U.S. patent application number 12/422882 was filed with the patent office on 2009-08-13 for astrocyte modulated genes and uses thereof.
Invention is credited to Paul B. Fisher, Dong-chul Kang, Zhao-zhong Su.
Application Number | 20090203885 12/422882 |
Document ID | / |
Family ID | 31891153 |
Filed Date | 2009-08-13 |
United States Patent
Application |
20090203885 |
Kind Code |
A1 |
Fisher; Paul B. ; et
al. |
August 13, 2009 |
Astrocyte Modulated Genes And Uses Thereof
Abstract
The present invention relates to Astrocyte Modulated Genes
(AMGs). AMGs are genes whose expression are modulated in human
astrocytes grown in primary cell culture following the exposure of
these cells to either the human immunodeficiency virus HIV-1 or to
the HIV-1 protein gp120. AMGs comprise both Astrocyte Enhanced
Genes (AEGs) and Astrocyte Suppressed Genes (ASGs). Thus, the
present invention further relates to Astrocyte Enhanced Genes
(AEGs), the expression of which are up-regulated in human
astrocytes grown in primary cell culture that are exposed to either
the human immunodeficiency virus HIV-1 or to the HIV-1 protein
gp120, and to Astrocyte Suppressed Genes (ASGs), the expression of
which are downregulated in human astrocytes grown in primary cell
culture that are exposed to either the human immunodeficiency virus
HIV-1 or to the HIV-1 protein gp120. Because they may play a role
in HIV-associated dementia ("HAD"), AMGs may be used as markers in
methods for screening for drugs that treat or prevent HAD.
Inventors: |
Fisher; Paul B.; (Scarsdale,
NY) ; Su; Zhao-zhong; (New York, NY) ; Kang;
Dong-chul; (Dongan-gu, KR) |
Correspondence
Address: |
BAKER BOTTS L.L.P.
30 ROCKEFELLER PLAZA, 44TH FLOOR
NEW YORK
NY
10112-4498
US
|
Family ID: |
31891153 |
Appl. No.: |
12/422882 |
Filed: |
April 13, 2009 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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10975280 |
Oct 27, 2004 |
7517973 |
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12422882 |
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PCT/US03/11887 |
Apr 17, 2003 |
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10975280 |
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60452629 |
Mar 6, 2003 |
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60376375 |
Apr 29, 2002 |
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Current U.S.
Class: |
530/350 |
Current CPC
Class: |
C07K 14/47 20130101 |
Class at
Publication: |
530/350 |
International
Class: |
C07K 14/16 20060101
C07K014/16 |
Goverment Interests
[0001] The subject matter described herein was supported in part by
National Institutes of Health Grant 5P01NS031492, so that the
United States Government has certain rights herein.
Claims
1-18. (canceled)
19. A protein encoded by a nucleic acid molecule, wherein the
sequence of the nucleic acid molecule comprises nucleotides
220-1968 of SEQ ID NO:1.
20. (canceled)
21. (canceled)
22. A protein encoded by a nucleic acid molecule, wherein the
nucleic acid molecule comprises residues which are at least 90
percent identical to nucleotides 220-1968 of SEQ ID NO:1.
23. (canceled)
24. (canceled)
25. A protein having an amino acid sequence as set forth in SEQ ID
NO:2.
26-31. (canceled)
32. A peptide which is a fragment of the protein of claim 19, and
which binds to an antibody which binds to a protein having an amino
acid sequence as set forth in SEQ ID NO:2.
33. (canceled)
34. (canceled)
35. A peptide which is a fragment of the protein of claim 22, and
which binds to an antibody which binds to a protein having an amino
acid sequence as set forth in SEQ ID NO:2.
36. (canceled)
37. (canceled)
38. A peptide which is a fragment of the protein of claim 25, and
which binds to an antibody which binds to a protein having an amino
acid sequence as set forth in SEQ ID NO:2.
39-63. (canceled)
Description
1. INTRODUCTION
[0002] The present invention relates to Astrocyte Modulated Genes
(AMGs). AMGs are genes whose expressions are modulated in human
astrocytes grown in primary cell culture and exposed to either the
human immunodeficiency virus HIV-1 or to the HIV-1 protein gp120.
AMGs comprise Astrocyte Enhanced Genes (AEGs) and Astrocyte
Suppressed Genes (ASGs) among those astrocyte genes whose
expression patterns are altered following exposure to HIV-1 or
gp120. Because they may play a role in HIV-associated dementia
("HAD"), AMGs may be used as markers in methods for screening for
drugs that treat or prevent HAD.
2. BACKGROUND OF THE INVENTION
[0003] Glial cells are the predominant cell type in the brain,
constituting more than 50% of the total cell count and outnumbering
neurons 10-fold (Schubert, 1984, Developmental Biology of Cultured
Nerve, Muscle, and Glia. Wiley and Sons, New York; Rutka et al.,
1997, J. Neurosurg. 87, 420-430). Two major subtypes of glia are
distinguished by their distinct functions in the nervous system:
oligodendrocytes, which form myelin sheaths around nerve cell
axons, and astrocytes, which maintain brain homeostasis and respond
to pathogens and brain injury (Benveniste, 1992, Am. J. Physiol.
263, C1-16; Verkhratsky et al., 1998, Physiol. Rev. 78,
100-130).
[0004] Historically, astrocytes were considered to provide mostly
passive support to neurons and the overall function of the nervous
system. Recent studies question this hypothesis and suggest that
astrocytes also play a critical role in several aspects of signal
transmission and that defects in these functions may lead to
neurodegeneration (Choi, 1988; Neuron 1, 623-634; Rothstein et al.,
1996, Neuron 16, 675-686; Verkhratsky et al., 1998, Physiol. Rev.
78, 100-130; Anderson & Swanson, 2000, Glia 32, 1-14). For
example, the high-affinity excitatory amino acid transport systems
acting through astrocyte-specific transporters EAAT1 and EAAT2 are
thought to be primarily responsible for maintenance of low levels
of free intrasynaptic L-glutamate, the major excitatory
neurotransmitter in the brain (Choi, 1988; Neuron 1, 623-634;
Benveniste, 1992, Am. J. Physiol. 263, C1-16; Anderson &
Swanson, 2000, Glia 32, 1-14). Defects that specifically abrogate
this function result in accumulation of extracellular glutamate in
synaptic clefts and overexcitation and death of neurons, a
phenomenon referred to as excitotoxicity (Choi, 1988; Neuron 1,
623-634; Gegelashvili, G. & Schousboe, A. (1997). Molec.
Pharmacol., 52, 6-15; Tanaka et al., 1997, Science 276,
1699-1702).
[0005] At least one neurodegenerative disease, Amyotropic Lateral
Sclerosis (ALS), has been linked to a significant decrease of
high-affinity sodium-dependent glutamate transport in synaptic
membranes (Rothstein et al., 1992, New Engl. J. Med. 326,
1464-1468) and a selective loss of the transporter EAAT2 (Bristol
& Rothstein, 1996, Ann. Neurol. 39, 676-679). Similar defects
are implicated in Alzheimer's disease, stroke/ischemia, epilepsy,
and HIV-1-associated dementia (HAD) (Choi, 1988, Neuron 1, 623-634;
Kaul et al., 2001, Nature 410, 988-994; Maragakis & Rothstein,
2001, Arch. Neurol., 58, 365-370).
[0006] In addition to their role in glutamate removal from
synapses, astrocytes significantly increase the number of synapses
and enhance synaptic efficacy by altering pre- and post-synaptic
functions in vitro (Oliet et al., 2001, Science 292, 923-926;
Ullian et al., 2001, Science 291, 657-660). Finally, astrocytes
appear to share many properties with neurons, including expression
of functional neuronal nicotinic acetylcholine receptors (nACHRs)
and competence for Ca.sup.++-dependent glutamate release, thus
permitting intercellular signaling between astrocytes and neurons
and, possibly, modulation of neuronal signal transmission by
astrocytes (Iino et al., 2001, Science 292, 926-929; Sharma &
Vijayaraghavan, 2001, Proc Natl Acad Sci USA 98, 4148-4153; Ullian
et al., 2001, Science 291, 657-660).
[0007] Overall, these studies suggest that astrocytes and neurons
are functionally integrated and that pathogenic stimuli that
adversely affect astrocytes will directly or indirectly impact on
neuronal function and survival.
[0008] Studies have focused on investigation of HIV-1 infection in
neural cells and the potential contribution of such infections to
neurodegeneration and HAD. The pathogenic events triggered by HIV-1
in the brain, which ultimately result in neuronal loss and CNS
dysfunction (Navia et al., 1986a, Ann. Neurol. 19, 525-535; Navia
et al., 1986, Ann. Neurol. 19, 517-524; reviewed in Lipton &
Gendelman, 1995, New Engl. J. Med. 233, 934-940), have not been
fully resolved. Neurons are rarely infected in vivo (Wiley et al.,
1986, Proc. Natl. Acad. Sci. USA 83, 7089-93) and it is unlikely
that neuronal loss in HIV-1 dementia is caused by cytopathic
infection of these cells.
[0009] Numerous neuropathology, immunocytochemistry, in situ
hybridization, and virus isolation studies indicate that
macrophages and microglial cells are the primary host cells for
productive HIV-1 infection in the CNS (Koening et al., 1986,
Science 233, 1089-1093; Brew et al., 1995, Ann. Neurol. 38,
563-570; reviewed in Lipton & Gendelman, 1995, New Engl. J.
Med. 233, 934-940). It has been suggested that HIV-1 infection and
subsequent activation of infected cells causes neuroinflammatory
responses involving production of chemokines, cytokines, nitric
oxide, and other factors, some of which were shown to be neurotoxic
in vitro (reviewed in Lipton & Gendelman, 1995, New Engl. J.
Med. 233, 934-940; Kaul et al., 2001, Nature 410, 988-994). Viral
products secreted by infected cells, including gp120 and Tat, can
also induce neurotoxicity in vitro and in animal models (Lipton
& Gendelman, 1995, New Engl. J. Med. 233, 934-940; Kaul et al.,
2001, Nature 410, 988-994).
[0010] Astrocytes also can be infected with HIV-1 in vitro and in
vivo, although with lower efficiency than T cells and macrophages
(Dewhurst et al., 1987, J. Virol. 61, 3774-3782, J. Virol. 61,
3774-3782; Tornatore et al., 1991, J. Virol. 65, 6094-6100; Saito
et al., 1994, Neurology 44, 474-481; Tornatore et al., 1994,
Neurology 44, 481-487; reviewed in Brack-Werner, 1999, AIDS 13,
1-22). The limited infection of astrocytes has been attributed to
various mechanisms including intracellular restrictions to virus
expression (Tornatore et al., 1994b, J. Virol. 68, 93-102; Gorry et
al., 1999, J. Virol. 73, 352-61; Ludwig et al., 1999, J. Virol. 73,
8279-8289) or, as has been shown recently, inefficient virus entry
(Bencheikh et al., 1999, J. Neurovirol. 5, 115-124; Canki et al.,
2001, J. Virol. 75, 7925-7933). There is general agreement,
however, that HIV-1 can persist in astrocytes for prolonged periods
in a low productive, non-cytolytic state, from which it can be
induced by physiologic stimuli such as tumor necrosis
factor-.alpha. (TNF-.alpha.) (Tornatore et al., 1991, J. Virol. 65,
6094-6100; Shahabuddin et al., 1992, Pathobiology 60, 195-205).
[0011] Surveys of autopsy tissues using in situ PCR and sensitive
immunocytochemistry techniques indicate that the frequency of
HIV-1-positive astrocytes in selected tissue sections from brains
of patients with dementia can achieve 1% (Saito et al., 1994,
Neurology 44, 474-481; Tornatore et al., 1994a, Neurology 44,
481-487; Takahashi et al., 1996, Ann. Neurol. 39, 705-711).
Considering that the number of astrocytes in the brain is between
10.sup.11 to 10.sup.12 cells (Verkhratsky et al., 1998, Physiol.
Rev. 78, 100-130), these cells clearly constitute a major target
for HIV-1 infection in the brain.
[0012] The consequences of this infection with respect to HAD
pathogenesis are unknown but they may be significant. Persistent,
non-cytolytic HIV-1 infection in culture alters gene expression in
lymphocytes (Shahabuddin et al., 1994, AIDS Res. Hum. Retroviruses
10, 1525-1529; Geiss et al., 2000, Virology 266, 8-16) and
astrocytes (Schneider-Schaulies et al., 1992, Virology 191,
765-772; He et al., 1997, Proc. Natl. Acad. Sci. USA 94,
3954-3959), indicating that such infections may affect cell
function. Exposure of astrocytes to recombinant HIV-1 envelope
glycoprotein gp120 alters cell physiology (Benos et al., 1994a,
Adv. Neuroimmunol. 4, 175-179), including a potential effect on
glutamate transport as indicated by increased D-aspartate efflux in
astrocytes treated with gp120 (Benos et al., 1994b, Proc. Natl.
Acad. Sci. USA 91, 494-498). Impairment of glutamate transport was
also observed after incubation of human astrocytes with TNF-.alpha.
(Fine et al., 1996, J. Biol. Chem. 271, 15303-15306) or
co-cultivation with T cells infected with human T cell leukemia
virus type I (HTLVI) (Szymocha et al., 2000, J. Virol. 74,
6433-6441), and similar defects were found in feline astrocytes
after infection with feline immunodeficiency virus (FIV) (Yu et
al., 1998, Proc. Natl. Acad. Sci. USA 95, 2624-2629).
[0013] More recent studies indicate that ligation of the HIV-1
coreceptor on astrocytes, CXCR4, by either stromal cell-derived
factor 1 (SDF-1) or gp120 can stimulate a novel signaling pathway
that involves Ca.sup.2+-dependent release of glutamate (Sharma
& Vijayaraghavan, 2001, Proc Natl Acad Sci USA 98, 4148-4153)
in a process including activation of the CXCR4 receptor, an
autocrine/paracrine TNF-.alpha.-dependent signaling, and
prostaglandin (Bezzi et al., 2001, Nat. Neurosci. 4, 702-710).
These results suggest that HIV-1, gp120, and other neuropathogenic
agents can alter specific signaling pathways in astrocytes in a way
that may impair important physiological functions of these cells in
neuronal signal transmission and response to brain injury.
3. SUMMARY OF THE INVENTION
[0014] The present invention relates to Astrocyte Modulated Genes
(AMGs). AMGs are genes whose expressions are modulated in human
astrocytes grown in primary cell culture following the exposure of
these cells to either the human immunodeficiency virus HIV-1 or to
the HIV-1 protein gp120. AMGs comprise Astrocyte Enhanced Genes
(AEGs) and Astrocyte Suppressed Genes (ASGs).
[0015] The invention is based, at least in part, on the results of
a series of experiments in which a rapid subtraction hybridization
(RaSH) method (Jiang et al., 2000, Proc. Natl. Acad. Sci. USA 97,
12684-12689) was used to globally identify human genes whose
expression in astrocytes display temporal alterations following
HIV-1 infection. Using this methodology, it was discovered that 15
AEGs, including 13 known and 2 novel genes, and 10 ASGs, including
9 known and 1 novel gene, displayed altered levels of expression in
early passage human fetal astrocytes as a consequence of HIV-1
infection or treatment with gp120. These AMGs may be used as
markers of HIV infection and/or pathology, and, as such, may be
used as reporter genes in screening assays for identifying agents
that inhibit or prevent such infection and/or pathology.
[0016] The invention is further based on the discovery that
upregulation of one particular AMG, termed AEG-1, results in the
downregulation of GLT-1, thereby increasing extracellular levels of
the potentially excitotoxic neurotransmitter L-glutamate. In
various embodiments of the invention, inhibition of AEG-1 may be
used to facilitate glutamate transport and thereby prevent or
decrease its toxic effects. As AEG-1 is constitutively present in
astrocytes, inhibition of AEG-1 may be useful in preventing or
decreasing glutamate toxicity in a variety of contexts, not limited
to HIV infection, but including other pathologies as well, such as
cerebral infarction and/or ischemia, Alzheimer's Disease, epilepsy,
and amyotrophic lateral sclerosis. AEG-1 may be inhibited, for
example, by antisense molecules or antagonists directed at the
expressed protein. Such antagonists may be identified using
screening assays which monitor AEG-1 levels, GLT-1 levels, and/or
glutamate transport capability.
3.1. Definitions
[0017] As used herein, the term "nucleic acid molecule" includes
both DNA and RNA and, unless otherwise specified, includes both
double-stranded and single-stranded nucleic acids. Also included
are molecules comprising both DNA and RNA, either DNA/RNA
heteroduplexes, also known as DNA/RNA hybrids, or chimeric
molecules containing both DNA and RNA in the same strand. Reference
to a nucleic acid sequence can also include modified bases as long
as the modification does not significantly interfere either with
binding of a ligand such as a protein by the nucleic acid or
Watson-Crick base pairing between two complementary nucleic acid
molecules.
[0018] As used herein, the term "gene" refers to a DNA molecule
that either directly or indirectly encodes a nucleic acid or
protein product that has a defined biological activity.
[0019] As used herein, a specific, non-limiting example of
stringent conditions for detecting hybridization of nucleic acid
molecules are as set forth in "Current Protocols in Molecular
Biology", Volume I. Ausubel et al., eds. John Wiley: New York N.Y.,
pp. 2.10.1-2.10.16, first published in 1989 but with annual
updating, wherein maximum hybridization specificity for DNA samples
immobilized on nitrocellulose filters may be achieved through the
use of repeated washings of a hybridized filter in a solution
comprising 0.1-2.times.SSC (15-30 mM NaCl, 1.5-3 mM sodium citrate,
pH 7.0) and 0.1% SDS (sodium dodecylsulfate) at temperatures of
65-68.degree. C. or greater. For DNA samples immobilized on nylon
filters, a stringent hybridization washing solution may be
comprised of 40 mM NaPO.sub.4, pH 7.2, 1-2% SDS and 1 mM EDTA.
Again, a washing temperature of at least 65-68.degree. C. is
recommended, but the optimal temperature required for a truly
stringent wash will depend on the length of the nucleic acid probe,
its GC content, the concentration of monovalent cations and the
percentage of formamide, if any, that was contained in the
hybridization solution. Stringent hybridizations are designed to
identify molecules with 80% identity or preferably 90% identity or
more preferably 95% identity over lengths of at least 15
nucleotides and preferably at least 50 nucleotides.
[0020] An Astrocyte Modulated Gene, or "AMG", as the term is used
herein, is a gene the expression of which is modified in astrocytes
as a result of HIV-1 infection and/or exposure to HIV-1 gp120
protein. AMGs comprise Astrocyte Enhanced Genes (AEGs), whose
expression levels are upregulated following exposure of cultured
human fetal astrocytes to HIV-1 and/or to the HIV-1 gp120 protein,
and Astrocyte Suppressed Genes (ASGs), whose expression levels are
down-regulated following exposure of cultured human fetal
astrocytes to HIV-1 and/or to the HIV-1 gp120 protein. AMGs also
comprise genes whose pattern of expression, e.g. the timing of
expression, is changed following exposure of cultured human fetal
astrocytes to HIV-1 and/or to the HIV-1 gp120 protein. Increased
expression is associated with higher levels of mRNA and/or protein,
while reduced expression is associated with lower levels of mRNA
and/or protein.
[0021] Examples of AEGs include: AEG-2 (G-binding protein), AEG-3
(GA17 protein) (Ryo et al., 2000, AIDS Res. Hum. Retroviruses 16,
995-1005), AEG-4 (unr/NRU) (Jeffers et al., 1990, Nucl. Acids. Res.
18, 4891-4899; Boussadia et al., 1993, Biochim. Biophys. Acta 1172,
64-72), AEG-5 (hGNT-IV-H) (Furukawa et al., 1999, J. Hum. Genet.
44, 397-401), AEG-6 (fibronectin), AEG-7 (human CTL2) (O'Regan et
al., 2000, Proc. Natl. Acad. Sci. USA 97, 1835-1840), AEG-8 (acidic
ribosomal phosphoprotein) (Rich & Stietz, 1987, Mol. Cell.
Biol. 7, 4065-4074), AEG-9 (calnexin) (Honore et al., 1994,
Electrophoresis 15, 482-490; Rubio & Wenthold, 1999, J.
Neurochem. 73, 942-948; Shi et al., 2001, Cell 105, 331-342),
AEG-10 (autotaxin) (Stracke et al., 1994, J. Biol. Chem. 267,
2524-2529; Kawagoe et al., 1997, Cancer Res. 57, 2516-25), AEG-12
(thymosin .beta.-4) (Gondo et al., 1987, J. Immunol. 139,
3840-3848), AEG-13 (human non-muscle .alpha.-actinin) (Youssoufian
et al., 1990, Am. J. Hum. Genet. 47, 62-72), AEG-14 (Schneider et
al., 1988, Cell 54, 787-793; Gonos, 1998, Ann. N.Y. Acad. Sci. 851,
466-469; Prieto et al., 1999, Brain Res. 816, 646-661) and AEG-15
(PGK-1) (Michelson et al., 1983, Proc. Natl. Acad. Sci. USA 80,
472-476; Tsukada et al., 1991, J. Gerontol. 46, B213-B216), and the
novel AEGs AEG-1 and AEG-11.
[0022] Examples of ASGs include: ASG-2 (human cDNA FLJ10705;
GenBank Acc. No. AK001567, ASG-3 (Platelet-endothelial cell
tetra-span antigen 3; CD151/PETA-3; Fitter et al., 1995, Blood 86,
1348-55; Yanez-Mo et al., 1998, J. Cell Biol. 141, 791-804), ASG-4
(guanine nucleotide-releasing factor; C3G; Schweighoffer et al,
1993, Oncogene 8, 1477-85; Tanaka et al, 1994, Proc. Natl. Acad.
Sci. USA 91, 3443-3447), ASG-5 (neuronatin; ASG-5; Dou &
Joseph, 1996, Brain Res. 723, 8-22; Usui et al, 1997, J. Mol.
Neurosci. 9, 55-60), ASG-6 (neuroendocrine differentiation factor;
CGI149; Wilson et al., 2001, J. Clin. Endocrinol. Metab. 86,
4504-11), ASG-7 (cysteine/glycine-rich protein 1; CSRP1; Liebhaber
et al. 1990, Nucleic Acids Res 18, 3871-3879), ASG-8 (MLL5;
Emerling et al, 2002, Oncogene 21, 4849-54), ASG-9 (human
mitochondrion encoding RNA), ASG-10 (signal recognition particle 9
kD; SRP9KD; (Lutcke, 1995, Eur. J. Biochem. 228, 531-550), and the
novel ASG ASG-1.
4. DESCRIPTION OF THE FIGURES
[0023] FIG. 1. Schematic representation of the RaSH approach to the
identification of AEGs as applied to HIV-1 infected early passage
human fetal astrocytes. For this scheme tester (HIV-1 infected, 6
h, 12 h, and 24 h, and 3 d and 7 d) and driver (control uninfected,
6 h, 12 h, 24 h, and 3 d and 7 d) early passage human fetal
astrocytes libraries were constructed followed by digestion of only
the tester library with XhoI. After hybridization, differentially
expressed sequences are cloned into XhoI-digested vectors,
resulting in a subtracted cDNA library enriched for AEGs displaying
reduced expression in human fetal astrocytes as a function of HIV-1
infection.
[0024] FIG. 2. Schematic representation of the RaSH approach to the
identification of ASGs as applied to HIV-1 infected early passage
human fetal astrocytes. For this scheme tester (control
(mock-infected), 6 h, 12 h, and 24 h, and 3 d and 7 d) and driver
(HIV-1-infected, 6 h, 12 h, 24 h, and 3 d and 7 d) early passage
human fetal astrocytes libraries were constructed followed by
digestion of only the tester library with XhoI. After
hybridization, differentially expressed sequences are cloned into
XhoI-digested vectors, resulting in a subtracted cDNA library
enriched for ASGs displaying elevated expression in human fetal
astrocytes as a function of HIV-1 infection.
[0025] FIG. 3A-B. Infection of human fetal astrocytes with HIV-1.
Astrocytes were infected with HIV-1 and tested for virus expression
as described in Materials and Methods. (A) HIV-1 expression at
various time points after infection. (B) Number of viable cells at
various time points after infection.
[0026] FIG. 4. Reverse Northern blot analysis of expressed sequence
tags upregulated by HIV-1 infection as identified by RaSH. Equal
quantities of PCR amplified products from random bacterial clones
of RaSH-derived libraries were loaded onto 1.2% agarose gels.
Samples were electrophoresed for 1 h under 100 V and transferred to
nylon membranes. The blots were hybridized with .sup.32P-labeled
putative AEGs cDNAs reverse transcribed RNA samples. Blots were
exposed for autoradiography. The lane numbers (1 to 10) indicate
the various upregulated ESTs, which were designated AEG-1 to
15.
[0027] FIG. 5. Reverse Northern blot analysis of expressed sequence
tags downregulated by HIV-1 infection as identified by RaSH. Equal
quantities of PCR amplified products from random bacterial clones
of RaSH-derived libraries were loaded onto 1.2% agarose gels.
Samples were electrophoresed for 1 h under 100 V and transferred to
nylon membranes. The blots were hybridized with .sup.32P-labeled
putative ASGs cDNAs reverse transcribed RNA samples. Blots were
exposed for autoradiography. The lane numbers (1 to 10) indicate
the various down-regulated ESTs, which were designated ASG-1 to
10.
[0028] FIG. 6. Confirmation of differential expression using
Northern blotting analysis of AEG-1 to AEG-5 displaying temporal
elevated expression following HIV-1 infection or treatment with gp
120. Early passage human fetal astrocytes were untreated (control),
infected with HIV-1 at a multiplicity of infection (MOI) of 1, or
treated with 1 nM gp120 for 6 h, 24 h, 3 d or 7 d, total RNA was
isolated and analyzed by Northern blotting. Membranes were probed
with the indicated radiolabeled [.sup.32P] AEG EST, identified by
RaSH, the blots were stripped and probed with a radiolabeled
[.sup.32P] gapdh cDNA probe. Expression was quantitated by
densitometric analysis.
[0029] FIG. 7. Confirmation of differential expression using
Northern blotting analysis of AEG-6 to AEG-10 displaying temporal
elevated expression following HIV-1 infection or treatment with
gp120. See "Materials and Methods" and FIG. 6 for experimental
details.
[0030] FIG. 8. Confirmation of differential expression using
Northern blotting analysis of AEG-11 to AEG-15 displaying temporal
elevated expression following HIV-1 infection or treatment with
gp120. See "Materials and Methods" and FIG. 6 for experimental
details.
[0031] FIG. 9. Confirmation of differential expression using
Northern blotting analysis of ASG-1 to ASG-4 displaying temporal
elevated expression following HIV-1 infection or treatment with
gp120. Early passage human fetal astrocytes were untreated
(control), infected with HIV-1 at a multiplicity of infection (MOI)
of 1, or treated with 1 nM gp120 for 6 h, 24 h, 3 d or 7 d, total
RNA was isolated and analyzed by Northern blotting. Membranes were
probed with the indicated radiolabeled [.sup.32P] ASG EST,
identified by RaSH, the blots were stripped and probed with a
radiolabeled [.sup.32P] gapdh cDNA probe. Expression was
quantitated by densitometric analysis.
[0032] FIG. 10. Confirmation of differential expression using
Northern blotting analysis of ASG-5 to ASG-7 displaying temporal
elevated expression following HIV-1 infection or treatment with
gp120. See "Materials and Methods" and FIG. 6 for experimental
details.
[0033] FIG. 11. Confirmation of differential expression using
Northern blotting analysis of ASG-8 to ASG-10 displaying temporal
elevated expression following HIV-1 infection or treatment with
gp120. See "Materials and Methods" and FIG. 6 for experimental
details.
[0034] FIG. 12. Temporal modulation of ASG expression in normal
human fetal astrocytes as a consequence of infection with HIV-1 or
exposure to gp120. Data presented reflects values obtained after
scanning and normalizing (to gapdh) the Northern blots shown in
FIGS. 9-11. Fold-decline in expression was determined as described
in the legend to Table 1.
[0035] FIG. 13A-B. Expression of AEG-6 and AEG-13 proteins in HIV-1
infected and control astrocytes as determined by Western blotting.
Astrocytes were infected with HIV-1 at a MOI of 1 or equal to 1 nM
gp120, and treated and untreated cells were cultured and sampled at
the designated times for immunoblot analysis as described in
"Materials and Methods". Asterisks indicate a double amount of cell
lysate loaded per lane. Panel A and Panel B represent studies using
two independently derived early passage human fetal astrocyte
cultures.
[0036] FIG. 14. Nucleic acid sequence of the AEG-1 cDNA (SEQ ID
NO:1).
[0037] FIG. 15. Amino acid sequence of the AEG-1 protein (SEQ ID
NO:2).
[0038] FIG. 16. Nucleic acid sequence of the AEG-11 DNA (SEQ ID
NO:3).
[0039] FIG. 17. Nucleic acid sequence of the ASG-1 DNA (SEQ ID
NO:4).
5. DETAILED DESCRIPTION OF THE INVENTION
[0040] The present invention relates to compositions and/or methods
which contain and/or utilize AMG nucleic acid molecules, proteins,
or related reagents. The nucleic acid molecules of the invention
may or may not comprise protein-encoding sequences. For clarity of
presentation, and not by way of limitation, the detailed
description is divided into the following subsections:
[0041] (i) AMG nucleic acids;
[0042] (ii) AMG proteins;
[0043] (iii) anti-AMG antibodies;
[0044] (iv) screening methods; and
[0045] (v) methods of diagnosis and treatment.
5.1. AMG Nucleic Acids
[0046] The present invention provides for nucleic acid molecules
corresponding to AMGs, which may comprise AEGs or ASGs. The nucleic
acid molecules of the invention include but are not limited to
"full-length" nucleic acid molecules which contain, in DNA or RNA
form, a complete protein coding sequence, in sense or antisense
orientation. The nucleic acid molecules of the invention also
include molecules which are not "full-length" but which represent,
in DNA or RNA form, a portion of an mRNA molecule, which may
constitute protein coding and/or untranslated regions in sense or
antisense orientation; such molecules may be useful as probes for
detecting RNA levels, as PCR primers or as antisense
inhibitors.
[0047] In particular non-limiting embodiments, the present
invention provides for nucleic acid molecules of the following
AEGs: AEG-2 (G-binding protein), AEG-3 (GA17 protein) (Ryo et al.,
2000, AIDS Res. Hum. Retroviruses 16, 995-1005), AEG-4 (unr/NRU)
(Jeffers et al., 1990, Nucl. Acids. Res. 18, 4891-4899; Boussadia
et al., 1993, Biochim. Biophys. Acta 1172, 64-72), AEG-5
(hGNT-IV-H) (Furukawa et al., 1999, J. Hum. Genet. 44, 397-401),
AEG-6 (fibronectin), AEG-7 (human CTL2) (O'Regan et al., 2000,
Proc. Natl. Acad. Sci. USA 97, 1835-1840), AEG-8 (acidic ribosomal
phosphoprotein) (Rich & Stietz, 1987, Mol. Cell. Biol. 7,
4065-4074), AEG-9 (calnexin) (Honore et al., 1994, Electrophoresis
15, 482-490; Rubio & Wenthold, 1999, J. Neurochem. 73, 942-948;
Shi et al., 2001, Cell 105, 331-342), AEG-10 (autotaxin) (Stracke
et al., 1994, J. Biol. Chem. 267, 2524-2529; Kawagoe et al., 1997,
Cancer Res. 57, 2516-25), AEG-12 (thymosin .beta.-4) (Gondo et al.,
1987, J. Immunol. 139, 3840-3848), AEG-13 (human non-muscle
.alpha.-actinin) (Youssoufian et al., 1990, Am. J. Hum. Genet. 47,
62-72), AEG-14 (Schneider et al., 1988, Cell 54, 787-793; Gonos,
1998, Ann. N. Y Acad. Sci. 851, 466-469; Prieto et al., 1999, Brain
Res. 816, 646-661) and AEG-15 (PGK-1) (Michelson et al., 1983,
Proc. Natl. Acad. Sci. USA 80, 472-476; Tsukada et al., 1991, J.
Gerontol. 46, B213-B216), and the novel AEGs AEG-1 and AEG-11. The
present invention further provides for nucleic acid molecules which
hybridize to these nucleic acid molecules under stringent
conditions, having lengths of at least 15 nucleotides and
preferably at least 50 nucleotides.
[0048] In other non-limiting embodiments, the present invention
provides for nucleic acid molecules encoding the novel AEGs AEG-1
and AEG-11. For example, the present invention provides for nucleic
acid molecules having the sequence set forth as SEQ ID NO:1 or as
SEQ ID NO:3, as depicted in FIG. 14 and FIG. 16, respectively, or
their complementary strands. The present invention further provides
for nucleic acid molecules that are between 15 and 500 nucleotides
in length, preferably between 50 and 1000 nucleotides in length,
and more preferably between 1000 and 10,000 nucleotides in length,
which hybridize to a molecule having SEQ ID NO: 1 or SEQ ID NO:3 or
their complementary strands under stringent conditions. The present
invention further provides for nucleic acid molecules which encode
a protein having an amino acid sequence as set forth as SEQ ID NO:2
and depicted in FIG. 15, their complementary strands, and nucleic
acid molecules which hybridize under stringent conditions to their
sense or antisense strands. No specific protein product has yet
been identified for the AEG-11 gene, consistent with a cellular
function for the RNA product of this gene.
[0049] In particular non-limiting embodiments, the present
invention also provides for nucleic acid molecules of the following
ASGs: ASG-2 (human cDNA FLJ10705; GenBank Acc. No. AK001567, ASG-3
(Platelet-endothelial cell tetra-span antigen 3; CD151/PETA-3;
Fitter et al., 1995, Blood 86, 1348-55; Yanez-Mo et al., 1998, J.
Cell Biol. 141, 791-804), ASG-4 (guanine nucleotide-releasing
factor; C3G; Schweighoffer et al., 1993, Oncogene 8, 1.477-85;
Tanaka et al., 1994, Proc. Natl. Acad. Sci. USA 91, 3443-3447),
ASG-5 (neuronatin; ASG-5, Dou & Joseph, 1996, Brain Res. 723,
8-22; Usui et al., 1997, J. Mol. Neurosci. 9, 55-60), ASG-6
(neuroendocrine differentiation factor; CGI149; Wilson et al.,
2001, J. Clin. Endocrinol. Metab. 86, 4504-11), ASG-7
(cysteine/glycine-rich protein 1; CSRP1; Liebhaber et al. 1990,
Nucleic Acids Res 18, 3871-3879), ASG-8 (MLL5; Emerling et al.,
2002, Oncogene 21, 4849-54), ASG-9 (human mitochondrion encoding
RNA), ASG-10 (signal recognition particle 9 kD; SRP9KD; (Lutcke,
1995, Eur. J. Biochem. 228, 531-550), and the novel ASG ASG-1. The
present invention further provides for nucleic acid molecules which
hybridize to these nucleic acid molecules under stringent
conditions, having lengths of at least 15 nucleotides and
preferably at least 50 nucleotides.
[0050] In other non-limiting embodiments, the present invention
provides for nucleic acid molecules for the novel ASG ASG-1. For
example, the present invention provides for nucleic acid molecules
having the sequences set forth as SEQ ID NO:4, as depicted in FIG.
17, or its complementary strand. The present invention further
provides for nucleic acid molecules that are between 15 and 500
nucleotides in length, preferably between 50 and 1000 nucleotides
in length, and more preferably between 1000 and 10,000 nucleotides
in length, which hybridize to molecules having SEQ ID NO:4 or its
complementary strand under stringent conditions. The present
invention further provides for nucleic acid molecules which encode
the protein products of the nucleic acid sequence set forth as SEQ
ID NO:4, its complementary strand, and nucleic acid molecules which
hybridize under stringent conditions to its sense or antisense
strands.
[0051] Any aforesaid nucleic acid molecule may be linked to a
heterologous nucleic acid, as discussed, for example, below.
[0052] For some purposes, an AMG may be engineered such that it is
in an "expressible form." An "expressible form" is one in which an
AMG is linked to one or more elements necessary or desirable for
transcription and/or translation. For example, the AMG nucleic acid
may be operatively linked to a suitable promoter element in an
expression cassette which may further comprise a transcription
initiation and termination site, nucleic acid encoding a nuclear
localization sequence, a ribosome binding site, a polyadenylation
site, and/or a mRNA stabilizing sequence, etc.
[0053] Examples of suitable promoter elements, include, but are not
limited to, the cytomegalovirus immediate early promoter, the Rous
sarcoma virus long terminal repeat promoter, the human elongation
factor-1.alpha. promoter, the human ubiquitin c promoter, etc. It
may be desirable, in certain embodiments of the invention, to use a
regulatable promoter. Non-limiting examples of regulatable
promoters include the murine mammary tumor virus promoter
(inducible with dexamethasone), commercially-available steroid- or
tetracycline-responsive promoters, or ecdysone-inducible promoters,
etc. It may further be desirable, in certain embodiments of the
invention, to use astrocyte-specific promoters. Non-limiting
examples of astrocyte-specific promoters include the specific glial
fibrillary acidic protein (GFAP) promoter, the murine
cytomegalovirus immediate-early (MCMV-IE) gene promoter, and the
alpha tubulin promoter. Other suitable constitutive, regulatable,
or cell- or tissue-specific promoter systems are known to those of
ordinary skill in the art.
[0054] A nucleic acid molecule of the invention, whether or not it
is to be expressed as a protein, may be inserted into a suitable
vector for duplication purposes. Suitable vectors include but are
not limited to plasmids, cosmids, phages, phagemids, artificial
chromosomes, replicons, and various virus-based vector systems
known in the art.
[0055] Where an AMG protein or peptide is to be expressed, suitable
expression vectors include virus-based vectors and non-virus based
DNA or RNA delivery systems. Examples of appropriate virus-based
gene transfer vectors include, but are not limited to, those
derived from retroviruses, for example Moloney murine
leukemia-virus based vectors such as LX, LNSX, LNCX or LXSN (Miller
and Rosman, 1989, Biotechniques 7, 980-989); lentiviruses, for
example human immunodeficiency virus ("HIV"), feline leukemia virus
("FIV") or equine infectious anemia virus ("EIAV")-based vectors
(Case et al., 1999, Proc. Natl. Acad. Sci. USA 96, 2988-2993;
Curran et al., 2000, Molecular Ther. 1, 31-38; Olsen, 1998, Gene
Ther. 5, 1481-1487); adenoviruses (Zhang, 1999, Cancer Gene Ther.
6, 113-138; Connelly, 1999, Curr. Opin. Mol. Ther. 1, 565-572), for
example Ad5/CMV-based E1-deleted vectors (Li et al., 1993, Human
Gene Ther. 4, 403-409); adeno-associated viruses, for example
pSub201-based AAV2-derived vectors (Walsh et al., 1992, Proc. Natl.
Acad. Sci. U.S.A. 89, 7257-7261); herpes simplex viruses, for
example vectors based on HSV-1 (Geller & Freese, 1990, Proc.
Natl. Acad. Sci. U.S.A. 87, 1149-1153); baculoviruses, for example
AcMNPV-based vectors (Boyce & Bucher, 1996, Proc. Natl. Acad.
Sci. U.S.A. 93, 2348-2352); SV40, for example SVluc (Strayer &
Milano, 1996, Gene Ther. 3, 581-587); Epstein-Barr viruses, for
example EBV-based replicon vectors (Hambor et al., 1988, Proc.
Natl. Acad. Sci. U.S.A. 85, 4010-4014); alphaviruses, for example
Semliki Forest virus- or Sindbis virus-based vectors (Polo et al.,
1999, Proc. Natl. Acad. Sci. U.S.A. 96, 4598-4603); vaccinia
viruses, for example modified vaccinia virus (MVA)-based vectors
(Sutter & Moss, 1992, Proc. Natl. Acad. Sci. U.S.A. 89,
10847-10851) or any other class of viruses that can efficiently
transduce human tumor cells and that can accommodate the nucleic
acid sequences required for therapeutic efficacy.
[0056] Non-limiting examples of non-virus-based delivery systems
that may be used according to the invention include, but are not
limited to, so-called naked nucleic acids (Wolff et al., 1990,
Science 247, 1465-1468), nucleic acids encapsulated in liposomes
(Nicolau et al., 1987, Methods in Enzymology 149, 157-176), nucleic
acid/lipid complexes (Legendre & Szoka, 1992, Pharmaceutical
Research 9, 1235-1242), and nucleic acid/protein complexes (Wu
& Wu, 1991, Biother. 3, 87-95).
[0057] AMGs may also be produced using nucleic acid contained in
plasmids, such as pCEP4 (Invitrogen, San Diego, Calif.), pMAMneo
(Clontech, Palo Alto, Calif.; see below), pcDNA3.1 (Invitrogen, San
Diego, Calif.), etc. Vectors useful in expressing AMG-1 in
bacterial systems include but are not limited to the GST vector
(Amersham) and the chitin binding domain vector (TYB-12) (New
England Biolabs).
[0058] In a preferred embodiment, an AMG vector comprises the AEG-1
gene of SEQ ID NO:1 operatively linked to a heterologous regulatory
element and a polyadenylation signal, both of which are active in
mammalian cells. The resulting expression cassette may be contained
within a plasmid or a virus-based vector. In preferred embodiments,
the expression cassette is contained within an adenovirus vector
derived from human adenovirus type 2 or 5, or within an
adeno-associated virus (AAV) vector derived from human AAV2.
5.2. AMG Proteins
[0059] The present invention provides for AMG proteins. These
include, but are not limited to, the following AEG proteins: AEG-2
(G-binding protein), AEG-3 (GA17 protein) (Ryo et al., 2000, AIDS
Res. Hum. Retroviruses 16, 995-1005), AEG-4 (unr/NRU) (Jeffers et
al., 1990, Nucl. Acids. Res. 18, 4891-4899; Boussadia et al., 1993,
Biochim. Biophys. Acta 1172, 64-72), AEG-5 (hGNT-IV-H) (Furukawa et
al., 1999, J. Hum. Genet. 44, 397-401), AEG-6 (fibronectin), AEG-7
(human CTL2) (O'Regan et al., 2000, Proc. Natl. Acad. Sci. USA 97,
1835-1840), AEG-8 (acidic ribosomal phosphoprotein) (Rich &
Stietz, 1987, Mol. Cell. Biol. 7, 4065-4074), AEG-9 (calnexin)
(Honore et al., 1994, Electrophoresis 15, 482-490; Rubio &
Wenthold, 1999, J. Neurochem. 73, 942-948; Shi et al., 2001, Cell
105, 331-342), AEG-10 (autotaxin) (Stracke et al., 1994, J. Biol.
Chem. 267, 2524-2529; Kawagoe et al., 1997, Cancer Res. 57,
2516-25), AEG-12 (thymosin .beta.-4) (Gondo et al., 1987, J.
Immunol. 139, 3840-3848), AEG-13 (human non-muscle .alpha.-actinin)
(Youssoufian et al., 1990, Am. J. Hum. Genet. 47, 62-72), AEG-14
(Schneider et al., 1988, Cell 54, 787-793; Gonos, 1998, Ann. N.Y.
Acad. Sci. 851, 466-469; Prieto et al., 1999, Brain Res. 816,
646-661) and AEG-15 (PGK-1) (Michelson et al., 1983, Proc. Natl.
Acad. Sci. USA 80, 472-476; Tsukada et al., 1991, J. Gerontol. 46,
B213-B216). The invention also encompasses peptide fragments of
these AEG proteins comprising at least 20 amino acids which
cross-react with an immunoglobulin which specifically binds to the
corresponding full-length protein.
[0060] In specific, non-limiting embodiments, the present invention
provides for proteins encoded by nucleic acids AEG-1 and AEG-11. In
one particular embodiment, the invention provides for a protein
encoded by a nucleic acid sequence as set forth in SEQ ID NO:1
(depicted in FIG. 14), and specifically for a protein having an
amino acid sequence as set forth in SEQ ID NO:2 (FIG. 15). The
invention further encompasses peptide fragments of such proteins
comprising at least 20 amino acids which cross-react with an
immunoglobulin which specifically binds to a full-length AEG-1 or
AEG-11 protein.
[0061] The present invention also provides for ASG proteins,
including, but not limited to, the following: ASG-2 (human cDNA
FLJ10705; GenBank Acc. No. AK001567, ASG-3 (Platelet-endothelial
cell tetra-span antigen 3; CD151/PETA-3; Fitter et al., 1995, Blood
86, 1348-55; Yanez-Mo et al., 1998, J. Cell Biol. 141, 791-804),
ASG-4 (guanine nucleotide-releasing factor; C3G; Schweighoffer et
al., 1993, Oncogene 8, 1477-85; Tanaka et al., 1994, Proc. Natl.
Acad. Sci. USA 91, 3443-3447), ASG-5 (neuronatin; ASG-5; Dou &
Joseph, 1996, Brain Res. 723, 8-22; Usui et al., 1997, J. Mol.
Neurosci. 9, 55-60), ASG-6 (neuroendocrine differentiation factor;
CG1149; Wilson et al., 2001, J. Clin. Endocrinol. Metab. 86,
4504-11), ASG-7 (cysteine/glycine-rich protein 1; CSRP 1; Liebhaber
et al. 1990, Nucleic Acids Res 18, 3871-3879), ASG-8 (MLL5;
Emerling et al., 2002, Oncogene 21, 4849-54), ASG-9 (human
mitochondrion encoding RNA), ASG-10 (signal recognition particle 9
kD; SRP9KD; (Lutcke, 1995, Eur. J. Biochem. 228, 531-550), and the
novel ASG ASG-1. The invention also encompasses peptide fragments
of these ASG proteins comprising at least 20 amino acids which
cross-react with an immunoglobulin which specifically binds to the
corresponding full-length protein.
[0062] In specific, non-limiting embodiments, the present invention
provides for proteins encoded by ASG-1 nucleic acids. In one
particular embodiment, the invention provides for a protein encoded
by nucleic acid sequences as set forth in SEQ ID NO:4 (depicted in
FIG. 17). The invention further encompasses peptide fragments of
such proteins comprising at least 20 amino acids which cross-react
with an immunoglobulin which specifically binds to a full-length
ASG-1 protein.
[0063] The AMG proteins and peptides of the invention may be
prepared by standard techniques, including recombinant DNA-related
techniques and chemical synthesis, or by collection from natural
sources. For recombinant DNA expression, a non-limiting list of
suitable expression vectors is set forth in the preceding
section.
[0064] Expression systems which may be used to produce AMG proteins
include prokaryotic and eukaryotic expression systems, including
eukaryotic cells, bacteria, fungi (e.g. yeast), insects, etc.
Depending on the expression system used, nucleic acid may be
introduced by any standard technique, including transfection,
transduction, electroporation, bioballistics, microinjection,
etc.
5.3. Anti-AMG Antibodies
[0065] The present invention also provides for antibody molecules
which react with AMG proteins and peptides. In specific,
non-limiting examples, the invention provides for antibody
molecules (as defined infra) which bind specifically to proteins
having an amino acid sequence as set forth in SEQ ID NO:2 (AEG-1).
According to the invention, an AMG protein or peptide, derivatives
(e.g. a histidine-tagged protein), or analogs thereof, may be used
as an immunogen to generate antibodies. Such antibodies include,
but are not limited to, polyclonal, monoclonal, chimeric, single
chain, Fab fragments, and a Fab expression library.
[0066] Various procedures known in the art may be used for the
production of polyclonal antibodies which specifically bind to an
AMG protein or peptide. For the production of antibodies, various
host animals can be immunized by injection with the protein or
peptide, including but not limited to rabbits, mice, rats, goats,
etc. Various adjuvants may be used to increase the immunological
response, depending on the host species, and including but not
limited to Freund's (complete or incomplete) adjuvant, mineral gels
such as aluminum hydroxide, surface active substances such as
lysolecithin, pluronic polyols, polyanions, peptides, oil
emulsions, keyhole limpet hemocyanins, dinitrophenol, and
potentially useful human adjuvants such as BCG (Bacille
Calmette-Guerin) and Corynebacterium parvum.
[0067] For preparation of monoclonal antibodies directed toward an
AMG protein or polypeptide fragments thereof, any technique which
provides for the production of antibody molecules by continuous
cell lines in culture also may be used. Examples of such techniques
include the hybridoma technique originally developed by Kohler
& Milstein (1975, Nature 256, 495-497), as well as the trioma
technique, the human B-cell hybridoma technique (Kozbor et al.,
Immunology Today 1983; 4, 72-79), and the EBV hybridoma technique
to produce human monoclonal antibodies (Cole et al., in Monoclonal
Antibodies and Cancer Therapy, Alan R. Liss, Inc., 1985, pp.
77-96). In an additional embodiment of the invention, monoclonal
antibodies can be produced in germ-free animals utilizing the
technology disclosed in International Patent Application
PCT/US89/02545. According to the invention, human antibodies may be
used and can be obtained by using human hybridomas (Cote et al.,
1983, Proc. Natl. Acad. Sci. U.S.A. 80, 2026-2030) or by
transforming human B cells with EBV virus in vitro (Cole et al., in
Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, 1985, pp.
77-96). Further, according to the invention, techniques developed
for the production of "chimeric antibodies" (Morrison et al., 1984,
Proc. Nat. Acad. Sci. U.S.A. 81, 6851-6855; Neuberger et al., 1984,
Nature 312, 604-608; Takeda et al., 1985, Nature 314, 452-454) by
splicing the genes from a mouse antibody molecule specific for an
AMG protein or peptide together with genes from a human antibody
molecule of appropriate biological activity may be used; such
antibodies are within the scope of this invention.
[0068] According to the invention, techniques described for the
production of single chain antibodies (U.S. Pat. No. 4,946,778) may
be adapted to produce AMG protein or peptide-specific single chain
antibodies. An additional embodiment of the invention utilizes the
techniques described for the construction of Fab expression
libraries (Huse et al., 1989, Science 246, 1275-1281) to allow
rapid and easy identification of monoclonal Fab fragments with the
desired specificity.
[0069] Antibody fragments which contain the idiotype of the
molecule can be generated by known techniques. For example, such
fragments include but are not limited to: the F(ab').sub.2,
fragment which can be produced by pepsin digestion of the antibody
molecule; the Fab' fragments which can be generated by reducing the
disulfide bridges of the F(ab').sub.2, fragment, the Fab fragments
which can be generated by treating the antibody molecule with
papain and a reducing agent.
5.4. Screening Methods
[0070] The present invention provides for methods of screening for
agents that counteract the effect of HIV infection on astrocytes.
Such methods comprise culturing early passage human fetal
astrocytes, contacting the cultured astrocytes with HIV-1, further
contacting the cultured astrocytes with an agent that is a
candidate for counteracting the effects of HIV-1 infection on
astrocytes, and monitoring the effect of the candidate agent on the
expression of AEGs or ASGs, and comparing the level of expression
of the AEGs, or ASGs in the presence and absence of the candidate
agent, wherein an agent that counteracts the effects of HIV-1
expression in astrocytes is one that prevents the enhancement of
expression of an AEG, or prevents the suppression of expression of
an ASG.
[0071] According to such methods, the effect of a test agent on the
expression of an AMG is monitored. Such methods are typically
carried out in vitro using cultured astrocytes. An AMG is defined
by a modulated level of expression, either upregulation or
downregulation, or an altered temporal expression pattern,
associated with HIV-1 infection and/or exposure to HIV-gp120;
however, it may be found that other viruses or viral proteins may
produce similar reductions in AMGs originally defined by an
HIV-1/gp120 system, in which case such viruses or proteins may be
used in the screening methods of the invention. For example, it may
be preferable to use a crippled virus (e.g. incapable of proper
packaging) rather than a wild-type virus to lessen the risks of
infection.
[0072] According to the screening methods of the invention, a test
agent may be evaluated for its ability to either prevent or
decrease the enhancement of AEG expression associated with such
viral infection or viral protein exposure, or to prevent or
increase the reduction of ASG expression associated with such viral
infection or viral protein exposure. The test agent may be
introduced into an cell culture either prior to, concurrent with,
or subsequent to introduction of virus or viral protein. The effect
of the test agent on AMG expression may be monitored, for example,
by measuring RNA and/or protein levels, using techniques known in
the art. In specific, non-limiting embodiments of the invention,
the AEGs to be monitored are AEG-1 through AEG-15, the ASGs to be
monitored are ASG-1 through ASG-10. RNA levels of AEGs, or ASGs may
be determined by Northern analysis or PCR. Protein levels may be
determined by Western Blot Analysis using anti-AEG, or anti-ASG
polyclonal or monoclonal antibodies. Similarly, other AMGs
described herein may be monitored using similar methods.
[0073] Other screening methods of the invention are based on the
discovery that enhanced AEG-1 expression decreases glutamate
transport. Accordingly, the methods set forth above may be modified
such that instead of measuring AEG levels directly, they may be
measured indirectly, by determining the glutamate transport
activity of the culture. For example, the amount of glutamate
uptake of the astrocytes may be measured, for example, using
detectably labeled glutamate. Intracellular levels of glutamate or
the level of glutamate in the culture medium may be determined.
[0074] In related embodiments, the invention provides for screening
methods for the identification of agents which increase glutamate
transport. Such methods have the same steps as those intended to
identify agents for treating or preventing HAD whereby either the
expression level of AEG-1 or the glutamate transport activity is
measured. Agents which increase glutamate transport may be useful
in treating or preventing a variety of neurological conditions,
including HAD, cerebral ischemia, amyotrophic lateral sclerosis,
epilepsy and Alzheimer's disease.
[0075] In still other embodiments, the present invention provides
for cell cultures and transgenic animals which overexpress an AEG
or underexpress an ASG, thereby producing a model system for
diseases associated with astrocyte pathology.
[0076] In the case of AEGs, the AEG may be placed under the control
of a strong promoter. In particular non-limiting embodiments, the
promoter may be inducible. Examples of suitable promoters are set
forth in Section 5.1. The promoters are desirably active in
astrocytes. In specific non-limiting embodiments of the invention,
cells in culture or in a transgenic animal are engineered to
contain AEG-1 under the control of a heterologous promoter, so that
AEG-1 may be overexpressed and/or inducibly expressed. Such cells
or animals may be used as model systems for study in HAD or other
neurological conditions associated with AEG-1 overexpression, and
may be used to identify agents which may treat or prevent such
conditions.
[0077] In the case of ASGs, the expression of the ASG may be
reduced in either the cell culture or transgenic animal model
systems using an antisense construct or a ribozyme directed against
the ASG mRNA sequence. Alternatively, triplex technology may also
be used to reduce transcription of the ASG gene. ASG knock-out or
knock-out/knock-in models may also be produced in which the ASG is
disrupted by insertion of heterologous DNA.
[0078] Such cells or animals may be used as model systems for study
in HAD or other neurological conditions associated with modulated
expression of AMGs, and may be used to identify agents which may
treat or prevent such conditions.
5.5. Methods of Diagnosis and Treatment
[0079] The present invention further provides for methods of
diagnosis and treatment of a disorder associated with a change in
expression of one or more AMG. In one set of embodiments, the
invention provides for methods of diagnosing HIV-1 infection and/or
HAD in a subject comprising determining whether the level of an AEG
is elevated in an astrocyte of the subject. The level of expression
may be determined directly, for example using a brain biopsy, or
indirectly, for example, by determining the level of an AEG protein
or a metabolic product thereof in a body fluid of the subject, such
as in serum or in cerebrospinal fluid. Metabolic imaging techniques
analogous to PET scan may also be employed. An indirect metabolic
product may also be used to detect increased AEG expression; for
example, since AEG-1 decreases glutamate transport, an increase in
cerebrospinal fluid levels of glutamate is consistent with enhanced
AEG-1 expression.
[0080] In a second set of embodiments, the invention provides for
methods of diagnosing HIV-1 infection and/or HAD in a subject
comprising determining whether the level of an ASG gene is reduced
in an astrocyte of the subject. The level of expression may be
determined directly, for example using a brain biopsy, or
indirectly, for example, by determining the level of an ASG protein
or a metabolic product thereof in a body fluid of the subject, such
as in serum or in cerebrospinal fluid. Metabolic imaging techniques
analogous to PET scan may also be employed. An indirect metabolic
product may also be used to detect decreased ASG expression.
[0081] Further, in view of the association between increased AEG-1
expression and decreased glutamate transport, the present invention
provides for methods of decreasing extracellular glutamate levels,
and hence treating or preventing conditions associated with
neuronal glutamate toxicity, comprising antagonizing the effects of
AEG-1. In one non-limiting embodiment, AEG-1 may be antagonized by
inhibiting its expression using antisense RNA molecules
complementary to all or a portion of AEG-1 nucleic acid, for
instance having the sequence set forth as SEQ ID NO:1. The design
of antisense molecules, including oligonucleotides, is known in the
art. Conditions which may be treated or prevented in this manner
include but are not limited to cerebral ischemia, amyotrophic
lateral sclerosis, and Alzheimer's disease. The present invention
further provides for the treatment or prevention of HAD by
antagonizing the effects of AEG-1, for example using antisense
molecules as set forth supra.
[0082] The present invention further provides for the treatment or
prevention of HAD by overexpression of ASGs, for example by using
vectors of various forms, both virus-based and non-virus-based, to
introduce additional expressible copies of ASGs into astrocytes.
Examples of such vectors for the expression of ASGs have been set
forth supra.
6. EXAMPLE
6.1. Materials and Methods
[0083] Human fetal astrocytes, other cells and cell culture
conditions. Fetal astrocytes were isolated from second trimester
(gestational age 16-19 wk) human fetal brains obtained from
elective abortions in fill compliance with NIH guidelines, as
previously described (Bencheikh et al., 1999, J. Neurovirol. 5,
115-124; Canki et al., 2001, J. Virol. 75, 7925-7933). Highly
homogenous preparations of astrocytes were obtained using
high-density culture conditions in the absence of growth factors in
F12 Dulbecco's Modified Eagle Medium (GIBCO-BRL, Gaithersburg, Md.)
containing 10% fetal bovine serum (FBS), penicillin, streptomycin,
and gentamycin. Cells were maintained in this medium at
2-5.times.10.sup.4 cells/cm.sup.2 and subcultured weekly up to six
times. For each experiment a single batch of astrocytes of similar
gestational age and passage was used. Cultures were regularly
monitored for expression of the astrocytic marker glial fibrillary
acidic protein (GFAP) and either HAM56 or CD68 to identify cells of
monocyte/macrophage lineage. Only cultures that
contained.gtoreq.99% GFAP positive astrocytes and rare or no
detectable HAM56- or CD68-positive cells were used in these
experiments (Canki et al., 2001, J. Virol. 75, 7925-7933). Other
cells used in this study were the human embryonal-kidney epithelial
cell line 293T (Graham et al., 1977, J. Gen. Virol. 36, 59-72),
used for HIV-1 propagation, and MAGI cells, a derivative of HeLa
carrying the .beta.-gal gene under the control of HIV-1 LTR and
expressing HIV-1 receptors (Kimpton & Emerman, 1992, J. Virol.
66, 2232-2239), used as indicator cells for HIV-1 titration. Both
cell lines were cultured in 90% DMEM/10% FBS supplemented with
antibiotics and, for MAGI cells, 0.2 mg/ml G-418. Culture media and
cells were tested for mycoplasma contamination using the Mycoplasma
PCR ELISA kit (Roche Molecular Biochemicals, Indianapolis, Ind.)
and found to be negative.
[0084] HIV-1 propagation. The HIV-1 strain used in this work was
NL4-3, a prototypical X4-tropic laboratory clone of HIV-1 that
expresses all known HIV-1 proteins (Adachi et al., 1986, J. Virol.
59, 284-91). Virus propagation was initiated by transfection of 15
.mu.g of NL4-3 DNA into 1.5.times.10.sup.6 293T cells as previously
described (Bencheikh et al., 1999, J. Neurovirol. 5, 115-124).
Culture supernatants were harvested 72 h after transfection,
filtered through a 0.45 .mu.m Millipore filters, and stored at
-80.degree. C. until use. Cell-free viral stock was tested for
HIV-1 p24 core antigen content by ELISA using HIV-1 Ag kit
according to the manufacturer's instructions (Coulter, Hialeah,
Fla.) and for titers of infectious virus by multinuclear activation
of a .beta.-galactosidase indicator (MAGI) assay (Kimpton &
Emerman, 1992, J. Virol. 66, 2232-2239). Culture supernatants
contained 1-2 .mu.g/ml of viral p24 protein and 1-2.times.10.sup.6
infectious units (I.U.) per ml. In our experience, a MOI of one for
CD4-positive T cells is approximately 1 pg of viral p24 per cell
(Dewhurst et al., 1987, J. Virol. 61, 3774-3782; Canki et al.,
2001, J. Virol. 75, 7925-7933)). Virus stocks were also tested for
mycoplasma contamination as described above and for endotoxin using
the E-TOXATE kit (Limulus Amebocyte Lysate, Sigma, St. Louis, Mo.),
and found to be negative in both tests.
[0085] HIV-1 infection of astrocytes with HIV-1 or exposure of the
cells to gp120, and preparation of samples for cellular RNA
analysis. Confluent cultures of human fetal astrocytes in 225
cm.sup.2 culture flasks were exposed to HIV-1 in 10 ml of medium at
1 pg p24 per cell for 2 h at 37.degree. C., washed 3 times in warm
PBS, and cultured in astrocyte culture medium as described. Control
astrocytes were treated as described above but without HIV-1. At 6
h, 12 h, 24 h, 3 d and 7 d after infection, culture supernatants
were removed, and control and infected cells were washed 3 times in
PBS and solubilized by addition of 10 ml of 4M guanidine
isothiocyanate directly to culture flasks. Cell lysates were stored
at -80.degree. C. until further use. To insure preparation of
sufficient amount of RNA for subsequent subtractive hybridization,
astrocyte cultures and HIV-1 infections were scaled up to
approximately 1.times.10.sup.8 cells per time point (infected or
control cells); the RNA yield was 5-10 .mu.g per 10.sup.6 cells.
Infection of astrocytes with HIV-1 was verified by testing the
levels of HIV-1 p24 antigen in culture supernatants by p24 ELISA as
described previously (Bencheikh et al., 1999, J. Neurovirol. 5,
115-124; Canki et al., 2001, J. Virol. 75, 7925-7933). gp120 used
for these experiments was a full-length, glycosylated protein from
HIV-1.sub.MN produced from baculovirus vector and purified by
ImmunoDiagnostics, and provided through the AIDS Research and
Reference Reagent Program (Rockville, Md.). For gp120 treatment of
astrocytes, large-scale cultures of cells prepared as described
above were treated with gp120 at 1 nM in 10 ml medium for 2 h, and
cells were washed, cultured, and extracted for RNA isolation as
described for HIV-1 infection.
[0086] RNA isolation and Northern blot analysis. Uninfected, HIV-1
infected and exposed gp120 astrocytes were treated with
4M-guanidiniurn and total RNA was isolated by the
guanidinium/phenol procedure and analyzed by Northern blotting as
described previously (Jiang & Fisher, 1993, Mol. Cell.
Different. 1, 285-299; Kang et al., 1998, Proc. Natl. Acad. Sci.
U.S.A. 95, 13788-13793; Kang et al., 2001, Gene 267, 233-242).
Northern blots were quantitated by densitometric analysis using a
Molecular Dynamics densitometer (Sunnyvale, Calif.). Relative
expression of the different AEGs or ASGs versus GAPDH expression
was determined at different time points for HIV-1 infected
(H-AEG/H-GAPDH or H-ASG/H-GAPDH), gp120-treated (G-AEG/G-GAPDH or
G-ASG/G-GAPDH) and control uninfected cultures (C-AEG/C-GAPDH or
C-ASG/C-GAPDH). Relative fold-change in expression of each AMG at 6
h, 12 h, 24 h, 3 d and 7 d was then determined for each condition
by dividing H-AEG/H-GAPDH or H-ASG/H-GAPDH by C-ASG/C-GAPDH or
C-ASG/C-GAPDH, respectively, to generate fold HIV modulation; or by
dividing G-AEG/G-GAPDH or G-ASG/G-GAPDH by C-AEG/C-GAPDH or
C-ASG/C-GAPDH to generate fold gp120 modulation. Poly(A) RNA was
purified using Oligo(dT) cellulose columns (GIBCO BRL).
[0087] AEG Protein Analysis by Immunoblotting. Analysis of AEG
protein products in astrocytes was determined by immunoblotting.
Astrocytes were infected with HIV-1 at an M.O.I. of 1, washed, and
cultured in parallel with uninfected controls as described above.
At the designated times, cells were counted and lysed in a buffer
containing 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate, 5 mM
iodoacetamide, 0.2 U/ml phenylmethylsulfonyl fluoride; cell lysates
corresponding to equivalent number of cells were resolved by
SDS-PAGE on 4-15% polyacrylamide ready gels (Bio-Rad, Hercules,
Calif.) and transferred onto a 0.2 .mu.m Trans-Blot nitrocellulose
membrane (Bio-Rad). The membranes were incubated in 5% (w/v) skim
milk in T-PBS (0.1% polyoxyethyline-sorbitan monolaurate in
phosphate buffered saline) and then stained with the indicated
primary antibodies followed by horseradish peroxidase-conjugated
second antibody. Protein bands were visualized on x-ray film after
luminescence reaction using an ECL kit (Amersham, Arlington, Ill.).
Samples were standardized by their .alpha.-tubulin content prior to
final evaluations. Antibodies used were: rabbit polyclonal
anti-fibronectin antibody (Abcam, Cambridge, UK), monoclonal
anti-.alpha.-actinin (Sigma), and monoclonal anti-.beta.-tubulin
(Sigma).
[0088] Primer design for RaSH procedure. The sequences of
oligonucleotides that were used are as follows: XDPN-18
CTGATCACTCGAGAGATC (SEQ ID NO:5), XDPN-14 CTGATCACTCGAGA (SEQ ID
NO:6), XDPN-12 GATCTCTCGAGT (SEQ ID NO:7). The adapters formed from
the two sets of oligonucleotides contained an XhoI recognition
site.
[0089] Preparation of PCR-based cDNA libraries used in RaSH
procedure. To clone AEG cDNAs expressed at elevated levels in early
passage human fetal astrocytes, 1 .mu.g of poly(A) RNA from
temporally spaced (6 h, 12 h, 24 h, and 3 d and 7 d) uninfected
astrocytes (driver) or temporally spaced (6 h, 12 h, 24 h, and 3 d
and 7 d) HIV-1 infected astrocytes (tester) prepared as described
above was used for double-stranded cDNA synthesis using standard
protocols (Gubler & Hoffman, 1983, Gene 25, 263-269). To clone
ASG cDNAs expressed at reduced levels in early passage human fetal
astrocytes, 1 .mu.g of poly(A) RNA from temporally spaced (6 h, 12
h, 24 h, and 3 d and 7 d) HIV-1-infected astrocytes (driver) or
temporally spaced (6 h, 12 h, 24 h, and 3 d and 7 d) mock-infected
astrocytes (tester) prepared as described above was used for
double-stranded cDNA synthesis using standard protocols (Gubler
& Hoffman, 1983, Gene 25, 263-269).
[0090] The resulting cDNAs were digested with DpnII (New England
Biolab, Beverly, Mass.) at 37.degree. C. for 3 h followed by
phenol/chloroform extraction and ethanol precipitation. The
digested cDNAs were mixed with primers XDPN-14/XDPN-12 (final
concentration 20 .mu.M) in 30 .mu.l of 1.times. ligation buffer
(GIBCO BRL), heated at 55.degree. C. for 1 min, and cooled down to
14.degree. C. within 1 h. After adding 3 .mu.l of T4 ligase (5
U/.mu.l) (GIBCO BRL) to the mixtures individually, ligation was
carried out at 14.degree. C. overnight. The mixtures were diluted
to 100 .mu.l with TE buffer (pH 7.0), and at least 40 .mu.l of the
mixtures were used for PCR amplification. The PCR mixtures were set
up as follows: 1 .mu.l of the cDNA mixture, 10 .mu.l 10.times.PCR
buffer, 1 mM MgCl.sub.2, 0.4 mM dNTPs, 1 .mu.M XDPN-18, and 1 U Taq
polymerase (GIBCO BRL). The parameters for PCR were one cycle for 5
min at 72.degree. C. followed by 25 cycles for 1 min at 94.degree.
C., 1 min at 55.degree. C., 1 min at 72.degree. C. preceded by one
cycle for 3 min at 72.degree. C. The PCR products were pooled and
purified using Centricon columns (Amicon, Bedford, Mass.). Ten
.mu.g of the tester PCR products were digested with XhoI followed
by phenol/chloroform extraction and ethanol precipitation.
[0091] Subtraction hybridization and generation of subtracted
libraries. One hundred ng of the tester cDNA were mixed with 3
.mu.g of the driver cDNA in 10 .mu.l of a hybridization solution
(0.5 M NaCl, 50 mM Tris pH 7.5, 0.2% SDS, 40% formamide), and after
boiling for 5 min, incubated at 42.degree. C. for 48 h. The
hybridization mixture was phenol/chloroform extracted, ethanol
precipitated, and dissolved in 20 .mu.l of TE buffer. One .mu.l of
the mixture was ligated with 1 .mu.g of XhoI-digested, CIP-treated
pCRII plasmids, overnight at 14.degree. C., and transformed into
Shot-1 bacteria.
[0092] Colony screening. Bacterial colonies were randomly picked
and PCR amplified. The PCR products were blotted onto filters and
reverse Northern blotting was performed to identify cDNAs
displaying differential expression in HIV-1 infected versus
uninfected early passage human fetal astrocytes (Kang et al., 1998,
Proc. Natl. Acad. Sci. U.S.A. 95, 13788-13793; Huang et al., 1999,
Gene 236, 125-131; Jiang et al., 2000, Proc. Natl. Acad. Sci. USA.
97, 12684-12689). cDNAs displaying elevated expression in HIV-1
infected fetal astrocytes versus uninfected fetal astrocytes were
designated AEG with a clone number of AEG-1 to AEG-15, while cDNAs
displaying reduced expression in HIV-1 infected fetal astrocytes
versus uninfected fetal astrocytes were designated ASG with a clone
number of ASG-1 to ASG-10. Appropriate expression of the AMG clones
identified by reverse Northern blotting was confirmed by Northern
blotting. The sequences of these clones were determined using
automated cycle sequencing at the DNA facility of Columbia
University.
6.2. Results
[0093] Infection of human astrocytes with HIV-1 and cloning of the
AMGs using the RaSH approach. Human fetal astrocytes were cultured
and infected with HIV-1 (NL4-3 clone) as previously described
(Bencheikh et al., 1999, J. Neurovirol. 5, 115-124; Canki et al,
2001, J. Virol. 75, 7925-7933). Mock-infected cells were cultured
and handled similarly in parallel as a control. HIV-1 infection of
astrocytes alters gene expression and cell function (He et al.,
1997, Proc. Natl. Acad. Sci. U.S.A. 94, 3954-3959; Kort, 1998, AIDS
Res. Hum. Retroviruses 14, 1329-1339). To define the repertoire of
genes modified as a consequence of infection of early passage fetal
astrocytes with HIV-1, the efficient and rapid cloning approach
RaSH was used (Jiang et al., 2000, Proc. Natl. Acad. Sci. USA. 97,
12684-12689; Kang et al., 2001, Gene 267, 233-242; Simm et al.,
2001, Gene 269, 93-101). A schematic of this approach as applied to
this fetal astrocyte model and HIV-1 infection is shown in FIGS. 1
and 2. For the current study, human fetal astrocytes were cultured
and infected with HIV-1 as previously described (Bencheikh et al.,
1999, J. Neurovirol. 5, 115-124; Canki et al., 2001, J. Virol. 75,
7925-7933) and pooled RNAs (6 h, 12 h, 24 h, and 3 d and 7 d)
extracted from uninfected and HIV-1 infected early passage fetal
astrocytes cultured in parallel were used in RASH to identify
cellular genes displaying modulated expression (AMGs) as a function
of HIV-1 infection. HIV-1 infection of astrocytes was confirmed by
following the levels of HIV-1 p24 core antigen in culture
supernatants (FIG. 3A). Consistent with previous results from this
and other laboratories (Tornatore et al., 1991, J. Virol. 65,
6094-6100; Canki et al., 2001, J. Virol. 75, 7925-7933), HIV-1
production by these cells peaked 3-4 d after infection at about 600
pg p24/ml and it declined thereafter (FIG. 3A).
[0094] In general, the RaSH procedure followed that described for
identification of cellular genes displaying elevated expression in
astrocytes, as shown in FIG. 1. For the identification of cellular
genes displaying reduced expression in infected astrocytes, the
tester and driver libraries were reversed, as shown schematically
in FIG. 2. The cDNA libraries were prepared by synthesizing
double-stranded cDNAs, digesting the cDNAs into small fragments
with the restriction enzyme DpnII, ligating the fragments to
adapters and amplifying by PCR. The DpnII-based RaSH approach was
previously shown to generate fragments of at least 256 bp in size
(Jiang et al., 2000, Proc. Natl. Acad. Sci. USA 97, 12684-12689).
Subtraction hybridization was then performed by incubating the
tester and driver PCR fragments without further PCR
amplification.
[0095] Selection of subtracted cDNAs for the identification of AEGs
was achieved by matching the ends of the cDNA fragments to the ends
of the plasmid vectors during ligation and construction of
subtracted libraries (FIG. 1). Selection of subtracted cDNAs for
the identification of ASGs was achieved by ligation of the common
XhoI restriction sites at the termini of the cDNA fragments and the
plasmid vectors (FIG. 2). Initial screening of a large number of
clones representing differential expression of cellular genes was
accomplished by reverse Northern hybridization, followed by
Northern blotting to confirm true differential expression of genes
in infected versus control cells (Kang et al., 1998, Proc. Natl.
Acad. Sci. U.S.A. 95, 13788-13793; Huang et al., 1999, Gene 236,
125-131). Previous studies document a high degree of conformity
(.about.89%) between reverse Northern and Northern expression of
RaSH-derived ESTs (Jiang et al., 2000, Proc. Natl. Acad. Sci.
U.S.A. 97, 12684-12689; Simm et al., 2001, Gene 269, 93-101).
[0096] Using this approach, 15 distinct AEGs displaying enhanced
expression (FIG. 4) and 10 distinct ASGs displaying reduced
expression (FIG. 5) following HIV-1 infection were identified by
reverse Northern blotting. Of the AEGs, thirteen were previously
identified AEGs and two were unknown AEGs (AEG-1 and AEG-11) not
reported in current databases (Table 1). RaSH identified AEG-1
(novel) in four independent analyses and AEG-2 (G-binding protein)
(Beals et al., 1987, Proc. Natl. Acad. Sci. U.S.A. 84, 7886-7890)
in two independent analyses, whereas the remaining AEGs represented
single cloning events. Of the ASGs, nine were previously identified
ASGs and one was an unknown ASG (ASG-1). ASG-4, -6, -7, and -9 were
isolated in two independent RaSH analyses, whereas the remaining
ASGs represented single cloning events (Table 2). Since only about
20% of the DpnII RaSH subtracted library was screened, it is
estimated that this library may contain more than 75 distinct
differentially expressed AEGs and 50 or more distinct
differentially expressed ASGs.
[0097] Sequence analysis of the RaSH-derived ESTs revealed nine
previously identified ASGs and one unknown ASG, designated ASG-1
(novel), not reported in current databases (Table 1). Among the
known ASG products are proteins involved in cell movement and cell
differentiation (ASG-3, platelet-endothelial tetraspan antigen 3
(PETA-3); ASG-5, neuronatin; and ASG-6, a neuroendocrine
differentiation factor), as well as intracellular regulators of
signal transduction and gene expression (ASG-4, a
guanine-nucleotide releasing protein C3 G; ASG-7, cysteine/glycine
rich protein 1 (CSRP1); and ASG-10, signal recognition particle 9
(SRP9).
[0098] In contrast, the AEGs included a wider range of genes,
including AEG-2 (G-binding protein), AEG-3 (GA 17 protein) (Ryo et
al., 2000, AIDS Res. Hum. Retroviruses 16, 995-1005), AEG-4
(unr/NRU) (Jeffers et al., 1990, Nucleic Acids Res. 18, 4891-4899;
Boussadia et al., 1993, Biochim. Biophys. Acta 1172, 64-72), AEG-5
(hGNT-IV-H) (Furukawa et al., 1999, J. Hum. Genet. 44, 397-401),
AEG-6 (fibronectin), AEG-7 (human CTL2) (O'Regan et al., 2000,
Proc. Natl. Acad. Sci. U.S.A. 97, 1835-1840), AEG-8 (acidic
ribosomal phosphoprotein) (Rich & Stietz, 1987, Mol. Cell.
Biol. 7, 4065-4074), AEG-9 (calnexin) (Honore et al., 1994,
Electrophoresis 15, 482-490; Rubio & Wenthold, 1999, J.
Neurochem. 73, 942-948; Shi et al., 2001, Cell 105, 331-342),
AEG-10 (autotaxin) (Stracke et al., 1994, J. Biol. Chem. 267,
2524-2529; Kawagoe et al., 1997, Cancer Res. 57, 2516-2521), AEG-12
(thymosin .beta.-4) (Gondo et al., 1987, J. Immunol. 139,
3840-3848), AEG-13 (human non-muscle .alpha.-actinin) (Youssoufian
et al., 1990, Am. J. Hum. Genet. 47, 62-71), AEG-14 (Schneider et
al., 1988, Cell 54, 787-793; Gonos, 1998, Ann. N.Y. Acad. Sci. 851,
466-469; Prieto et al., 1999, Brain Res. 816, 646-661) and AEG-15
(PGK-1) (Michelson et al., 1983, Proc. Natl. Acad. Sci. USA 80,
472-476; Tsukada et al., 1991, J. Gerontol. 46, B213-B216).
[0099] Expression analysis and characterization of AMGs. The
differential expression of the RaSH-derived AMGs was confirmed by
Northern blot analyses, shown in FIGS. 6 through 8 for AEGs and
FIGS. 9 through 11 for the ASGs. The results of densitometry
analyses of ASG expression are shown in FIG. 12. RNAs were isolated
after 6 h, 12 h, 24 h, and 3 d and 7 d from uninfected, HIV-1
infected, and HIV-1 glycoprotein (gp120)-treated early passage
human fetal astrocytes. gp120 treatment was included in this
analysis because gp120 alone (without infection with intact HIV-1)
induces marked functional and gene-expression changes in human
astrocytes (Schneider-Schaulies et al., 1992; Benos et al., 1994b;
He et al., 1997;). RNA levels were quantitated by Northern
blotting, first by probing with a random-primed [.sup.32P]-labeled
ASG or ASG cDNAs, followed by stripping of the blot and reprobing
with a probe for the transcript of the human house-keeping enzyme,
glyceraldehyde-3-phosphate dehydrogenase (gapdh). After
autoradiography, relative hybridization intensity was determined by
densitometry comparisons of AEG/gadph or ASG/gadph for the various
temporal uninfected samples relative to HIV-1 infected or
gp120-treated normalized (AEG/gadph or ASG/gadph) RNA samples, as
described above in the Materials and Methods. The experiments and
analyses were repeated two to three times using different
preparations of astrocytes and virus or gp120 and qualitatively
similar results were observed.
[0100] Compared to their expression in control cells, an
.about.1.5- to .about.4.5-fold temporal increase in expression of
the various AEGs was apparent following HIV-1 infection and an
.about.1.5- to .about.3.2-fold temporal increase was seen following
gp120 treatment (Table 1; see below). The expression of specific
ASGs was reduced at defined time-points up to .about.5.1-fold by
HIV-1 infection and up to .about.4.2-fold following gp120 treatment
(Table 2 and FIG. 12).
[0101] In general, the temporal pattern and magnitude of enhanced
AEG expression was similar in cells infected with HIV-1 or treated
with gp120. However, expression of AEG-2 (G-binding protein), AEG-5
(hGNT-IV-H) (Furukawa et al., 1999), AEG-7 (human CTL2) (O'Regan et
al., 2000), AEG-8 (acidic ribosomal phosphoprotein) (Rich &
Stietz, 1987) and AEG-15 (PGK-1) (Michelson et al., 1983, Proc.
Natl. Acad. Sci. USA 80, 472-476; Tsukada et al., 1991, J.
Gerontol. 46, B213-B216) was elevated in fetal astrocyte cultures
more rapidly following HIV-1 infection than gp120 treatment (FIGS.
6 through 8). AEG-2 (G-binding protein), AEG-5 (hGNT-IV-H) and
AEG-7 (human CTL2) expression was elevated by 3 d post-infection
with HIV-1, whereas enhanced expression of these genes was not
apparent until 7 d after gp120 treatment. In the case of AEG-8
(acidic ribosomal phosphoprotein) and AEG-15 (PGK-1) increased
expression was apparent by 24 h following infection with HIV-1 and
by 3 d following gp120 treatment. Quantitative differences in
enhancement were also apparent with HIV-1 causing a greater
increase than gp120 in the case of AEG-2 (G-binding protein), AEG-3
(GA17 protein) (Ryo et al., 2000), AEG-7 (human CTL2), AEG-10
(autotaxin) (Stracke et al., 1994, J. Biol. Chem. 267, 2524-2529;
Kawagoe et al., 1997, Cancer Res. 57, 2516-21) and AEG-13 (human
non-muscle .alpha.-actinin) (Youssoufian et al., 1990, Am. J. Hum.
Genet. 47, 62-72).
[0102] AEG-3 (GA17 protein), AEG-11 (novel) and AEG-13 (human
non-muscle .alpha.-actinin) appear to be early response genes,
which are elevated by 6 h after infection with HIV-1 or treatment
with gp120 (FIGS. 6 through 8). In contrast, AEG-1 (novel), AEG-2
(G-binding protein), AEG-4 (unr/NRU) (Jeffers et al., 1990, Nucl.
Acids. Res. 18, 4891-4899; Boussadia et al., 1993, Biochim.
Biophys. Acta 1172, 64-72), AEG-5 (hGnT-IV-H), AEG-7 (human CTL2),
AEG-8 (acidic ribosomal phosphoprotein), AEG-9 (calnexin) (Honore
et al., 1994, Electrophoresis 15, 482-490; Rubio & Wenthold,
1999, J. Neurochem. 73, 942-948; Shi et al., 2001), Cell 105,
331-342, AEG-10 (autotaxin) and AEG-(Schneider et al., 1988, Cell
54, 787-793; Gonos, 1998, Ann. N.Y. Acad. Sci. 851, 466-469; Prieto
et al., 1999, Brain Res. 816, 646-661), represent late response
genes that display elevated expression by 3 to 7 d post-treatment
(FIGS. 6 through 8). Specific AEGs had unique temporal expression
patterns. In the case of AEG-6 (fibronectin) (Niquet et al., 1994,
Neurosci. Lett. 180, 13-16), elevated expression was predominantly
detected 24 h after exposure to HIV-1 or gp120 (FIG. 7). In the
case of AEG-12 (thymosin .beta.-4) (Gondo et al., 1987, J. Immunol.
139, 3840-3848), enhanced activity was observed following HIV-1 and
gp120 treatment for 24 h and 7 d, whereas no enhancement was
apparent at 6 h or 3 d (FIG. 8). While most of the AEGs hybridized
with a single mRNA species, AEG-11 (novel) and AEG-13 (human
non-muscle .alpha.-actinin) hybridized with two separate mRNA
species, which may reflect alternate processing of the same
gene.
[0103] HIV-1 infection and gp120 treatment of early passage fetal
astrocytes enhances fibronectin and .alpha.-actinin protein
expression. To determine if elevated AEG mRNA correlated with
enhanced protein expression, we obtained commercially available
antibodies to fibronectin (AEG-6), calnexin (AEG-9) and human
non-muscle .alpha.-actinin (AEG-13) and tested expression of these
proteins in HIV-1 infected and control astrocytes by Western
blotting. The results of analyses of fibronectin and
.alpha.-actinin protein expression in two independently derived
primary human fetal astrocyte cultures are shown in FIG. 13.
Although fibronectin (AEG-6) levels were elevated in both astrocyte
cultures following HIV-1 infection, the kinetics of increase was
different for the two different astrocyte cultures. In the cultures
analyzed in FIG. 13A, enhancement was only apparent after 24 h,
which correlated with a similar increase in AEG-6 mRNA in these
cells following HIV-1 infection (FIG. 7). However, in the astrocyte
cultures analyzed in FIG. 13B, increases in fibronectin were
apparent after 6 h, 24 h and 3d. Similarly, .alpha.-actinin
(AEG-13) levels were elevated after HIV-1 infection only at 6 h in
the astrocytes in FIG. 13A, whereas an increase at 6 h and a small
increase at 24 h were apparent in the astrocytes in FIG. 13B. The
difference in the temporal kinetics of expression between these two
cultures following HIV-1 infection may reflect cellular
heterogeneity or the growth status of these astrocyte cultures.
However, since the cultures in FIG. 13B were not examined for
fibronectin or .alpha.-actinin mRNA it is not known if mRNA
synthesis displayed similar kinetic changes in these cells
following HIV-1 infection. Calnexin (AEG-13) could not be detected
in astrocytes under any conditions. Thus, for two of the AEG
products, fibronectin (AEG-6) and .alpha.-actinin (AEG-13), HIV-1
induced elevations in mRNA expression correlate with increases in
their respective protein levels.
[0104] Similar to differentially expressed gene products described
above for AEGs, the ASGs also display distinct patterns of
expression over time in response to HIV-1 infection of astrocytes,
again stratifying into early and sustained versus late responders
(Table 2, FIGS. 9-11). Six out of the 10 ASGs, (ASG-1, -2, -3, -5,
-6, and -9) can be considered late-response genes with changes
apparent in their expression only 3 d and 7 d after HIV-1 exposure
(FIGS. 9-11). A temporal decrease in expression of ASG-1, -5, and
-9 was apparent over the seven days of observation. ASG-4, -7, and
-8 were early, sustained response genes whose expression was
suppressed within 6-24 h and remained suppressed for the rest of
the period of observation (FIGS. 10 and 11). ASG-4, the signal
transduction protein C3G, showed an interesting pattern including
an increase in expression 6 h after infection followed by
decreasing expression relative to control cells from 1d to 7 d
after infection, suggesting several levels of transcriptional
control of C3G. ASG-6 reaches a nadir of expression 3 d after
infection with HIV-1, followed by a rebound in expression at 7 d.
The observation that the extent and time course of perturbation of
cellular gene expression by a given agent is specific to the
particular transcript examined reproduces the cellular response
pattern seen previously in astrocytes (Su et al., 2002, Oncogene
21, 3592-3602) and in terminally-differentiated human melanoma
cells (Huang et al., 1999, Gene 236, 125-131), and in work by
others (Corbeil et al., 2001, Genome Res 11, 1198-1204),
underscoring the generality of this phenomenon and the importance
of assessing RNA levels at multiple times after a single stimulus
or multiple stimuli.
TABLE-US-00001 TABLE 1 General Characteristics of Genes Enhanced in
HIV-1-infected and gp120-treated astrocytes (ASGs) Maximum
upregulation* Gene HIV-1 gp120 Approx. size # of Clonal designation
infected treated of mRNA (kb) isolates** Sequence homology AEG-1
2.3 (7 d) 2.7 (7 d) ~3.0 4 Novel sequence AEG-2 4.1 (3 d, 7 d) 3.0
(3 d) 1.6 2 Human G-binding protein (coupling protein G(s)
alpha-subunit (alpha-S1) (stimulatory regulatory component Gs of
adenyl cyclase) AEG-3 4.2 (6 h, 24 h) 2.3 (6 h, 24 h) 1.7 1 GA17
protein AEG-4 2.4 (3 d, 7 d) 2.5 (3 d, 7 d) 3.6 1 unr/NRU (gene
closely linked to N-ras) AEG-5 2.9 (3 d, 7 d) 1.8 (7 d) 3.8 1
UDP-N-acetylglucosamine; alpha-1,3-D-mannoside beta-1,4-N-
acetylglucosaminyltransferase IV (hGnT-IV-H) AEG-6 3.2 (24 h) 3.2
(24 h) 7.7 1 Fibronectin AEG-7 3.8 (3 d, 7 d) 2.3 (3 d, 7 d) 2.8 1
Human CTL2 AEG-8 2.0 (24 h, 3 d, 7 d) 2.0 (3 d, 7 d) 1.1 1 Acidic
ribosomal phosphoprotein AEG-9 2.3 (3 d, 7 d) 2.2 (3 d, 7 d) 4.0 1
Calnexin (human integral membrane protein) AEG-10 4.5 (3 d, 7 d)
1.6 (3 d, 7 d) 3.2 1 Autotaxin (phosphodiesterase I) AEG-11 2.8 (6
h) 2.2 (6 h) ~2.0 & 3.0 1 Novel sequence AEG-12 3.2 (24 h, 7 d)
3.0 (24 h, 7 d) 0.459 1 Thymosin .beta.4 (interferon-inducible
protein) AEG-13 3.5 (6 h) 2.4 (6 h) ~2.0 & 3.0 1 Human
non-muscle .alpha.-actinin AEG-14 2.8 (3 d, 7 d) 2.6 (3 d, 7 d) 2.4
1 gas-6 (growth arrest specific 6 gene) AEG-15 2.6 (24 h, 3 d, 7 d)
2.8 (3 d, 7 d) 1.6 1 PGK-1 (phosphoglycerate kinase 1) *Maximum
upregulation of ASG mRNA determined in HIV-1-infected or
gp120-treated human fetal astrocytes relative to the same time
point in control (untreated) cells. Fold HIV-1 upregulation =
C-ASG/C-gapdh divided by H-ASG/H-gapdh. Fold gp120 upregulation =
C-ASG/C-gapdh divided by G-ASG/G-gapdh. Time of maximum fold
upregulation indicated in parenthesis. h = hour; d = days. When
up-regulation was apparent at different time points, maximum
upregulation is indicated by underlining. Average fold change from
2 to 3 independent experiments using different primary human fetal
astrocyte cultures. **Reverse Northern blotting identified
potential upregulated AEGs. Sequencing indicated that AEG-1 had
been cloned two times and AEG-2 had been cloned two times, whereas
all of the other AEGs were identified one time in the ~20% of the
subtracted library screened by reverse Northern blotting.
TABLE-US-00002 TABLE 2 General Characteristics of Genes Suppressed
in HIV-1-infected and gp120-treated astrocytes (ASGs) Maximum
downregulation* Gene HIV-1 Approx. size # of Clonal designation
infected gp120 treated of mRNA (kb) isolates** Sequence homology
ASG-1 4.2 .+-. 0.5 (7 d).dagger. 4.8 .+-. 0.9 (3 d).dagger. ~4.0
& ~2.0 1 Novel sequence (maps to 11q23) ASG-2 3.2 .+-. 0.5 (3
d).dagger. 2.1 .+-. 0.1 (3 d).dagger. 3.0 & 1.5 1 Human cDNA
FLJ10705 (unpublished) (mRNA from NT2 neuronal precursor cell after
2 wk RA induction) ASG-3 3.4 .+-. 0.8 (7 d).dagger. 3.3 .+-. 0.4 (7
d).dagger. 1.5 1 Platelet-endothelial cell tetra-span antigen 3
mRNA (CD151/PETA-3) ASG-4 3.5 .+-. 0.1 (7 d).dagger. 2.9 .+-. 0.5
(3 d).dagger. 4.1 2 Guanine nucleotide-releasing factor, C3G ASG-5
4.1 .+-. 0.1 (7 d).dagger. 2.9 .+-. 0.5 (7 d).dagger. 1.3 1
Neuronatin ASG-6 3.2 .+-. 0.5 (3 d).dagger. 3.2 .+-. 0.4 (7
d).dagger. 3.0, 1.0 & 0.7 2 Neuroendocrine differentiation
factor; CGI149 mRNA ASG-7 3.5 .+-. 0.1 (3 d).dagger. 3.0 .+-. 0.7
(3 d).dagger. 1.8 2 Cysteine/glycine rich protein 1 mRNA (CSRP1)
ASG-8 3.1 .+-. 0.6 (24 h).dagger. 3.0 .+-. 0.1 (24 h).dagger. ~3.0
& ~1.5 1 MML5 gene ASG-9 5.3 .+-. 0.4 (3 d).dagger. 3.0 .+-.
0.7 (7 d).dagger. 16.5 2 Human mitochondrion (encoding rRNA) ASG-10
3.7 .+-. 0.3 (7 d).dagger. 1.6 .+-. 0.2 (7 d) 2.5 & 1.5 1
Signal recognition particle 9 KD (SRP9KD), human clone 45620
*Maximum downregulation of ASG mRNA determined in HIV-1-infected or
gp120-treated human fetal astrocytes relative to the same time
point in control (untreated) cells. Fold HIV-1 downregulation =
C-ASG/C-gapdh divided by H-ASG/H-gapdh. Fold gp120 downregulation =
C-ASG/C-gapdh divided by G-ASG/G-gapdh. Time of maximum fold
downregulation indicated in parenthesis. Average fold change from 2
independent experiments using different primary human fetal
astrocyte cultures .+-. S.D. Statistical analysis of the gene
expression changes between 6 h control and the time point showing
maximum change were analyzed using the Student's t-test.
.dagger.Indicates that the change in expression is significant (p
< 0.05). All maximum changes indicated in this table were
significant (p < 0.05) with the exception of ASG-10 in
gp120-treated PHFA at day 7. Qualitatively similar results were
obtained in an additional experiment (data not shown). **Reverse
Northern blotting identified potential downregulated ASGs.
Sequencing indicated that ASG-4, ASG-6, ASG-7 and ASG-9 had been
cloned two times, whereas the rest were identified one time in the
~20% of the subtracted library screened by reverse Northern
blotting.
[0105] Although the ASG library was constructed on the basis of
transcripts from HIV-1 infected astrocytes (FIG. 2), all of the
ASGs thus far identified were also suppressed in expression in
astrocytes treated with gp120. Neither can the extent of
suppression distinguish specific genes modulated by HIV-1 versus gp
120-treatment. The maximum suppression observed was induced by
HIV-1 in seven of the cases, ASG-3, -4, -5, -6, -7, -9, and -10 but
was maximally reduced by gp120 in the remaining three cases, ASG-1,
-2, and -8. The only transcript markedly more sensitive to HIV-1
than to gp120 is ASG-5. It is perhaps most interesting that the
suppression of gene expression was as sustained in cells exposed to
gp120 as in HIV-1-infected cells, for example ASG-8 was suppressed
by both agents for seven days of observation. This result is
similar to the findings for the AEGs discussed supra. The similar
patterns of altered cellular gene expression in infected and
gp120-treated astrocytes indicate that intact HIV-1 and gp120
activate similar cellular pathways leading to transcriptional
modulation of these genes. It should be noted, however, that since
the RaSH library was based on HIV-1 infected cells, these data
couldn't identify cellular genes whose expression might be affected
by gp120 but not by HIV-1.
[0106] Another implication of these results is that some modulatory
effects of HIV-1 on cellular gene expression in human astrocytes,
like the ones represented by changes in AEG and ASG, are
independent of virus replication because they can be reproduced to
a significant extent by treatment of cells with recombinant gp120
in the absence of other viral products (FIGS. 6-11). A similar
conclusion was reached in a recent study on the regulation of
glutamate transporters and glutamate transport in human astrocytes
by HIV-1. This conclusion is less surprising if one considers that
productive infection of astrocytes by HIV-1 is inefficient and only
a small proportion of infected cells, about 1%, express virus
products that could possibly affect host cell physiology (Tornatore
et al., 1991, J. Virol. 65, 6094-6100; Takahashi et al., 1996, Ann
Neurol 39, 705-711; Bencheikh et al., 1999, J. Neurovirol. 5,
115-124). However, the viral effects observed here are "global",
that is, they must occur in a majority of cells in order to be
detectable. For example, the .about.4-fold decline in ASG-7
expression at 24 h after HIV-1 exposure (FIG. 10) can be explained
only by a total loss of ASG-7 expression in 75% of affected cells
or 75% loss incurred by all the cells in the population.
[0107] The replication-independent modulation of cellular gene
expression by HIV-1 is likely mediated by efficient viral
interaction with surface receptors on astrocytes. Although these
cells lack surface CD4, the canonical HIV-1 receptor on T cells and
macrophages (Klatzman et al., 1984, Nature 312, 767-768;
Cheng-Mayer et al., 1987, Proc. Natl. Acad. Sci. USA 84,
3526-3530), they do express a high-molecular weight protein of
about 260 kDa which was shown to bind gp120 with high affinity (Ma
et al., 1994, J. Virol. 68, 6824-6828) and which may be responsible
for binding intact HIV-1 as well. Astrocytes also express the
chemokine receptors CXCR4 and CCR5 (Andjelkovic et al., 1999, Glia
28, 225-235; Rezaie et al., 2002, Glia 37, 64-75) as well as
galactocerebroside (Harouse et al., 1989, J. Virol. 63, 2527-2533),
all of which can bind HIV-1 envelope. Further studies are needed to
determine the identity of the membrane receptors on astrocytes that
may mediate the HIV-1 effects on cellular gene expression observed
here and in our other studies.
[0108] Although RNA transcripts of a single size is apparent in
astrocytes after probing with ASG-3, -4, -5, -7 and -9, multiple
hybridizing RNA species are apparent in Northern blots after
probing astrocyte RNAs with ASG-1, -2, -6, -8 and -10 (FIGS. 9-11).
The presence of multiple RNAs may indicate alternative processing
of the respective gene resulting in multiple sized transcripts or
it could reflect a cloning artifact resulting from two sequences
being cloned together in a single RaSH-derived clone. In cases
where a decrease is apparent in the multiple RNA species as a
consequence of infection with HIV-1 or treatment with gp120, such
as ASG-1 and -6, differential processing of a single gene is a more
plausible explanation for the different sized mRNAs. However, in
the case of ASG-2, -8 and -10, further analysis was performed to
determine if chimeric clones have been produced during the RaSH
procedure. Use of RaSH in its presently described form highlighted
two potential problems that can readily be addressed by minor
modifications in the protocol as addressed by Kang et al. (Kang et
al., 2002, In: Analysing Gene Expression, Lorkowski S, Cullen P,
eds, In press: Wiley-VCH Verlag GmbH, Germany). As indicated above,
although the majority of ASG RaSH clones contained single inserts,
some clones contained more than one insert that were ligated in
tandem (Jiang et al., 2000, Proc. Natl. Acad. Sci. USA 97,
12684-12689). Multiple inserts can obscure differential expression
in screening procedures, such as reverse Northern hybridization.
Moreover, if the gene has not been reported previously, the hybrid
molecule can inappropriately serve as a basis for attempting to
clone a spurious molecule. Careful consideration, especially with
respect to the presence internally of the restriction site used in
library construction (DpnII) in the RaSH clone could be used to
circumvent this problem. This inspection has been done for the ASGs
described in the present study, indicating that those genes
hybridizing to multiple transcripts are not chimera genes, i.e.,
produced as a result of cloning artifacts during the RaSH
procedure. Additionally, digestion of a cDNA with a frequent cutter
could increase additional redundancy due to cloning different parts
of the same gene. Immobilization of the 3'-end by using
biotinylated RT primer with a cloning site (e.g., XhoI) and
ligation of adapter with another cloning site (BamHI) may prove
useful in ameliorating this problem of redundant clone isolation
and the isolation of clones containing multiple inserts. This
modification in the original protocol will also enhance the cloning
efficiency of the differentially expressed insert into the vector
(Kang et al., 2002, Proc. Natl. Acad. Sci. U.S.A. 99, 637-642).
[0109] HIV-1-mediated dysregulation of cellular gene expression in
human astrocytes and neuropathogenesis. The major implication of
the findings presented here is that HIV-1 has profound, global
effects on expression of a broad array of cellular genes in
astrocytes, suggesting that this may be one route through which
HIV-1 infected astrocytes contribute to HAD. Overall, 25 genes were
identified as differentially expressed in astrocytes as a result of
HIV-1 exposure, 15 of these were upregulated in their expression
(AEGs) and 10 suppressed (ASGs). Based on the size of the
RaSH-derived EST libraries utilized in these studies, the number of
differentially expressed genes in this system may exceed 100. The
magnitude of this HIV-1 effect on astrocyte biology is more
remarkable as it occurs despite relatively inefficient viral
expression in these cells (Tornatore et al., 1991, J. Virol. 65,
6094-6100; Canki et al., 2001, J. Virol. 75, 7925-7933; Su et al.,
2002, Oncogene 21, 3592-3602) and it can be reproduced by treatment
of astrocytes with isolated HIV-1 envelope glycoprotein (FIG. 12).
As shown by recent gene array and RaSH analyses, HIV-1 also exerts
profound effects on cellular gene expression during infection of T
lymphocytes (Nye & Pinching, 1990, AIDS 4, 41-45; Shahabuddin
et al., 1994, AIDS Res. Hum. Retroviruses 10, 1525-1529; Swingler
et al., 1999, Nat Med 9, 997-103; Geiss et al., 2000, Virology 266,
8-16; Corbeil et al., 2001, Genome Res 11, 1198-1204; Simm et al.,
2001, Gene 269, 93-101). Notably, in the study of Corbeil et al.
(Corbeil et al., 2001, Genome Res 11, 1198-1204), HIV-1 infection
of CEM cells was associated with a 30% decline in overall cellular
RNA expression, replacement of cellular RNA by viral transcripts,
and increased expression of proapoptotic genes and selected
caspases; these molecular changes are consistent with the known
cytopathic course of HIV-1 infection in T cells that culminates in
cell death. In contrast, primary astrocytes are not killed by HIV-1
infection in vitro (Tornatore et al., 1991, J. Virol. 65,
6094-6100; (Bencheikh et al., 1999, J. Neurovirol. 5, 115-124) and
the major morphological change observed in astrocytes during HIV-1
infection in vivo is gliosis (Budka, 1991, Brain Pathol. 1,
163-175; Sharer, 1992, J. Neuropath. Exp. Neur. 51, 3-11), which
represents activation and possible expansion of the glial tissue.
These findings lend themselves to an interpretation of the current
findings as indicating that exposure of astrocytes to HIV-1 or
gp120 may induce long-lasting effects on cell physiology and
functions rather than affecting cell viability, as with other HIV-1
host cells. Some of these functions, such as maintenance of ionic
equilibrium in the synapse and transport of the neurotransmitter
L-glutamate, impact neurons directly and their impairment has been
shown to cause neurotoxicity (Choi, 1988; Neuron 1, 623-634;
Maragakis & Rothstein, 2001, Arch. Neurol., 58, 365-370).
Recent data also indicate that astrocytes are a critical functional
component of the synapse and play a role in signal transmission
((Iino et al., 2001, Science 292, 926-929; Oliet et al., 2001,
Science 292, 923-926; Beattie et al., 2002, Science 295,
2282-2285); disruption of these functions could impair the function
of the nervous system and lead to neurodegeneration. Finally, there
are indications that astrocytes may serve as immune effector cells
in the brain (Dong & Benveniste, 2001, Genomics 33, 292-297);
disruption of this function could weaken immune responses against
HIV-1, particularly the recruitment of macrophages into the brain
(Lipton & Gendelman, 1995, New Engl. J. Med. 233, 934-940).
[0110] The AEGs (Table 1) and ASGs (Table 2) identified thus far
can provide leads for investigation of cellular pathways co-opted
by HIV-1 in astrocytes. One important indicator is the time of gene
activation or suppression relative to HIV-1 infection. Early
modulation of cellular genes (6 h and 24 h after HIV-1 infection in
our studies) may indicate direct cellular responses to HIV-1
mediated by signal transduction mechanisms activated by virus
interaction with cell surface receptors or by disruption of the
plasma membrane integrity during virus-cell fusion. Such responses
were observed after HIV-1 exposure in T cells (Gupta &
Vayuvegula, 1987, J Clin Immun 7, 486-489; Fermin & Garry,
1992, Virology 191, 941-946; Miller et al., 1993, Virology 196,
89-100), macrophages (Zheng et al., 1999, J Virol 73, 8256-8267;
Choe et al., 2001, J Virol 75, 10738-10745), and neurons (Zheng et
al., 1999, J Virol 73, 8256-8267). The early-responder genes are
represented by ASG-4, -7, and -8, all of which show a decline in
expression within 6-24 h after HIV-1 infection (FIG. 12). ASG-4 is
of particular interest in the context of HIV-1 infection. This gene
codes for C3G, a guanine nucleotide releasing (exchange) protein
that was originally identified as one of the two major proteins
binding to the Src homology-3 (SH3) domain of the Crk adaptor
protein (Tanaka et al., 1994, Proc. Natl. Acad. Sci. USA 91,
3443-3447). The Crk-C3G complex transduces signals from
tyrosine-phosphorylated receptors (RTKs) in the plasma membrane to
Rap 1, a member of Ras-family G-proteins (York et al., 1998, Nature
392, 622-626), and subsequently to Jun kinase, JNK (Tanaka et al.,
1997, Science 276, 1699-1702). The exact role of the
RTK/Crk/C3G/Rap 1 signaling pathway has not been fully
characterized (Tanaka et al., 1997, Science 276, 1699-1702), but
published data indicate that it may exert pluripotent effects on
cellular gene expression, possibly depending on the initiating
signal. For example, Rap 1 was shown to antagonize Ras-mediated
cell transformation and MAP kinase activation in Rat-1 fibroblasts
and cloned rat embryo fibroblasts (CREF), (Cook et al., 1993, EMBO
J. 12, 3475-3485; Su et al., 1993, Oncogene 8, 1211-1219) but,
conversely, it mediated sustained MAP kinase activation and cell
differentiation in response to NGF in PC12 cells (York et al.,
1998, Nature 392, 622-626) and C3G-dependent Rap1 activation
promoted adhesion of mouse embryonic fibroblasts (Ohba et al.,
2001, EMBO J. 20, 3333-3341). Of note, the Crk/C3G pathway was
recently shown to serve as a downstream effector for the
latency-associated protein LMP2A of Epstein-Barr virus, a major
human pathogen (Engels et al., 2001, J. Exp. Med. 194, 255-264).
The observed downregulation of C3G in astrocytes by HIV-1 may thus
disrupt an important cellular signaling pathway and the functions
it controls including, if cell adhesion is affected (Ohba et al.,
2001, EMBO J. 20, 3333-3341), the activity of astrocytes as
antigen-presenting cells (Dong & Benveniste, 2001, Genomics 33,
292-297). Of the other two early-response genes in HIV-1 infected
astrocytes, ASG-8 is a homolog of the Drosophila trithorax gene and
ASG-7 is a partially characterized transcription regulation factor
that belongs to the zinc finger protein family (Liebhaber et al.
1990, Nucleic Acids Res 18, 3871-3879). Sequence analysis of ASG-8
suggests that it contains four putative zinc fingers, which may
have evolved by duplication of a preexisting two-finger unit
(Liebhaber et al. 1990, Nucleic Acids Res 18, 3871-3879). This
human cysteine-rich protein gene is highly conserved and was
detected in every nucleated tissue and cell line tested (Liebhaber
et al. 1990, Nucleic Acids Res 18, 3871-3879). Although further
experimentation is necessary, changes in expression of this
transcription factor could affect expression of target genes in
astrocytes that may contribute to normal astrocyte physiology.
[0111] Similar to the AEG series of astrocyte genes, the majority
of ASGs described here (6 out of 10) appear to be late-response
genes, that is, their expression declined maximally only 3 d to 7 d
after HIV-1 infection. These genes include ASG-1 (novel), ASG-3
(platelet-endothelial cell tetra-span antigen 3, or CD 151/PETA-3;
Fitter et al., 1995, Blood 86, 1348-55; Yanez-Mo et al., 1998, J
Cell Biol. 141, 791-804), ASG-5 (neuronatin; Dou & Joseph,
1996, Brain Res. 723, 8-22; Usui et al., 1997, J. Mol. Neurosci. 9,
55-60), ASG-6 (neuroendocrine differentiation factor; CGI149;
Wilson et al., 2001, J. Clin. Endocrinol. Metab. 86, 4504-11),
ASG-9 (human mitochondrion genomic DNA, this fragment is homologous
to the 952 to 1232-bp region of genomic DNA which encodes 16s rRNA
(from the 650 to 1603-bp region of genomic DNA), and ASG-10 (signal
recognition particle, SRP9; Lutcke, 1995, Eur. J. Biochem. 228,
531-550). PETA-3 (ASG-3) is a glycoprotein of 253 amino acids that
belongs to the tetraspanin family of surface proteins (Fitter et
al., 1995, Blood 86, 1348-55; Testa et al., 1999, Cancer Res 59,
3812-3820). PETA-3 RNA is downmodulated almost 4-fold in infected
versus uninfected astrocytes (Table 2). Although PETA-3 was
originally identified as a platelet surface protein, recent data
indicate that it functions as a component of integrin signaling
complexes on endothelial cells and it may be involved in regulation
of cell motility (Yanez-Mo et al., 1998, J. Cell Biol. 141,
791-804; Testa et al, 1999, Cancer Res 59, 3812-3820).
Dowmnodulation of PETA-3 in astrocytes by HIV-1 may affect
astrocyte function in maintaining the integrity of the
blood-brain-barrier (BBB) by reducing both the flexibility and
adhesion strength of the astrocytic underlayer of the BBB (Morgello
et al., 1995, Glia 14, 43-54). Neuronatin (ASG-5) is a
brain-specific human protein that is selectively expressed during
development (Dou & Joseph, 1996, Genomics 33, 292-297; Dou
& Joseph, 1996, Brain Res. 723, 8-22) and therefore it is
unlikely to play a role in the adult disease such as HAD. The
five-fold downmodulation of neuronatin observed here is of interest
because of the proposed function of the protein as a regulator of
anion channels (Dou & Joseph, 1996, Brain Res. 723, 8-22), an
activity that may be functionally related to the observed
downregulation of glutamate transport in these cells (Wang et al.,
2002, J. Virol., in press).
[0112] The neuroendocrine differentiation factor, NEDF (ASG-6), has
been recently identified by a yeast two-hybrid screening as a novel
intracellular protein that interacts with the IGF-binding
protein-related protein-1 (IGFBP-rP1) and proposed to act together
with IGFBP-rP1 in inducing neuroendocrine cell differentiation in
response to IGF (Wilson et al., 2001, J. Clin. Endocrinol. Metab.
86, 4504-11). ASG-6 is of interest in the context of HIV-1
infection because IGF-like growth factors appear to be protective
during CNS injury (Bondy & Lee, 1993, Ann. N. Y. Acad. Sci.
692, 33-43) and HIV-1 disease correlates with defects in the
insulin-like growth factor system (Frost et al., 1996, Clin.
Endocrinol. 44, 501-514; Jain et al., 1998, Endocr. Rev. 5,
625-646). Also of note, it has been suggested that the NEDF
(ASG-6)/IGFBP-rP1 complex acts through the Ras/MAPK signaling
pathway (Wilson et al., 2001, J. Clin. Endocrinol. Metab. 86,
4504-11), an alternative to the Crk/C3G/Rap1 pathway discussed
earlier in the context of observed downmodulation of C3G (ASG-4).
Thus two genes in astrocytes whose expression is reduced by HIV-1,
one early (ASG-4) and one late (ASG-6) after infection, encode
products that transduce signals from RTKs, indicating that this
signal transduction pathway is a major target for HIV-1 mediated
dysregulation of astrocyte physiology. Experiments are now under
way to investigate this possibility.
[0113] ASG-10 encodes a signal recognition particle SRP9, a
component of Alu RNA binding heterodimer SRP9/14 (Lutcke, 1995,
Eur. J. Biochem. 228, 531-550). SRP targets secretory and membrane
proteins to rough endoplasmic reticulum in a complex,
co-translational process that includes a temporary arrest of
elongation (Lutcke, 1995, Eur. J. Biochem. 228, 531-550). In the
CNS, the heterodimer SRP9/14 was found to be an integral part of
the brain-specific BC200 RNA, a small non-messenger RNA that is a
constituent of a ribonucleoprotein complex in neurons and that is
believed to regulate protein biosynthesis in dendrites
(Kremerskothen et al., 1998, Neurosci. Lett. 245, 123-126).
Downmodulation of SRP9 expression by HIV-1 may impair the formation
and function of the SRP9/14 heterodimer and, consequently, affect
synthesis of secretory and membrane proteins by astrocytes. The
virus could adopt this mechanism as a means for reducing exposure
of infected astrocytes to immune recognition, similar in an outcome
to the Nef-mediated downmodulation of HLA-Class I in T lymphocytes
(Collins et al., 1998, Nature 391, 397-401).
[0114] Various publications are cited herein, the contents of which
are hereby incorporated by reference in their entireties.
Sequence CWU 1
1
713611DNAHomo sapiens 1tcctggcggc ggcggagtga ggctgacagc ggggaacctg
ggagacccct ccgccctccc 60cgcggtggca gcggccgatc cccggctccg gcgcgaggga
cggccgcgat gcgctcggcc 120tgaggttacc cggcccggcc cttcctcgct
tccctcgact attccactgc gtctccgcgc 180cccggcgtca tcctgcgagt
ccctctgacg ggagggaaga tggctgcacg gagctggcag 240gacgagctgg
cccagcaggc cgaggagggc tcggcccggc tgcgggaaat gctctcggtc
300ggcctaggct ttctgcgcac cgagctgggc ctcgacctgg ggctggagcc
gaaacggtac 360cccggctggg tgatcctggt gggcactggc gcgctcgggc
tgctgctgct gtttctgctg 420ggctacggct gggccgcggc ttgcgccggc
gcccgcaaaa agcggaggag cccgccccgc 480aagcgggagg aggcggcggc
cgtgccggcc gcggcccccg acgacctggc cttgctgaag 540aatctccgga
gcgaggaaca gaagaagaag aaccggaaga aactgtccga gaagcccaaa
600ccaaatgggc ggactgttga agtggctgag ggtgaagctg ttcgaacacc
tcaaagtgta 660acagcaaagc agccaccaga gattgacaag aaaaatgaaa
agtcaaagaa aaataagaag 720aaatcaaagt cagatgctaa agcagtgcaa
aacagttcac gccatgatgg aaaggaagtt 780gatgaaggag cctgggaaac
taaaattagt cacagagaga aacgacagca gcgtaaacgt 840gataaggtgc
tgactgattc tggttcattg gattcaacta tccctgggat agaaaatacc
900atcacagtta ccaccgagca acttacaacc gcatcatttc ctgttggttc
caagaagaat 960aaaggtgatt ctcatctaaa tgttcaagtt agcaacttta
aatctggaaa aggagattct 1020acacttcagg tttcttcagg attgaatgaa
aacctcactg tcaatggagg aggctggaat 1080gaaaagtctg taaaactctc
ctcacagatc agtgcaggtg aggagaagtg gaactccgtt 1140tcacctgctt
ctgcaggaaa gaggaaaact gagccatctg cctggagtca agacactgga
1200gatgctaata caaatggaaa agactgggga aggagttgga gtgaccgttc
aatattttct 1260ggcattgggt ctactgctga gccagtttct cagtctacca
cttctgatta tcagtgggat 1320gttagccgta atcaacccta tatcgatgat
gaatggtctg ggttaaatgg tctgtcttct 1380gctgatccca actctgattg
gaatgcacca gcagaagagt ggggcaattg ggtagacgaa 1440gaaagagctt
cacttctaaa gtcccaggaa ccaattcctg atgatcaaaa ggtctcagat
1500gatgataaag aaaagggaga gggagctctt ccaactggga aatccaaaaa
gaaaaaaaag 1560aaaaagaaga agcaaggtga agataactct actgcacagg
acacagaaga attagaaaaa 1620gagattagag aagaccttcc agtgaatacc
tctaaaaccc gtccaaaaca ggaaaaagct 1680ttttccttga agaccataag
cactagtgat ccagccgaag tactcgtcaa aaatagccag 1740cctatcaaga
ctcttccacc tgctacttct accgagccat ctgtaatctt atcaaaaagt
1800gattctgaca agagctcttc ccaagtgccg ccaatactac aagagacaga
taaatccaag 1860tcaaatacca agcaaaatag tgtgcctcct tcacagacca
agtctgaaac tagctgggaa 1920tctcccaaac aaataaaaaa gaagaaaaaa
gccagacgag aaacgtgaaa ttttttttcc 1980tgaattggac atgtgtttgc
aaacacttgt cttgaagatt atgctgttta tgcaataatt 2040tgtgaacatg
tacagagttt tatataaatt taaaccaatt tttaaaacaa aactgcggac
2100accaccataa aaatggaatc aaaagaaagt taatttatga aattaagagg
tcagcagaat 2160atactcagtg atggaagaca cttgggaaag tctttttaat
agaacaagaa cgatcttaat 2220ttaagaatat tatcctggtt taacaacagt
gccctgttta caacagattg tgccctatct 2280catctgcagc cgaggaataa
aggattctga ttagaaagag ggttgcctac agattagtaa 2340gcaattcctt
ggatcttatg cacagaactt gtaccatttg aatctgtttt atgcttaaat
2400caaagtgctt tgatcaaatg cataacctgc catatcttta catatttgtt
ggtagcaatt 2460tgtattaaag aaatcacaag tgcaaataaa aagtcattta
tcatttgttt aactaaactg 2520tcatggttta gtttacaatt tttaaaaagt
tcttaaaata ctgaaaatgc agttgacact 2580tgtgtatggc ttatgaagtt
atttttgata gtcttacatt acttgaattg ttcaaagtac 2640agtatatttt
aaattaagaa aagtgaacta tatgtatttg ttttatacat ttaaggctta
2700gactcataaa taatgctatt gtttatgatt tgaaaacttt caggcaaaat
ccaatttaca 2760tttttccctt ccctagcaat tacttttttc cagcttcaac
tcttcttagt tactaatact 2820ttgttgactt taaaaatgaa atcattcaca
aacttttggt atatgatgga gaatgaaaaa 2880ctagagtcag acagctttaa
ttgacattgt caacacctcc agttatcagg aatacatttt 2940tttactgcct
taacctgtag tgcgtagaat atgcatcaat ttcttgaagg agattcatgt
3000ttttataaga attttcatgt aattattgca attgtggtca aataaggaac
gtttcctgct 3060tgaaattata ttgatttaaa tgatgtgtga gatgtttcac
cattttcagg cactgtgtaa 3120ttctattgta ataaactggc aggtatcttt
gtaactataa atagtgcatg ctcagccatg 3180tacactgtaa atagccttta
ccaaacgtgt ttgacaagga ccataattaa catcacttag 3240tgaattgtga
taaagaaaaa aaagccatga tttattcgat gtgattggct tgtttttatg
3300tggcgccaag aacgaacctg tttaacagct gtaaccaatg gtactgatct
atccatccaa 3360tgttgtcatt atatttgact gtggttcaac agtattgcgt
tgtcagacta ggaaagctaa 3420acgaacaaaa tggttttagt tttgctgaag
actggcctta ttaatggaca gctttcctaa 3480caagagatta ttaactttta
tcaggtgtta acatctgttt caggaacatg gcagtatgtt 3540tacatgtcag
aagttttgtt taattctatg gtatttctaa attgacttgt ttaaataaat
3600tcagcaaatg g 36112582PRTHomo sapiens 2Met Ala Ala Arg Ser Trp
Gln Asp Glu Leu Ala Gln Gln Ala Glu Glu1 5 10 15Gly Ser Ala Arg Leu
Arg Glu Met Leu Ser Val Gly Leu Gly Phe Leu20 25 30Arg Thr Glu Leu
Gly Leu Asp Leu Gly Leu Glu Pro Lys Arg Tyr Pro35 40 45Gly Trp Val
Ile Leu Val Gly Thr Gly Ala Leu Gly Leu Leu Leu Leu50 55 60Phe Leu
Leu Gly Tyr Gly Trp Ala Ala Ala Cys Ala Gly Ala Arg Lys65 70 75
80Lys Arg Arg Ser Pro Pro Arg Lys Arg Glu Glu Ala Ala Ala Val Pro85
90 95Ala Ala Ala Pro Asp Asp Leu Ala Leu Leu Lys Asn Leu Arg Ser
Glu100 105 110Glu Gln Lys Lys Lys Asn Arg Lys Lys Leu Ser Glu Lys
Pro Lys Pro115 120 125Asn Gly Arg Thr Val Glu Val Ala Glu Gly Glu
Ala Val Arg Thr Pro130 135 140Gln Ser Val Thr Ala Lys Gln Pro Pro
Glu Ile Asp Lys Lys Asn Glu145 150 155 160Lys Ser Lys Lys Asn Lys
Lys Lys Ser Lys Ser Asp Ala Lys Ala Val165 170 175Gln Asn Ser Ser
Arg His Asp Gly Lys Glu Val Asp Glu Gly Ala Trp180 185 190Glu Thr
Lys Ile Ser His Arg Glu Lys Arg Gln Gln Arg Lys Arg Asp195 200
205Lys Val Leu Thr Asp Ser Gly Ser Leu Asp Ser Thr Ile Pro Gly
Ile210 215 220Glu Asn Thr Ile Thr Val Thr Thr Glu Gln Leu Thr Thr
Ala Ser Phe225 230 235 240Pro Val Gly Ser Lys Lys Asn Lys Gly Asp
Ser His Leu Asn Val Gln245 250 255Val Ser Asn Phe Lys Ser Gly Lys
Gly Asp Ser Thr Leu Gln Val Ser260 265 270Ser Gly Leu Asn Glu Asn
Leu Thr Val Asn Gly Gly Gly Trp Asn Glu275 280 285Lys Ser Val Lys
Leu Ser Ser Gln Ile Ser Ala Gly Glu Glu Lys Trp290 295 300Asn Ser
Val Ser Pro Ala Ser Ala Gly Lys Arg Lys Thr Glu Pro Ser305 310 315
320Ala Trp Ser Gln Asp Thr Gly Asp Ala Asn Thr Asn Gly Lys Asp
Trp325 330 335Gly Arg Ser Trp Ser Asp Arg Ser Ile Phe Ser Gly Ile
Gly Ser Thr340 345 350Ala Glu Pro Val Ser Gln Ser Thr Thr Ser Asp
Tyr Gln Trp Asp Val355 360 365Ser Arg Asn Gln Pro Tyr Ile Asp Asp
Glu Trp Ser Gly Leu Asn Gly370 375 380Leu Ser Ser Ala Asp Pro Asn
Ser Asp Trp Asn Ala Pro Ala Glu Glu385 390 395 400Trp Gly Asn Trp
Val Asp Glu Glu Arg Ala Ser Leu Leu Lys Ser Gln405 410 415Glu Pro
Ile Pro Asp Asp Gln Lys Val Ser Asp Asp Asp Lys Glu Lys420 425
430Gly Glu Gly Ala Leu Pro Thr Gly Lys Ser Lys Lys Lys Lys Lys
Lys435 440 445Lys Lys Lys Gln Gly Glu Asp Asn Ser Thr Ala Gln Asp
Thr Glu Glu450 455 460Leu Glu Lys Glu Ile Arg Glu Asp Leu Pro Val
Asn Thr Ser Lys Thr465 470 475 480Arg Pro Lys Gln Glu Lys Ala Phe
Ser Leu Lys Thr Ile Ser Thr Ser485 490 495Asp Pro Ala Glu Val Leu
Val Lys Asn Ser Gln Pro Ile Lys Thr Leu500 505 510Pro Pro Ala Thr
Ser Thr Glu Pro Ser Val Ile Leu Ser Lys Ser Asp515 520 525Ser Asp
Lys Ser Ser Ser Gln Val Pro Pro Ile Leu Gln Glu Thr Asp530 535
540Lys Ser Lys Ser Asn Thr Lys Gln Asn Ser Val Pro Pro Ser Gln
Thr545 550 555 560Lys Ser Glu Thr Ser Trp Glu Ser Pro Lys Gln Ile
Lys Lys Lys Lys565 570 575Lys Ala Arg Arg Glu Thr58031594DNAHomo
sapiensmisc_feature115n = A,T,C or G 3gcggaaataa acaaaatgag
tagacatggg aacataaaat acattgtgga atatccaagt 60aataatgcat tatgacatta
tctaatgttt ctgaagaaca tatattatca agggnaatgg 120ccgcattctt
ttcatgccca tggcagacag agcaaaccta aatgtgcaag catatatcaa
180gcttctgtag agtcaggttt ctcaaattat cattgaccaa aataagttac
atggccaagg 240ccaagctcta catcaataat caggaaacag ctgggtgcag
tggctcacac tattttcata 300ttattagtat taaaattccc atttgcatat
ggttatagat ttcttaccaa cttccatatt 360ttcaaactga aaaaatgtaa
aactaaattc ttttttagaa actccatgtg tgataaataa 420aattatttaa
cattttggtg taatatttac aaatcctgca aaatagcacc cagatccttg
480gttgatgcca gagtcactct gcattatgca atgacacttt aggcaaaaag
taataataat 540agctgcaaag aaacattcaa gaatactgta ctaaatgcaa
ctaaactcta caataaatgc 600aaagtatcag cacttaagtg gattagttga
agtgtgccat tatcatgcca gggtgacata 660acacaacaca atggcatata
ccattgtatc agagtagagg aaacctttct ctccttttca 720catttgcata
tggttacgtt taaattacca cctattttat ataataactg gcaggtgtta
780tgataaagat attaaataac attccatttt ttcattaaag aaatcaaaat
atagaatagc 840cctaaagaaa ctgagagttg tgtacaggtt ttatgagcac
agaagcactt ttaaatatgt 900aacaggggac atgcaaacat tggaacatca
gaattagcca ccgtaattct ctaatctcca 960tgtttctatt tgtatataat
ctgaaacagg gaaactatac tcacaatcag ggaaaactac 1020tggtaaattg
ttcatgccat tgcataataa aatggctaaa gtagttaaaa agtaatgaat
1080ctggaagtct ttagcagaca tattcagtgt aacttatgtc acacttaaaa
gagaaaaata 1140gtattaaaaa ttagagatct tttatcttcc cttagtaaat
aaccttttct atcgaaacaa 1200aattacttga gtctaaatcc tgttataagc
acagtgttag atatcttaca tatattattt 1260cattgaatac taacaaaaaa
ccctttgata aacatggtat tcttcccagt ttatagagat 1320ggaaacatag
aaattaggta atgtgcccat gtttgtacag gtaataagtg gtagacctgg
1380atctaaaata catattaggc ttcaaactaa ttcatactct aaaagaatat
ttgccaaagt 1440acataataca atttgctaac agatgctaag gtaaacaaaa
aattgattat gatagttaat 1500ttacacaagg ataagttata agctaaaata
tttttttccc ttaacaaaat attatgtatc 1560atttatttat ttattttttt
gaaacggagt ctcg 159442720DNAHomo sapiens 4gagagctctc attttccccc
cttgctcggg atggtgccac aggaggctgt gcgggccccg 60ctccgcttcg aatggttgtg
ttgatgttcc tttcagctgg tcctatagta cccctcctca 120ggaatgtctc
cccagtgcaa ggacaaagac tgaagagact gctatattga tggactctca
180agccaactat gaagttgaaa caaagaaagt gatcacctga agacacctcc
tctgctaaga 240aacaccccca aattgtgcag cttctgccac tagaactctc
agaacaagag acaatctttt 300caagaaacag aaaaactcaa taatgacatc
tagattttca tgagccaaga actttccctt 360cctcatgtgt attcctctgt
ttgtacttaa attcatgtga cattcatttt tttcctagta 420tggatatgct
tattaatgca cttgtttcaa aatcccaaat tgcacaaatg tgttaatatt
480ttaagaaaca aaatgaatcc tacaaggaga atgattttta gccacacata
gggttggatc 540ttgagagtga cctacagaat aaaagtactt ttaaaataaa
gtagtcagag gctattcaaa 600gggtaaaata atcatagtac cacattggtc
cacttgacac taaccaatcg atcatttttt 660tttaatcaag aaagctagat
tctatcagat aaaatcactg cttctaaaga gtttaaatct 720agttagaaaa
agttatagaa atgtttgcaa agataagtaa cagatagagt cagtagagga
780taagatcaaa aacaaaacca agcaaaagat gagttcaggg gagtttgcca
tcaagttggc 840aaaactgact tacttaggga agaaagttat aaaacaggaa
aatatgagat gaaccttgag 900tgatgtggaa gatttagata aatggaaagg
aaggagaaaa tggagttctt taggtggttg 960taattggagg aggaaatgaa
tacacacatc ttgttgactt aaacccagac attcagcagc 1020tctctataca
tatctggaaa agactgcaca gtcacctcct gtctctcacc ccaggtatta
1080cttagaatta ttatcatatt tcccttcctt taaagtaagt aagggtgatt
ggtgacaata 1140tggagaacta tgatttttcc attaacctaa taataattgg
tatttattga gttctgttaa 1200gcattttaca tattaactca cttaagcctt
tcaacagcct tgcaaaatag gtattattat 1260ccccatttta caggcaagaa
aactgaggtt taagtaactt gccgaagtgc catatacagg 1320gctcacattc
agtattgcag ttgcaaagct catgatctat agtgccaagt tgcaatattg
1380tagtcaatgt cacaattatt accccttttt atattccttg atatttttcc
atggcaaaca 1440attagctatt tcatttaata atcacctaaa acttttcagt
cttctgatta aaattacgct 1500ggagtgatag aatgtatttt catgatagaa
attgggaaaa aaaatgggga atgaagttta 1560tcagcatttc agacttgttt
tttttttttt ttttttgcaa gactttgatg agattgttca 1620cttttgtcta
tgtaaaatcc caaatccttg agaataaaaa agggggaggt ttaagtcact
1680tgttgcaatg ccctttttaa tagaggcaat aaatctaaag gccataaatt
tagagtgact 1740tacagaagat cgaactttgg agtgtggcag agtaagggat
ggaaaccggg ccctccagtt 1800cactatcagt agcttttgca ctggtctgcc
cttcctaaat taagtatgca cttcaatttg 1860atgagtggaa acagtctatc
tgggcagtaa ccagggagct ttgtgcctag tagattgctt 1920ctgttctgca
cttctttggt ttcccacctc aatgtaaaaa atagctagca atgaagtcca
1980gaagttgtca atggttcatc cccagaagaa tgcataatgt ccaaagttgt
atgtgtatga 2040tgtcttcaat ggtattaagt tatttcaaat tcttagttca
cctacataaa tcatttctaa 2100caagcatctt cttaaccaac tttatgcaca
gtgtatgttt gtaagtgctt ctgcacgaat 2160gtttatacat gactgtttcc
atagtactta tgtttttaaa aatattcagt catttcctac 2220tataatcctc
atgtatccat gtaactgact caaaaatact tcagccacag aaagctaaaa
2280ctgagcaaat ctcattcttc ttttccatcc cctttgcatg tggctggcat
ttagtaatga 2340ttaataatat ggccagctga ataacagagg tttgagacac
aattctttct caaaggagtc 2400agctaagctg ggtctactta tggacaaaca
tctaaatgtg tggaagtatc tgatatttga 2460caatggtaaa tttccactta
gctagctagc attgtcagac ttcaatctcc tcatggctct 2520ggccgtcctg
ttttaagcat gataattgtt ggccacatct cacatagttc tcattgagtg
2580agttcataaa taaacagggt tttttttttt tttaaagagc agccaagcac
aaagtgtgac 2640tttgttgaca ttttatgtga ctttgtcata tgttcctaac
ccccaataaa agcaatgttg 2700caccaaaaaa aaaaaaaaaa 2720518DNAHomo
sapiens 5ctgatcactc gagagatc 18614DNAHomo sapiens 6ctgatcactc gaga
14712DNAHomo sapiens 7gatctctcga gt 12
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