCTCTCAAAATTAGTTTCTCTTTCACATACCAAAGTAACCT TGAGCTTTCTAGCCTGCAAATTTCTCTCCCTTAATATTCTTTCTCTGTTCCGTTC CACTGAAAGTGATGTCACAGTGGTGTAGTTAGAGTCTGGGTTACTCTTTCCTGC AGAACTGTTCTTCAGTACCTCTAGATAGAAAATAGTCCAACATCAAGTCTTGC ATGAGTTTTCCTTTACCAAAGATCTAGTAGTCTAGAAGATATTTAAAAATCACA TTATTGAGCCCACATCTGCAAAAAGGAGAAGTATATACAATATTCTTAGGACT CAATACATATTACTTGTGTGCTTGTCTTTGCATGGACATGTATGTGTTTTAATTT CTCTTGCGTAAACACTTAGGGTTGGCATTGCTGACCCACATGGTAAGTGTATGT TTAACTTTATAAGCA
Human genome Map Ilpl5 (2160 bp sequence) (SEQ ID No. 12) 29A
TACTGCTTCATCCTTGAGTTCTACAAAAACACACACACACAAAAACCAACAAA ACTTAACTATAGGCTGGGTGAAGTGGCTCATGCCTGTAATCCCAGTGCTGTGG GAAGATCTTTTGAAGCTAGGAGGTTTAAAATCAGCCTGGGCATCAAGGCAAGA CCCCATCTCTACAAAAAAAAAAAAAAGCCAGGCATGGTAGTGCACACCTGCA GTCCTAGCTACTCAGAAGGCTGAGGTAGGAGGATCACTTGAACCCAGCAGTTT GAGATTGCAGTAAGCCATGATCACATTACTGCACTCCAGCCTGGCTGACAGAA CAAAACACCACCTCTAAAAATAAAAATATAAAATAAATAAAAAAATTTAAAA ACCTAAACATAGCTGCACTTTACTCAATATATTTACAGTTCTACATATGTAAAA ACTTGTATATTGACTATGTTTTAAATGTGTAGGGGAAGTTTCTCACCTAAAGGA GTCCCATAGTGAACATTTAAGAGCAAATGATTCCTTTTTTATTTGTATTTTTGGT TTTGCCTCTAGCACATCAGGTATTCTTTAAGAAGGCTATGCCTCTGAGGTTGCA TGATCATTAACTAATTCATAATTTCCCTTGCATATATTTGGGTATTTTGGTGTTT CAGCCTTTCCCACACTTTTTTTATTTGCATGTCTTCACGATCACCATTATATCTT TGTTCCACCTGTACTATTATTTACTCACTCTTTGTCTTTAAATCAAATCACGTTT CTTACTCAAGTAGATTTAGTTTTAAGACAAACCTTATGGCCGGGCACAGTGGCT CACACCTGTAATCCCAGCACTTTGGGAGGCCAAGGCGGGTGGATCATGAGGTC AGGAGTTTGAGACCAGCCTGGCCAACGTAATGAAACCCCGTCCCTACTAAAAA TACAAAAAATTAGCTGGGCGTGGTGGCGGGCACCTGTAATCCCAGCTACTTGG GAGGCTGAGGCAGGAGAATCACTTGAACCCGGGGGGGCAGAGGTTGCAGTGA GCCGAGATCGTGCCACTGCACTCCAGCCCAGGCAACAACGCGAGACTCTGTCT CAAAAAAAGAAAAAAAGGAACTTTATGTCGCTACCATAAATGTGAAATTACT AGAACTCACAATAAATAGAAGTTAGTAAAGACACTGAATTCTAACTAGACGCT ATTGCTTGTTGAAGGCTTTGATCTTAGGAGGATTAGAAAGCATTCTAGGCCAG GCACGGTGGCTTCCTGTGTGTAATCCCAGCAGTTGGAGAGGCTGAGGCAGGCG GGTTGCTTGAGCTCAGGAATTTGAGACCAGCCTGGGCAACATGGCAAGACCCT GTCTCTACAAAAACATACAAAACTTAGCCAGGCGTGGTGATGGCCACGTATGG TCCCAGCTACTCAGGTGGCTGAGGCAGGAGGATTGATGAACCTGGGAGGCTAA GGCTCTAGTGAGCCATGATCACACCACTGCACTCCAGCCTGGGTGACAGAGCC ACACCCTGTCTCAAAGGAAAAAAAAAAAAAAAAAAGAATTCTAGTGGTGTGG TGTGGAAGACACATTCTCAGCAGACTAAGGTTGTATCTTTATAACCACAAGGA TTGAAAAAGAACGGAAGGACAATAACTTTCTCATAAGGTGATTCAATGTTATT TAGTGCTGTTTCTGTGTACCATCAAAAATCCTCTTACTACACACAGAATATTAT AACACCATCTCATTGTCCACATGAGCTCAGAAATTGGTCATCAAAGCAGAAAA GTCTTTAAAACATTGATCTCCGGCCGGGCGTGGTGGCTCACACCTGTAATCCCA GCACTTTGGGAGGCTGAGGCGGGCGGATCACAAGGTCAAGAGATCGAGACCA TCCTGGCCAACATGGTGAAATCCCATCTCTACTAAAAATACAAAAATTAGCTG GGTGCAGTGGCAGACGCCTGTAATCCCAAGCTACTCGGGAGGCTGAGGCAGG AGAATTGCTTGAACCCAGGAGGCAGAAGTTGCAGTGAGCCGAGATCACGCCA CTGCACTCCAGCCTGGGCAACAGAGCCAGACTCCATCTCGAAAAATAAAATAA AATAAAACATTGATCTCCAAGAAAGTAGATCATATCTGCTCTCTATCTGACCAC ATTGTTAAACTTGGTTATGTTTGCAGGTTAAAG
Human Genome Map Xp21 (2040 bp sequence) (SEQ ID No. 13) 3OA
CAAAGACAGGCCAGTGCTCTACTCCTTGCTTCCTGGGCTCCCCAAAAGGGAGC TGACTCCTCATCCCTCAACCTGGAGAACCAGTTCAGCTCTTCTTTTCACCAGAA TCCTTTCCCTGCTTCCGACTCATCTTCTTTTTCTCAAAGCTGTTGTAACTGTATT GTTCTCACCTGCTTTGCCCAAGACAGATTCCCCAGTCCTCCCCATCAGTGTTTG GCATTTATTCTGGGTGTTCTACTAGTAATGCCCAGCCCCGGTCCTGGGCTTCCT GCTGTTTCTATTGCATCTCCCTAACTCTTACATCCACCCCAACTCAGTGTTTTTG GCCTTCCTCAGCAACCAGGAATCTAAACCACCCTCCACCCCATAGCACCCTAT GGATGACGGAGCCTTAGTTCTTGATGGTGATGCAGACACCTTGAGGTGTGGCC ATGACATTCACTCAGCCCTTGGCCTGGTAGCAGCAATTTTCCCTGATAAGGTCC CCAAACTGACCCTCAGTTGTCCCCTGCAGTCCCATTAGGGCCTGTGGAATTTAC GACTTCCATACACAGCACCAGGAAGTTGAGGATGGCTCCACGTGCTAGCTCAG TCTCTTTGCCCTCTCTCTGCCTGTGGCAGATTGTATTTTCCAAAGATGACTGCAC CAAAATATTCCACCCCATGTTATCTTCTTAAATGTGAAGTTCACACTAATTCTT CAAGAAATGGGGCCTCTGTTTACACCTGCTGAATCTTGGCAGGCCTATAATTAT AGTGGTTATGATTCTAGTGATGCTATATGACTTCTGAGACCATAAAAAGACAA TACAGCTTCCACCTGGTCCTATTGGAACAGTCATTCTTGGAACCAAGCCACCAT GTTGTGAGAAAACCCAGCCCACATGGGAAGGTCACATGTAGGGATGACAGTCC CCACTGAGCCCCAGCCAATAGCCGGCATCAACTGCAAGACATGTGAGTAAGCG AACCCTCAGATGATTCCAGCCCCCAGCCTTTGAGCTGCCCCAACTGATGCTTTG TGGAACAGAGAAAAGCTGTCCCCATTGAGCTCTGCTCAGATTTCACATTTATG GTAAAAATCTATATGGTCCTTACTTTAAGTTACTAAATTTGGGGATGCTTTCTT TACATAGCAGTAGGTAATTAGAACACTGCCTGATCAAACTGCACTGCAACTTT TACTCGGCTGCTAACTATATGGCTATAGCCGAGCATCATGGGGCCACCGTGTCT GGCAGTCCCCACATCCGAGTTCCAAATGCGGAGCACAAAAGTCCCACTGTCAC TGATCTTCCCTTCCACTCTCAGAATCTCAGTCTAGTATGGGGAAGCAAGGGTCG AACCATGTGCTTCCCCCGTCAGGGCAGATGGTTCTCTTCCTGCCTGGAAGGAAT TCCCTCTACATAAAAGCCTCTTTCCACCAGGTATGGTGGCTCAAGCCTGTAATC CCAGCACTTTGGGAGGTGAAGTGGGCAGATCACCTGAGGTCAGGAGTTTGAGA CCAGCCTGGCCAACATGGTGAAACCCCATCTCTACTAAAAATATAAAAATTAG CTGGGCATGGTGGTGGGCGCCTGTAATTCCAGCTACTCGGGAAGCTGAGGCAG GAGAATCACTTGAACTCGGGAGGCGGAGGTTGCCGTGAGCCGAGATCATGCCA CTGCACTCCAGCCTGGGCAACAGAGTGGGACTCCATCTCAAAAAAGTAATAAT AACAAAAAATTTTTAAAAAGTGTTCTTCTTCCCAAGAAAGCAGACATCAGACA TCTTTCCCCCTTCATTGGGGCCTTAATTGCAGATGGGACTCTGGAAGAGATACT GACATAAGCATAAAAGTAGGTCCAAGAATATTTAACCTCTACATAAGAATTCA AATAAGCTATTGACCTTATGAGAGAGTCACAATGATGGACACCTTCAAAGGAA GGAGAAGCACCATGGAGGGCAGAGGAGAAAGACCATACCTGCTTGACTTGGT GTGAGAGGCATTCTAGATGTTATTGACATCATATAATAGACAGATGACAAGCA TAGAAG Human Genome Map 12q21 (2100 bp sequence) (SEQ ID No. 14) 31A
TGTACATGCATTCATTTTGCTATCCTGCATTTGTTCTTTCTTCCTCAGACCTTTTC AGTCCCTAAGAAAATGGTATCATTCTGGGTAGGACCTAAGATCAGTGATGAAA TAACAGAATGCAGGATGGCAAGACTCTATATGGAGAGGGAAATTTTACAGAAT CTAAACCTGGGGATACTAAATTAGATCAATTGAGTATAGGCAATATCAGAGGG TAATAAAATAGTTTAAGAATACATAGATGTTTTTTGTTTGTTGGTTAGTTTTTGC TTTTCTTGTAATTCAGGTTAAAGATGTGACATTTCCTAACAGCACCAAGGAAGG AGCACCAGAGAACATGAAAAGGCACTCAAGCTAAGTGGTCTGTTGAACTTTG CATATCTTCCTCTTTGTCAATGAAAGAAGGCAAAACCAAACCTAAATGAAACA AAACAAAGAACTACTTACAAGGAGTTTTAAAATATCTCAATCTCTGCGTTACTT ATGTAACCCACAGAGTATATAGATGACCACTTAGAGGTATTCCTACTATTAAC AAAGGTATTAGTATCTGTGTTTTACCCATGAAAAATTAGAAACTTAGAAAAAT ATCTTGTTCAGTTTCACACTGGTACAATATGAGGGTATGAAAGTTGTAACTGAC TCTAAAGCTTGAGCTATTTTCTTATAGATATAATTTAAATAATGTCTATCAACT TTCTTGAAATATTCTCATTGTTACCTAAGAATTTAAAATATTGATATGCAATTG ATCTAAGAGAGGTTAAACATGAATTGGAAATTCCTTCTCCGATATCAGGTTTG ATTTTTCACAAATATCAATGTTTATGAAAGTATTCCTAAAATTTCAGGTAAAAC CACCCAATAATATGACTGTGAAAAATTATGCTTCTTCCTCTATGAGCTACTACA TTCTTAAATTATCTGAGCATTCTATTAAAACTTAAAAAAATGCTTAACTTGAGT CTGCATGAATCTGAATTCCCTGCATATTTAATTTTAAAGAAAATAGTTTATTTT TTTGTTAGACCAATACTTTACAAACTTCCCCAACCAATAAGAAAGAACAAAGA GGAGAACATGAATATCCCTGGGTATTTGTGAGTAAATCCCCAAGAGACTAACC ATAAATGTGAAATTTCTTTATAATTGTATGTCTTCTTCTAAAATATTCATGTGG ATTGTGCATTCTATTCTATCTTCTTATTTTAATAAAATCTGTTTTAAATTATTTA CTTCCTGGAAACAAATCTCCCTGTTGTGTTGGTTTATGAACATGGTTCTATTGC CTTCAGTCTATTGTCGGAAATAAAAACAGTCCTGCAGTTGTTGATTGAGTGTAC TATGCCTTTAAGAAGTCATGGCACTCATGCAACAGCCATGTAGTTGTTGATTGA GAGTACTGTGTCTTAAAAAAAGAACTTTTGCTAAATAAACTGACTCTGTGAGC AGCCCTTCATCATTTAAGTGAGAAATGTATTGAATTAAGTTACCTTGATATTGC CTTTTGTTATATTTTTATTTCTTTGATACAAAAGAGTAACAATTTAATTCGAAAT TTGAAATCCCTGAATTGCCTATCCTCTCCAGTAAGTCACTACACACCTGTATAG GGGAGCAGCCTTTCAGAATATTTTTCCTGAACATGAGAATATAAAGCAGGAGG TGGTCATATTTGTTTGAGTAGCACCTCCTGATACCATTAATCTGAGCAGAAGAG TATGGGTCCATACTAGAACAGGATATGACTAGGAAAATGAAGAAGAAATGAA AGCAAAGTATTCAACAGAAACATCTATGCTTTTTGCCATTAGCTGAATGTGAC ATAAGAGTATAGAATGATTTGACACGATTCCAAATCTAAATGTAACCAAGGAA CTTTAAATATTATTAATGAGCATGGCAAAGTTTAGGGTCAGGGGGAACAAATT TAAAAACTATGAGCATTCTCATGACATGAGTTAAAATGCAAAGACGTAAGTTA AAACATAACTAATGACATTAATAAAGTGATTGAAGCTCTATGTCACTTAAAGA TAAGAAGGTATGATAGTTTAGACATTGTTCTAAAGGCCAATCTAAGTGAAAAA AGTTTTCAGG Human Genome Map 17q21 (2100 bp sequence) (SEQ ID No. 15)
32A
AGTTAGAATTCTGTGAGGTTTGTATAAAAGGAATAGAGTGGGGGCCCAAAAAC CAGTAAGATGAGAAAGTACTGTTTGCTCAGTTCTAGGATCCATGAAATAAATA ATAAATAAATAAAAAAGAGAAAGTAGTGTTTCCTGCCACTTTAGAGGAAGGAC TCACATATCCTACCTTCCATCAGCCTTGAAGGAGATGAGTGCCCTCTCTCCAAC ACCTGGTGGCCTTCCCTACCCCTTCCCCAAAGCCTCCAAGAAGGCCCCTGGCCT AGCCTGATGCCCACTATCAGCAGGAACAGGCACGACAAACTTTCCCCTTCCTA TCCCTCCCCACCTCTGGAAAGGGCTGGGGACAGCAGATGTGTCCTTGTTAGTTC CATCCATTTCAGCTTTGGCTGGGGAGCTAATTTCACTGGAGCCAGGATAAGCA TTAGGGTAAGTAACTATTTTTCCTGTCTTGGGCAGTTTCCTCACTGACAAATGA GGGCAGAGTTCTAAGCTCTCTTCTAATTCTAAAATTCTAATGTAAAAATTGCCA GACTAGTGGTGGCGCAAGCCTGTAATCCCAGATACTCAGGAGGCTTAGGCAGG AGAATCGCTTGAACCCAGGAGGCGGAGGTTGCGGTGAGCTGAGATCGCGCCAT TGTACTCCAGCCTGGCAACAAGAGGGAACTCCATCTCAAAAAAAAAAAAAAT CACCAGACTAATATTTACCTTGAGTGTTATGCGCATCCATGTGAAGAGACCAC CAAACAGGCTTTGTGTGAGCAATAGTTTTTTAATCACCTGGAGTCAGCAAAAG GAGATGGGGTGGGGCAGTTTTATAGGATTTGGGTAGGTAGTGGAAAAATTACA GTTAACGTGCGTTTTCTCTTGTGGGCAGGGGTGGGGGTAACAAGGTGCTTGGT GAGGAGCTCCTGAGACTCATTGTCCAGGAGAAGGAATGTCACAAGATCAATTG ATCAGTTAGGGTGGAGCAGGAACAAATCACAATGGTGGAATGTCATCAGTTA AGGCAGGAACTGGCTATTTCACGTTTATGGTTCTTCAGTTGCTTCAGGCCATC TGGATGTATATGTGCAGGTCACAGGGTTATGATGGCTTAGCTTGGGCTCAGGG GCCTGACATTGAGGATTCTTTTTTATCTTCCTCTGATGCTCTTCTATAAGAATGA CTCTGTTTTGGAAGAAAACGCAATTAAGATTTTCCATCACAACAACCACTATCT CCAAATCTGTATTCATTCCTTTTAATTCATTATAAGTCTCATCTACCTAATGAGA TAACTTTTTTGAAGACAGGAATTGTATGCTGTTTAACAGTGCTTTGTTTCTTCCA TAGTTCAGTCATCCTTGATATTTTGCGGGGGACTGGTTCTAGGATACTGCCCCC ACACACCAGAATCTGTGAACGCTCAATCCCTTACATATAATGGTGCAGTATTT GAATATAACCAACACACATCCCCCCGCCACCACCCAATTAACTTTTTTACTTTT TTTCCCCCCCGAGACAGAGTCTTGCCCTGTCGCCCAGGCTGGAGTGCAGTGGC ACGATCTCGGCTCACTGCAAGCTCTGCCTCCTAGGTTCATGCCATTCTCCTGCC TCAGCCTCCCGAGTAGCTGGGATTACAGGTGCCCGCCACCACACCGGGCTAGT TTTTTTTTTTTTTTTTTTCTTTGTATTTTTAGTAGAGATGGGGTTTCACCATGTTA GCGGGTGGACCTTGTGATCCGCCCACCTTGGCCTCCCAAAGTGCTGGGATTAC AGGTGTGAGCCACCATGCCCAGCCAATTTTTGTATTTTTAGTAGAGACGGGGTT TCACCATGTTGGCCAGGCTGGTCTCGAACACCTGACATCAAGTGATCCGCCCA CCTTGGCCTCCCAAAGTGCTAGGATTACAGGCATGAGCCACCGCACCCAGCCT CAGACTAAACTATAATAAAAGAGAAAGCAGAGAGAGTAAGAGCACCTCATAT GGAATCACCTACATTTCAGAAGCTGGAAAGAAAGAGAGTGGTCTACTTGATGA TATGAAGCATGATCAATCAGTATCAATACTAGCTTTAGGGTGAAGGCATAGCC AAATTGGAAACTGTGGA
Human Genome Map Iq32.2 (2100 bp sequence) (SEQ ID No. 16) 33A
AAAAAAAACCTTGTCCAGGCACAGTGGCTCACATCTGTAATCCCAACACTTTG GGAGGCCAAGGTGGGCTGATCACTTGAGGCCAGGATTTTGAAACCAGCCTGGC CGACATTGCAAAACCCCACAAAAACTAGCCGGGTGTGGTGGCACACACCTGTA ATTCCAGCTACTTGGGAGGCCGAGGCACAAGAATCACCTGAAGCTGGGAGGC AAAGGTTGCAGTGAACCAAGATCATCCCACTGCACTCCAGCTTGTGACACAGT GAGACTGTCTCAAAAAATAATAACGAAAATAAAATAATCTAAAATTTAAAAA AACCCTAATTCATAGTTATGGAATTATTGAGCATATTAAATAAGATAATGCAT GCAAAGTACTTAACAGCATCGGACATATTTTAAGCACTCACTGATGCTTGCTAT ATAGTTAAATTATATAGCTATATGTATGTGTATATACATGCAAAGATCAGGAG ATATGCTGACATAGAATGACTATGGCAGGGTCCTGAAAGAGACCACAAAAGA GAGAAGTTTTCACACTGGTTCTTTCCTTTTGGGGGATGCTGACAGTTCCCCTAG AAGGCAGCAGACTTTGCCTCTGGGAGCAAGCCACTGGCCTGGCCCAGCACTCC TGAGATGAGCAGAAATGGGCAGAGGAGAGCTCAAGACAGAGCACAGGCCAGA CTAATGTCTTCCTGAGAGGAGAGCAGTGGGAGGAAAAAGGGGGCAGCAAAGA GACAAGAGATTGCCTCCTTCACCTCCACCAATGTTACCTAAGCTAAAACCCCTC TGTCTACCAAATCAGCCCTGGTCACAAACTAAAACCCAAACCCAACAGGAGGC TTACTTATCCACGCCAATCTGAATTTCTCCATGACATGGACCAGGTGGGACTGT GGGTTTGGTGCCATGTACATGACCTGTGACTTAGTGGATGGAGTTCCTTAGGCC ACAGCAGCCTCTGGCTCAATGAAGCTTGATCTACTGAGTACCTGGACCACATG GGGCTCTAGCAGCAGTCCTATCTTGAGCCCAGAACAGTAACTTTCAATAGAAT ATACACTGGTATGTTGAATCAGAAGTTCAAACGCCCTTCACCTTTATGGTGA CTTTTCTCTCAAGGACCTCCACTGCTTTCTTCTACTATGCCTTCGTATCATTGAC CATTCCATCAGTGAGGGCCACCACAGTCCCTCAGAATTCTTTAAGACTAACTA GGGGGAGATTAGAGTACCAATCCTTCTAAACCTTTCAAAAGGCTTCTTTTGAAC CCTTTTCAAAAGATTTCTTCACTTAGCACCCTGGAACCAAATGGAAGTGAATAT TTTTGAGAAGACGTGACATCTTTCTCCTGGGCCTTGCCCAGCCAAAAATGTTCT GTTATCTGTTGCAATTAAAAGAGAGCAAAGAGTAAGAAGTCTCTTTCCTTAAA GTTTCTTTGGCCACTTGAGCGGAGCTTCCCAGAGCAGTAAACTCCTTTAGGATA GGGACTGTTGGAATTAAATGAGCTGGGGAACCACAACCTAGAAACTGGACTTC AGCTTTGTAAACTCCGAAACTCATTATCACTGTGATGGTTAATTTGATGTGTCA ACTTGAGTAGGCCATAGGGTGCCCAGATTAAATGTTGTTCTGGGTGTGTCCATG AGGATGTTTCCAGATGAGAATAGCATTTGAATTGGTAGACTCGGCAAAGTAG ATTGCCCTCCTCAGTGTGGCTGGGCATTGAAAAGGCCGAAGAAAGAATTTGCC CCTCTTTTCCTGCCTCACTATTGAGCTGGGATATCTCATTTCACCTTCTCCTGCC CTCAAACTGGGATTTACATCATCAGCACCCCTGGCTCTCAGGCCTTTGGACTT GGACCAAACTGCATCACTGACTTTCCTCCATCTCCAGCTTGCAGACACAGATTA TTGGACTTCTCAACCTCCATAATCATGTGAGCCAACTACTCATAATAAATAAAT AAATAGGCTGGGTGTGGTGGCTCATGCCTGTAATCCCAGCACTTTGGGAGGCC CAGGTGGGCAGATCACGAGGTCAGGAGTTTGAGACCAGCCTGGCCAATATGGT GAAACCCCCATCTCTACTAAAAA
Human Genome Map 5ql5 (2040 bp sequence) (SEQ ID No. 17) 34A
TACAGGATGAAGGTAACAATAAGAGAAAGTGATCATAATAGTCAATATTTAAT ACATATTTAATATATATATTTTTAGATTTAACAACTGGTTAAATCTATTAACCA TATATCATCTAACCATTTCTATACCTTCCTATCACTCTTCTTTTCCTATTCTCTCT TAATTCCAATTTTCCTACAACACACACACTCATATACACATACGCACACATGCA CCCACTATCCATAAGACCATCACGTCTGGGGATTTTGCACATACAGAGCCTAA TAAAATTCAGCAACAAAGATCATTCAAATTCATAACTCAAAAATTATCAGGTA CAGAAATACATGAACTGAGGTGAAGAAAAGCCAAAATACTGGGAGAAGTAGA ATATCTTTCACAAAAGACCTTCTAGGAAGATCATATGGTACTGTTCTGCAAAT CTTTCCTTTTACAACATTGAATGTTTTAATGTGAGCTTTGCAGATTCAGTTTCAA ATCTTATCACTAATCCTGCCCTTAAGCAAAGCTGTAAAGAAGGTGAAATTAAT TTTATACATTTCCTATTCTGCATTCTGTCATCCTCATCTTCCTTTGAGGGTCTAA CAACTTCCTACCACTTTCTGCTTGTGCCCATTACAACCCAGATTTTCATCTTTT GTACCTGGAACAGGCCTGGCCTTCCACAATGCTCTATGTGCTATGAAAGTCAG TTTCTGCTATTATCATTGTGTTCTATTATTTCATGTATTTTCTAATAGCCTTAAA GATTACTTTGAATAGGCCTGGAATCTCTAACACAATTAAATACTATAGCAGGC ATATAGTATTACCAAGGAAAGTAAAGCAAGATATAGAACAAATTGAATGATG AAAACTGAGACAAACTGGAGACAGGGACCATGTCCAAAATGGACATCCATTA TTCAACTGTAGCCTCATGTTATCATGTGGGAATGAAGGCCTAGTGTTGTAAGAT TTTAGATTTTTCAAGAGGAGCCAGAAATTTGTACCTTCATACAAAAATTTCAAT ATTTGTAAAACACCATAAAAGTGAAAAAAATACCTACAAATCAGAATTTAGA CTATGTTGACATTAACTTGCTACCTCTGTGATAAGCGATTTTAGAATAAGATTT TATTCTTGCTTAATTCTTCTCTTCAGAGATACCTAGTGTAGTAGGAGAATCTAA CACTTAAAAACAGGTCAGGTATGGTGTCTCACACCTATAATCCCAGCACTTTTG GAGGACAAGGTGGGTGGATTGCTTGAGCCTAGGAGTTTGAGAGCAGCCTGGGC AACAGAATGAGACCCTGTCTCTACAAAAAATACAGAAATTAGTCAGCTGTAGT GGCCTACGCCTGTACTCCCAGCCATTTAGGATGCTGAGGTGGGAGAACACTTG AGCCCAGGAGGTCAAGTCTGCAGTGAGCCATGATCATGCTACTGCACTCCAGG CTGGGCAAGAGAGCAAGATCCTGTCTCATATAATAATAATAATAGTAATAATA ATAACAATTTTGTGATGGGTGATAAATATCATAGGGGCAAAATGTCATGGGAG CACAGAGATGGGAGAGGGGACTTTCATAAGCCTAGAATGTTCTCAAGGAGTAT GTCTCAGAGAATATGAATCTCAAATGAAAGTAGGCCTTTACCAGAGAGAGAGA AAATAAAGAGTATTCCAGAGAGTTGTGTGTGGGAAGATGCAGACATAAGAAA CAGCAGCATTTACTTGGGGAAAAAATAGTTTAGGTTCTGTTCCCAGATAAGTG GAATTATATCAGATACAGTTTTTAAGGAGAGTCTATTTGGGGCAGGAGGGCCT CTTGAGTTCTTATTAATAGTTTTAAAATGTGAACACACCTTACTGCACATTAAG CACATGTACCCCAGAACTTAAAGTATAATAAAAAAAAATTTTAAAAAAAGAA AAAAAATGTGAACACACCTCTATTTCTCTCTCCAGGTAATTTTAACATGACCTT GCTACTCCCTTGGATGAAAGGATCATCACGAAGTTTTACAACAAACTTTATGGT TATGGAAGTTCT
Human Genome Map 8pll.2 (2100 bp sequence) (SEQ ID No. 18) 35A
TCTAAGGCTTCTGGACCTGAACTGAGCCATGCTACCAGTATTTCAGGATGTTCA
GCTTGCAGATAGCCTGTCGCGGAACTTCTCAGCCTCTAGAATCACATGAGTCA
ATTCCCCTAATAAATCTCCTTTTATCTATCTGAACATCTCTCTTCATCTCTCCAT CCATCCACTCATGTGTCCATCCATCCATCCATCTATTGCTATCTATCTATCCATC CATGCATCCATCCATTCAACCATCCATCCACCCATCCATCCATCCCTGTGCCAT CTATATCTATCTATCTATATATCTATCTATCCATGCATCCATCCATCCATCTATC CATCTATCCATCCATCACTATCTATCCATGCATCCACCCATCCACCCATCCATC CATCCATCCATCCATCCATCACTATCTATCCATCCATGCATGCATGCATCCATC CATCCATCCATCCATCCATCCATCCATCCATTTATCGCTATCTATCTATCCATCC ATGTATCCATCCATCCATCTGTTCATCTATCACTGTCTATATATCTATGTATCTA TCTATCCATCCATCCATGCATCCATCCATGCATCCATGCATCCATCTATCACTA TCCATCCATCCATCCATCCATCCATTCATCCATCTATCTGTCTTCTACCTACCTA CCTATCTAACTCTCTGGAGAACTCTGACTAATAAACTAGCTTTATAAACATGTT ATTCTCTCTCTGCAATGTCTATTGCTTTATCTTCAGGAACATTCCACACATCCTG TAAGACTTCAGTTAAATTATCTCTCTGTTTCTTCTCCAATCATCCTCTGCCTTCC CTAGTCTCCTAACGTACTTTGTACATCTGTCACAAACCCCTCATCATATTTACT GTAATTTTTTTCCTACAGATTTGGATAGGAATTGAGCCATTTTTTTAATTTCAC TTTTATGGTTGTTACAAATAAAAGAGCAAGCAGGCCCCTCACTGTAATTCACC TGTATTTGCATTTAACTTATTAACCAAGGCATACTATTTCAAATAATCTAATAT AGTATTTCCTATTTAATAACCAAACATACAGAACAGTTCCAAGCACATGTAAC CATGTGATACATTTTCCTCTTTGAATAATAAATATATTTCTTATAATTAATATGT GATAAAATTGCAATATTTTTAATCTCCTACATCCTTCTCTTTTAATCAGGTTTCC TTATCAACTGGTTCCTATCTCACGGGGTTGTTGCAGAGATGAGGAAAAAAAGT ATTCTATTGGTTCATGCATCTCAAAATAGGCAGATTCTTTTCTCTGCTTCTTCCT TCATTGGCTCAGGTGTGGAGTGCTTCTCCCAATTATATGTGCCAGCCTTGGTAT GTTCTCATTGCTGTACCACACTGCCTGAGACATCCAAGACCACATCTTCCTTTG GGGGCACATTGGACCTTTGTCATTGGCACTGGCAGGGAAGCTTTTATTTCACCA GGTCTAAGGCAATTCTTCCAAAAAAATCCCAAATAGTGAAAGAATTGATTTAT TCTTCTAATATTTAAGCAAATGTAAAAAAAAAGTTACATTAGTTATGTTTTTTT CAGATTTTGGATCAGTGAGACTTCATTAAAACACTTTGAGGTTATAAAGCAAG TAATTTTTGTTTCCAGAAAAGTTAGTTTCCTTTGGCTGAAGGGACATCTCTATG CAGGCCAGATCAAGACAAAAATAACTTTTAAGAAGGGAAATGAGGGAATGGA GTTTGGAAAACATAAATCCCACAGCAAAGTACGTCACCAACAATAAGAGTCAT CTCTTTCACAGAGGCCTTTCCTAGAAAAGCCCTGACAGACTAGGAGTCCAATC TTCGGCTCCCATAGCACCCATGCCTGCTTCCACTCTGGAGCTTACTACTTTGCG TTGAAATTAATTTTTACATGTCTATGGCTTCTATTACAAATAGCTTATTGAAAA GAGAACCATGTACATTACAAATACTTTTTTAGAGTTGCTGAACTGAACAAATC AGTACCTACGGGGTTAGTATGCTGGCTTCTATTCCAGCAGGGTTTTGAGCCATG AGATTTTGAATGCTCCCGACATTGTTAGTTCAGGATGATTAAAAATAT
Human Genome Map 3pl2 (2040 bp sequence) (SEQ ID No. 19) 36A
TGAGAAAATGCAAGAAAGGAAGCCAGAGACGTTGTGGACTGCAGGCTCCTTC CCCATCATGTCACTCAAACACAATGTTTCATTTGTAAAACATATTTTAAAAGAT TATGAATGCTATTTAAAAGAACACTAAAGGCCGGGCACAGTGACTCACTCTTG TAATTCCAGCACTTTGGGAGGCTGAGGTGGGCAGATTACTTGAGTTTAGGAGT TTGAGACCAGCCTGGCCAACATGGCAAGACCCTGTGTCTACTAAAAATACAAA AAACAAACAAACAAACAAACCCAACATGGTGATGTGTGCCTGTGGTCCCCACT ACTTGAGAGGCTGAAGTGGGAAGATCACTTGAGCCTAGTAGGTGGAGGTTGCA GTGAGCCAAGATCACACCACTGCACTCCAGCCTGGGTGACAGAGCAAGAATA CATATATATATATATATATATATATATATATATATATATATATATATGTATATA TATATATATATATATATATATGTATATATATATATATACAGCAATCAACCAAAC AACAGCAGCAAAACATTAAAGTGACATGCTGACTCCTTGGAAAGGTGGTAAGC TCTTTAGGCTTCCTAATCAGAGAAGGCAGATAAAAAGAGTAATTTAACATCTT TCTAATCTACCCCAATGGAATTTGCAGTGATTTTCTTTCCATTTTTTCTCATTGT TTTTCAACCTGATCACTAATAGGTAGCTGAAATGGAGTCACTTAATGGTTTTTG CTTTTTAAGAATTCCAAACTCACAGTTTGAAAATTATAGTTCTTGCATTTGAAG TTTTTCTTCATCGTGCTTGGCCTGCCTGGTGGCTTTTTGTTTTGTTTTGTTTTGGT TTGGTTTGGTTTGTGGTCTCTTTCATCTACCCAAAGCCAGTTGAAATAAATCAA AAGTTGTCTCACTCAGGAATTGTCTAAAGTAAATAAATGAAAAAAAAAAAAA AAACAAGACTAATAATTAGGCAACTCATTGAGTAGGCTGTTGAACCAGCTAAA GTGGGAAAGAAATTATTCAAGTTCTAAACCTTTCTACTTGCAAATTAGCCAAA ATCAATTGCATTTTAAGCACTGCATCACCTTGATTGATTTTTAAAATGGATAGC ACTTTGCTGTTCACATTTATGGTGAGCCGTGAAGGACTTGGCAATGGGCATC TTTTCTACCGTGTTCTGCATTAAACTCTTTAAATAGCTTCTGCTTCTTAATGTTG AATGAACTTTACTGCAGACTGAGTCTGAGGGTTTTTTTTTCAAGGTTGAAATAC ATTCTTCAGACTTTACTTTTTGCATGTAGCATTCTTCTTACTTAAAATACCTAAG AGTTTCTAACTAATTTCTTTCAGCCACAGGAAATATTTCTGTAATTTTAGGGTT AAAATGGAGAACTATAGGATGAAAATATAATACAAGAAAATGAATTAAACCC TAAAATTTATTATTATATGTGGGTAAAAGTAGGGGGGAAAATCACTGGTTTTG AAATTAAAAGATGAAAATTGTGAACACTTGCATGGAGACCCTATTTGTAAACT TGGACAAGCTTCAGTGCCCTCTCTCTGCATCTTCTGTCATTATTATTTTTCCTCA AGGCATCGTTTTGAGGATTAAATTAGAAAATGTGAACTAGCTTGAGAGTACAG TCAGCACTTGAACAGCATGGGTTGGAACTGCAAGAGTCCACTTATACACAAAC TGTTTTCAAACAAGCTCTGATCTAAAATACAATATTTGTAGGATCTAAAACACG TGTATACAGAGGGCCAACTTTTCACATATGAAGGTTCTGCAGGGCCAGCTGCA GGACTTGAATGTGCCTGGATTTGGGTATAAACAGGTAGTCCTGGAACCGATAC ACCAAGTATACTGAGGGACAATTGTAATAGGCACAAAAATGTCGATTGATAGA TTTTGTTCTTTTTCCTGAAATGCAAACCAGGACACTTACAAAACTAAATGAATA ATTACTTATACATTTAGTGTCTCTGTGCTTCTCTCTTTTACCTTTTCCTACTTCTT CCGTAT Human Genome Map 14ql3 (2100 bp sequence) (SEQ ID No. 20)
37A AAGTACAATTGGCCAGGCGTGGTGGCTCATGCCTTTAATCCCAGTACTTTGGG AGGCGAAGGTGGGCGGATCACTTGAGGTCAGGAGTTCAAGACCAGCCTGGTC AACATGGTGAAACCCCGTCTCTACTAAACATACAAAAATTGGCTAGGCGTGGT GGTGGCACCTGTAATCCAAACTACTCGGGAGGCTGAGGCAGGAGAATGGCTTA AACTCAGGAGGCGGAAGTTGCAGTGAGCTGAGATCGCGCCGCTGCATTCTAGC CTGGGCAACAGAGCAAGACTCTGTCTCAACAAAAAAAAAAAAAAAAAAAAAA GTACAATCAGTGTTCTGTTGTGTTTTGTTGTTGGTTTTTTTTTTTTTTTTTTCTTTT TTTAGACAGGATCTTGCTCTGTTGCCCAGGCTACTATGCAGTGGCACAAGAAC AGCTTACTGCAGCCTTGACCTCCTGGGCTCAAGTGATCCTCCCACCTCAGCATC CATAATAGCTGGGACTACAAGTGCACACCACCACACCCAGCCAATTTTTTAAT TTTTTTGTAGAGACAGGGTCTTACTATGTTGCCCAGGCTGGTCTCGAACTCCTA GGCTCAAGTGATCCTCCCACCTCGGCTTCCCAAAGTACTGAGATTACATGCATG AGCCACCATGCCCAGCTCTATTGTGTTTCTTGTTTTTTAGCTTGACAGGAGGGT GTCAGGGATTTCCTGGCTTACAGAAATGATATGAATTTGCAGAAGAAACTAAA ACTGTTAACAAACATTTGAAAAAAGTTAGCCCCATTATTTATTTTTTAAATGCA AATTAAAACAACATACCTCAGATTTAATGTAACTTCTATCAAAATCTCTGCTGG CTTCTTTGTAGAAATTGACAAACTGATTCTAAAATTCACATGGAAATTCAAGG GACCAAGAATAGCCAAAACAACTTAGAAAAGGAACAAAATTGGAGGGCCCAC ACTTTCTGACTTCAAAACTTGCTAGAAAGCTACACTAATTAAGACTGTGTGGTA CTGGCATAAAGACAGACAAACAGATCAATGGAATAAAATTGAGAGTCCAGAA ATAAACCTTCACATTTATGGTGAATTCATTTTTAAGAAGGGTACCAAGATAA TTCAAGTAGGAGAAAAATAGTCTTTTCAACAAATAGTGCCAGGACAACTAGAT ATCCATATACCAAAAAGTGAAGTTGGACTCATACCAAGTACAAAAATCAACTC AAAGAGAAAAAAAACCTAAATGTAAAACTATAAAACTCCTAGAAAAAAAGAT GTAAATCATTGTTACTTTGGATTAAACAATGGTTTCTTTTCTTTTTTTTTTTTTTT TTGAGACAGAGTCTTGCTCTGTCACCCAGGCCGGAGTGCAGTGACACAATCTT GGTTCACTTCAACCTTTGCCTCCCAGGTTCAAGTGATTCTCCTGCCTTAGCCTCC CAAATAGCTGGGATTACAGGTGCCTGCCACCACGCCAGGTTAATTTTTGTATTT TCAGTAGAGATGGGGTTTCACCATGTTGGCAAGGCTGGTCTAGAACTCCTGAC CTCAGGTGATCTGCCCACCTTGGCCTCCCAAAGTGCTGGGATTACAGGCGTGA GCCACTGCGCCCACCCTAGACAATGGTTTCTTAAGGTACAAAACCAAAAAGAC AAGTTATGAAAGAAAAAAAATAGATTGGACATCATCACAATTAAAAACTTTTG TGCTTCAAAAGTCATCTTCAAGAAAGTGAAGCCAAAAACAGAATGGGGGGAA AATTTTGCAAATATATATCTGAGAAGGACCTAATATCCAAAATGCATAAAGAA CTCTTACAATTCAATAATAAAAGAAAATCAATCCAATTTTAAAATAGGCAAAG GATCTGAATGGACATTTCTCCATAGACTATACACAAATGGCTAATAAGCATAT TAAAAGATGCTCAACATCATTAGCCATCAGAGAAACACGAGTCAAAAATCACT TACGATACCACTTCACACCTATTAGTACGACTATAATAAAAAAGACAGTTAAC AACAAGTGCTAGCAAGGATATGGAGAAATTAGATCCTTTATATACTGCTGGTG AGAATGTAGAATGGTACAGCCCT
Human Genome Map 4pl5.31 (2100 bp sequence) (SEQ ID No. 21) 38A TAGAAATATTGCAATGGAAACTTCAGAAGTAAAAATGATTATAACGGGCTATT ATAAACAATTATGTGCAATATTTAATCAGGAAGAAATAGAAAGCTTGAATGGA CCAATAAAAATTAAGAGATTGAAATATGAATTCAAAACTTTGCAACAAAGAAA AGCCCAGGACGAGATGGCTTCATGAATGAATTCTACTAAACATTCAAAGAAGT ATTACCAATATTTAAATTCTCCCAACAAATAGAGATAGAAGAAATACCTGCAA ACACATTTTACAAGGCAAGCATCACCTTGATCCCTAAGCCAATGACATCACAA AAAAGAAAACTATAGGCCAATATCTCTGATGAACATTGATGGAAAAATTCTCA ATAAAATATTAGCAAACAAAATTCAACATCACATCAAAAAGATTATACATCAT GACCAATAGGATTTATCCCTAGCATGCAAGGCTGGTTTAACATACACGAATGA AACAATGTGACACATCACATTAACAGGATGAAAGATAAAAAACACAGAATTTT CTCAATCAACACAGAAAAAGCATTTGACAAAGTTCAGCATCCTTTCCTGATAA AAACTCTTAACAGTTTATGTATAGAAAGAAAATTTCTCAACATAATATAATAA AGGTGATTTATGAAAAATCCACAGCTAACATAATAATCAGTGGGAAACAGTTG AAAGCTTTTTCACTAAGATCCAGTGCAAAGCACAAATGCCCACTTTTGCTACTT CTATTCCACATAATATTGGAAGTACTAGCAATAGCAATCAGACCAGAGAAAGA AATAAAAAGCATTTAAGTCAGAAAGAAGAAAAAGTAAAATTATCTCTATTTG CAGATGATATAATCCCTTATGTAGAAAACCCTAAAGATTCCACAAAAAACTGA CAGAATGAATTAATTCAGTAAACTTGCAGGATACAAAATCAACATACAAAAAT CAGTAGCATTTTTATACACTAATAACAACATATCTGAAAAAGACGCTTTAAAA TCCCATTTATGAAAGCATAAAAATAGTTAGAAATAAATTTAACCATAAAGGT GAAATATTTGTATACCGATAACTATAAACCTTTGATAAAAAAAGTTGAAGAA GACACATATAAATAGAATAATATTCTGTGTTCATGAATCAAAAAATTTAACAA TGTTAAAATGTCTGTATTAACCAAAGCAATATACAAATTCAATGCAATTTCTAT CAAAATTTCAAGGATATGCATCACAGAAATAGAAAAAAAATTCTTGAAATTCA TATGGAACCACAGACACATAAAAACAGAATAGGCAAAGGAACAATGAGAAAG CAAAACAAAGCTTGAGGCATCACACTTCCTAAGTTAAAATTATATTGCAAAGC TACAGTAATCAAAAACAGTATACAAATGGCATGAAAACGAAAATGTGGACCA ACGGAACAGAATATAGAGAGCCAGAAACTTAACTAATTTTCAACAAGGGTAC CAACAGGACACCCTGAAGTAAAGATAGTTTCTTCAATAAATGATTCTGGGAAA ATTGGATTGCAACATGCAGAAGAATGAAATTGGACCCTAATCTTGCACCATAT ACAAAAATGGACTCAAAATAGATAGGAGACCTAAATGTAAGATGTGAAACCA TAAAACTCCTAGAGAAGAACATAGGGGGAAAAATTCCTTGATATTGGCCTTGG AGATGATTTTTGGATATCACACCAAAAGCTTAGGCTACAGAATCGAAAATAAA TAAATGGAACTACATCAAACTACAAAGTGTCTGCACAGTAAAGGAATCAATCA ACCAAATAAAAAGGCAACATACAGACTGGGAAATATATTTTCACACAGCATAT CTCCTAAGAGGCTAATATTCAACATTTGTAAAGAACACTTACAAATGAGTAAC AGAAACAACAAACAGCTTGATTAAAAACAGGCAAGGGACCTGAACATACTTTT CTCCAAAGGAGAAATAATGGCTAACAGGATATGAAAAGGTATACAACATTGCT AATCATTAGGGAAACACAAATGAAAACCACTATGAGATATCACCCTTCACCCA TTAGGATGGCTATTATAAAAAAAAAAAAAGACAAGAGG
Human Genome Map Xpll.3 (2100 bp sequence) (SEQ ID No. 22) 39A AGTAAGTGGTGGAGCCAAGAATTGAACCACAATGTTCAGGATTCATAGAATGA TTAGAATATAGTGAAAACAAAGCAAGAGATAAATTAGAGAGCTGGCAGGCGG GGCCCACTCATGAGAGATGCATGTGTACGCCATGTAGGATACCCAGGTTTTCTT CTTTTGGTGAACGGAATCTCTTGAAGGGTGTTGAGCAAGACAGCAATATGAGC AGAGAGTTAGAAGCCCCACCACTCAGGAAGCAATGCAGAGGTAGGCACGGGA GTGCATCTGAGATGAAGAGAATGGGCTGAGGTTGCAGCTCAGAGGCATAAACT AGAATTGAATGTGCTGGGCCTCGGATGGACTCTAAGCCTCTTGTTGAGTGTTAG GGAGACAGGGCCATCATGGAAGCCAGCTTTGGACAGCAGGAGGGACACCTTTT CTACCGAGATGAAAAAGGAATGGCAAGTTGTGGATGTGGGGGCATGGGTGTG GTTTACATCTCAGGATCTCTATATGCTCTATTAACCTGGAGATGAGGTTTTCTG TTATAAATTGGAGGAAAATGGTGAAGTTAGGAGCTTAAGAGTGAGAAAGTCTG AAATATCCATTGTGGAGAGTGGAAACAGGTACAACTAGGCCTTGATTCCCTCC TTAACTGCTTCTTCATCCTCAGCTACTCACTGATCCATCAGTTGTCCAGTTTGTC ATCTGTGACTTTGATTTCTATTGGCCCTTTCCTTTTGGACCTTATTGGTCCTTTC CTTTCAGCATATAGAATGTTCATGTCTCTCTCAACTTAAAACAACAGCAAAACC CAGCCCATGACCTTCTGCCCGGTAAAGTCATCCTGGTAGTGTCCACCAGCCTG GCAGAACCTGTCTTCCACAGCAGCCTGGTGACAACAAGAAGCCACAGCAGCA GTGTTTGGCAGTGGTCTGGCCCTCTCCAGACAAAATAAGCATCCCTGGGGTTCC TGGACAATGAAGGGCAAAGGAAAAGAGAACAGGTCTCAAGAATATACTGACA AATCTCCAAGAGGAATTCAAGATAATATGGCAAATGGGAATTAAATGAGAAA GTAAAAGAGAGGTGACCATATGCCAATCTGGAGCAGGTGGAATGTGTCATGA TGGTTCAAGGGCAATCAAGAGTTCACATTTATGGTGAGGATTTCTCTTGATTT TTTTTCCTCCTGTCATCTTATTTTGTGTTTTCAGTTTTTCATGTATTGTTACCATT ACCTTATATTGATTGCTTCTTGTCTTTTGCAATACATCCTAAATATAGCAGGTTC TTGAATAACATCACCTCGTTCAACATCATTTTATGATAATGTTGATGGGGAAAA AAAAATGGCTCCAGGCTGGAGCCACTGTCTGCATGAAGTTTGTACTTTCTCTCC ATGTCTGCGTGGGTTTTCTCTGGTTTCTTCTCACATCCCAAAGCTGTGCACATG AGGTGAGCTGGCATGTCTATATGGTCCCAGTGTGACTGAGTGTGGATGTGTGA GTGCACCCTGCGATGGGATGGTGTCCTGTCCAGGGCTGGTTCCCACCTTGTACC CTGAGCTGCTGGGACAGGCTCCAGCCACCCATGACCTTGAACTGGAATAAGCA CATTGGAAAATGAATGAACAAATGAATACAAATTAGGATAAAATAAAAACTC ATCAAGTCTATGACAATAAAGGACATGGGACAAAAGCGCTCAGCAAGCCTGCT CTACTTGTGATTTTTTGGTTTTGAACTGCAAGGTGGGAAAAGATGCTCCTTACA ATGTTCGCTCTGCAAACATTTATTCCCTGATTTAACCCATCACTACCATGGCCA CTGCCACTCACTGATTCACCAAAATTGGGTAAATCATTGTCTTGTTTTTATTAA TTTTTTTTTTTGAGACAGAGTTTTACTCTTGTTGCCCAGGCTGGAGTGCAATGGT GTGATCTCAGCTCACTGTAACCTCCGCCTCCCGGGTTTAAGCGATTCTCCTGCC TCAGTCTCCCGAGTAGCTGGAATTACAGGCGCCCGCCACCACACCCAGCTAAT TTTTTGTATTTTTCGTAGAGATGGGGTTTCACCATGTTGGCCAGGCTGGTCTCG AACTCCTGACCTTAGG
Human Genome Map Ilpl5 (2100 bp sequence) (SEQ ID No. 23) 4OA AAAGGCTTCCTTCCCTCAACAAAAAGGATCTCACCATTCTTTATATTCCAGGTT TACTTTCTGATTTACCCGTACAGTATCATAGCCTGGAGGCTCCTGAGATGCATT CTTTTTTGGAAGGGGCCTGATTTTGAAGTCTGACTCAGCCACTGATTAGTGAGC AGGCTTTCCTAAATCTTCCTTTCCTCTCTAGTAAAGTAGGTCAGATCGTAATCT CTTCCTCACAGGACCATTGTGCAAATTAAAGGAGATAACGTGAATGGTTTGGT AGCAACTCAATGAATGTATCTGAATCCTCCCGTCAATTTACTCTGCTGTCCATT ATAGTCAGTTATGTATCTGTTTTATCCTGTCTGGTCGAGAATGGAATCTGAGGA CCAAAACTCCTCTCTTACTCATCTCTGTGCATTTTCCTCTTTGCCCCTGCCCCTT CCTCCAACCCAGTACCTAGCATGGTTTCTTCATTTATGTGTAAATTTTATCAAA GTGGTGTCAGTCTGTGCAAATCATCTGCTGAAGTCTGTTAAGAACGTTCAGCA ATATACACTTCAACAGTAACAGGGACAGTGTGGAAAACCCTTGTTTTCTTCCTA CTCTCCAATTCTTCTGCCTCTTTCCACTGCTTTGGAGAGGAGGTTACAATCAGA GTCATTTCACCATAAGAAAATCATCAATTTTCTAATGAAAACCCCCCTCCTTAA ATATTCAGCCGTGGCACAATCCCTAAAGGAGAGAACACATTAATGCAATGCAC TTGGCCAATTTGTGAGCCCTGACTTGTTGAGCACAGACTCTTCCCTGACCCTGG AAGACAATCGGCAGGACCCCTGAATAATGAAGTATGAATGCTCACCATTGTGC TATCTCTAGCTACACCAGCTTCTACCTAATCTTTTTTTTCCTTTTCATTGCTCTCC TTTGCAACTTCCTAAGGATTTGGCCATTTTCCACTGTGTCCAACCTACGAAGCT GTAAATACCTGACTGAGAAACACAAAATGTGTTTATAGGACTTCTGATGGCTT CTCATCTAAACCATAAATGTGAAATGCATTTCAACATTTCTCAGAAAACACCC CATCCCCCAGATTCAATTGGCAGCACAAATTTACTTCTCAGAAGACAGCACCA AAATCTCAACATTGGCATTTTGAATCAAGCACACACACGCAGCTGCATTGTGTT AACAAAAGTGAAACATTATTAGGTCGCATACTCTGAGTGACAATATCCTCGAA TGATCATTTCTGTGAGAAATTAAGCTATGAGAAATTCAATTTGGTAGCATTTGG GCATTACAGACAGCTAGTCTGTTTATATCAGCAAGGCTTTGATGTTAAGACTCT GTAACTGCAGCCACAGGAAAAGCAGACTGAATACAGGGTGGATAAGGTCACA GATATAAAAATCAGATAGAGTTCTGTTCTATTATCTACATAGTGTGTACTTTGG GAAGTTACTTAATATTTCTAAGCCTCAGTTTCCTCATAAAAATAAAAATGGCAA GCAATATGAAAACTATCTAATAGAATATTTGTGACACTAAATTGTAATAATGT ATATAAATCACTTAGCCTAGTATGTGGCATTTATTAACACTCAAGCAAAAGTGT AATTTTTTAAAAAAAGACTCTTATATCCCTTACATGACAGAAATATTAAGACC AAAATGGTTACTGAGCCCTCAAAGGTATTATCTCATTCTGGCTGAGCTATCTGG ACCTGGAGAAAAGTCTAGAAAGACTCTATTTCACTCCAAGTTTCTTGACCCTAT CTTTATTTTTTATCTTCTATCCACTAGGACCTGTGATCAGGCCAGGATTAACCA GTGTTCTCTAGGATTAACGTTTTTGGCAGCTGGGGATGATTGCCTAAGATAATT GTTTTTGTGTCTGCCTCTCCTGCTAGAATGCAAACTCTCAAGGGCAGGACATAT GTCTTTCTTTATCCTACCTGTTAGTGATCAACAGGAGAAGGCCACTGCTTAACT GTTAGTGTCAGGTCAGCTCCAAGCTGGTACTTCTTAGGAACTCTTTTCTTTTCTT TTTCTTTTTCCTTCCTTCCTTCCATTTCTTTCTCTTTCTTCCTTCCTTCCTTCTGT
Human Genome Map 2q31 (2100 bp sequence) (SEQ ID No. 24) 41A
CACGTGAGGACACAGCATAATAGTAGCCACCTGCAAGCAAAGGAGAGAGGCC CAGGAGAAACAAGCCCTGCCAGCACCTTACTTAATCTTAGACTTCCAGCCTCC AGAATTGTGAGAAAATAAATTTCTGTTGTTTAAGCTATCCAGCCTGTGATATTT TGTTATGGCAGCCCCAATAAACTAGTATGTGTATAATGAAGCCCTAGACAACA AGGGACTCTCATTTCTCCGCATATTTGTAGAACTCATCCCAATTATATAGAGCT CCTACTCTGAGTGCTAGACACAGTGTTAAACACTTTCCCTGTGTTATCTCGTTT AACCATTAAGCTGAATCCTCCAAAACCCTTTGGAAATCAGACTTATCTAAGAA ACTCACTATTGTAGTGAAGCTGTTTTAAAGAAGAATTGAAGGTATTTTTCTTTA TCTTATAATCTGTTACATTGTGTTACATTTTAAGATAATACTAATCTAAGGACT GATAACAATTTAATTTGCCAGAATCATTAAACCAAATAACATCTTTAACAGTG GCTGCTAGACAGGGGCAGCTGTATATTTTAATGCCATATTTGGGGGAAAAAAA ACAGGGTAGCAAACATATCTATAAATAAGAATTAATTGCTACAAATTACCTGG GAAGGGAAAAATGTCAAGTTCATATAAAGAATATTATTGACCCATGGATTTAC AGCTATATAATAATTTGGTACCTGGTTTATTTCTTAAAGACCTAGCACGTTTCT TGTTTTCTCCTGCTATATTACGTGTACATGGCGTTTCAATAATCAAGCAAAAAA GATGTATGCACTATCTTAGTCTTTGTTGTCTAATTAAAACTTTTTATGCATAGCA ATTGCTTACCATTTTGCATTATCACCAGAGCTCATTTCTCATGGAAAAAAAATT AGCATCAGTTTAAAAGAATATTTCTTTAATCAACAGTTCTGATTGTCAGTAGTA CCATTTTGTAGATAGTTTTTAGCTGACTAACAAATCTTTTATTTTATTGGCTGTC TCATTTTGCTCTCTTGCATATTTCACATTTATGGTCTATTCAGACATTCTCCTG TTTTGTTAAGTGGAAATCTGTGTGGTCTTTGATGTAAGACATAATTTATTTGAC AAGGAAATATGAGTCTGTGCCCTGAATCCACATTTAACTGATGGATTGAGAAA TTTTAAAATTGCAACAAGATAGACTCTCCTCCAGATTGCCGTACTACTTGCATT TTGCTTATCTATTTGGGAGTGAATTTACATATGTGTGTCTATATACGAATATAT AGAGAGTCATACAACCATGCAGCTGTACTTGTGCAATTTTTCTACTTTGTTAAT AGAAAATGCAGTCTCATTTTGTTAGTCATTAATGGTTCCTATAGAAAATTTTTA AAGAATTTTTTTCTGAAATTAAATTCAAGATACTTATTATGTTTTATCTTCATAT AGATAGCTTTATAAAGAGAGTGATGTCTTCAAGTCTGTACTGCTCGCTTCTCAG CCTAGTAAATGGAAGTTTTGTTAGCATTTCAAGATTTATATATTTCATATGTTCT CCCAAGTCTATGGCCCAGTTCTGGTAATTGGAAACTTACTTTCAGCTCATTCCC TCTGCTCAGACTACTTGTCAATTAACCTTTGCAAAATGATAGTTTTAAAAAATA TGACTTTCATATTTCAATCATGTTCATTTTCAATCATCTCAAAATGTAGAAATT GAATAACACCCGGGGTTCTACAGTGCTTTTTACATATCATTTAAGGTTTAAAAC ATCTCTTTGATGTTCAAATATGACTGCCATTTATATTCAATGGATGAGATTAAG TGGTTAAAATTACTTGTACTGGGCATGCCCCTGCTTTGTTTATAGGTATGAACA AAACACTAAGGATTTTTCATAAATATGCACCATTTCCATTGATGTTTTTGACTG CTGTCTGTGACACACTAGGTAGGCCATATTAAGTAATGGGGAAGAAATCATAG GTCCTACTGTGATATTAAAAATTTACATTTTGATGAATTAAATAGAGTTGTTGA CCATTCTACACTGTTGATTATATGAAGGGAAAAAGCTAACAACTTCTAAGAAT AA
Human Genome Map 3pl2.3 (2040 bp sequence) (SEQ ID No. 25) 42A
AAAGGTAAAAGAGAGAAGCACAGAGACACTAAATGTATAAGTAATTATTCAT TTAGTTTTGTAAGTGTCCTGGTTTGCATTTCAGGAAAAAGAACAAAATCTATC AATCGACATTTTTGTGCCTATTACAATTGTCCCTCAGTATACTTGGTGTATCGG TTCCAGGACTACCTGTTTATACCCAAATCCAGGCACATTCAAGTCCTGCAGCTG GCCCTGCAGAACCTTCATATGTGAAAAGTTGGCCCTCTGTATACACGTGTTTTA GATCCTACAAATATTGTATTTTAGATCAGAGCTTGTTTGAAAACAGTTTGTGTA TAAGTGGACTCTTGCAGTTCCAACCCATGCTGTTCAAGTGCTGACTGTACTCTC AAGCTAGTTCACATTTTCTAATTTAATCCTCAAAACGATGCCTTGAGGAAAAAT AATAATGACAGAAGATGCAGAGAGAGGGCACTGAAGCTTGTCCAAGTTTACA AATAGGGTCTCCATGCAAGTGTTCACAATTTTCATCTTTTAATTTCAAAACCA GTGATTTTCCCCCCTACTTTTACCCACATATAATAATAAATTTTAGGGTTTAATT CATTTTCTTGTATTATATTTTCATCCTATAGTTCTCCATTTTAACCCTAAAATTA CAGAAATATTTCCTGTGGCTGAAAGAAATTAGTTAGAAACTCTTAGGTATTTTA AGTAAGAAGAATGCTACATGCAAAAAGTAAAGTCTGAAGAATGTATTTCAAC CTTGAAAAAAAAACCCTCAGACTCAGTCTGCAGTAAAGTTCATTCAACATTAA GAAGCAGAAGCTATTTAAAGAGTTTAATGCAGAACACGGTAGAAAAGATGCC CATTGCCAAGTCCTTCACGGCTCACCATAAATGTGAACAGCAAAGTGCTATC CATTTTAAAAATCAATCAAGGTGATGCAGTGCTTAAAATGCAATTGATTTTGG CTAATTTGCAAGTAGAAAGGTTTAGAACTTGAATAATTTCTTTCCCACTTTAGC TGGTTCAACAGCCTACTCAATGAGTTGCCTAATTATTAGTCTTGTTTTTTTTTTT TTTTTCATTTATTTACTTTAGACAATTCCTGAGTGAGACAACTTTTGATTTATTT CAACTGGCTTTGGGTAGATGAAAGAGACCACAAACCAAACCAAACCAAAACA AAACAAAACAAAAAGCCACCAGGCAGGCCAAGCACGATGAAGAAAAACTTCA AATGCAAGAACTATAATTTTCAAACTGTGAGTTTGGAATTCTTAAAAAGCAAA AACCATTAAGTGACTCCATTTCAGCTACCTATTAGTGATCAGGTTGAAAAACA ATGAGAAAAAATGGAAAGAAAATCACTGCAAATTCCATTGGGGTAGATTAGA AAGATGTTAAATTACTCTTTTTATCTGCCTTCTCTGATTAGGAAGCCTAAAGAG CTTACCACCTTTCCAAGGAGTCAGCATGTCACTTTAATGTTTTGCTGCTGTTGTT TGGTTGATTGCTGTATATATATATATATACATATATATATATATATATATATAT ATATATATATACATATATATATATATATATATATATATATATATATATATATAT ATATATATATATGTATTCTTGCTCTGTCACCCAGGCTGGAGTGCAGTGGTGTGA TCTTGGCTCACTGCAACCTCCACCTACTAGGCTCAAGTGATCTTCCCACTTCAG CCTCTCAAGTAGTGGGGACCACAGGCACACATCACCATGTTGGGTTTGTTTGTT TGTTTGTTTTTTGTATTTTTAGTAGACACAGGGTCTTGCCATGTTGGCCAGGCTG GTCTCAAACTCCTAAACTCAAGTAATCTGCCCACCTCAGCCTCCCAAAGTGCTG GAATTACAAGAGTGAGTCACTGTGCCCGGCCTTTAGTGTTCTTTTAAATAGCAT TCATAATCTTTTAAAATATGTTTTACAAATGAAACATTGTGTTTGAGTGACATG ATGGGGAAGGAGCCTGCAGTCCACAACGTCTCTGGCTTCCTTTCTTGCATTTTC TCAG Human Genome Map 4ql3.3 (2040 bp sequence) (SEQ ID No. 26) 43A
CTCAGTTTTAAATGTTTCCATCAATCAGTAATTCAGCTCCAGAGTTGCCATAGA AGGTTATGGGAAAAAAATCCTTCTGCTTTTCCAATATCAAAGAGAGAGATGTT CTGGAAGGTTTTATTTTTGCCACCCTGTTTCTAATGCATTTCGCCTTAAGAATA ATATTACTCATCTCCAGCAATCAGGCTCAAGGAGGAAATTTGATACCATTCTGT GGGTCATCCCCAGATCTCTGCAGGCTTCTGGCAGATGTATGATGTAGTGGGCA CCACTAACTTTGTCTGCTAGGGTATTAGTAGGACGTGGTCTACTGAATACCGGA CAGACATTTGGAAATTAAACTAATCAAAATAGGCTAATTTCACGTGCTAAGCA ATGCAATTTCCCTGAATTTGTAGTTCCATGATCTACTTTTTCTTCTACATTTCTC TTACTCCCTCTCTTCTCCGTTTACAACACAAAATTCAACAAACTGCTAACTCTA GCTATTAATATCCCATAGTATTTCTCTAAGCAGCTTCATAGTCACAGTTTCAGT TAAGGCTCTAGGGCTTTCATTCCTGAACTACGTGTACTACAGATCCCAGTTAAA ATCTCTATCCTTCCCACAACAGTGGTTGACCACCTTCCTCAACATTGGATTTGG GGTGATAATGACAACATTCTGAACAAAGCTCATTTTTTCCCCTAACCCCCAGCT AAATAGAAGAAATGTTAATGTTACTCCTCTTTAGATTTTTTTAATTAAGAATAT TTTTAAAGGATTTTTTTATGATTTAATGGGACACAATGAAAAGATATTTTGACA AAGGTAAACATCTGAAACTGAGGCAAAGAATTTTAGATGTTGCCTGTGTAACC ATTATTCCATGATCAAAAGCCCAATGTTTAACATATCCTGATTTCATCATTAAA CAAGCATAAGAAAAAAAAAAAAGAAAACCATAAAAGTGAAATAGATTACAT CTTGTAATAATACAGCTATGAAATTCTGACCAGAATGAAAATATGAGTATGAA GAGAGTAATCATTTGCTTATTAATTCAAGGAACAATTTGCCATTTTTCAAGTAT TATGAAAATAAGAGACTGTTGGACTCTTTTTAAACACGCAGGTTTTTCAAATGT ATGTACAGTAATATTATAGCTCTGGTGAAAATTTTGATGAAAACAAAATTTTCT GTCTTCTTTTACTTACCTTGCCCTTTTCAAAAAATAGTGCTTAATGTTAACCAAT GGACAGTCTAACTACCTGAAGCTTTTCATTCAGCTTTATTTTCTCAGCAACTAT GGTTGAAACTGACAAGTTAGGTGAAAGGTTGTGTAAGTATCCAGGCAGGGGCA AAAATATCGAGTTATCCCCAAATACTAACAAGCACATAGGTAGAATATTTCTA CCAAGTTAAAGAGAATAAAGGAACCACATTGAGCAGAGCTACCTTATTCAAGG ACCTCATTATCTCAAGGCACCCCAATTGAATAAGTGTACCATTATTCCCTTCGT TCTGTGCAAACAGCAGACGTAGAGCACAAAGAGAGACGATTTCAGTGAATCA CACTGTAATTATAAATGCCACATTAAAAAACGGAACAAAAGCAACAGCAGGA CACTGTAACGTCGATGGTTAAGGAGGGCAAACAGAGAAACATTCTCAAAGGC CACAATAAATTACATGATCCAGTTCTTTGTTACAGGCAAATTATTGGACAATAA GAGAGACACTGAACACAACTTATCAGTGGTAAAGTAACTTGCACACATTCTCC TACCACTGAAATATTCCTGTTACCTACACAACTGCATTTGTATATACAAGCAAG AATTTTGATGCACTAAGTAATTAATATAGCTCTCTAGTATTTTTTTTACCTCTGT GCTTATTCTATCTGGGCAAGGTGTGGTAATAGCACCTTAAAAAATAAGATCAG ATTTAGGAGTGAAGAACACTGCATTGGAAAAGGTAATTGCTAATTTTATTGTA TTTTAATTATTTGACCATTTGTGCACAAAATTAAATAATACTTGCTTCATCTCTA TATT
Human Genome Map 4ql3.3 (2160 bp sequence) (SEQ ID No. 27) 44 A
GTGTATTGCTGTGAAAAAAGTCACTTCAAATTTAGTAGCTTCAACCAACAATC ATTTTATTTGCTACCAATTTTTTGGGTGAGAAGTTTGTGCTGGGCTCAGTTGGA CAGTTCTACTGATTTCCCTGTATATACCTCATGCAGCAAAAGTCATCCTGGTGG TTCTATGTGATGGTTAATTTTTGTATCGACTTGGCTGGATGATAGTCCCCAGAT ATTTGGTCAAACGTCATTCTAAATATTTCTGTCTATGTGTTTTGGGAATGAGAT AATATTTTACTGGATTATTCTTCACAATGCGGGTGGGCCTCATCCAGTTAGTTG AAGGCCTTAATAGACAATAATTGACATCAGACCTAAGTAAGAAGGAATTCTGC CAGCAGACTGCTTTTGGACTCAAATTGCAACTCTTCCCTGAGTCTCAAGCCTGC TGGCCTACCTTGCATATTTTGGACTTACCAAGCTTCCACGACTGTATGAAGCAT TTCCTTAAAATAAATGTACATATCTGTCTCTCTACACACATACATATATACACA CTCTCACACACATCCTGTTGGTTCTCTTTCTCTGGAGAACCCTAACACACTCCA CTCAGACAGATTGTCTAAGGTGGCTTCACTTAAGTTTTGGATATCGACTCTTGC CGTCACTCTCTTGAAACAATGTTCTTAGACTCTCATGTAAAGAAACTCCATCTA GCTGCACCCTGGCACTGTCCCAAAAAAGTGAGGATAATACCTGCAAGGTTTCT TAAGGATTATGCCTTGAAGTCATATTTTACTTCTGTAGAGTTCTATTAGATCAA ACAAGTTACCTGGCCAAGCCTAGGGTCATAGTGGGAGAATTTTGAGGGCCATA TGGCAAACAGCCCACCATACAATACATTCTCAAATGGCTTCTCAAATTTTACAT TCTTGTGAATGATTCTCTCTTCTTGTGATTAATTTTACACACATTACTTCAATAT AAATTTTTATCTCATTGCATTTTCAGTTTTTTTGTAATTTCACATTTATGGTAC TCTTACAATGTGCCATGTGCTATTCTAAGTATTTTATGTAGATTAATTTAATTCT TACAACAACCTTTTGAGGTAGGTAATACTATTTTGTTCCCATTTTCAGATGAAA ATGCCAAGGCATAGAAAGCTTATGTAACTTGCCCAGTAACACTCAGAGACTTA ATGTCACAACCAGTACTTAAATGTATACTATCTGACTACAGGGTATGCATGCTT AGTCATAATGTTATTAAAATATCATTTGTGATGACTGAGGCATCATGGCAGAT AGGAGGCAGGACTAGATTGCAGTTCCAGACAGAGCAGGAGACAGAGGCTTGC ACATTGAATTTTAGCTCCAGATCGACTGCAAGAGCAAACCGGTAATCCTGAGA GGACCCACAGATCCTCTGCCGGAAGCAGACTGTTTCTGCAGGACCAAGGAGAC ACCACAGATACTGTGGGTGTCCCAACTGCAGAAATTGGAAAGGGAGACCCTTC TCTTCCAAACACACACCCCACTGGAGAAGCTGTTTCTGACTTTACCTGGAGCTG AGTCAAGTTAGAGAGCTGAGCCAAGTGAAATACAGGGGTAGGGGAAGTAGCG GAAAGACCCTGGGAGCTCGCTGGGTCCCCCAAGCAGCCCATACCTGCCTGGCA CCACAGGGATCCACTGGGAGGTTGGCCAGAGAAGTAGGGGGTAAAATACCAC AGGCAGAAGGAATTCTCTAGCTAAACTCTGTAACAATTTGAACGGGGCATGAA GCCTCCTGGCCAGTACTTCAAGGAGGGTGTGAATCCAGCATGCAGACCTCACA GGCAGGGGGGAAAACTAAAGCCCTTTTCTTTGGCAGCCGGGAGGTGGAAAGC CTCAGGCAAGTTTTCAAGCAGGGCTCACCCTCCACCTGGAAACAGACTCCAGG TTGTTGAGGGGGACACGGTGGGAGTGAGACTGGCCCTTCAGCTAGCATGTGAA CTAGGTGAGGCCTGTGACTGCTGGCTTTCCCCTACTTACCTGACAACCTACATG ACTCAGCAGAGGCAGCCGTACTTCTCCTAGTGTGTCCGGAATTGGTGGGTTCTT GGTCTCGCTGTCTTCAAGGATGAAGCCGCGGACCCTCACGGTGAGTGTTACAG TTCTTAAAGATGGTGTGTCCG Human Genome Map 4pl4 (2100 bp sequence) (SEQ ID No. 28) 45A
CCTCCCACAAGGTCCAACTCAATCAGAACCAAAAGGGAGATCACAGCATATCC ATGCAATCCCTGGCTGGACAGACGGGGCACCCTAGGGCCTGGAGTTACGCAGC TGACTGGCAGAGGTCAGTACCCAGTTCTACCCAGTGTGGCCACCCGATCCAAC TCTGTGACTCATTTTAAATCATAAGTAGTTTGAACAAAGACTTAAAATTAACGG GTTTGGTCATCATTAAAGTTTATTTTTAAACGTCAAGTAATTTGGCATTTTACTA ACAACAACTTGAGGATATCCACAGTGTATGAAACACCAACTCTTGTTTCAATA AAAGTCTAATGAAAGTTTCTCAAATTCTGAAACCTAATTCCAGGATTCTTATTT GGAACTGTTTTTTTGTTTTTGTTTTTGTTTTTGTTTTATCAGAGTACATTAACTCA GGGGGAAAATGAGATTATCTTTTGATTCAGAGAGAAACAGAACATTCCACTGA TAGTTTAAAAATAACACAGTGACCACAGATAACTGTAGTTCAGCAATTTTCAA ATTCAGATTCTGGGTCAGGGCACAAGATTATGCGTCTTTAACAAGCACAACTG ATTATAATGCTGATGGTCTAGAGGAAACTTGATGGGAAACACTGATCTATTCA ACACTTTTTAAAAGTACACTTTAGTACTATTTCTACCTAGTCAATAAAATAAAG AAGGAAGGAGAAAGGAGGACGAATAAGAGAGAATCCGAAAGACACACTACC CAGACAGACTACTCAGACAGACGAGACAGTCAGGCAGACATGGTGCTACCCG TCAAGCATGCAGGAAAGCAGCTTTCATGGAATAACATTCCTAAACTCTTGCCT AATAAATTATGCTGAGAGCTGCTGCTAAGAATTTTTTAAACCAACTCAAAGCA AAAAAGGAGCTATTCACCCAATCAACAGGTGAAATCAAGTCACAGACTAGTAT AGGGTTTGGCAGATTTCAGGCCCTCCAGAAATATCTGTTTAATTGAGAAGCAA CTCCAGCTCTAGCTAGAAATCTATTTAACCATAAAAGTGAAATCATAATGAAT TTGGTCGTATCTTATTTTTCCCCTTTGTTTGTTTCCTCGGGGCATCTATAATGGC TGAATTGGAAATGGAACCACAAGTATTATAACAACATTTGTTGGAAAGTTCAT CCTGTATTTTAGTAGTACATAAGTTGACAGATATGGCTTTATGAATTGTTCTCA GAGACTTAAAAAAAAAAAAACCCTGAACTTTGTAAAAATTACATCCATTATCC ACCAAGTAACATTTGCAAGCAAAACCCTCTACTAGAAAAAATGGGTGCGAAA ATAGGAAAAGGAGAAGAACAGGAGGAAGAAAAGGAGAACAGGACGTACAAT TAATTGAGGGGAAAAAAATCATGAGTAAAGAAGTCAGAAATAAATGTAGCTA AAAATACAAACTGCTACTTTATGGTCCAGATATTGTAATATATCATTTTTAACA TAAAAGAAAAACAAATCCTCAACAGACTTCCTATAAACGAAATTATCAGAGTT CCCGAGTACACCGGGGGTCGAGGGAAGAATCTCCATGTGCTCCGAGTATCGAT AGCCAGTCCAGCTTCATTCACTCATTCATTTCTTTTCTTTCATTTCAGGAGAACA TTTAGCAGTGTTTTGTTTTATTTATTTTATTCAAAGGGAAATCCTCATGTGACAC TAGCGGTGAAAATAACTTGTATTTGTAAGTTAATGTCTGCTGTACATCTGAGTA CACAATTGTCTTTCACAGAAGATGGAGCAAAGTATTACGGAAAGTTCATTGGC TTCTGAGTCTGAGAGAAATGGGTTCAAATCCTGAATACGTTCCTTATCTGTGTG ATCTTAAGACTCATCATTTAATATTCTGAGTCAGTTTCCTCCTCTATAAAACAA GAATCAGACGGGGCACAGTGGCTCACGCCTGTAATCTCAGCACTTTGTGAGGC CAAGACGGATGAATCATCTGAGGTTAGGAGTTCGAGACCAGCCGGACTAACAT GGGAAAACCCCCGTCTCTACTAAAAATACAAAATTAGCCGGGAATGGTGGTGT ACGCCTGCAATCCCAGCTACT
Human Genome Map 2q22.1 (2040 bp sequence) (SEQ ID No. 29)
47A
TTGATTCATGGGATGTTTATGTGGATAATTCCTTTGAAATCCAGCTTGATTTAT GAACAATCTTCTCTGCTCTATTGAGCCATTAAATCCAGAGTATTAGTGCATTTG GAATACACAGAGATGATAATGACATCCAAAGAAGAGTCCAGCAAAACTTATT TCCATGAGGACTTTTTCAGAGGGATGAAGTAACATTAGCTATACAGGTTAGCA TTATAAGACTTCCCAAGTGTAGATGAGAATAATGGCAACTCTGTGGTCCTAAG GATGAATATTGCTCTGGAATATGCATTTTACACTATATGAAAGAAATTAGGAT CGATATAAGCTCACTTATCTTTGCCTTATTCCTCCTCATGTTGTTTTTGTCTAGA TTGTCTCAGCCACTTGTTTTATTTTACTTAAATTTTAATTTCATCTTATTGTAAA CCTCCATTCCTTCAGAAACAGGTCAAGAAACATGTCAATCTACCTAAGTGAAT AACTAATATTAACAATTAAATAATAAATAGTACTGAATGAATATACGAATACA AGAATAAATAAAAATAAAATGTATTACTTCATCGATGGATTTCCTAGTAGATG GGGAAACGGTGAGAGGATATGAGCTTCAATAAGAAAAATGGTGCAAAATAAG GCAGAAGCAAATGCCCAAAACAAATCAACACATTCACAATTTTTCCAAGGACC TGTCATGTATATATTTTTTTCTTTTTTTAACCATTTGTGGCCCCTTTTTTTAACCA TTTGTGGCCCCTTTCTTATATCTCATTTCTCTCTTTTGTAAGGCTTCTGTGTTAAT TGACAGCATGTTCAGATATAAATCCATCACAGGAATGTGATGAAATTAGCCAT TCAGACCCCTGATATTAAGAAATTCAAAGAAATGGATAGAGTATCCAACCAGT GAGGAATTAAAGAAGAAAGAAGAGAAAGAGAAGGAGAGAGAAACTAGCTGT AAAGTTGGGATGGGTCGGGGGTGGTGAAGAAAACCAATTATTCATTGAAGGTG CCAGAAGGAAAATTGATGGCATGAATCCATAGCTTCTCACCATAAAGGTGAA TAATGACACAGACACTTAGATTGGGGAATGAGAAAAAAAAGGTGCATGCAAG GTTCTTCTATTTATATCTGATTAAGATATGAAAAGAAAATGAGAGACTGGATT ACTAAAGAAAAATTCCAGACAGTTAAGCAATTTTAGGAATGATTCATTTTAAG ATATGGCCATCAATTATTTATAAGGGTTAATAAATAGATTTATAAGCAAGAGG TACATGGAATCTAGAAATACATAAATGCTCTTCAAATTATTTACAGCTCTGACA AGTCATAACACATGAACTACTACCAAAAACACCATTTACTTGACTTTAAAATTT GCACCATAAACTATAAATGGACCAGTTATGGAGCATCAGCCATTTGTAATGTG CCATGCAATATTTAACATCAACTAAATGTGTTTTCACTAGCTGCTGACCACTTG GATTAATTTAATAAGCATGCCTAGTGCCTAATGATTTATTTGTGGGTAAATGAT CATAACTATTTAATGGCCTTAATATTACAGATGTAATTCTGAAATAAAATATCA TAACTTGGATTTAGTACATCCAGTTAAATAACAAGCATCGACATTTTTAAAAA ATAATAAAAACAGTGGCCAGAAAAAGAAATTAAAGCACTTGCTAGTCATATGT CCCCATAGGTTTCCAGCTTCATATTGGCTTTATTTCTTTTTTTCCTTTCATTTAGG TCACCCATTAATTTTCTTTCTTCATTTGCACACCCTCTTCCATTTCCTGTACTATC TTTTGTTCTAATCCTCTAGTAATTCCCCAGTGAGCTCTCAGCTTCCAAAGGGCA CTCTATTTCTATTAAGCATGGCAGTCAACAAGTGGAAATAGTCCTTGGTTGTCC TGCTTTCTGGGTGAATAGCAGAGTCCCTTTGCATCACCTCAAAGACTCTGATTC TCATGATCCTCAGTCTGGTGCTGAATTGTGCTTTTGCTCATCCACACACATCCC CTACC
Human Genome Map Ilq24.2 (2100 bp sequence) (SEQ ID No. 30) 48A
ACTCAGCAATGGGTAGCTATACTTGAGATAATAGGCGGGATTTTATGTGCAGC AATGTAGAGGATGCAGGGGCCCAGGATGGGCTGCCAGGTCTTCCAGAGAGTG CTAAGGTATCCACCAAGGATCATGAATGTGAACAAGATAATGAATCACTGTCT ACTTACTCTTTTGGAAAAGCTTCCATATCTCTGCCAATTGAATCACACTATAAC CAGTCCCAGGCAATTCAGGATGACAAGTTCCACTTCGAACAGTTCTGGGAGTC ATCCTGAGGGTCCCTGTGTATAGACATAAAAAGTTCCATTTGTTCTTACACAGT GAAAATGACAGAACAAATATTATGGGGATTATGCCTGGGGAAAAAAAATCTG TCTCTGGATATTCCTGACACTATGGAGAGAAAATCAGCAAAATTTAGAATCTT GGATCTCTTCCACTCACACTAGGATGTTGTTTCTAGAAATCTCCCTGAAGTATG GTACTGACTCTTGGTGGTAAAAGTGGAGAGGCTTAGAACTGAAATCTGGTCAG TAGAAGACTGAGGGTTAAAAGTGGACGGTCAACCCATTGAATGAAGGCCTAG CAGGAAATAGAGAGACAAAAATACAGGCATTAAGGGAATAATAGCTGAATAG TAATAATAATACATTATGTCAACAGCGGTGACAAAGGAAACACTCAATGTATT TATAGAGCTAAATAAACGGCAGATCTAGGTCCTACGTTTTGACTCTGAACAAC CTTCTCGGTTGGATTTTGCTTCTGCCTAAGGATTATTTTGGAAAGAGCTATTATT ATCCGTGATTTATCACGCTGCACTGGGGGGAACTCATACTTTCCACGGAGACA ATTACTGAATTCTCACTGGAGGCGCTTAAAGGAGCCAGGACCTGTTCTGAGGG TTCAGGTGGGAAAGGTGTGCCAGCAGGGGACTGCAGCCTGGCACCATGGGAC GTGTGTGCTGTTGACCACTTCTGTGCCCAGATGCCTCAGGCGCTTTCTCATTAG ATGCACCCTTCAATCTCCTGGTTATTGAAACAGGACTGGGGAGAGGAGTTCA CATTTATGGTGAGCCCATGCAAGAAGACCCTCCGACAGGTGCCTGTCACCCCT GAGGAGTCACTGGTTGCAGCCCGTTCTGAAGTGTCATTGAGATAGAAACCAAG TCAAAGCCGTGGCCTAGAAAGAGAGTCTGGGCAGAATTCTGCAAGCAGATTCT TTATTTGAGTAAGTATTCCTTGAAGAAGCCCAGTTGTGCAGCTGTGTTTGGGTG GAGGTCATCAGAGGTTTAGAAAAAGAGAGAAGTCATGGTTAATATTAGAAAA GAACTCTGAGAATCTGGAGGAAGGAAAATGCATTACTAGTTCTAAGCAACAAC TGTGGAAATAAACAATGATAAATACCGTATTAAATCTAAAGAGTTACGTTAAT AGATAATAACAAGTAGGAGAGCTAATAGCTAGCCATTAATACAGGCCAATTTA TTATTTAAAACATTTATTAAGATTTAACAATAGTCAAATAATTTTTTTGTGAAA CAGTTATTAAACTGAATCTCTGCATACATTAATCAACTGATATTTATCATTCAG AATGTATCTCATTATATCCAAAAGGGTGTGTGTATAGGCTTCAAAAACAAACT GGAAGATTTAAAATGAACTGTAGTTCATTTTTGCAAAGTGTAGATGTGTAAAG ATTATTATGTTTGCCAGCTGGGTAGCCAGACAGTGAAGTGGACTTGTCTAATTA GGAACAATCGCTGATAAATCAATTCTTTCCTTTTATAGGACAATTACAGTTTGT GTGTATATGTGATTGTGTTTTAAATTCTAATTCGATTTTGTGCATTGTTCTGTAA CCAAGTTAATTCTTTGAAGCCTTTTTAAATGGTACAAATTTTCCATAAAATATA AATAGGTTTATTGCTGTTTTATCAGTCACGCAAATAATCCAAGATCCATCTATT CACATAATTCAGGCATTAACTGTGTATAATTACTCACATGAAGTCTTCAGTCTG GTTTACTATACGGAACCCCAAATATGACTTTAAATTGCTCGCCTCCTCTTTTCCT CTGTTATTTCTCTCCCTCTCTT
Human Genome Map 21q21 (2103 bp sequence) (SEQ ID No. 31) 49A
TTGCATCATCTGACCTCTCTTCGAGTCCAAAGGACTGAGAACTAGAAGAACTA CTGCTGTAAGTTCCAAAGTCCCAAGGCCCCTGAACTAAGATCTCCAATGTATG AGAGCAGGAAAAGATGGATATCCCAATTTAAGGAGAGAGAAAGAGAAAATTT GCCCTTCTTGTTCTATTTAGATCCTCAAGGAATTGGAATGCCCAACCACATTGG TGAGTGTGGGTCTTCTTTACTTATTCTACTGATTCAAATGTTAACAGATTCTGG AAACGTTCTCACAGATACACACAGAAATAATGTTTTGCCAGCTCTCTGGGCAG CTCTTAGCCCAGTCAAGTTTACATATAAGAATAATCAACCCAGCTTTTTATAAT CATCTTAGTATTTAATCAAGGAAATGATATCAGCTATCTACTACCACAAAAAT AATTTTAAAAATTGGCTTATAAAAGATGACTTAGTGGCCCTCAGCTGGGACAG CTTGTCACTATCCAACCAGCCTTTAAATCCTTCCAAGAAGCAATCCTGGCTTGT TCTCCTGAAAACTGGGTAGTTTTTCAAGAGATTGAGCAGAAGCATACAAGGCC TCCTGACACCTAGGTGTGAGATGGGCACACAACCACTCCTGTCAAATTCTATTG GCCAAAGCAAGTGACAAAGCCAATGCAGATTTAAGAGGTGGTGGAAAAAAAC CCTAAAAATAGAAGTTGTTGAAAAGTCATATTTCAAAAGTCATTGGTATAAAG TAGTGAAAAATTTGACATTTTTGCAATCAAGCTTATCAAACAATATTATCCCAA AATATAACAATACACTCAGTTTGCACACTTGTTTACCTTTTGCAAAACAGGTAA GACAGTAGGACAAAGCAGGTGCTTTATGTTGTTTCAATCATTCAGGATTTGGA CAGTTTGGATATTTTCTGTATCACTATAATTGATAAATACTCAGATGATTCTAT AGTTAAGTTAGAATGGAAATTTTGGGTATAGTAACAAATACTACTTTAATTTAA ACTTACATGTAAACAGTTTCCCTAAAGCAGTTAGAAGTGTGACCATAAAAGT GAAATGGTTTAAATACATGCATTTACATCTGTCCTAGAGTGATTAATGTAACTT TATTATAAAACTACTAATTTTGTGATTACATACACCCTTCCAAAGATACATTAT ACATTCCTATGTACACTCAAATATTATTTTTAAACTTCCATTCCAATCATTAAG TAGAAATGCATTTAAGAATCATGATTTTTTTAGAGTAAGTCTATAGGTGGTACT TTTATATTATAGATAACATTTCCTATACCCTTTCCACATAAACACAAGAACATT ATGCTATAGATTGAAAATTCCTGTAAACACTAAGCAGAGCTTTTGTACATAAA CTTGTAAAAACTCTACATAAATGTATTCAGAAATACATGCTATTAAAATATTTT ATTGTATATTACTGTTTGGAAGTTTTCAGCTTAAATATTTTTATTTGATGATCAA TAAGATCTAGTATTAAATGGTCTTATTTATTAACCATTAAGTTAAATACATGGA GAAATCCACTATGTCTTTTCCTCCAGCCTGTAAGTAACACAGGGTTGCATTTCT AATATTAACTAAGTTACATGTATTTTCCATTGAGAAGAGTGCTATCGAACTCAT CCATGTTAAATCACTCTTATGTGGAAAAGGCTAACATATAAATAAAAAAACTA GAAAATTTAAAAAAGGATAAAGAAAGAAGAAAAATGAACAGAATTTAACAGC AGTGCAACAGTAGTCTCTTCCTACCTTTCCTGGGCATCTTCCAATTTTATGGTG GTCTGATAAGCTTTCCAAAACACTTTGCTCATTTCCAGCACTGGACATTTACAC TCAAGACTGCAGACTCGAGGAGTCACACACTCAGCATCTTTTAGCTGTATGTTG TCAAGTTCAGACTACTCAAAGTGGCATGTCTTTAAATTAGAATGTGTCAAGTG GGTCTAGTAACTGCACCGAAATATTTTAATAGTCATATTAATCATTAATAAGTC AGGAAACATGTTTTTCTAATTTTCAGATCCCAATACACATGACTGATATGGTTT GCATCTGTGTCCCTC
Human Genome Map 21q21 (2100 bp sequence) (SEQ ID No. 32) 5OA
AAGGTTTGGAAAGCTTAAGTCAAAATGTGTTGTTCATAAATACGGTCTGAATA ATTTGAACATTTTCTGTTAATGGTATTTGTTCAACTATAATGATATTTTCCAGCC AAGATATAATTGGCAATGTCAAAGTCACACACAGATGGGTAAAATGGCCAATG TCTCTGGAAAATCTTGATAATAACTTTTTAGTATCTCTGGTGCAAGGTCACTTA AATTCAGAAAATAGCACCCAAGGAAAAAATAGCCATATTCAAAAAAATAAGC TCCATATATTTAGATGTAGATATAAATTTGGGGTGATTTATTTCTTATTAGACA CTAATATTTTTTAAAACAGAGAATGACAAATAAGGAAATTTTGCAGTTAACTA TGTCCTAATGAAAAAGGGTAGTAGTTTTACAAGAAAGATATAATTCATCAAAA AGGCAGGGAAGCATTCAGACTAAACATTGAGTATGTTTGGAAATAATAAAAAT TATTGTTTCTTTTACCAACATCAACAATCTTTTCAAATTAATTTATAAAACTGTC ATCTCTGTTCACTAATTTTGAATTACTCATATTATTTTTAATTTTGAATACTTAT AATATTACTTACTAATTTTTAATTAATTTTGATATACCTATATCACTGTTTTGAA TTGATCTATGAATGATCTAGAAATGACTTTGCCTGTTTTTTTTTTTGACTCATGG GTATTTACTTTTCATTAGGTAATTTTAATGTATTGTTAACTAGAAAAATAAGAT GAAGAAAAAAACATTTTAAATGCAAAATATAAATTTAAAGAACTTCAAAAGA ATAAAATTTCAGTTTTATGTCTTTCAAGTAAATTTGCTGTTTTCAAAATTATTTT TTGTTACAAACCTATTTTATTTCAAAAAATATGCTATTGTTTTTAACCTATAATT TTTAAATATCTGACAGCATTGTAGGACTTAAAGCTATTAAATATATAAAGATAT AATAGAACTTATTGGAAATATTCAAGGAAAAACTAACATATTCTTTAAAAACA TTTTAATTTTTAAATTCTATGTTAATTGACTTTTTGATACATATTTTACTTTTCCT TCACTTCTTTTGTCAATTCTTAAAAATGTCTTTCTTCATAATTTTTGGCAATTAG TTTTTACACTTTAATAGCAAACATTGCCATAAAAGTGAAATTAAGCATTAATT AATTTTATGTCTGCAGGCAGAGTGATTTCCTTAGGGAATCAATTTAATAGAGA GAACTATGTTTGTACCTGGCAGGATATTCACAGAAATAAAATATTTATTGGCC ATCTACTTTGTTTAAGACCTCTTAACAAACCATAACTTATTAAAGCATAAAGTA ACATACATAGTAAATACTTTTAAAATCTGTAAACAACTAATTCCTTTCTTCTTG TGAAGTCTTGTTTAGATCATTAAAGTAATAGCAGATTTTCTCACAACAGGTTTG TGAATATTGTCTGTTTAACATGAAAACTATAAAAAAATTAAAGACAATTGATA TATATTTATTCAACTATGTCAACTCAAAGATGATCTGCAATTGTTTTCTGAATA ACTTATTAATAATGCTTAGGCCCCTTTGTTGAACATGCTTTTATTTGTGTAAATA AGAATTCATTTAAAAATACATTGTACAACTTCAACACATTGTGTGTCCCTGAAG GTACTCTGAGATTTTGCAGTTATAGTATAAATGAGACAAAACGGCAGAGAAAA TATTCCCCATGTGTAATTCTTTCTACATTTATTTCCCACATCAATCTCACAAGTG TTTTTATTTCACACTGATTGATATCATTGAGCACATACCTCAATATCTATTATCA CAAAAACTATCATTATCAACAAGGACTTTAAAAAATATCTAAACATTATTATCT GGGTAGCAACTCTATACTCCATTTTATCCATTAATTTTGTCTAATTAGTAAAGA AGTACTTATGGTAAAAACAAATTAAAAATAGTACAGAAAACATACTCCTGTAT GCAATTATTACAAATATTTTATTTAGTTCCTATAAAGTATTTACATAGCTGAGA TCACTATATAATATTATACTCATGTTACTTTATGTCCTAACTTTATATCA
Human Genome Map Ilq23.2-q24.2 (2040 bp sequence) (SEQ ID No. 33) 51A
CGGCTCTCCTGGCCTCGCGCTGCACATTCTCTCCTGGCGGCGGCGCCACCTGCA GTAGCGTTCGCCCGAACATGGCGACACGGAGCAGCAGGAGGGAGTCGCGACT CCCGTTCCTATTCACCCTGGTCGCACTGCTGCCGCCCGGAGCTCTCTGCGAAGT CTGGACGCAGAGGCTGCACGGCGGCAGCGCGCCCTTGCCCCAGGACCGGGGC TTCCTCGTGGTGCAGGGCGACCCGCGCGAGCTGCGGCTGTGGGCGCGCGGGGA TGCCAGGGGGGCGAGCCGCGCGGACGAGAAGCCGCTCCGGAGGAAACGGAGC GCTGCCCTGCAGCCCGAGCCCATCAAGGTGTACGGACAGGTGAGCAGTTTTGC AACCCGCCTCCCTCCAGTTTTTTCCTCTCCCTGCACTTCCTCACCCCCGCATCCA TCCGTTGCAGTCGCCTCCTAGGTGCAGGCACCACTGGGGACTTCCCGGCTTGC ATTTGTTTTTTTCCTTCACGAGTACAACCGTCAGCACTTGAATCGCATTGATCTT TCCTTCTTCCTGTCGATTTAGTAAACGTATTCCAGGTAACTCGCCGGGTGCAGT GCGTATTACCCCAGGGTGTGTGCAGAGAGATGTAGTTTCCGGCAGGTATAGGA GGGGTGCAGCTTCATTTTACATCTGGATAAAAAACGGGCTTTCTTTAGTGTATC ATCAGTTGGCAGTGGAGGCGAGCACCCTGCAGTTGCGGTACACTTACACAGAA CAGCACGAGGTGGGGGTTTCCACACTTAGCATTATTAGCACAATAAAAGTGGG CAAACCTGAAAGCTTGTCGACTATCTCTGTACAGTCAGACAAGAGGTGTGTGT ATGTGTGTGCGTGTGTAAAGGCTGAATTTTTAATTTTTAATTTTTGGCGAGCGT GTGAGATGCTCTCCATTCCTTCTTCCCCACCCTTCAAGATGCTGACTCTCCCAC CCCCGTCAAGATAACTTTATTTTGGAGAGGAATACCCCTCATGGCACTTGGAG ATTTGAAAGGACTGCAGGAAATTTGGTGGGCATTATTATTCTATAAGTGATTTA TTTCTACCCAGGCAATAGGTTTATTAGATCATAAGTAACGTGAATTTCACTTT TATGGTCAGACTTACTGCGAGGAATTGCAGATGGAGTTTGTAGGTTAGGATCA GCACTGGCAAAATTAATTTGACCGTGTTATTGCCTCATGAGACTCCCAGTCCTG CAGTTAAGATTGACATCAGCAAAAGTATAAGGTCGGTGGGGGAGAAAAAGTA GGACCAGAGGAGGGGGTAAATACACTTGTTTTCTAGAGTCAAATTGTTCCTTTT GAAGTAGAAATTATTAATAAAAGATTACCCTGAGTTCTGCCTTTTCTCACTAAT TTCACTTTAGCCATTTCTTCAGGAAATACAGAGTTAAATGTTCAACCCTTGGAT CCAGGACGAACCTTGTAAACATATCACCCTATTGTGTCATTTTGTTGGTGAAGA AACTGAAGCGTGGAATGGTGAAGTGACTAGTCCAAGGTCATACCGGGAAGGT GGCCTGCTCTCTAGTTTTTGTCTGCATTGTCTCAGTGACCTTTGCTTGACTGCAG TCACCCTGTCTTTATGCAATGCTGCTGAAATACCTCCTTTCTAAAATAAAATAG ATCTGGTATAAAGGGGGAAAGGATGGTGGTGACTGGGTGGGAGCGTTGGATTT CCCTCCACTATTGGTCCCTGGGCAAGAATGTGTGCCCCAGGGCATGTAACTAA TGGTGGCCACAGGCTGCAGGAACCTGCATGCTCAGTTCCTCTTGGGCCCAGAT CCTTGTCCCCCTGTCCCCACCCCATATGACAAATATGTGTATGAACAAAAAGA AGTCATCAAGGTCCTTGCTCTTAACAGCGACACCAGCATGGGGCTGATGGAGG GTGGGAGAAGGAGGAGGAGTGGCCCACTTCTTCATTGGGCCTCCGCAGTCAGC CCAGCTCTGCTGTGCTCTTGAATCAGCATTCTGGGAACTGGGAGTTGGGGGCT GGTGGGAGACAA
Human Genome Map 8pll.2 (2100 bp sequence) (SEQ ID No. 34)
52A ACAAAAGGCAAATTGGTGTCTCTGTCCTGGAGTCCTTACTCCTCATCTTGTGCT TAGACATGAAATTACACATCTCCAGCCTTGGGATTCCAGGACTTACACCAGTA GCATGCCTATGTTCTAAGGCCTTTGGCCTGGGACTGAGAATTACACCATCAGCT TTTCTGGTTCTAAGGCTTCTGGACCTGAACTGAGCCATGCTACCAGTATTTCAG GATGTTCAGCTTGCAGATAGCCTGTCGCGGAACTTCTCAGCCTCTAGAATCACA TGAGTCAATTCCCCTAATAAATCTCCTTTTATCTATCTGAACATCTCTCTTCATC TCTCCATCCATCCACTCATGTGTCCATCCATCCATCCATCTATTGCTATCTATCT ATCCATCCATGCATCCATCCATTCAACCATCCATCCACCCATCCATCCATCCCT GTGCCATCTATATCTATCTATCTATATATCTATCTATCCATGCATCCATCCATCC ATCTATCCATCTATCCATCCATCACTATCTATCCATGCATCCACCCATCCACCC ATCCATCCATCCATCCATCCATCCATCACTATCTATCCATCCATGCATGCATGC ATCCATCCATCCATCCATCCATCCATCCATCCATCCATTTATCGCTATCTATCTA TCCATCCATGTATCCATCCATCCATCTGTTCATCTATCACTGTCTATATATCTAT GTATCTATCTATCCATCCATCCATGCATCCATCCATGCATCCATGCATCCATCT ATCACTATCCATCCATCCATCCATCCATCCATTCATCCATCTATCTGTCTTCTAC CTACCTACCTATCTAACTCTCTGGAGAACTCTGACTAATAAACTAGCTTTAAAA CATGTTATTCTCTCTCTGCAATGTCTATTGCTTTATCTTCAGGAACATTCCACAC ATCCTGTAAGACTTCAGTTAAATTATCTCTCTGTTTCTTCTCCAATCATCCTCTG CCTTCCCTAGTCTCCTAACGTACTTTGTACATCTGTCACAAACCCCTCATCATA TTTACTGTAATTTTTTTCCTACAGATTTGGATAGGAATTGAGCCATTTTTTTAAT TTCACTTTTATGGTTGTTACAAATAAAAGAGCAAGCAGGCCCCTCACTGTAA TTCACCTGTATTTGCATTTAACTTATTAACCAAGGCATACTATTTCAAATAATC TAATATAGTATTTCCTATTTAATAACCAAACATACAGAACAGTTCCAAGCACAT GTAACCATGTGATACATTTTCCTCTTTGAATAATAAATATATTTCTTATAATTA ATATGTGATAAAATTGCAATATTTTTAATCTCCTACATCCTTCTCTTTTAATCAG GTTTCCTTATCAACTGGTTCCTATCTCACGGGGTTGTTGCAGAGATGAGGAAAA AAAGTATTCTATTGGTTCATGCATCTCAAAATAGGCAGATTCTTTTCTCTGCTT CTTCCTTCATTGGCTCAGGTGTGGAGTGCTTCTCCCAATTATATGTGCCAGCCT TGGTATGTTCTCATTGCTGTACCACACTGCCTGAGACATCCAAGACCACATCTT CCTTTGGGGGCACATTGGACCTTTGTCATTGGCACTGGCAGGGAAGCTTTTATT TCACCAGGTCTAAGGCAATTCTTCCAAAAAAATCCCAAATAGTGAAAGAATTG ATTTATTCTTCTAATATTTAAGCAAATGTAAAAAAAAAGTTACATTAGTTATGT TTTTTTCAGATTTTGGATCAGTGAGACTTCATTAAAACACTTTGAGGTTATAAA GCAAGTAATTTTTGTTTCCAGAAAAGTTAGTTTCCTTTGGCTGAAGGGACATCT CTATGCAGGCCAGATCAAGACAAAAATAACTTTTAAGAAGGGAAATGAGGGA ATGGAGTTTGGAAAACATAAATCCCACAGCAAAGTACGTCACCAACAATAAG AGTCATCTCTTTCACAGAGGCCTTTCCTAGAAAAGCCCTGACAGACTAGGAGT CCAATCTTCGGCTCCCATAGCACCCATGCCTGCTTCCACTCTGGAGCTTACTAC TTTGCGTTGAAATTAATTTTTACATGTCTATGGCTTCTATTACAAA
In addition, the authors propose in the present invention, a novel model of Alzheimer's Disease generation that involves four novel pol Ill-dependent ncRNA molecules called 17A, 38A, 45A, 51A and their respective Pol II genes GPR51, KCNIP4, APBB2 and SORLl, respectively. Besides the fact that the present invention provides four novel proteins to be investigated in an Alzheimer's Disease-related context, it has to be noted that the model proposed implies that the regulation of the alternative splicing events has to be ascribed to the specific expression modulation of four correspondent Pol III small RNAs whose transcription regulation is under the control of Pol Ill-specific promoters. Although it is not yet possible to predict if the perturbation of the ncRNAs mentioned here is causative of the disease onset or maintenance, it has to be emphasized that the regulation of their synthesis lies upstream to the regulation of the synthesis of their Pol II protein counterparts. Altogether these findings open a novel way of investigating Alzheimer 's disease and provide novel elements for its diagnosis (such as, for example, the Variant IV/Variant I ratio of the KCNIP4 gene). The development of a novel specific drug treatment inhibiting the AD-associated Pol III RNA promoter cluster is possible. In addition 17 A, 38 A, 45 A, 51A promoters (that are likely to regulate their Pol II counterparts) constitute valuable targets for AD studies and therapeutic applications. Therefore it is an object of the present invention a nucleic acid molecule comprising sequentially: a) a 7SL small-RNA derived sequence comprising at least the binding domain to srp9 and srpl4 proteins of the 7SL ribonucleocomplex b) a sequence identical or complementary to a target sequence; c) a pol III type 3 promoter. Preferably, the 7SL small-RNA derived sequence is an Alu-derived sequence. More preferably, the Alu-derived sequence is a 29A derived sequence. Preferably, the Alu- derived sequence is a 21 A derived sequence.
Still preferably, the sequence identical or complementary to a target sequence is of a length of at least 15 nucleotides. More preferably, it is of a length of at least 50 nucleotides. Yet preferably, the pol III type 3 promoter has a sequence comprised in the group of SEQ ID No.l to SEQ ID No.34.
It is an object of the present invention an expression vector comprising the nucleic acid molecule as described above. A further object of the invention is a host cell transformed with the expression vector as described above.
Another object of the invention is a non human transgenic animal bearing the nucleic acid molecule as described above.
It is an object of the present invention the use of the nucleic acid molecule as described above, or of the vector as described above or of the host cell as described above to modulate the expression of the target sequence in vivo or in vitro. Preferably, the target sequence is involved in a pathological state.
It is a further object of the invention, a nucleic acid molecule comprised in at least one of the following sequence: Seq ID No. 68, Seq ID No. 69, Seq ID No. 70 and Seq ID No. 71 for the diagnosis of an age-related pathology. Preferably, the age-related pathology is a neurodegenerative disease. More preferably, the neurodegenerative disease is Alzheimer's disease.
It is also an object of the present invention a nucleic acid molecule comprised in at least one of the following sequence: Seq ID No. 68, Seq ID No. 69, Seq ID No. 70 and Seq ID No. 71 for medical use. Another object of the invention is a molecule able to be vehiculated into the CNS and to bind to the promoter activating region of a nucleic acid molecule comprised in at least one of the following sequence: Seq ID No. 68, Seq ID No. 69, Seq ID No. 70 and Seq ID No. 71. It is a further object of the invention, the use of the transcribed region of the nucleic acid molecule comprised in at least one of the following sequence: Seq ID No. 68, Seq ID No. 69, Seq ID No. 70 and Seq ID No. 71 for molecular diagnosis of an age-related pathology. Preferably, the age-related pathology is a neurodegenerative disease. More preferably, the neurodegenerative disease is Alzheimer's disease.
The invention will be now described by non limiting examples referring to the following figures:
FIG.l : Promoter activity transfection assay. A specific luciferase-silencing hairpin is transcribed by four PSE/DSE-dependent promoters (21A, U6, Hl, 29A). A view of the silencing constructs enclosing the hairpin sequence is enclosed. The promoter region encompasses the putative pol III Type 3 regulatory regions (PSE, DSE and TATA). pMock is a negative control being a luciferase hairpin construct lacking the entire PSE/DSE- dependent promoter region, thus resulting transcriptionally inactive. A) Transfection in HeLa cells; B) Transfection in a NIH 3T3 mouse cell line. FIG. 2: TaD/AM Structure. A = Srp9 Binding Site; B = Srpl4 Binding Site; T = TATA box; P: PSE; D: DSE; 1 : AIu Module ; 2: Target Determinant; 3: 29A Pol III Type 3 Promoter.
FIG.3: GFP-specific Antisense TaD/AM transfection in HeLa cells. A: Construct structures. B: Representative fluorescence microscope fields showing the silencing effects. C: Graphical quantitative determination of GFP silencing by different constructs.
FIG. 4: GFP-specific Sense TaD/AM transfection in HeLa cells. A: Construct structures. B: Representative fluorescence microscope field showing the silencing effects. C: Graphical quantitative determination of GFP silencing by different constructs. FIG. 5: Mouse Gene expression silencing by TaD/AM. A: Construct structures. B: Representative fluorescence microscope field showing the silencing effects.
FIG. 6: Luciferase gene expression silencing by TaD/AM in HeLA cells. Quantitative determination of luminescence emission after constructs transfection. FIG. 7: Nucleic acid sequence phylogenetic tree of 2 Kbp of the whole collection of promoters. The Alzheimer's disease associated promoters are indicated by the arrows. FIG. 8: KCNIP4 N-terminal fragment variants. In bold underlined is indicated the predicted signal peptide with IEA-GLED as cleavage site indicated by the arrow. VARIANT-I, Ace. N°: NM025221 (gene 1-2361; CDS 121-873); VARIANT-II Ace. N°: NM147181 (gene 1-2259; CDS 121-771); VARIANT-III Ace. N°: NM147182 (gene 1- 2371; CDS 317-883) and VARIANT-IV Ace. N°: NM147183 (gene 1-2178; CDS 1-690). NCBI conserved Domains Database Analysis:
123-187 (e-9)Efh: EF-hand, calcium binding motif; A diverse superfamily of calcium sensors and calcium signal modulators; most examples in this alignment model have 2 active canonical EF hands. Ca2+ binding induces a conformational change in the EF-hand motif, leading to the activation or inactivation of target proteins.
160-231 (e-8)EH: Epsl5 homology domain; found in proteins implicated in endocytosis, vesicle transport, and signal transduction. The alignment contains a pair of EF-hand motifs, typically one of them is canonical and binds to Ca2+, while the other may not bind to Ca2+.
79-238 (e-18) FRQl: Ca2+-binding protein (EF-Hand superfamily) [Signal transduction mechanisms / Cytoskeleton / Cell division and chromosome partitioning / General function prediction only] FIG. 9: Promoter activity transfection assay. A specific luciferase-silencing hairpin is transcribed by three PSE/DSE-dependent promoter elements (pMock, pU6-ffl, p38A-ffl). A view of the silencing constructs including the hairpin nucleotide sequence is enclosed. The promoter region encompasses the putative pol III type 3 regulatory regions (TATA box, PSE, and DSE). pU6-ffl is the canonical pol III promoter used as positive control; pMock contains no promoter thus resulting transcriptionally inactive.
FIG. 10: Graphical representation of 38A expression in AD patients and controls. mRNA product of the housekeeping gene GAPDH (Glyceraldehyde 3 Phosphate Dehydrogenase) was measured and subsequently all the 38A expression values were normalized to their GAPDH counterparts. FIG. 11 : A) Dissociation curves. The merge of several analysis of the amplification product dissociation curves evidences that the two KCNIP4 splice variants [here referred to as Variant I (I) and Variant IV (IV)] are unambiguously distinguishable by two picks at different dissociation temperatures. The splice variant I is characterized by a pick at 83.3 + 0.5 0C while the alternative splice variant IV is dissociated at 80.8 0C in the same conditions. B) Graphical representation of the different KCNIP4 Variant IV/Variant I ratio in Alzheimer's disease patients (full columns) versus healthy controls (striped columns). Results are normalized to the lowest value (sample Ctr 4-2). FIG. 12: In vitro transcription analysis of 17A and 38A. a: In vitro transcription of 17A (lanes 2, 7) or empty vector (lanes 1, 4). Transcription products were either radiolabeled during synthesis, gel- fractionated and directly visualized (lanes 1, 2) or subjected to primer extension analysis (lanes 4-7; lane 5, no DNA during in vitro transcription; lane 6, no reverse transcriptase during primer extension). Radiolabeled RNA size markers were loaded on lane 3. The most abundant 17A-specifϊc primer extension product is indicated by a red arrowhead. Shown in lanes 8-11 are the results of sequencing reactions primed with the same oligonucleotide utilized for primer extension, b: The in vitro transcription experiment was identical to the one reported in panel a, except that the specific template was the 38A transcription unit plus regulatory regions, and primer extension analysis was conducted using a 38 A- specific primer. The migration positions and length (in nt) of RNA size markers run in parallel are indicated on the left, c: The in vitro transcription experiment was identical to the one reported in panel A, except that the specific templates were the human 7SK gene (lanes 1 and 10), promoter- less 7SK (lanes 3 and 9), the 7SK transcribed region fused with the 38A promoter region (lanes 2 and 8). Reported below panels a-c are the sequences of 17A, 38A and hybrid 38A-7SK transcription units, respectively. The PSE, TATA and terminator sequences are in red, the bases corresponding to the transcription start sites identified by primer extension are in blue, the transcribed regions are underlined. In panel c, the 38A-derived upstream sequence is in italic character. FIG. 13: ncRNA- induced alternative splicing analysis. a,b: Real Time RT-PCR detection of KCNIP4 and GPR51 splice variants synthesis in SH-SY5Y (full bars) and SKNBE (striped bars) cell lines transiently transfected with either 17A or 38A expression plasmids. a) In both the cell lines results show that the overexpression of 17A ncRNA specifically leads to an increased splice variant ratio of GPR51 variant 2 + 1 vs the physiologically expressed variant 1 whereas non-specific splice variants perturbation of KCNIP4 was not detected, b) Similarly in both the cell lines the transient overexpression of 38A ncRNA caused an increased KCNIP4 splice variant ratio where favoring the synthesis of the alternatively spliced variant IV vs the physiologically synthesized variant I. Altogether these results indicate that each ncRNA specifically influences the splicing of its corresponding protein-coding gene. N. D.: Not Detectable, c-f: Immuno fluorescent detection of GPR51 (c,d) and KCNIP4 (e,f) alternative splicing. Antibodies raised against GPR51 and KCNIP4 N-terminal fragments show a clearly detectable signal in GFP- expressing cells only in the absence of a concomitant 17A/38A overexpression (arrows in c for GPR51 and e for KCNIP4) whereas the signal is absent or very weak in cells transfected with a bicistronic construct coexpressing 17A and 38 A together with GFP: in this case the overexpression of the ncRNA leads to the synthesis of an alternatively spliced form of GPR51 or KCNIP4 inefficiently recognized by the same IgGs (arrows in d for GPR51 and f for KCNIP4). Cells that do not express GFP and 17A/38A are positive for GPR51/KCNIP4 (arrowhead in d and f) according with a 17A/38A-dependent change in the corresponding protein biochemical structures.
FIG. 14: ncRNA expression and alternative splicing in AD cases, a: 17A and 38A expression in AD cases (orange columns) and non-AD control individuals (green columns) as determined by Real-Time RT-PCR. AD cases 124 and 1024 have a familiar origin. The amplification product dissociation curves unambiguously distinguish the RNAs of interest by peaks at specific temperatures (insets). The averaged results are also reported. Asterisks indicate the concomitant highest expression of 17A and 38A in the same individuals keeping in line with a common deregulation of the members of the AP-cluster. b: Real- Time RT-PCR quantitative detection of KCNIP4 and GPR51 splice variants synthesis in AD patients (orange columns) and non-AD control individuals (green columns). Results are reported as splice variants ratio (KCNIP4 Variant IV vs Variant I and GPR51 Variant 2 vs Variant 1) so that an increased ratio indicates an alternative splicing shift. The averaged ratio are also reported. The dissociation curves (insets) show that the splice variants of interest are unambiguously distinguishable by peaks at specific temperatures. FIG. 15: Expression analysis of 38A a sequence variation in three different cell lines. The lucif erase emission after 38Aa promoter-dependent transcription of the luciferase silencing hairpin is expressed as the result obtained by 38Aa normalized to that obtained in the same cell line by 38A wild type promoter. FIG. 16: ncRNA-induced perturbation of Aβsecretion a: Increased amyloid β secretion and perturbation of Aβ42/Aβ40 ratio in 17A/38A-overexpressing SHSY5Y cells. X axis: transfected plasmids. Y axis: quantitative determination of Aβ (pg/ml) secreted in the medium 48 hours after transfection as determined by sandwich ELISA (results were normalized to the pMock-transfected cell line). The resulting Aβx-42/Aβ x-40 ratio is reported. Full columns: untreated cell samples. Striped columns: cell samples treated with 20 μm ML60218 pol Ill-specific inhibitor, b: Demonstration that Pol III transcription is specifically inhibited in ML-60218-treated cells. The 5 S rRNA, 7SK RNA, 17A, 38A and c-Myc transcript levels were analysed by Real-time RT-PCR in the same cells treated with 20μM ML-60218 and in untreated controls used for the experiment in panel a. Only the expression of the Pol II-transcribed gene (c-Myc) was unaffected by inhibitor treatment. All the measures were referred to a ML-60218 unaffected pol II housekeeping gene (G3PDH).
FIG. 17: Schematic view of GPR51 (G protein-coupled receptor 51) locus. Variant I is referred to as Variant a in AceView,http://www.ncbi.nlm.nih.gov/IEB/Research/Acembly and Variant II represents Variant b as referred to Aceview). The alternatively spliced Variant II sequence is homologous to Pan troglodytes GPR51 (XP 520146.2) except for the additional 20 residues long predicted sequence stretch (underlined) most likely constituting a misread protein product. Both the variants encompass a predicted ANF receptor domain (receptor family ligand binding domain). Additionally Variant I contains a 7tm 3 (7 transmembrane receptor, metabotropic glutamate domain) missed in Variant II. GPR51 was reminescent of a possible involvement in AD generation due to the fact that: i) Alterations in signal transduction pathway of G-protein-coupled receptors (GPCRs) were found in the cerebrocortex and in the peripheral cultured tissues of AD patients (Leosco D. et AL, 2007) and ii) the membrane G protein-coupled receptors Kinases were significantly reduced in brain cortices of an early-onset AD transgenic model (Suo Z. et al., 2004). FIG. 18: Schematic view of KCNIP4 locus. Protein sequences were analyzed in silico showing that the alternative Variant IV (variant b in AceView) harbors a predicted endoplasmic reticulum localization due to a predicted signal peptide (Signal IP) whereas Variant I (Variant a in Aceview) lacks this structural feature (see Aceview annotations at www.ncbi.nlm.nih.gov/IEB/Research/Acembly). As shown, 38A maps in intron I of KCNIP4 gene (GC04M020407, I lpl5.31) in a region where alternative splicing events give rise to the distinct I and IV protein variants. Together with the other proteins of the same protein family, KCNIP4 regulates the electrophysiological properties of Kv Kb channel with a specific role of the splice variant IV in determining an altered kinetics of potassium channels (Holmqvist, M. H et al., 2002). KCNIP4 is a Presenilin 1 and 2 protein interactor (Morohashi H. et al., 2002). FIG. 19: Cerebral cortex samples. FIG. 20: Speculative model of the AP cluster contribution to Alzheimer's disease.
EXAMPLE 1: TARGET DETERMINANT/ALU MODULE (TAD/AM) GENE EXPRESSION REGULATORY CONFIGURATION Materials and Methods
Databases and searches
All the sequence searches and alignments were carried out taking advantage Basic Local Alignment Search Tool of the National Center for Biotechnology Informations (www.ncbi.nlm.nih.gov/BLAST/).
Cell culture, transfection and Luciferase assay
For transient transfections HeIa cells (grown in DMEM supplemented with 10% FCS), were grown in multiwell Petri dishes 16 hours before transfection. The expression constructs containing the regions of interest cloned in the pTopo vectors (Invitrogen) were introduced into the cells using the Fugene 6 transfection reagent (Roche) according to the manufacturer's instructions. A plasmid Expressing Luciferase (pGL3) and one expressing Renilla (pRL) were used as control of transfection efficiency (to which all the results were normalized). 24, 48 and 72 hours after transfection cells were harvested and both firefly luciferase and Renilla luciferase activities were measured by Dual-Luciferase reporter assay system (Promega) according to manufacturer's protocol. RNAi-silencing assay
In order to test the promoter activity of the novel transcription units, the authors prepared four plasmid constructs expressing a firefly luciferase silencing hairpin (obtained by Gregory Hannon's Laboratory-Cold Spring Harbor Laboratories) which transcription was driven by the Hl, U6, 21A, 29A promoters, respectively. The hairpin sequence [targeting a firefly luciferase mRNA from a co-transfected expression plasmid (Promega)] is:5'GGAUUCCAUUCAGCGGAGCCACCUGAUGAAGCUUGAUCGGGUCUCGCUG AGUUGGAAUCCAUU-3' (SEQ ID No. 35). Oligos used to subclone the novel Pol III Type 3 promoters within Not I/HinD III restriction sites (in capital) were the following: HlFprom Not I: 5 '-ATGCGCGGCCGCATTTGCATGTCGCTATGTG-S ' (SEQ ID NO. 36)
HlRprom HinDIII:5 '-GATCAAGCTTCATCAGGTGGCTCCCGCTGAATTGGAATC CACGCACTCAGCTCGTG-3' (SEQ ID No. 37) 21AFprom Not I: 5 '-ATGCGCGGCCGCACAGCTGTAGCAGATGCT-S XSEQ ID NO. 38)
21ARprom HinDIII:5'-GATCAAGCTTCATCAGGTGGCTCCCGCTGAATTGGAAT CCACCAC ACTTGGTCAACT AT-3 '(SEQ ID No. 39) 29AFprom Not I: 5 '-ATGCGCGGCCGCTTCTCACCTAAAGGAGTC-S ' (SEQ ID NO.
40)
29ARprom HinDIII:5 '-GATCAAGCTTCATCAGGTGGCTCCCGCTGAATTGGAAT
CCTTCT AATCCTCCT AAGATCA-3' (SEQ ID No. 41). In this analysis the above constructs were co-transfected with a pGL3 plasmid
(Promega) expressing Firefly (ffl) Luciferase as target to be silenced and with a pRL plasmid (Promega) expressing a Renilla Luciferase to which all the determinations were normalized. 24, 48 and 72 hours after transfection cells were harvested and firefly/Renilla luciferase activities were measured by Dual-Luciferase reporter assay system (Promega) according to the manufacturer's protocol.
Plasmid constructs generation and sequencing
The original plasmid constructs p21A and p29A were generated amplifying from a genomic DNA preparation the regions of interest; the PCR products were then subcloned into the pNEB193 vector. The oligos used to generate p21A and p29A PCR fragments were the fo Ho wing :
21 A Forward: 5 '-GGAAATCTTACCTTCCTGCC-S ' (SEQ ID NO. 42) 21 A Reverse: 5 '-TGGCTAGGTCATGTGACCAT-S ' (SEQ ID NO. 43) 29A Forward: 5 '-CATAGCTGCACTTTACTCA-S ' (SEQ ID NO. 44) 29A Reverse: 5 '-ACATTGAATCACCTTATGAG-S ' (SEQ ID NO. 45) The insert obtained was then subcloned in pTOPO vector (Invitrogen) following manufacturer's instructions. Prior to transfection all the plasmids were sequenced by DNA
Sequencing Kit (Applied Biosystems) following manufacturer's instructions.
The TaD/ AM constructs were generated by ligating together in the pSHAG vector the three elements of their construction (the 29A and or U6 promoter, the GFP sense Target Determinant, the GFP antisense Target Determinant, The Luciferase sense Target
Determinant) obtained by PCR as follows:
29A Promoter:
- Forward: 5 '-CATAGCTGCACTTTACTCA-S ' (SEQ ID NO. 46)
- Reverse: 5 '-AGTCAAGCTTTCAGTGTCTTTACTAACTTC-S ' (SEQ ID NO. 47) U6 Promoter:
- Forward: 5 '-AAGCTTGGCTGCAGGTCG-S ' (SEQ ID NO. 48)
- Reverse: 5'- GGTGTTTCGTCCTTTCCACAA-3' (SEQ ID No. 49) GFP sense Target Determinant:
- Forward: 5 '-AGTCAAGCTTAGGTTATGTACAGGAACGCA-S ' (SEQ ID NO. 50)
- Reverse: 5 '-AGTCAAAAAAATCTTCAATGTTGTGGCGAAT-S ' (SEQ ID NO. 51) GFP antisense Target Determinant: - Forward: 5 '-AGTCGGATCC AGGTT ATGTAC AGGAACGC A-3 ' (SEQ ID No. 52)
- Reverse: 5 '-AGTCAAGCTTATCTTCAATGTTGTGGCGAAT-S ' (SEQ ID NO. 53) Luciferase antisense Target Determinant:
- Forward: 5 '-ATGCGGATCCACGTGAATTGCTCAACAGTATG -3' (SEQ ID No. 54)
- Reverse: 5 '-ATGCAAGCTTGTCCCTATCGAAGGACTCTG -3' (SEQ ID No. 55) Luciferase sense Target Determinant:
- Forward: 5 '-ATGCAAGCTTACGTGAATTGCTCAACAGTATG-S ' (SEQ ID NO. 56)
- Reverse: 5 '-ATGCAAAAAAGTCCCTATCGAAGGACTCTG-S ' (SEQ ID NO. 57)
Results Pol III Type 3 TaD/ AM promoters.
In order to identify the most efficient pol III Type 3 promoter of the authors collection for the construction of the Ta/DAM molecules, the authors fused the two promoters of the Alu/7SL-derived transcription units (2 IA and 29A) with an hairpin able to down-regulate the expression of a co-transfected Luciferase plasmid (pGL3) by a mechanism of RNA interference. After transfection in HeLa cells, a very high 21 A/29 A expression level would lead to an efficient luciferase silencing detectable by a decreased luminescence emission, while the unefficient transcription of the hairpin by the examined Type 3 promoter would lead to a very high luciferase signal. As negative control a luciferase hairpin construct lacking the entire promoter region was prepared (here referred to as pMock). As positive controls two similar constructs were prepared substituting the 21 A and/or 29 A promoters with that of two well known Pol III Type 3 transcription unit, Hl and U6 respectively. After co -transfection followed by normalization to the untransfected sample, the luciferase emission was 0.5 + 0.1 for the 21A-driven construct, 0.1 + 0.005 for the U6-driven sample, 0.2 + 0.01 for the Hl-driven construct and 0.1 + 0.02 for the 29A-driven construct (Fig. IA). Therefore, based on the above results the authors decided to use 29 A as the most efficient Pol III Type 3 promoter for the construction of an artificial TaD/ AM construct to be used in primate cells (Fig. IA). Since 29A and 21A are Alu-derived elements and Alus are known to be primate- specific, the authors also tested the efficiency of the above mentioned promoters transcription in NIH 3T3 mouse cell line in order to identified the best promoter to be used in mouse. Results, as normalized with pMock, showed that both 21 A and 29A are inefficient in mouse while U6 and Hl are very active (Fig. IB). Altogether these results suggested the use of U6 as the most suitable type 3 promoter for the development of a specific gene expression downregulation system useful in the two species and for the development of TaD/AM-dependent transgenic mouse models. Gene expression silencing by GFP-specifϊc Antisense TaD/ AM configuration In order to test the hypothesis of a useful stabilization of the silencing molecule by adopting a TaD/ AM strategy, the authors designed a first primate-specific GFP TaD/ AM theoretically useful for the down-regulation of an exogenously expressed Green Fluorescent Protein (GFP) in mammalian cells. The authors fused the 29 A promoter (encompassing a TATA box, a Proximal Sequence Element, PSE, and a Distal Sequence Element, DSE) with a 246 base pairs long antisense fragment of GFP 47 followed by the 191 base pairs long 21 A AIu module expected to bind srp9 and srp 14 proteins (Fig. 2). All the constructs were then sequenced in order to assess the correct cloning procedures.
48 hours after the Antisense TaD/ AM and GFP 47 co-transfection in HeLa cell, the authors analyzed the samples by a fluorescence microscope and counted several microscope fields for GFP positive cells evidencing that: a) the promoter without TaD/ AM shows a rate of positive cells (1.1 + 0.09) comparable to the unsilenced sample transfected with the GFP 47 plasmid. This indicates that an eventual effect observed further to transfection of the cells with TaD/ AM would be an insert specific effect; b) The cells transfected with the TaD (a construct enclosing the GFP 246 bp long Antisense Target Determinant without the AIu Module) were slightly silenced showing decrease in the number of GFP positive cells (0.8 + 0.1) indicating that the antisense sequence alone is not enough to induce an efficient GFP targeting, c) The samples transfected with the TaD/ AM configuration enclosing the AS Target Determinant fused with the AIu Module showed a dramatically decreased number of GFP-positive cells (0.3 + 0.03) indicating a very efficient GFP targeting (Fig 3). The further observation of macroscopic cell parameters such as cell viability and/or morphology did not evidence differences between the TaD/AM-transfected and the other samples, thus suggesting absence of toxic effects induced by the TaD/AM molecule. The authors also measured the OAS (2'-5' o- Adenylate Synthetase) level by Real Time RT PCR in all the samples, comparable results were obtained suggesting that antiviral response activation could be excluded.
Therefore based on the above experiments the authors conclude that the AIu Module fused to the Target Determinant confers stability to the silencer molecule most likely acting as ribonucleoparticle able to evade the enzymatic degradation of the molecule. Gene expression silencing by GFP-speciβc Sense TaD/ AM configuration
It is now well known that mRNA molecules are almost always folded following specific rules. Internal molecular sequence pairing of specific stretches together with proteins bound to the nucleic acid is at the base of the proper secondary structure determination. In this context, the authors considered that the disruption of the proper mRNA secondary structure constitutes per se a mechanism of gene expression downregulation that deserves a detailed investigation. Therefore they subcloned in the GFP-specifϊc TaD/ AM construct previously used, the same GFP Target determinant in Sense configuration. A different version lacking the AIu Module of the same construct was then prepared and used as control of the stability conferred by the AIu module. All the construct were then sequenced in order to assess the correct cloning procedures. 48 hours after co-transfection in HeLa cells of these constructs with a plasmid expressing GFP 47, the authors measured the rate of GFP positive cells in several microscope fields after normalizing the results to the unsilenced sample (transfected with pGFP without the silencer construct). Results showed that: a) As previously demonstrated, the promoter alone does not induce variation in the number of GFP positive cells (1.1 + 0.2) when compared to the unsilenced sample; b) The construct enclosing the Target Determinant in Sense configuration butr lacking the AIu module inhibits with low efficiency the GFP expression (0.86 + 0.2); c) Strikingly, the GFP-sense TaD/ AM construct is the most efficient configuration for gene expression silencing (0.25 + 0.03). This could be explained by a conjugating and unexpected silencer effect of the sense configuration with the stability conferred by the AIu Module (Fig. 4). The constructs did not induce any other changes that could be associated with a possible antiviral response.
Mouse Gene expression silencing by TaD/ AM
In order to test the possible use of TaD/ AM configuration to silence genes in mouse, the authors substituted the 29A primate-specific promoter with the U6 mouse- readable promoter in the above GFP-(AS)TaD/AM construct. After sequencing, the constructs were transfected in GFP transgenic mouse osteoblast primary cells. 48 hours after transfection, the cells were examined for their fluorescence emission. Results resembled what previously observed in human cells and show that sense TaD/AM configuration is the most efficient in silencing endogenously expressed GFP (Fig. 5). Luciferase gene expression silencing by TaD/AM
In order to assess the silencing efficacy and the reproducibility of the silencing effect of the sense TaD/AM configuration on a different gene, the authors prepared a Luciferase-specifϊc Sense TAD/AM construct. A 270 bp long luciferase fragment was subcloned in the GFP TaD/AM construct substituting the GFP sense sequence moiety. After sequencing, the construct was co-transfected with a plasmid expressing luciferase (pGL3) and a plasmid expressing Renilla luminescent protein (pRL) in HeLa cell. As negative control a pMock plasmid was used as above. 48 hours after transfection, the luminescence of both Luciferase and Renilla were measured by a luminometer. The luciferase emission was then normalized to that of Renilla in order to eliminate artifacts due to different transfection efficiencies in different samples. Normalized results showed that, taken the pMock-transfected cells luciferase emission as 1 + 0.28 the correspondent value in pLuc-TaD/AM was 0.14 + 0.02. The result demonstrates a very efficient TaD/AM-dependent silencing of the luciferase gene expression (Fig. 6). Again, no morphological differences (and/or a decrease in cell viability) were detected between the luciferase-silenced samples and the negative control. Therefore this result further strengthens the efficacy of the novel TaD/AM configuration in silencing technology.
EXAMPLE 2: POL III-DEPENDENT NONCODING RNAS IN ALZHEIMER'S DISEASE
Materials and Methods
Databases and searches
All the sequence searches and alignments were carried out taking advantage of the Basic Local Alignment Search Tool of the National Center for Biotechnology Informations (www.ncbi.nlm.nih.gov/BLAST/).
Cell culture, transfection and Luciferase assay
For transient transfections, HeIa cells (grown in DMEM supplemented with 10% FCS) were grown in multiwell Petri dishes 16 hours before transfection. The expression constructs containing the regions of interest cloned in the pTopo vectors (Invitrogen) were introduced into the cells using the Fugene 6 transfection reagent (Roche) according to the manufacturer's instructions. A plasmid Expressing Luciferase (pGL3) and one expressing Renilla (pRL) were used as control of transfection efficiency (to which all the results were normalized). 24, 48 and 72 hours after transfection cells were harvested and both firefly luciferase as well as Renilla luciferase activities were measured by Dual-Luciferase reporter assay system (Promega) according to manufacturer's protocol. RNAi-silencing assay.
In order to test the promoter activity of the novel transcription units we prepared two plasmid constructs expressing a firefly luciferase silencing hairpin (obtained by Gregory Hannon's Laboratory-Cold Spring Harbor Laboratories) which transcription was driven by the U6, 38 A, promoters respectively. The hairpin sequence [targeting a firefly luciferase mRNA from a co-transfected expression plasmid (Promega)] is: 5 ' GGAUUCCAUUCAGCGGAGCCACCUGAUGAAGCUUGAUCGGGUCUCGCUGA GUUGGAAUCCAUU-3' (SEQ ID No. 58). Oligos used to subclone the novel Pol III Type III promoters within Not I/HinD III restriction sites (in capital) were the following: 38AFprom Not I: 5 '-ATGCGCGGCCGCTTCACTAAGATCCAGTGC-S ' (SEQ ID NO. 59) 38ARprom HinDIII:5 '-GATCAAGCTTCATCAGGTGGCTCCCGCTGAATTGGAAT CCGATTCATGAAC ACAGAAT ATT-3' (SEQ ID No. 60).
In this analysis the above constructs were co-transfected with a pGL3 plasmid (Promega) expressing Firefly (ffl) Luciferase as target to be silenced and with a pRL plasmid (Promega) expressing a Renilla Luciferase to which all the determinations were normalized. 24, 48 and 72 hours after transfection cells were harvested and firely/Renilla luciferase activities were measured by Dual-Luciferase reporter assay system (Promega) according to the manufacturer's protocol. Plasmid constructs generation and sequencing
The original plasmid construct p38A was generated amplifying from a genomic DNA preparation the regions of interest; the PCR products were then subcloned into the pNEB193 vector. The oligos used to generate p38A PCR fragments were the following: 38A Forward: 5 '-AGCAATAGCAATCAGACCAG-S ' (SEQ ID NO. 61); 38A Reverse: 5 '-GTCCTGTTGGTACCCTTGT-S ' (SEQ ID NO. 62). The insert obtained was then subcloned in pTOPO vector (Invitrogen) following manufacturer's instructions. Prior to transfection all the plasmids were sequenced by DNA Sequencing Kit (Applied Biosystems) following manufacturer's instructions. Real-Time Quantitative RT-PCR Total RNA preparations of 8 post-mortem brain biopsis derived from Alzheimer's disease patients and 8 derived from healthy donors (no correlation with AD) were subjected to reverse transcription by Superscript II First Strand Synthesis Kit (Invitrogen) following manufacturer's instructions. The cDNA obtained was measured by real-time quantitative RT-PCR using SYBR GREEN gene expression quantization, on PE ABI PRISM@ 7700 Sequence Detection System (Perkin Elmer). The sequences of forward and reverse primers as designed by the Primer Express 1.5 software were KCNIP4 Sybr For (varl) 5'-ATGAAGCTCTTGCCCTGCTC-S', (SEQ ID NO. 63); KCNIP4 Sybr For (var4) 5'-TGGAACAGTTTGGGCTGATTG-S' (SEQ ID NO. 64); and KCNIP4 Sybr Rev (varl/4) 5'-CGGTGGCCATCTCCAGTT-S' (SEQ ID NO. 65). Threshold cycle, CT, which correlates inversely with the target mRNA levels, was measured as the cycle number at which the reporter fluorescent emission increases above a threshold level. For endogenous control, the expression of Glyceraldehyde 3 phosphate dehydrogenase (GAPDH) gene was examined by quantitative RT-PCR as described above. The sequences for human GAPDH primers were 5'-GAAGGTGAAGGTCGGAGTC-S' (SEQ ID NO. 66), 5'- GAAGATGGTGATGGGATTTC-3' (SEQ ID No. 67). Relative transcript levels were determined from the relative standard curve constructed from stock cDNA dilutions, and divided by the target quantity of the calibrator following manufacturer's instructions. Results Co-evolution of the four AD-associated Pol III promoter elements. In order to gain insights into Pol Ill-driven promoters and with the final aim to identify novel regulatory regions, the authors aligned by MegAlign software (www.dnastar.com/products/megalign.php) 2 Kbp of the sequence of each promoter of the whole Pol III Type 3 collection (the sequences are indicated in Table 1).
Table 1: Sequences of 2000 bp of 17A, 38A, 45A, 51A nucleic acid promoter sequences; the PSE elements are in bold, the TATA boxes are underlined. Promoters are followed by the transcript sequences indicated in italics. Transcript sequences are followed by 600 bp of downstream regions. 17A promoter (2000bp) Seq ID No. 68
(embl AL 445495.51) (From 58423 to 56424) (reverse complement feature)
GGGGCCCACCTGTATCCAGAAGGATTTGAGACAGCAGAAATAAACTCTAATG GAAAAGTTAACTTTTTCCCCCCATTGACTAGTTACCAATCTTCCATCAAATTGA GGTAAAAACGAAGGATGGACAAGGTTCACGCTGACTGTGACAGCATCTTTCT TTTTACAAAACATTGATCACAAGGCATTTCAAGCATTCAGAATAAAATAAACAC CCAAGGACTTGCTAGCCAGAATGATTACATGTTCACATTTGCTCCATTTGCTT CATATTTCTGTCAAAGAAATAAAAGATGGCAGCTCCCATCACAGTCTGTTTTG TCTCCCTTCCTTGGTGTTGACCATCCTGTGACATTTAAGGCCAGATTATCCAA AATGCTCTGATGTATACATTACAAGGATCCACCAAGTGTTGAAATAAGAAGTT CTTTTTTATTGTGATACAATATACATAGCAGAATATTTATCATTTTAGCCATTTA TAAGGGTACAGTTCAGTAGCAGTATGTTCACATGTTTGTGCAACTATCACTAC TGCATCTGCAGAACTTTTTTTTTCATTCCACACTGAAACTCTGTGCCCATTAAA CAATCACTCCCCACCCCCTCCTTCCCTCAGCTGCTGGTAACCACCATTCTACT CTCTGTCTCTATAAATGTGACTAACCTGAGTATCTCATATGAATAGAGTCATAC AATATTTGTCCTTTTGTGTCTGCCTTATTTCATGTAGCATGATGTCTTCAAGGC TCATCCATGTTGTAGCATGTGTCAGAATTTTATTCCTTTTGAAGGCTGAATAGT ATTTCACTGTCTGTATACACCACAATTCATTTATCCATTCATCGAGCAGTGAAC GTTTGGGCTGCTTCTGCCTTTTAGCTGCAGTGAACAAATGTTCTTATGAACAT GGGTGTAAAATGTCTGTCAAGTCCCTGCTTCCAATTCTTTTGGGTACGTGCCC AGAAGTGGAATTGCTGGATCATGTGGTAATTTTATATTTATTTTTTTGAGGGGC TGCTATACTGTTTTCCACAGCAGTGGCAGCATTTTACATTTCCACCAGCAACA CACAAGGGTTCCAGTTTCTCCACATCTTCACCAACACTTGTGGTTTTGCTTTTT GGTAATAGCCATGCTAATGGGTGTGAACAAGAAGTGCTTTAAGCATCTCCTAA AGCGGAAGAAACTGAGGCCCAGAGAAGGGAAGAATCACACGAGAGATTGAG GTCACAAGCAAGTCAGTGATAGAGCAGGACCTGGAAGCTGGATCCCCTAACC CCAGCCTAGTTCTTGCTACTAAAACCCAAAATCCAGTTTCCATTGCTATATGTC AGAGGGTGCACAGCCATGGCCACAGGCCAGATACAGACTTTAAGTTTATTTG GTTTGATCCTTACTTTCTTTTTTTTTTAATTCAAAATAGTATCAACATTTAAAAAT TAGGGGATTTTATATTTTAAAAGTCTAAATTTCTGATTTCTCCCCTCAAAAATCA GAAGGTCTGGTAACCCTTGACCCACATTCTAACTCAGCAACCAACTATTACTG TCTTTTGTTTTGTTTTGTTCTGTTGAGACAAGGTCTTGTTCTGTCACCCAGGCT GGAGTACGGTGGCGTGATCACGGCTCACTGCAGTCTTGAACTCCTGGGCTC AAGCAAGCCCCCCGTCTTGGCCTCCCAAAGCTCTGGGATTACAGGTGTGAGC CCACGCCCAGCCCTATCATTCTGTAATATCCTTCCACACAGGCTAGTTCACAC ACTGGCTGGTCCTGGTAACACTGGAGTTTGCAGCCCTTTGCTTTTCACATCCA TAGATATTCCTCATTCTGAGTGTCAGTAGACACATAGTTACGTGTAACATCATA GGCAGGTTCCATACTTCTTTCCTCTTTCCTTTACTCTATATTGTCTTTGAATAT CTTAGCTATTTCCTCACCATAAAAGTGAAATAATGTTGCAAATAAATAGTGCA AAATATTAACAAAGACACAATTGAATAG
CCTGA TTTGGAGACAGTGTGATGTGGTGGAGAAACACCAGCAAAGGTGCTAA TCTTGACTCTTGCAGGAACTCCCTGGGCAATCTTGGAAAGTCCCTTCCCTGTC TGGCCTCCTTGTATCCATGTGTCAGTTGCAGGGTGGGACTGGGGGATCCTCA AG
TTTTTGTGGGGCGGTCTAGTTAATAATGTAAAACAAAACCACTCACTTTGCATT GTCGGTAGAGAAAATCGCTTTGCCCAATTGACTTCCCCAGGGTGACCCCTCG GAACTGGCCCTTCCTGTGGTCCCCAGCACACCCACCCTCTGCTTTCTCTTTG CTGACAGGTGCGGAATGACCTGACTGGAGTTCTGTATGGCGAGGACATTGAG ATTTCAGACACCGAGAGCTTCTCCAACGATCCCTGTACCAGTGTCAAAAAGCT GAAGGTAGGTGTGGCTAGCCACAGGGTGAATGGCAGACTGATCTCAATGCC CTGGGTGCCCAAATTACCTGACTATGAAAGAGCCTCAAGTTTGGTCTGGGTC TCAAGTTCATTTCCACCTTGGCATCCCAGACAGGAGAAGCTCCCAGGAATTG GCTTCCCTTGGCTTTCCCTTCCTTGCTCTCCAACACTGGCCAGAGGTGCTGG CTGCAGGTGCACAGCCACTGAGCACTTTCCTGATGGTTTTATATCTCTCAACG CAGCCTCAGGGGCAAGCAGGGCACACATGATTGTCATGTCTGTTTTGAAGAG TCACAGAGAGAGGAACTGTCTTTCA
38A promoter (2000bp) Seq ID No. 69 gb AC109636 (From 8647 to 10647)
AATGCAACAGCATCTATCCCACTATTTTTGTCTAAGCACTGCTATATTAAAAAG CTTATTGTTTCTAGGGCTTGTTAATCTTGAAGACATGATTTATTTCTATGGGAC TAATTTATTCAATTCGAGCAGGGATGATAGAAAATACTAGAATAAAAGCAAATT ACTTTATGGATATGTGCCTGTGTGTGTAATTGGACACAAAGAACAATTAGAGG CATAAAAATGTTCAAATAAATGCATTTTTTCAGAGTATGAAAAATATATGGCAT TTTTAATGTCACCTATAAAACTACGTATAATCTATAAATTATATTATCCTTATTG GGACTATTCATTGATCATCTAAACAGACCAATACAAATTTTCTAAGATAGTTTC TTTTTTTTTTTGTATTTTATTCTTCCTATTCTACCTTGATTTACCAAGATATGAAA CTACAAATCAATAACATGGGGAACTTGGAAAATCCACAAATACATGAAAATTA AACCACATGCTCCTGAATGACCAATAAATTAAAGAAAAACTCAAAAGGGAAGT TAAAAAATATTTTGAAACAAATGACAACACAACATATCAAAATCTATGGGATGC AGCAAAAACAGTTCTAGGATAAAGGTTTATAACAATAAATGCCTACATTAAAAA AGAAGGTAGATTCCAAACAAATAGCCTAACATTATGCCTCAAGAAACTAAAAA AAAGAAGAGCAAACTAAACCCAAAGTTAGTGGAAAAAAGAAAATAATAAAGAC TAGAATGAAATAAAAGGGAGCAGAAAAACCATTTAAAAATAGTAAAACCAAGA ATTAGTTGTATGGATAGGTAAACAAAATTCACAAACTCCTAACTACCTAAGGA AAAAGACTCAAATAAATAAAATCAGAAATAAAAGTAGAAATATTGCAATGGAAA CTTCAGAAGTAAAAATGATTATAACGGGCTATTATAAACAATTATGTGCAATAT TTAATCAGGAAGAAATAGAAAGCTTGAATGGACCAATAAAAATTAAGAGATTG AAATATGAATTCAAAACTTTGCAACAAAGAAAAGCCCAGGACGAGATGGCTTC ATGAATGAATTCTACTAAACATTCAAAGAAGTATTACCAATATTTAAATTCTCC CAACAAATAGAGATAGAAGAAATACCTGCAAACACATTTTACAAGGCAAGCAT CACCTTGATCCCTAAGCCAATGACATCACAAAAAAGAAAACTATAGGCCAATA TCTCTGATGAACATTGATGGAAAAATTCTCAATAAAATATTAGCAAACAAAATT CAACATCACATCAAAAAGATTATACATCATGACCAATAGGATTTATCCCTAGCA TGCAAGGCTGGTTTAACATACACGAATGAAACAATGTGACACATCACATTAAC AGGATGAAAGATAAAAAACACAGAATTTTCTCAATCAACACAGAAAAAGCATT TGACAAAGTTCAGCATCCTTTCCTGATAAAAACTCTTAACAGTTTATGTATAGA AAGAAAATTTCTCAACATAATATAATAAAGGTGATTTATGAAAAATCCACAGCT AACATAATAATCAGTGGGAAACAGTTGAAAGCTTTTTCACTAAGATCCAGTGC AAAGCACAAATGCCCACTTTTGCTACTTCTATTCCACATAATATTGGAAGTACT AGCAATAGCAATCAGACCAGAGAAAGAAATAAAAAGCATTTAAGTCAGAAAGA AGAAAAAGTAAAATTATCTCTATTTGCAGATGATATAATCCCTTATGTAGAAAA CCCTAAAGATTCCACAAAAAACTGACAGAATGAATTAATTCAGTAAACTTGCA GGATACAAAATCAACATACAAAAATCAGTAGCATTTTTATACACTAATAACAAC ATATCTGAAAAAGACGCTTTAAAATCCCATTTATGAAAGCATAAAAATAGTTAG AAATAAATTTAACCATAAAGGTGAAATATTTGTATACCGATAACTATAAACCT TTGATAAAAAAAGTTGA
A GAA GA CA CA TA TAAA TA GAA TAA TA TTC TG TG TTCA TGAA TCAAAAAA TTTAA CAA TG TTAAAA TG TC TG TA TTAACCAAA G CAA TA TA CAAA TTCAA TG CAA TTTC TA TCAAAA TTTCAAGGA TA TGCA TCACA GAAA TA GAAAAAAAA TTCTTGAAA TT CATATGGAACCACAGACACATAAAAACAGAATAGGCAAAGGAACAATGAGAA AGCAAAACAAAGCTTGAGGCATCACACTTCCTAAGTTAAAATTATATTGCAAA GCTACAGTAATCAAAAACAGTATACAAATGGCATGAAAACGAAAATGTGGACC AACGGAACAGAATATAGAGAGCCAGAAACTTAACTAA
TTTTCAACAAGGGTACCAACAGGACACCCTGAAGTAAAGATAGTTTCTTCAAT
AAATGATTCTGGGAAAATTGGATTGCAACATGCAGAAGAATGAAATTGGACCC
TAATCTTGCACCATATACAAAAATGGACTCAAAATAGATAGGAGACCTAAATG TAAGATGTGAAACCATAAAACTCCTAGAGAAGAACATAGGGGGAAAAATTCCT TGATATTGGCCTTGGAGATGATTTTTGGATATCACACCAAAAGCTTAGGCTAC AGAATCGAAAATAAATAAATGGAACTACATCAAACTACAAAGTGTCTGCACAG TAAAGGAATCAATCAACCAAATAAAAAGGCAACATACAGACTGGGAAATATAT TTTCACACAGCATATCTCCTAAGAGGCTAATATTCAACATTTGTAAAGAACACT TACAAATGAGTAACAGAAACAACAAACAGCTTGATTAAAAACAGGCAAGGGAC CTGAACATACTTTTCTCCAAAGGAGAAATAATGGCTAACAGGATATGAAAAGG TATACAACATTGCTAATCATTAGGGAAACACAAATGAAAACCACTATGAGATAT CACCCTTCACCCATT 45A (2000bp) gb AC096712.31 (5341-7341) Seq ID No. 70
ATAATAAAAATATTTATTACTGAGGATTTGTGAGAAATGTTTCCTCTAAATAGA GGTCCATAAATTACTGAGATTTAAAAAACAATGCTCTTTATATACTTCACCACC CTTGATACATTTATCATTCTCTCCATAAATGAATGGAGAGAAATGGAACACAAT CGCACATGCAAGAAATCATTTGCTAAACTCCAATTTCTCCCCAGTTCTCCAGC TGTTTGAATTATGTCATTACTGATTAACCTTTGGAGGCAGCAGGCCTTGTTTTT TTCCTCCAATAGAGATGGCTTCTAATAGGAAAAATGACTGGAAACACTTCTCT GCCCAGCTCCTACCATTATACCCGCTTCTACTAGAATTTGTAAAATAACACAA ATGCACCTGGCAATAGTACAACCAATCAAATGTCTCCAACAAAGGATGGTCGT CCTTGCCTTAAGGAAGTCCATTTCCTGGTTGCGTTTCCATAACCAGGAATTTT GGGAAATATGGCATTGTTTTTCTGAACGAGTAAAGAAACTACCAATCATTTCTT TTAAACTTCTAGATTTTTATTCTCTATGGACTGTAAACTTTCACAGCTTCATTTT AAAAGTCCTGTGCCATAATTTTACCCTACAATTCCTGTCTCCCTGAAATGTGTA AAACCAAGCTGTAGCCCAACTCTCTCATGCACGCTTTATCAGGATCTCTTGAA ATTGTGTAACCTAGACCACAGTCACTTATACTGGTTCAGAATAAACCTCTTTAG ACATTTTGGCAGAATTTGGATTTTTCCCATATTGAGTGTTTTTTGGTCCCAAAA CTGATTAAAAATTACCTTGTGTTTTAAAAGTCCTATGCCAAGATAAAACTTTCT CTCTCCAACATATGTGCTCATGTATCAACATCTATGTTTCTCCTCCCACAAGGT CCAACTCAATCAGAACCAAAAGGGAGATCACAGCATATCCATGCAATCCCTG GCTGGACAGACGGGGCACCCTAGGGCCTGGAGTTACGCAGCTGACTGGCA GAGGTCAGTACCCAGTTCTACCCAGTGTGGCCACCCGATCCAACTCTGTGAC TCATTTTAAATCATAAGTAGTTTGAACAAAGACTTAAAATTAACGGGTTTGGTC ATCATTAAAGTTTATTTTTAAACGTCAAGTAATTTGGCATTTTACTAACAACAAC TTGAGGATATCCACAGTGTATGAAACACCAACTCTTGTTTCAATAAAAGTCTAA TGAAAGTTTCTCAAATTCTGAAACCTAATTCCAGGATTCTTATTTGGAACTGTT TTTTTGTTTTTGTTTTTGTTTTTGTTTTATCAGAGTACATTAACTCAGGGGGAAA ATGAGATTATCTTTTGATTCAGAGAGAAACAGAACATTCCACTGATAGTTTAAA AATAACACAGTGACCACAGATAACTGTAGTTCAGCAATTTTCAAATTCAGATTC TGGGTCAGGGCACAAGATTATGCGTCTTTAACAAGCACAACTGATTATAATGC TGATGGTCTAGAGGAAACTTGATGGGAAACACTGATCTATTCAACACTTTTTA AAAGTACACTTTAGTACTATTTCTACCTAGTCAATAAAATAAAGAAGGAAGGA GAAAGGAGGACGAATAAGAGAGAATCCGAAAGACACACTACCCAGACAGACT ACTCAGACAGACGAGACAGTCAGGCAGACATGGTGCTACCCGTCAAGCATG CAGGAAAGCAGCTTTCATGGAATAACATTCCTAAACTCTTGCCTAATAAATTAT GCTGAGAGCTGCTGCTAAGAATTTTTTAAACCAACTCAAAGCAAAAAAGGAGC TATTCACCCAATCAACAGGTGAAATCAAGTCACAGACTAGTATAGGGTTTGGC AGATTTCAGGCCCTCCAGAAATATCTGTTTAATTGAGAAGCAACTCCAGCTCT AGCTAGAAATCTATTTAACCATAAAAGTGAAATCATAATGAATTTGGTCGTAT CTTATTTTTCCCCTTTGTTTGTTTCC
TCGGGGCATCTATAATGGCTGAATTGGAAATGGAACCACAAGTATTATAACAA CATTTGTTGGAAAGTTCATCCTGTA TTTTAGTAGTACATAAGTTGACAGATATGGCTTTATGAATTGTTCTCAGAGACT TAAAAAAAAAAAAACCCTGAACTTTGTAAAAATTACATCCATTATCCACCAAGT AACATTTGCAAGCAAAACCCTCTACTAGAAAAAATGGGTGCGAAAATAGGAAA AGGAGAAGAACAGGAGGAAGAAAAGGAGAACAGGACGTACAATTAATTGAG GGGAAAAAAATCATGAGTAAAGAAGTCAGAAATAAATGTAGCTAAAAATACAA ACTGCTACTTTATGGTCCAGATATTGTAATATATCATTTTTAACATAAAAGAAA AACAAATCCTCAACAGACTTCCTATAAACGAAATTATCAGAGTTCCCGAGTAC ACCGGGGGTCGAGGGAAGAATCTCCATGTGCTCCGAGTATCGATAGCCAGT CCAGCTTCATTCACTCATTCATTTCTTTTCTTTCATTTCAGGAGAACATTTAGC AGTGTTTTGTTTTATTTATTTTATTCAAAGGGAAATCCTCATGTGACACTAGCG GTGAAAATAACTTGTATTTGTAAGTTAATGTCTGCTGTACATCTGAGTACACAA TTGTCTTTCACAGAA
51A (2000 bp) Seq ID No. 71 dbj AP000977.41 (From 7549 to 9549) (reverse complement feature)
TGGACAGTGTCTGAGCCCACCACCCCTTATCTCTCCATCTCCCATGCG CTGCATGTCACACAGCCCGTTATCACACCATTGTAGAGTTGTGGCTGG TTTCTTGTGACCATGTTTCCTGGTTCCCATCCTGGTACATTGACATGCC TGTATCTGCATCTGCTTGCTCTCCTCCTTAAATTGTAAATTTCCTAAAAG CAGGAATGAAATTTTATTCCCCAGCTCACTGCACCTCCATAGAACCACA CTCAAGTCAAGCAGTACTGTTGGAAAACATCACCATTCCTTAGGTTTAA ACGTTGAAGGGGAAAGTCGTGATTACAAAGTGGAACTTGGCTTAAGGC TAAAAGTAGGACCATATAATTTATTGTCACATCTTGACACTTTTGAGAGT GAAGGGGGTGCTATTCATAACCACCAGAGACAACTTAGAAACCAGGTC TGCCCCAGGGAAACACAGAGGCAATGGTCATCCTTGCTAGAAAGATCA TTCCAAATAGATGTTAGTTACTCCAGAGTTCCAAGAGGGGCACCTCAAA CCCCTTCAGTCACCAACCTGTGCTTGCATTCTGAAGGGTCTGCACCAG ATAGAACTTGTGGCAGAAAGCCCAGTACAGGGAAGTTCTTCCTTTTATC TGACTCAAATCCTTACAGATTCAAATTAATTCCACTTCTTATTAACCTCA GAGCAAGAGGCAACAATGGCATAGGAAGGCAGCCACAGAGAAGAAAA ATCTCACCCTTGTACCCTCACCCTCCGGTTAGGAATTCCTCATTATGAG TTTGACTGCTAGTTCTCCATCCTTCAGCTGAACCAAACTCAATCTGGCT GGCCCAGAAACTCAAAAGCCCAGGGTTTGGGAGTCACTCCCAGTGAG AACAGAGTTCATGTCCAACCTGCCCCACATCCCCTGGAGCTTTTCATAA ACCAAACCCAAGAAAGAAATGGCACATGTACACATATTTTTGAAACCAG AAAACTTTTTCCTCAACCTAAACTATAGCTTCAGGGGACCTGAGCACTG GGAGGGATTGTCTCCCACCAGCCCCCAACTCCCAGTTCCCAGAATGCT GATTCAAGAGCACAGCAGAGCTGGGCTGACTGCGGAGGCCCAATGAA GAAGTGGGCCACTCCTCCTCCTTCTCCCACCCTCCATCAGCCCCATGC TGGTGTCGCTGTTAAGAGCAAGGACCTTGATGACTTCTTTTTGTTCATA CACATATTTGTCATATGGGGTGGGGACAGGGGGACAAGGATCTGGGC CCAAGAGGAACTGAGCATGCAGGTTCCTGCAGCCTGTGGCCACCATTA GTTACATGCCCTGGGGCACACATTCTTGCCCAGGGACCAATAGTGGAG GGAAATCCAACGCTCCCACCCAGTCACCACCATCCTTTCCCCCTTTATA CCAGATCTATTTTATTTTAGAAAGGAGGTATTTCAGCAGCATTGCATAAA GACAGGGTGACTGCAGTCAAGCAAAGGTCACTGAGACAATGCAGACAA AAACTAGAGAGCAGGCCACCTTCCCGGTATGACCTTGGACTAGTCACT TCACCATTCCACGCTTCAGTTTCTTCACCAACAAAATGACACAATAGGG TGATATGTTTACAAGGTTCGTCCTGGATCCAAGGGTTGAACATTTAACT CTGTATTTCCTGAAGAAATGGCTAAAGTGAAATTAGTGAGAAAAGGCAG AACTCAGGGTAATCTTTTATTAATAATTTCTACTTCAAAAGGAACAATTT GACTCTAGAAAACAAGTGTATTTACCCCCTCCTCTGGTCCTACTTTTTC TCCCCCACCGACCTTATACTTTTGCTGATGTCAATCTTAACTGCAGGAC TGGGAGTCTCATGAGGCAATAACACGGTCAAATTAATTTTGCCAGTGCT GATCCTAACCTACAAACTCCATCTGCAATTCCTCGCAGTAAGTCTGACC ATAAAAGTGAAATTCACGTTACTTATGATCTAATAAACCTATTGCCTGG GTAGAAA
TAAA TCA C TTA TA GAA TAA TAA TGCCCA CCAAA TTTCC TGCA G TCC TTTCAAA T CTCCAAGTGCCATGAGGGGTATTCCTCTCCAAAATAAAGTTATCTTGACGGG GGTGGGAGAGTCAGCATCTTGAAGGGTGGGGAAGAAGGAATGGAGAGCATC TCACACGCTCGCCAAAAATTAAAAATTAAAAATTCAGCCTTTACACACGCACA CACATACACACACCTCTTGTCTGACTGTACAGAGATAGTCGACAAGCTTTCAG GTTTGCCCAC
TTTTATTGTGCTAATAATGCTAAGTGTGGAAACCCCCACCTCGTGCTGTTCTG TGTAAGTGTACCGCAACTGCAGGGTGCTCGCCTCCACTGCCAACTGATGATA CACTAAAGAAAGCCCGTTTTTTATCCAGATGTAAAATGAAGCTGCACCCCTCC TATACCTGCCGGAAACTACATCTCTCTGCACACACCCTGGGGTAATACGCAC TGCACCCGGCGAGTTACCTGGAATACGTTTACTAAATCGACAGGAAGAAGGA AAGATCAATGCGATTCAAGTGCTGACGGTTGTACTCGTGAAGGAAAAAAACA AATGCAAGCCGGGAAGTCCCCAGTGGTGCCTGCACCTAGGAGGCGACTGCA ACGGATGGATGCGGGGGTGAGGAAGTGCAGGGAGAGGAAAAAACTGGAGG GAGGCGGGTTGCAAAACTGCTCACCTGTCCGTACACCTTGATGGGCTCGGG CTGCAGGGCAGCGCTCCGTTTCCTCCGGAGCGGCTTCTCGTCCGCGCGGCT CGCCCCCCTGGCATCCCCGCGCGCCCACAGCCGCAGCTCGCGCGGGTCGC CCTGCACCACGAGGAAGCCCCGGTCCTGGGGCA
Results evidenced a very high sequence identity between 17 A, 38 A, 45 A, 51A promoters. Taking advantage of the same bioinformatics tool, a phylogenetic tree was traced showing that altogether 17 A, 38 A, 45 A and 51A promoter sequences constitute a specific branch of the tree and suggesting a process of co-evolution at the base of their phylogenetic origin (Fig. 7). Such association suggested their putatively coordinated gene expression modulation. A splice variant of KCNIP 4 gene contains a potential signal peptide region
In order to experimentally test a role of these promoters as AD-associated regulatory elements, the authors choose one of the four transcription units (38A) as an experimental model for further detailed analysis. Since 38A maps in the first intron of its putative gene target (KCNIP4, Uniprot/SWISSPROT Acc:Q6PIL6; from human gene ENSGOOOOO 185774, mapping at 4pl5.31) in antisense configuration and being, once expressed, most likely able to modulate its splicing mechanism, the authors postulated a 38A-specific role in the regulation of KCNIP4 mRNA splicing. In fact, several alternatively spliced variants of KCNIP4 mRNA were found in NCBI databases (http://www.ncbi.nlm.nih.gov) and at least four of them (called Variant I to IV, respectively) result from differential hnRNA processing associated to the intron I genomic region. Once translated, such alternatively spliced variants provide proteins with different N-terminal fragments. A bioinformatic analysis of the N-termini of these alternatively spliced KCNIP4 polypeptides showed that two of them are of particular interest: i) Variant I being the longest, highly expressed and prevalent form and ii) Variant IV because it potentially contains a N-terminal Signal Peptide (Fig 8). As KNIP4 does not contain a KDEL sequence motif in its C-terminal moiety (that would redirect the protein into the endoplasmic reticulum), the authors deduced that this splice variant most likely migrates in a different cellular compartment with respect to the predominant Variant I whose amino acidic sequence is not compatible with a sorting via endoplasmic reticulum. Therefore, following their hypothesis, being the intron I the site of the I to IV alternative splicing shift and considering that in the same chromosomal region 38A can be actively expressed in antisense configuration most likely masking splicing sites, the authors deduced that the determination of KCNIP4 subcellular localization might be dependent on 38A expression and subjected to its regulatory action. 38A is actively transcribed in SK-NBE neuron-like cells.
In order to assess 38A transcription, the authors fused its promoter to a luciferase- specific silencer hairpin; 48 hours after co-transfection of sk-NBE neuron-like cells with this construct together with a plasmid expressing luciferase (pGL3), the authors detected a significant decrease in luciferase activity (0.27 fold of the negative control emission, a DNA construct harboring the same hairpin without promoter, here referred to as pMock) thus demonstrating an efficient hairpin transcription driven by 38A Pol III promoter. As positive control in the same experiment, a canonical Pol III promoter (U6) was fused to the luciferase-specific hairpin and co-transfected with pGL3 in the same conditions. Results showed a luminescence emission of 0.08 fold of the pMock-transfected samples, to which results were normalized (Fig. 9). Altogether these data support the conclusion that 38A transcription unit is controlled by an active Pol III promoter. 38A is specifically overexpressed in Alzheimer's Disease patients
In order to test their hypothesis of an Alzheimer's Disease-associated 38A expression, the authors tested by quantitative real Time RT-PCR its RNA amount in post mortem AD patients' brain tissues and in healthy subjects negative controls. A previous control showed that the PCR reaction dissociation curve resembles that of a specific amplification product characterized by a single specific peack, thus ruling out the occurrence of an artifactual cross-amplification. In the samples the mRNA product of the housekeeping gene GAPDH (Glyceraldehyde 3 Phosphate Dehydrogenase) was measured and subsequently all the 38A expression values were normalized to their GAPDH counterparts. Results showed that the tissue samples derived from AD patients exhibit a 38A expression level significantly higher than those obtained in control samples. This result demonstrates a specific association of 38A small RNA expression modulation with the Alzheimer's disease phenotype (Fig. 10).
KCNIP4 Variant IV is specifically expressed in Alzheimer's Disease patients.
Since the authors speculated that the 38A overexpression modulation in AD patients might be responsible for the synthesis of a different KCNIP4 splice variant (Variant IV) and that this protein shift might be one of the key events of Alzheimer's Disease generation, they analyzed the same samples as above by Real Time RT PCR amplification. The two most different variants of this protein coding mRNAs: Splice Variant I, as the longest, canonically expressed protein and Splice Variant IV, as the one in which a signal peptide is predicted by several computer-based analysis to constitute the N- terminal fragment, were measured. After normalization to GAPDH expression, all the values obtained for the alternative Splice Variant IV were divided by those obtained for the Splice Variant I so that their expression Ratio (hereafter referred to as Var IV/V ar I Ratio) represents in all samples a clear indication of a possible alternative splicing event in AD patients. Results showed that in all the control samples the amount of the two polypeptides is rather similar. By contrast, in Alzheimer's disease patients the Splice Variant IV is significantly overexpressed (Fig. 11 panel B). Therefore, considering that: i) 38A expression is AD-specific; ii) 38A is expressed in Antisense configuration in the intron I of KCNIP4 alternative splicing site; iii) the KCNIP4 Variant IV is specifically synthesized in AD patients and iv) the whole phenomenon is strictly associated with Alzheimer's Disease samples, the authors concluded that the KCNIP4 Variant IV is associated with the generation and/or the maintenance of AD phenotype. The mechanism most likely involves an alternative subcellular localization/sorting of the polypeptide and takes origin from a 38A expression modulation. The dissociation curves of the two Splice variants constitute a diagnostic hallmark of Alzheimer 's disease.
In order to have an unambiguous profile of KCNIP4 amplification products, the authors analyzed the dissociation curves of such products. Results indicated that the amplification fragment of the variant I shows a specific dissociation pick at 83.2 0C. By contrast, the alternatively spliced Variant IV is characterized by a pick at 80.80C. Therefore the two curves are clearly distinguishable and their specific profile in different samples can be used as diagnostic tool for AD onset (Fig. 11, panel A).
EXAMPLE 3: RNA POLYMERASE III TRANSCRIPTS CONTRIBUTE TO ALZHEIMER'S DISEASE Material and methods
In vitro transcription
Transcription reactions were carried out in a final volume of 25 μl in the presence of 2 μg of template DNA and HeLa cell nuclear extract (100 μg) supplemented with 50 ng of recombinant human TBP. The standard transcription mix contained: 5 mM creatine phosphate, 70 mM KCl , 5 mM MgCl2, 20 mM Tris/HCl pH 8, 1 mM DTT, 2 μg/ml α- amanitin, 0.5 mM CTP,ATP,GTP, 25 μM/ 10 μCi UTP /[α-32P]UTP, SUPERase IN (Ambion, 10 U), glycerol 10 % (v/v). The reactions were incubated for Ih at 30 0C. The products were phenol extracted, precipitated with ammonium acetate, gel-fractionated and visualized by phosphorimaging using a Personal Molecular Imager FX (Bio-Rad). Primer extension reaction
A double scale transcription reaction was performed as described, without including radiolabeled UTP. The purified transcripts were resuspended in a final volume of 12 μl in the presence of 0.5 mM dNTPs and 1 pmole of specific 5 '-end-radio labeled probe. The mixture was heated at at 650C for 5 min and a mixture providing 50 mM Tris/HCl pH 8, 75 mM KCl, 3 mM MgCl2,5 mM DTT, SUPERase IN (Ambion, 10 U) and 200 U Superscript III reverse transcriptase (Invitrogen) was added to a final volume of 20 μl. The reactions were incubated for Ih at 60 0C and subsequently for 15 min at 700C to inactivate the enzyme. The products were precipitated with ammonium acetate and gel- fractionated. DNA templates
The following primers were utilized to produce 17A and 38A fragments subsequently cloned into pGEM-Teasy vector (Promega): 5 '-CTCTATATTGTCTTTGAATATCTTAGC (Seq ID No. 72) and
5 '-GTTTTGTTTTACATTATTAACTAGACC (Seq ID No. 73) for the 17A region; 5 '-TAATAACAACATATCTGAAAAAGACGC (Seq ID No. 74) and 5'-TTCAGGGTGTCCTGTTGGTACC (Seq ID No. 75) for the 38A region. The inserts were digested with Sphl-Sacl and subcloned into pNEB193 vector. Real Time RT PCR primers
The sequences of 17A forward and reverse primers, as designed by the Primer Express 1.5 software, were 5'-CCACCCTGCAACTGACACAT-S' (Seq ID No. 76) and 5'- GCAAAGGTGCTAATCTTGACTCTTG-3' (Seq ID No. 77). The sequences of 38A forward and reverse primers were 5'-CTATCAAAATTTCAAGGATATGCATCA-S' (Seq ID No. 78) and 5'-GATGCCTCAAGCTTTGTTTTGC-S' (Seq ID No. 79). The sequences of GPR51 (Vl) forward and reverse primers were 5'-TCCGTCACATCCATCATTGC-S' (Seq ID No. 80) and 5'-GCTGGATTCACCGCATTGTCT-S' (Seq ID No. 81). The sequences of GPR51 (V2) forward and reverse primers were 5'- GCC ATTCTGAAGTTGCTC AAGC-3' (Seq ID No. 82) and 5'- GAGAAGCTCTCGGTGTCTGAAATC-3' (Seq ID No. 83). The sequences of KCNIP4 (Vl) forward and reverse primers were 5'-ATGAAGCTCTTGCCCTGCTC-S' (Seq ID No. 84) and 5'-CGGTGGCCATCTCCAGTT-S' (Seq ID No. 85). The sequences of KCNIP4 (V4) forward and reverse primers were 5'-TGGAACAGTTTGGGCTGATTG-S' (Seq ID No. 86) and 5'-CGGTGGCCATCTCCAGTT-S' (Seq ID No. 87). For endogenous control the expression of Glyceraldehyde 3 phosphate dehydrogenase (G3PDH) gene was examined. The sequences for human G3PDH primers were 5'- GAAGGTGAAGGTCGGAGTC-3' (Seq ID No. 88) and 5'- GAAGATGGTGATGGGATTTC-3'(Seq ID No. 89) . The sequences for human 5s rRNA primers were 5'-TACGGCCATACCACCCTGAA-S' (Seq ID No. 90) and 5'- GCGGTCTCCCATCCAAGTAC-3' (Seq ID No. 91). The sequences for human 7SK RNA primers were 5'-AGGACCGGTCTTCGGTCAA-S' (Seq ID No. 92) and 5'- TCATTTGGATGTGTCTGCAGTCT-3' (Seq ID No. 93). The sequences for human c- Myc primers were 5'-CGTCTCCACACATCAGCATAA-S' (Seq ID No. 94) and 5'- GACACTGTCCAACTTGACCCTCTT-3' (Seq ID No. 95). Relative transcript levels were determined from the relative standard curve constructed from stock cDNA dilutions, and divided by the target quantity of the calibrator following manufacturer's instructions. Immunofluorescence detection 17A and 38A transfected cells were washed in PBS and fixed for 10 min with 4% paraformaldehyde in PBS buffer (0.1 M, pH 7.4), permeabilized 5 min with 0,25% Triton XlOO in PBS and blocked for 30 min with 3% bovine serum albumin in PBS. Cells were subsequently incubated respectively with rabbit polyclonal anti-GABAβR2 (H-300) (1 :250) (Santa Cruz Biotechnology Inc., Europe) or goat polyclonal KChIP4(N-14) antibody (1 :100) (Santa Cruz Biotechnology inc., Europe). The incubation was performed overnight at 4°C in 0,5% BSA in PBS. The day after the cells were labeled with secondary antibodies for 45 min in 0,5% BSA/PBS solution: anti-rabbit rhodamine-TRITC (1 :100) (Jackson ImmunoResearch) and anti-goat Rhodamine-TRITC (1 :50) (Jackson ImmunoResearch) for GPR51 and KCNIP4 respectively. Cells were then incubated with DAPI for 5 min and mounted with Mowiol (Invitrogen). Immunostained cells were observed with the appropriate filters on Axiovert 200 M (Zeiss) microscope and captured at the same adjustments of laser intensity and photomultiplier sensitivity using Axio Vision software. A$ detection in SHSY5Y cells Media of 17A/38A permanently transfected SHS Y5 Y cells were diluted in EIA buffer and processed using a kit specific for both Aβ species, following the indications of the manufacturer. The kits are solid phase sandwich ELISA using plates pre-coated with the specific polyclonal anti-human Aβ antibody (raised against residues 38-42 or 35-40). An HRP-conjugated monoclonal human anti-Aβantibody (11-28) was also supplied. Both assays show a linear reactivity within the range of concentration 7-1000 pg/ml for both Aβspecies. Aβconcentration was determined using Benchmark Microplate Reader and evaluated by Microplate Manager Version 5.1 Software (Biorad, Hercules, CA, USA).
Results
The central event of Alzheimer's Disease (AD) is the formation in the brain of beta- amyloid (Aβ) soluble aggregates10'20. Gene mutations determine overproduction of Aβ in familial early-onset AD3°. On the contrary, in the sporadic late-onset form of AD, the genetic causes of increased Aβ deposition are unknown40. The authors found that Aβ overproduction can be induced by a small set of RNA polymerase (Pol) III -transcribed small non-coding (nc) RNAs specifically overexpressed in brains of AD patients. These RNAs originate from transcription units mapping in the intronic regions of protein-coding genes of brain- specific proteins: Potassium Channel Interacting Protein, KCNIP45c 7c; G Protein Coupled Receptor 51, GPR518c l lc; Sortilin-Related Receptor 1, SORLl 12c l4c and Amyloid Peptide Beta Binding 2, APBB215c'16c. The authors here show that two of these Pol III transcripts used as experimental models drive AD-specific alternative splicing events leading to the synthesis of distinct protein isoforms and influence Aβ secretion. The authors also show that at least one of these elements (38A) is present in three different genetic variants associated to its promoter that are transcriptionally deregulated and thus provide a further genetic support to the model here proposed where the overexpression of the ncRNAs ultimately leads to AD-specific overproduction of Aβ peptides.
The authors have recently identified a set of approximately 30 RNA Polymerase (Pol) Ill-dependent non-coding RNAs (ncRNAs), and proposed that they regulate gene expression through specific Pol Ill/Pol II cogene/gene pairs17c'18c. By multiple alignments of the promoter sequences of these transcripts, we noted a significant similarity among four members of the collection (referred to as 17A (SEQ. ID No. 68) 38A (SEQ. ID No. 69) 45A (SEQ. ID No. 70) and 51A (SEQ. ID No. 71)) clustered in two adjacent branches in the promoter phylogenetic tree. By in silico genome analysis the authors found that these four ncRNA transcription units map in intronic portions of genes whose expression is specifically associated to brain: Potassium Channel Interacting Protein, KCNIP4; G Protein Coupled Receptor 51, GPR51; Sortilin-Related Receptor 1, SORLl and Amyloid Peptide Beta Binding 2, APBB2. Interestingly, each of the four clustered ncRNAs maps in a chromosomal region where alternative splicing events take place (see Sl and S2 online for details about GPR51 and KCNIP4). Due to their intronic location, the authors hypothesized that these ncRNAs might perturbate the balance between alternatively spliced mRNA forms potentially associated to brain physiopathology. To test this hypothesis they selected two of them [17A (GPR51 intron 1) and 38A (KCNIP4 intron I)] for a more detailed investigation.
First, they verified 17A and 38 A activity as Pol III templates in vitro, using a HeLa cell nuclear extract. In the case of 17A, a single transcript of the expected size and transcription start site was produced (Figure 12a). Transcription of 38A produced RNAs of different sizes, with some transcription initiating, as expected, about 30 bp downstream of the TATA box (Fig. 12b). The 38A promoter region was also found to be active in directing faithful 7SK gene transcription in fusion constructs (Fig. 12c).
The authors then tested if the overexpression of 17A and 38A causes a gene- specific alternative splicing shift of GPR51 and KCNIP4 respectively. To this purpose, they transiently transfected SHSY5Y and SKNBE neuroblastoma cells19c"20c with constructs expressing these transcripts (driven by their natural promoters) and measured changes in KCNIP4 and GPR51 alternative splicing occurred upon transfection. Strikingly, after 48 hours of transfection, a gene-specific alteration of splicing isoforms ratio was detected (0.16 vs "not detected" and 21.1 vs 1 for GPR51 and KCNIP4 respectively in SHS Y5 Y; similar results were obtained in SKNBE cells) indicating that the expression of these elements causes an alternative splicing shift of those brain-specific proteins (Fig. 13a,b).
The Pol III transcript-driven alternative splicing was also evidenced at protein level by immunofluorescence microscopy. By the use of a polyclonal antibody raised against GPR51 N-terminal epitopes the authors observed a well-marked immuno fluorescent signal in pMock-transfected SHSY5Y cells (Fig. 13c); in contrast, a strongly decreased signal was detected in cells overexpressing 17A where the alternatively spliced GPR51 protein form (lacking the long N-terminal portion) is synthesized and not recognized by the same IgGs (FIG. 13d). The same experiment was performed challenging 38A-transfected cells with a KCNIP4-specific antibody raised against the N-terminus of the protein. Again results showed that the overexpression of 38A RNA leads to the synthesis of an alternative KCNIP4 splicing form not recognized by the antibody (Fig. 13e,f). Although the precise subcellular localization of the alternative splice variants will be accurately determined only with antibodies specifically raised against the alternatively spliced N-terminal regions of GPR51 and KCNIP4, altogether the above results are in agreement with the model of a 17A/38A-driven alternative splicing.
As GPR51 and KCNIP4 might be involved in Alzheimer's disease (AD) phenotype the authors postulated a possible active role of the ncRNA cluster in this neurodegenerative disorder. In order to test this hypothesis the authors measured by quantitative Real Time RT-PCR 17A and 38A transcript amounts in post-mortem cerebral cortex samples obtained from 18 AD patients and from 10 non-Alzheimer control individuals (Fig. 19). AD cases exhibit a significantly increased expression level of 17A and 38A (13.3-fold and 10.5-fold on average, respectively) with respect to the non-AD control individuals suggesting the existence of a correlation between the expression of these ncRNAs and the AD phenotype (Fig. 14a). Interestingly, the highest expression of 17A and 38A was detected in the same AD samples (cases AD94-392, AD02-11, AD08-01) keeping in line with their likely co- regulated expression suggested by their common phylogenetic origin. The unaltered transcription of 5 S rRNA in the same samples indicated that the phenomenon is due to a specific overexpression of 17A and 38A rather then a general increase in Pol III transcriptional activity (data not shown).
To verify ex vivo if the overexpression of 17A and 38A in AD patients determined the synthesis of distinct GPR51 and KCNIP4 splice variants, the authors investigated the alternatively spliced forms of their corresponding mRNAs. In particular, the authors measured in cortical extracts from AD and non-AD control individuals the relative amount of: 1) GPR51 splice variants 1 and 2 (the latter lacking a long N-terminal portion)210 and 2) KCNIP4 splice variants I and IV (the latter harboring a predicted N-terminal signal peptide)220. These experiments demonstrated that the overexpression of 17A and 38A in AD patients is accompanied by an alternative splicing shift of GPR51 and KCNIP4 that leads to the enrichment of their alternatively spliced variants to the detriment of those canonically expressed (Fig. 14b). Altogether these results confirmed in pathologic post mortem samples what previously evidenced in vitro, i.e. a functional association between the expression of the ncRNA cluster and the occurrence of alternative splicing events. Therefore, considering: i) the AD-associated ncRNA-cluster transcripts enhanced synthesis; ii) their chromosomal location in intronic regions where the alternative splicing events occur; iii) the AD-specific occurrence of alternative splicing in the corresponding genes that leads to the synthesis of alternative polypeptides, the authors concluded that GPR51 Variant 2 and KCNIP4 Variant IV are associated to the AD phenotype with a mechanism that, based on the lack of distinct protein domains in the alternatively spliced forms, might involve their unusual subcellular localization and/or a different protein- protein interaction behaviour that brings to the ultimate AD pathological manifestations. Notably, preliminary sequence data of 16 out of 22 AD cases exhibited specific heterozygous sequence variations in 38A promoter that support their altered transcription in AD pathologic manifestations (here referred to as Allele CC, β and γ, indicated in Table II) whereas only 5 out of 11 non-AD controls harbor these alleles.
Table II: 38A AD-associated genetic variations, 38A promoter sequence analysis of AD cases. The three 38A novel alleles are indicated with α, β and γ, and compared to the wild type promoter sequence. The functional promoter elements (DSE, PSE and TATA box) are indicated in italics. The base substitutions are indicated in red and bold whereas the point deletions are indicated by a star. The underscored sequence corresponds to the transcribed region.
38 A Allele wt (fragment of SEQ ID No. 69)
AGTAAAATTATCTCT^ TTTGCA GATGATATAATCCCTTATGTAGAAAACCCTAAAGATTCCACA
AAAAACTGACAGAATGAATTAATTCAGTAAACTTGCAGGATACAAAATCAACATACAAAAATC AGTAGCATTTTTATACACTAATAACAACATATCTGAAAAAGACGCTTTAAAATCCCATTTATGA AAGCATAAAAATAGTTAGAAATAAATTTAACCATA4AGGTGA4ATATTTGTATACCGATAACTA Z^^CCTTTGATAAAAAAAGTTGAAGAAGACACATATAAATAGAATAATATTCTGTGTTCATGA ATCAAAAAATTTAACAATGTTAAAATGTCTGTATTAACCAAAGCAATATACAAATTCAATGCAA TTTCTATCAAAATTTCAAGGATATGCATCACAGAAATAGAAAAAAAATTCTTGAAATTCATATG GAACCACAGACACATAAAAACAGAATAGGCAAAGGAACAATGAGAAAGCAAAACAAAGCTTG AGGCATCACACTTCCTAAGTTAAAATTATATTGCAAAGCTACAGTAATCAAAAACAGTATACAA ATGGCATGAAAACGAAAATGTGGACCAACGGAACAGAATATAGAGAGCCAGAAACTTAACTA ATTTTCAACA
38 A Allele α (fragment of SEQ ID No. 69, but * )
AAAATTATCTCT,4 TTTGCA GATGATATAATCCCTTT -\ * G * G GAAAACCCTAAAGATTCCACAA AAAACTGACAGAATGAATTAATTCAGTAAACTTGCAGGATACAAAATCAACATACAAAAATCA GTAGCATTTTTATACACTAATAACAACATATCTGAAAAAGACGCTTTAAAATCCCATTTATGAA AGCATAAAAATAGTTAGAAATAAATTZ^ CCA TAAA GGTGAAA ZATTTGTATACCGATAACZ4Z4 A4CCTTTGATAAAAAA ♦ GTTGAAGAAGACACATATAAATAGAATAATATTCTGTGTTCATGAAT CAAAAAATTTAACAATGTTAAAATGTCTGTATTAACCAAAGCAATATACAAATTCAATGCAATT TCTATCAAAATTTCAAGGATATGCTTCACAGAAATAGAAAAAAAATTCTTGAAATTCATATGGA ACCACAGACACATAAAAACAGAAfAGGCAAAGGAACAATGAGAAAGCAAAACAAAGCTTGAG GCATCACACTTCCTAAGTTAAAATTATATTGCAAAGCTACAGTAATCAAAAACAGTATACAAAT GGCATGAAAACGAAAATGTGGACCAACGGAACAGAATATAGAGAGCCAGAAACTTAACTAATT TTCAACA
38 A Allele β (fragment of SEQ ID No. 69, but * ) AAAATTATCTCTΛ TTTGCA GATGATATAATCCCTTATGTAGAAAACCCTAAAGATTCCACAAAA AACTGACAGAATGAATTAATTCAGTAAACTTGCAG'VATACAAAATCAACATACAAAAATCAGT AGCATTTTTATACACTAATAACAACATATCTGAAAAAGACGCTTTAAAATCCCATTTATGAAAG CATAAAAATAGTTAGAAATAAATTJΛΛ CCA TAAA GGTGAAA TATTTGTATACCGATAACTX TAAA CCTTTGATAAAAAA * GTTGAAGAAGACACATATAAATGGAATAATATTCTGTGTTCATGAATC AAAAAATTTAACAATGTTAAAATGTCTGTATTAACCAAAGCAATATACAAATTCAATGCAATTT CTATCAAAATTTCAAGGATATGCTTCACAGAAATAGAAAAAAAATTCTTGAAATTCATATGGAA CCACAGACACATAAAAACAGAATAGGCAAAGGAACAATGAGAAAGCAAAACAAAGCTTGAGG CATCACACTTCCTAAGTTAAAATTATATTGCAAAGCTACAGTAATCAAAAACAGTATACAAATG GCATGAAAACGAAAATGTGGACCAACGGAACAGAATATAGAGAGCCAGAAACTTAACTAATTT TCAACA 38 A Allele γ (fragment of SEQ ID No. 69, but * )
AGTAAAATTATCTCT^ TTTGCA GATGATATAATCCCTTATGTAGAAAACCCTAAAGATTCCACA AAAAACTGACAGAATGAATTAATTCAGTAAACTTGCAGGATACAAAATCAACATACAAAAATC AGTAGCATTTTTATACACTAATAACAACATATCTGAAAAAGACGCTTTAAAATCCCATTTATGA AAGCATAAAAATAGTTAGAAATAAATTZ^ CCA TAAA GGTGAAA TATTTGTATACCGATAACT^ 7^/I^CTTTTGATAAAAAAAGTTGAAGAAGACACATATAAATAGAATAATATTCTGTGTTCATGA ATCAAAAAATTTAACAATGTTAAAATGTCTGTATTAACCAAAGCAATATACAAATTCAATGCAA TTTCTATCAAAATTTCAAGGATATGCATCACAGAAATAGAAAAAAAATTCTTGAAATTCATATG GAACCACAGACACATAAAAACAGAATAGGCAAAGGAACAATGAGAAAGCAAAACAAAGCTTG AGGCATCACACTTCCTAAGTTAAAATTATATTGCAAAGCTACAGTAATCAAAAACAGTATACAA ATGGCATGAAAACGAAAATGTGGACCAACGGAACAGAATATAGAGAGCCAGAAACTTAACTA ATTTTCAACA
In order to determine if the novel AD-associated sequence variations can be at the base of a deregulated expression of 38A in AD cases the authors fused the 38 Aa promoter to a luciferase silencer hairpin; 48 hours after co-transfection of this construct with a plasmid expressing luciferase in HeLa cells they detected a very strong decrease of luciferase activity in the samples promoted by the α genetic variant and a less pronounced luciferase inhibition in the sample transfected with a hairpin construct which expression was promoted by the wild type allele, thus, demonstrating an increased hairpin transcription driven by the novel altered 38Aα Pol III promoters. The experiment was repeated in parallele in three different cell lines providing the same result (Fig. 15).
In light of the above findings, and considering that the proteolytic processing of APP has a central role in AD aethiology through the formation of neurotoxic β-amyloid peptides (Aβ23c'24(^), the authors decided to investigate whether the selective overexpression of the ncRNAs would affect also the formation of Aβ x-42 and/or Aβ x-40 species, measuring their relative amount in the medium of SHS Y5 Y cells permanently transfected with 17A and/or 38A. Samples of 48h conditioned culture medium were tested for their Aβ peptide composition. Results showed that both 17A as well as 38A significantly enhanced the levels of Aβ x-42 (to 1.75-fold and 1.91 -fold with respect to the pMock-transfected control respectively). The effect of 17A and 38A overexpression on the secretion of the soluble Aβ x-40 peptide was more modest (up to 1.3 and 1.4 with respect to the pMock- transfected control respectively). Altogether these results indicated that the concerted action of 17A and 38A changes on a quantitative as well as on a quality level the Aβ composition. To further demonstrate the Pol Ill-dependency of this phenomenon, a parallel experiment was performed in presence of 20μM ML60218, a membrane-soluble Pol Ill- specific inhibitor250; in this condition the ncRNA cluster-dependent secretion increase of both Aβx-42andAβ x-40 was abolished, thus confirming the pivotal role of the AP-cluster ncRNAs in Aβ peptide ipersecretion (Fig. 16). In this experiment the occurrence of the Pol Ill-specific inhibition was confirmed by a decreased synthesis of Pol Ill-transcribed RNAs (5S rRNA, 7SK RNA, 17A RNA, 38A RNA) and a concomitantly unaffected expression of Pol II-dependent transcripts (cMyc, Glyceraldehyde Phosphate Dehydrogenase) (Fig. 16).
Altogether these results demonstrate that the overexpression of 17A and 38A give rise to an unbalanced Aβ secretion increasing the Aβx-42 more toxic isoform. Therefore a cascade of reactions triggered by the expression of Pol Ill-dependent ncRNAs ultimately brings to the generation of a neurotoxic composition of the secreted amyloid. In conclusion the authors propose that newly identified ncRNA molecules transcribed by RNA Pol III may control splicing events of protein-coding genes, and that this novel mechanism of cogene/gene regulation has a strong association with sporadic late-onset AD. The association with the AD phenotype is proposed based on the fact that the here described ncRNAs: i) significantly up-regulates the synthesis of alternatively spliced transcript variants specifically in the brain of AD subjects; ii) causes a perturbation of the subcellular localization and/or abundance of the canonical splice variants; iii) increase the Aβ secretion and generate an unbalanced Aβ ratio, favoring the production of Aβx-42 species.
The molecular mechanism by which these transcripts act in pathology is at present unclear and needs further analysis, although the experiments on Aβ suggest that their role may be in line of the amyloid hypothesis. In this view it may be relevant to determine whether and how the ncRNA cluster affects Aβ x-42/Aβ x-40 formation or its degradation. Finally, in the light of the 38A promoter sequence variation exhibited by AD cases the authors' findings provide novel insights to understand the genetic origin of sporadic AD. As the genetic variations here identified are mainly associated to individuals that developed late onset Alzheimer's disease and that show an increased expression of 38A transcript it is reasonable to conclude that the AD-specifϊc 38A alleles here identified are at the base of a genetic risk to AD manifestation. These results therefore are of particular importance in order to set up a novel early diagnostic tool based on 38A genetics that provides the first possibility to bring to light Alzheimer's disease individual risk. In addition, the identification of possible 38A-specific inhibitors provide the first pharmacological therapy useful for a very early treatment.
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Claims

1- A nucleic acid molecule comprising sequentially: a) a 7SL small-RNA derived sequence comprising at least the binding domain to srp9 and srpl4 proteins of the 7SL ribonucleocomplex; b) a sequence identical or complementary to a target sequence; c) a pol III type III promoter.
2- The nucleic acid molecule according to claim 1 wherein the 7SL small-RNA derived sequence is an Alu-derived sequence. 3- The nucleic acid molecule according to claim 2 wherein the Alu-derived sequence is a 29A derived sequence.
4- The nucleic acid molecule according to claim 2 wherein the Alu-derived sequence is a 21 A derived sequence.
5- The nucleic acid molecule according to any of previous claims wherein the sequence identical or complementary to a target sequence is of a length of at least 15 nucleotides.
6- The nucleic acid molecule according to claim 5 wherein the sequence identical or complementary to a target sequence is of a length of at least 50 nucleotides.
7- The nucleic acid molecule according to any of previous claims wherein the pol III type 3 promoter has a sequence comprised in the group of SEQ ID No. 1 to SEQ ID No. 34. 8- An expression vector comprising the nucleic acid molecule according to any one of the previous claims.
9- A host cell transformed with the expression vector according to claim 8.
10- A non human transgenic animal bearing the nucleic acid molecule according to claim 1 to 7. 10- Use of the nucleic acid molecule according to claims 1 to 7, or of the vector according to claim 8 or of the host cell according to claim 9 to modulate the expression of the target sequence in vivo or in vitro.
11- Use according to claim 10 wherein the target sequence is involved in a pathological state. 12- A nucleic acid molecule comprised in at least one of the following sequence: Seq ID No. 68, Seq ID No. 69, Seq ID No. 70 and Seq ID No. 71 for the diagnosis of an age- related pathology. 13- The nucleic acid molecule according to claim 12 wherein the age-related pathology is a neurodegenerative disease.
14- The nucleic acid molecule according to claim 13 wherein the neurodegenerative disease is Alzheimer's disease. 15- A nucleic acid molecule comprised in at least one of the following sequence: Seq ID No. 68, Seq ID No. 69, Seq ID No. 70 and Seq ID No. 71 for medical use. 16- A molecule able to be vehiculated into the CNS and to bind to the promoter activating region of a nucleic acid molecule according to claim 15.
17-Use of the transcribed region of the nucleic acid molecule according to claim 15 for molecular diagnosis of an age-related pathology.
18- Use according to claim 17 wherein the age-related pathology is a neurodegenerative disease.
19- Use according to claim 18 wherein the neurodegenerative disease is Alzheimer's disease. 20-An inhibitor of the nucleic acid molecule comprised in at least one of the following sequence: Seq ID No. 68, Seq ID No. 69, Seq ID No. 70 and Seq ID No. 71.
21 -The inhibitor according to claim 20 for use as a medicament.
22-The inhibitor according to claim 20 for use in the treatment of a neurodegenerative disease. 23 -The inhibitor according to claim 22 wherein the neurodegenerative disease is
Alzheimer's disease.
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JP2017169573A (en) * 2011-03-30 2017-09-28 国立研究開発法人理化学研究所 Functional nucleic acid molecule and use thereof
JP2021006047A (en) * 2011-03-30 2021-01-21 トランサイン セラピューティクス リミテッド Functional nucleic acid molecule, and utilization of the same
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