Saturday, December 11, 2010

Gene therapy From Wikipedia, the free encyclopedia

http://en.wikipedia.org/wiki/Gene_therapy
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Wednesday, December 01, 2010

Scientists Decode Memory-Forming Brain Cell Conversations

http://www.sciencedaily.com/releases/2009/12/091215202322.htm

Scientists Decode Memory-Forming Brain Cell Conversations

ScienceDaily (Dec. 16, 2009) — The conversations neurons have as they form and recall memories have been decoded by Medical College of Georgia scientists.

The breakthrough in recognizing in real time the formation and recollection of a memory opens the door to objective, thorough memory studies and eventually better therapies, said Dr. Joe Tsien, neuroscientist and co-director of MCG's Brain & Behavior Discovery Institute. He is corresponding author on the study published Dec. 16 inPLoS ONE.

"It's a beginning, a first glimpse of a memory," Dr. Tsien said. "For the first time it gives us the ability to look at the brain dynamic and tell what kind of memory is formed, what are the components of the memory and how the memory is retrieved at the network level." The finding could help pinpoint at what stage memory formation is flawed and whether drugs are improving it.

For their studies, MCG scientists combined new technology and computational methods with century-old Pavlovian conditioning.

In the memory center of the brain, they used 128 electrodes capable of monitoring a handful of neurons each to simultaneously record the conversations of 200 to 300 neurons as mice learned to associate a certain tone with a mild foot shock 20 seconds later.

A computational algorithm translated the neuronal chatter into a discernable and dynamic activity pattern that provided scientists a trace or picture of what the memory looked like as it was formed and recalled.

"By listening to the neuronal activity we were able to decipher the real-time dynamic pattern and the meaning of those conversations so this is really satisfying," said Dr. Tsien, the Georgia Research Alliance Eminent Scholar in Cognitive and Systems Neurobiology.

The trace changed slightly each time it was recalled -- likely as the mood or situation of the rodent changed -- but still remained recognizable as a specific memory.

The scientists later correlated retrieval of the memory with the mice's actions, such as freezing upon hearing the tone or returning to the chamber where the foot shock occurred. They found the traces tightly correlated with memory scores: the mice that had lower scores predictably had a fainter trace and those with stronger traces had better behavioral performance, such as freezing in anticipation of a shock. "At the behavioral level he is just frozen, but with this technique of decoding the real-time memory, it will tell you exactly what he is thinking," Dr. Tsien said.

As expected, when mice were returned one hour later to the chamber where conditioning took place, they repeatedly froze, on average, 1.4 seconds after the recall pattern emerged in the brain. When placed in a setting with no history of the foot shock, the mice would freeze after they heard the tone.

One of the most surprising findings was that the memory trace of the foot shock was the sole memory that emerged in their brains 20 seconds after hearing the tone: just when the mild shock would have followed. "You think we are the only ones that can tell time?" Dr. Tsien said of this unexpected evidence of memory of time.

Problems with memory, the most fundamental cognitive function, can occur at any level -- learning, consolidating, storing or retrieving. The ability to watch memories being made in real time should help pinpoint where problems lie, enabling more targeted research and eventual treatment, Dr. Tsien said.

"If you don't know the basic biomarkers such as blood glucose or insulin level, it's hard to assess and study diabetes. Without knowing what memory traces are, you really don't have the precise physiological biomarkers to study memory and to reliably evaluate the effectiveness of treatment of memory disorders. We all know that behavior can be quite misleading sometime." The ability to tell what memory is produced and how good that memory is could also dramatically shape development of machines that are controlled directly by the mind, rather than those using hands as an intermediary, he noted.

Studies were done in the CA1 region of the hippocampus, a well-documented center for forming associative memories. Similar tests are needed in other memory regions, Dr. Tsien said.

Related technology is already advancing patient care. For instance, primate studies of essential motor neuron connections are advancing the development of mind-controlled prosthetic limbs and electrodes can help identify where seizures originate. But it will take time and improved non-invasive recording technology before Dr. Tsien's memory tests can be done on humans.

Dr. Guifen Chen, former MCG postdoctoral fellow, is first author on the paper, and Dr. L. Phillip Wang, MCG research scientist, is a co-author. The research was funded by the National Institutes of Health and the Georgia Research Alliance.

Disclaimer: This article is not intended to provide medical advice, diagnosis or treatment. Views expressed here do not necessarily reflect those of ScienceDaily or its staff.

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The above story is reprinted (with editorial adaptations by ScienceDaily staff) from materials provided by Medical College of Georgia, via EurekAlert!, a service of AAAS.

Journal Reference:

  1. Guifen Chen, L. Phillip Wang, Joe Z. Tsien. Neural Population-Level Memory Traces in the Mouse Hippocampus. PLoS ONE, 2009; DOI:10.1371/journal.pone.0008256
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MLA
Medical College of Georgia (2009, December 16). Scientists decode memory-forming brain cell conversations. ScienceDaily. Retrieved December 1, 2010, from http://www.sciencedaily.com/releases/2009/12/091215202322.htm

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Coliphage phi-X174, complete genome




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    Coliphage phi-X174, complete genome

    GenBank: J02482.1

    FASTA Graphics

    LOCUS       PX1CG                   5386 bp ss-DNA     circular PHG 16-JUN-2004 DEFINITION  Coliphage phi-X174, complete genome. ACCESSION   J02482 M10348 M10379 M10714 M10749 M10750 M10866 M10867 M24859             V01128 VERSION     J02482.1  GI:216019 KEYWORDS    . SOURCE      Enterobacteria phage phiX174   ORGANISM  Enterobacteria phage phiX174             Viruses; ssDNA viruses; Microviridae; Microvirus. REFERENCE   1  (bases 1047 to 1094)   AUTHORS   Ziff,E.B., Sedat,J.W. and Galibert,F.   TITLE     Determination of the nucleotide sequence of a fragment of             bacteriophage phiX 174 DNA   JOURNAL   Nature New Biol. 241 (106), 34-37 (1973)    PUBMED   4349156 REFERENCE   2  (bases 2370 to 2421)   AUTHORS   Robertson,H.D., Barrell,B.G., Weith,H.L. and Donelson,J.E.   TITLE     Isolation and sequence analysis of a ribosome-protected fragment             from bacteriophage phiX 174 DNA   JOURNAL   Nature New Biol. 241 (106), 38-40 (1973)    PUBMED   4572838 REFERENCE   3  (bases 2370 to 2420)   AUTHORS   Barrell,B.G., Weith,H.L., Donelson,J.E. and Robertson,H.D.   TITLE     Sequence analysis of the ribosome-protected bacteriophase phiX174             DNA fragment containing the gene G initiation site   JOURNAL   J. Mol. Biol. 92 (3), 377-393 (1975)    PUBMED   1095758 REFERENCE   4  (bases 2365 to 2591)   AUTHORS   Air,G.M., Blackburn,E.H., Sanger,F. and Coulson,A.R.   TITLE     The nucleotide and amino acid sequences of the N (5') terminal             region of gene G of bacteriophage phiphiX 174   JOURNAL   J. Mol. Biol. 96 (4), 703-719 (1975)    PUBMED   1081600 REFERENCE   5  (bases 2263 to 2421)   AUTHORS   Fiddes,J.C.   TITLE     Nucleotide sequence of the intercistronic region between genes G             and F in bacteriophage phiX174 DNA   JOURNAL   J. Mol. Biol. 107 (1), 1-24 (1976)    PUBMED   826639 REFERENCE   6  (bases 1017 to 1081)   AUTHORS   Sedat,J., Ziff,E. and Galibert,F.   TITLE     Direct determination of DNA nucleotide sequences. Structure of             large specific fragments of bacteriophage phiX174 DNA   JOURNAL   J. Mol. Biol. 107 (4), 391-416 (1976)    PUBMED   1003475 REFERENCE   7  (bases 730 to 903)   AUTHORS   Blackburn,E.H.   TITLE     Transcription and sequence analysis of a fragment of bacteriophage             phiX174 DNA   JOURNAL   J. Mol. Biol. 107 (4), 417-431 (1976)    PUBMED   826641 REFERENCE   8  (bases 1017 to 1762)   AUTHORS   Air,G.M., Blackburn,E.H., Coulson,A.R., Galibert,F., Sanger,F.,             Sedat,J.W. and Ziff,E.B.   TITLE     Gene F of bacteriophage phiX174. Correlation of nucleotide             sequences from the DNA and amino acid sequences from the gene             product   JOURNAL   J. Mol. Biol. 107 (4), 445-458 (1976)    PUBMED   1088826 REFERENCE   9  (bases 2395 to 2922)   AUTHORS   Air,G.M., Sanger,F. and Coulson,A.R.   TITLE     Nucleotide and amino acid sequences of gene G of omegaX174   JOURNAL   J. Mol. Biol. 108 (3), 519-533 (1976)    PUBMED   1088827 REFERENCE   10 (bases 4137 to 4207)   AUTHORS   Mansfeld,A.D., Vereijken,J.M. and Jansz,H.S.   TITLE     The nucleotide sequence of a DNA fragment, 71 base pairs in length,             near the origin of DNA replication of bacteriophage 0X174   JOURNAL   Nucleic Acids Res. 3 (10), 2827-2844 (1976)    PUBMED   995652 REFERENCE   11 (bases 4505 to 5374)   AUTHORS   Brown,N.L. and Smith,M.   TITLE     The sequence of a region of bacteriophage phiX174 DNA coding for             parts of genes A and B   JOURNAL   J. Mol. Biol. 116 (1), 1-28 (1977)    PUBMED   592379 REFERENCE   12 (bases 1 to 5375)   AUTHORS   Sanger,F., Air,G.M., Barrell,B.G., Brown,N.L., Coulson,A.R.,             Fiddes,C.A., Hutchison,C.A., Slocombe,P.M. and Smith,M.   TITLE     Nucleotide sequence of bacteriophage phi X174 DNA   JOURNAL   Nature 265 (5596), 687-695 (1977)    PUBMED   870828 REFERENCE   13 (bases 5022 to 5132)   AUTHORS   Brown,N.L. and Smith,M.   TITLE     DNA sequence of a region of the phi X174 genome coding for a             ribosome binding site   JOURNAL   Nature 265 (5596), 695-698 (1977)    PUBMED   859573 REFERENCE   14 (bases 5346 to 5386; 1 to 159)   AUTHORS   Smith,M., Brown,N.L., Air,G.M., Barrell,B.G., Coulson,A.R.,             Hutchison,C.A. III and Sanger,F.   TITLE     DNA sequence at the C termini of the overlapping genes A and B in             bacteriophage phi X174   JOURNAL   Nature 265 (5596), 702-705 (1977)    PUBMED   859575 REFERENCE   15 (sites)   AUTHORS   Fiddes,J.C.   TITLE     The nucleotide sequence of a viral DNA   JOURNAL   Sci. Am. 237 (6), 54-67 (1977)    PUBMED   929160 REFERENCE   16 (bases 1 to 5386)   AUTHORS   Sanger,F., Coulson,A.R., Friedmann,T., Air,G.M., Barrell,B.G.,             Brown,N.L., Fiddes,J.C., Hutchison,C.A. III, Slocombe,P.M. and             Smith,M.   TITLE     The nucleotide sequence of bacteriophage phiX174   JOURNAL   J. Mol. Biol. 125 (2), 225-246 (1978)    PUBMED   731693 REFERENCE   17 (bases 1290 to 1302; 1340 to 1430; 1510 to 1570; 1600 to 1750)   AUTHORS   Air,G.M., Coulson,A.R., Fiddes,J.C., Friedmann,T., Hutchison,C.A.             III, Sanger,F., Slocombe,P.M. and Smith,A.J.   TITLE     Nucleotide sequence of the F protein coding region of bacteriophage             phiX174 and the amino acid sequence of its product   JOURNAL   J. Mol. Biol. 125 (2), 247-254 (1978)    PUBMED   731694 REFERENCE   18 (bases 4256 to 4317)   AUTHORS   Langeveld,S.A., van Mansfeld,A.D., de Winter,J.M. and Weisbeek,P.J.   TITLE     Cleavage of single-stranded DNA by the A and A* proteins of             bacteriophage phi X174   JOURNAL   Nucleic Acids Res. 7 (8), 2177-2188 (1979)    PUBMED   160544 REFERENCE   19 (bases 4248 to 4332)   AUTHORS   Heidekamp,F., Langeveld,S.A., Baas,P.D. and Jansz,H.S.   TITLE     Studies of the recognition sequence of phi X174 gene A protein.             Cleavage site of phi X gene A protein in St-1 RFI DNA   JOURNAL   Nucleic Acids Res. 8 (9), 2009-2021 (1980)    PUBMED   6253953 REFERENCE   20 (bases 436 to 490; 630 to 669; 930 to 979)   AUTHORS   Takeshita,M., Kappen,L.S., Grollman,A.P., Eisenberg,M. and             Goldberg,I.H.   TITLE     Strand scission of deoxyribonucleic acid by neocarzinostatin,             auromomycin, and bleomycin: studies on base release and nucleotide             sequence specificity   JOURNAL   Biochemistry 20 (26), 7599-7606 (1981)    PUBMED   6173064 REFERENCE   22 (bases 1064 to 1757)   AUTHORS   Merville,M.P., Piette,J., Lopez,M., Decuyper,J. and van de Vorst,A.   TITLE     Termination sites of the in vitro DNA synthesis on single-stranded             DNA photosensitized by promazines   JOURNAL   J. Biol. Chem. 259 (24), 15069-15077 (1984)    PUBMED   6239864 REFERENCE   23 (bases 2380 to 2512; 2593 to 2786; 2788 to 2947)   AUTHORS   Air,G.M., Els,M.C., Brown,L.E., Laver,W.G. and Webster,R.G.   TITLE     Location of antigenic sites on the three-dimensional structure of             the influenza N2 virus neuraminidase   JOURNAL   Virology 145 (2), 237-248 (1985)    PUBMED   2411049 REFERENCE   24 (bases 449 to 482; 504 to 598; 1047 to 1111)   AUTHORS   Ueda,K., Morita,J. and Komano,T.   TITLE     Sequence specificity of heat-labile sites in DNA induced by             mitomycin C   JOURNAL   Biochemistry 23 (8), 1634-1640 (1984)    PUBMED   6232949 COMMENT     On Apr 28, 2004 this sequence version replaced gi:15535.             [8]  intermittent sequences.             [15]  review; discussion of complete genome.             Double checked with sumex tape.             Single-stranded circular DNA which codes for eleven proteins.             Replicative form is duplex, icosahedron, related to s13 & g4. [21]             indicates that mitomycin C reduced with sodium borohydride induced             heat-labile sites in DNA most preferentially at dinucleotide             sequence 'gt' (especially 'Pu-g-t').             Bacteriophage phi-X174 single stranded DNA molecules were             irradiated with near UV light in the presence of promazine             derivatives, after priming with restriction fragments or synthetic             primers [22].  The resulting DNA fragments were used as templates             for in vitro complementary chain synthesis by E.coli DNA polymerase             I [22].  More than 90% of the observed chain terminations were             mapped one nucleotide before a guanine residue [22].  Photoreaction             occurred more predominantly with guanine residues localized in             single-stranded parts of the genome [22].  These same guanine             residues could also be damaged when the reaction was performed in             the dark, in the presence of promazine cation radicals [22]. FEATURES             Location/Qualifiers      source          1..5386                      /organism="Enterobacteria phage phiX174"                      /mol_type="genomic DNA"                      /db_xref="taxon:10847"      CDS             join(3981..5386,1..136)                      /function="viral strand synthesis"                      /note="rf replication"                      /codon_start=1                      /transl_table=11                      /product="A"                      /protein_id="AAA32570.1"                      /db_xref="GI:216020"                      /translation="MVRSYYPSECHADYFDFERIEALKPAIEACGISTLSQSPMLGFH                      KQMDNRIKLLEEILSFRMQGVEFDNGDMYVDGHKAASDVRDEFVSVTEKLMDELAQCY                      NVLPQLDINNTIDHRPEGDEKWFLENEKTVTQFCRKLAAERPLKDIRDEYNYPKKKGI                      KDECSRLLEASTMKSRRGFAIQRLMNAMRQAHADGWFIVFDTLTLADDRLEAFYDNPN                      ALRDYFRDIGRMVLAAEGRKANDSHADCYQYFCVPEYGTANGRLHFHAVHFMRTLPTG                      SVDPNFGRRVRNRRQLNSLQNTWPYGYSMPIAVRYTQDAFSRSGWLWPVDAKGEPLKA                      TSYMAVGFYVAKYVNKKSDMDLAAKGLGAKEWNNSLKTKLSLLPKKLFRIRMSRNFGM                      KMLTMTNLSTECLIQLTKLGYDATPFNQILKQNAKREMRLRLGKVTVADVLAAQPVTT                      NLLKFMRASIKMIGVSNLQSFIASMTQKLTLSDISDESKNYLDKAGITTACLRIKSKW                      TAGGK"      CDS             join(4497..5386,1..136)                      /function="shut off host DNA synthesis"                      /codon_start=1                      /transl_table=11                      /product="A*"                      /protein_id="AAA32571.1"                      /db_xref="GI:216021"                      /translation="MKSRRGFAIQRLMNAMRQAHADGWFIVFDTLTLADDRLEAFYDN                      PNALRDYFRDIGRMVLAAEGRKANDSHADCYQYFCVPEYGTANGRLHFHAVHFMRTLP                      TGSVDPNFGRRVRNRRQLNSLQNTWPYGYSMPIAVRYTQDAFSRSGWLWPVDAKGEPL                      KATSYMAVGFYVAKYVNKKSDMDLAAKGLGAKEWNNSLKTKLSLLPKKLFRIRMSRNF                      GMKMLTMTNLSTECLIQLTKLGYDATPFNQILKQNAKREMRLRLGKVTVADVLAAQPV                      TTNLLKFMRASIKMIGVSNLQSFIASMTQKLTLSDISDESKNYLDKAGITTACLRIKS                      KWTAGGK"      CDS             join(5075..5386,1..51)                      /function="capsid morphogenesis"                      /codon_start=1                      /transl_table=11                      /product="B"                      /protein_id="AAA32572.1"                      /db_xref="GI:216022"                      /translation="MEQLTKNQAVATSQEAVQNQNEPQLRDENAHNDKSVHGVLNPTY                      QAGLRRDAVQPDIEAERKKRDEIEAGKSYCSRRFGGATCDDKSAQIYARFDKNDWRIQ                      PAEFYRFHDAEVNTFGYF"      variation       23                      /note="in am18 and am35 [14]"                      /replace="t"      variation       25                      /note="ts116 [14]"                      /replace="c"      CDS             51..221                      /codon_start=1                      /transl_table=11                      /product="K"                      /protein_id="AAA32573.1"                      /db_xref="GI:216023"                      /translation="MSRKIILIKQELLLLVYELNRSGLLAENEKIRPILAQLEKLLLC                      DLSPSTNDSVKN"      variation       57                      /note="am6 [14]"                      /replace="c"      variation       117                      /note="am6 [14]"                      /replace="a"      CDS             133..393                      /note="DNA maturation"                      /codon_start=1                      /transl_table=11                      /product="C"                      /protein_id="AAA32574.1"                      /db_xref="GI:216024"                      /translation="MRKFDLSLRSSRSSYFATFRHQLTILSKTDALDEEKWLNMLGTF                      VKDWFRYESHFVHGRDSLVDILKERGLLSESDAVQPLIGKKS"      mRNA            358..3975                      /product="major transcript"      mRNA            358..991                      /product="minor transcript"      CDS             390..848                      /function="capsid morphogenesis"                      /codon_start=1                      /transl_table=11                      /product="D"                      /protein_id="AAA32575.1"                      /db_xref="GI:216025"                      /translation="MSQVTEQSVRFQTALASIKLIQASAVLDLTEDDFDFLTSNKVWI                      ATDRSRARRCVEACVYGTLDFVGYPRFPAPVEFIAAVIAYYVHPVNIQTACLIMEGAE                      FTENIINGVERPVKAAELFAFTLRVRAGNTDVLTDAEENVRQKLRAEGVM"      CDS             568..843                      /function="cell lysis"                      /codon_start=1                      /transl_table=11                      /product="E"                      /protein_id="AAA32576.1"                      /db_xref="GI:216026"                      /translation="MVRWTLWDTLAFLLLLSLLLPSLLIMFIPSTFKRPVSSWKALNL                      RKTLLMASSVRLKPLNCSRLPCVYAQETLTFLLTQKKTCVKNYVRKE"      CDS             848..964                      /note="core protein; DNA condensation"                      /codon_start=1                      /transl_table=11                      /product="J"                      /protein_id="AAA32577.1"                      /db_xref="GI:216027"                      /translation="MSKGKKRSGARPGRPQPLRGTKGKRKGARLWYVGGQQF"      CDS             1001..2284                      /note="major coat protein"                      /codon_start=1                      /transl_table=11                      /product="F"                      /protein_id="AAA32578.1"                      /db_xref="GI:216028"                      /translation="MSNIQTGAERMPHDLSHLGFLAGQIGRLITISTTPVIAGDSFEM                      DAVGALRLSPLRRGLAIDSTVDIFTFYVPHRHVYGEQWIKFMKDGVNATPLPTVNTTG                      YIDHAAFLGTINPDTNKIPKHLFQGYLNIYNNYFKAPWMPDRTEANPNELNQDDARYG                      FRCCHLKNIWTAPLPPETELSRQMTTSTTSIDIMGLQAAYANLHTDQERDYFMQRYHD                      VISSFGGKTSYDADNRPLLVMRSNLWASGYDVDGTDQTSLGQFSGRVQQTYKHSVPRF                      FVPEHGTMFTLALVRFPPTATKEIQYLNAKGALTYTDIAGDPVLYGNLPPREISMKDV                      FRSGDSSKKFKIAEGQWYRYAPSYVSPAYHLLEGFPFIQEPPSGDLQERVLIRHHDYD                      QCFQSVQLLQWNSQVKFNVTVYRNLPTTRDSIMTS"      CDS             2395..2922                      /note="major spike protein"                      /codon_start=1                      /transl_table=11                      /product="G"                      /protein_id="AAA32579.1"                      /db_xref="GI:216029"                      /translation="MFQTFISRHNSNFFSDKLVLTSVTPASSAPVLQTPKATSSTLYF                      DSLTVNAGNGGFLHCIQMDTSVNAANQVVSVGADIAFDADPKFFACLVRFESSSVPTT                      LPTAYDVYPLNGRHDGGYYTVKDCVTIDVLPRTPGNNVYVGFMVWSNFTATKCRGLVS                      LNQVIKEIICLQPLK"      CDS             2931..3917                      /function="adsorption"                      /note="minor spike protein"                      /codon_start=1                      /transl_table=11                      /product="H"                      /protein_id="AAA32580.1"                      /db_xref="GI:216030"                      /translation="MFGAIAGGIASALAGGAMSKLFGGGQKAASGGIQGDVLATDNNT                      VGMGDAGIKSAIQGSNVPNPDEAAPSFVSGAMAKAGKGLLEGTLQAGTSAVSDKLLDL                      VGLGGKSAADKGKDTRDYLAAAFPELNAWERAGADASSAGMVDAGFENQKELTKMQLD                      NQKEIAEMQNETQKEIAGIQSATSRQNTKDQVYAQNEMLAYQQKESTARVASIMENTN                      LSKQQQVSEIMRQMLTQAQTAGQYFTNDQIKEMTRKVSAEVDLVHQQTQNQRYGSSHI                      GATAKDISNVVTDAASGVVDIFHGIDKAVADTWNNFWKDGKADGIGSNLSRK"      misc_feature    3962                      /note="transcription start site"      rep_origin      4306                      /note="origin of viral strand synthesis"      misc_feature    4899                      /note="transcription start site" ORIGIN               1 gagttttatc gcttccatga cgcagaagtt aacactttcg gatatttctg atgagtcgaa        61 aaattatctt gataaagcag gaattactac tgcttgttta cgaattaaat cgaagtggac       121 tgctggcgga aaatgagaaa attcgaccta tccttgcgca gctcgagaag ctcttacttt       181 gcgacctttc gccatcaact aacgattctg tcaaaaactg acgcgttgga tgaggagaag       241 tggcttaata tgcttggcac gttcgtcaag gactggttta gatatgagtc acattttgtt       301 catggtagag attctcttgt tgacatttta aaagagcgtg gattactatc tgagtccgat       361 gctgttcaac cactaatagg taagaaatca tgagtcaagt tactgaacaa tccgtacgtt       421 tccagaccgc tttggcctct attaagctca ttcaggcttc tgccgttttg gatttaaccg       481 aagatgattt cgattttctg acgagtaaca aagtttggat tgctactgac cgctctcgtg       541 ctcgtcgctg cgttgaggct tgcgtttatg gtacgctgga ctttgtggga taccctcgct       601 ttcctgctcc tgttgagttt attgctgccg tcattgctta ttatgttcat cccgtcaaca       661 ttcaaacggc ctgtctcatc atggaaggcg ctgaatttac ggaaaacatt attaatggcg       721 tcgagcgtcc ggttaaagcc gctgaattgt tcgcgtttac cttgcgtgta cgcgcaggaa       781 acactgacgt tcttactgac gcagaagaaa acgtgcgtca aaaattacgt gcggaaggag       841 tgatgtaatg tctaaaggta aaaaacgttc tggcgctcgc cctggtcgtc cgcagccgtt       901 gcgaggtact aaaggcaagc gtaaaggcgc tcgtctttgg tatgtaggtg gtcaacaatt       961 ttaattgcag gggcttcggc cccttacttg aggataaatt atgtctaata ttcaaactgg      1021 cgccgagcgt atgccgcatg acctttccca tcttggcttc cttgctggtc agattggtcg      1081 tcttattacc atttcaacta ctccggttat cgctggcgac tccttcgaga tggacgccgt      1141 tggcgctctc cgtctttctc cattgcgtcg tggccttgct attgactcta ctgtagacat      1201 ttttactttt tatgtccctc atcgtcacgt ttatggtgaa cagtggatta agttcatgaa      1261 ggatggtgtt aatgccactc ctctcccgac tgttaacact actggttata ttgaccatgc      1321 cgcttttctt ggcacgatta accctgatac caataaaatc cctaagcatt tgtttcaggg      1381 ttatttgaat atctataaca actattttaa agcgccgtgg atgcctgacc gtaccgaggc      1441 taaccctaat gagcttaatc aagatgatgc tcgttatggt ttccgttgct gccatctcaa      1501 aaacatttgg actgctccgc ttcctcctga gactgagctt tctcgccaaa tgacgacttc      1561 taccacatct attgacatta tgggtctgca agctgcttat gctaatttgc atactgacca      1621 agaacgtgat tacttcatgc agcgttacca tgatgttatt tcttcatttg gaggtaaaac      1681 ctcttatgac gctgacaacc gtcctttact tgtcatgcgc tctaatctct gggcatctgg      1741 ctatgatgtt gatggaactg accaaacgtc gttaggccag ttttctggtc gtgttcaaca      1801 gacctataaa cattctgtgc cgcgtttctt tgttcctgag catggcacta tgtttactct      1861 tgcgcttgtt cgttttccgc ctactgcgac taaagagatt cagtacctta acgctaaagg      1921 tgctttgact tataccgata ttgctggcga ccctgttttg tatggcaact tgccgccgcg      1981 tgaaatttct atgaaggatg ttttccgttc tggtgattcg tctaagaagt ttaagattgc      2041 tgagggtcag tggtatcgtt atgcgccttc gtatgtttct cctgcttatc accttcttga      2101 aggcttccca ttcattcagg aaccgccttc tggtgatttg caagaacgcg tacttattcg      2161 ccaccatgat tatgaccagt gtttccagtc cgttcagttg ttgcagtgga atagtcaggt      2221 taaatttaat gtgaccgttt atcgcaatct gccgaccact cgcgattcaa tcatgacttc      2281 gtgataaaag attgagtgtg aggttataac gccgaagcgg taaaaatttt aatttttgcc      2341 gctgaggggt tgaccaagcg aagcgcggta ggttttctgc ttaggagttt aatcatgttt      2401 cagactttta tttctcgcca taattcaaac tttttttctg ataagctggt tctcacttct      2461 gttactccag cttcttcggc acctgtttta cagacaccta aagctacatc gtcaacgtta      2521 tattttgata gtttgacggt taatgctggt aatggtggtt ttcttcattg cattcagatg      2581 gatacatctg tcaacgccgc taatcaggtt gtttctgttg gtgctgatat tgcttttgat      2641 gccgacccta aattttttgc ctgtttggtt cgctttgagt cttcttcggt tccgactacc      2701 ctcccgactg cctatgatgt ttatcctttg aatggtcgcc atgatggtgg ttattatacc      2761 gtcaaggact gtgtgactat tgacgtcctt ccccgtacgc cgggcaataa cgtttatgtt      2821 ggtttcatgg tttggtctaa ctttaccgct actaaatgcc gcggattggt ttcgctgaat      2881 caggttatta aagagattat ttgtctccag ccacttaagt gaggtgattt atgtttggtg      2941 ctattgctgg cggtattgct tctgctcttg ctggtggcgc catgtctaaa ttgtttggag      3001 gcggtcaaaa agccgcctcc ggtggcattc aaggtgatgt gcttgctacc gataacaata      3061 ctgtaggcat gggtgatgct ggtattaaat ctgccattca aggctctaat gttcctaacc      3121 ctgatgaggc cgcccctagt tttgtttctg gtgctatggc taaagctggt aaaggacttc      3181 ttgaaggtac gttgcaggct ggcacttctg ccgtttctga taagttgctt gatttggttg      3241 gacttggtgg caagtctgcc gctgataaag gaaaggatac tcgtgattat cttgctgctg      3301 catttcctga gcttaatgct tgggagcgtg ctggtgctga tgcttcctct gctggtatgg      3361 ttgacgccgg atttgagaat caaaaagagc ttactaaaat gcaactggac aatcagaaag      3421 agattgccga gatgcaaaat gagactcaaa aagagattgc tggcattcag tcggcgactt      3481 cacgccagaa tacgaaagac caggtatatg cacaaaatga gatgcttgct tatcaacaga      3541 aggagtctac tgctcgcgtt gcgtctatta tggaaaacac caatctttcc aagcaacagc      3601 aggtttccga gattatgcgc caaatgctta ctcaagctca aacggctggt cagtatttta      3661 ccaatgacca aatcaaagaa atgactcgca aggttagtgc tgaggttgac ttagttcatc      3721 agcaaacgca gaatcagcgg tatggctctt ctcatattgg cgctactgca aaggatattt      3781 ctaatgtcgt cactgatgct gcttctggtg tggttgatat ttttcatggt attgataaag      3841 ctgttgccga tacttggaac aatttctgga aagacggtaa agctgatggt attggctcta      3901 atttgtctag gaaataaccg tcaggattga caccctccca attgtatgtt ttcatgcctc      3961 caaatcttgg aggctttttt atggttcgtt cttattaccc ttctgaatgt cacgctgatt      4021 attttgactt tgagcgtatc gaggctctta aacctgctat tgaggcttgt ggcatttcta      4081 ctctttctca atccccaatg cttggcttcc ataagcagat ggataaccgc atcaagctct      4141 tggaagagat tctgtctttt cgtatgcagg gcgttgagtt cgataatggt gatatgtatg      4201 ttgacggcca taaggctgct tctgacgttc gtgatgagtt tgtatctgtt actgagaagt      4261 taatggatga attggcacaa tgctacaatg tgctccccca acttgatatt aataacacta      4321 tagaccaccg ccccgaaggg gacgaaaaat ggtttttaga gaacgagaag acggttacgc      4381 agttttgccg caagctggct gctgaacgcc ctcttaagga tattcgcgat gagtataatt      4441 accccaaaaa gaaaggtatt aaggatgagt gttcaagatt gctggaggcc tccactatga      4501 aatcgcgtag aggctttgct attcagcgtt tgatgaatgc aatgcgacag gctcatgctg      4561 atggttggtt tatcgttttt gacactctca cgttggctga cgaccgatta gaggcgtttt      4621 atgataatcc caatgctttg cgtgactatt ttcgtgatat tggtcgtatg gttcttgctg      4681 ccgagggtcg caaggctaat gattcacacg ccgactgcta tcagtatttt tgtgtgcctg      4741 agtatggtac agctaatggc cgtcttcatt tccatgcggt gcactttatg cggacacttc      4801 ctacaggtag cgttgaccct aattttggtc gtcgggtacg caatcgccgc cagttaaata      4861 gcttgcaaaa tacgtggcct tatggttaca gtatgcccat cgcagttcgc tacacgcagg      4921 acgctttttc acgttctggt tggttgtggc ctgttgatgc taaaggtgag ccgcttaaag      4981 ctaccagtta tatggctgtt ggtttctatg tggctaaata cgttaacaaa aagtcagata      5041 tggaccttgc tgctaaaggt ctaggagcta aagaatggaa caactcacta aaaaccaagc      5101 tgtcgctact tcccaagaag ctgttcagaa tcagaatgag ccgcaacttc gggatgaaaa      5161 tgctcacaat gacaaatctg tccacggagt gcttaatcca acttaccaag ctgggttacg      5221 acgcgacgcc gttcaaccag atattgaagc agaacgcaaa aagagagatg agattgaggc      5281 tgggaaaagt tactgtagcc gacgttttgg cggcgcaacc tgtgacgaca aatctgctca      5341 aatttatgcg cgcttcgata aaaatgattg gcgtatccaa cctgca //

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