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Medical Forum / Diseases and Disorders / AIDS / May 2004

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Human immunodeficiency virus 1, complete genome.

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Baby Peanut - 15 May 2004 04:28 GMT
LOCUS       NC_001802               9181 bp ss-RNA     linear   VRL 17-APR-2004
DEFINITION  Human immunodeficiency virus 1, complete genome.
ACCESSION   NC_001802
VERSION     NC_001802.1  GI:9629357
KEYWORDS    .
SOURCE      Human immunodeficiency virus 1 (HIV-1)
 ORGANISM  Human immunodeficiency virus 1
           Viruses; Retroid viruses; Retroviridae; Lentivirus; Primate
           lentivirus group.
REFERENCE   1  (bases 1 to 9181)
 AUTHORS   Martoglio,B., Graf,R. and Dobberstein,B.
 TITLE     Signal peptide fragments of preprolactin and HIV-1 p-gp160 interact
           with calmodulin
 JOURNAL   EMBO J. 16 (22), 6636-6645 (1997)
 MEDLINE   98031891
  PUBMED   9362478
REFERENCE   2  (bases 1 to 9181)
 AUTHORS   Petropoulos,C.J.
 TITLE     Appendix 2: Retroviral taxonomy, protein structure, sequences, and
           genetic maps
 JOURNAL   (in) Coffin,J.M. (Ed.);
           RETROVIRUSES: 757;
           Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York,
           NY, USA (1997)
REFERENCE   3  (bases 1 to 9181)
 AUTHORS   Chappey,C.
 TITLE     Direct Submission
 JOURNAL   Submitted (15-MAR-1999) NIH, NLM, Rockville Pike, Bethesda, MD
           20894, USA
 REMARK    Sequence update by submitter
REFERENCE   4  (bases 1 to 9181)
 AUTHORS   Chappey,C.
 TITLE     Direct Submission
 JOURNAL   Submitted (12-NOV-1997) NIH, NLM, Rockville Pike, Bethesda, MD
           20894, USA
COMMENT     REVIEWED REFSEQ: This record has been curated by NCBI staff. The
           reference sequence was derived from AF033819.
           The annotation of this sequence was corrected and updated with the
           kind help of Dr. Colombe Chappey (ViroLogic Inc., South San
           Francisco, CA USA) and Roger Ptak (Southern Research Institute,
           Frederick, MD USA).
FEATURES             Location/Qualifiers
    source          1..9181
                    /organism="Human immunodeficiency virus 1"
                    /mol_type="genomic RNA"
                    /db_xref="taxon:11676"
                    /note="strain for reference annotation"
    misc_feature    1..96
                    /note="repeat; positions of RNA transcription
                    initialization and polyadenylation; Region: R"
    polyA_signal    73..78
                    /note="both 5' and 3' poly A signals are transcribed into
                    RNA, but the 5' one is suppressed"
    5'UTR           97..181
    primer_bind     182..199
    gene            336..4642
                    /gene="gag-pol"
                    /locus_tag="HIV1gp1"
                    /db_xref="GeneID:155348"
                    /db_xref="LocusID:155348"
    CDS             join(336..1637,1637..4642)
                    /gene="gag-pol"
                    /locus_tag="HIV1gp1"
                    /note="fusion protein consisting of the viral structural
                    proteins and enzymes; cleaved by the viral protease into
                    individual mature proteins; The processing products of the
                    Gag and Gag-Pol polyproteins were annotated with the help
                    of Pettit et al., 2003 and references therein; Pr160;
                    ribosomal slippage at slippery sequence tttttta
                    (1631..1637)"
                    /codon_start=1
                    /product="Gag-Pol"
                    /protein_id="NP_057849.4"
                    /db_xref="GI:28872819"
                    /db_xref="GeneID:155348"
                    /db_xref="LocusID:155348"
                    /translation="MGARASVLSGGELDRWEKIRLRPGGKKKYKLKHIVWASRELERF
                    AVNPGLLETSEGCRQILGQLQPSLQTGSEELRSLYNTVATLYCVHQRIEIKDTKEALD
                    KIEEEQNKSKKKAQQAAADTGHSNQVSQNYPIVQNIQGQMVHQAISPRTLNAWVKVVE
                    EKAFSPEVIPMFSALSEGATPQDLNTMLNTVGGHQAAMQMLKETINEEAAEWDRVHPV
                    HAGPIAPGQMREPRGSDIAGTTSTLQEQIGWMTNNPPIPVGEIYKRWIILGLNKIVRM
                    YSPTSILDIRQGPKEPFRDYVDRFYKTLRAEQASQEVKNWMTETLLVQNANPDCKTIL
                    KALGPAATLEEMMTACQGVGGPGHKARVLAEAMSQVTNSATIMMQRGNFRNQRKIVKC
                    FNCGKEGHTARNCRAPRKKGCWKCGKEGHQMKDCTERQANFLREDLAFLQGKAREFSS
                    EQTRANSPTRRELQVWGRDNNSPSEAGADRQGTVSFNFPQVTLWQRPLVTIKIGGQLK
                    EALLDTGADDTVLEEMSLPGRWKPKMIGGIGGFIKVRQYDQILIEICGHKAIGTVLVG
                    PTPVNIIGRNLLTQIGCTLNFPISPIETVPVKLKPGMDGPKVKQWPLTEEKIKALVEI
                    CTEMEKEGKISKIGPENPYNTPVFAIKKKDSTKWRKLVDFRELNKRTQDFWEVQLGIP
                    HPAGLKKKKSVTVLDVGDAYFSVPLDEDFRKYTAFTIPSINNETPGIRYQYNVLPQGW
                    KGSPAIFQSSMTKILEPFRKQNPDIVIYQYMDDLYVGSDLEIGQHRTKIEELRQHLLR
                    WGLTTPDKKHQKEPPFLWMGYELHPDKWTVQPIVLPEKDSWTVNDIQKLVGKLNWASQ
                    IYPGIKVRQLCKLLRGTKALTEVIPLTEEAELELAENREILKEPVHGVYYDPSKDLIA
                    EIQKQGQGQWTYQIYQEPFKNLKTGKYARMRGAHTNDVKQLTEAVQKITTESIVIWGK
                    TPKFKLPIQKETWETWWTEYWQATWIPEWEFVNTPPLVKLWYQLEKEPIVGAETFYVD
                    GAANRETKLGKAGYVTNRGRQKVVTLTDTTNQKTELQAIYLALQDSGLEVNIVTDSQY
                    ALGIIQAQPDQSESELVNQIIEQLIKKEKVYLAWVPAHKGIGGNEQVDKLVSAGIRKV
                    LFLDGIDKAQDEHEKYHSNWRAMASDFNLPPVVAKEIVASCDKCQLKGEAMHGQVDCS
                    PGIWQLDCTHLEGKVILVAVHVASGYIEAEVIPAETGQETAYFLLKLAGRWPVKTIHT
                    DNGSNFTGATVRAACWWAGIKQEFGIPYNPQSQGVVESMNKELKKIIGQVRDQAEHLK
                    TAVQMAVFIHNFKRKGGIGGYSAGERIVDIIATDIQTKELQKQITKIQNFRVYYRDSR
                    NPLWKGPAKLLWKGEGAVVIQDNSDIKVVPRRKAKIIRDYGKQMAGDDCVASRQDED"
    mat_peptide     join(1632..1637,1637..1798)
                    /gene="gag-pol"
                    /product="Gag-Pol Transframe peptide"
                    /note="the Glu-Asp-Leu tripeptide (positions 4-6) is a
                    specific inhibitor of the HIV-1 protease. Involved in
                    regulation of the protease-mediated polyprotein
                    processing; p6*; alternative p6 protein"
                    /protein_id="NP_787043.1"
                    /db_xref="GI:28872821"
    mat_peptide     1655..4639
                    /gene="gag-pol"
                    /locus_tag="HIV1gp1"
                    /product="Pol"
                    /note="unprocessed Pol polyprotein; includes part of the
                    transframe peptide, protease, reverse transcriptase and
                    integrase domains."
                    /protein_id="NP_789740.1"
                    /db_xref="GI:28872823"
    mat_peptide     1799..2095
                    /gene="gag-pol"
                    /locus_tag="HIV1gp1"
                    /product="protease"
                    /note="The proteinase domain of Gag-Pol (in the form of
                    homodimer) mediates all the cleavages in the polyprotein.
                    Cleaves itself from the polyprotein late in particle
                    assembly; aspartic peptidase"
                    /protein_id="NP_705926.1"
                    /db_xref="GI:25121906"
    mat_peptide     2096..3775
                    /gene="gag-pol"
                    /locus_tag="HIV1gp1"
                    /product="reverse transcriptase"
                    /note="transcribes single stranded viral RNA genome into
                    double stranded proviral DNA; HIV-1 reverse transcriptase
                    is composed of the p66 subunit (this protein) and the p51
                    subunit that lacks the RNAse H domain of the larger
                    subunit"
                    /protein_id="NP_705927.1"
                    /db_xref="GI:25121907"
    mat_peptide     2096..3415
                    /gene="gag-pol"
                    /locus_tag="HIV1gp1"
                    /product="reverse transcriptase p51 subunit"
                    /note="HIV-1 reverse transcriptase is composed of the p66
                    subunit and the p51 subunit (this protein) that lacks the
                    RNAse H domain of the larger subunit"
                    /protein_id="NP_789739.1"
                    /db_xref="GI:28872822"
    mat_peptide     3776..4639
                    /gene="gag-pol"
                    /locus_tag="HIV1gp1"
                    /product="integrase"
                    /note="mediates integration of the viral DNA into the
                    infected cell chromosome"
                    /protein_id="NP_705928.1"
                    /db_xref="GI:25121908"
    gene            336..1838
                    /gene="gag"
                    /locus_tag="HIV1gp2"
                    /db_xref="GeneID:155030"
                    /db_xref="LocusID:155030"
    CDS             336..1838
                    /gene="gag"
                    /locus_tag="HIV1gp2"
                    /note="Pr55; The processing products of the Gag and
                    Gag-Pol polyproteins were annotated with the help of
                    Pettit et al., 2003 and references therein"
                    /codon_start=1
                    /product="Gag"
                    /protein_id="NP_057850.1"
                    /db_xref="GI:9629360"
                    /db_xref="GeneID:155030"
                    /db_xref="LocusID:155030"
                    /translation="MGARASVLSGGELDRWEKIRLRPGGKKKYKLKHIVWASRELERF
                    AVNPGLLETSEGCRQILGQLQPSLQTGSEELRSLYNTVATLYCVHQRIEIKDTKEALD
                    KIEEEQNKSKKKAQQAAADTGHSNQVSQNYPIVQNIQGQMVHQAISPRTLNAWVKVVE
                    EKAFSPEVIPMFSALSEGATPQDLNTMLNTVGGHQAAMQMLKETINEEAAEWDRVHPV
                    HAGPIAPGQMREPRGSDIAGTTSTLQEQIGWMTNNPPIPVGEIYKRWIILGLNKIVRM
                    YSPTSILDIRQGPKEPFRDYVDRFYKTLRAEQASQEVKNWMTETLLVQNANPDCKTIL
                    KALGPAATLEEMMTACQGVGGPGHKARVLAEAMSQVTNSATIMMQRGNFRNQRKIVKC
                    FNCGKEGHTARNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSYKGRPGNFLQ
                    SRPEPTAPPEESFRSGVETTTPPQKQEPIDKELYPLTSLRSLFGNDPSSQ"
    mat_peptide     336..731
                    /gene="gag"
                    /locus_tag="HIV1gp2"
                    /product="matrix"
                    /note="viral structural protein; forms the outer
                    structural shell of HIV-1 virions; involved in the nuclear
                    import of the HIV-1 preintegration complex; p17"
                    /protein_id="NP_579876.2"
                    /db_xref="GI:28876542"
    mat_peptide     732..1424
                    /gene="gag"
                    /locus_tag="HIV1gp2"
                    /product="capsid"
                    /note="viral structural protein; forms the core of HIV-1
                    virions; p24"
                    /protein_id="NP_579880.1"
                    /db_xref="GI:19172948"
    mat_peptide     1425..1466
                    /gene="gag"
                    /locus_tag="HIV1gp2"
                    /product="p2"
                    /note="Processing of Gag-Pol by the protease domain dimer
                    starts with cleavage between the p2 and nucleocapsid
                    proteins."
                    /protein_id="NP_579882.1"
                    /db_xref="GI:19172950"
    mat_peptide     1467..1631
                    /gene="gag"
                    /locus_tag="HIV1gp2"
                    /product="nucleocapsid"
                    /note="viral structural protein; coats the genomic RNA
                    inside the virion core; binds and delivers full-length
                    viral RNAs into assembling HIV-1 virions; p7"
                    /protein_id="NP_579881.1"
                    /db_xref="GI:19172949"
    mat_peptide     1632..1679
                    /gene="gag"
                    /locus_tag="HIV1gp2"
                    /product="p1"
                    /note="important for virus infectivity, protein
                    processing, and genomic RNA dimer stability"
                    /protein_id="NP_787042.1"
                    /db_xref="GI:28872820"
    mat_peptide     1680..1835
                    /gene="gag"
                    /locus_tag="HIV1gp2"
                    /product="p6"
                    /note="important for incorporation of Vpr into assembling
                    HIV-1 virions; helps mediate efficient virus particle
                    release from infected cells"
                    /protein_id="NP_579883.1"
                    /db_xref="GI:19172951"
    gene            4587..5165
                    /gene="vif"
                    /locus_tag="HIV1gp3"
                    /db_xref="GeneID:155459"
                    /db_xref="LocusID:155459"
    CDS             4587..5165
                    /gene="vif"
                    /locus_tag="HIV1gp3"
                    /note="p23; viral infectivity factor; viral accessory
                    protein important for virus replication in vivo"
                    /codon_start=1
                    /product="Vif"
                    /protein_id="NP_057851.1"
                    /db_xref="GI:9629361"
                    /db_xref="GeneID:155459"
                    /db_xref="LocusID:155459"
                    /translation="MENRWQVMIVWQVDRMRIRTWKSLVKHHMYVSGKARGWFYRHHY
                    ESPHPRISSEVHIPLGDARLVITTYWGLHTGERDWHLGQGVSIEWRKKRYSTQVDPEL
                    ADQLIHLYYFDCFSDSAIRKALLGHIVSPRCEYQAGHNKVGSLQYLALAALITPKKIK
                    PPLPSVTKLTEDRWNKPQKTKGHRGSHTMNGH"
    gene            5105..5396
                    /gene="vpr"
                    /locus_tag="HIV1gp4"
                    /db_xref="GeneID:155807"
                    /db_xref="LocusID:155807"
    CDS             join(5105..5319,5321..5396)
                    /gene="vpr"
                    /locus_tag="HIV1gp4"
                    /note="p15; viral protein R; viral accessory protein
                    important for virus replication in vivo; involved in the
                    nuclear import of the HIV-1 preintegration complex;
                    induces G2 cell cycle arrest; influences mutation rates
                    during viral DNA synthesis; An artificial frameshift
                    eliminating the orf-disrupting nucleotide at position 5320
                    is introduced to obtain the typical HIV-1 Vpr protein
                    sequence. For this particular HIV-1 strain, HXB2, only a
                    short (78 amino acid long) variant of the Vpr sequence can
                    be obtained by translation of nucleotides 5105 through
                    5341 without the frameshift"
                    /codon_start=1
                    /exception="artificial frameshift"
                    /product="Vpr"
                    /protein_id="NP_057852.2"
                    /db_xref="GI:28872817"
                    /db_xref="GeneID:155807"
                    /db_xref="LocusID:155807"
                    /translation="MEQAPEDQGPQREPHNEWTLELLEELKNEAVRHFPRIWLHGLGQ
                    HIYETYGDTWAGVEAIIRILQQLLFIHFRIGCRHSRIGVTRQRRARNGASRS"
    gene            5377..7970
                    /gene="tat"
                    /locus_tag="HIV1gp5"
                    /db_xref="GeneID:155871"
                    /db_xref="LocusID:155871"
    CDS             join(5377..5591,7925..7970)
                    /gene="tat"
                    /locus_tag="HIV1gp5"
                    /note="p14; transcriptional activator; viral regulatory
                    protein required for virus replication; transactivates the
                    viral LTR promoter through interactions with cellular
                    transcription factors; associated with pathogenic effects
                    of the virus; the length of Tat varies depending on virus
                    strain or clade"
                    /codon_start=1
                    /product="Tat"
                    /protein_id="NP_057853.1"
                    /db_xref="GI:9629358"
                    /db_xref="GeneID:155871"
                    /db_xref="LocusID:155871"
                    /translation="MEPVDPRLEPWKHPGSQPKTACTNCYCKKCCFHCQVCFITKALG
                    ISYGRKKRRQRRRAHQNSQTHQASLSKQPTSQPRGDPTGPKE"
    gene            5516..8199
                    /gene="rev"
                    /locus_tag="HIV1gp6"
                    /db_xref="GeneID:155908"
                    /db_xref="LocusID:155908"
    CDS             join(5516..5591,7925..8199)
                    /gene="rev"
                    /locus_tag="HIV1gp6"
                    /note="p19; regulator of expression of virion proteins;
                    prevents splicing of viral RNA; shuttles unspliced viral
                    RNA to the cytoplasm for expression of viral proteins and
                    incorporation of full length viral genomic RNA into
                    virions"
                    /codon_start=1
                    /product="Rev"
                    /protein_id="NP_057854.1"
                    /db_xref="GI:9629359"
                    /db_xref="GeneID:155908"
                    /db_xref="LocusID:155908"
                    /translation="MAGRSGDSDEELIRTVRLIKLLYQSNPPPNPEGTRQARRNRRRR
                    WRERQRQIHSISERILGTYLGRSAEPVPLQLPPLERLTLDCNEDCGTSGTQGVGSPQI
                    LVESPTVLESGTKE"
    gene            5608..5856
                    /gene="vpu"
                    /locus_tag="HIV1gp7"
                    /db_xref="GeneID:155945"
                    /db_xref="LocusID:155945"
    CDS             5608..5856
                    /gene="vpu"
                    /locus_tag="HIV1gp7"
                    /note="p16; viral protein U; viral accessory protein
                    important for virus replication in vivo; promotes
                    degradation of CD4 and down-regulates cell surface
                    expression of MHC class I proteins; helps mediate
                    efficient virus particle release from infected cells;
                    reported to induce apoptosis by suppressing the nuclear
                    factor kappaB-dependent expression of antiapoptotic
                    factors; may attenuate the level of Env precursor(gp160)
                    biosynthesis; Vpu and gp160 are translated from different
                    reading frames of the same bicistronic mRNA"
                    /codon_start=1
                    /product="Vpu"
                    /protein_id="NP_057855.1"
                    /db_xref="GI:9629366"
                    /db_xref="GeneID:155945"
                    /db_xref="LocusID:155945"
                    /translation="MQPIPIVAIVALVVAIIIAIVVWSIVIIEYRKILRQRKIDRLID
                    RLIERAEDSGNESEGEISALVEMGVEMGHHAPWDVDDL"
    gene            5771..8341
                    /gene="env"
                    /locus_tag="HIV1gp8"
                    /db_xref="GeneID:155971"
                    /db_xref="LocusID:155971"
    CDS             5771..8341
                    /gene="env"
                    /locus_tag="HIV1gp8"
                    /note="gp160; envelope glycoprotein; envelope polyprotein;
                    cleaved by cellular proteases into mature proteins gp120
                    and gp41"
                    /codon_start=1
                    /product="Envelope surface glycoprotein gp160, precursor"
                    /protein_id="NP_057856.1"
                    /db_xref="GI:9629363"
                    /db_xref="GeneID:155971"
                    /db_xref="LocusID:155971"
                    /translation="MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTVYYGVPV
                    WKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEVVLVNVTENFNMWKNDMVE
                    QMHEDIISLWDQSLKPCVKLTPLCVSLKCTDLKNDTNTNSSSGRMIMEKGEIKNCSFN
                    ISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYKLTSCNTSVITQACPKVSFEPIPIHYC
                    APAGFAILKCNNKTFNGTGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSVN
                    FTDNAKTIIVQLNTSVEINCTRPNNNTRKRIRIQRGPGRAFVTIGKIGNMRQAHCNIS
                    RAKWNNTLKQIASKLREQFGNNKTIIFKQSSGGDPEIVTHSFNCGGEFFYCNSTQLFN
                    STWFNSTWSTEGSNNTEGSDTITLPCRIKQIINMWQKVGKAMYAPPISGQIRCSSNIT
                    GLLLTRDGGNSNNESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKRRVVQR
                    EKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQLLSGIVQQQNNLLRAIEAQQHLL
                    QLTVWGIKQLQARILAVERYLKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWN
                    HTTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWFNITNWLWYI
                    KLFIMIVGGLVGLRIVFAVLSIVNRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGER
                    DRDRSIRLVNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKYWWN
                    LLQYWSQELKNSAVSLLNATAIAVAEGTDRVIEVVQGACRAIRHIPRRIRQGLERILL
                    "
    sig_peptide     5771..5854
                    /gene="env"
                    /protein_id="NP_579893.2"
                    /db_xref="GI:28876543"
    mat_peptide     5855..7303
                    /gene="env"
                    /locus_tag="HIV1gp8"
                    /product="Envelope surface glycoprotein gp120"
                    /note="mediates binding of HIV-1 to CD4 and cellular
                    co-receptors; cooperates with gp41 to mediate fusion of
                    viral membrane with cellular membrane during virus entry
                    into cells; Envelope surface unit; SU"
                    /protein_id="NP_579894.2"
                    /db_xref="GI:28876544"
    mat_peptide     7304..8338
                    /gene="env"
                    /locus_tag="HIV1gp8"
                    /product="Envelope transmembrane glycoprotein gp41"
                    /note="cooperates with gp120 to mediate fusion of viral
                    membrane with cellular membrane during virus entry into
                    cells; Envelope transmembrane domain; TM"
                    /protein_id="NP_579895.1"
                    /db_xref="GI:19172954"
    gene            8343..8963
                    /gene="nef"
                    /locus_tag="HIV1gp9"
                    /db_xref="GeneID:156110"
                    /db_xref="LocusID:156110"
    CDS             8343..8963
                    /gene="nef"
                    /locus_tag="HIV1gp9"
                    /note="p27; negative factor; viral accessory protein;
                    important for virus replication in vivo; determinant of
                    HIV-1 pathogenesis; down-regulates cell surface CD4 and
                    MHC class I molecules; enhances virus infectivity through
                    interactions with multiple cellular signaling proteins;
                    This particular nucleotide sequence has a premature stop
                    codon in place of a well-conserved tryptophan codon at
                    position 8712-8714 that truncates the HIV1 Nef protein
                    sequence to a 123 amino acids-long N-terminal portion (not
                    shown)"
                    /codon_start=1
                    /transl_except=(pos:8712..8714,aa:Trp)
                    /product="Nef"
                    /protein_id="NP_057857.2"
                    /db_xref="GI:28872818"
                    /db_xref="GeneID:156110"
                    /db_xref="LocusID:156110"
                    /translation="MGGKWSKSSVIGWPTVRERMRRAEPAADRVGAASRDLEKHGAIT
                    SSNTAATNAACAWLEAQEEEEVGFPVTPQVPLRPMTYKAAVDLSHFLKEKGGLEGLIH
                    SQRRQDILDLWIYHTQGYFPDWQNYTPGPGVRYPLTFGWCYKLVPVEPDKIEEANKGE
                    NTSLLHPVSLHGMDDPEREVLEWRFDSRLAFHHVARELHPEYFKNC"
    3'UTR           8631..9085
    misc_feature    9086..9181
                    /note="repeat; positions of RNA transcription
                    initialization and polyadenylation; Region: R"
    polyA_signal    9158..9163
                    /note="both 5' and 3' poly A signals are transcribed into
                    RNA, but the 5' one is suppressed"
ORIGIN
Yana - 18 May 2004 16:47 GMT
Anyone can tell just by looking at that that it is not the genome of HIV
they have presented.  

It is obviously a green tree frog.  
abinkum@yahoo.com - 19 May 2004 13:56 GMT
CAn you please describe the source of the RNA for this analysis, and
the methodology used.
Robert S. Holzman - 19 May 2004 23:57 GMT
> CAn you please describe the source of the RNA for this
> analysis, and the methodology used.

Could not locate a descriptive paper for that particular clone
in medline, but the two papers below are the original reports
of HIV-1 cloning.  Hope it is close enough for what you want.

Li, Y., J. C. Kappes, et al. (1991). "Molecular
characterization of human immunodeficiency virus type 1 cloned
directly from uncultured human brain tissue: identification of
replication-competent and -defective viral genomes." J Virol
65(8): 3973-85.
    All presently available replication-competent proviral
clones of human immunodeficiency virus type 1 (HIV-1) are
derived from cell culture-amplified virus. Since tissue
culture is highly selective for viral strains with an in vitro
growth advantage, such clones may not be representative of the
biologically relevant virus present in vivo. In this study, we
report the molecular cloning and genotypic characterization of
10 HIV-1 genomes directly from uncultured brain tissue of a
patient with AIDS dementia complex. Targeting unintegrated
circular HIV-1 molecules for recombinant lambda phage cloning,
we obtained four full-length genomes with one or two long
terminal repeats (LTRs), three defective genomes with internal
deletions, two rearranged genomes with inverted LTR sequences,
and one integrated proviral half with flanking cellular
sequences. Nucleotide sequence analysis of these clones
demonstrated chromosomal integration, circle formation,
genomic inversion, and LTR-mediated autointegration of HIV-1
genomes in vivo. Comparison of a 510-bp hypervariable envelope
region among 8 lambda phage-derived and 12 polymerase chain
reaction-derived clones from the same brain specimen
identified a predominant viral form as well as genetically
divergent variants. Variability among 19 of 20 clones ranged
between 0.2 and 1.2%. One clone exhibited 8.2% nucleotide
sequence differences consisting almost exclusively of G-to-A
changes. Transfection of the four full-length HIV-1 genomes
identified one clone (YU-2) as replication competent and
exhibiting growth characteristics similar to those of tissue
culture-derived macrophage tropic strains of HIV-1. These
results demonstrate, for the first time, that replication-
competent HIV-1 genomes, complex mixtures of defective viral
forms, and chromosomally integrated provirus persist in vivo.
In addition, the brain-derived viral clones are expected to
prove valuable for future studies of macrophage and
neurotropism as well as for the analysis of other viral
properties that are subject to in vitro selection pressures.

Li, Y., H. Hui, et al. (1992). "Complete nucleotide sequence,
genome organization, and biological properties of human
immunodeficiency virus type 1 in vivo: evidence for limited
defectiveness and complementation." J Virol 66(11): 6587-600.
    Previous studies of the genetic and biologic
characteristics of human immunodeficiency virus type 1 (HIV-1)
have by necessity used tissue culture-derived virus. We
recently reported the molecular cloning of four full-length
HIV-1 genomes directly from uncultured human brain tissue (Y.
Li, J. C. Kappes, J. A. Conway, R. W. Price, G. M. Shaw, and
B. H. Hahn, J. Virol. 65:3973-3985, 1991). In this report, we
describe the biologic properties of these four clones and the
complete nucleotide sequences and genome organization of two
of them. Clones HIV-1YU-2 and HIV-1YU-10 were 9,174 and 9,176
nucleotides in length, differed by 0.26% in nucleotide
sequence, and except for a frameshift mutation in the pol gene
in HIV-1YU-10, contained open reading frames corresponding to
5'-gag-pol-vif-vpr-tat-rev-vpu-env-nef-3' flanked by long
terminal repeats. HIV-1YU-2 was fully replication competent,
while HIV-1YU-10 and two other clones, HIV-1YU-21 and HIV-1YU-
32, were defective. All three defective clones, however, when
transfected into Cos-1 cells in any pairwise combination,
yielded virions that were replication competent and
transmissible by cell-free passage. The cellular host range of
HIV-1YU-2 was strictly limited to primary T lymphocytes and
monocyte-macrophages, a property conferred by its external
envelope glycoprotein. Phylogenetic analyses of HIV-1YU-2 gene
sequences revealed this virus to be a member of the North
American/European HIV-1 subgroup, with specific similarity to
other monocyte-tropic viruses in its V3 envelope amino acid
sequence. These results indicate that HIV-1 infection of brain
is characterized by the persistence of mixtures of fully
competent, minimally defective, and more substantially altered
viral forms and that complementation among them is readily
attainable. In addition, the limited degree of genotypic
heterogeneity observed among HIV-1YU and other brain-derived
viruses and their preferential tropism for monocyte-
macrophages suggest that viral replication within the central
nervous system may differ from that within the peripheral
lymphoid compartment in significant and clinically important
ways. The availability of genetically and biologically well
characterized HIV-1 clones from uncultured human tissue should
facilitate future studies of virus-cell interactions relevant
to viral pathogenesis and drug and vaccine development.
Yana - 21 May 2004 07:14 GMT
“All presently available replication-competent proviral clones of human
immunodeficiency virus type 1 (HIV-1) are derived from cell
culture-amplified virus. Since tissue culture is highly selective for
viral strains with an in vitro growth advantage, such clones may not be
representative of the biologically relevant virus present in vivo.”

May I repeat that last statement…..

“.... Such clones may not be representative of the biologically relevant
virus present in vivo………..”

==========================================
Additionally, why do they need to use cell cultured-amplified virus to
clone?  Why not use the isolated virus itself?

Oh I remember…because there IS no isolated virus!  

So again I ask, what are they cloning?  (It still could be a green tree
grog for all we know)
Nick Bennett - 27 May 2004 18:00 GMT
> “All presently available replication-competent proviral clones of human
> immunodeficiency virus type 1 (HIV-1) are derived from cell
[quoted text clipped - 6 lines]
> “.... Such clones may not be representative of the biologically relevant
> virus present in vivo………..”

This is a simple statement of the limitations of the process.  It's well
known that HIV exists as a mixture of quasispecies, in the same way as
most humans differ in their genetic make-up over the planet.  Some clones
isolated from infected people are in fact defective: not always
immediately obvious from culture (the genes aren't required) but
sometimes.  The odds are though that anything you find from a patient with
HIV will be a dominant strain (the numbers game) and presumably such a
strain will be replication competant.  It's not always been true, at least
one strain that I'm aware of in common lab usage has a deletion from true
"wild type" HIV-1.

> ==========================================
> Additionally, why do they need to use cell cultured-amplified virus to
> clone?  Why not use the isolated virus itself?

Not enough, basically.  However you do it, you'll have to amplify the
genetic material.  Either through PCR or through culture.  Culture has the
advantage that the genetic code is less likely to be corrupted through
normal error-generation mechanisms, and that it'll happily deal with a
full-length construct.  It does however have the disadvantage that you
might grow out a clone that is defective for a gene that is required in
vivo but not in vitro (see above).

I have read one report which I'm fairly sure used sequences derived from
peripheral blood samples of an infected patient, but due to the
limitations of PCR (in order to get enough DNA to work with) they had to
clone seperate chunks and then line them up in order to get the full
sequence.  This is the same process used in detecting a gene from a
linkage study (chromosome walking) but with a different aim.  Not ideal
really, but it was done from a patient's sample as I recall.

> Oh I remember…because there IS no isolated virus!

*sigh*.  When are you going to listen to the science, and try to learn how
it works, instead of repeating rubbish ad nauseum?  You don't need an
isolate to find new genetic material (if you did, then the entire
DNA-based forensics business would collapse overnight!), just the
material.  You can then use the genetic "isolate" to produce virus
isolates, true purified virus, to confirm your suspicion that the material
produces virus.

> So again I ask, what are they cloning?  (It still could be a green tree
> grog for all we know)

The methods used for HIV don't differ from any used for other viruses like
herpes, adenovirus, measles etc etc.  "Isolating" it usually involves some
method of amplification, either PCR or culture.

If you were to fill a grain of salt (1mm cube) with HIV particles you
would have over 1 trillion of them.  That would be enough to fill the
blood stream of 200 AIDS patients with viral loads of 1 million per ml.
So...in order to get enough virus to even begin to attempt to isolate RNA
directly from it, are you suggesting we bleed several hundred people dry,
in the name of science?

I don't think so.  Seriously, those are the numbers we're talking about.
Viruses are small.  My back-of-the-envelope math says that the total RNA
you'd get from that would be 64ng.  Just enough to even see on an
ethidium gel (typical amounts you might run from, say, a plasmid prep are
1 microgram, or 15 times as much).  From 200 fully bled AIDS patients.
Sheesh.

THAT puts it into perspective.  If you want to isolate your genome using
that method, go right ahead.  I won't hold my breath.

Bennett
Dr. Phillip S. Duke - 20 May 2004 01:42 GMT
> CAn you please describe the source of the RNA for this analysis, and
> the methodology used.

That is the NCBI "Reference Sequence" for HIV-1.  It is from
the Lambda-HXB-2 proviral genomic clone produced by
George Shaw and coworkers in the Gallo lab in 1984 (1),
sequenced in 1985 (3) and 1987 (4) after proving to be an infectious
molecular clone in 1985 (2).  The complete methodology is
clearly spelled out in those papers.  The source was proviral
DNA, not viral RNA.

1:  Shaw GM, Hahn BH, Arya SK, Groopman JE, Gallo RC,
Wong-Staal F.
Molecular characterization of human T-cell leukemia
(lymphotropic) virus type III in the acquired immune
deficiency syndrome.
Science. 1984 Dec 7;226(4679):1165-71.

2:  Fisher AG, Collalti E, Ratner L, Gallo RC, Wong-Staal F
A molecular clone of HTLV-III with biological activity.
Nature. 1985 Jul 18-24;316(6025):262-5.

3: Ratner L, Haseltine W, Patarca R, Livak KJ, Starcich B,
Josephs SF,  Doran ER, Rafalski JA, Whitehorn EA,
Baumeister K, et al.
Complete nucleotide sequence of the AIDS virus, HTLV-III.
Nature. 1985 Jan 24-30;313(6000):277-84.  

4: Ratner L, Fisher A, Jagodzinski LL, Mitsuya H, Liou RS,
Gallo RC, Wong-Staal F.
Complete nucleotide sequences of functional clones of
the AIDS virus.
AIDS Res Hum Retroviruses. 1987 Spring;3(1):57-69.
Baby Peanut - 20 May 2004 19:10 GMT
> CAn you please describe the source of the RNA for this analysis, and
> the methodology used.

Oh that's easy.  I used blatent copyright violation to aquire the data.

BASE COUNT     3272 a   1642 c   2225 g   2042 t
ORIGIN      
       1 ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac
      61 tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt
     121 gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca
     181 gtggcgcccg aacagggacc tgaaagcgaa agggaaacca gaggagctct ctcgacgcag
     241 gactcggctt gctgaagcgc gcacggcaag aggcgagggg cggcgactgg tgagtacgcc
     301 aaaaattttg actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa
     361 gcgggggaga attagatcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat
     421 ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg
     481 gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc
     541 agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc
     601 atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa
     661 acaaaagtaa gaaaaaagca cagcaagcag cagctgacac aggacacagc aatcaggtca
     721 gccaaaatta ccctatagtg cagaacatcc aggggcaaat ggtacatcag gccatatcac
     781 ctagaacttt aaatgcatgg gtaaaagtag tagaagagaa ggctttcagc ccagaagtga
     841 tacccatgtt ttcagcatta tcagaaggag ccaccccaca agatttaaac accatgctaa
     901 acacagtggg gggacatcaa gcagccatgc aaatgttaaa agagaccatc aatgaggaag
     961 ctgcagaatg ggatagagtg catccagtgc atgcagggcc tattgcacca ggccagatga
    1021 gagaaccaag gggaagtgac atagcaggaa ctactagtac ccttcaggaa caaataggat
    1081 ggatgacaaa taatccacct atcccagtag gagaaattta taaaagatgg ataatcctgg
    1141 gattaaataa aatagtaaga atgtatagcc ctaccagcat tctggacata agacaaggac
    1201 caaaggaacc ctttagagac tatgtagacc ggttctataa aactctaaga gccgagcaag
    1261 cttcacagga ggtaaaaaat tggatgacag aaaccttgtt ggtccaaaat gcgaacccag
    1321 attgtaagac tattttaaaa gcattgggac cagcggctac actagaagaa atgatgacag
    1381 catgtcaggg agtaggagga cccggccata aggcaagagt tttggctgaa gcaatgagcc
    1441 aagtaacaaa ttcagctacc ataatgatgc agagaggcaa ttttaggaac caaagaaaga
    1501 ttgttaagtg tttcaattgt ggcaaagaag ggcacacagc cagaaattgc agggccccta
    1561 ggaaaaaggg ctgttggaaa tgtggaaagg aaggacacca aatgaaagat tgtactgaga
    1621 gacaggctaa ttttttaggg aagatctggc cttcctacaa gggaaggcca gggaattttc
    1681 ttcagagcag accagagcca acagccccac cagaagagag cttcaggtct ggggtagaga
    1741 caacaactcc ccctcagaag caggagccga tagacaagga actgtatcct ttaacttccc
    1801 tcaggtcact ctttggcaac gacccctcgt cacaataaag ataggggggc aactaaagga
    1861 agctctatta gatacaggag cagatgatac agtattagaa gaaatgagtt tgccaggaag
    1921 atggaaacca aaaatgatag ggggaattgg aggttttatc aaagtaagac agtatgatca
    1981 gatactcata gaaatctgtg gacataaagc tataggtaca gtattagtag gacctacacc
    2041 tgtcaacata attggaagaa atctgttgac tcagattggt tgcactttaa attttcccat
    2101 tagccctatt gagactgtac cagtaaaatt aaagccagga atggatggcc caaaagttaa
    2161 acaatggcca ttgacagaag aaaaaataaa agcattagta gaaatttgta cagagatgga
    2221 aaaggaaggg aaaatttcaa aaattgggcc tgaaaatcca tacaatactc cagtatttgc
    2281 cataaagaaa aaagacagta ctaaatggag aaaattagta gatttcagag aacttaataa
    2341 gagaactcaa gacttctggg aagttcaatt aggaatacca catcccgcag ggttaaaaaa
    2401 gaaaaaatca gtaacagtac tggatgtggg tgatgcatat ttttcagttc ccttagatga
    2461 agacttcagg aagtatactg catttaccat acctagtata aacaatgaga caccagggat
    2521 tagatatcag tacaatgtgc ttccacaggg atggaaagga tcaccagcaa tattccaaag
    2581 tagcatgaca aaaatcttag agccttttag aaaacaaaat ccagacatag ttatctatca
    2641 atacatggat gatttgtatg taggatctga cttagaaata gggcagcata gaacaaaaat
    2701 agaggagctg agacaacatc tgttgaggtg gggacttacc acaccagaca aaaaacatca
    2761 gaaagaacct ccattccttt ggatgggtta tgaactccat cctgataaat ggacagtaca
    2821 gcctatagtg ctgccagaaa aagacagctg gactgtcaat gacatacaga agttagtggg
    2881 gaaattgaat tgggcaagtc agatttaccc agggattaaa gtaaggcaat tatgtaaact
    2941 ccttagagga accaaagcac taacagaagt aataccacta acagaagaag cagagctaga
    3001 actggcagaa aacagagaga ttctaaaaga accagtacat ggagtgtatt atgacccatc
    3061 aaaagactta atagcagaaa tacagaagca ggggcaaggc caatggacat atcaaattta
    3121 tcaagagcca tttaaaaatc tgaaaacagg aaaatatgca agaatgaggg gtgcccacac
    3181 taatgatgta aaacaattaa cagaggcagt gcaaaaaata accacagaaa gcatagtaat
    3241 atggggaaag actcctaaat ttaaactgcc catacaaaag gaaacatggg aaacatggtg
    3301 gacagagtat tggcaagcca cctggattcc tgagtgggag tttgttaata cccctccctt
    3361 agtgaaatta tggtaccagt tagagaaaga acccatagta ggagcagaaa ccttctatgt
    3421 agatggggca gctaacaggg agactaaatt aggaaaagca ggatatgtta ctaatagagg
    3481 aagacaaaaa gttgtcaccc taactgacac aacaaatcag aagactgagt tacaagcaat
    3541 ttatctagct ttgcaggatt cgggattaga agtaaacata gtaacagact cacaatatgc
    3601 attaggaatc attcaagcac aaccagatca aagtgaatca gagttagtca atcaaataat
    3661 agagcagtta ataaaaaagg aaaaggtcta tctggcatgg gtaccagcac acaaaggaat
    3721 tggaggaaat gaacaagtag ataaattagt cagtgctgga atcaggaaag tactattttt
    3781 agatggaata gataaggccc aagatgaaca tgagaaatat cacagtaatt ggagagcaat
    3841 ggctagtgat tttaacctgc cacctgtagt agcaaaagaa atagtagcca gctgtgataa
    3901 atgtcagcta aaaggagaag ccatgcatgg acaagtagac tgtagtccag gaatatggca
    3961 actagattgt acacatttag aaggaaaagt tatcctggta gcagttcatg tagccagtgg
    4021 atatatagaa gcagaagtta ttccagcaga aacagggcag gaaacagcat attttctttt
    4081 aaaattagca ggaagatggc cagtaaaaac aatacatact gacaatggca gcaatttcac
    4141 cggtgctacg gttagggccg cctgttggtg ggcgggaatc aagcaggaat ttggaattcc
    4201 ctacaatccc caaagtcaag gagtagtaga atctatgaat aaagaattaa agaaaattat
    4261 aggacaggta agagatcagg ctgaacatct taagacagca gtacaaatgg cagtattcat
    4321 ccacaatttt aaaagaaaag gggggattgg ggggtacagt gcaggggaaa gaatagtaga
    4381 cataatagca acagacatac aaactaaaga attacaaaaa caaattacaa aaattcaaaa
    4441 ttttcgggtt tattacaggg acagcagaaa tccactttgg aaaggaccag caaagctcct
    4501 ctggaaaggt gaaggggcag tagtaataca agataatagt gacataaaag tagtgccaag
    4561 aagaaaagca aagatcatta gggattatgg aaaacagatg gcaggtgatg attgtgtggc
    4621 aagtagacag gatgaggatt agaacatgga aaagtttagt aaaacaccat atgtatgttt
    4681 cagggaaagc taggggatgg ttttatagac atcactatga aagccctcat ccaagaataa
    4741 gttcagaagt acacatccca ctaggggatg ctagattggt aataacaaca tattggggtc
    4801 tgcatacagg agaaagagac tggcatttgg gtcagggagt ctccatagaa tggaggaaaa
    4861 agagatatag cacacaagta gaccctgaac tagcagacca actaattcat ctgtattact
    4921 ttgactgttt ttcagactct gctataagaa aggccttatt aggacacata gttagcccta
    4981 ggtgtgaata tcaagcagga cataacaagg taggatctct acaatacttg gcactagcag
    5041 cattaataac accaaaaaag ataaagccac ctttgcctag tgttacgaaa ctgacagagg
    5101 atagatggaa caagccccag aagaccaagg gccacagagg gagccacaca atgaatggac
    5161 actagagctt ttagaggagc ttaagaatga agctgttaga cattttccta ggatttggct
    5221 ccatggctta gggcaacata tctatgaaac ttatggggat acttgggcag gagtggaagc
    5281 cataataaga attctgcaac aactgctgtt tatccatttt cagaattggg tgtcgacata
    5341 gcagaatagg cgttactcga cagaggagag caagaaatgg agccagtaga tcctagacta
    5401 gagccctgga agcatccagg aagtcagcct aaaactgctt gtaccaattg ctattgtaaa
    5461 aagtgttgct ttcattgcca agtttgtttc ataacaaaag ccttaggcat ctcctatggc
    5521 aggaagaagc ggagacagcg acgaagagct catcagaaca gtcagactca tcaagcttct
    5581 ctatcaaagc agtaagtagt acatgtaatg caacctatac caatagtagc aatagtagca
    5641 ttagtagtag caataataat agcaatagtt gtgtggtcca tagtaatcat agaatatagg
    5701 aaaatattaa gacaaagaaa aatagacagg ttaattgata gactaataga aagagcagaa
    5761 gacagtggca atgagagtga aggagaaata tcagcacttg tggagatggg ggtggagatg
    5821 gggcaccatg ctccttggga tgttgatgat ctgtagtgct acagaaaaat tgtgggtcac
    5881 agtctattat ggggtacctg tgtggaagga agcaaccacc actctatttt gtgcatcaga
    5941 tgctaaagca tatgatacag aggtacataa tgtttgggcc acacatgcct gtgtacccac
    6001 agaccccaac ccacaagaag tagtattggt aaatgtgaca gaaaatttta acatgtggaa
    6061 aaatgacatg gtagaacaga tgcatgagga tataatcagt ttatgggatc aaagcctaaa
    6121 gccatgtgta aaattaaccc cactctgtgt tagtttaaag tgcactgatt tgaagaatga
    6181 tactaatacc aatagtagta gcgggagaat gataatggag aaaggagaga taaaaaactg
    6241 ctctttcaat atcagcacaa gcataagagg taaggtgcag aaagaatatg cattttttta
    6301 taaacttgat ataataccaa tagataatga tactaccagc tataagttga caagttgtaa
    6361 cacctcagtc attacacagg cctgtccaaa ggtatccttt gagccaattc ccatacatta
    6421 ttgtgccccg gctggttttg cgattctaaa atgtaataat aagacgttca atggaacagg
    6481 accatgtaca aatgtcagca cagtacaatg tacacatgga attaggccag tagtatcaac
    6541 tcaactgctg ttaaatggca gtctagcaga agaagaggta gtaattagat ctgtcaattt
    6601 cacggacaat gctaaaacca taatagtaca gctgaacaca tctgtagaaa ttaattgtac
    6661 aagacccaac aacaatacaa gaaaaagaat ccgtatccag agaggaccag ggagagcatt
    6721 tgttacaata ggaaaaatag gaaatatgag acaagcacat tgtaacatta gtagagcaaa
    6781 atggaataac actttaaaac agatagctag caaattaaga gaacaatttg gaaataataa
    6841 aacaataatc tttaagcaat cctcaggagg ggacccagaa attgtaacgc acagttttaa
    6901 ttgtggaggg gaatttttct actgtaattc aacacaactg tttaatagta cttggtttaa
    6961 tagtacttgg agtactgaag ggtcaaataa cactgaagga agtgacacaa tcaccctccc
    7021 atgcagaata aaacaaatta taaacatgtg gcagaaagta ggaaaagcaa tgtatgcccc
    7081 tcccatcagt ggacaaatta gatgttcatc aaatattaca gggctgctat taacaagaga
    7141 tggtggtaat agcaacaatg agtccgagat cttcagacct ggaggaggag atatgaggga
    7201 caattggaga agtgaattat ataaatataa agtagtaaaa attgaaccat taggagtagc
    7261 acccaccaag gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg gaataggagc
    7321 tttgttcctt gggttcttgg gagcagcagg aagcactatg ggcgcagcct caatgacgct
    7381 gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca atttgctgag
    7441 ggctattgag gcgcaacagc atctgttgca actcacagtc tggggcatca agcagctcca
    7501 ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg
    7561 ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt ggagtaataa
    7621 atctctggaa cagatttgga atcacacgac ctggatggag tgggacagag aaattaacaa
    7681 ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga
    7741 acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa
    7801 ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat
    7861 agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt
    7921 tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg
    7981 tggagagaga gacagagaca gatccattcg attagtgaac ggatccttgg cacttatctg
    8041 ggacgatctg cggagcctgt gcctcttcag ctaccaccgc ttgagagact tactcttgat
    8101 tgtaacgagg attgtggaac ttctgggacg cagggggtgg gaagccctca aatattggtg
    8161 gaatctccta cagtattgga gtcaggaact aaagaatagt gctgttagct tgctcaatgc
    8221 cacagccata gcagtagctg aggggacaga tagggttata gaagtagtac aaggagcttg
    8281 tagagctatt cgccacatac ctagaagaat aagacagggc ttggaaagga ttttgctata
    8341 agatgggtgg caagtggtca aaaagtagtg tgattggatg gcctactgta agggaaagaa
    8401 tgagacgagc tgagccagca gcagataggg tgggagcagc atctcgagac ctggaaaaac
    8461 atggagcaat cacaagtagc aatacagcag ctaccaatgc tgcttgtgcc tggctagaag
    8521 cacaagagga ggaggaggtg ggttttccag tcacacctca ggtaccttta agaccaatga
    8581 cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc
    8641 taattcactc ccaaagaaga caagatatcc ttgatctgtg gatctaccac acacaaggct
    8701 acttccctga ttagcagaac tacacaccag ggccaggggt cagatatcca ctgacctttg
    8761 gatggtgcta caagctagta ccagttgagc cagataagat agaagaggcc aataaaggag
    8821 agaacaccag cttgttacac cctgtgagcc tgcatgggat ggatgacccg gagagagaag
    8881 tgttagagtg gaggtttgac agccgcctag catttcatca cgtggcccga gagctgcatc
    8941 cggagtactt caagaactgc tgacatcgag cttgctacaa gggactttcc gctggggact
    9001 ttccagggag gcgtggcctg ggcgggactg gggagtggcg agccctcaga tcctgcatat
    9061 aagcagctgc tttttgcctg tactgggtct ctctggttag accagatctg agcctgggag
    9121 ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt
    9181 c
//

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