U.S. patent application number 10/179766 was filed with the patent office on 2003-10-09 for eukaryotic genes involved in adult lifespan regulation.
This patent application is currently assigned to The Regents of the University of California. Invention is credited to Apfeld, Javier, Dillin, Andrew, Garigan, Delia, Hsu, Ao-Lin A., Kenyon, Cynthia, Lehrer-Graiwer, Josh, Murphy, Coleen.
Application Number | 20030190312 10/179766 |
Document ID | / |
Family ID | 27404746 |
Filed Date | 2003-10-09 |
United States Patent
Application |
20030190312 |
Kind Code |
A1 |
Kenyon, Cynthia ; et
al. |
October 9, 2003 |
Eukaryotic genes involved in adult lifespan regulation
Abstract
The present invention relates to regulation of adult lifespan in
eukaryotes. More particularly, the present invention is directed to
methods of assaying for genes, gene products, and genes in pathways
controlled by such genes and gene products, using RNAi and
microarray analysis, that regulate lifespan (e.g., extend or
truncate adult lifespan) in eukaryotes such as invertebrates (e.g.,
C. elegans), plants, and mammals, e.g., humans. For example, the
present invention is directed to genes encoding components of the
mitochondrial respiratory chain and genes encoding glycolysis
enzymes, which are involved in lifespan regulation, and genes and
gene products in pathways controlled by such genes. Other genes and
gene products identified as regulating aging and aging pathways
include a gene encoding a GTPase; a transcriptional activator;
novel genes: llw-1, llw-2, llw-3, and llw-4; genes encoding
cytochrome P450 proteins (involved in steroid biosynthesis); a
melatonin synthesis gene; genes encoding insulin and insulin-like
peptides; genes encoding heat shock factors; genes encoding
catalases; stress-response genes; and metabolic genes. The
invention further relates to methods for identifying and using
agents, including small molecule chemical compositions, antibodies,
antisense nucleic acids, and ribozymes, that regulate, e.g.,
enhance, adult lifespan via modulation of aging associated
proteins; as well as to the use of expression profiles, markers,
and compositions in diagnosis and therapy related to lifespan
extension, life expectancy, and aging. The present invention also
relates to gene therapy involving lifespan associated genes.
Inventors: |
Kenyon, Cynthia; (San
Francisco, CA) ; Apfeld, Javier; (San Francisco,
CA) ; Dillin, Andrew; (Oakland, CA) ; Garigan,
Delia; (San Francisco, CA) ; Hsu, Ao-Lin A.;
(Albany, CA) ; Lehrer-Graiwer, Josh; (San
Francisco, CA) ; Murphy, Coleen; (San Francisco,
CA) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER
EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
The Regents of the University of
California
|
Family ID: |
27404746 |
Appl. No.: |
10/179766 |
Filed: |
June 24, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60300577 |
Jun 22, 2001 |
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60301052 |
Jun 25, 2001 |
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60373975 |
Apr 18, 2002 |
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Current U.S.
Class: |
424/130.1 ;
435/6.1; 435/7.2; 800/8 |
Current CPC
Class: |
C12Q 1/6883 20130101;
A61P 43/00 20180101; A01K 67/0336 20130101; C07K 14/43545 20130101;
C12N 2320/12 20130101; A01K 2217/05 20130101; C12Q 1/34 20130101;
C12Q 1/32 20130101; G01N 2500/10 20130101; A01K 2217/075 20130101;
C12Q 1/527 20130101; C12N 2310/14 20130101; C12Q 1/533 20130101;
C12Q 2600/158 20130101; C12Q 1/26 20130101; C12N 15/111 20130101;
C12N 2330/31 20130101 |
Class at
Publication: |
424/130.1 ;
800/8; 435/6; 435/7.2 |
International
Class: |
C12Q 001/68; G01N
033/53; G01N 033/567; A01K 067/033; A61K 039/395 |
Goverment Interests
[0002] This invention was made with government support under Grant
No. NIH AG11816, awarded by the NIH. The government has certain
rights in this invention.
Claims
What is claimed is:
1. A method for identifying a compound that modulates aging, the
method comprising the steps of: (i) contacting the compound with a
polypeptide, wherein the polypeptide is encoded by a nucleic acid
that hybridizes under stringent conditions to a nucleic acid
encoding a polypeptide comprising an amino acid sequence selected
from the group consisting of: cytochrome C.sub.1 (SEQ ID NO: 1),
NADH oxidoreductase (SEQ ID NO: 2), ATP synthase (SEQ ID NO: 3),
cytochrome C oxidase (SEQ ID NO: 4), phosphoglucose isomerase (SEQ
ID NO: 5), GTPase (SEQ ID NO: 6), LLW-1 (SEQ ID NO: 7), LLW-2 (SEQ
ID NO: 8), LLW-3 (SEQ ID NO: 9), LLW-4 (SEQ ID NO: 10) and HSF-1
(SEQ ID NO: 11), and human homologues thereof; and (ii) determining
the functional effect of the compound upon the polypeptide.
2. The method of claim 1, wherein the polypeptide is encoded by a
nucleic acid that hybridizes under stringent conditions to a
nucleic acid encoding a polypeptide comprising an amino acid
sequence selected from the group consisting of: cytochrome C.sub.1
(SEQ ID NO: 1), NADH oxidoreductase (SEQ ID NO: 2), ATP synthase
(SEQ ID NO: 3), cytochrome C oxidase (SEQ ID NO: 4), phosphoglucose
isomerase (SEQ ID NO: 5), GTPase (SEQ ID NO: 6), LLW-1 (SEQ ID NO:
7), LLW-2 (SEQ ID NO: 8), LLW-3 (SEQ ID NO: 9), and LLW-4, (SEQ ID
NO: 10) or human homologues thereof.
3. The method of claim 1, wherein the polypeptide is encoded by a
nucleic acid that hybridizes under stringent conditions to a
nucleic acid encoding a polypeptide comprising an amino acid
sequence of HSF-1 (SEQ ID NO: 11), or human homologues thereof.
4. The method of claim 1, wherein the functional effect is
determined in vitro.
5. The method of claim 4, wherein the functional effect is
determined by measuring enzymatic activity.
6. The method of claim 4, wherein the functional effect is
determined by measuring ligand, substrate, or cofactor binding to
the polypeptide.
7. The method of claim 4, wherein the functional effect is
determined by measuring binding to a promoter sequence by the
polypeptide.
8. The method of claim 1, wherein the polypeptide is expressed in a
mitochondrial extract or an isolated mitochondrial membrane.
9. The method of claim 1, wherein the polypeptide is expressed in a
eukaryotic host or host cell and the polypeptide is contacted with
the compound in a living cell.
10. The method of claim 9, wherein the host cell is derived from C.
elegans, mouse, rat, or human.
11. The method of claim 9, wherein the host is C. elegans, mouse,
rat, or human.
12. The method of claim 9, wherein the functional effect is a
determined by measuring ligand, substrate, or cofactor binding to
the polypeptide.
13. The method of claim 9, wherein the functional effect is
determined by measuring transcriptional activation.
14. The method of claim 9, wherein the functional effect is
determined by evaluating age-associated parameters.
15. The method of claim 9, wherein the functional effect is
determined by evaluating expression of an age-associated gene.
16. The method of claim 14, wherein the age-associated parameter is
lifespan.
17. The method of claim 1, wherein the modulation is inhibition of
aging.
18. The method of claim 17, wherein inhibition of aging occurs by
activation of an HSF polypeptide encoded by a nucleic acid that
hybridizes under stringent conditions to a nucleic acid encoding a
polypeptide comprising an amino acid sequence of HSF-1 (SEQ ID NO:
11), or human homologues thereof.
19. The method of claim 17, wherein inhibition of aging occurs by
inhibition of a polypeptide encoded by a nucleic acid that
hybridizes under stringent conditions to a nucleic acid encoding a
polypeptide comprising an amino acid sequence selected from the
group consisting of: cytochrome C.sub.1 (SEQ ID NO: 1), NADH
oxidoreductase (SEQ ID NO: 2), ATP synthase (SEQ ID NO: 3),
cytochrome C oxidase (SEQ ID NO: 4), phosphoglucose isomerase (SEQ
ID NO: 5), GTPase (SEQ ID NO: 6), LLW-1 (SEQ ID NO: 7), LLW-2 (SEQ
ID NO: 8), LLW-3 (SEQ ID NO: 9), and LLW-4 (SEQ ID NO: 10) or human
homologues thereof.
20. The method of claim 1 wherein the polypeptide is
recombinant.
21. The method of claim 1, wherein the compound is an antibody, an
antisense molecule, or a small molecule.
22. A method for identifying a compound that modulates aging, the
method comprising the steps of: (i) contacting the compound with a
polypeptide, wherein the polypeptide is encoded by a nucleic acid
that hybridizes under stringent conditions to a nucleic acid
encoding a polypeptide comprising an amino acid sequence selected
from the group consisting of: cytochrome C.sub.1 (SEQ ID NO: 1),
NADH oxidoreductase (SEQ ID NO: 2), ATP synthase (SEQ ID NO: 3),
cytochrome C oxidase (SEQ ID NO: 4), phosphoglucose isomerase (SEQ
ID NO: 5), GTPase (SEQ ID NO: 6), LLW-1 (SEQ ID NO: 7), LLW-2 (SEQ
ID NO: 8), LLW-3 (SEQ ID NO: 9), LLW-4 (SEQ ID NO: 10) and HSF-1
(SEQ ID NO: 11), and human homologues thereof, (ii) determining the
functional effect of the compound upon the polypeptide; and (iii)
contacting a host or host cell expressing the protein and
evaluating an age-associated parameter in a host or host cell,
thereby identifying a compound that modulates aging.
23. The method of claim 22, wherein the polypeptide is
recombinant.
24. The method of claim 22, wherein the functional effect is a
physical effect.
25. The method of claim 22, wherein the functional effect is a
chemical effect.
26. The method of claim 22, wherein the functional effect is a
phenotypic effect.
27. The method of claim 22, wherein the functional effect is
determined in vitro.
28. The method of claim 22, wherein the functional effect is
determined in a eukaryotic host organism or host cell.
29. The method of claim 22, wherein the age-associated parameter is
lifespan.
30. A method for identifying a compound that modulates aging, the
method comprising the steps of: (i) contacting the compound with a
polypeptide that is a component of the mitochondrial respiratory
chain; and (ii) determining the functional effect of the compound
upon the polypeptide.
31. The method of claim 30, wherein the polypeptide is expressed in
a mitochondrial extract or an isolated mitochondrial membrane.
32. A method for identifying a compound that modulates aging, the
method comprising the steps of: (i) contacting the compound with a
polypeptide, wherein the polypeptide is encoded by a nucleic acid
that hybridizes under stringent conditions to a nucleic acid listed
in Table 5 or Table 6, or a nucleic acid encoding a polypeptide
listed in Table 5 or Table 6, or human homologues thereof; and (ii)
determining the functional effect of the compound upon the
polypeptide.
33. A method for identifying a compound that modulates aging, the
method comprising the steps of: (i) contacting the compound with a
polypeptide, wherein the polypeptide is encoded by a nucleic acid
that hybridizes under stringent conditions to a nucleic acid listed
in Table 5 or Table 6, or a nucleic acid encoding a polypeptide
listed in Table 5 or Table 6, or human homologues thereof (ii)
determining the functional effect of the compound upon the
polypeptide; and (iii) contacting a host or host cell expressing
the protein and evaluating an age-associated parameter in a host or
host cell, thereby identifying a compound that modulates aging.
34. A compound that modulates an aging process, wherein the
compound is identified by the method of claim 1.
35. The compound of claim 34, wherein the compound is an antibody,
an antisense molecule, or a small molecule.
36. The compound of claim 34, wherein the compound is antimycin or
an analog thereof.
37. A method of increasing lifespan or treating premature aging in
a subject, the method comprising the step of administering to the
subject an effective amount of a compound identified using the
method of claim 1.
38. The method of claim 37, wherein the compound is antimycin.
39. A method of increasing lifespan or treating premature aging in
a subject, the method comprising the step of administering to the
subject an effective amount of a compound that modulates the
expression of a polypeptide comprising an amino acid sequence
selected from the group consisting of: cytochrome C.sub.1 (SEQ ID
NO: 1), NADH oxidoreductase (SEQ ID NO: 2), ATP synthase (SEQ ID
NO: 3), cytochrome C oxidase (SEQ ID NO: 4), phosphoglucose
isomerase (SEQ ID NO: 5), GTPase (SEQ ID NO: 6), LLW-1 (SEQ ID NO:
7), LLW-2 (SEQ ID NO: 8), LLW-3 (SEQ ID NO: 9), LLW-4 (SEQ ID NO:
10) and HSF-1 (SEQ ID NO: 11), and human homologues thereof.
40. The method of claim 37 or 39, wherein the aging process is
abnormal.
41. The method of claim 40, wherein the abnormal aging process is
selected from Werner syndrome, Hutchinson-Guilford disease, Bloom's
syndrome, Cockayne's syndrome, ataxia telangiectasia, and Down's
syndrome.
42. The method of claim 37 or 39, wherein the aging process is
normal.
43. The method of claim 37 or 39, further comprising the step of
evaluating an age-associated parameter of the subject.
44. A method of identifying a gene or gene product that modulates
aging, the method comprising the steps of: (i) providing a library
of nucleic acids, each nucleic acid of the library comprising a
segment of a gene of an organism; and (ii) for each member of the
library, (a) generating double-stranded RNA (dsRNA) from the
respective member of the library, (b) providing the dsRNA to a cell
or one or more cells of the organism to provide a ds-RNA treated
cell or ds-RNA treated organism, and (b) monitoring an
age-associated parameter of the ds-RNA treated cell or the ds-RNA
treated organism.
45. A method of identifying a gene or gene product that modulates
aging, the method comprising the steps of: (i) evaluating a
relationship between presence or abundance for each species of a
plurality of RNA or protein species with respect to age of a cell
or organism; (ii) producing or delivering a double-stranded RNA to
a cell or one or more cells of an organism, the RNA corresponding
to a species whose presence or abundance is correlated with age;
and (iii) monitoring an age-associated parameter of the cell or the
one or more cells or the organism.
46. A method of identifying a compound that modulates aging, the
method comprising the steps of: (i) contacting a test compound to a
living or biochemical system that comprising a C. elegans target
protein selected from the group consisting of: a respiratory chain
component, a heat shock promoter activator, LLW-1, LLW-2, LLW-3,
LLW-4, a small GTPase, a protein in Table 5, and a protein in Table
6; and (ii) evaluating a property associated with the target
protein; and (iii) evaluating an aging-associated parameter of a C.
elegans organism contacted with the test compound.
47. A method of identifying a gene or gene product that modulates
aging, the method comprising the steps of: (i) providing a nematode
in which activity of a target protein is reduced in the organism by
RNA interference; (i) expressing a gene encoding a candidate
mammalian protein that is heterologous to the organism; and (iii)
evaluating an age associated parameter of the organism.
48. A C. elegans nematode that (1) expresses a heterologous gene in
at least some cells, the heterologous gene encoding a respiratory
chain component or glycolysis component that is non-identical to
the corresponding endogenous respiratory chain component or
glycolysis component, or a functional domain thereof; and (2) is
deficient in at least some cells for an endogenous activity
provided by the corresponding endogenous respiratory chain
component or glycolysis component.
49. A method comprising: assessing an age-associated parameter of
the nematode of claim 48.
50. A C. elegans nematode that (1) has a deficiency in at least
some cells for an endogenous activity, the deficiency generated by
dsRNA in the cells, and (2) has an average lifespan of at least 40%
greater than an otherwise identical nematode without the
deficiency.
51. A method of identifying a gene or gene product that modulates
aging, the method comprising the steps of: (i) providing the
nematode of claim 50; (ii) introducing a heterologous gene that
encodes a heterologous polypeptide into the nematode; (iii)
expressing the heterologous gene in the nematode or a progeny of
the nematode under conditions wherein the heterologous polypeptide
is produced; and (iv) monitoring an age-associated parameter of the
nematode or the progeny of the nematode.
52. The method of claim 51, further comprising contacting a test
compound to the nematode or the progeny prior to or during the
monitoring.
Description
CROSS REFERENCES TO RELATED APPLICATIONS
[0001] The present application is related to U.S. Serial No.
60/300,577, filed Jun. 22, 2001, U.S. Serial No. 60/301,052, filed
Jun. 25, 2001, and U.S. Serial No. 60/373,975, filed Apr. 18, 2002,
each herein incorporated by reference in their entirety.
FIELD OF THE INVENTION
[0003] The present invention relates to regulation of lifespan in
eukaryotes. More particularly, one aspect of the present invention
is directed to methods of assaying for genes, gene products, and
genes in pathways controlled by such genes and gene products, using
RNAi and microarray analysis, that regulate lifespan (e.g., extend
or truncate adult lifespan) in eukaryotes such as invertebrates
(e.g., C. elegans), plants, and mammals, e.g., humans. For example,
one aspect of the present invention is directed to genes, in
particular human genes, encoding components of the mitochondrial
respiratory chain and genes encoding glycolysis enzymes, which are
involved in lifespan regulation, and genes and gene products in
pathways controlled by such genes. Other genes and gene products
identified as regulating aging and aging pathways include a gene
encoding a GTPase; a transcriptional activator; novel genes: llw-1,
llw-2, llw-3, and llw-4; genes encoding cytochrome P450 proteins
(involved in steroid biosynthesis); a melatonin synthesis gene;
genes encoding insulin and insulin-like peptides; genes encoding
heat shock factors; genes encoding catalases; stress-response
genes; and metabolic genes. The invention further relates to
methods for identifying and using agents, including small molecule
chemical compositions, antibodies, antisense nucleic acids, and
ribozymes, that regulate, e.g., enhance, adult lifespan via
modulation of aging associated proteins; as well as to the use of
expression profiles, markers, and compositions in diagnosis and
therapy related to lifespan extension, life expectancy, and aging.
The present invention also relates to gene therapy involving
lifespan associated genes.
BACKGROUND OF THE INVENTION
[0004] Previously, classic genetic screens have been used to
identify genes involved in the C. elegans development. In one
example, inhibition of mitochondrial respiratory chain genes such
as NADH ubiquinone oxidoreductase and ATP synthase in C. elegans
larva was found to impair larval development and cause arrest in
the third larval stage (see, e.g., Tsang et al., JBC
276:33240-33246 (2001)). In other examples, classical genetic
screens have been used to identify C. elegans genes involved in a
variety of processes, including dauer formation, and embryonic
development. Some of these genes, for example the daf-2 and daf-16
genes, have been implicated in the regulation of lifespan see,
e.g., Kenyon et al., Nature 366:461-464 (1993); Morris et al.,
Nature 382:536-539 (1996); Kimura et al., Science 277:942-946
(1997); Paradis et al., Genes Dev. 12:2488-2498 (1998); Paradis et
al., Genes Dev. 13:1438-1452 (1999); Off & Ruvkun, Mol. Cell
2:886-893 (1998); Guarente & Kenyon, Nature 408:255-262 (2000);
Ogg et al., Nature 389:994-999 (1997); and Lin et al., Science
278:1319-1322 (1997)).
[0005] Classical genetic screens are frequently time consuming,
both in identification of interesting mutants and in cloning a gene
associated with a mutation. Classical genetic screens can include
labor intensive backcrosses to eliminate mutations unlinked to the
phenotype of interest. Classical genetic screens also may require
the extra step of cloning the gene of interest, by complementation
of the mutation.
[0006] Many different genes likely regulate the process of aging in
eukaryotes and their identification will aid in understanding the
process. Regulation of biological processes is frequently conserved
between divergent organisms. For example, cell cycle proteins and
their mechanism of action are conserved between organisms as
divergent as yeast and humans. Thus, regulatory mechanisms
identified in a genetically tractable organism can be used to
predict and identify homologous genes and gene products that
regulate similar biological processes in higher eukaryotes.
However, even in genetically tractable organisms, such as C.
elegans, classical genetic methods are frequently labor intensive
and cumbersome for identification of interesting mutants and for
isolation of a gene of interest. The present invention solves these
and other problems.
SUMMARY OF THE INVENTION
[0007] The present invention provides, for the first time, methods
of assaying for aging associated genes and proteins using RNAi, as
well as aging associated genes and proteins so identified.
Double-stranded RNA libraries are administered to a test organism
or cell, e.g., C. elegans. For administration to C. elegans, the
RNAi library can be expressed in a bacterial cell and fed to the C.
elegans. Age-associated mutants (those that live longer or age
faster) are identified by measuring lifespan increase or decrease,
or an age-associated parameter such as stress resistance or a
Nomarski analysis indicia, e.g., yolk accumulation, loss of ability
to chew and expel (distended oral and anal cavities), necrotic
cavities in tissue, curdled appearing tissue, and germ cell
appearance (graininess, large, well separated nuclei, fewer nuclei,
and cavities).
[0008] Other methods can be used to identify age-associated genes
and gene products, including microarray analysis, database
profiling with known age-associated genes, mammalian
complementation assays, yeast two hybrid assays,
immunoprecipitation, etc.
[0009] The use of dsRNA has identified a number of genes and gene
products which, when inhibited by dsRNA, results in longer
lifespans, e.g., phosphoglucose isomerase, cytochrome C1, NADH
oxidoreductase, ATP synthase, cytochrome C oxidase, a GTPase, and
four novel genes, llw1-4 (see also Tables 5 and 6). Thus, compounds
that inhibit such genes and gene products, as well as genes in a
pathway controlled by such genes and gene products, would be useful
for increasing lifespan and modulating the aging process in
eukaryotes, e.g., plants, mammals, humans. The genes identified
herein include any mammalian or human homologs thereof.
[0010] In addition, the use of dsRNA has identified genes and gene
products which, when inhibited by dsRNA, results in a shorter
lifespan, e.g., heat shock protein or heat shock factor (HSF) (see
also Table 5 and Table 6). Overexpression of HSF has also been
shown to increase lifespan in C. elegans. Thus, compounds that
activate heat shock proteins or genes such as HSF-1 or HSF targets,
or genes or gene products controlled by HSF, or genes that respond
to stress and activate HSF would be useful for increasing lifespan
and modulating the aging process in eukaryotes, e.g., plants,
mammals, humans. In one example, compounds such as zinc finger
proteins (naturally occurring or recombinant) can be used to bind
to HSF binding regions on genes and alter expression of
HSF-controlled genes (see, e.g., U.S. Pat. Nos. 5,789,538 and
6,242,568).
[0011] Microarrays were used to analyze gene expression profiles in
daf-2 and daf-6 mutants and identify genes and gene products
involved in lifespan regulation. The expression of a number of
genes varied (by over- or under-expression) in long-lived (daf-2)
or short-lived (daf-16 and daf-16; daf-2) C. elegans mutants (see
Table 5). The activity of genes identified using microarray
analysis was then modulated using RNAi (Table 6). The genes thus
identified include hormones that activate the daf-2 pathway,
several encoding cytochrome P450 proteins (involved in steroid
biosynthesis), the melatonin synthesis gene, insulin and
insulin-like peptides, heat shock factors, catalases,
stress-response genes, and metabolic genes. These genes and gene
products, and genes and gene products controlled by these genes
(e.g., steroid hormones, melatonin) are therefore useful for
developing drugs to regulate aging in eukaryotes, e.g., plants,
mammals, humans. In addition, these genes can be used as markers
for the insulin/IGF system activity, as markers for the aging
process, and as markers that indicate the likely longevity of an
individual.
[0012] In one aspect of the invention, nucleic acids from C.
elegans and corresponding mammalian genes (e.g., human) encoding
glycolysis proteins, e.g., phosphoglucose isomerase, and
mitochondrial respiratory chain proteins, e.g., cytochrome C1
component of complex III (CYC1), NADH oxidoreductase (NUO2), ATP
synthase (ATP3, a member of the ATP synthase delta family), and
cytochrome C oxidase (CCO1), are provided, as are nucleic acids
from C. elegans and corresponding mammalian genes encoding a GTPase
associated with aging; a transcriptional activator, heat shock
factors, e.g., HSF-1; novel proteins LLW-1, LLW-2, LLW-3, and
LLW-4; cytochrome P450 proteins, a melatonin synthesis gene,
insulin and insulin-like peptides, heat shock factors, catalases,
stress-response genes, and other genes listed in Tables 5 and 6. In
another aspect, the present invention provides nucleic acids, such
as probes, antisense oligonucleotides, and ribozymes, that
hybridize to glycolysis genes, e.g., phosphoglucose isomerase
(GPI-1); mitochondrial respiratory chain genes, e.g., cytochrome
C1, NADH oxidoreductase, ATP synthase, and cytochrome C oxidase; a
GTPase associated with aging; a transcriptional activator, heat
shock factors, e.g., HSF-1; novel genes llw-1, llw-2, llw-3, and
llw-4; cytochrome P450 proteins, a melatonin synthesis gene,
insulin and insulin-like peptides, heat shock factors, catalases,
stress-response genes, and other genes listed in Tables 5 and
6.
[0013] In another aspect, the invention provides expression vectors
and host cells comprising nucleic acids encoding glycolysis
proteins, e.g., phosphoglucose isomerase; mitochondrial respiratory
chain proteins, e.g., cytochrome C1, NADH oxidoreductase, ATP
synthase, and cytochrome C oxidase; a GTPase associated with aging;
a transcriptional activator, heat shock factors, e.g., HSF-1; novel
genes llw-1, llw-2, llw-3, and llw-4; cytochrome P450 proteins, a
melatonin synthesis gene, insulin and insulin-like peptides, heat
shock factors, catalases, stress-response genes, and other genes
listed in Tables 5 and 6. In another aspect, the present invention
provides glycolysis proteins, e.g., phosphoglucose isomerase;
mitochondrial respiratory chain proteins, e.g., cytochrome C 1,
NADH oxidoreductase, ATP synthase, and cytochrome C oxidase; a
GTPase associated with aging; a transcriptional activator, the heat
shock factor; novel proteins LLW-1, LLW-2, , LLW-3, LLW-4;
cytochrome P450 proteins, a melatonin synthesis protein, insulin
and insulin-like peptides, heat shock factors, catalases,
stress-response proteins, and other gene products listed in Tables
5 and 6 and antibodies thereto.
[0014] In another embodiment, the invention provides heterologous
constructs comprising an age-associated gene as described herein,
and a heterologous sequence such as a regulatory region, a reporter
gene, a purification tag, e.g., for production of a fusion protein,
for purification of a gene product, for more efficient expression
of the gene or gene product, or for regulated expression of the
gene.
[0015] In one embodiment, methods known to those of skill in the
art such as RT-PCR, northern, Southern analysis, cDNA and genomic
library cloning, etc. can be used to identify eukaryotic orthologs,
e.g., invertebrate, vertebrate, plant, mammalian, and human
orthologs, of the age-associated proteins provided herein. In
another embodiment, computer sequence analysis can be used to
identify orthologs. Such methods optionally include the step of
assessing an age associated parameter in a cell in which the
suspected ortholog is perturbed.
[0016] In one embodiment, endogenous or recombinant gene products
of the age associated genes described herein are purified using the
methods described herein, to at least about 50% purity, preferably
60%, 70%, 80%, 90% or higher purity. In another embodiment, the
present invention provides a reaction mixture comprising an
age-associated protein and another component such as a test
compound, an antibody, a peptide, etc.
[0017] In another aspect, the present invention provides a method
for identifying a compound that modulates adult aging, the method
comprising the steps of: (i) contacting the compound with a
glycolysis protein, e.g., phosphoglucose isomerase, or a
mitochondrial respiratory chain protein, e.g., cytochrome C1, NADH
oxidoreductase, ATP synthase, and cytochrome C oxidase or a GTPase
associated with aging; a novel proteins LLW-1, LLW-2, LLW-3, LLW-4;
and (ii) determining the functional effect, e.g., lifespan effect
or another age-associated parameter of the compound upon the
polypeptide.
[0018] In another aspect, the present invention provides a method
for identifying a compound that modulates adult aging, the method
comprising the steps of: (i) contacting the compound with protein
encoded by a gene listed in Table 5 or 6; and (ii) determining the
functional effect, e.g., lifespan effect or another age-associated
parameter of the compound upon the polypeptide.
[0019] In another aspect, the present invention provides a method
for identifying a compound that modulates adult aging, the method
comprising the steps of: (i) contacting the compound with a heat
shock factor and (ii) determining the functional effect, e.g.,
lifespan effect or another age-associated parameter, of the
compound upon the polypeptide.
[0020] In one embodiment, the functional effect is a physical
effect or a chemical effect. In another embodiment, the functional
effect is a phenotypic effect. In one embodiment, the polypeptide
is expressed in a eukaryotic host or host cell, e.g., C. elegans.
In another embodiment, the functional effect is determined by
measuring longevity, average lifespan, or mean lifespan of an
organism contacted with a compound. In one embodiment, the
functional effect is determined by measuring enzymatic activity. In
one embodiment, the functional effect is determined by measuring
transcriptional activation. In one embodiment, the organism is C.
elegans. In another embodiment, the organism is mammalian host or
cell, e.g., a mouse, a rat, a guinea pig, a monkey, or a human.
[0021] In another embodiment, compounds that modulate aging are
identified using computer programs that model age-associated
protein structure and determining compounds that bind or interact
with the modeled protein. Optionally, the effect of the compound
can be validated by examining its effect on a cell or organism
expressing the modeled age-associated protein.
[0022] In another embodiment, the method comprises providing a
sequence comprising an age-associated protein, altering the
sequence, e.g., by mutagenesis, and assaying the protein encoded by
the altered sequence.
[0023] In another aspect, the present invention provides a method
of modulating lifespan in a subject, the method comprising the step
of contacting the subject with an therapeutically effective amount
of a compound identified using the methods described herein, e.g.,
a compound such as antimycin. In one embodiment, the subject is C.
elegans. In another embodiment, the subject is a mammalian subject,
e.g., a mouse, a rat, a guinea pig, a monkey, or a human. In one
embodiment, the subject is a plant. In one embodiment, the compound
is antimycin or an analog thereof.
[0024] In another aspect, the present invention provides a method
of detecting the presence of a lifepan associated protein described
herein, and the genes encoding such proteins in eukaryotic tissue,
the method comprising the steps of: (i) isolating a biological
sample; (ii) contacting the biological sample with a specific
reagent that selectively associates with the protein of choice;
and, (iii) detecting the level of specific reagent that selectively
associates with the sample.
[0025] In one embodiment, the specific reagent is selected from the
group consisting of: antibodies, oligonucleotide primers, and
nucleic acid probes.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIG. 1 shows RNAi of respiratory chain and glycolysis genes
increasing lifespan.
[0027] FIG. 2 shows daf-2(e1370) worms treated with dsRNAs of
respiratory and glycolytic genes.
[0028] FIG. 3 shows antimycin increases lifespan.
[0029] FIG. 4 shows daf-16(mu86) mutant worms treated with dsRNAs
of respiratory and glycolytic genes.
[0030] FIG. 5 shows certain genes identified using microarray
analysis that are upregulated in daf-2(-) worms.
[0031] FIG. 6 shows that hsp-12.6 RNAi reduces daf-2 longevity.
[0032] FIG. 7 shows that mtl-1 RNAi reduces daf-2 longevity.
[0033] FIG. 8 shows that cytochrome P450 shortens daf-2
lifespan.
[0034] FIG. 9 shows certain genes identified using microarray
analysis that are downregulated in daf-2 mutants.
[0035] FIG. 10 shows that RNAi of vitellogenins increases
lifespan.
[0036] FIG. 11 shows that RNAi of ASMLT (melatonin synthesis gene)
prolongs lifespan.
[0037] FIG. 12 shows certain genes that modulate lifespan.
DETAILED DESCRIPTION OF THE INVENTION
Introduction
[0038] Double-stranded RNA-mediated interference (RNAi) provides a
sequence specific mechanism for inhibiting gene expression in C.
elegans (see, e.g., Fire et al., Nature 391:806-811 (1998) and WO
99/32619)). This technique is also useful for functional genomics
analysis of C. elegans genes (see, e.g., Fraser et al., Nature
408:325-330 (2000); Kamath et al., Genome Biol. 2:RESEARCH0002
(2000)).
[0039] Surprisingly, RNAi can be used to identify genes and gene
products involved in lifespan and aging mechanisms in adults, by
feeding bacteria expressing a dsRNA library over long-term time
periods to C. elegans and measuring, e.g., mean and median lifespan
increase or decrease in adults. Such assays can be used, in
addition, to determine if two or more genes function together in a
similar aging pathway, to determine if gene function is cell
autonomous, and to determine if drug compounds known to alter the
aging process function in the same or different pathways.
Microarray assays can also be used to identify genes and gene
products involved in lifespan and aging (see, e.g., Table 5 and
Table 6).
[0040] Microarray and RNAi assays are also useful in combination,
e.g., by identifying a gene or gene product involved in lifespan
using RNAi and then examining its expression pattern in old and
young adults using microarray analysis, or by identifying a
differentially expressed gene or gene product involved in lifespan
using microarray analysis and then examining the effects of gene or
gene product inhibition using RNAi. Microarray analysis can also be
performed on animals treated with RNAi of genes identified using
microarray analysis.
[0041] Using the technique of double-stranded RNA inhibition
(RNAi), two types of metabolic genes and gene products have been
found to regulate the adult lifespan of a model eukaryote, C.
elegans. Inhibition of genes and gene products that function in the
mitochondrial respiratory chain, including genes encoding
cytochrome C1, cytochrome C oxidase and ATP synthetase, lengthen
lifespan, as does inhibition of a gene that functions in
glycolysis, glucose phosphate isomerase. However, these
perturbations produce different phenotypes. Inhibition of
respiratory chain components cause a Clk (clock) phenotype,
including slow growth to adulthood, and, in adults, slow movement,
and decreased pumping and defecation rates. In contrast, inhibition
of glucose phosphate isomerase extends adult lifespan without
producing a Clk phenotype. Together these findings indicate that at
least two distinct pathways can regulate lifespan in response to
changes in metabolic gene activities in eukaryotes. These
RNAi-mediated phenotypes are examples of "reverse phenotypes,"
i.e., suppression or inhibition of the gene lengthens lifespan. For
RNAi interventions that decrease lifespan, restoring gene
expression should restore a normal lifespan. Therefore, under- and
over-expression of lifespan genes, gene products and pathways, as
well as activation or inhibition of lifespan gene, gene products,
and pathways, can be used to modulate aging in a subject.
[0042] The present invention therefore provides nucleic acids from
C. elegans encoding cytochrome C1 component of complex III, gene
C54G4.8 (CYC1, SEQ ID NO: 1, Accession No. CAA99820.1, which is 50%
identical to human ortholog CYC1); NADH oxidoreductase, gene
T10E9.7 (NUO2, SEQ ID NO: 2, Accession No. AAB522474.1, which is
56% identical to human ortholog NDUFS3, Accession No. NM.sub.13
004551); ATP synthase (delta family), gene F27C1.7 (ATP3, SEQ ID
NO: 3, Accession No. AAB37654.1, 43% identical to human ortholog
ATP50); and cytochrome C oxidase, gene F26E4.9 (CCO1, SEQ ID NO: 4,
Accession No. CAB03002.1, 35% identical to human ortholog COX5B,
Accession No. NM.sub.13 001862), which are protein components of
the mitochondrial respiratory chain. The present invention also
provides nucleic acids from C. elegans encoding glucose phosphate
isomerase, gene Y87G2A.8 (GPI-1, SEQ ID NO: 5, Accession No.
CAB60430.1, 68% identical to human ortholog GPI, Accession No.
NM.sub.13 000175). The present invention also provides gene and
gene products shown in Tables 5 and 6, and human homologs thereof.
The present invention also provides homologs of these conserved
proteins that are found in mammals, such as humans, and which are
known to those of skill in the art. The invention therefore
provides methods of screening for compounds, e.g., small molecules,
antibodies, antisense molecules, and ribozyme, that are capable of
modulating lifespan in adult eukaryotes, in particular, in mammals,
e.g., for lifespan enhancement and treatment of premature
aging.
[0043] Furthermore, it was discovered that administration of
antimycin, a mitochondrial respiration inhibitor, substantially
increased lifespan in the model C. elegans.
[0044] The present invention includes other genes and gene products
that regulate the aging process in C. elegans. One such gene,
encodes a GTPase, gene T23H2.5; (SEQ ID NO: 6, Accession No.
AAC48200.1). Inhibition of GTPase results in a longer lifespan.
[0045] Four other genes and gene products that regulate aging in C.
elegans are also provided: llw-1, gene Y54G11A.8 (SEQ ID NO: 7,
Accession No. CAA22452); llw-2, gene F59E12.10 (SEQ ID NO: 8,
Accession No. AAB54251); and llw-3, gene Y48E1B.1 (SEQ ID NO: 9,
Accession No. CAB07688). Also included is llw4, gene F45H10.4 (SEQ
ID NO: 10; Accession No. CAB04386). Inhibition of these genes
results in a Long-Lived Worm (llw) phenotype.
[0046] Also using the technique of double stranded RNA inhibition,
a gene encoding a heat shock factor (HSF) or heat shock protein
(HSP) was found to regulate aging in C. elegans. Inhibition of the
HSF gene and gene product resulted in worms that display the
objective characteristic of aging prematurely (described below) and
have short lifespans compared to control worms. HSF is a
transcriptional activator of stress related genes. The present
invention provide nucleic acids from C. elegans encoding HSF-1,
gene Y53C10A.12 (SEQ ID NO: 11, Accession No. CAA22146). As
described above, for RNAi treatments that decrease lifespan,
restoring gene expression should restore a normal lifespan.
Furthermore, overexpression of this gene in C. elegans extends
lifespan.
[0047] It has been discovered that old worms have a number of
characteristic features that are highly recognizable and diagnostic
when the animals are viewed with Nomarski optics. These
characteristics allow for a determination of whether of not a worm
is aging prematurely, aging normally, aging slowly, or not aging at
all, e.g., has a mutation that causes a sick rather than an aging
phenotype. Thus, these objective characteristics can be used as an
assay, in combination with RNAi, for genes, gene products, and
drugs involved in lifespan regulation. The present invention
therefore also provides genes, identified using RNAi, that regulate
the aging process in adult eukaryotes.
[0048] As described above, the present invention provides a method
of using RNAi to identify genes and gene products involved in
lifespan regulation, by screening, e.g., for increased or decreased
mean and median lifespan. The present invention also demonstrates
that Nomarski microscopy provides a powerful way to monitor the
aging process in cells and tissues of C. elegans. This technique
permits an objective of the quality of cells and tissues that make
up most of the body mass. It was found that the tissues of aging
animals have a very characteristic appearance, using a number of
objective criteria, e.g., yolk accumulation, loss of ability to
chew and expel (distended oral and anal cavities), bacterial
packing in the intestine (constipation), necrotic cavities in
tissue, curdled appearing tissue, and germ cell appearance
(graininess, large, well separated nuclei, fewer nuclei, and
cavities). These characteristics allow for a determination of
whether of not a worm is aging prematurely (progeria), aging
normally, aging slowly, or not aging at all, e.g., has a mutation
that causes a sick rather than an aging phenotype. Thus, these
objective characteristics can be used, as an assay for genes and
drugs involved in lifespan regulation. In one embodiment, the
characteristics are used to evaluate an organism or cell that is
treated with RNAi. In another embodiment, the characteristics are
used to evaluate an organism or cell that is genetically mutated.
Using the technique of double stranded RNA inhibition, a gene
encoding Heat Shock Factor (HSF) was found to prevent premature
aging in C. elegans.
[0049] In one aspect, the invention features a method that
includes: (1) providing a library of nucleic acids, each nucleic
acid of the library comprising a segment of a gene of an organism;
and (2) for each member of the library, (a) generating
double-stranded RNA (dsRNA) from the respective member of the
library, (b) providing the dsRNA to a cell or one or more cells of
an organism to provide a dsRNA treated cell or a dsRNA treated
organism, and (b) monitoring an age-associated parameter of the
dsRNA treated cell or the dsRNA treated organism. Exemplary
dsRNA-treated organisms include a nematode, C. elegans, an organism
other than a nematode, Drosophila, zebrafish, and a mammal, e.g., a
mouse. The providing of dsRNA can include feeding the organism the
dsRNA or a bacterium that expresses the dsRNA. Exemplary
dsRNA-treated cells include a Drosophila cell or a mammalian cell,
e.g., a murine, canine, bovine, primate, or human cell. The cell
can be culture in vitro. The age-associated parameter can be any
age-associated parameter, e.g., a parameter described herein such
as lifespan.
[0050] The method can be repeated for a cohort of dsRNA treated
organisms and the age-associated parameter can include a survival
curve for the cohort. The method can further include, for each
member of the library indicated as affecting lifespan of the
organism, evaluating abundance of an RNA or a protein corresponding
to indicated library member or an RNA or protein corresponding to a
gene listed in Tables 5 and 6. The abundance can be evaluated, for
example, in a defined set of cells or in organisms of defined age.
The abundance can be evaluated using an array, e.g., a nucleic acid
microarray. The method can include other features described
herein.
[0051] In another aspect, the invention features a method that
includes: evaluating a relationship between presence or abundance
for each species of a plurality of RNA or protein species with
respect to age of a cell or organism; producing or delivering a
double-stranded RNA to a cell or one or more cells of an organism,
the RNA corresponding to a species whose presence or abundance is
correlated with age; and monitoring an age-associated parameter of
the cell or the one or more cells or the organism. Evaluating the
relationship can include transcriptional profiling using a nucleic
acid array. The plurality of RNA or protein species can include one
or more species corresponding to a gene listed in Tables 5 and 6.
The plurality can include at least 10, 20, 40, 50, 60, 70% or 100%
of the genes listed in Tables 5 and 6. In one embodiment, the
organism is an invertebrate. In another embodiment, the organism is
a vertebrate, e.g., a mammal. Similarly, a cell can be a cell of a
vertebrate (e.g., a mammal, e.g., a human) or an invertebrate,
e.g., a nematode.
[0052] In another aspect, the invention features a method that
includes: contacting a test compound to a living or biochemical
system that includes a target protein selected from the group
consisting of: a respiratory chain component, a heat shock promoter
activator (e.g., an HSF protein or is an artificial chimeric
transcription factor), LLW-1, LLW-2, LLW-3, LLW-4, a small GTPase,
a protein encoded by a gene listed in Table 5 and 6, and mammalian
(e.g., human) homologues thereof; and evaluating a property
associated with the target protein. The method can be used, e.g.,
to evaluating a test compound, e.g., for an effect on lifespan or a
lifespan-related process. In one embodiment, the target protein is
a C. elegans protein, e.g., a protein that includes SEQ ID NO: 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, or 11. In another embodiment, the
target protein is a mammalian protein.
[0053] The living or biochemical system can be a mammalian or
non-mammalian system. In one embodiment, the system includes a cell
extract, e.g., a lysate or fraction of a cell, e.g., a membrane
preparation, a cytoplasmic preparation, or a partially or
completely purified preparation. In another embodiment, the system
includes isolated mitochondria, e.g., a cell extract or fraction
enriched in mitochondria. In still another embodiment, the system
includes a living cell, e.g., cultured cells, e.g., primary cell or
transformed cells. In yet another embodiment, the system includes a
living organism. The organism includes a cell that can express the
target protein.
[0054] The method can further include contacting the test compound
to an organism (e.g., C. elegans, Drosophila, or a mammal, e.g., a
mouse) and evaluating an age-associated parameter of the
organism.
[0055] Exemplary properties include a catalytic parameter;
structural conformation; post-translational modification; redox
state; physical interaction of the target protein with another
protein; metabolite formation or consumption; subcellular
localization of the target protein; in vivo half-life of the target
protein or target protein activity; transcription of a gene
encoding the target protein or translation of the target
protein.
[0056] In one embodiment, the catalytic parameter describes the
catalytic properties of an enzyme, other than the target protein.
For example, the target protein is a substrate of the enzyme. In
one embodiment, the post-translational modification is a
modification of the target protein. In another embodiment, the
post-translational modification is catalyzed by the target protein.
Exemplary post-translational modifications include phosphorylation,
ubiquitination, methylation, acetylation/deacetylation,
geranygeranylation, farnesylation, or proteolytic modification. In
one embodiment, where the property relates to metabolite production
or consumption, the metabolite can be a direct substrate or direct
product of a reaction catalyzed or effected by the target protein.
In another embodiment, where the property relates to metabolite
production or consumption, the metabolite can be an indirect
substrate or indirect product of a reaction catalyzed or effected
by the target protein.
[0057] A culture cell used in the method can include a heterologous
nucleic acid that encodes and expresses the target protein. The
method can further include assessing whether the test compound
directly interacts with the target protein.
[0058] In one embodiment, the target protein is operably linked to
a reporter protein and the evaluating comprises evaluating the
reporter protein.
[0059] In one embodiment, the target protein is a cytochrome, and
the property is redox state of the cytochrome.
[0060] In another aspect, the invention features a method that
includes providing a nematode in which activity of a target protein
is reduced in the organism by RNA interference; expressing a gene
encoding a protein (e.g., candidate protein) that is heterologous
to the organism; and evaluating an age associated parameter of the
organism. The method can further include, prior to the evaluating,
contacting the organism with a test compound. The candidate protein
can be, for example, a mammalian protein. The candidate protein can
be, for example, a respiratory chain component. The method can
include other features described herein.
[0061] In another aspect, the invention features a method that
includes: providing a cell in which activity of a target protein is
reduced in the organism by RNA interference; expressing a gene
encoding a protein (e.g., candidate protein) that is heterologous
to the organism; and evaluating an age associated parameter of the
organism. The method can include other features described
herein.
[0062] In still another aspect, the invention features a method
that includes providing a cell or an organism (e.g., a nematode) in
which activity of a target protein is reduced in the cell or one or
more cells of the organism by RNA interference; contacting the
organism or the cell with a test compound; and evaluating an age
associated parameter of the organism.
[0063] In another aspect, the invention features a method that
includes assessing an age-related parameter of a nematode that (1)
expresses a heterologous gene in at least some cells; and (2) is
deficient in at least some cells for an endogenous activity
provided by a respiratory chain component. In one embodiment, the
heterologous gene is from a non-nematode species, e.g., a mammalian
species. In a related embodiment, the heterologous gene encodes a
variant of a mammalian protein, the variant having between one and
ten substitutions, insertions, or deletions. The heterologous gene
can encode a domain of at least 30 amino acids from a mammalian
protein or a variant thereof, having between one and six
substitutions, insertions, or deletions.
[0064] In one embodiment, the endogenous activity is provided by a
respiratory chain component (e.g., cytochrome C.sub.1 (SEQ ID NO:
1), NADH oxidoreductase (SEQ ID NO: 2), ATP synthase (SEQ ID NO:
3), cytochrome C oxidase (SEQ ID NO: 4), phosphoglucose isomerase
(SEQ ID NO: 5)), a GTPase (SEQ ID NO: 6), LLW-1 (SEQ ID NO: 7),
LLW-2 (SEQ ID NO: 8), LLW-3 (SEQ ID NO: 9); LLW-4 (SEQ ID NO: 10);
HSF-1 (SEQ ID NO: 11), or a protein listed in Table 5 or 6.
[0065] The method can include contacting the organism with a test
compound, e.g., prior to the assessing. The method can include
other features described herein.
[0066] In another aspect, the invention features a method of
characterizing a protein, the method includes: providing a nucleic
acid that encodes a protein having a subject amino acid sequence
that contains at least one substitution, insertion, or deletion
relative to a reference amino acid sequence, wherein the subject
amino acid sequence and the reference amino acid sequence are at
least 70% identical; expressing the nucleic acid in a culture cell
or in an invertebrate cell; and evaluating an age-associated
parameter of the cell, or an organism that includes the cell. The
reference amino acid sequence can be selected from a sequence
described herein, e.g., one of the following sequences cytochrome
C.sub.1 (SEQ ID NO: 1), NADH oxidoreductase (SEQ ID NO: 2), ATP
synthase (SEQ ID NO: 3), cytochrome C oxidase (SEQ ID NO: 4),
phosphoglucose isomerase (SEQ ID NO: 5), GTPase (SEQ ID NO: 6),
LLW-1 (SEQ ID NO: 7), LLW-2 (SEQ ID NO: 8), LLW-3 (SEQ ID NO: 9),
LLW-4 (SEQ ID NO: 10); HSF-1 (SEQ ID NO: 11), the amino acid
sequences of proteins listed in Table 5 and 6, and human
homologues.
[0067] In another aspect, the invention features a C. elegans
nematode that (1) expresses a heterologous gene in at least some
cells, the heterologous gene encoding a heterologous protein (e.g.,
a protein described herein (e.g., a mammalian gene described
herein) that is non-identical to a corresponding endogenous
protein, or a functional domain thereof; and (2) is deficient in at
least some cells for an endogenous activity provided by the
corresponding endogenous protein. For example, the nematode can (1)
express a heterologous gene in at least some cells, the
heterologous gene encoding a respiratory chain component that is
non-identical to the corresponding endogenous respiratory chain
component, or a functional domain thereof; and (2) be deficient in
at least some cells for an endogenous activity provided by the
corresponding endogenous respiratory chain component.
[0068] In one embodiment, the heterologous gene encodes a mammalian
protein. In another embodiment, the heterologous gene encodes a
variant of a mammalian protein, the variant having between one and
ten substitutions, insertions, or deletions. For example, the
heterologous gene encodes at least a domain of at least 30 amino
acids from a mammalian protein or a variant thereof, having between
one and six substitutions, insertions, or deletions. The deficiency
can be mediated by dsRNA, e.g., by RNA interference.
[0069] The invention also features a method that includes assessing
an age-associated parameter of a nematode described herein, e.g., a
nematode described above. In one embodiment, the method further
includes, prior to the assessing, contacting the nematode to a test
compound.
[0070] In another aspect, the invention features a C. elegans
nematode that (1) is deficient in at least some cells for an
endogenous activity, the deficiency generated by dsRNA in the
cells, and (2) has an average lifespan of at least 24, 26, or 28
days (e.g., in the N2 background) or an average lifespan of at
least 25% greater than the average lifespan of an otherwise
identical nematode not contacted with the dsRNA. The nematode is
such that, absent the deficiency, the nematode has an average
lifespan of less than 22, 20, 18, or 16 days. In one embodiment,
the nematode has functional genes for dauer pathway and/or
functional genes for clk-1, gro-1, and/or another gene described
herein. For example, the nematode can be wild-type with respect to
a laboratory standard, e.g., the N2 background or another
background. The dsRNA may include a strand that is complementary to
a nucleic acid encoding a protein described herein, e.g.,
comprising an amino acid sequence selected from the group
consisting of: cytochrome C.sub.1 (SEQ ID NO: 1), NADH
oxidoreductase (SEQ ID NO: 2), ATP synthase (SEQ ID NO: 3),
cytochrome C oxidase (SEQ ID NO: 4), phosphoglucose isomerase (SEQ
ID NO: 5), GTPase (SEQ ID NO: 6), LLW-1 (SEQ ID NO: 7), LLW-2 (SEQ
ID NO: 8), LLW-3 (SEQ ID NO: 9); LLW-4 (SEQ ID NO: 10); and HSF-1
(SEQ ID NO: 11), proteins listed in Table 5 and 6, and human
homologues corresponding to all of the above.
[0071] In another aspect, the invention features a method that
includes: providing a nematode described herein, e.g., a nematode
described above; introducing a heterologous gene that encodes a
polypeptide into the nematode; expressing the heterologous gene in
the nematode or a progeny of the nematode under conditions wherein
the polypeptide is produced; and monitoring an age-associated
parameter of the nematode or the progeny of the nematode. The
heterologous gene can be, e.g., a nematode gene or a mammalian
gene.
[0072] In another aspect, the invention features a method includes:
providing a library of a test agents; contacting each test agent of
the library to cells; evaluating expression of a HSF-regulated gene
in the contacted cells; for each test agent that alters the
expression of a HSF-regulated gene, contacting a test organism with
the test compound; and evaluating an age-associated parameter of
the test organism. Many genes regulated by HSF are known or can be
readily identified. See, e.g., GuhaThakurta et al. Genome Res
12(5):701-12 (2002) and Christians et al. Crit Care Med 2002
Jan;30(1 Suppl):S43-50 (2002).
[0073] In another aspect, the invention features a nucleic acid
vector that includes (1) a coding sequence encoding an amino acid
sequence comprising SEQ ID NO: 7, 8, 9, or 10 or a fragment
thereof, and (2) one or more of the following: (a) a promoter that
is operably linked and heterologous to the coding sequence, and (b)
a second coding sequence encoding a reporter protein or protein
tag, the second coding sequence being operably linked and
heterologous to the coding sequence. The invention also features a
dsRNA (e.g., an isolated dsRNA) that comprises a nucleic acid
segment that is complementary to a C. elegans gene that encodes SEQ
ID NO: 7, 8, 9, or 10, wherein the dsRNA, when administered to a C.
elegans or cell thereof, causes an extension of lifespan of at
least 20%. In one embodiment, the dsRNA is not complementary to a
homologue (e.g., a homologue described herein) of the C. elegans
gene that encodes SEQ ID NO: 7, 8, 9, or 10. The invention also
provides a method that includes providing (e.g., feeding) an adult
nematode a dsRNA described herein. The invention also features an
antibody that binds to SEQ ID NO: 7, 8, 9, or 10. In a related
aspect, the invention features a nucleic acid vector that includes
(1) a coding sequence encoding an amino acid sequence comprising to
a gene listed in Table 5 or 6; and (2) one or more of the
following: (a) a promoter that is operably linked and heterologous
to the coding sequence, and (b) a second coding sequence encoding a
reporter protein or protein tag, the second coding sequence being
operably linked and heterologous to the coding sequence.
[0074] The invention also features a method that includes:
providing a cell, organism, or biochemical system that includes a
subject protein described herein (e.g., a protein comprising an
amino acid sequence selected from the group consisting of:
cytochrome C.sub.1 (SEQ ID NO: 1), NADH oxidoreductase (SEQ ID NO:
2), ATP synthase (SEQ ID NO: 3), cytochrome C oxidase (SEQ ID NO:
4), phosphoglucose isomerase (SEQ ID NO: 5), GTPase (SEQ ID NO: 6),
LLW-1 (SEQ ID NO: 7), LLW-2 (SEQ ID NO: 8), LLW-3 (SEQ ID NO: 9);
LLW-4 (SEQ ID NO: 10); and HSF-1 (SEQ ID NO: 11), and proteins
described in Tables 5 and 6, or human homologues thereof);
contacting an antibody that binds to the subject protein or
antigen-binding fragment thereof to the cell or the organism; and
evaluating an age-associated parameter of the cell, organism, or
biochemical system.
Definitions
[0075] As is known to those of skill in the art, "oxidative
phosphorylation" or "chondrial respiratory chain protein" and
"glycolysis" proteins are involved in metabolic energy pathways
that are universal in eukaryotic biological systems. The terms
"oxidative phosphorylation or mitochondrial respiratory chain
protein," e.g., cytochrome C1 (SEQ ID NO: 1 Accession No.
CAA99820.1); NADH oxidoreductase (SEQ ID NO: 2 Accession No.
AAB522474.1); ATP synthase (SEQ ID NO: 3 Accession No. AAB37654.1);
and cytochrome C oxidase (SEQ ID NO: 4 Accession No. CAB03002.1);
or a "glycolysis protein," e.g., phosphoglucose isomerase, hexose
isomerase, or glucose phosphate isomerase (SEQ ID NO: 5 Accession
No. CAB60430.1); or "GTPase" e.g., (SEQ ID NO: 6, Accession No.
AAC48200.1); or "heat shock factor," e.g. HSF-1 (SEQ ID NO: 1 1,
Accession No. CAA22146); or "Long-lived worm protein," e.g. LLW-1
(SEQ ID NO: 7, Accession No. CAA22452), LLW-2 (SEQ ID NO: 8,
Accession No. AAB54251), LLW-3 (SEQ ID NO: 9, Accession No.
CAB07688), LLW-4 (SEQ ID NO: 10, Accession No. CAB04386), or a
nucleic acid encoding a "mitochondrial respiratory chain protein,
e.g., cytochrome C1, cytochrome C oxidase, NADH oxidoreductase, and
ATP synthase" or a "glycolysis protein, e.g., phosphoglucose
isomerase," or a "GTPase" or a "heat shock factor" or a "long-lived
worm protein, e.g., llw-1, llw-2, llw-3, llw-4" as well as the
genes and gene products listed in Table 5 and Table 6 refer to
eukaryotic and mammalian nucleic acids and polypeptide polymorphic
variants, alleles, mutants, and interspecies homologs that: (1)
have an amino acid sequence that has greater than about 50% amino
acid sequence identity, preferably 60%, 65%, 70%, 75%, 80%, 85%,
90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 99% or greater amino
acid sequence identity, preferably over a region of over a region
of at least about 25, 50, 100, 200, 500, 1000, or more amino acids,
to an amino acid sequence encoded by the C. elegans genes provided
herein; (2) bind to antibodies, e.g., polyclonal antibodies, raised
against an immunogen comprising an amino acid sequence of a C.
elegans mitochondrial respiratory chain, glycolysis protein,
GTPase, heat shock factor, or long-lived worm protein, or gene
product listed in Table 5 or 6 provided herein, and conservatively
modified variants thereof; (3) specifically hybridize under
moderately stringent hybridization conditions to an anti-sense
strand corresponding to a nucleic acid sequence encoding an C.
elegans mitochondrial respiratory chain, glycolysis protein,
GTPase, heat shock factor, or long-lived worm protein or gene
listed in Table 5 or Table 6 as provided herein, and conservatively
modified variants thereof; (4) have a nucleic acid sequence that
has greater than about 50%, preferably greater than about 60%, 65%,
70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98% or 99% or higher nucleotide sequence identity, preferably
over a region of at least about 25, 50, 100, 200, 500, 1000, or
more nucleotides, to a C. elegans mitochondrial respiratory chain,
glycolysis protein, GTPase, heat shock factor, or long-lived worm
protein as provided herein; and/or (5) sequences that genetically
complement the C. elegans loss of function of a mitochondrial
respiratory chain, glycolysis protein, GTPase, heat shock factor,
or long-lived worm protein or gene listed in Table 5 or Table 6, as
provided herein. A polynucleotide or polypeptide sequence is
typically from a eukaryote, e.g., an invertebrate, vertebrate, or
plant, preferably a mammal including, but not limited to, primate,
e.g., human; rodent, e.g., rat, mouse, hamster; cow, pig, horse,
sheep, or any mammal. The nucleic acids and proteins of the
invention include both naturally occurring or recombinant
molecules. The sequence of the C. elegans genome can be found in
Science 282:2012-2018 (1998).
[0076] The definition explicitly includes the human or mammalian
homologues or counterparts of each C. elegans aging associated gene
or protein described herein, e.g., cytochrome C1, gene C54G4.8 (SEQ
ID NO: 1, Accession No. CAA99820.1), NADH oxidoreductase, gene
T10E9.7 (SEQ ID NO: 2, Accession No. AAB522474.1), ATP synthase,
gene F27C1.7 (SEQ ID NO: 3, Accession No. AAB37654.1), cytochrome C
oxidase, gene F26E4.9 (SEQ ID NO: 4, Accession No. CAB03002.1),
GTPase, gene T23H2.5; (SEQ ID NO: 6, Accession No. AAC48200.1)
llw-1, gene Y54G11A.8 (SEQ ID NO: 7, Accession No. CAA22452);
llw-2, gene F59E12.10 (SEQ ID NO: 8, Accession No. AAB54251);
llw-3, gene Y48E1B.1 (SEQ ID NO: 9, Accession No. CAB07688); llw-4,
gene F45H10.4 (SEQ ID NO: 10, Accession No. CAB04386); HSF-1, gene
Y53C10A.12 (SEQ ID NO: 11, Accession No. CAA22146), and the genes
and gene products listed in Tables 5 and 6.
[0077] The phrase "functional effects" in the context of assays for
testing compounds that modulate activity of aging associated genes
and proteins includes the determination of a parameter that is
indirectly (e.g., upstream or downstream biochemical or genetic
effects) or directly under the influence of aging associated
proteins, e.g., a chemical or phenotypic effect, such as the
ability to increase or decrease lifespan (see, e.g., Kenyon et al.,
Nature 366:461-464 (1993); Hsin & Kenyon, Nature 399:362-366
(1999); Apfeld & Kenyon, Cell 95:199-210 (1998); and Lin et
al., Nature Genet. 28:139-145 (2001)) or, e.g., a physical effect
such as ligand, cofactor or substrate binding or inhibition of
ligand, cofactor or substrate binding. A functional effect
therefore includes ligand, cofactor and substrate binding activity;
changes in gene expression and gene expression levels in cells;
changes in post transcriptional modification of a protein, e.g.,
phosphorylation or glycosylation; reporter gene or marker
expression; changes in abundance and cellular localization;
enzymatic activity; cellular half life; redox state; and structural
conformation, etc.; and age-associated parameters, i.e.,
characteristics of young or old cells or organisms such as stress
resistance, lifespan, doubling time, telomere length, physiological
characteristics, appearance, disease states, etc. "Functional
effects" include in vitro, in vivo, and ex vivo activities. The
functional effect can be measured in a host cell, organelle (e.g.,
isolated mitochondria), host cell membrane, isolated organelle
membrane (e.g., isolated mitochondrial membrane), cellular extract,
organelle extract (e.g., mitochondrial extract) or host
organism.
[0078] By "determining the functional effect" is meant assaying for
a compound that increases or decreases a parameter that is
indirectly or directly under the influence of aging associated
proteins or genes, e.g., measuring physical and chemical or
phenotypic effects. Such functional effects can be measured by any
means known to those skilled in the art, e.g., changes in
spectroscopic characteristics (e.g., fluorescence, absorbance,
refractive index); hydrodynamic (e.g., shape); chromatographic; or
solubility properties for the protein; measuring inducible markers
or transcriptional activation of the protein; measuring binding
activity or binding assays, e.g. binding to antibodies; measuring
changes in ligand binding activity; measuring cellular
proliferation or lifespan; measuring cell surface marker
expression; measurement of changes in protein levels for associated
sequences; measurement of RNA stability; phosphorylation or
dephosphorylation; signal transduction, e.g., receptor-ligand
interactions, second messenger concentrations (e.g., cAMP, IP3, or
intracellular Ca.sup.2+); identification of downstream or reporter
gene expression (CAT, luciferase, .beta.-gal, GFP and the like),
e.g., via chemiluminescence, fluorescence, colorimetric reactions,
antibody binding, inducible markers, and ligand binding assays.
[0079] "Ligand" refers to a molecule that is specifically bound by
a protein.
[0080] "Substrate" refers to a molecule that binds to an enzyme and
is part of a specific chemical reaction catalyzed by the
enzyme.
[0081] "Cofactor" refers to an additional component required for
activity of an enzyme. (Leninger, Principles of Biochemistry
(1984); Stryer, Biochemistry (1995)). A cofactor may be inorganic
such as Fe, Cu, K, Ni, Mo, Se, Zn, Mn or Mg ions, or an organic
molecule also known as a coenzyme. Coenzymes include flavin adenine
dinucleotide (FAD), nicotinamide adenine dinucleotide (NAD),
nicotinamide adenine dinucleotide phosphate (NADP), heme, coenzyme
A, pyrodoxal phosphate, thiamine pyrophosphate,
5'-deoxyadenosylcobalamine, biocytin, tetrahydrofolate, retinal,
and 1,25-dihydroxycholecalciferol. A co-factor can also include a
protein subunit bound to the co-factor.
[0082] "Inhibitors", "activators", and "modulators" of aging
associated genes and proteins are used to refer to activating,
inhibitory, or modulating molecules identified using in vitro and
in vivo assays of aging associated proteins and genes. Inhibitors
are compounds that, e.g., bind to, partially or totally block
activity, decrease, prevent, delay activation, inactivate,
desensitize, or down regulate the activity or expression of aging
associated proteins and genes, e.g., antagonists. "Activators" are
compounds that increase, open, activate, facilitate, enhance
activation, sensitize, agonize, or up regulate aging associated
proteins. Inhibitors, activators, or modulators also include
genetically modified versions of aging associated proteins and
genes, e.g., versions with altered activity, as well as naturally
occurring and synthetic ligands, antagonists, agonists, antibodies,
antisense molecules, ribozymes, small chemical molecules and the
like. Such assays for inhibitors and activators include, e.g.,
expressing aging associated proteins in vitro, in cells, or cell
membranes, applying putative modulator compounds, and then
determining the functional effects on activity, as described
above.
[0083] Samples or assays comprising aging associated proteins and
genes that are treated with a potential activator, inhibitor, or
modulator are compared to control samples without the inhibitor,
activator, or modulator to examine the extent of inhibition.
Control samples (untreated with inhibitors) are assigned a relative
protein or gene activity value of 100%. Inhibition of aging
associated proteins or genes is achieved when the activity value
relative to the control is about 80%, preferably 50%, more
preferably 25-0%. Activation of aging associated proteins or genes
is achieved when the activity value relative to the control
(untreated with activators) is 110%, more preferably 150%, more
preferably 200-500% (i.e., two to five fold higher relative to the
control), more preferably 1000-3000% higher.
[0084] The term "test compound" or "drug candidate" or "modulator"
or grammatical equivalents as used herein describes any molecule,
either naturally occurring or synthetic, e.g., protein,
oligopeptide (e.g., from about 5 to about 25 amino acids in length,
preferably from about 10 to 20 or 12 to 18 amino acids in length,
preferably 12, 15, or 18 amino acids in length), small organic
molecule, polysaccharide, lipid, fatty acid, polynucleotide,
oligonucleotide, etc., to be tested for the capacity to directly or
indirectly modulation lymphocyte activation. The test compound can
be in the form of a library of test compounds, such as a
combinatorial or randomized library that provides a sufficient
range of diversity. Test compounds are optionally linked to a
fusion partner, e.g., targeting compounds, rescue compounds,
dimerization compounds, stabilizing compounds, addressable
compounds, and other functional moieties. Conventionally, new
chemical entities with useful properties are generated by
identifying a test compound (called a "lead compound") with some
desirable property or activity, e.g., inhibiting activity, creating
variants of the lead compound, and evaluating the property and
activity of those variant compounds. Often, high throughput
screening (HTS) methods are employed for such an analysis.
[0085] A "small organic molecule" refers to an organic molecule,
either naturally occurring or synthetic, that has a molecular
weight of more than about 50 daltons and less than about 2500
daltons, preferably less than about 2000 daltons, preferably
between about 100 to about 1000 daltons, more preferably between
about 200 to about 500 daltons. "Biological sample" include
sections of tissues such as biopsy and autopsy samples, and frozen
sections taken for histologic purposes. Such samples include blood,
sputum, tissue, cultured cells, e.g., primary cultures, explants,
and transformed cells, stool, urine, etc. A biological sample is
typically obtained from a eukaryotic organism, e.g., C. elegans,
most preferably a mammal such as a primate e.g., chimpanzee or
human; cow; dog; cat; a rodent, e.g., guinea pig, rat, mouse; or a
rabbit.
[0086] The terms "identical" or percent "identity," in the context
of two or more nucleic acids or polypeptide sequences, refer to two
or more sequences or subsequences that are the same or have a
specified percentage of amino acid residues or nucleotides that are
the same (i.e., about 50% identity, preferably 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or
higher identity over a specified region (e.g., the C. elegans
proteins provided herein), when compared and aligned for maximum
correspondence over a comparison window or designated region) as
measured using a BLAST or BLAST 2.0 sequence comparison algorithms
with default parameters described below, or by manual alignment and
visual inspection. Such sequences are then said to be
"substantially identical." This definition also refers to, or may
be applied to, the compliment of a test sequence. The definition
also includes sequences that have deletions and/or additions, as
well as those that have substitutions. As described below, the
preferred algorithms can account for gaps and the like. Preferably,
identity exists over a region that is at least about 25 amino acids
or nucleotides in length, or more preferably over a region that is
50-100 amino acids or nucleotides in length.
[0087] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are entered into a computer, subsequence coordinates are
designated, if necessary, and sequence algorithm program parameters
are designated. Preferably, default program parameters can be used,
or alternative parameters can be designated. The sequence
comparison algorithm then calculates the percent sequence
identities for the test sequences relative to the reference
sequence, based on the program parameters.
[0088] A "comparison window", as used herein, includes reference to
a segment of any one of the number of contiguous positions selected
from the group consisting of from 20 to 600, usually about 50 to
about 200, more usually about 100 to about 150 in which a sequence
may be compared to a reference sequence of the same number of
contiguous positions after the two sequences are optimally aligned.
Methods of alignment of sequences for comparison are well-known in
the art. Optimal alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith &
Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment
algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),
by the search for similarity method of Pearson & Lipman, Proc.
Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized
implementations of these algorithms (GAP, BESTFIT, FASTA, and
TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group, 575 Science Dr., Madison, Wis.), or by manual
alignment and visual inspection (see, e.g., Current Protocols in
Molecular Biology (Ausubel et al., eds. 1995 supplement)).
[0089] A preferred example of algorithm that is suitable for
determining percent sequence identity and sequence similarity are
the BLAST and BLAST 2.0 algorithms, which are described in Altschul
et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J.
Mol. Biol. 215:403-410 (1990), respectively. BLAST and BLAST 2.0
are used, with the parameters described herein, to determine
percent sequence identity for the nucleic acids and proteins of the
invention. Software for performing BLAST analyses is publicly
available through the National Center for Biotechnology
Information. This algorithm involves first identifying high scoring
sequence pairs (HSPs) by identifying short words of length W in the
query sequence, which either match or satisfy some positive-valued
threshold score T when aligned with a word of the same length in a
database sequence. T is referred to as the neighborhood word score
threshold (Altschul et al., supra). These initial neighborhood word
hits act as seeds for initiating searches to find longer HSPs
containing them. The word hits are extended in both directions
along each sequence for as far as the cumulative alignment score
can be increased. Cumulative scores are calculated using, for
nucleotide sequences, the parameters M (reward score for a pair of
matching residues; always >0) and N (penalty score for
mismatching residues; always <0). For amino acid sequences, a
scoring matrix is used to calculate the cumulative score. Extension
of the word hits in each direction are halted when: the cumulative
alignment score falls off by the quantity X from its maximum
achieved value; the cumulative score goes to zero or below, due to
the accumulation of one or more negative-scoring residue
alignments; or the end of either sequence is reached. The BLAST
algorithm parameters W, T, and X determine the sensitivity and
speed of the alignment. The BLASTN program (for nucleotide
sequences) uses as defaults a wordlength (W) of 11, an expectation
(E) of 10, M=5, N=-4 and a comparison of both strands. For amino
acid sequences, the BLASTP program uses as defaults a wordlength of
3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see
Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915
(1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and
a comparison of both strands. "Nucleic acid" refers to
deoxyribonucleotides or ribonucleotides and polymers thereof in
either single- or double-stranded form. The term encompasses
nucleic acids containing known nucleotide analogs or modified
backbone residues or linkages, which are synthetic, naturally
occurring, and non-naturally occurring, which have similar binding
properties as the reference nucleic acid, and which are metabolized
in a manner similar to the reference nucleotides. Examples of such
analogs include, without limitation, phosphorothioates,
phosphoramidates, methyl phosphonates, chiral-methyl phosphonates,
2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).
[0090] Unless otherwise indicated, a particular nucleic acid
sequence also implicitly encompasses conservatively modified
variants thereof (e.g., degenerate codon substitutions) and
complementary sequences, as well as the sequence explicitly
indicated. Specifically, degenerate codon substitutions may be
achieved by generating sequences in which the third position of one
or more selected (or all) codons is substituted with mixed-base
and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res.
19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608
(1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The
term nucleic acid is used interchangeably with gene, cDNA, mRNA,
oligonucleotide, and polynucleotide.
[0091] A particular nucleic acid sequence also implicitly
encompasses "splice variants." Similarly, a particular protein
encoded by a nucleic acid implicitly encompasses any protein
encoded by a splice variant of that nucleic acid. "Splice
variants," as the name suggests, are products of alternative
splicing of a gene. After transcription, an initial nucleic acid
transcript may be spliced such that different (alternate) nucleic
acid splice products encode different polypeptides. Mechanisms for
the production of splice variants vary, but include alternate
splicing of exons. Alternate polypeptides derived from the same
nucleic acid by read-through transcription are also encompassed by
this definition. Any products of a splicing reaction, including
recombinant forms of the splice products, are included in this
definition.
[0092] The terms "polypeptide," "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residue is an artificial chemical mimetic of a
corresponding naturally occurring amino acid, as well as to
naturally occurring amino acid polymers and non-naturally occurring
amino acid polymer.
[0093] The term "amino acid" refers to naturally occurring and
synthetic amino acids, as well as amino acid analogs and amino acid
mimetics that function in a manner similar to the naturally
occurring amino acids. Naturally occurring amino acids are those
encoded by the genetic code, as well as those amino acids that are
later modified, e.g., hydroxyproline, .gamma.-carboxyglutamate, and
O-phosphoserine. Amino acid analogs refers to compounds that have
the same basic chemical structure as a naturally occurring amino
acid, i.e., an .alpha. carbon that is bound to a hydrogen, a
carboxyl group, an amino group, and an R group, e.g., homoserine,
norleucine, methionine sulfoxide, methionine methyl sulfonium. Such
analogs have modified R groups (e.g., norleucine) or modified
peptide backbones, but retain the same basic chemical structure as
a naturally occurring amino acid. Amino acid mimetics refers to
chemical compounds that have a structure that is different from the
general chemical structure of an amino acid, but that functions in
a manner similar to a naturally occurring amino acid.
[0094] Amino acids may be referred to herein by either their
commonly known three letter symbols or by the one-letter symbols
recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
Nucleotides, likewise, may be referred to by their commonly
accepted single-letter codes. "Conservatively modified variants"
applies to both amino acid and nucleic acid sequences. With respect
to particular nucleic acid sequences, conservatively modified
variants refers to those nucleic acids which encode identical or
essentially identical amino acid sequences, or where the nucleic
acid does not encode an amino acid sequence, to essentially
identical sequences. Because of the degeneracy of the genetic code,
a large number of functionally identical nucleic acids encode any
given protein. For instance, the codons GCA, GCC, GCG and GCU all
encode the amino acid alanine. Thus, at every position where an
alanine is specified by a codon, the codon can be altered to any of
the corresponding codons described without altering the encoded
polypeptide. Such nucleic acid variations are "silent variations,"
which are one species of conservatively modified variations. Every
nucleic acid sequence herein which encodes a polypeptide also
describes every possible silent variation of the nucleic acid. One
of skill will recognize that each codon in a nucleic acid (except
AUG, which is ordinarily the only codon for methionine, and TGG,
which is ordinarily the only codon for tryptophan) can be modified
to yield a functionally identical molecule. Accordingly, each
silent variation of a nucleic acid which encodes a polypeptide is
implicit in each described sequence with respect to the expression
product, but not with respect to actual probe sequences.
[0095] As to amino acid sequences, one of skill will recognize that
individual substitutions, deletions or additions to a nucleic acid,
peptide, polypeptide, or protein sequence which alters, adds or
deletes a single amino acid or a small percentage of amino acids in
the encoded sequence is a "conservatively modified variant" where
the alteration results in the substitution of an amino acid with a
chemically similar amino acid. Conservative substitution tables
providing functionally similar amino acids are well known in the
art. Such conservatively modified variants are in addition to and
do not exclude polymorphic variants, interspecies homologs, and
alleles of the invention.
[0096] The following eight groups each contain amino acids that are
conservative substitutions for one another: 1) Alanine (A), Glycine
(G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N),
Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I),
Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F),
Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8)
Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins
(1984)).
[0097] Macromolecular structures such as polypeptide structures can
be described in terms of various levels of organization. For a
general discussion of this organization, see, e.g., Alberts et al.,
Molecular Biology of the Cell (3.sup.rd ed., 1994) and Cantor and
Schimmel, Biophysical Chemistry Part I: The Conformation of
Biological Macromolecules (1980). "Primary structure" refers to the
amino acid sequence of a particular peptide. "Secondary structure"
refers to locally ordered, three dimensional structures within a
polypeptide. These structures are commonly known as domains, e.g.,
extracellular domains, transmembrane domains, and cytoplasmic
domains. Domains are portions of a polypeptide that form a compact
unit of the polypeptide and are typically 15 to 350 amino acids
long. Typical domains are made up of sections of lesser
organization such as stretches of .beta.-sheet and .alpha.-helices.
"Tertiary structure" refers to the complete three dimensional
structure of a polypeptide monomer. "Quaternary structure" refers
to the three dimensional structure formed by the noncovalent
association of independent tertiary units. Anisotropic terms are
also known as energy terms.
[0098] A "label" or a "detectable moiety" is a composition
detectable by spectroscopic, photochemical, biochemical,
immunochemical, chemical, or other physical means. For example,
useful labels include .sup.32P, fluorescent dyes, electron-dense
reagents, enzymes (e.g., as commonly used in an ELISA), biotin,
digoxigenin, or haptens and proteins which can be made detectable,
e.g., by incorporating a radiolabel into the peptide or used to
detect antibodies specifically reactive with the peptide.
[0099] The term "recombinant" when used with reference, e.g., to a
cell, or nucleic acid, protein, or vector, indicates that the cell,
nucleic acid, protein or vector, has been modified by the
introduction of a heterologous nucleic acid or protein or the
alteration of a native nucleic acid or protein, or that the cell is
derived from a cell so modified. Thus, for example, recombinant
cells express genes that are not found within the native
(non-recombinant) form of the cell or express native genes that are
otherwise abnormally expressed, under expressed or not expressed at
all.
[0100] The term "heterologous" when used with reference to portions
of a nucleic acid indicates that the nucleic acid comprises two or
more subsequences that are not found in the same relationship to
each other in nature. For instance, the nucleic acid is typically
recombinantly produced, having two or more sequences from unrelated
genes arranged to make a new functional nucleic acid, e.g., a
promoter from one source and a coding region from another source.
Similarly, a heterologous protein indicates that the protein
comprises two or more subsequences that are not found in the same
relationship to each other in nature (e.g., a fusion protein).
[0101] The phrase "stringent hybridization conditions" refers to
conditions under which a probe will hybridize to its target
subsequence, typically in a complex mixture of nucleic acids, but
to no other sequences. Stringent conditions are sequence-dependent
and will be different in different circumstances. Longer sequences
hybridize specifically at higher temperatures. An extensive guide
to the hybridization of nucleic acids is found in Tijssen,
Techniques in Biochemistry and Molecular Biology--Hybridization
with Nucleic Probes, "Overview of principles of hybridization and
the strategy of nucleic acid assays" (1993). Generally, stringent
conditions are selected to be about 5-10.degree. C. lower than the
thermal melting point (T.sub.m) for the specific sequence at a
defined ionic strength pH. The T.sub.m is the temperature (under
defined ionic strength, pH, and nucleic concentration) at which 50%
of the probes complementary to the target hybridize to the target
sequence at equilibrium (as the target sequences are present in
excess, at T.sub.m, 50% of the probes are occupied at equilibrium).
Stringent conditions may also be achieved with the addition of
destabilizing agents such as formamide. For selective or specific
hybridization, a positive signal is at least two times background,
preferably 10 times background hybridization.
[0102] Exemplary stringent hybridization conditions can be as
following: 50% formamide, 5.times.SSC, and 1% SDS, incubating at
42.degree. C., or, 5.times.SSC, 1% SDS, incubating at 65.degree.
C., with wash in 0.2.times.SSC, and 0.1% SDS at 65.degree. C. For
PCR, a temperature of about 36.degree. C. is typical for low
stringency amplification, although annealing temperatures may vary
between about 32.degree. C. and 48.degree. C. depending on primer
length. For high stringency PCR amplification, a temperature of
about 62.degree. C. is typical, although high stringency annealing
temperatures can range from about 50.degree. C. to about 65.degree.
C., depending on the primer length and specificity. Typical cycle
conditions for both high and low stringency amplifications include
a denaturation phase of 90.degree. C.-95.degree. C. for 30 sec.-2
min., an annealing phase lasting 30 sec.-2 min., and an extension
phase of about 72.degree. C. for 1-2 min. Protocols and guidelines
for low and high stringency amplification reactions are provided,
e.g., in Innis et al. (1990) PCR Protocols, A Guide to Methods and
Applications, Academic Press, Inc. N.Y.).
[0103] Nucleic acids that do not hybridize to each other under
stringent conditions are still substantially identical if the
polypeptides which they encode are substantially identical. This
occurs, for example, when a copy of a nucleic acid is created using
the maximum codon degeneracy permitted by the genetic code. In such
cases, the nucleic acids typically hybridize under moderately
stringent hybridization conditions. Exemplary "moderately stringent
hybridization conditions" include a hybridization in a buffer of
40% formamide, 1 M NaCl, 1% SDS at 37.degree. C., and a wash in
1.times.SSC at 45.degree. C. A positive hybridization is at least
twice background. Those of ordinary skill will readily recognize
that alternative hybridization and wash conditions can be utilized
to provide conditions of similar stringency. Additional guidelines
for determining hybridization parameters are provided in numerous
reference, e.g., and Current Protocols in Molecular Biology, ed.
Ausubel, et al
[0104] "Antibody" refers to a polypeptide comprising a framework
region from an immunoglobulin gene or fragments thereof that
specifically binds and recognizes an antigen. The recognized
immunoglobulin genes include the kappa, lambda, alpha, gamma,
delta, epsilon, and mu constant region genes, as well as the myriad
immunoglobulin variable region genes. Light chains are classified
as either kappa or lambda. Heavy chains are classified as gamma,
mu, alpha, delta, or epsilon, which in turn define the
immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
Typically, the antigen-binding region of an antibody will be most
critical in specificity and affinity of binding.
[0105] An exemplary immunoglobulin (antibody) structural unit
comprises a tetramer. Each tetramer is composed of two identical
pairs of polypeptide chains, each pair having one "light" (about 25
kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each
chain defines a variable region of about 100 to 110 or more amino
acids primarily responsible for antigen recognition. The terms
variable light chain (V.sub.L) and variable heavy chain (V.sub.H)
refer to these light and heavy chains respectively.
[0106] Antibodies exist, e.g., as intact immunoglobulins or as a
number of well-characterized fragments produced by digestion with
various peptidases. Thus, for example, pepsin digests an antibody
below the disulfide linkages in the hinge region to produce
F(ab)'.sub.2, a dimer of Fab which itself is a light chain joined
to V.sub.H-C.sub.H 1 by a disulfide bond. The F(ab)'.sub.2 may be
reduced under mild conditions to break the disulfide linkage in the
hinge region, thereby converting the F(ab)'.sub.2 dimer into an
Fab' monomer. The Fab' monomer is essentially Fab with part of the
hinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993).
While various antibody fragments are defined in terms of the
digestion of an intact antibody, one of skill will appreciate that
such fragments may be synthesized de novo either chemically or by
using recombinant DNA methodology. Thus, the term antibody, as used
herein, also includes antibody fragments either produced by the
modification of whole antibodies, or those synthesized de novo
using recombinant DNA methodologies (e.g., single chain Fv) or
those identified using phage display libraries (see, e.g.,
McCafferty et al., Nature 348:552-554 (1990))
[0107] For preparation of antibodies, e.g., recombinant,
monoclonal, or polyclonal antibodies, many technique known in the
art can be used (see, e.g., Kohler & Milstein, Nature
256:495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983);
Cole et al., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy,
Alan R. Liss, Inc. (1985); Coligan, Current Protocols in Immunology
(1991); Harlow & Lane, Antibodies, A Laboratory Manual (1988);
and Goding, Monoclonal Antibodies: Principles and Practice (2d ed.
1986)). Techniques for the production of single chain antibodies
(U.S. Pat. No. 4,946,778) can be adapted to produce antibodies to
polypeptides of this invention. Also, transgenic mice, or other
organisms such as other mammals, may be used to express humanized
antibodies. Alternatively, phage display technology can be used to
identify antibodies and heteromeric Fab fragments that specifically
bind to selected antigens (see, e.g., McCafferty et al., Nature
348:552-554 (1990); Marks et al., Biotechnology 10:779-783
(1992)).
[0108] A "chimeric antibody" is an antibody molecule in which (a)
the constant region, or a portion thereof, is altered, replaced or
exchanged so that the antigen binding site (variable region) is
linked to a constant region of a different or altered class,
effector function and/or species, or an entirely different molecule
which confers new properties to the chimeric antibody, e.g., an
enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the
variable region, or a portion thereof, is altered, replaced or
exchanged with a variable region having a different or altered
antigen specificity.
[0109] In one embodiment, the antibody is conjugated to an
"effector" moiety. The effector moiety can be any number of
molecules, including labeling moieties such as radioactive labels
or fluorescent labels, or can be a therapeutic moiety. In one
aspect the antibody modulates the activity of the protein.
[0110] The phrase "specifically (or selectively) binds" to an
antibody or "specifically (or selectively) immunoreactive with,"
when referring to a protein or peptide, refers to a binding
reaction that is determinative of the presence of the protein,
often in a heterogeneous population of proteins and other
biologics. Thus, under designated immunoassay conditions, the
specified antibodies bind to a particular protein at least two
times the background and more typically more than 10 to 100 times
background. Specific binding to an antibody under such conditions
requires an antibody that is selected for its specificity for a
particular protein. For example, polyclonal antibodies raised to
aging associated proteins, polymorphic variants, alleles,
orthologs, and conservatively modified variants, or splice
variants, or portions thereof, can be selected to obtain only those
polyclonal antibodies that are specifically immunoreactive with
aging associated proteins and not with other proteins. This
selection may be achieved by subtracting out antibodies that
cross-react with other molecules. A variety of immunoassay formats
may be used to select antibodies specifically immunoreactive with a
particular protein. For example, solid-phase ELISA immunoassays are
routinely used to select antibodies specifically immunoreactive
with a protein (see, e.g., Harlow & Lane, Antibodies, A
Laboratory Manual (1988) for a description of immunoassay formats
and conditions that can be used to determine specific
immunoreactivity).
Assays for Genes and Gene Products that Regulate Aging
[0111] Genetic and other models can be used to identify mutants,
phenotypes (mediated by mutants and by RNAi), genes, and gene
products in the aging process, e.g., RNAi analysis; microarray
analysis; chemical mutagenesis; mammalian complementation assays
for age-associated proteins; yeast two hybrid assays,
immunoprecipitation; alteration in age-associated reporter gene
expression or localization (e.g., daf-2 or daf-16); overexpression,
underexpression, or knockout of gene expression, etc. Suitable
controls include organisms with altered lifespan, e.g., by mutation
or RNAi. These assays can be used with eukaryotic organisms, cells,
and organelles such as mitochondria. The genes and gene products
associated with a mutation are then identified and used to analyze
the aging process at a molecular level. Genes and gene products
that regulate the aging process can be identified under normal
aging conditions. Patterns of gene expression that correlate with
normal or abnormal aging can also be used to identify genes
associated with aging. The aging process has likely been conserved
throughout evolution. Thus, genes and gene products that regulate
the aging process in one species will be useful to identify similar
or orthologous genes and gene products in divergent species.
[0112] A. Manifestations of the Aging Process
[0113] The most obvious disruption of the aging process is a change
in lifespan of an individual. Lifespan can either be increased or
decreased by a mutation in a gene that participates in the aging
process or, as shown here, by another intervention, e.g., RNAi
mediated silencing of such a gene. In addition, for all eukaryotic
organisms other physical characteristics can be used to distinguish
young individuals from older individuals. Thus, at an organismal
level, a mutation that affects the aging process will usually
affect the lifespan of an individual and may also affect other
aging characteristics of that individual. Such manifestations of
the aging process are known as "age-associated parameters," e.g.,
indicia from Nomarski analysis, stress resistance, appearance,
physiological changes, disease states, loss of doubling capacity,
changes in differentiated phenotype, indirect effects such as
fusion protein expression and localization or posttranscriptional
modification, etc., are described in more detail below.
[0114] Those of skill in the art will recognize that the aging
process can also be manifested at an organismal level or at a
cellular level. While a list of characteristics of aging is
provided below, it is not exhaustive and other characteristics of
the aging process may also be analyzed within the scope of the
present invention.
[0115] Characteristics of aging can be distinguished at the
organismal level and may be species specific. For example,
characteristics of older human individuals include skin wrinkling,
graying of the hair, baldness, cataracts, hypermelanosis,
osteoporosis, cerebral cortical atrophy, lymphoid depletion, thymic
atrophy, increased incidence of diabetes type II, atherosclerosis,
cancer, and heart disease (Nehlin et al., Annals NY Acad. Sci.,
980:176-179 (2000)). Other characteristics of mammalian aging
include the following: weight loss; lordokyphosis (hunchback
spine); absence of vigor; lymphoid atrophy; decreases in bone
density, dermal thickness, and subcutaneous adipose tissue;
decreased ability to tolerate stresses, such as wound healing,
anesthesia, and response to hematopoietic precursor cell ablation;
sparse hair; liver pathologies; atrophy of intestinal villi; skin
ulceration; amyloid deposits; and joint diseases (Tyner et al.,
Nature 415:45-53 (2002)).
[0116] Careful observation reveals characteristics of the aging
process in other eukaryotes, including invertebrates. For example,
characteristics of aging in the model nematode C. elegans as
observed by Nomarski analysis include slow movement, flaccidity,
yolk accumulation, intestinal autofluorescence (lipofuscin), loss
of ability to chew and expel (distended oral and anal cavities),
necrotic cavities in tissue, curdled appearing tissue, and germ
cell appearance (graininess, large, well separated nuclei, fewer
nuclei, and cavities).
[0117] Characteristics of aging can also be observed in cultured
cells and also in mitochondria. Note that many of these
characteristics can also be observed in animals. Normal eukaryotic
cells have a defined lifespan when taken from the organism grown in
culture. These "primary" tissue culture cells are cells that have
neither been immortalized nor acquired a transformed phenotype. The
primary cells will divide a defined number of times in culture and
then die (reviewed in Campisi, Exper. Geron. 36:6-7-618 (2001)).
Cellular aging is also characterized by changes other than loss of
doubling capability, e.g. changes in apoptotic death and changes in
differentiated phenotypes (Id.). In some cases, cellular
characteristics of aging can also be observed in immortalized or
transformed cell lines. Aging cells also show stress resistance,
e.g., free radical generation and H.sub.2O.sub.2 resistance.
Age-related bio-markers, gene, and protein expression patterns may
also be used to determine or measure aging.
[0118] Finally, aging can be assessed indirectly, by an aging
related functional effects (phenotypic, physical, and chemical
effects), e.g., gene expression (e.g., transcript abundance),
protein abundance/localization/modification state, chromatin
structure, signal transduction, second messenger levels, marker
expression, phosphorylation, posttranscriptional modification,
reporter gene expression, reporter or fusion protein localization,
etc. Such effects can often be monitored when examining upstream or
downstream genetic or biochemical pathways of an aging associated
gene. Such effects can also be monitored using the aging associated
gene.
[0119] In one embodiment, a test compound is contacted to one or
more cells of an organism or one or more culture cells, and the one
or more cells, or the entire organism is evaluated. In particular,
a characteristic of aging (e.g., a direct observation or an
aging-related functional effect) can be evaluated to determine the
test compound has an affect on aging or an aging-related process
such as stress resistance or metabolism.
[0120] B. Isolation of Genes Associated with Aging
[0121] Those of skill in the art will recognize that aging
associated nucleic acids and proteins may be conserved in divergent
species. Thus, the sequence of a nucleic acid or protein associated
with aging in one species can be used to identify aging associated
nucleic acids and proteins from other species, as well as genetic
and biochemical pathways for the aging associated genes. For
example, using methods described in this specification, aging
associated genes identified in C. elegans can be used to identify
aging associated genes or proteins in humans or other higher
eukaryotes.
[0122] Isolation of Genes and Gene Products Associated with Aging
Using Classical Genetic Methods.
[0123] Using classical genetic methods (random genomic
mutagenesis), aging mutants are be generated by mutagenesis. The
mutagenesis protocol will depend on the organism. For example, some
eukaryotic organisms can be randomly mutagenized chemically by
treatment with compounds like ethane methyl sulfonate (EMS) or can
be mutagenized by exposure to UV or gamma irradiation. Preferably,
these compounds would be used on organisms such as mammalian cells,
yeast, C. elegans, Drosophila melanogaster, or zebrafish.
[0124] Mutants in the aging process will preferably be
characterized by an increase or a decrease in lifespan. Mutants in
the aging process will also preferably exhibit a temporal change in
expression of an aging characteristic, including those listed
above. Those of skill in the art will recognize that mutants can be
generated in many ways depending on the organism and phenotypes
studied. Typically, the mutagenesis process decreases, increases,
or changes gene activity. Examples of such mutants include age-1,
daf-2, and daf-16 in C. elegans.
[0125] Isolation of Genes and Gene Products Associated with Aging
Using Gene Inactivation.
[0126] In another embodiment, aging mutants are made by
inactivation of a gene of interest, using methods other than
classical genetic mutagenesis methods. The gene of interest can be
inactivated, e.g., using dsRNA inhibition, by using antisense
technology, or can be inactivated by homologous recombination. The
inactivation can take place in a multicellular organism or in
cultured cells. For example, the p66 gene has been removed from
mice using homologous recombination, creating a mouse with a longer
lifespan than wildtype. Transgenic mice of interest which show
lifespan increase include Ames dwarf mutant mice, p66(-/-) knockout
mice, alpha MUPA and MGMT transgenic mice (see, e.g., Anisimov,
Mech Aging Dev. 122:1221-1255 (2001); Lithgow & Andersen,
Bioessays 22:410-413 (2000)).
[0127] dsRNA inhibition can also be used to screen a large number
of genes for a phenotype. The method is preferably done in an
organism whose genome has been sequenced. DNA fragments
corresponding to predicted genes are cloned into a vector between
two bacterial promoters in inverted orientation. The library is
then transformed into a bacterial strain capable of expressing the
DNA fragments. The transformed bacteria or the library DNA alone is
then introduced into the experimental organism. If desired,
inducible promoters can be used and expression of the inhibitory
dsRNA can be induced during a particular time of development or
under desired conditions.
[0128] A preferred embodiment uses a library whose members each
include a DNA fragment from C. elegans. Each library member is
transformed into E. coli and the E. coli fed to the worms. The DNA
fragments are under the control of T7 promoters. The bacteria
express a T7 polymerase that is inducible by IPTG, rendering
expression of the inhibitory dsRNA inducible by IPTG.
[0129] Isolation of Genes and Gene Products Associated with Aging
Using Overexpression.
[0130] In another embodiment, aging mutants are made by
overexpressing a gene associated with aging, using methods other
than classical genetic mutagenesis methods. The gene associated
with aging is cloned into a vector under the control of a promoter
appropriate for the experimental system. The expression vector is
then introduced into the experimental system. The overexpression
can take place in either a multicellular organism or in cultured
cells.
[0131] Isolation of Genes and Gene Products Associated with Aging
Using Naturally Occurring Mutants.
[0132] Aging mutants can also occur naturally. Those of skill in
the art will recognize that such mutants do exist and can be used
in the present invention. For example, in humans, several premature
aging syndromes have been characterized including Werner syndrome,
Hutchinson-Guilford disease, Bloom's syndrome, Cockayne's syndrome,
ataxia telangiectasia, and Down's syndrome. Where appropriate,
cells from an individual afflicted with an aging syndrome can be
studied, rather than the whole organism.
[0133] Isolation of Genes and Gene Products Associated with Aging
Using Genetic or Biochemical Pathways Known to Regulate Aging.
[0134] Genetic analysis can also be used to delineate regulatory
pathways and determine functional relationships between genes and
gene products. In the case of a complex biological process such as
aging, more than one regulatory pathway may regulate the aging
process. Those of skill in the art will recognize that genetic
analysis of mutants can be used to characterize regulatory pathways
and determine relationships between genes. Of course, it also
possible to use RNA interference to modulate gene activity in
analyzing the regulatory pathways and relationships.
[0135] An example of genetic analysis of a regulatory pathway is
found in C. elegans. The daf-2 gene encodes an insulin/IGF-1
receptor homologue. Mutations that lower the level of daf-2 result
in animals that have enhanced lifespans. (For review see Guarente
and Kenyon, Nature 408:255-262 (2000)). daf-16 encodes a forkhead
transcription factor homologue that acts downstream of daf-2 and is
required for daf-2 activity. daf-16 mutants have short lifespans.
Newly isolated mutations can be analyzed for interaction with the
daf2/daf16 pathway. In that way, genes and gene products can be
assigned to a regulatory pathway.
[0136] In addition, genes that interact with the pathway can be
identified by using an appropriate mutant screen. For example, the
C. elegans protein DAF-16 is a transcriptional activator. A fusion
protein between DAF-16 and green fluorescent protein (DAF-16/GFP)
can be used to identify the cellular location of the protein. In
wild-type animals the protein is localized throughout cells. In
long-lived daf-2 mutants, DAF-16 is localized to the nucleus.
[0137] Those of skill in the art will recognize that the
localization of DAF-1 6/GFP can be used to identify mutants that
perturb the daf2/daf16 pathway. Localization of DAF-16/GFP to the
nucleus can be used to screen for drugs that enhance lifespan or
mutations that enhance lifespan.
[0138] A similar fusion using an end product of the pathway,
superoxide dismutase (SOD-3), was constructed. Long lived mutants
of the daf-2/daf-16 pathway expressed SOD-3/GFP at a higher level
than wild-type worms. Levels of fluorescence from SOD-3/GFP can be
followed by microscopy. Those of skill in the art will recognize
that expression of SOD-3/GFP can be used to screen for long-lived
mutants.
[0139] Isolation of Genes and Gene Products Associated with Aging
Using Changes in Expression Levels.
[0140] Those of skill in the art will recognize that levels of
messenger RNA can be measured during the aging process. For aging
associated proteins, changes in mRNA levels can be detected either
during normal aging process or when comparing an aging mutant to a
wild-type individual. Changes in mRNA levels can be measured using
techniques known to those of skill in the art, including
microarrays, northern blots, and RT PCR.
[0141] Aging associated genes can be identified through the use of
microarrays where changes in expression of mRNA levels under
different conditions or at different times of development can be
assayed (see Tables 5 and 6). mRNA levels can also be analyzed in
aging mutants to identify genes that are affected by increases or
decreases in lifespan.
[0142] Microarrays are made by methods known to those of skill in
the art, or are purchased. Gene expression profiles for the genes
described herein can be generated and used for comparison to
identify other age-associated genes. The profile can be generated
using a microarray, or by other means. The profiles can be derived
from animals, cells, mitochondria, or other suitable sources
expressing the genes of interest, e.g., RNAi treated cells or
animals. Such profiles can be stored as computer files and analyzed
or compared to identify additional genes using algorithms known to
those of skill in the art.
[0143] Moreover, a gene identified by any method, e.g., transcript
or protein profiling, RNAi, or genetic mutation, can then by
analyzed by one of the other methods. For example, the activity of
a gene whose transcription is correlated with aging can altered
using RNAi. Further, chromosomal deficiencies and genetic mutations
can be identified in the gene of interest. These exemplary
alterations can be used to evaluate the contribution of a gene or
gene product to the aging phenotype. The functional relevance of
genes so identified can be tested with mutants or RNAi.
Computer Assisted Drug Design and Profiling
[0144] Yet another assay for compounds that modulate aging involves
computer assisted drug design, in which a computer system is used
to generate a three-dimensional structure of an aging associated
protein based on the structural information encoded by the amino
acid or nucleic acid sequence. The input amino acid sequence
interacts directly and actively with a pre-established algorithm in
a computer program to yield secondary, tertiary, and quaternary
structural models of the protein. The models of the protein
structure are then examined to identify regions of the structure
that have the ability to bind, e.g., ligands, substrates,
cofactors, etc. These regions are then used to identify ligands
that bind to the protein.
[0145] The three-dimensional structural model of the protein is
generated by entering an aging associated protein amino acid
sequences of at least 25, 50, 75 or 100 amino acid residues or
corresponding nucleic acid sequences encoding an aging associated
protein into the computer system. The amino acid sequence
represents the primary sequence or subsequence of each of the
proteins, which encodes the structural information of the protein.
At least 25, 50, 75, or 100 residues of the amino acid sequence (or
a nucleotide sequence encoding at least about 25, 50, 75 or 100
amino acids) are entered into the computer system from computer
keyboards, computer readable substrates that include, but are not
limited to, electronic storage media (e.g., magnetic diskettes,
tapes, cartridges, and chips), optical media (e.g., CD ROM),
information distributed by internet sites, and by RAM. The
three-demensional structural model of the aging-associated protein
is then generated by the interaction of the amino acid sequence and
the computer system, using software known to those of skill in the
art. The resulting three-dimensional computer model can then be
saved on a computer readable substrate. For example,
three-dimensional models of the structures of a number of proteins
described here are known and can be used to model homologs and
interactions with other chemical compounds. See, e.g., Damberger et
al. Protein Sci. 1994 Oct;3(10):1806-21. (HSF structure); Harrison
et al. Science. 1994 Jan 14;263(5144):224-7 (HSF structure); Lange
et al. Proc Natl Acad Sci USA. 2002 Mar 5;99(5):2800-5 (cytochrome
bcl complex); Iwata, et al., Science. 1998 Jul 3;281(5373):64-71;
Gibbons et al., Nat Struct Biol. 2000 Nov;7(11):1055-61
(F(1)-ATPase); Faig et al., (2001) Structure (Camb). 2001
Aug;9(8):659-67 (NAD(P)H:quinone oxidoreductase 1); Ingelman et al.
Biochemistry. 1999 Jun 1;38(22):7040-9. (NAD(P)H:flavin
oxidoreductase).
[0146] The amino acid sequence represents a primary structure that
encodes the information necessary to form the secondary, tertiary
and quaternary structure of the aging associated protein. The
software looks at certain parameters encoded by the primary
sequence to generate the structural model. These parameters are
referred to as "energy terms," or anisotropic terms and primarily
include electrostatic potentials, hydrophobic potentials, solvent
accessible surfaces, and hydrogen bonding. Secondary energy terms
include van der Waals potentials. Biological molecules form the
structures that minimize the energy terms in a cumulative fashion.
The computer program is therefore using these terms encoded by the
primary structure or amino acid sequence to create the secondary
structural model.
[0147] The tertiary structure of the protein encoded by the
secondary structure is then formed on the basis of the energy terms
of the secondary structure. The user at this point can enter
additional variables such as whether the protein is membrane bound
or soluble, its location in the body, and its cellular location,
e.g., cytoplasmic, surface, or nuclear. These variables along with
the energy terms of the secondary structure are used to form the
model of the tertiary structure. In modeling the tertiary
structure, the computer program matches hydrophobic faces of
secondary structure with like, and hydrophilic faces of secondary
structure with like.
[0148] Once the structure has been generated, potential ligand and
substrate binding regions are identified by the computer system.
Three-dimensional structures for potential ligands are generated by
entering amino acid or nucleotide sequences or chemical formulas of
compounds, as described above. The three-dimensional structure of
the potential ligand is then compared to that of the aging
associated protein to identify ligands that bind to the aging
associated protein, orthologs thereof, etc. Binding affinity
between the protein and ligands is determined using energy terms to
determine which ligands have an enhanced probability of binding to
the protein.
[0149] Computer systems are also used to screen for mutations,
polymorphic variants, alleles and interspecies homologs of the
aging associated protein or gene. Such mutations can be associated
with disease states. Once the variants are identified, diagnostic
assays can be used to identify patients having such mutated genes
associated with disease states. Identification of the mutated aging
associated protein involves receiving input of a first nucleic
acid, e.g., SEQ ID NOS: 1-11, and genes disclosed in Tables 5 and 6
and orthologs or conservatively modified versions thereof. The
sequence is entered into the computer system as described above.
The first nucleic acid or amino acid sequence is then compared to a
second nucleic acid or amino acid sequence that has substantial
identity to the first sequence. The second sequence is entered into
the computer system in the manner described above. Once the first
and second sequences are compared, nucleotide or amino acid
differences between the sequences are identified. Such sequences
can represent allelic differences in aging associated protein,
e.g., human genes and mutations associated with disease states. The
first and second sequences described above can be saved on a
computer readable substrate.
[0150] Nucleic acids encoding aging associated proteins can be used
with high density oligonucleotide array technology (e.g.,
GeneChip.TM.) to identify family members and homologs, orthologs,
alleles, conservatively modified variants, and polymorphic variants
in this invention. In the case where the homologs being identified
are linked to a known disease, they can be used with GeneChip.TM.
as a diagnostic tool in detecting the disease in a biological
sample, see, e.g., Gunthand et al., AIDS Res. Hum. Retroviruses 14:
869-876 (1998); Kozal et al., Nat. Med. 2:753-759 (1996); Matson et
al., Anal. Biochem. 224:110-106 (1995); Lockhart et al., Nat.
Biotechnol. 14:1675-1680 (1996); Gingeras et al., Genome Res.
8:435-448 (1998); Hacia et al., Nucleic Acids Res. 26:3865-3866
(1998).
[0151] In another aspect, the invention features a computer medium
having a plurality of digitally encoded data records. Each data
record includes a value representing the level of expression of one
or more age-associated gene as described herein in a sample, and a
descriptor of the sample. The level of expression can relate to
mRNA level and/or protein levels. The record can further include an
aging-associated parameter as described herein. The descriptor of
the sample can be indicate a subject from which the sample was
derived (e.g., a patient, a mutant animal), a treatment (e.g., RNAi
treatment), or a location of the sample. In one embodiment, the
data record further includes values representing the level of
expression of genes and proteins other than an age-associated gene
of the invention (e.g., other genes associated with an
aging-disorder, or other genes on an array). The data record can be
structured as a table, e.g., a table that is part of a database
such as a relational database (e.g., a SQL database of the Oracle
or Sybase database environments).
[0152] Also featured is a method of evaluating a sample. The method
includes providing a sample, e.g., from the subject, and
determining an expression profile of the sample, wherein the
profile includes a value representing the level expression of an
age-associated gene described herein. The method can further
include comparing the value or the profile (i.e., multiple values)
to a reference value or reference profile. The gene expression
profile of the sample can be obtained by any of the methods
described herein (e.g., by providing a nucleic acid from the sample
and contacting the nucleic acid to an array). The method can be
used to infer a longevity-associated phenotype in a subject wherein
an increase or decrease expression of an age-associated gene
described herein is an indication that the subject has or is
disposed to having an altered longevity-associated phenotype. The
method can be used to monitor a treatment for an aging in a
subject. For example, the gene expression profile can be determined
for a sample from a subject undergoing treatment. The profile can
be compared to a reference profile or to a profile obtained from
the subject prior to treatment or prior to onset of the disorder
(see, e.g., Golub et al., Science 286:531 (1999)).
Isolation of Nucleic Acids Encoding Aging Associated Proteins
[0153] This invention can include use of routine techniques in the
field of recombinant genetics. Basic texts disclosing the general
methods of use in this invention include Sambrook et al., Molecular
Cloning, A Laboratory Manual (2nd ed. 1989); Kriegler, Gene
Transfer and Expression: A Laboratory Manual (1990); and Current
Protocols in Molecular Biology (Ausubel et al., eds., 1994)).
[0154] Aging associated protein-encoding nucleic acids, polymorphic
variants, orthologs, and alleles can be isolated using the C.
elegans genes provided herein using, e.g., moderate or low
stringent hybridization conditions, by screening libraries, by
analyzing a sequence database, and/or by synthetic gene
construction. Alternatively, expression libraries can be used to
clone aging associated proteins, polymorphic variants, orthologs,
and alleles by detecting expressed homologs immunologically with
antisera or purified antibodies made against C. elegans or
mammalian aging associated proteins or portions thereof or by
complementation, e.g., of a C. elegans phenotype. In a preferred
embodiment, human nucleic acid libraries are screened for
homologues of C. elegans genes or proteins that are associated with
aging.
[0155] To make a cDNA library, one can choose a source that is rich
in the RNA of choice. The mRNA is then made into cDNA using reverse
transcriptase, ligated into a recombinant vector, and transfixed
into a recombinant host for propagation, screening and cloning.
Methods for making and screening cDNA libraries are well known
(see, e.g., Gubler & Hoffman, Gene 25:263-269 (1983); Sambrook
et al., supra; Ausubel et al., supra).
[0156] For a genomic library, the DNA is extracted from the tissue
and either mechanically sheared or enzymatically digested to yield
fragments of about 12-20 kb. The fragments are then separated by
gradient centrifugation from undesired sizes and are constructed in
bacteriophage lambda vectors. These vectors and phage are packaged
in vitro. Recombinant phage are analyzed by plaque hybridization as
described in Benton & Davis, Science 196:180-182 (1977). Colony
hybridization is carried out as generally described in Grunstein et
al., Proc. Natl. Acad. Sci. USA., 72:3961-3965 (1975).
[0157] An alternative method of isolating aging associated
protein-encoding nucleic acid and their orthologs, alleles,
mutants, polymorphic variants, and conservatively modified variants
combines the use of synthetic oligonucleotide primers and
amplification of an RNA or DNA template (see U.S. Pat. Nos.
4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and
Applications (Innis et al., eds, 1990)). Methods such as polymerase
chain reaction (PCR) and ligase chain reaction (LCR) can be used to
amplify nucleic acid sequences of aging associated protein-encoding
genes directly from mRNA, from cDNA, from genomic libraries or cDNA
libraries. Degenerate oligonucleotides can be designed to amplify
homologs using the sequences provided herein. Restriction
endonuclease sites can be incorporated into the primers. Polymerase
chain reaction or other in vitro amplification methods may also be
useful, for example, to clone nucleic acid sequences that code for
proteins to be expressed, to make nucleic acids to use as probes
for detecting the presence of aging associated protein encoding
mRNA in physiological samples, for nucleic acid sequencing, or for
other purposes. Genes amplified by the PCR reaction can be purified
from agarose gels and cloned into an appropriate vector.
[0158] Gene expression of aging associated proteins can also be
analyzed by techniques known in the art, e.g., reverse
transcription and amplification of mRNA, isolation of total RNA or
poly A.sup.+ RNA, northern blotting, dot blotting, in situ
hybridization, RNase protection, high density polynucleotide array
technology, e.g., and the like.
[0159] Nucleic acids encoding aging associated proteins can be used
with high density oligonucleotide array technology (e.g.,
GeneChip.TM.) to identify aging associated proteins, orthologs,
alleles, conservatively modified variants, and polymorphic variants
in this invention. In the case where the homologs being identified
are linked to modulation of aging associated proteins, they can be
used with GeneChip.TM. as a diagnostic tool in detecting the
disease in a biological sample, see, e.g., Gunthand et al., AIDS
Res. Hum. Retroviruses 14: 869-876 (1998); Kozal et al., Nat. Med.
2:753-759 (1996); Matson et al., Anal. Biochem. 224:110-106 (1995);
Lockhart et al., Nat. Biotechnol. 14:1675-1680 (1996); Gingeras et
al., Genome Res. 8:435-448 (1998); Hacia et al., Nucleic Acids Res.
26:3865-3866 (1998).
[0160] The gene for aging associated proteins are typically cloned
into intermediate vectors before transformation into prokaryotic or
eukaryotic cells for replication and/or expression. These
intermediate vectors are typically prokaryote vectors, e.g.,
plasmids, or shuttle vectors.
Expression in Prokaryotes and Eukaryotes
[0161] To obtain high level expression of a cloned gene, such as
those cDNAs encoding aging associated proteins, one typically
subclones aging associated protein encoding nucleic acids into an
expression vector that contains a strong promoter to direct
transcription, a transcription/translation terminator, and a
ribosome binding site for translational initiation. Suitable
bacterial promoters are well known in the art and described, e.g.,
in Sambrook et al., and Ausubel et al, supra. Bacterial expression
systems for expressing aging associated proteins are available in,
e.g., E. coli, Bacillus sp., and Salmonella (Palva et al., Gene
22:229-235 (1983); Mosbach et al., Nature 302:543-545 (1983). Kits
for such expression systems are commercially available. Eukaryotic
expression systems for mammalian cells, yeast, and insect cells are
well known in the art and are also commercially available.
[0162] Selection of the promoter used to direct expression of a
heterologous nucleic acid depends on the particular application.
The promoter is preferably positioned about the same distance from
the heterologous transcription start site as it is from the
transcription start site in its natural setting. As is known in the
art, however, some variation in this distance can be accommodated
without loss of promoter function.
[0163] In addition to the promoter, the expression vector typically
contains a transcription unit or expression cassette that contains
all the additional elements required for the expression of aging
associated protein encoding nucleic acid in host cells. A typical
expression cassette thus contains a promoter operably linked to the
nucleic acid sequence encoding aging associated proteins and
signals required for efficient polyadenylation of the transcript,
ribosome binding sites, and translation termination. Additional
elements of the cassette may include enhancers and, if genomic DNA
is used as the structural gene, introns with functional splice
donor and acceptor sites.
[0164] In addition to a promoter sequence, the expression cassette
should also contain a transcription termination region downstream
of the structural gene to provide for efficient termination. The
termination region may be obtained from the same gene as the
promoter sequence or may be obtained from different genes.
[0165] The particular expression vector used to transport the
genetic information into the cell is not particularly critical. Any
of the conventional vectors used for expression in eukaryotic or
prokaryotic cells may be used. Standard bacterial expression
vectors include plasmids such as pBR322 based plasmids, pSKF,
pET23D, and fusion expression systems such as MBP, GST, and LacZ.
Epitope tags can also be added to recombinant proteins to provide
convenient methods of isolation, e.g., c-myc.
[0166] Expression vectors containing regulatory elements from
eukaryotic viruses are typically used in eukaryotic expression
vectors, e.g., SV40 vectors, papilloma virus vectors, and vectors
derived from Epstein-Barr virus. Other exemplary eukaryotic vectors
include pMSG, pAV009/A.sup.+, pMTO10/A.sup.+, pMAMneo-5,
baculovirus pDSVE, and any other vector allowing expression of
proteins under the direction of the CMV promoter, SV40 early
promoter, SV40 later promoter, metallothionein promoter, murine
mammary tumor virus promoter, Rous sarcoma virus promoter,
polyhedrin promoter, or other promoters shown effective for
expression in eukaryotic cells.
[0167] Expression of proteins from eukaryotic vectors can be also
be regulated using inducible promoters. With inducible promoters,
expression levels are tied to the concentration of inducing agents,
such as tetracycline or ecdysone, by the incorporation of response
elements for these agents into the promoter. Generally, high level
expression is obtained from inducible promoters only in the
presence of the inducing agent; basal expression levels are
minimal. Inducible expression vectors are often chosen if
expression of the protein of interest is detrimental to eukaryotic
cells.
[0168] Some expression systems have markers that provide gene
amplification such as thymidine kinase and dihydrofolate reductase.
Alternatively, high yield expression systems not involving gene
amplification are also suitable, such as using a baculovirus vector
in insect cells, with mitochondrial respiratory chain protein
encoding sequences and glycolysis protein encoding sequence under
the direction of the polyhedrin promoter or other strong
baculovirus promoters.
[0169] The elements that are typically included in expression
vectors also include a replicon that functions in E. coli, a gene
encoding antibiotic resistance to permit selection of bacteria that
harbor recombinant plasmids, and unique restriction sites in
nonessential regions of the plasmid to allow insertion of
eukaryotic sequences. The particular antibiotic resistance gene
chosen is not critical, any of the many resistance genes known in
the art are suitable. The prokaryotic sequences are preferably
chosen such that they do not interfere with the replication of the
DNA in eukaryotic cells, if necessary.
[0170] Standard transfection methods are used to produce bacterial,
mammalian, yeast or insect cell lines that express large quantities
of aging associated proteins, which are then purified using
standard techniques (see, e.g., Colley et al., J. Biol. Chem.
264:17619-17622 (1989); Guide to Protein Purification, in Methods
in Enzymology, vol. 182 (Deutscher, ed., 1990)). Transformation of
eukaryotic and prokaryotic cells are performed according to
standard techniques (see, e.g., Morrison, J. Bact. 132:349-351
(1977); Clark-Curtiss & Curtiss, Methods in Enzymology
101:347-362 (Wu et al., eds, 1983).
[0171] Any of the well-known procedures for introducing foreign
nucleotide sequences into host cells may be used. These include the
use of calcium phosphate transfection, polybrene, protoplast
fusion, electroporation, biolistics, liposomes, microinjection,
plasma vectors, viral vectors and any of the other well known
methods for introducing cloned genomic DNA, cDNA, synthetic DNA or
other foreign genetic material into a host cell (see, e.g.,
Sambrook et al., supra). It is only necessary that the particular
genetic engineering procedure used be capable of successfully
introducing at least one gene into the host cell capable of
expressing aging associated proteins.
[0172] After the expression vector is introduced into the cells,
the transfected cells are cultured under conditions favoring
expression of aging associated proteins, which is recovered from
the culture using standard techniques identified below.
[0173] Expression vectors with appropriate regulatory sequences can
also be used to express a heterologous gene in a nematode. In one
example, the expression vector is injected in the gonad of the
nematode, and the vector is incorporated, e.g., as an
extra-chromosomal array in progeny of the nematode. The vector can
further include a second gene (e.g., a marker gene) that indicates
the presence of the vector. For example, the heterologous gene can
be a mammalian gene, e.g., a mammalian cDNA, or a fragment
thereof.
Purification of Aging Associated Proteins
[0174] Either naturally occurring or recombinant aging associated
proteins can be purified for use in functional assays. Naturally
occurring aging associated proteins can be purified, e.g., from
human tissue. Recombinant aging associated proteins can be purified
from any suitable expression system.
[0175] Aging associated proteins may be purified to substantial
purity by standard techniques, including selective precipitation
with such substances as ammonium sulfate; column chromatography,
immunopurification methods, and others (see, e.g., Scopes, Protein
Purification: Principles and Practice (1982); U.S. Pat. No.
4,673,641; Ausubel et al., supra; and Sambrook et al., supra).
[0176] A number of procedures can be employed when recombinant
aging associated proteins are being purified. For example, proteins
having established molecular adhesion properties can be reversible
fused to aging associated proteins. With the appropriate ligand,
aging associated proteins can be selectively adsorbed to a
purification column and then freed from the column in a relatively
pure form. The fused protein is then removed by enzymatic activity.
Finally, aging associated proteins could be purified using
immunoaffinity columns.
[0177] A. Purification of Aging Associated Proteins from
Recombinant Bacteria
[0178] Recombinant proteins are expressed by transformed bacteria
in large amounts, typically after promoter induction; but
expression can be constitutive. Promoter induction with IPTG is one
example of an inducible promoter system. Bacteria are grown
according to standard procedures in the art. Fresh or frozen
bacteria cells are used for isolation of protein.
[0179] Proteins expressed in bacteria may form insoluble aggregates
("inclusion bodies"). Several protocols are suitable for
purification of aging associated protein inclusion bodies. For
example, purification of inclusion bodies typically involves the
extraction, separation and/or purification of inclusion bodies by
disruption of bacterial cells, e.g., by incubation in a buffer of
50 mM TRIS/HCL pH 7.5, 50 mM NaCl, 5 mM MgCl.sub.2, 1 mM DTT, 0.1
mM ATP, and 1 mM PMSF. The cell suspension can be lysed using 2-3
passages through a French Press, homogenized using a Polytron
(Brinkman Instruments) or sonicated on ice. Alternate methods of
lysing bacteria are apparent to those of skill in the art (see,
e.g., Sambrook et al., supra; Ausubel et al., supra).
[0180] If necessary, the inclusion bodies are solubilized, and the
lysed cell suspension is typically centrifuged to remove unwanted
insoluble matter. Proteins that formed the inclusion bodies may be
renatured by dilution or dialysis with a compatible buffer.
Suitable solvents include, but are not limited to urea (from about
4 M to about 8 M), formamide (at least about 80%, volume/volume
basis), and guanidine hydrochloride (from about 4 M to about 8 M).
Some solvents which are capable of solubilizing aggregate-forming
proteins, for example SDS (sodium dodecyl sulfate), 70% formic
acid, are inappropriate for use in this procedure due to the
possibility of irreversible denaturation of the proteins,
accompanied by a lack of immunogenicity and/or activity. Although
guanidine hydrochloride and similar agents are denaturants, this
denaturation is not irreversible and renaturation may occur upon
removal (by dialysis, for example) or dilution of the denaturant,
allowing re-formation of immunologically and/or biologically active
protein. Other suitable buffers are known to those skilled in the
art. Aging associated proteins are separated from other bacterial
proteins by standard separation techniques, e.g., with Ni-NTA
agarose resin.
[0181] Alternatively, it is possible to purify aging associated
proteins from bacteria periplasm. After lysis of the bacteria, when
the aging associated proteins are exported into the periplasm of
the bacteria, the periplasmic fraction of the bacteria can be
isolated by cold osmotic shock in addition to other methods known
to skill in the art. To isolate recombinant proteins from the
periplasm, the bacterial cells are centrifuged to form a pellet.
The pellet is resuspended in a buffer containing 20% sucrose. To
lyse the cells, the bacteria are centrifuged and the pellet is
resuspended in ice-cold 5 mM MgSO.sub.4 and kept in an ice bath for
approximately 10 minutes. The cell suspension is centrifuged and
the supernatant decanted and saved. The recombinant proteins
present in the supernatant can be separated from the host proteins
by standard separation techniques well known to those of skill in
the art.
[0182] B. Standard Protein Separation Techniques for Purifying
Aging Associated Proteins
[0183] Solubility Fractionation
[0184] Often as an initial step, particularly if the protein
mixture is complex, an initial salt fractionation can separate many
of the unwanted host cell proteins (or proteins derived from the
cell culture media) from the recombinant protein of interest. The
preferred salt is ammonium sulfate. Ammonium sulfate precipitates
proteins by effectively reducing the amount of water in the protein
mixture. Proteins then precipitate on the basis of their
solubility. The more hydrophobic a protein is, the more likely it
is to precipitate at lower ammonium sulfate concentrations. A
typical protocol includes adding saturated ammonium sulfate to a
protein solution so that the resultant ammonium sulfate
concentration is between 20-30%. This concentration will
precipitate the most hydrophobic of proteins. The precipitate is
then discarded (unless the protein of interest is hydrophobic) and
ammonium sulfate is added to the supernatant to a concentration
known to precipitate the protein of interest. The precipitate is
then solubilized in buffer and the excess salt removed if
necessary, either through dialysis or diafiltration. Other methods
that rely on solubility of proteins, such as cold ethanol
precipitation, are well known to those of skill in the art and can
be used to fractionate complex protein mixtures.
[0185] Size Differential Filtration
[0186] The molecular weight of the aging associated proteins can be
used to isolate it from proteins of greater and lesser size using
ultrafiltration through membranes of different pore size (for
example, Amicon or Millipore membranes). As a first step, the
protein mixture is ultrafiltered through a membrane with a pore
size that has a lower molecular weight cut-off than the molecular
weight of the protein of interest. The retentate of the
ultrafiltration is then ultrafiltered against a membrane with a
molecular cut off greater than the molecular weight of the protein
of interest. The recombinant protein will pass through the membrane
into the filtrate. The filtrate can then be chromatographed as
described below.
[0187] Column Chromatography
[0188] The aging associated proteins can also be separated from
other proteins on the basis of its size, net surface charge,
hydrophobicity, and affinity for ligands. In addition, antibodies
raised against proteins can be conjugated to column matrices and
the proteins immunopurified. All of these methods are well known in
the art. It will be apparent to one of skill that chromatographic
techniques can be performed at any scale and using equipment from
many different manufacturers (e.g., Pharmacia Biotech).
Immunological Detection of Aging Associated Proteins
[0189] In addition to the detection of aging associated genes and
gene expression using nucleic acid hybridization technology, one
can also use immunoassays to detect aging associated proteins of
the invention. Such assays are useful for screening for modulators
of aging associated proteins, e.g., for regulation of lifespan, as
well as for therapeutic and diagnostic applications. Immunoassays
can be used to qualitatively or quantitatively analyze aging
associated proteins. A general overview of the applicable
technology can be found in Harlow & Lane, Antibodies: A
Laboratory Manual (1988).
[0190] Methods of producing polyclonal and monoclonal antibodies
that react specifically with the aging associated proteins are
known to those of skill in the art (see, e.g., Coligan, Current
Protocols in Immunology (1991); Harlow & Lane, supra; Goding,
Monoclonal Antibodies: Principles and Practice (2d ed. 1986); and
Kohler & Milstein, Nature 256:495-497 (1975). Such techniques
include antibody preparation by selection of antibodies from
libraries of recombinant antibodies in phage or similar vectors, as
well as preparation of polyclonal and monoclonal antibodies by
immunizing rabbits or mice (see, e.g., Huse et al., Science
246:1275-1281 (1989); Ward et al., Nature 341:544-546 (1989)).
[0191] A number of immunogens comprising portions of aging
associated proteins may be used to produce antibodies specifically
reactive with an aging associated protein. For example, recombinant
protein or an antigenic fragment thereof, can be isolated as
described herein. Recombinant protein can be expressed in
eukaryotic or prokaryotic cells as described above, and purified as
generally described above. Recombinant protein is the preferred
immunogen for the production of monoclonal or polyclonal
antibodies. Alternatively, a synthetic peptide derived from the
sequences disclosed herein and conjugated to a carrier protein can
be used an immunogen. Naturally occurring protein may also be used
either in pure or impure form. The product is then injected into an
animal capable of producing antibodies. Either monoclonal or
polyclonal antibodies may be generated, for subsequent use in
immunoassays to measure the protein.
[0192] Methods of production of polyclonal antibodies are known to
those of skill in the art. An inbred strain of mice (e.g., BALB/C
mice) or rabbits is immunized with the protein using a standard
adjuvant, such as Freund's adjuvant, and a standard immunization
protocol. The animal's immune response to the immunogen preparation
is monitored by taking test bleeds and determining the titer of
reactivity to the beta subunits. When appropriately high titers of
antibody to the immunogen are obtained, blood is collected from the
animal and antisera are prepared. Further fractionation of the
antisera to enrich for antibodies reactive to the protein can be
done if desired (see, Harlow & Lane, supra).
[0193] Monoclonal antibodies may be obtained by various techniques
familiar to those skilled in the art. Briefly, spleen cells from an
animal immunized with a desired antigen are immortalized, commonly
by fusion with a myeloma cell (see, Kohler & Milstein, Eur. J.
Immunol. 6:511-519 (1976)). Alternative methods of immortalization
include transformation with Epstein Barr Virus, oncogenes, or
retroviruses, or other methods well known in the art. Colonies
arising from single immortalized cells are screened for production
of antibodies of the desired specificity and affinity for the
antigen, and yield of the monoclonal antibodies produced by such
cells may be enhanced by various techniques, including injection
into the peritoneal cavity of a vertebrate host. Alternatively, one
may isolate DNA sequences which encode a monoclonal antibody or a
binding fragment thereof by screening a DNA library from human B
cells according to the general protocol outlined by Huse, et al.,
Science 246:1275-1281 (1989).
[0194] Monoclonal antibodies and polyclonal sera are collected and
titered against the immunogen protein in an immunoassay, for
example, a solid phase immunoassay with the immunogen immobilized
on a solid support. Typically, polyclonal antisera with a titer of
10.sup.4 or greater are selected and tested for their cross
reactivity against non-specific proteins, using a competitive
binding immunoassay. Specific polyclonal antisera and monoclonal
antibodies will usually bind with a K.sub.d of at least about 0.1
mM, more usually at least about 1 .mu.M, preferably at least about
0.1 .mu.M or better, and most preferably, 0.01 .mu.M or better.
Antibodies specific only for a particular ortholog, such as a human
ortholog, can also be made, by subtracting out other cross-reacting
orthologs from a species such as a non-human mammal.
[0195] Once the specific antibodies against aging associated
proteins are available, the protein can be detected by a variety of
immunoassay methods. In addition, the antibody can be used
therapeutically as aging associated protein modulators, e.g., to
enhance and extend lifespan or to prevent premature aging. For a
review of immunological and immunoassay procedures, see Basic and
Clinical Immunology (Stites & Terr eds., 7.sup.th ed. 1991).
Moreover, the immunoassays of the present invention can be
performed in any of several configurations, which are reviewed
extensively in Enzyme Immunoassay (Maggio, ed., 1980); and Harlow
& Lane, supra.
[0196] It is also possible to use protein arrays to detect an aging
associated protein, e.g., to concurrently detect a plurality of
aging associated proteins. Exemplary methods for producing protein
arrays are provided in De Wildt et al. (2000) Nat. Biotechnol.
18:989-994; Lueking et al. (1999) Anal. Biochem. 270:103-111; Ge
(2000) Nucleic Acids Res. 28, e3, I-VII; MacBeath and Schreiber
(2000) Science 289:1760-1763; WO 0/98534, WO01/83827, WO02/12893,
WO 00/63701, WO 01/40803 and WO 99/51773. In some implementations,
polypeptides (including peptides) are spotted onto discrete
addresses of the array, e.g., at high speed, e.g., using
commercially available robotic apparati, e.g., from Genetic
MicroSystems or BioRobotics. The array substrate can be, for
example, nitrocellulose, plastic, glass, e.g., surface-modified
glass. The array can also include a porous matrix, e.g.,
acrylamide, agarose, or another polymer.
Assays for Modulators of Aging Associated Proteins
[0197] A. Assays
[0198] Modulation of aging associated proteins and genes can be
assessed using a variety of in vitro and in vivo assays, as
described herein, and, such assays can be used to test for
inhibitors and activators of aging associated proteins. Such
modulators of aging associated proteins and genes, which are
involved in aging, are useful for enhancing lifespan or treating
premature aging. Modulators of aging associated proteins and genes
are tested using either recombinant or naturally occurring,
preferably C. elegans, mouse, rat, guinea pig, monkey, or human
aging associated proteins.
[0199] An example of a modulator of the mitochondrial respiratory
chain is antimycin. Antimycin inhibits the transfer of electrons
from complex 2 to complex 3 during mitochondrial respiration. This
drug has been found to extend the lifespan of C. elegans.
Administration of antimycin to adult worms increased lifespan,
while administration of antimycin to worms from hatching through
adulthood substantially increases lifespan. Mean lifespan of
untreated worms was 17 days while animals grown in the presence of
0.1 micromolar antimycin in ethanol had a mean lifespan of 21
days.
[0200] Preferably, the aging associated proteins or genes will have
a C. elegans or a mammalian, e.g., a rat, mouse, guinea pig,
rabbit, monkey, or human sequence. Alternatively, the aging
associated proteins or genes of the assay will be derived from a
eukaryote and include an nucleic acid or amino acid subsequence
having sequence identity to the C. elegans genes and gene products
described herein. Generally, the sequence identity will be at least
30%, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65%, 70%,
75%, 80%, 85%, or 90%, most preferably at least 95%.
[0201] Measurement of modulation of aging phenotype with aging
associated proteins or cells expressing aging associated proteins
or genes, either recombinant or naturally occurring, can be
performed using a variety of assays, in vitro, in vivo, and ex
vivo. A suitable physiological change that affects activity can be
used to assess the influence of a test compound on the polypeptide
or nucleic acid of this invention. When the functional effects are
determined using intact cells or animals, one can also measure a
variety of effects such as, increases or decreases in lifespan,
cellular proliferation, or in the case of signal transduction,
hormone release, transcriptional changes to both known and
uncharacterized genetic markers (e.g., northern blots), changes in
cell metabolism such as cell growth or pH changes, and changes in
intracellular second messengers such as cGMP.
[0202] In a preferred embodiment, aging associated protein or gene
modulators are assayed in vivo by screening in C. elegans or in a
mammalian model system (cellular or animal) for changes in mean and
median lifespan.
[0203] Some aging associated proteins have measurable enzymatic
activity. Thus, enzymatic assays can be performed to identify
compounds that modulate the enzymatic activity. Enzymatic activity
can encompass a chemical reaction carried out by a protein, as well
as binding of substrates, cofactors, regulatory compounds, or
ligands to the protein. It may also be useful to monitor the affect
of a test compound on other properties of the aging associated
protein, e.g., a structural property (e.g., conformation,
oligomerization state, stability, mobility, and the like) or a
cellular property (e.g., cellular localization, accessibility,
clustering, and the like).
[0204] The protein activity and binding capabilities assayed will
depend on the aging associated protein. For example, the following
proteins with known enzymatic activities have been associated with
aging in C. elegans: cytochrome C.sub.1 (gene C54G4.8, SEQ ID NO: 1
Accession No. CAA99820.1), NADH oxidoreductase (gene T10E9.7, SEQ
ID NO: 2 Accession No. AAB522474.1), ATP synthase (gene F27C1.7,
SEQ ID NO: 3 Accession No. AAB37654.1), cytochrome C oxidase (gene
F26E4.9, SEQ ID NO: 4 Accession No. CAB03002.1), phosphoglucose
isomerase (gene Y87G2A.8, SEQ ID NO: 5 Accession No. CAB60430.1), a
GTPase (gene T23H2.5, SEQ ID NO: 6 Accession No. AAC48200.1), and
HSF-1 (gene Y53C10A.12, SEQ ID NO: 11 Accession No. CAA22146), and
the genes and gene products listed in Table 5 and Table 6
[0205] For activity of cytochrome C and C1, binding to the cofactor
heme is important. (Lehninger, Principles of Biochemistry (1984);
Stryer, Biochemistry (1995)). Thus, changes in ability to bind heme
may correlate with a mutant phenotype or change during aging.
Cytochrome C1 is an electron-transferring protein that contains
heme and is found in cytochrome reductase, a membrane-associated
proton pump. Cytochrome C is a water soluble protein that transfers
electrons from cytochrome reductase to cytochrome oxidase. Changes
in electron transport may also be seen during aging or as a result
of an aging mutation. It is possible to monitor the redox state of
the heme cofactor, for example, by spectroscopy.
[0206] NADH oxidoreductase is an oligomeric enzyme complex located
in the inner mitochondrial membrane. (Lehninger, Principles of
Biochemistry (1984); Stryer, Biochemistry (1995)). In C. elegans
inhibition of the 30 Kd subunit resulted in enhanced lifespan.
Thus, assays of NADH oxidoreductase activity could be associated
with aging. It is possible to monitor the redox state of NADH, for
example, by spectroscopy.
[0207] Cytochrome C oxidase catalyzes the transfer of electrons
from cytochrome C to molecular oxygen. (Lehninger, Principles of
Biochemistry (1984); Stryer, Biochemistry (1995)). Those of skill
in the art will recognize that this activity can be assayed
spectrophotometrically. In addition to this activity, changes in
binding to substrates cytochrome C and oxygen may also be
assayed.
[0208] ATP synthase catalyzes the synthesis of ATP from ADP and
orthophosphate. (Lehninger, Principles of Biochemistry (1984);
Stryer, Biochemistry (1995)). The energy of a proton gradient is
used to release the ATP product from its binding site. The ATP
synthase protein is a multisubunit enzyme. Inhibition of the C.
elegans homologue of the delta subunit of ATP synthase resulted in
an enhanced lifespan phenotype. Activity of the delta subunit is
sensitive to the drug oligomycin. Stryer, page 546.
[0209] Phosphoglucose isomerase catalyzes the reversible
isomerization of glucose-6-phosphate and fructose-6-phosphate.
(Lehninger, Principles of Biochemistry (1984); Stryer, Biochemistry
(1995)). The enzyme is involved in glycolysis in most higher
organisms and in gluconeogenesis in mammals. An extracellular role
has been reported for the protein as a nerve growth factor and
cytokine. (Jeffery et al., Biochem. 39:955-964 (2000)).
[0210] A GTPase enzyme catalyzes the hydrolysis of GTP to GDP. The
reaction can be followed using radioactively labeled GTP an
separating the reaction products from the reaction substrates.
[0211] HSF, a transcription factor, activates transcription by
forming a tri-mer, binding to a specific DNA sequence called a
promoter and then recruiting RNA polymerases to begin
transcription. Stress response and heat shock response proteins are
known as chaperoning. Activation of transcription and binding to a
target DNA site can measured in vitro using purified or partially
purified components. Activation of transcription can also be
measured in vivo using a reporter gene construct with these
proteins.
[0212] The functional activities described above do not represent
all of the enzymatic activities that could be found in aging
associated proteins. For example, some aging proteins could act to
down regulate transcription of messenger RNA. Still other aging
proteins may functional, e.g., as a structural scaffold or adaptor
protein, e.g., they may or may not have an enzymatic activity.
[0213] Assays to identify compounds with modulating activity can be
performed in vitro, e.g., in a test tube, or using isolated
membranes, e.g., mitochondrial membranes, or using cellular or
mitochondrial extracts. Exemplary assays can include, for example,
methods described or referenced in Al-Awqati, Annu. Rev. Cell Biol.
2:179-199 (1986); Brand et al., Biol. Rev. Cambridge Philsophic
Soc. 62:141-193 (1987); Capaldi et al., FEBS Lett 138:1-7 (1982);
Casey, Biochim. Biophys. Acta 768:319-347 (1984); Erecinska et al.,
J. Membr. Biol. 70:1-14 (1982); Fillingame, Annu. Rev. Biochem.
49:1079-1113 (1980); Hamamoto, Proc. Natl. Acad. Sci. USA
82:2570-2573 (1985); Hatefi, Annu. Rev. Biochem. 54:1015-1070
(1985); Klingenberg, Trends Biochem. Sci. 4:249-252 (1979); LaNoue
et al., Annu. Rev. Biochem. 48:871-922 (1979); Mitchell, Nature
191:144-148 (1961); Prince, Trends Biochem. Sci. 13:159-160 (1988);
Slater, Trends Biochem. Sci. 8:239-242 (1983); Srere, Trends
Biochem. Sci. 7:375-378 (1982); Tzagoloff, Mitochondria, New York:
Plenum (1982); Weiss et al., Biochem. Soc. Trans. 15:100-102
(1987).
[0214] For example, the aging associated protein or gene is first
contacted with a potential modulator and incubated for a suitable
amount of time, e.g., from 0.5 to 48 hours. In one embodiment,
aging associated protein or gene expression levels are determined
in vitro by measuring the level of protein or mRNA. The level of
protein or nucleic acid is measured using immunoassays such as
western blotting, ELISA and the like with an antibody that
selectively binds to the polypeptide or a fragment thereof. For
measurement of mRNA, amplification, e.g., using PCR, LCR, or
hybridization assays, e.g., northern hybridization, RNAse
protection, dot blotting, are preferred. The level of protein or
mRNA is detected using directly or indirectly labeled detection
agents, e.g., fluorescently or radioactively labeled nucleic acids,
radioactively or enzymatically labeled antibodies, and the like, as
described herein.
[0215] Alternatively, a reporter gene system can be devised using
an aging associated protein promoter operably linked to a reporter
gene such as chloramphenicol acetyltransferase, firefly luciferase,
bacterial luciferase, .beta.-galactosidase and alkaline
phosphatase. Furthermore, the gene or protein of interest can be
used as an indirect reporter via attachment to a second reporter
such as green fluorescent protein (see, e.g., Mistili &
Spector, Nature Biotechnology 15:961-964 (1997)). The reporter
construct is typically transfected into a cell. After treatment
with a potential modulator, the amount of reporter gene
transcription, translation, or activity is measured according to
standard techniques known to those of skill in the art.
[0216] B. Modulators
[0217] The compounds tested as modulators of aging associated
proteins and genes can be any small chemical compound, or a
biological entity, such as a protein, e.g., an antibody, a sugar, a
nucleic acid, e.g., an antisense oligonucleotide or a ribozyme, or
a lipid. Alternatively, modulators can be genetically altered
versions of an aging associated proteins and genes. Typically, test
compounds will be small chemical molecules and peptides, or
antibodies, antisense molecules, or ribozymes. Essentially any
chemical compound can be used as a potential modulator or ligand in
the assays of the invention, although most often compounds can be
dissolved in aqueous or organic (especially DMSO-based) solutions
are used. The assays are designed to screen large chemical
libraries by automating the assay steps and providing compounds
from any convenient source to assays, which are typically run in
parallel (e.g., in microtiter formats on microtiter plates in
robotic assays). It will be appreciated that there are many
suppliers of chemical compounds, including Sigma (St. Louis, Mo.),
Aldrich (St. Louis, Mo.), Sigma-Aldrich (St. Louis, Mo.), Fluka
Chemika-Biochemica Analytika (Buchs Switzerland) and the like.
[0218] In one preferred embodiment, high throughput screening
methods involve providing a combinatorial chemical or peptide
library containing a large number of potential therapeutic
compounds (potential modulator or ligand compounds). Such
"combinatorial chemical libraries" or "ligand libraries" are then
screened in one or more assays, as described herein, to identify
those library members (particular chemical species or subclasses)
that display a desired characteristic activity. The compounds thus
identified can serve as conventional "lead compounds" or can
themselves be used as potential or actual therapeutics.
[0219] A combinatorial chemical library is a collection of diverse
chemical compounds generated by either chemical synthesis or
biological synthesis, by combining a number of chemical "building
blocks" such as reagents. For example, a linear combinatorial
chemical library such as a polypeptide library is formed by
combining a set of chemical building blocks (amino acids) in every
possible way for a given compound length (i.e., the number of amino
acids in a polypeptide compound). Millions of chemical compounds
can be synthesized through such combinatorial mixing of chemical
building blocks.
[0220] Preparation and screening of combinatorial chemical
libraries is well known to those of skill in the art. Such
combinatorial chemical libraries include, but are not limited to,
peptide libraries (see, e.g., U.S. Pat. No. 5,010,175, Furka, Int.
J. Pept. Prot. Res. 37:487-493 (1991) and Houghton et al., Nature
354:84-88 (1991)). Other chemistries for generating chemical
diversity libraries can also be used. Such chemistries include, but
are not limited to: peptoids (e.g., PCT Publication No. WO
91/19735), encoded peptides (e.g., PCT Publication No. WO
93/20242), random bio-oligomers (e.g., PCT Publication No. WO
92/00091), benzodiazepines (e.g., U.S. Pat. No. 5,288,514),
diversomers such as hydantoins, benzodiazepines and dipeptides
(Hobbs et al., Proc. Nat. Acad. Sci. USA 90:6909-6913 (1993)),
vinylogous polypeptides (Hagihara et al., J. Amer. Chem. Soc.
114:6568 (1992)), nonpeptidal peptidomimetics with glucose
scaffolding (Hirschmann et al., J. Amer. Chem. Soc. 114:9217-9218
(1992)), analogous organic syntheses of small compound libraries
(Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)), oligocarbamates
(Cho et al., Science 261:1303 (1993)), and/or peptidyl phosphonates
(Campbell et al., J. Org. Chem. 59:658 (1994)), nucleic acid
libraries (see Ausubel, Berger and Sambrook, all supra), peptide
nucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083),
antibody libraries (see, e.g., Vaughn et al., Nature Biotechnology,
14(3): 309-314 (1996) and PCT/US96/10287), carbohydrate libraries
(see, e.g., Liang et al., Science, 274:1520-1522 (1996) and U.S.
Pat. No. 5,593,853), small organic molecule libraries (see, e.g.,
benzodiazepines, Baum C&EN, January 18, page 33 (1993);
isoprenoids, U.S. Pat. No. 5,569,588; thiazolidinones and
metathiazanones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Pat.
Nos. 5,525,735 and 5,519,134; morpholino compounds, U.S. Pat. No.
5,506,337; benzodiazepines, U.S. Pat. No. 5,288,514, and the
like).
[0221] Devices for the preparation of combinatorial libraries are
commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem
Tech, Louisville Ky., Symphony, Rainin, Woburn, Mass., 433A Applied
Biosystems, Foster City, Calif., 9050 Plus, Millipore, Bedford,
Mass.). In addition, numerous combinatorial libraries are
themselves commercially available (see, e.g., ComGenex, Princeton,
N.J., Asinex, Moscow, Ru, Tripos, Inc., St. Louis, Mo., ChemStar,
Ltd, Moscow, RU, 3D Pharmaceuticals, Exton, Pa., Martek
Biosciences, Columbia, Md., etc.).
[0222] In one embodiment, the invention provides solid phase based
in vitro assays in a high throughput format, where the cell or
tissue expressing aging associated proteins is attached to a solid
phase substrate. In the high throughput assays of the invention, it
is possible to screen up to several thousand different modulators
or ligands in a single day. In particular, each well of a
microtiter plate can be used to run a separate assay against a
selected potential modulator, or, if concentration or incubation
time effects are to be observed, every 5-10 wells can test a single
modulator. Thus, a single standard microtiter plate can assay about
96 modulators. If 1536 well plates are used, then a single plate
can easily assay from about 100- about 1500 different compounds. It
is possible to assay many plates per day; assay screens for up to
about 6,000, 20,000, 50,000, or 100,000 or more different compounds
are possible using the integrated systems of the invention.
[0223] C. Solid State and Soluble High Throughput Assays
[0224] In one embodiment the invention provides soluble assays
using aging associated proteins or genes, or a cell or tissue
expressing aging associated proteins or genes, either naturally
occurring or recombinant. In another embodiment, the invention
provides solid phase based in vitro assays in a high throughput
format, where the aging associated protein or gene is attached to a
solid phase substrate.
[0225] In the high throughput assays of the invention, it is
possible to screen up to several thousand different modulators or
ligands in a single day. In particular, each well of a microtiter
plate can be used to run a separate assay against a selected
potential modulator, or, if concentration or incubation time
effects are to be observed, every 5-10 wells can test a single
modulator. Thus, a single standard microtiter plate can assay about
100 (e.g., 96) modulators. If 1536 well plates are used, then a
single plate can easily assay from about 100- about 1500 different
compounds. It is possible to assay many plates per day; assay
screens for up to about 6,000, 20,000, 50,000, or more than 100,000
different compounds are possible using the integrated systems of
the invention.
[0226] The protein of interest, or a cell or membrane comprising
the protein of interest can be bound to the solid state component,
directly or indirectly, via covalent or non covalent linkage e.g.,
via a tag. The tag can be any of a variety of components. In
general, a molecule which binds the tag (a tag binder) is fixed to
a solid support, and the tagged molecule of interest is attached to
the solid support by interaction of the tag and the tag binder.
[0227] A number of tags and tag binders can be used, based upon
known molecular interactions well described in the literature. For
example, where a tag has a natural binder, for example, biotin,
protein A, or protein G, it can be used in conjunction with
appropriate tag binders (avidin, streptavidin, neutravidin, the Fc
region of an immunoglobulin, etc.) Antibodies to molecules with
natural binders such as biotin are also widely available and
appropriate tag binders; see, SIGMA Immunochemicals 1998 catalogue
SIGMA, St. Louis Mo.). Similarly, any haptenic or antigenic
compound can be used in combination with an appropriate antibody to
form a tag/tag binder pair.
[0228] Synthetic polymers, such as polyurethanes, polyesters,
polycarbonates, polyureas, polyamides, polyethyleneimines,
polyarylene sulfides, polysiloxanes, polyimides, and polyacetates
can also form an appropriate tag or tag binder. Many other tag/tag
binder pairs are also useful in assay systems described herein, as
would be apparent to one of skill upon review of this
disclosure.
[0229] Common linkers such as peptides, polyethers, and the like
can also serve as tags, and include polypeptide sequences, such as
poly gly sequences of between about 5 and 200 amino acids. Such
flexible linkers are known to persons of skill in the art. For
example, poly(ethelyne glycol) linkers are available from
Shearwater Polymers, Inc. Huntsville, Ala. These linkers optionally
have amide linkages, sulfhydryl linkages, or heterofunctional
linkages.
[0230] Tag binders are fixed to solid substrates using any of a
variety of methods currently available. Solid substrates are
commonly derivatized or functionalized by exposing all or a portion
of the substrate to a chemical reagent which fixes a chemical group
to the surface which is reactive with a portion of the tag binder.
For example, groups which are suitable for attachment to a longer
chain portion would include amines, hydroxyl, thiol, and carboxyl
groups. Aminoalkylsilanes and hydroxyalkylsilanes can be used to
functionalize a variety of surfaces, such as glass surfaces. The
construction of such solid phase biopolymer arrays is well
described in the literature. See, e.g., Merrifield, J. Am. Chem.
Soc. 85:2149-2154 (1963) (describing solid phase synthesis of,
e.g., peptides); Geysen et al., J. Immun. Meth. 102:259-274 (1987)
(describing synthesis of solid phase components on pins); Frank
& Doring, Tetrahedron 44:60316040 (1988) (describing synthesis
of various peptide sequences on cellulose disks); Fodor et al.,
Science, 251:767-777 (1991); Sheldon et al, Clinical Chemistry
39(4):718-719 (1993); and Kozal et al., Nature Medicine 2(7):753759
(1996) (all describing arrays of biopolymers fixed to solid
substrates). Non-chemical approaches for fixing tag binders to
substrates include other common methods, such as heat,
cross-linking by UV radiation, and the like.
[0231] Another example of a high-throughput assay does not require
immobilizing a target protein. Such examples include homogenous
assays such as fluorescence resonance energy transfer and
fluorescence polarization. Spectroscopy can also be used in a
variety of ways. Assays can also be used to generate
structure-activity relationships (SAR). A method of analyzing an
aging associated protein can also include assays that may not be
traditionally associated with a particular throughput, e.g.,
certain NMR binding assays (e.g., SAR by NMR), calorimetry,
crystallization, and so forth.
Cellular Transfection and Gene Therapy
[0232] The present invention provides the nucleic acids of aging
associated proteins for the transfection of cells in vitro and in
vivo. These nucleic acids can be inserted into any of a number of
well-known vectors for the transfection of target cells and
organisms as described below. The nucleic acids are transfected
into cells, ex vivo or in vivo, through the interaction of the
vector and the target cell. The nucleic acid, under the control of
a promoter, then expresses a protein of the present invention,
thereby mitigating the effects of absent, partial inactivation, or
abnormal expression of the gene of interest, or increasing lifespan
in a subject with normal gene expression. For example, as described
herein, overexpression of HSF-1 extends adult lifespan. The
compositions are administered to a patient in an amount sufficient
to elicit a therapeutic response in the patient. An amount adequate
to accomplish this is defined as "therapeutically effective dose or
amount."
[0233] Such gene therapy procedures have been used to correct
acquired and inherited genetic defects. The ability to express
artificial genes in humans facilitates the prevention and/or cure
of many important human diseases, including many diseases which are
not amenable to treatment by other therapies (for a review of gene
therapy procedures, see Anderson, Science 256:808-813 (1992); Nabel
& Felgner, TIBTECH 11:211-217 (1993); Mitani & Caskey,
TIBTECH 11:162-166 (1993); Mulligan, Science 926-932 (1993);
Dillon, TIBTECH 11: 167-175 (1993); Miller, Nature 357:455-460
(1992); Van Brunt, Biotechnology 6(10):1149-1154 (1998); Vigne,
Restorative Neurology and Neuroscience 8:35-36 (1995); Kremer &
Perricaudet, British Medical Bulletin 51(1):31-44 (1995); Haddada
et al., in Current Topics in Microbiology and Immunology (Doerfler
& Bohm eds., 1995); and Yu et al., Gene Therapy 1:13-26
(1994)).
Pharmaceutical Compositions and Administration
[0234] Pharmaceutically acceptable carriers are determined in part
by the particular composition being administered (e.g., nucleic
acid, protein, modulatory compounds or transduced cell), as well as
by the particular method used to administer the composition.
Accordingly, there are a wide variety of suitable formulations of
pharmaceutical compositions of the present invention (see, e.g.,
Remington's Pharmaceutical Sciences, 17.sup.th ed., 1989).
Administration can be in any convenient manner, e.g., by injection,
oral administration, inhalation, transdermal application, or rectal
administration.
[0235] Formulations suitable for oral administration can consist of
(a) liquid solutions, such as an effective amount of the packaged
nucleic acid suspended in diluents, such as water, saline or PEG
400; (b) capsules, sachets or tablets, each containing a
predetermined amount of the active ingredient, as liquids, solids,
granules or gelatin; (c) suspensions in an appropriate liquid; and
(d) suitable emulsions. Tablet forms can include one or more of
lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn
starch, potato starch, microcrystalline cellulose, gelatin,
colloidal silicon dioxide, talc, magnesium stearate, stearic acid,
and other excipients, colorants, fillers, binders, diluents,
buffering agents, moistening agents, preservatives, flavoring
agents, dyes, disintegrating agents, and pharmaceutically
compatible carriers. Lozenge forms can comprise the active
ingredient in a flavor, e.g., sucrose, as well as pastilles
comprising the active ingredient in an inert base, such as gelatin
and glycerin or sucrose and acacia emulsions, gels, and the like
containing, in addition to the active ingredient, carriers known in
the art.
[0236] The compound of choice, alone or in combination with other
suitable components, can be made into aerosol formulations (i.e.,
they can be "nebulized") to be administered via inhalation. Aerosol
formulations can be placed into pressurized acceptable propellants,
such as dichlorodifluoromethane, propane, nitrogen, and the
like.
[0237] Formulations suitable for parenteral administration, such
as, for example, by intraarticular (in the joints), intravenous,
intramuscular, intradermal, intraperitoneal, and subcutaneous
routes, include aqueous and non-aqueous, isotonic sterile injection
solutions, which can contain antioxidants, buffers, bacteriostats,
and solutes that render the formulation isotonic with the blood of
the intended recipient, and aqueous and non-aqueous sterile
suspensions that can include suspending agents, solubilizers,
thickening agents, stabilizers, and preservatives. In the practice
of this invention, compositions can be administered, for example,
by intravenous infusion, orally, topically, intraperitoneally,
intravesically or intrathecally. Parenteral administration and
intravenous administration are the preferred methods of
administration. The formulations of commends can be presented in
unit-dose or multi-dose sealed containers, such as ampules and
vials.
[0238] Injection solutions and suspensions can be prepared from
sterile powders, granules, and tablets of the kind previously
described. Cells transduced by nucleic acids for ex vivo therapy
can also be administered intravenously or parenterally as described
above.
[0239] The dose administered to a patient, in the context of the
present invention should be sufficient to effect a beneficial
therapeutic response in the patient over time. The dose will be
determined by the efficacy of the particular vector employed and
the condition of the patient, as well as the body weight or surface
area of the patient to be treated. The size of the dose also will
be determined by the existence, nature, and extent of any adverse
side-effects that accompany the administration of a particular
vector, or transduced cell type in a particular patient.
[0240] In determining the effective amount of the vector to be
administered in the treatment or prophylaxis of conditions owing to
diminished or aberrant expression of the protein of choice, the
physician evaluates circulating plasma levels of the vector, vector
toxicities, progression of the disease, and the production of
anti-vector antibodies. In general, the dose equivalent of a naked
nucleic acid from a vector is from about 1 .mu.g to 100 .mu.g for a
typical 70 kilogram patient, and doses of vectors which include a
retroviral particle are calculated to yield an equivalent amount of
therapeutic nucleic acid.
[0241] For administration, compounds and transduced cells of the
present invention can be administered at a rate determined by the
LD-50 of the inhibitor, vector, or transduced cell type, and the
side-effects of the inhibitor, vector or cell type at various
concentrations, as applied to the mass and overall health of the
patient. Administration can be accomplished via single or divided
doses.
[0242] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended claims.
All publications, patents, and patent applications cited herein are
hereby incorporated by reference in their entirety for all
purposes.
EXAMPLES
[0243] The following examples are provided by way of illustration
only and not by way of limitation. Those of skill in the art will
readily recognize a variety of noncritical parameters that could be
changed or modified to yield essentially similar results.
Example 1:
[0244] Objective Characteristics of Aging Identified Using Nomarski
Differential Interference Contrast Microscopy
[0245] A. Introduction
[0246] In this study, the tissues of wild-type animals were
compared to those of long-lived and short-lived insulin/IGF-1
signaling mutants during the course of their lives. We have found
that Nomarski differential interference contrast (DIC) microscopy
provides an effective, rapid and convenient means of visualizing
many features of tissue aging. Using this method, we have found
that extensive tissue deterioration takes place during aging, not
only in the post-mitotic somatic tissues of the animal, but also in
a mitotic lineage, the germline. Our findings indicate that
insulin/IGF-1 signaling influences lifespan by changing the rate at
which the tissues age, and that this pathway governs the aging not
only of the post-mitotic somatic cells, but mitotic lineages as
well. Interestingly, the majority of animals we have examined whose
adult lifespans were shortened by mutations or certain RNAi
treatments did not look prematurely "old" upon observation with
Nomarski microscopy. However, animals with lifespan mutations that
caused shorter lifespans due to premature aging looked prematurely
old (e.g., hsp). Thus, this technique aids in distinguishing
mutated animals who die young, and mutated animals with premature
aging, even though both classes of animals have shortened adult
lifespans.
[0247] B. Methods
[0248] The following strains were used: N2, daf-16(mu86)I,
daf-2(e1370)III, daf-2(mu150)III, daf-2(e1370)III, deg-1(u38)X,
ced-3(n1286)IV, DH1033 bIs1[vit-2::gfp; rol-6]; sqt-1(sc103),
daf-2(e1370)III, him-5(e1490)V.
[0249] Lifespan Analysis: Wild-type, daf-2 and daf-16 animals
raised at 20.degree. C. were shifted to 25.degree. C. at the L4
molt, and transferred to new plates every other day thereafter
until progeny production ceased. Animals were judged to be dead
when they no longer responded to gentle prodding. Animals that
crawled off the plate, became desiccated on the sides of the plate,
displayed extruded internal organs or died from internally-hatched
progeny were censored. Censored animals were incorporated into the
data set until the day of their disqualification, as described
previously (Apfeld & Kenyon Nature 402:804-9 (1999). Lifespan
analysis of ced-3 mutant animals was conducted at 25.degree. C.
using isogenic N2 from the Horvitz lab, kindly provided by Cori
Bargmann, as a control. Lifespans of daf-2; deg-1 animals were
assayed at 20.degree. C. Statview 5.0.1 (SAS) software was used to
construct lifespan curves, and to determine means and percentiles.
Ages given refer to days of adulthood.
[0250] Lipofuscin: Endogenous gut fluorescence was photographed
using a 525 nm bandpass filter. Images were collected without
automatic gain control in order to preserve the relative intensity
of different animals' fluorescence. 2-, 5- and 10-day old adults
were photographed on the same day in order to avoid effects of
light source variation on apparent fluorescence intensity.
[0251] Visualization of Yolk: The vit-2::GFP fusion strain (see
above) was a kind gift of David Hersch and Barth Grant. Animals
were allowed to age and were photographed using both Nomarski
optics and epifluorescence (525 nm).
[0252] Nomarski Analysis: Animals were placed on a 2% agarose pad
in M9 buffer with 2 mM sodium azide and covered with a coverslip.
Control experiments indicated that sodium azide did not affect the
age-related phenotypes we observed. Delicate older animals of all
genotypes occasionally ruptured and were lost during this process
(approximately 10%). Images were captured using a CCD camera
coupled to Universal Imaging Corporation's MetaMorph Imaging System
(version 3.6). Image files were contrast-balanced and rotated when
necessary using PhotoShop 5.0.
[0253] Quantification of Tissue Damage: C. elegans heads were
photographed as described above. In a blind experiment, photographs
of heads were given a score of 1-5, with 1 representing a youthful,
unsullied appearance, and 2 through 4 denoting low, medium and high
levels of overall deterioration. A rare score of 5 was assigned to
animals so deteriorated as to be nearly unrecognizable.
[0254] Photographs of germ cells in the distal gonad also were
rated on a scale of 1-5 based on these criteria. In addition, these
photographs were also assigned a cumulative value that represented
the presence and extent of several of the correlates of aging,
e.g., graininess; large, well-separated nuclei; cavities; and a
shriveled appearance. Each correlate of aging was given a separate
score, which contributed to the final score. Animals could thus be
compared not only by the extent of age-related degeneration, but
also by the type of changes they exhibited.
[0255] Scores were assigned without knowledge of the age or
genotype of the worm in the photograph. Overall scores were
re-evaluated at least once, and a naive observer was asked to score
a selection of photographs in a double-blind experiment.
[0256] Statistical Analysis of Tissue damage: Nonparametric
analysis of head scores was conducted, using the Kruskal-Wallis
Test to determine if there were significant differences between
multiple groups, followed by a pairwise comparison, the
Mann-Whitney test. All statistical analysis was conducted using
Statview 5.0.1 (SAS) software.
[0257] C. Results
[0258] Decline of Tissue Integrity in Aging Wild-Type Animals
[0259] Nomarski optics are commonly used to observe the development
of C. elegans. In young animals, the nuclei and nucleoli of all
cells are readily visualized using this method. It is also possible
to see the boundaries of certain cells and tissues, such as the
muscles, gonad, epidermal seam cells, and certain neurons. We
observed that in young adults, the cells and tissues appeared
similar to those of late juvenile stages, except that the nuclear
boundaries were less distinct. This lack of definition became more
pronounced as the animals grew older. For example, by day 10 of
adulthood, it was very difficult to see the nuclei of the epidermal
cells. Neuronal nuclei, which have a wrinkled, "raisin-like"
appearance, remained visible throughout the life of the animal,
although they, too, grew less distinct with time. In young animals,
the cytoplasm and nucleoplasm of most cells are smooth and uniform.
However, as the animals grew older, both began to show signs of
deterioration. Necrotic cavities of various sizes appeared, often
containing vibrating particles that appeared to display Brownian
motion. Tissues often acquired a curdled texture.
[0260] In older animals, the pharynx, as well as the anterior and
anal regions of the intestine were frequently distended and packed
with the bacteria. We wondered whether these animals were failing
to chew or expel their food, or whether they might be succumbing to
bacterial infection. To test this, we looked for constipation in
animals grown on plates seeded with bacteria killed by
UV-irradiation. We found that worms grown on killed bacteria also
become highly constipated shortly before death. This suggests that
very aged animals lose the ability to chew or expel their food.
[0261] Older animals also accumulated shiny, mobile patches of a
substance that appeared be yolk. In young animals, yolk is
transported from its site of synthesis in the intestine into the
gonad, where it is incorporated into embryos. It is possible that
yolk accumulates in old animals when the production of embryos
ceases. We examined animals expressing a GFP-tagged yolk protein,
and confirmed that this substance was in fact yolk. We also
observed shiny but less-mobile material in the bodies of worms that
did not show GFP fluorescence. Finally, we also observed increased
intestinal autofluorescence, which is thought to be caused by
lysosomal deposits of lipofuscin. Lipofuscin is a pigment that
progressively accumulates in aging eukaryotic tissues as a result
of the oxidative degradation and autophagocytosis of cellular
components. Its ubiquitous occurrence in a variety of organisms
makes it a universal marker of aging.
[0262] The cellular deterioration that we observed during aging was
widespread. We chose to study the head of the worm in more detail,
since it is a particularly informative and compact area composed of
multiple tissue types. The head contains the pharynx, a
neuromuscular pump that ingests and grinds bacteria, as well as
nervous tissue, muscle, and surrounding epidermis. Using Nomarski
optics, we were able to evaluate the general character of this body
region, but, because cellular boundaries are often indistinct, we
were not able to resolve individual tissue types with
certainty.
[0263] To quantify the changes we observed, we analyzed photographs
of 84 individual wild-type worm heads and assigned each animal a
numerical value that represented the extent of damage and
deterioration it exhibited (see Methods). At every age, some
animals exhibited more extensive tissue deterioration than others,
consistent with the fact that some animals live longer than others.
Nevertheless, the average level of tissue deterioration in the
overall population increased steadily with age in a
statistically-significant manner. Together these findings indicate
that the tissues of C. elegans deteriorate in a progressive fashion
as the animals grow older. This tissue decline can account for the
decreased mobility and the flaccid appearance of old worms that are
visualized with a low power-dissecting microscope, and is likely to
contribute to the death of the animals.
[0264] Tissue Deterioration Occurs in the Germline of Aging
Wild-Type Animals
[0265] The only cells that are able to divide in C. elegans adults
are the germline stem cells. Thus it was particularly interesting
to ask whether signs of age-related tissue deterioration were
present in this tissue. We found that the germlines of older
animals showed dramatic signs of aging. The germline of C. elegans
is a multinucleate syncytium; however, in young animals the
boundaries of individual nuclei are easy to see. We found that
older animals often had fewer nuclei within the gonad, that the
boundaries of these nuclei were often ragged. In addition, the
surrounding tissue often acquired a granular texture. Occasionally
the nuclei were enlarged and appeared to be cellularized,
suggesting that they might have entered meiosis. These changes
became apparent at approximately the fifth day of adulthood and
increased with age. Our findings indicate that integrity of the
germline declines during the life of the animal. Germline
deterioration was quantified as described above, by assigning
photographs of individual animals a value that reflected the
condition of the tissue. Two-day, five-day and ten-day-old animals
exhibited progressively more extensive degeneration, and these
differences were statistically significant (p=0.0003).
[0266] Mutations in the Insulin/IGF-1 Pathway Change the Rate at
which Both Mitotic and Post-Mitotic Tissues Age
[0267] To ask how mutations in the insulin/IGF-1 signaling system
influence tissue aging, we examined long-lived daf-2(e1370) and
daf-2(mu150) mutants and short-lived daf-16(mu86) mutants at
different ages. The daf-2(e1370) allele has been characterized
previously (Dorman et al., Genetics 141:1399-1406 (1995); Gems et
al., Genetics 150:129-155 (1998); Larsen et al., Genetics
139:1567-1583 (1995)). At 25.degree., this mutant is uncoordinated
and produces progeny late in life (up to day 40; data not shown).
It appears dark when viewed with a dissecting microscope. We found
that these animals were shorter than normal animals (1.0 vs 1.4 mm,
p<0.0001), and their bodies were thinner (47 .mu.m vs 54 .mu.m,
p=0.002). In contrast, the mu150 allele, which we isolated in a
screen for long-lived mutants, is also long lived but appears much
healthier than e1370. Unlike daf-2(e1370) animals, which all become
dauers at 25.degree. C., only approximately 30% of daf-2(mu150)
animals become dauers at this temperature. In addition, mu150
animals moved normally and did not produce progeny late in life.
Their bodies were not dark like e1370 mutants, but resembled wild
type, indicating that fat production and longevity can be
uncoupled. They were only slightly shorter than normal (1.2 vs 1.4
mm, p=0.01), and not thinner (53 .mu.m vs 54 .mu.m, p=0.45).
Overall, this mutant appears remarkably similar to wild type in
terms of its behavior and morphology. We also examined short-lived
animals carrying the daf-16 null mutation mu86. We were
particularly interested to learn whether the signs of aging that we
observed in wild-type animals were present in these mutants, or
whether they might age in an entirely new way. In addition, since
these mutations change the lifespan of the animal, we wanted to
know whether they might change the rate at which the tissues
aged.
[0268] We found that the quality of tissue deterioration in both
daf-2 and daf-16 mutants resembled that of wild type, suggesting
that both types of mutants age in a normal way but at a different
rate. All of the mutants displayed increased lipofuscin-like
intestinal fluorescence at relatively old ages. We found that both
the somatic tissues and the germlines of daf-2(mu150) and daf-16
mutants exhibited age-related damage that appeared identical to
that seen in wild-type. daf-2(e1370) mutants also aged in a manner
similar to wild type, but they exhibited high levels of
autofluorescence throughout life, and had less yolk in the body
cavity than wild type, even at advanced ages (perhaps because yolk
was still exported into progeny). Also, the frequency of
constipation and bacterial packing in the pharynx was markedly
reduced in daf-2(e1370) mutants, although it was present in some
infirm individuals.
[0269] Although the character of the tissue deterioration we
observed in daf-2 mutants was similar to that of wild type, we
found that the rate of this deterioration was dramatically slowed.
For example, in daf-2(e1370) mutants, the outlines of epidermal
nuclei were clearly visible until at least 20 days of adulthood,
compared to approximately 5 days in wild type. In addition, it was
not until daf-2 animals were approximately 20 days old that we
began to see the cavities and "curdled" tissues that were so common
in 5-day old wild-type animals.
[0270] We quantified the extent of tissue damage by scoring
photographs of individual heads and germ cells in the distal gonad.
Nonparametric analysis of scores assigned to daf-2 and wild type
animals of the same age confirmed our impression that daf-2 animals
exhibited far less age-related damage in both tissues. We also
examined the tissues of daf-16 mutants, whose lifespans are
slightly shorter than normal at 25.degree. C. In general, daf-16
animals did not look dramatically older than age-matched wild type
animals. On the second day of adulthood, wild type and daf-16 heads
looked marginally older than heads of daf-2 mutants (p=0.04). By
the fifth day of adulthood, the difference in head scores between
wild type or daf-16 and either daf-2 allele was highly significant
(p<0.0001). daf-16 and wild-type heads and germ cells were
indistinguishable at early ages; it was only on the tenth day of
adulthood that daf-16 mutant animals began to look significantly
older than wild type when scores of heads (p=0.01) and germ cells
(p=0.02) were compared. daf-16 animals at this age will all be dead
within two days, whereas wild type worms have a longer life
expectancy.
[0271] The cavities we saw in old wild-type animals resembled the
necrotic cell death seen in certain neurodegeneration mutants, such
as the deg-1 sodium-channel mutants. If daf-2 mutations delay the
appearance of necrotic cavities during aging, they might be
expected to suppress or ameliorate these degenerations in deg-1
animals. However, we found that daf-2; deg-1 double mutants
displayed both an abundance of necrotic cavities at a young age and
a long, daf-2-like lifespan. This suggests that daf-2 may not
directly suppress necrotic cell death, but instead may act to delay
an age-dependent process that causes necrosis.
[0272] Apoptosis is Unlikely to Influence Aging in C. Elegans
[0273] In vertebrates, apoptosis, or programmed cell death, occurs
throughout life and plays an important role in development and
cellular growth control. In C. elegans, 131 cells undergo
apoptosis, during development. It is reasonable to think that
apoptosis, could potentially influence the lifespan of the
organism. We looked for apoptotic cells, which have a
characteristic refractile appearance (Sulston et al., Developmental
Biology 56:110-156 (1977)), in the somatic tissues of wild type and
mutant animals during aging, and failed to observe them. Apoptotic
cell death does occur in early meiotic cells of the germline
(Gumienny et al., Development 126:1011-1022 (1999)). To test the
role of apoptosis in a more definitive way, we examined the
lifespans of ced-3 mutants, which lack a caspase that is required
for apoptosis in C. elegans. In ced-3 mutants, cells that should
die instead remain alive and, at least in some cases, differentiate
into functional cells. We reasoned that if programmed cell death
influences organismal death, mutants defective in apoptosis should
have abnormal, possibly extended, lifespans. However, we found that
ced-3 mutants had lifespans that were indistinguishable from those
of wild type. We conclude that apoptosis does not play a
significant role in the regulation of wild-type lifespan.
Example 2
[0274] C. elegans Mutants that Extend Lifespan
[0275] Using libraries representing double stranded RNAs from
Chromosome I (Fraser et al., Nature 408:325-330 (2000)) and
Chromosome II, genes that extend the lifespan of C. elegans when
inhibited were identified. The gene encoded include mitochondrial
respiratory proteins, glycolytic proteins, a GTPase, and three
genes of unknown function.
[0276] A. Screen for Lifespan Enhancing Genes.
[0277] Animals were cultured on bacterial strains expressing double
stranded RNA in a bacterial feeding library. (Kamath et al., Genome
Biol. 2:research0002.1-0002.10 (2000); Fraser et al., Nature
408:325-330 (2000)). The library was made as follows. The sequence
of the C. elegans genome is known and has been used to identify
predicted genes. DNA fragments corresponding to predicted genes
were cloned into a feeding vector L4440 between two T7 bacterial
promoters in inverted orientation. The library was then transformed
into a bacterial strain carrying IPTG-inducible expression of T7
polymerase. Expression of the dsRNA was induced by addition of
IPTG.
[0278] C. elegans were cultured on bacterial strains expressing
dsRNA corresponding to predicted gene sequences from Chromosomes I
or II. Worm lifespan was monitored. A sterile C. elegans strain
(fem-1, fer-2 double mutant) was used to allow the lifespan of a
single generation to be analyzed. To analyze lifespan, after
reaching adulthood worms were transferred to new plates at least
once every seven days. Worms were scored as dead if they failed to
respond to being prodded three times with a wire loop. Lifespan
data was entered into the Statview statistical data management
program for analysis.
[0279] B. Inhibiting Metabolic Pathways Results in Enhanced
Lifespan.
[0280] In the present invention, several metabolic genes were
identified that influence the lifespan of C. elegans in an RNAi
screen of genes located on chromosome I. All of the genes on
chromosome I were expressed as double-stranded RNA in a bacterial
feeding library (see, e.g., Fraser et al., Nature 408:325-330
(2000)). C. elegans were cultured on each of these bacterial
strains, and lifespan was monitored. Inhibition of any of four
metabolic genes extended lifespan significantly.
[0281] Respiratory Chain Mutants
[0282] During a systematic screen of a C. elegans Chromosome I RNAi
library it was discovered that animals grown on bacteria expressing
atp-3 double-stranded RNA lived much longer than normal (Fraser et
al., Nature 408:325 (2000)). Lifespan analysis was conducted at
25.degree. C. as described previously (Kenyon et al., Nature
366:461 (1993)). The pre-fertile period of adulthood was used as
t=0 for lifespan analysis. Strains were grown at 20.degree. C. for
at least two generations before use in lifespan analysis. Statview
5.0.1 (SAS) software was used for statistical analysis and to
determine means and percentiles. In all cases, p values were
calculated using the logrank (Mantel-Cox) method.
[0283] atp-3 encodes a component of the mitochondrial ATP synthase
(Complex V). In addition, we found that RNAi of three genes
encoding components of the mitochondrial respiratory chain also
extended lifespan. These were nuo-2, which encodes a component of
complex I (NADH/ubiquinone oxiodoreductase), cyc-1, which encodes a
component of complex III (cytochrome C reductase) and cco-1, which
encodes a component of complex IV (cytochrome C oxidase). Results
are shown in Table 1.
1TABLE 1 Statistical analysis of adult lifespans Mean .+-. 75th
Number of s.e.m. percentile animals that Strain/treatment (days)
(days)* died/total.sup..dagger. P N2 animals grown at 25.degree. C.
on bacteria containing: Vector (.alpha.) 15.2 .+-. 0.4 18 48/52
daf-2 dsRNA 20.1 .+-. 0.9 26 55/71 <0.0001.sup..sctn. Complex I
(nuo-2) dsRNA 21.6 .+-. 0.9 25 51/54 <0.0001.sup..sctn. Complex
IV (cco-1) dsRNA 24.5 .+-. 0.7 27 67/69 <0.0001.sup..sctn.
Complex V (atp-3) dsRNA 22.1 .+-. 0.9 30 54/54
<0.0001.sup..sctn. Vector (.beta.) 13.6 .+-. 0.5 17 65/69
Complex III (cyc-1) dsRNA 25.4 .+-. 0.9 29 65/69
<0.0001.sup..cent. gpi-1 dsRNA 18.2 .+-. 0.5 22 63/67
<0.0001.sup..cent. Antimycin A Treatments fer-15(b26);
fem-1(hc17) + ethanol 16.6 .+-. 0.6 20 57/61 fer-15(b26);
fem-1(hc17) + 0.1 .mu.M 20.4 .+-. 0.7 25 51/61
<0.0001.dagger-dbl. Antimycin A Lifespans of fer-15(b26); fem-
1(hc17) animals grown at 25.degree. C. on bacteria containing the
following dsRNA for the indicated times: Vector, larval and adult
16.6 .+-. 0.6 20 57/64 Complex III (cyc-1) dsRNA, 24.0 .+-. 1.0 29
50/68 <0.0001.sup..DELTA. larval and adult Complex III (cyc-1)
dsRNA, 16.9 .+-. 0.6 20 62/63 0.6585.sup..DELTA. adult only Complex
V (atp-3) dsRNA, 25.2 .+-. 0.8 29 61/80 <0.0001.sup..DELTA.
larval and adult Complex V (atp-3) dsRNA, adult 15.7 .+-. 0.5 18
55/64 0.1714.sup..DELTA. only gpi-1 dsRNA, larval and adult 20.2
.+-. 0.8 25 68/71 <0.0001.sup..DELTA. gpi-1 dsRNA, adult only
16.3 .+-. 0.7 20 61/63 0.6075.sup..DELTA. daf-16(mu86) animals
grown at 25.degree. C. on bacteria containing: Vector (.alpha.) 9.8
.+-. 0.2 11 45/47 daf-2 dsRNA 9.7 .+-. 0.2 10 55/62 0.0792.sup.#
Complex I (nuo-2) dsRNA 13.7 .+-. 0.3 15 59/60 <0.0001.sup.#
Complex IV (cco-1) dsRNA 14.6 .+-. 0.4 18 60/60 <0.0001.sup.#
Complex V (atp-3) dsRNA 14.5 .+-. 0.6 18 35/37 <0.0001.sup.#
Vector (.beta.) 9.2 .+-. 0.3 10 60/69 Complex III (cyc-1) dsRNA
16.1 .+-. 0.3 17 43/53 <0.0001.sup..infin. gpi-1 dsRNA 10.8 .+-.
0.3 12 57/64 <0.0001.sup..infin. daf-2(e1370) animals shifted to
25.degree. C. as L4 and grown on bacteria containing: Vector 25.1
.+-. 1.2 34 55/58 Complex I (nuo-2) dsRNA 48.5 .+-. 1.4 54 47/60
<0.0001.sup..paragraph. Complex III (cyc-1) dsRNA 41.5 .+-. 1.8
54 52/60 <0.0001.sup..paragraph. Complex IV (cco-1) dsRNA 48.3
.+-. 1.4 56 50/62 <0.0001.sup..paragrap- h. Complex V (atp-3)
dsRNA 40.8 .+-. 3.2 54 46/59 <0.0001.sup..paragraph. gpi-1 dsRNA
34.5 .+-. 1.0 39 58/60 <0.0001.sup..paragraph. Alteration of the
reproductive system of N2 animals grown at 20.degree. C. on
bacteria containing: intact + vector 22.3 .+-. 0.5 24 62/80 intact
+ Complex IV (cco-1) dsRNA 28.9 .+-. 1.4 38 63/80
<0.0001.sup..Yen. germline ablation + Complex IV 44.8 .+-. 1.9
53 44/46 <0.0001.sup..Yen. (cco-1) dsRNA whole-gonad ablation +
Complex IV 31.7 .+-. 1.5 40 41/44 <0.0001.sup..Yen. (cco-1)
dsRNA Alteration of the reproductive system of clk-1(qm 30) animals
grown at 20.degree. C.: N2 19.8 .+-. 0.5 24 132/159 clk-1(qm30)
31.0 .+-. 1.2 37 67/80 <0.0001.sup..check mark. clk-1(qm30)
germline ablation 37.5 .+-. 2.4 49 49/55 <0.0001.sup..check
mark. clk-1(qm30) whole-gonad ablation 19.9 .+-. 0.8 23 45/50
0.4549.sup..check mark. *The 75th percentile is the age when the
fraction of animals alive reaches 0.25. .sup..dagger.The total
number of observations equals the number of animals that died plus
the number censored. Animals that crawled off the plate, exploded
or bagged were censored at the time of the event. This step
incorporated those worms into the data set until the censor date,
and was necessary to avoid the loss of information; for example, if
a 50-day-old animal crawls off the plate, it is important to
include that information in the data set, as that animal was long #
lived. Control and experiment animals were cultured in parallel and
plates were changed at the same time. The logrank (Mantel-Cox) test
was used to test the hypothesis that the survival functions among
groups were equal. P values were calculated for individual
experiments, each consisting of control and experimental animals
examined at the same time. .sup..sctn.Compared with N2 worms
cultured on HT115 bacteria containing an empty plasmid vector at
25.degree. C. Vector (.alpha.), daf-2, Complex I, IV and V dsRNA
experiments were conducted at the same time. .sup..cent.Compared
with N2 worms cultured on HT115 bacteria containing an empty
plasmid vector at 25.degree. C. Vector (.beta.), Complex III, and
gpi-1 dsRNA experiments were conducted at the same time.
.sup..dagger-dbl.Compared with control fer-15(b26); fem-1(hc17)
double mutants grown on equivalent amount of solvent (ethanol) at
25.degree. C. .sup..DELTA.Compared with control fer-15(b26);
fem-1(hc17) double mutants cultured on bacteria containing an empty
plasmid vector at 25.degree. C. .sup.#Compared with daf-16(mu86)
mutants cultured on bacteria containing an empty plasmid vector at
25.degree. C. Vector (.alpha.), daf 2, Complex I, IV and V dsRNA
experiments were conducted at the same time. .sup..infin.Compared
with daf-16(mu86) mutants cultured on bacteria containing an empty
plasmid vector at 25.degree. C. Vector (.beta.), Complex III and
gpi-1 experiments were conducted at the same time.
.sup..paragraph.Compared with daf-2(e1370) mutants cultured on
bacteria containing an empty plasmid vector shifted to 25.degree.
C. as L4 larvae. .sup..Yen.Compared to N2 worms with an intact
reproductive system cultured on bacteria containing an empty
plasmid vector at 20.degree. C. .sup..check mark.Compared to N2
worms with an intact reproductive system cultured on OP-50 bacteria
at 20.degree. C.
[0284] The protein ATP3 (Genbank # AAB37654.1) is a member of the
ATP synthase delta family and is 43% identical to human ATP50. NUO2
(Genbank # AAB52474.1) protein is 56% identical to human NDUFS3.
CYC1 (Genbank # CAA99820.1) is 50% identical to human CYC1. CCO1
(Genbank # CAB03002.1) is 35% identical to human COX5B. GPI-1
(Genbank # CAB60430.1) is 68% identical to human GPI.
[0285] We also found that treatment of wild-type animals with the
drug antimycin A, which inhibits complex III (Slater, Trends
Biochem Sci. 8:239 (1983)), increased lifespan as well.
[0286] OP-50 bacteria were spread onto NG plates and allowed to
grow for two days at room temperature. 50 .mu.l of 0.01 M Antimycin
A (Sigma) dissolved in 100% ethanol was dropped onto bacterial lawn
and allowed to dry overnight in a fume hood at room temperature.
Eggs of fer-15(b26); fem-1(hc17) double mutants, which become
sterile adults at 25.degree. C., were added to the dried plates and
incubated at 25.degree. C. Animals were transferred to new
Antimycin A plates every 4 days. Control worms (ethanol only) were
handled in the same manner) (Table 1).
[0287] Because RNAi reduces the level of wild-type mRNA, these
treatments were all predicted to decrease the rate of electron
transport and ATP production. We measured ATP levels as follows.
For each time point, 750 fer-15(b26); fem-1(hc17) double mutants
were grown at 25.degree. C., to inhibit progeny production. Each
time point was repeated in triplicate. The collected worms were
washed four times with S-basal buffer, resuspended in cell lysis
buffer and quickly frozen in liquid N.sub.2. All samples were
stored at -80.degree. C. and processed on the same day. A Roche ATP
Bioluminscent HSII kit was used to measure ATP concentrations with
an OPTOCOMP I luminometer. ATP concentrations were normalized to
absolute protein concentrations. A BioRad protein assay kit was
used to measure protein concentrations using a Perkin Elmer MBA2000
spectrophotometer.
[0288] ATP levels were reduced 60-80% in animals subjected to
complex III (cyc-1) or ATP synthase (atp-3) RNAi, and 40-60% in
animals treated with complex I (nuo-2) or complex IV (cco-1) RNAi.
Thus, these perturbations all decrease the rate of mitochondrial
respiration.
[0289] In addition to extending lifespan, respiratory-chain RNAi
affected growth and behavior. First, the animals were smaller than
normal, suggesting that mitochondrial respiration promotes growth.
Because the small animals were well proportioned, it seems likely
that a metabolite whose level is regulated by mitochondrial
respiration acts as a signal to control body size. Small body size
itself is unlikely to cause lifespan extension, because mutants
defective in daf-4, which encodes a Smad protein (Estevez et al.,
Nature 365:644 (1993)), are small but not long lived (Tissenbaum et
al., Nature 410:227 (2001)). Respiratory-chain RNAi also decreased
the rate of growth to adulthood, as well as the rates of pharyngeal
pumping (eating) and defecation (Table 2). In addition, the animals
moved much more slowly than normal (Table 2). Mutations in mev-1,
gas-1, clk-1 and isp-1 all reduce the rates of development and
movement (Ishii et al., Nature 394:694 (1998); Hartman et al., Mech
Aging Dev. 122:1187 (2001); Wong et al., Genetics 139:1247 (1995);
Feng et al., Developmental Cell 1:663 (2001)) but do not affect
body size, suggesting that the level of respiratory activity in our
RNAi-treated animals may be lower than in these mutants.
2TABLE 2 Timing of biological processes in RNAi-treated and clk-1
mutant animals Length of Defecation post- Feeding Rate Rate
(seconds/ Mobility embryonic dsRNA or Other (pumps/min. .+-.
defecation .+-. (swim strokes/ development Treatment s.d.)*
s.d.).sup..dagger. min .+-. s.d.).sup.# (hours .+-.
s.d.).sup..dagger-dbl. Vector 201 .+-. 9 41 .+-. 3 130 .+-. 6 43
.+-. 2 Complex I(nuo-2) 159 .+-. 8 80 .+-. 4 90 .+-. 2 69 .+-. 3
Complex III(cyc-1) 161 .+-. 10 81 .+-. 1 75 .+-. 13 88 .+-. 2
Complex IV(cco-1) 144 .+-. 9 79 .+-. 2 94 .+-. 3 68 .+-. 2 Complex
V(atp-3) 124 .+-. 17 87 .+-. 5 90 .+-. 9 96 .+-. 2 gpi-1 198 .+-. 8
43 .+-. 3 127 .+-. 6 43 .+-. 2 clk-1(qm30) intact 161 .+-. 36 69
.+-. 6 77 .+-. 9 clk1(qm30) 140 .+-. 20 50 .+-. 8 91 .+-. 7
germline-ablated clk-1(qm30) gonad- 117 .+-. 22 63 .+-. 9 93 .+-. 5
ablated *Mean .+-. s.d. The number of pharyngeal pumps observed in
a one-day-old adult animal in 1 minute at 20.degree. C. (n .gtoreq.
8). .sup..dagger.Mean .+-. s.d. The time required to complete one
defecation cycle. A minimum of three consecutive defecations was
recorded for each trial at 20.degree. C. (n .gtoreq. 5). .sup.#Mean
.+-. s.d. The number of swim cycles completed in 1 minute of a
one-day-old adult animal at 20.degree. C. (n .gtoreq. 5).
.sup..dagger-dbl.Mean .+-. s.d. Time (hours) to adulthood at
20.degree. C. (n .gtoreq. 25).
[0290] Because RNAi can be administered at different times during
the life of an animal, we were able to ask whether mitochondrial
respiration acts in an ongoing fashion throughout life to influence
aging. To do this, we transferred animals onto bacteria expressing
respiratory-chain dsRNA at different ages (Dillin et al., Nature,
submitted). Surprisingly, we found that if we allowed the animals
to grow to adulthood on normal bacteria, and then shifted them as
young adults onto bacteria expressing respiratory-chain dsRNA,
their lifespans were not extended (Table 1). To determine whether
RNAi lowered respiratory-chain activity in these animals, we
assayed their ATP levels and found that they were reduced to the
same extent as in animals exposed to respiratory-chain dsRNA from
hatching. Thus respiratory-chain RNAi does decrease mitochondrial
activity in adult animals. Together these findings indicate that
the respiratory chain acts during development and not during
adulthood to influence lifespan.
[0291] We also asked whether initiating RNAi during adulthood
decreased behavioral rates. The rate of movement we observed was
the same as that of control animals, but the rate of pumping was
decreased (Table 3). Interestingly, however, whereas animals
treated with RNAi from the time of hatching pumped in a slow but
steady fashion, animals treated as young adults exhibited bursts of
rapid pumping followed by pauses. Possibly these pauses were caused
by transient depletion of ATP.
3TABLE 3 Pumping rates of larva and adults treated with
respiratory-chain RNAi Feeding Rate (pumps/min .+-. s.d.)* RNAi
Treatment Day 2 Day 3 Day 4 Day 5 Vector 118 .+-. 15 140 .+-. 21
145 .+-. 24 131 .+-. 49 (0/8) (0/8) (0/8) (1/10) Complex III
(cyc-1) 97 .+-. 21 83 .+-. 12 84 .+-. 12 75 .+-. 12 larval + adult
(0/10) (0/10) (0/10) (0/10) Complex III (cyc-1) 79 .+-. 62 112 .+-.
80 80 .+-. 59 69 .+-. 45 adult only (5/12) (3/12) (4/12) (4/12)
Complex V (atp-3) 98 .+-. 15 87 .+-. 10 80 .+-. 9 75 .+-. 8 larval
and adult (0/10) (0/10) (0/10) (0/10) Complex V (atp-3) 88 .+-. 80
98 .+-. 64 83 .+-. 59 89 .+-. 54 adult only (6/14) (5/14) (4/14)
(4/14) *Mean .+-. s.d. The number of pharyngeal pumps observed in
an adult animal in 1 minute at 25.degree. C. Number in parentheses
is the number of animals that produced less than 50 pumps per
minute/total number of animals tested.
[0292] Interaction of Respiratory Chain Mutants with the DAF 16
Pathway.
[0293] The insulin/IGF-1 signaling pathway affects lifespan
(Guarente et al., Nature 408:25 (2000)). Reducing the activity of
DAF-2, an insulin/IGF-1 receptor homolog, or downstream signaling
components, extends lifespan approximately two-fold. This lifespan
extension requires activity of the forkhead-family transcription
factor DAF-16.
[0294] However, we found that daf-16(mu86) null mutants lived much
longer than normal when subjected to respiratory-chain RNAi (Table
1). In addition, we found that the already long lifespans of
daf-2(e1370) mutants were nearly doubled again when the animals
were cultured on any of the four bacterial strains expressing
respiratory chain dsRNA (Table 1). Moreover, unlike reduction of
respiratory chain activity, reduction of insulin/IGF-1 signaling is
known to cause a significant increase in ATP levels (Braeckman et
al., Curr Biol, 9:493 (1999)).
[0295] Finally, both daf-2 and daf-16 act exclusively in adults to
regulate lifespan (Dillin et al., Nature). Together these findings
indicate that respiratory-chain RNAi does not increase lifespan by
inhibiting the DAF-2 pathway.
[0296] Interaction of Respiratory Chain Mutants with the
Reproductive Pathway.
[0297] In C. elegans, removing the germline extends lifespan (Hsin
et al., Nature 399:362 (1999)). This lifespan extension is not
caused by sterility per se, because animals in which the somatic
gonad as well as the germline are removed have normal lifespans
(Hsin et al., Nature 399:362 (1999); Kenyon et al., Nature 366:461
(1993)).
[0298] We found that animals treated with respiratory-chain RNAi
were either sterile or had small broods. The longevity of these
animals was not likely to be caused by reduced germline activity,
however, because the lifespan extension caused by germline ablation
is daf-16 dependent (Hsin et al., Nature 399:362 (1999)).
[0299] To test whether the reproductive system could influence
lifespan in these RNAi-treated animals, we removed either their
germlines or the entire reproductive systems. The germlines or the
entire reproductive systems of complex IV (cco-1) RNAi-treated
animals were removed by killing Z2 and Z3 or Z1 and Z4,
respectively. Laser ablations were performed on newly hatched
animals using a nitrogen-pumped dye laser, which produces a
wavelength of 440 nm. Successful ablation was confirmed by
examining the reproductive systems of adult animals with a
dissecting microscope. Worms were anaesthetized with 1.0-1.5 M
NaN.sub.3 during ablation, a treatment that had no effect on
lifespan. The germline and whole-gonad of ablation clk-1 (qm30)
mutants was performed as described above, in two experiments, with
similar results.
[0300] In addition to the laser ablation experiments described
above, the germlines of respiratory-chain RNAi-treated animals were
ablated genetically using glp-1 (e2141ts) mutant worms, which, at
25.degree. C., produce only a few germ cells and live longer than
wild type. Mean lifespans of glp-1 (e2141ts) mutant worms grown at
25.degree. C. on bacteria expressing the following dsRNA were:
Control (empty vector), 15.0.+-.0.7; Complex I (nuo-2) dsRNA,
19.2.+-.0.7 (p<0.0001); Complex III (cyc-1) dsRNA, 19.7.+-.1.0
(p<0.0001); Complex IV (cco-1) dsRNA, 22.9.+-.0.6 (p<0.0001);
and Complex V (atp-3) dsRNA, 23.0.+-.0.6 (p<0.0001)). As with
wild type, germline ablation further extended lifespan, whereas
whole-gonad ablation did not (Table 1). Thus the reproductive
system of these animals appeared to influence lifespan in a normal
way.
[0301] We also examined the role of the reproductive system in
clk-1 mutants. As with animals subjected to respiratory-chain RNAi,
ablating the germlines of clk-1 mutants caused an increase in
lifespan. However, removing the whole gonad of clk-1 (qm30) mutants
produced an unexpected result; it completely abolished their
lifespan extensions (Table 1). Thus the longevity of these animals
is entirely dependent on the presence of the reproductive system.
Interestingly, ablation of the gonad did not rescue the slow growth
or behavioral phenotypes of clk-1 (qm30) mutants (Table 2). This
finding is significant, because it indicates that the longevity of
these mutants is not likely to be caused by their slow rates of
growth or behavior.
[0302] Glycolytic Pathway Mutants
[0303] In our Chromosome I RNAi screen, we also found that lowering
the activity of a gene that functions in glycolysis, glucose
phosphate isomerase (gpi-1), increased lifespan approximately 20%
(Table 1). GPI-1 (Genbank # CAB60430.1) is 68% identical to human
GPI. The C. elegans genome contains only one glucose phosphate
isomerase, implying that these animals have lowered rates of
glycolysis. Consistent with this, we found that these animals had
reduced levels of ATP.
[0304] In vertebrates, PGI is required not only for glycolysis but
also for the survival of sensory neurons (Chaput et al., Nature
332:454 (1988); Faik et al., Nature 332:455 (1988)). Sensory
neurons are known to influence the lifespan of C. elegans (Apfeld
et al., Nature 402:804 (1999)). However, when we used a fluorescent
dye (DiI) to visualize many sensory neurons, we failed to detect
any abnormalities (data not shown). In addition, whereas the
lifespan extension of sensory mutants is largely daf-16 dependent
(Apfeld et al., Nature 402:804 (1999)), this was not the case for
animals treated with gpi-1RNAi (Table 1). Together these findings
suggest that gpi-1 RNAi does not increase lifespan by affecting
sensory neurons.
[0305] Like respiratory chain RNAi, gpi-1 RNAi further extended the
lifespans of daf-2(e1370) mutants (Table 1). Interestingly, unlike
animals treated with respiratory-chain RNAi, these animals appeared
healthy and were normal in size. They had wild-type rates of growth
to adulthood and rates of behavior (Table 2). In addition, animals
treated with gpi-1 RNAi as adults were not long-lived (Table 1),
suggesting that, like respiratory-chain activity, gpi-1 may
function during development to affect lifespan.
[0306] C. Inhibiting a GTPase Results in Enhanced Lifespan.
[0307] Using the screen described above, a Rab-like GTPase was
identified from a Chromosome I library. The name of the gene is
T23H2.5 and its accession number is AAC482001. Analysis of the
mutant demonstrated that inhibition of the gene extended lifespan
by 25%. The inhibition of lifespan was independent of the Daf2/16
pathway.
[0308] D. Inhibiting llw Genes Results in an Enhanced Lifespan.
[0309] An RNAi screen of a chromosome II library identified three
novel genes that extend lifespan when inhibited. llw-1, gene
Y54G11A.8, contains a TPR-like domain and its accession number is
CAA22452 (SEQ ID NO: 7). llw-3, gene Y48E1B.1, contains a proline
-rich domain and its accession number is CAB07688 (SEQ ID: 9).
Inhibition of llw-2, gene F59E12.10, also resulted in enhanced
lifespan, and its accession number is AAB54251 (SEQ ID NO: 8).
Table 1 shows the result of dsRNA expression corresponding to these
genes.
4TABLE 4 Inhibition of llw genes enhances C. elegans lifespan Gene
dsRNA expressed % worms dead at day 20 Vector only 88% llw-1
Y54G11A.8 llw-2 F59E12.10 37% llw-3 Y48E1B.1 52% llw-4 F45H10.4
35%
Example 3
[0310] C. elegans Mutants that Shorten Lifespan
[0311] Using the Chromosome I library described previously, C.
elegans were fed bacteria expressing dsRNA and screened for
shortened lifespan. A cursory examination using objective aging
criteria allowed immediate discarding of 6 out of 7 short-lived
RNAi-treated strains from further consideration as possible
progeric animals. Moreover, the one remaining clone had a dramatic
phenocopy of normal aging.
[0312] The progeric RNAi candidate encoded a homologue of HSF
(heat-shock factor), a transcription factor that activates
expression of heat-shock genes in response to heat shock and
oxidative stress. The HSF-1 gene is Y53C10.12 and the accession
number is CAA22146. As heat shock factor genes are highly
conserved, this family of genes therefore is shown to be involved
in aging, including human homologs of HSF family genes, e.g.,
HSF-1.
[0313] In a second experiment, over-expression of HSF-1 gene in C.
elegans caused an increased adult lifespan. The HSF-1 gene has been
cloned and constructed into a plasmid that can be used to inject
into the worm. The HSF-1 gene was ectopically over-expressed in the
worms that carry this plasmid. Compared to wild type worms, these
worms have an increased lifespan.
Example 4
[0314] Screening for Genes that Affect Aging Using Microarrays
[0315] In C. elegans, aging is regulated in one instance by an
insulin/IGF-1 pathway. An insulin receptor homolog, DAF-2 activates
a PI 3 kinase pathway which in turn appears to inhibit the function
of DAF-16, a forkhead-family transcription factor. daf-2 mutants
are long lived, whereas daf-16 and daf-16; daf-2 mutants are short
lived. Previous experiments have shown that this daf-2 pathway
functions non-cell autonomously, meaning that it must regulate the
expression of one or more genes that either encode or regulate the
production of a downstream signal or hormone. In addition, there
must be a cellular pathway that responds to this second hormone,
and finally genes that encode proteins directly involved in the
aging process.
[0316] Microarrays were constructed using C. elegans DNA primers
purchased from Research Genetics. The arrays were then used to
analyze gene expression profiles in daf-2 and daf-6 mutants. The
expression of a number of genes varied in long-lived (daf-2) or
short-lived (daf-16 and daf-16; daf2) C. elegans mutants (see Table
5). The activity of genes identified using microarray analysis was
then inhibited using RNAi (Table 6). The genes thus identified
include hormones that activate the daf-2 pathway, several encoding
cytochrome P450 proteins, e.g., genes involved in lipid and steroid
biosynthesis, the melatonin synthesis gene (human homolog ASMTL,
Accession No. NM.sub.--004192), insulin and insulin-like peptides
(e.g., ins-7), heat shock factors, catalases, stress-response
genes, neuronal genes, oxidative stress protection genes,
antibacterial and antifungal genes, vitellogenins, and metabolic
genes (see FIGS. 5-12).
Example 5
[0317] Screening for Genes and Compounds that Affect Aging Using a
DAF-16/GFP Fusion Protein.
[0318] A DAF16-GFP fusion was constructed as described in Lin et
al., Nature Gen. 28:139-145(2001). DAF16 is a transcription factor
and in normal animals is localized throughout cells. Using the
DAF16-GFP fusion protein, localization of DAF-16 in long lived
animals was studied. In animals with extended lifespans, the
DAF-16/GFP fusion was localized to the nuclei.
[0319] The following extended lifespan mutants were analyzed: daf-2
mutants and germline ablated animals. DAF-2 is an insulin/IGF-1
receptor homologue. In daf-2 mutants, lifespan is extended in a
daf-16 dependent manner. When expressed in daf-2 mutants, the
DAF-16/GFP fusion was localized in most nuclei throughout larval
development and in adults. In germline ablated animals, the
DAF-16/GFP fusion was localized in the nuclei of intestinal cells
and to a lesser extent the nuclei of other cell types also.
[0320] Localization of DAF-16/GFP is used to screen for drugs or
mutations that extend lifespan. Localization of DAF-16/GFP is also
used to determine whether a mutation extends lifespan by perturbing
the insulin/IGF-1 pathway, germline pathway, or other aging
regulatory pathway.
Example 6
[0321] Screening for Genes and Compounds that Affect Aging Using an
SOD-3/GFP Fusion Protein
[0322] The C. elegans SOD-3 (superoxide dismutase) gene is
regulated by the insulin/IGF-1 system. Mutants in the insulin/IGF1
pathway overexpress SOD-3. When an SOD-3/GFP was expressed in
long-lived mutants of the insulin/IGF1 pathway, the mutants had a
higher level of GFP fluorescence, especially in the intestine, than
did wild-type worms expressing the same fusion protein.
[0323] Worms expressing the SOD-3/GFP fusion protein are screened
for extended lifespan by assaying for intense fluorescence
patterns. Screens are done for mutants in the insulin/IGF-1
pathway, for long-lived germline signaling mutants, and for
respiratory chain mutants. Screens are also done to identify drugs
that affect the aging process.
5TABLE 5 CF512 WL Sept description mean std err % vector % avg V
C04F6.1 vit-5 15.92 0.626 121.5267 103.7134 C17G1.4 muc-1 11.38
0.477 86.87023 74.13681 C17G10.5 Ent. His. 13.53 0.846 103.2824
88.14332 N-acetylmuraminidase C18B2.3 12.22 0.495 93.28244 79.60912
C40H5.1 12.68 0.736 96.79389 82.60586 C44E4.2 13.39 0.79 102.2137
87.23127 C54G4.6 ASMTL 16.92 0.758 129.1603 110.228 C55B7.4
AcylCoADH 13.5 0.885 103.0534 87.94788 Y54G11A.6 ctl-1 16.3 0.748
124.4275 106.1889 Y54G11A.5b ctl-2 13.53 0.808 103.2824 88.14332
Daf-16 13 0.519 99.23664 84.69055 Daf-2 17.1 0.834 130.5344
111.4007 F08B1.1 vhp-1 phosphatase 15.5 0.644 118.3206 100.9772
F21F3.3 farnesyl cystein carboxyl 12.73 0.501 97.17557 82.9316
methyltransferase F32A5.5 aquaporin 14.37 0.63 109.6947 93.61564
F35H12.2 PTB/PID GAP 14 0.562 106.8702 91.20521 F48D6.4 14.07 1.06
107.4046 91.66124 F56G4.3 F-box domain sim. to pes-2 14.84 0.684
113.2824 96.67752 F59D8.2 vit-4 12.5 0.541 95.41985 81.43322
H16D19.1 C-type lectin 13.22 0.778 100.916 86.12378 H22K11.1 asp-3
15 0.708 114.5038 97.71987 K07A1.7 15.12 0.617 115.4198 98.50163
K10B3.8 gpd-2 14.05 0.679 107.2519 91.53094 K11D2.2 ASAH acid
ceramidase 14.86 1.024 113.4351 96.80782 R03E9.1 mdl-1 tf 15.8
0.522 120.6107 102.9316 T01A4.1 Npr1-like 13.19 0.734 100.687
85.92834 T07D10.4 C-type lectin 12.96 1.03 98.9313 84.42997 T10B9.1
CytP450 10.9 0.443 83.20611 71.00977 T25C12.2 like T08A9.9
antibacterial 13.72 0.874 104.7328 89.38111 T25C12.3 EGF/lectin
14.34 1.02 109.4656 93.4202 vector 13.1 0.532 100 85.34202 ZK1
320.2 14.63 0.459 111.6794 95.30945 ZK270.2 FERM domain/band 4.1
10.54 0.536 80.45802 68.6645 ZK355.E 15.2 0.585 116.0305 99.0228
CF512adult description mean std err % vector % avg V C04F6.1 vit-5
17.15 0.587 103.1269 111.7263844 C17G1.4 muc-1 18.67 0.411 112.267
121.6286645 C17G10.5 Ent. His. 15.724 0.471 94.55201 102.4364821
N-acetylmuraminidase C18B2.3 15.83 0.544 95.18942 103.1270358
C40H5.1 18.2 0.542 109.4408 118.5667752 C44E4.2 19.33 0.536
116.2357 125.9283388 C54G4.6 ASMTL 18.41 0.803 110.7035 119.9348534
C55B7.4 AcylCoADH 17.27 0.676 103.8485 112.5081433 Y54G11A.6 ctl-1
16.71 0.567 100.4811 108.8599349 Y54G11A.5b ctl-2 16.38 0.341
98.49669 106.7100977 Daf-16 14.53 0.49 87.37222 94.65798046 Daf-2
21.36 0.953 128.4426 139.1530945 F08B1.1 vhp-1 phosphatase 13.43
0.506 80.75767 87.49185668 F21F3.3 farnesyl cystein carboxyl 15.86
0.449 95.36981 103.3224756 methyltransferase F32A5.5 aquaporin
15.43 0.468 92.78413 100.5211726 F35H12.2 PTB/PID GAP 18.87 0.972
113.4696 122.9315961 F48D6.4 17 0.567 102.2249 110.7491857 F56G4.3
F-box domain sim. to pes-2 18.58 0.791 111.7258 121.0423453 F59D8.2
vit-4 17.22 0.657 103.5478 112.1824104 H16D19.1 C-type lectin 15.34
0.541 92.24293 99.93485342 H22K11.1 asp-3 17.36 0.498 104.3897
113.0944625 K07A1.7 15.68 0.559 94.28743 102.1498371 K10B3.8 gpd-2
17.36 0.629 104.3897 113.0944625 K11D2.2 ASAH acid ceramidase 15.77
0.607 94.82862 102.7361564 R03E9.1 mdl-1 tf 16.96 0.536 101.9844
110.4885993 T01A4.1 Npr1-like 17.25 0.48 103.7282 112.3778502
T07D10.4 C-type lectin 14.93 0.496 89.77751 97.26384365 T10B9.1 Cyt
P450 15.83 0.448 95.18942 103.1270358 T25C12.3 like T08A9.9
antibacterial 17.8 0.83 107.0355 115.9609121 vector EGF/lectin
16.63 0.468 100 108.3387622 ZK1320.2 11.72 0.435 70.47505
76.35179153 ZK270.2 18.3 0.771 110.0421 119.218241 ZK355.E FERM
domain/band 4.1 15.52 0.462 93.32532 101.1074919 CF512 Dec.
description mean std err % vector % avg V AC3.7 UDP-glucuronosyl
14.78 0.365 101.2329 96.28664 transferase B0213.15 Cyt P450 14.35
0.365 98.28767 93.48534 B0554.1 12.67 0.417 86.78082 82.54072
B0554.6 like AK6.11 12.43 0.4 85.13699 80.9772 C04F6.1 vit-5 12.52
0.372 85.75342 81.56352 C17G1.4 muc-1 12.21 0.41 83.63014 79.54397
C17H12.8 15.16 0.325 103.8356 98.76221 C24B9.4 DUF32 domain 14.12
0.376 96.71233 91.98697 C32F10.9 13.59 0.381 93.08219 88.5342
C32H11.10 DUF141 12.92 0.493 88.49315 84.16938 C32H11.12 DUF141
12.1 0.38 82.87671 78.82736 C46F4.2 long-chain fatty acid- 13.33
0.386 91.30137 86.84039 Coenzyme A ligase 4 C54D10.1 GST 13.56
0.371 92.87671 88.33876 C54G4.6 ASMTL/Maf-like 12.81 0.422 87.73973
83.45277 C55B7.4 Acyl-Co ADH 13.04 0.431 89.31507 84.95114 Daf-16
8.34 0.288 57.12329 54.33225 Daf-2 16.017 0.235 109.7055 104.3453
F10D2.9 fat-7 14.71 0.33 100.7534 95.83062 F11A5.12 short-chain
DH-reductase; 13.93 0.407 95.41096 90.74919 hydroxysteroid 17-beta
DH F28D1.3 thaumatin 12.28 0.442 84.10959 80 F28D1.5 thaumatin
12.29 0.463 84.17808 80.06515 F38E11.2 hsp-12.6 14.79 0.383
101.3014 96.35179 F49A5.6 thaumatin 14.43 0.341 98.83562 94.00651
F55G11.5 12.1 0.356 82.87671 78.82736 K04E7.2 pep-2 10.6 0.315
72.60274 69.05537 K06A11.1 15.16 0.374 103.8356 98.76221 K07C6.4
Cyt P450 12.13 0.411 83.08219 79.0228 K10D11.1 14.29 0.337 97.87671
93.09446 K11G9.6 mtl-1 13.16 0.429 90.13699 85.7329 K12G11.3
Alcohol DH 12.58 0.396 86.16438 81.9544 T10B9.1 Cyt P450 10.77
0.309 73.76712 70.16287 T13F2.1 fat-4 13.12 0.369 89.86301 85.47231
T16G12.1 aminopeptidase 14.25 0.367 97.60274 92.83388 T20G5.7 15.7
0.314 107.5342 102.2801 T22G5.2 lbp-7 14.116 0.349 96.68493
91.96091 Vector 14.6 0.372 100 95.11401 W06D12.3 fat-5 13.69 0.429
93.76712 89.18567 W08D2.4 fat-3 12.13 0.241 83.08219 79.0228
Y38H6C.5 Zn knuckle domain 13.25 0.37 90.75342 86.31922 Y49E10.8
15.85 0.291 108.5616 103.2573 ZK1251.2 ins-7 16.4 0.19 112.3288
106.8404 ZK384.4 12.6 0.401 86.30137 82.08469 CF596 description
mean std err % vector CF596 % vector B0213.15 Cyt P450 26 0.5
111.9242 B0213.15 111.9242 C02A12.4 N-acetylmuraminidase 18.63
0.855 80.19802 vector 100 C05E4.4 18.933 0.913 81.50237 K12G11.3
91.08911 C24B9.4 DUF23 18.59 0.816 80.02583 F10D2.9 82.5226 Daf-16
11.47 0.423 49.37581 C05E4.4 81.50237 Daf-2 13.88 0.659 59.75032
C02A12.4 80.19802 F10D2.9 fat-7 19.17 0.885 82.5226 T20G5.7
80.19802 F28D1.3 thaumatin 18.4 0.927 79.20792 C24B9.4 80.02583
F38E11.2 hsp-12.6 18.42 0.78 79.29402 K11G9.6 79.85364 K11G9.6
mtl-1 18.55 1.02 79.85364 F38E11.2 79.29402 K12G11.3 Alcohol DH
21.16 1.05 91.08911 F28D1.3 79.20792 T10B9.1 Cyt P450 15.36 0.813
66.12139 T10B9.1 66.12139 T20G5.7 18.63 0.938 80.19802 Daf-2
59.75032 vector 23.23 1.02 100 Daf-16 49.37581 rrf-3 20C
description mean std err % vector RNAi % vector C04F6.1 vit-5 17.47
0.763 116.4667 daf-2 207.3333 C32H11.12 DUF141 19.7 0.676 131.3333
ZK1251.2 155.2 C42D8.2 vit-2 18.15 0.576 121 C54G4.6 132.38 C54G4.6
ASMTL 19.857 0.658 132.38 C32H11.12 131.3333 daf-16 14.84 0.476
98.93333 ZK896.1 125.4667 daf-2 31.1 0.749 207.3333 C42D8.2 121
vector 15.02 0.613 100.1333 C04F6.1 116.4667 ZK1251.2 ins-7 23.28
0.512 155.2 vector 100.1333 ZK896.8 gcy-18 18.82 0.731 125.4667
daf-16 98.93333 CF51225 description mean std err % vector % avg. V
C04F6.1 vit-5 14.7 0.586 86.47059 95.76547 C32H11.12 16.5 0.635
97.05882 107.4919 C42D8.2 vit-2 15.42 0.58 90.70588 100.456 C54G4.6
ASMTL 18.71 0.637 110.0588 121.8893 daf-16 11.2 0.279 65.88235
72.96417 daf-2 18.13 0.76 106.6471 118.1107 vector 17.06 0.574
100.3529 111.1401 ZK1251.2 ins-7 17.05 0.606 100.2941 111.0749
ZK896.8 gcy-18 16.41 0.695 96.52941 106.9055 vector 16.63 0.468
vector 17.06 0.574 Vector 14.6 0.372 vector 13.1 0.532 15.3475
[0324]
6TABLE 6 Upregulated in daf-2 mutants and with daf-2 RNAi,
downregulated with daf-16 RNAi Gene Gene product/homology C15H9.1
C15H9.1: Protein of unknown function C53B7.3 C53B7.3: Protein with
weak similarity to EGF-like repeats, has moderate similarity to C.
elegans F46C8.4 C34C6.7 C34C6.7: Protein of unknown function
E04F6.9 E04F6.9: Protein of unknown function, has moderate
similarity to C. elegans E04F6.8 C50F7.5 C50F7.5: Protein of
unknown function, has moderate similarity to H. sapiens MUC1 gene
product, a transmembrane mucin Y15E3B.f Y15E3B.fY15E3B.f F09F7.7
F09F7.7: Protein of unknown function T23G7.3 T23G7.3: Protein of
unknown function, has weak similarity to S. cerevisiae Ygr280p
F08B12.4 F08B12.4: Protein of unknown function F47H4.10 F47H4.10:
Member of an uncharacterized protein family H14N18.1 H14N18.1:
unc-23: Highly similar to mammalian BAG-2, BCL2- associated
athanogene 2, a chaperone regulator C40H1.5 C40H1.5: Member of an
uncharacterized protein family T23B3.2 T23B3.2: Protein of unknown
function, has weak similarity to C. elegans F47B7.1 C25E10.8
C25E10.8: Member of an uncharacterized protein family Y105C5A.12
Y105C5A.12: Protein of unknown function B0507.8 B0507.8: Protein of
unknown function, has similarity to C. elegans B0507.7 C25E10.9
C25E10.9: Member of an uncharacterized protein family F38E11.1
F38E11.1: Member of the small heat shock protein family Y51A2D.11
Y51A2D.11: Member of an uncharacterized protein family Y51A2B.1
Y51A2B.1: Protein of unknown function, has moderate similarity to
C. elegans C07G3.2 C08E8.4 C08E8.4: Protein of unknown function,
has weak similarity to C. elegans C07G3.2 C44H9.5 C44H9.5: Protein
of unknown function K10E9.1 K10E9.1: Protein of unknown function
C08F11.3 C08F11.3: Protein of unknown function, has moderate
similarity to C. elegans F56G4.1 C01H6.6 C01H6.6: Protein of
unknown function, has weak similarity to S. cerevisiae Yo1060p
C24A11.8a C24A11.2 C24A11.2 W06D12.3 W06D12.3: fat-5: Likely a
palmitoyl-CoA delta-9 fatty acid desaturase, specific for medium
chain (14 T02B5.1 T02B5.1: Member of the carboxylesterase protein
family C05E4.9 C05E4.9: Putative ortholog of S. cerevisiae MLS1
gene product (Malate synthase 1, functions in glyoxylate cycle, has
near identity to Dal7p) ZC395.5 ZC395.5: Protein of unknown
function H24O09.c H24O09.c H24O09.c C24B9.9 C24B9.9: Protein of
unknown function, has moderate similarity to C. elegans T04C12.1
T20G5.7 T20G5.7: Protein of unknown function, has moderate
similarity to C. elegans T20G5.8 K07C6.4 K07C6.4: Member of the
P450 heme-thiolate protein family B0213.15 B0213.15: Member of the
P450 heme-thiolate protein family F09F7.6 F09F7.6: Protein of
unknown function R03E9.1 R03E9.1: mdl-1: Member of the MAD family
of putative transcription factors, interacts with C. elegans MAX-1
B0238.1 B0238.1: Member of the carboxylesterase protein family
E01G4.3 E01G4.3: Protein of unknown function M02D8.4 M02D8.4:
Member of the asparagine synthetase protein family Y6E2A.3 Y6E2A.3:
Protein of unknown function, has weak similarity to C. elegans
Y6E2A.5 F38E11.2 F38E11.2: hsp-12.6: Member of the small heat shock
protein family F17B5.1 F17B5.1: Protein with strong similarity to
C. elegans T20D4.7 gene product [Member of the thioredoxin protein
family] K07A1.7 K07A1.7: Protein with similarity to D. melanogaster
HDC (headcase) protein, a branching inhibitor produced by
specialized tracheal cells F48D6.4 F48D6.4: Protein of unknown
function F11A5.12 F11A5.12: Protein with similarity to estradiol
17-beta-dehydrogenases; strongly related to C. elegans C06B3.4,
C06B3.5, F25G6.5, and C56G2.6 C06B3.4 C06B3.4: Possible estradiol
17 beta-dehydrogenase, member of a protein family H10D18.2
H10D18.2: Putative paralog of C. elegans H10D18.4 Y40B10A.6
Y40B10A.e Y40B10A.e ZK355.3 ZK355.3: Protein of unknown function
K11G9.6 K11G9.6: mtl-1: Metallothionein-related, cadmium-binding
intestinal protein C02A12.4 C02A12.4: Member of an uncharacterized
protein family with weak similarity to Entemeba histolytica
N-acetylmuraminidase AC3.7 AC3.7: Member of the
UDP-glucuronosyltransferase protein family C54D10.3 C54D10.3:
Member of an uncharacterized protein family Upregulated in daf-2
mutants ZK973.7 ZK973.7: Protein of unknown function, putative
paralog of C. elegans ZK973_14.I ZK973.7 ZK973.7: Protein of
unknown function, putative paralog of C. elegans ZK973_14.I ZK973.7
ZK973.7: Protein of unknown function, putative paralog of C.
elegans ZK973_14.I W10G6.3 W10G6.3: ifa-2: Putative intermediate
filament protein ZK270.2a ZK270.2 ZK270.2 F16H6.7 F16H6.7: Member
of an uncharacterized protein family F57H12.7 F57H12.7: Protein of
unknown function, has weak similarity to C. elegans W06B11.1
F32A5.5 F32A5.5: Protein with strong similarity to human AQP9, an
aquaporin; putative paralog of C. elegans C01G6.1 and Y69E1A.G
C17G1.4 C17G1.4: Protein with moderate similarity to H. sapiens
MUC1 gene product, a transmembrane mucin K12G11.4 K12G11.4: Member
of the alcohol dehydrogenase protein family K12G11.3 K12G11.3:
Member of the alcohol dehydrogenase protein family F21F3.3 F21F3.3:
Protein with strong similarity to S. cerevisiae Ste14p, a farnesyl
cysteine T07D10.4 T07D10.4: Member of the C-type lectin protein
family H16D19.1 H16D19.1: Member of the C-type lectin protein
family K11D2.2 K11D2.2: Putative acid ceramidase, has strong
similarity to human ASAH, acid ceramidase (N-acylsphingosine
amidohydrolase) C52E4.1 C52E4.1: gcp-1: Cysteine protease expressed
in the intestine W08D2.4 W08D2.4: fat-3: Protein with delta6-fatty
acid-desaturase activity F53F4.13 F53F4.13: Protein of unknown
function, has weak similarity to C. elegans F20A1.10 C26C6.3
C26C6.3: Member of the zinc metalloprotease protein family, has
strong similarity to C. elegans TOH-2, has similarity to D.
melanogaster and human TGF-beta-like growth factors K08F4.7
K08F4.7: gst-4: Member of the glutathione S-transferase protein
family, has similarity to human and D. melanogaster glutathione
S-transferases VC5.3 VC5.3: Ladder protein VC5.3 VC5.3: Ladder
protein E01A2.8 E01A2.8: Putative paralog of C. elegans E01A2.7
K10B3.8 K10B3.8: gpd-2: Glyceraldehyde-3-phosphate dehydrogenase
R09B5.6 R09B5.6: Member of the hydroxyacyl-CoA dehydrogenase
protein family C56A3.2 C56A3.2: Member of an uncharacterized
protein family H22K11.1 H22K11.1: asp-3: Probable aspartyl protease
and an ortholog of human cathepsin D C06G8.1 C06G8.1: Protein of
unknown function, has strong similarity to C. elegans K02D7.5, has
similarity to D. melanogaster SLV (saliva), a putative
transmembrane protein Dowuregulated in daf-2 mutants and with daf-2
RNAi, upregulated with daf-16 RNAi M60.1 M60.1: Putative seine
proteinase, has strong similarity to human placental serine
proteinase, P11 ZK6.10 ZK6.10: Protein of unknown function,
putative paralog of C. elegans ZK6.11 C49C3.9 C49C3.9: Protein of
unknown function F55G11.7 F55G11.7: Member of an uncharacterized
protein family F55G11.8 F55G11.8: Member of an uncharacterized
protein family K10D11.1 K10D11.1: Member of an uncharacterized
protein family C32H11.4 C32H11.4: Member of an uncharacterized
protein family Y46C8_103.a Y46C8_103.a Y46C8_103.a F56D6.2 F56D6.2:
Member of the phospholipase A2 receptor protein family B0365.6
B0365.6: Member of the C-type lectin protein family ZK1127.10
ZK1127.10: Member of the cystathionine gamma-lyase protein family
C04F12.3 C04F12.3: Protein with similarity to human B-cell
CLL/lymphoma 3 (BCL3) T05A12.3 T05A12.3: Protein of unknown
function, has weak similarity to C. elegans R07G3.3 C17H12.8
C17H12.8: Member of an uncharacterized protein family C17B7.1
C17B7.1: G protein-coupled receptor, member of a large subfamily
that contains ODR-10 odorant response protein, no homolog found in
human or D. melanogaster F15E11.1 F15E11.1 F15E11.1 F15E11.12
F15E11.12 F15E11.12 F22A3.6 F22A3.6: Possible lysozyme, member of
an uncharacterized protein family F58F6.2 F58F6.2: Putative
collagen, has similarity to human COL9A1, alpha-1 collagen, type IX
B0478.1 B0478.1: jnk-1: Neuronally expressed serine/threonine
protein kinase of the MAP kinase subfamily F59D8.2 F59D8.2: vit-4:
Member of the vitellogenin protein family; expressed only in C.
elegans intestinal cells F59D8.1 F59D8.f F59D8.f ZK757.1 ZK757.1:
Protein of unknown function with similarity to a DHHC zinc finger
domain, putative paralog of C. elegans B0546.5 protein R11G1.3
R11G1.3: Member of the glutathione S-transferase protein family,
has similarity to H. sapiens and D. melanogaster glutathione
S-transferases C04F6.1 C04F6.1: vit-5: 170 kDa yolk protein F41A4.1
F41A4.1: Protein with weak similarity to C. elegans let-653
(Putative mucin-like protein required for development beyond the
late L1 larval stage or early L2 larval stage) C54G4.6 C54G4.6:
Protein with similarity to human ASMTL protein, acetylserotonin
N-methyltransferase-like protein Y19D10A.9 F15E11.9 F15E11.9
Y19D10A.9 Y19D10A.j Y19D10A.j Y19D10A.9 F56A4.j F56A4.j K08D8.5
K08D8.5: Member of an uncharacterized protein family C32H11.1
C32H11.1: Member of an uncharacterized protein family T03E6.7
T03E6.7: Member of the thiol protease protein family R09H10.5
R09H10.5: Member of the EGF-repeat protein family T25C12.3
T25C12.3: Member of the EGF-repeat protein family, member of the C-
type lectin family K12H4.7 K12H4.7: Member of the carboxypeptidase
protein family F28H7.3 F28H7.3: Member of the lipase protein family
Y38H6C.5 Y38H6C.5: Putative Zinc finger, CCHC class protein F57F4.3
F57F4.3: Protein of unknown function, putative paralog of C.
elegans F57F4.4 F57F4.4 F57F4.4: Protein of unknown function,
putative paralog of C. elegans F57F4.3 C12C8.2 C12C8.2: Member of
the cystathionine gamma-lyase protein family W01A11.4 W01A11.4:
Member of the galactoside-binding lectin protein family C08F11.8
CO8F11.8: Member of the UDP-glucuronosyltransferase protein family
Y38H6C.1 Y38H6C.1: Protein of unknown function, has weak similarity
to C. elegans M02H5.B K06A4.5 K06A4.5: Putative
3-hydroxyanthranilate 3,4-dioxygenase, has strong similarity to
human Hs.108441 gene product (3-hydroxyanthranilic acid
dioxygenase) F23H11.7 F23H11.7: Protein of unknown function F52G3.4
F52G3.4: Protein with weak similarity to C. elegans F33H12.6
F46E10.1 F46E10.1 F46E10.1 C39E9.1 C39E9.1: Member of the
testis-specific protein TPX-1 like protein family C25F6.3 C25F6.3:
Member of the 4Fe-4S ferredoxins and related iron-sulfur cluster
binding protein family F52E1.5 F52E1.5: Protein of unknown function
F28B4.3 F28B4.3: Member of the EGF-repeat protein family Y106G6H.10
Y106G6H.10: Protein of unknown function, putative paralog of C.
elegans Y106G6H.9 Y106G6H.9 Y106G6H.9: Protein of unknown function,
putative paralog of C. elegans Y106G6H.10 K06A4.1 K06A4.1: Putative
zinc metalloprotease with similarity over the N- terminus to human
and D. melanogaster TOLLOID-like proteins, has strong similarity
over the middle region to C. elegans F56A4.K and Y19D10A.K B0281.5
B0281.5: Putative potassium voltage-gated channel, Shaw-related
subfamily F13A7.9 F13A7.9: Protein with moderate similarity to S.
cerevisiae Skp1p, a component of the kinetochore complex and a
component of SCF (Skp1p- cullin-F-box) complexes that target
proteins for ubiquitin-dependent degradation; also has strong
similarity to several other C. elegans proteins Y51H7BR.2
Y51H7BR.2: Protein of unknown function, putative paralog of C.
elegans Y51H7BR.1 Y38E10A.14 Y38E10A.n Y38E10A.n Y46H3C_14.c
Y46H3C_14.c Y46H3C_14.c Y9D1A.1 Y9D1A.1: Protein with moderate
similarity to C. elegans Y9D1A.A Y49E10.8 Y49E10.8: Protein of
unknown function F56G4.3 F56G4.3: Protein of unknown function, is
identical to C. elegans PES-2 F56G4.2 F56G4.2: pes-2: Protein of
unknown function, is identical to C. elegans F56G4.3 C52D10.9
C52D10.9: Protein with similarity to the SKP1 family of proteins,
nearly identical to C. elegans C52D10.7 C52D10.7 C52D10.7: Protein
with similarity to the SKP1 family of proteins, nearly identical to
C. elegans C52D10.9 Y56A3A.15 Y56A3A.15: Member of an
uncharacterized protein family C31A11.5 C31A11.5: G protein-coupled
receptor, member of unnamed subfamily with distant homology to SRG
subfamily, no homolog found in human or D. melanogaster F49E11.7
F49E11.7: Member of the protein phosphatase protein family
C32H11.10 C32H11.10: Member of an uncharacterized protein family
C32H11.9 C32H11.9: Member of an uncharacterized protein family
ZK6.11 ZK6.11: Protein of unknown function, putative paralog of C.
elegans ZK6.10 B0554.6 B0554.6: Member of an uncharacterized
protein family F55G11.5 F55G11.5: Member of an uncharacterized
protein family C32H11.12 C32H11.12: Member of an uncharacterized
protein family F35E12.5 F35E12.5: Member of an uncharacterized
protein family C08F8.5 C08F8.5: Protein of unknown function, has
weak similarity to C. elegans F38H4.2 Y62H9A.3 Y62H9A.3: Protein of
unknown function, has weak similarity to C. elegans Y62H9A.5
Y62H9A.4 Y62H9A.4: Protein of unknown function, has weak similarity
to C. elegans Y62H9A.6 Y62H9A.6 Y62H9A.6: Protein of unknown
function, has weak similarity to C. elegans Y62H9A.4 Y62H9A.5
Y62H9A.5: Protein of unknown function, has weak similarity to C.
elegans Y62H9A.3 F49E12.2 F49E12.2: Member of the calpain protease
protein family ZK896.8 ZK896.8: gcy-18: Protein with a cytoplasmic
receptor tyrosine kinase domain and a guanylate cyclase domain, has
strong similarity to human natriuretic peptide receptor NPR1 and
photoreceptor-specific membrane retina guanylyl cyclase RetGC-2
T24B8.5 T24B8.5: Protein of unknown function, has weak similarity
to C. elegans F49F1.7 K02H11.2 K02H11.2: G protein-coupled
receptor, member of a large subfamily that contains ODR-10 odorant
response protein, no homolog found in human or D. melanogaster
Y22F5A.5 Y22F5A.5: Member of an uncharacterized protein family with
weak similarity to Entemeba histolytica N-acetylmuraminidase
C25B8.3 C25B8.3: cpr-6: Member of the Cathepsin B-like Cysteine
Protease family Y55B1AR.1 Y55B1AR.1: Protein with weak similarity
to C. elegans W09H1.6 (Galactoside-binding lectin) F49C12.7
F49C12.7: Member of an uncharacterized protein family ZK896.5
ZK896.5: Member of an uncharacterized protein family W02D9.7
W02D9.7: Protein of unknown function F49F1.1 F49F1.1: Member of an
uncharacterized protein family
[0325]
Sequence CWU 1
1
12 1 285 PRT Caenorhabditis elegans cytochrome C1 component of
complex III, gene C54G4.8 (CYC1) 1 Met Gln Arg Ala Val Val Gln Gly
Ser Lys Arg Gly Leu Ala Ala Leu 1 5 10 15 Ala Gly Val Thr Ala Ala
Ser Gly Met Gly Leu Val Tyr Ala Leu Glu 20 25 30 Asn Ser Val Ser
Ala Ser Gly Asp Asn Val His Pro Tyr Ala Leu Pro 35 40 45 Trp Ala
His Ser Gly Pro Phe Ser Ser Phe Asp Ile Ala Ser Val Arg 50 55 60
Arg Gly Tyr Glu Val Tyr Lys Gln Val Cys Ala Ala Cys His Ser Met 65
70 75 80 Lys Phe Leu His Tyr Arg His Phe Val Asp Thr Ile Met Thr
Glu Glu 85 90 95 Glu Ala Lys Ala Glu Ala Ala Asp Ala Leu Ile Asn
Asp Val Asp Asp 100 105 110 Lys Gly Ala Ser Ile Gln Arg Pro Gly Met
Leu Thr Asp Lys Leu Pro 115 120 125 Asn Pro Tyr Pro Asn Lys Lys Ala
Ala Ala Ala Ala Asn Asn Gly Ala 130 135 140 Ala Pro Pro Asp Leu Ser
Leu Met Ala Leu Ala Arg His Gly Gly Asp 145 150 155 160 Asp Tyr Val
Phe Ser Leu Leu Thr Gly Tyr Leu Glu Ala Pro Ala Gly 165 170 175 Val
Lys Val Asp Asp Gly Lys Ala Tyr Asn Pro Tyr Phe Pro Gly Gly 180 185
190 Ile Ile Ser Met Pro Gln Gln Leu Phe Asp Glu Gly Ile Glu Tyr Lys
195 200 205 Asp Gly Thr Pro Ala Thr Met Ser Gln Gln Ala Lys Asp Val
Ser Ala 210 215 220 Phe Met His Trp Ala Ala Glu Pro Phe His Asp Thr
Arg Lys Lys Trp 225 230 235 240 Ala Leu Lys Ile Ala Ala Leu Ile Pro
Phe Val Ala Val Val Leu Ile 245 250 255 Tyr Gly Lys Arg His Ile Trp
Ser Phe Thr Lys Ser Gln Lys Phe Leu 260 265 270 Phe Lys Thr Val Lys
Gly Arg Glu Pro Pro Lys Ala Gln 275 280 285 2 401 PRT
Caenorhabditis elegans NADH oxidoreductase component of complex I
(NADH/ubiquinone oxidoreductase), gene T10E9.7 (NUO2) 2 Met Asp Ser
Glu Ile Leu Asn Tyr Pro Ile His Arg Phe Leu Asp Ser 1 5 10 15 Ser
Leu Leu Gly Gln Phe Ile Gly Pro Glu Gly Ser Asn Ile Tyr Lys 20 25
30 Ile Glu Lys Tyr Asn Lys Val Ala Leu Asp Ile Trp Lys Asn Asp Glu
35 40 45 Glu Asn Ser Asn Val Arg Ile Thr Gly Pro Tyr Trp Asn Leu
Lys Ser 50 55 60 Ala Leu Asn Asp Val Leu Glu Leu Val Ser Thr Ile
Arg Asn Lys Asn 65 70 75 80 Gln Arg Tyr Lys Phe Glu Met Pro Ser Lys
Asp Ile Gly Phe Leu Ile 85 90 95 Gly Lys Asn Gly Ala Lys Ile Asn
Glu Ile Lys Leu Ser Ser Asn Val 100 105 110 Asp Val His Phe Glu Arg
Asn Asn Glu Asn Arg Asp Asn Gly Glu Thr 115 120 125 Asp Gly Arg Val
Lys Met Leu Gly Ser Val Ile Arg Gln Ala Val Ser 130 135 140 Arg Gln
Ile Val Arg Asn Ser Pro Ile Ser Thr Thr Ala Ala Val Ala 145 150 155
160 Gln Thr Asn Gln Thr Gly Asp Lys Lys Glu Ser Pro Lys Lys Pro Thr
165 170 175 Ile Trp Lys Ile Asp Glu His Lys Arg Glu Arg Leu Ala Asn
Phe Gly 180 185 190 Lys Tyr Ala Ala Glu Cys Leu Pro Lys Phe Val Gln
Lys Val Gln Phe 195 200 205 Ala Ala Gly Asp Glu Leu Glu Leu Leu Ile
His Pro Ser Gly Val Val 210 215 220 Pro Val Leu Ser Phe Leu Lys Gly
Asn His Ser Ala Gln Phe Thr Asn 225 230 235 240 Leu Thr Phe Ile Thr
Gly Met Asp Val Pro Thr Arg Lys Asn Arg Leu 245 250 255 Glu Val Ile
Tyr Ser Leu Tyr Ser Val Arg Phe Asn Ala Arg Val Arg 260 265 270 Val
Arg Thr Tyr Thr Asp Glu Ile Ala Pro Ile Asp Ser Ala Thr Pro 275 280
285 Val Phe Lys Gly Ala Asp Trp Phe Glu Arg Glu Val Tyr Asp Met Tyr
290 295 300 Gly Val Trp Phe Asn Asn His Pro Asp Leu Arg Arg Ile Leu
Thr Asp 305 310 315 320 Tyr Gly Phe Glu Gly His Pro Phe Arg Lys Asp
Tyr Pro Leu Ser Gly 325 330 335 Tyr Asn Glu Val Arg Tyr Asp Pro Glu
Leu Lys Arg Val Val Tyr Glu 340 345 350 Pro Ser Glu Leu Ala Gln Glu
Phe Arg Lys Phe Asp Leu Asn Thr Pro 355 360 365 Trp Glu Thr Phe Pro
Ala Phe Arg Asn Gln Ser Ile Thr Ser Gly Tyr 370 375 380 Glu Thr Ile
Leu Glu Val Ala Glu Pro Thr Pro Ala Thr Pro Gln Asn 385 390 395 400
Lys 3 207 PRT Caenorhabditis elegans ATP synthase component of
complex V (delta family), gene F27C1.7 (ATP3) 3 Met Ala Gln Leu Met
Lys Arg Gly Phe Ser Thr Ser Ala Ala Leu Ala 1 5 10 15 Lys Ala Gln
Leu Val Lys Thr Pro Ile Gln Val His Gly Val Glu Gly 20 25 30 Arg
Tyr Ala Ala Ala Leu Tyr Ser Ala Gly His Lys Gln Asn Lys Leu 35 40
45 Asp Gln Ile Ser Thr Asp Leu Asn Asn Val Arg Ser Val Tyr Lys Asp
50 55 60 Asn Lys Lys Phe Gln Glu Phe Val Leu Asp Pro Thr Leu Lys
Ala Asn 65 70 75 80 Lys Lys Lys Thr Ala Ile Glu Ala Ile Ser Thr Lys
Leu Gly Leu Thr 85 90 95 Lys Glu Thr Gly Asn Phe Leu Gly Leu Leu
Ala Glu Asn Gly Arg Leu 100 105 110 Asn Lys Leu Glu Ser Val Val Ser
Ser Phe Glu Ser Ile Met Arg Ala 115 120 125 His Arg Gly Glu Leu Phe
Val Gln Val Thr Ser Ala Glu Glu Leu Ser 130 135 140 Ser Ser Asn Gln
Lys Ala Leu Ser Asp Ala Leu Ser Lys Ile Gly Lys 145 150 155 160 Ser
Gly Gln Lys Leu Thr Val Thr Tyr Ala Val Lys Pro Ser Ile Leu 165 170
175 Gly Gly Leu Val Val Thr Ile Gly Asp Lys Tyr Val Asp Leu Ser Ile
180 185 190 Ala Ser Arg Val Lys Lys Tyr Lys Asp Ala Leu Ala Thr Ala
Ile 195 200 205 4 132 PRT Caenorhabditis elegans cytochrome C
oxidase component of complex IV, gene F26E4.9 (CCO1) 4 Met Ala Gln
Leu Ala Lys Thr Ala Val Ala Ala Leu Ser Lys Lys Leu 1 5 10 15 Val
Ala Pro Ala Ala Val Ala Arg Arg Thr Leu Ala Thr Glu Ala Ser 20 25
30 Pro Glu Asp Tyr Gly Tyr Tyr Pro Asp Pro Leu Glu His Ala Thr Gly
35 40 45 Arg Glu Lys Lys Met Leu Leu Ala Arg Leu Ala Gly Asp Asp
Arg Tyr 50 55 60 Glu Pro Lys Val Tyr Tyr Arg Ala Glu Ala Ser Thr
Lys Gln Lys Pro 65 70 75 80 Asn Leu Val Pro Ser His Tyr Asp Phe Arg
Ile Ile Gly Cys Met Cys 85 90 95 Glu Gln Asp Ser Gly His Val Asn
Phe Met Thr Ile Arg Lys Gly Asp 100 105 110 Pro Lys Arg Cys Glu Cys
Gly His Trp Phe Lys Gly Val Asp Ala Asp 115 120 125 Pro Glu Ser Ile
130 5 551 PRT Caenorhabditis elegans glucose phosphate isomerase
(phosphoglucose isomerase), gene Y87G2A.8 (GPI-1) 5 Met Ser Leu Ser
Gln Asp Ala Thr Phe Val Glu Leu Lys Arg His Val 1 5 10 15 Glu Ala
Asn Glu Lys Asp Ala Gln Leu Leu Glu Leu Phe Glu Lys Asp 20 25 30
Pro Ala Arg Phe Glu Lys Phe Thr Arg Leu Phe Ala Thr Pro Asp Gly 35
40 45 Asp Phe Leu Phe Asp Phe Ser Lys Asn Arg Ile Thr Asp Glu Ser
Phe 50 55 60 Gln Leu Leu Met Arg Leu Ala Lys Ser Arg Gly Val Glu
Glu Ser Arg 65 70 75 80 Asn Ala Met Phe Ser Ala Glu Lys Ile Asn Phe
Thr Glu Asn Arg Ala 85 90 95 Val Leu His Val Ala Leu Arg Asn Arg
Ala Asn Arg Pro Ile Leu Val 100 105 110 Asp Gly Lys Asp Val Met Pro
Asp Val Asn Arg Val Leu Ala His Met 115 120 125 Lys Glu Phe Cys Asn
Glu Ile Ile Ser Gly Ser Trp Thr Gly Tyr Thr 130 135 140 Gly Lys Lys
Ile Thr Asp Val Val Asn Ile Gly Ile Gly Gly Ser Asp 145 150 155 160
Leu Gly Pro Leu Met Val Thr Glu Ser Leu Lys Asn Tyr Gln Ile Gly 165
170 175 Pro Asn Val His Phe Val Ser Asn Val Asp Gly Thr His Val Ala
Glu 180 185 190 Val Thr Lys Lys Leu Asn Ala Glu Thr Thr Leu Phe Ile
Ile Ala Ser 195 200 205 Lys Thr Phe Thr Thr Gln Glu Thr Ile Thr Asn
Ala Glu Thr Ala Lys 210 215 220 Glu Trp Phe Leu Ala Lys Ala Gly Asp
Ala Gly Ala Val Ala Lys His 225 230 235 240 Phe Val Ala Leu Ser Thr
Asn Val Thr Lys Ala Val Glu Phe Gly Ile 245 250 255 Asp Glu Lys Asn
Met Phe Glu Phe Trp Asp Trp Val Gly Gly Arg Tyr 260 265 270 Ser Leu
Trp Ser Ala Ile Gly Leu Ser Ile Ala Val His Ile Gly Phe 275 280 285
Asp Asn Tyr Glu Lys Leu Leu Asp Gly Ala Phe Ser Val Asp Glu His 290
295 300 Phe Val Asn Thr Pro Leu Glu Lys Asn Ile Pro Val Ile Leu Ala
Met 305 310 315 320 Ile Gly Val Leu Tyr Asn Asn Ile Tyr Gly Ala Glu
Thr His Ala Leu 325 330 335 Leu Pro Tyr Asp Gln Tyr Met His Arg Phe
Ala Ala Tyr Phe Gln Gln 340 345 350 Gly Asp Met Glu Ser Asn Gly Lys
Phe Val Thr Arg His Gly Gln Arg 355 360 365 Val Asp Tyr Ser Thr Gly
Pro Ile Val Trp Gly Glu Pro Gly Thr Asn 370 375 380 Gly Gln His Ala
Phe Tyr Gln Leu Ile His Gln Gly Thr Arg Leu Ile 385 390 395 400 Pro
Ala Asp Phe Ile Ala Pro Val Lys Thr Leu Asn Pro Ile Arg Gly 405 410
415 Gly Leu His His Gln Ile Leu Leu Ala Asn Phe Leu Ala Gln Thr Glu
420 425 430 Ala Leu Met Lys Gly Lys Thr Ala Ala Val Ala Glu Ala Glu
Leu Lys 435 440 445 Ser Ser Gly Met Ser Pro Glu Ser Ile Ala Lys Ile
Leu Pro His Lys 450 455 460 Val Phe Glu Gly Asn Lys Pro Thr Thr Ser
Ile Val Leu Pro Val Val 465 470 475 480 Thr Pro Phe Thr Leu Gly Ala
Leu Ile Ala Phe Tyr Glu His Lys Ile 485 490 495 Phe Val Gln Gly Ile
Ile Trp Asp Ile Cys Ser Tyr Asp Gln Trp Gly 500 505 510 Val Glu Leu
Gly Lys Gln Leu Ala Lys Val Ile Gln Pro Glu Leu Ala 515 520 525 Ser
Ala Asp Thr Val Thr Ser His Asp Ala Ser Thr Asn Gly Leu Ile 530 535
540 Ala Phe Ile Lys Asn Asn Ala 545 550 6 201 PRT Caenorhabditis
elegans GTPase, gene T23H2.5 6 Met Ala Arg Arg Pro Tyr Asp Met Leu
Phe Lys Leu Leu Leu Ile Gly 1 5 10 15 Asp Ser Gly Val Gly Lys Thr
Cys Ile Leu Tyr Arg Phe Ser Asp Asp 20 25 30 Ala Phe Asn Thr Thr
Phe Ile Ser Thr Ile Gly Ile Asp Phe Lys Ile 35 40 45 Lys Thr Ile
Glu Leu Lys Gly Lys Lys Ile Lys Leu Gln Ile Trp Asp 50 55 60 Thr
Ala Gly Gln Glu Arg Phe His Thr Ile Thr Thr Ser Tyr Tyr Arg 65 70
75 80 Gly Ala Met Gly Ile Met Leu Val Tyr Asp Ile Thr Asn Ala Lys
Ser 85 90 95 Phe Asp Asn Ile Ala Lys Trp Leu Arg Asn Ile Asp Glu
His Ala Ser 100 105 110 Glu Asp Val Val Lys Met Ile Leu Gly Asn Lys
Cys Asp Met Ser Asp 115 120 125 Arg Arg Val Val Ser Arg Glu Arg Gly
Glu Lys Ile Ala Gln Asp His 130 135 140 Gly Ile Ser Phe His Glu Thr
Ser Ala Lys Leu Asn Val His Val Asp 145 150 155 160 Thr Ala Phe Tyr
Asp Leu Ala Glu Ala Ile Leu Ala Lys Met Pro Asp 165 170 175 Ser Thr
Asp Glu Gln Ser Arg Asp Thr Val Asn Pro Val Gln Pro Gln 180 185 190
Arg Gln Ser Ser Ser Gly Gly Cys Cys 195 200 7 396 PRT
Caenorhabditis elegans llw-1 (long-lived worm protein), gene
Y54G11A.8 7 Met Leu Pro Ser Met Ser Lys Leu Cys Thr Ser Thr Val Arg
Pro Val 1 5 10 15 Ala Ala Ala Phe Ser Thr Gly Thr Thr Arg Gln His
His Ser Ser Gly 20 25 30 Arg Ser Arg Gln Gln His His Arg His Gly
Gly Ser Gly Gly Lys Thr 35 40 45 Asn Gly Gly Gly Arg Trp Ser Arg
Tyr Gly Lys Ser Ala Ala Thr Gly 50 55 60 Gly Thr Thr Val Leu Ala
Leu Ser Trp Met Thr Thr Ile Lys Asp Val 65 70 75 80 Leu Gly Ile Glu
Lys Val Gln Leu Asp Ala Asp Pro Leu Lys Glu Lys 85 90 95 Val Lys
Gln Ser Trp Leu Tyr Arg Lys Arg Arg Gln Tyr Asp Asp Ala 100 105 110
Ile Gln Val Leu Gln Leu Ala Leu Glu Glu Ala Glu Glu Arg Lys Glu 115
120 125 Asp Met Pro Ile Thr Arg Val Tyr Asp Glu Met Ala Asn Thr Phe
Tyr 130 135 140 Glu Lys Met Asn Leu Asp Glu Ala Asp Lys Tyr Phe Arg
Ile Val Ile 145 150 155 160 Gln Arg Leu Val Gln Leu His Gly Lys Lys
Asp Phe Asp Pro Glu Phe 165 170 175 Ile Gly Val Ser Leu Lys Leu Ala
Asp Ile Leu Ala His Arg Gly Asp 180 185 190 Leu Glu Ser Ala Glu Ser
Gly Phe Lys His Cys Val Arg Arg Gln Met 195 200 205 Lys Val Met Glu
Glu His Met Lys Lys Phe Ser Val Ala His Gly Ala 210 215 220 Leu Val
Glu Asp Arg His Thr Val Asp Thr Phe Gly Pro Met Tyr Thr 225 230 235
240 Asp Pro Ile Ala Leu Phe Gly Met Thr Leu Glu Ala Tyr Ala Asn Phe
245 250 255 Leu Ile Asn Tyr Cys Gly Glu Thr Arg Met Ala Glu Val Glu
Glu Tyr 260 265 270 Ile Asp Glu Val Met Lys Ile Ser Tyr Gln Ile Tyr
Gly Ala Ser Ser 275 280 285 Ala His Thr Ile Asn Met Leu Asn Asn Phe
Gly Ala Thr Leu Val Leu 290 295 300 Lys Asn Arg Phe Glu Leu Ala Lys
Lys Tyr Leu Ala Ile Gly Val Asp 305 310 315 320 Arg Ile Leu Tyr Val
Asn Glu Cys Ala His Met Leu Pro Gly Tyr Tyr 325 330 335 Cys Asn Tyr
Ala Glu Ser Leu Phe His Thr Gly Gln Lys Asn Glu Ala 340 345 350 Leu
Glu Phe Ala Arg Lys Ala Val Gln Met Ser Arg Ser Gly Asp Asp 355 360
365 Arg Val Arg His Tyr Thr Gln Asn Phe Leu Asn Asp Leu Glu Lys Asp
370 375 380 Ile Asn Arg Gly Lys Pro Lys Ser Trp Trp Phe Phe 385 390
395 8 189 PRT Caenorhabditis elegans llw-2 (long-lived worm
protein), gene F59E12.10 8 Met Asn Ala Ser Ser Arg Thr Lys Pro Ala
Ile Asp Leu Asn Lys Val 1 5 10 15 Pro Pro Ile Asp His His Arg Thr
Ala Val Thr Phe Asn Cys Leu Ile 20 25 30 Met Lys Met Thr Glu Met
Leu Asn Asn Phe Gly Asn Lys Met Glu Asp 35 40 45 Ile Leu Glu Lys
Ala Glu Gln Ser Leu Asp Thr Ala Asp Arg Lys Leu 50 55 60 Arg Leu
Met Glu Ser Lys Leu Ala Gly Met Ser Leu Glu Asp Lys Ser 65 70 75 80
Thr Thr Ala Thr Pro Ser Ser Ala Pro Glu Ile Asp Glu Ile His Glu 85
90 95 Ser Asn Pro Ser Ser Ser Gln Ile Val Glu Glu Thr Val Glu Glu
Lys 100 105 110 Pro Glu Glu His Thr Thr Thr Val Leu Ile Lys Asp Asp
Pro Ala Tyr 115 120 125 Ser Lys Tyr Phe Lys Met Leu Lys Leu Gly Val
Leu Glu Ala Gly Val 130 135 140 Ile Gln Lys Met Lys Ser Glu Gly Val
Asp Pro Ser Ile Leu Lys Arg 145 150 155 160 Gly Asp
Glu Pro Ser Arg Pro Gln Ala Gln Thr Ser Arg Asn Tyr Glu 165 170 175
Ser Ser Gly Glu Ser Thr Ala Ser Phe Ser Asp Ser Asp 180 185 9 497
PRT Caenorhabditis elegans llw-3 (long-lived worm protein), gene
Y48E1B.1 9 Met Tyr His Val Pro Leu Ile Pro Arg Asp Ala Gly Arg Glu
Glu Thr 1 5 10 15 Ile Phe Arg Ile Asn Gln Ser Leu Gln Lys Leu Leu
Arg Val Ser Asp 20 25 30 Glu Ile Phe Asp Arg Val Glu His Arg Ile
Thr Arg Ile His Gly Lys 35 40 45 Ala Glu Ala Ile Asp Arg Arg Thr
Glu Val Leu Glu Lys Lys Leu Glu 50 55 60 Ser Leu Gln Glu Ser Asp
Lys Val Ile Thr Phe Thr Leu Pro Arg Gln 65 70 75 80 Leu Pro Lys Leu
Pro Glu Glu Pro Pro Thr Ser Thr Ser Leu Phe Arg 85 90 95 Ile Asn
Ile Asp Thr Glu His Phe Pro Gly Ser Glu Glu Leu Pro Ala 100 105 110
Phe Arg Arg Ala Asp Asp His Val Leu Arg Pro Cys Glu Pro Ile Asp 115
120 125 Phe Thr Tyr Glu Leu Asn Lys Pro Asp Lys Phe Phe Leu Thr Ser
Gln 130 135 140 Val Leu Lys Glu Tyr Glu Gln Lys Gly Trp Glu Arg Tyr
Lys Lys Arg 145 150 155 160 Leu Leu Gly Gly Leu Arg Glu Leu Ser Arg
Ser Pro Glu His Ile Ala 165 170 175 Glu Leu Phe Tyr Ala Gly Thr Ser
Ile Pro Ala Phe Glu Gly Val Ser 180 185 190 Gly Asp Phe Ser Lys Lys
Ala Leu Asp Ala Asp Asp Asp Gly Gly Thr 195 200 205 Ser Arg Ser Gly
Arg Thr Thr Asp Glu Leu Ala Gln Leu Arg Leu His 210 215 220 Glu Gln
Leu Leu Glu Asp Thr Ala Leu Ser Ser Thr Leu Met Gln Glu 225 230 235
240 Asp Ser Leu Asp Asp Asn His Pro Leu Ala Phe Arg Ile Asn Phe Asn
245 250 255 Glu Lys Lys Lys Lys Thr Ala Lys Met Val Glu Met Pro Asp
Ser Leu 260 265 270 Pro Asn Leu Lys Gly His Ala His Asp Phe Thr Leu
Arg Asp Pro Glu 275 280 285 Ile Asp Glu Asp Arg Leu Leu Asp Ile Leu
Pro Ala Asp Asp Gln Ile 290 295 300 Pro Glu Ala Ser Glu Pro Thr Glu
Ala Glu Ala Asp Ala Pro Thr Thr 305 310 315 320 Phe Ile Leu Pro Pro
Pro Pro Pro Pro Met Lys Leu Asp Pro Ser Pro 325 330 335 Gln Pro Ala
Ala Thr Pro Val Glu Ile Thr Glu Ile Pro Pro Ile Ile 340 345 350 Ser
Pro Pro Ala Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro 355 360
365 Pro Pro Gln Thr Pro Ser Ala Ser Ser Ser Val Thr Phe Ser Pro Thr
370 375 380 Lys Ser Val Asp Gly Gly Arg Ser Asp Leu Met Ala Ala Ile
Arg Ala 385 390 395 400 Ala Gly Gly Ala Gly Asn Ala Lys Leu Ser Arg
Ile Ala Glu Lys Pro 405 410 415 Lys Arg Lys Gly Lys Phe Asp Gly Ile
Leu Glu Ser Ser Ala Leu Leu 420 425 430 Gly Ala Ser Glu Thr Pro Arg
Asn Ser Ala Pro Ala Pro Asp Gly Gly 435 440 445 Gly Gly Gly Gly Asp
Leu Met Ser Ala Leu Ser Lys Ala Leu Asp Ala 450 455 460 Arg Arg Lys
Ala Ile Asn Gly Lys Val Glu Ala Gln Pro Pro Ala Lys 465 470 475 480
Val Ser Ser Thr Ile Pro Ala Pro Pro Asn Phe Asp Asp Glu Glu Trp 485
490 495 Asp 10 884 PRT Caenorhabditis elegans llw-4 (long-lived
worm protein), gene F45H10.4 10 Met Arg Ile Ile Ala Cys Ser Leu Leu
Ile Ala Ser Leu Ile Pro Thr 1 5 10 15 Val Ile Gly Leu Lys Ser Asn
Arg Thr Ser Cys Phe His Tyr Val Ser 20 25 30 Cys Leu Glu Ala Thr
Glu Ala Asn Leu Lys Gln Cys Ala Gly Gly Thr 35 40 45 Ala Ile Ser
Leu Thr Leu Glu Ala Lys Asp Val Asn Ile Arg Asp Leu 50 55 60 Val
Lys Tyr Arg Ala Leu Glu Phe Val Gly Cys Gln Asp Arg Leu Leu 65 70
75 80 Lys Glu Val Val Asp Phe Glu Ser Leu Gln Ile Leu Val Asn Glu
Asp 85 90 95 Ala Arg Glu Cys Phe Glu Lys Leu Pro Glu Ser Ser Lys
Arg Val Glu 100 105 110 Glu Phe Thr Asp Ser Cys Asp Tyr Val Gln Pro
Val Ser Arg Asn Ala 115 120 125 Ser Lys Gly Asp Ala Leu Gln Cys Leu
Ile Glu Phe Lys Gln Asp Arg 130 135 140 Glu Tyr Cys Glu Ser Leu Leu
Glu Cys Cys Pro Asp His Thr Arg Cys 145 150 155 160 Gly Glu Arg Met
Asn Ala Val Ser Leu Ser Tyr Gln Asn Ala Arg Val 165 170 175 Lys Ala
Glu Gln Ile Val Tyr Ser Met Ile Ser Cys Ile Val Val Asn 180 185 190
Asp Pro Arg Phe Leu Arg Glu Gly Ala Arg Leu Gln Ser Leu Arg Asp 195
200 205 Pro Tyr Arg Asn Ala Gly Val Pro Phe Leu Arg Pro Asp Ala Tyr
Thr 210 215 220 Glu Ala Arg Ile Thr Arg Arg Leu Ala Leu Thr Ser Thr
Ala Thr Leu 225 230 235 240 Ser Gln Arg Arg Glu Arg Phe Leu Lys Lys
Tyr Ser Gln Ile Arg Gln 245 250 255 Val Val Ala Gln Asn Leu Phe Gly
Gln Gln Gln Arg Leu Thr Arg Pro 260 265 270 Val Glu Asp Leu Val Thr
Glu Thr Ser Ser Lys Leu Ala Val Glu Glu 275 280 285 Ala Pro Glu Glu
Thr Thr Thr Gln Glu Glu Thr Thr Thr Asp Ala Ser 290 295 300 Glu Val
Thr Thr Thr Lys Ala Val Glu Glu Ala Thr Glu Glu Val Thr 305 310 315
320 Glu Glu Ala Thr Glu Ala Thr Glu Ala Pro Val Ala Thr Thr Lys Glu
325 330 335 Ser Ser Glu Met His Val Asn Thr Ile Arg Asn Met Ile Arg
Ser Ala 340 345 350 Ser Glu Lys Asp Leu Ser Lys Tyr Val Thr Leu Ile
Ser Glu Gly Lys 355 360 365 Phe Ser Glu Leu Phe Glu Leu Ala Glu Gln
Lys Lys Leu Thr Leu Thr 370 375 380 Ser Lys Phe Asp Glu Lys Leu Ser
Ser Lys Met Ala Lys Leu Lys Asp 385 390 395 400 Leu Ile Asn Glu Ala
Leu Ser Glu Lys Glu Lys Ser Gly Glu Ile Glu 405 410 415 Gln Ala Met
Glu Lys Phe Glu Lys Pro Glu Lys Ser Glu Leu Val Ala 420 425 430 Met
Glu Asp Lys Asp Thr Pro Ala Val Phe Thr Ile Ser Asp Ser Leu 435 440
445 Lys His Lys Lys Ala Glu Ala Lys Leu Ala His Thr Ile Val Ser Arg
450 455 460 Asn Val Val Glu Ala Glu Asn Ala Ile Glu Lys Glu Val Val
Glu Pro 465 470 475 480 Lys Ala Glu Glu Lys Lys Val Lys Glu Glu Asp
Val Lys Ala Val Ala 485 490 495 Glu Glu Lys Lys Glu Glu Lys Lys Pro
Gly Lys Leu Pro Met Lys Ile 500 505 510 Glu Lys Leu Glu Lys Pro Val
Asp Thr Lys Ser Glu Asn His Glu Leu 515 520 525 Lys Lys Val Leu Asp
Asp Lys Glu Arg Ala Leu Leu Val Glu Ser Glu 530 535 540 Ile Lys Asn
Thr Ala Glu Glu Thr Lys Pro Lys Val Glu Ser Phe Lys 545 550 555 560
Ser Glu Glu Thr Thr Val Ala Ile Asp Asp Met Pro Ala Leu Glu Lys 565
570 575 Glu Glu Ser Ala Glu Lys Lys Glu Thr Thr Gly Glu Pro Thr Thr
Thr 580 585 590 Glu Ala Ala Val Glu Thr Thr Glu Ala Ser Glu Thr Pro
Lys Pro Glu 595 600 605 Ala Lys Pro Glu Leu Leu Ser Asn Leu Glu Asp
Val Leu Thr Leu Thr 610 615 620 Thr Pro Glu Thr Glu Thr Ile Glu Gly
Ser Gly Glu Arg Glu Glu Pro 625 630 635 640 Thr Thr Ser Ala Pro Ala
Ala Glu Ala Thr Ser Glu Ile Thr Leu Leu 645 650 655 Lys Ser Ser Ser
Asp Val Ala Val Ile Glu Asn Val Lys Arg Ile Arg 660 665 670 Pro Arg
Thr Glu Gln Thr His Cys Gln Gln Tyr Ala Ser Cys Trp Gln 675 680 685
Thr Val Leu Asp Tyr Glu Gln Gln Cys Asp Arg Lys Tyr Ser Thr Glu 690
695 700 Val Leu Ser His Gly Ile Asp Asp Ser Glu Ile Leu Asn Ile Leu
His 705 710 715 720 Asn Ser Ser Ile Ser His His Glu Ile Val Leu Lys
Ala Cys Leu Arg 725 730 735 Pro Leu Asp Arg Ser Val His Ser Thr Leu
Lys Gln Leu Leu Val Ile 740 745 750 Gln Arg Gly Val Arg Lys Ala Cys
Leu Glu Leu Gly Arg Asn Lys Ile 755 760 765 Ala Val Thr Asp Ser Glu
Glu Ala Leu Cys Asn Thr Glu Ile Pro Ser 770 775 780 Thr Ala Ala Ile
Asp Glu Phe Ile Ser Ser Glu His Val Arg Ser Gln 785 790 795 800 Ser
Asn His Leu Thr Cys Arg Ala Lys Leu Glu Pro Ile Arg Glu Thr 805 810
815 Cys Ser Ile Val Arg Asn Cys Cys Ala Ser Val Asp Thr Cys Asp Asn
820 825 830 Tyr Ile Ser Ser Ser Pro Val Lys Lys Leu Glu Thr Glu Ala
Ile Arg 835 840 845 Arg Leu Val Lys Lys Gln Asn Asp Cys Glu Thr Lys
Met Leu Gln Thr 850 855 860 Leu Ser Tyr Ile His Glu Gln Leu Ser Asn
Pro Ser Arg Arg Arg Arg 865 870 875 880 Phe Tyr Tyr His 11 671 PRT
Caenorhabditis elegans HSF-1 (heat shock factor), gene Y53C10A.12
11 Met Gln Pro Thr Gly Asn Gln Ile Gln Gln Asn Gln Gln Gln Gln Gln
1 5 10 15 Gln Leu Ile Met Arg Val Pro Lys Gln Glu Val Ser Val Ser
Gly Ala 20 25 30 Ala Arg Arg Tyr Val Gln Gln Ala Pro Pro Asn Arg
Pro Pro Arg Gln 35 40 45 Asn His Gln Asn Gly Ala Ile Gly Gly Lys
Lys Ser Ser Val Thr Ile 50 55 60 Gln Glu Val Pro Asn Asn Ala Tyr
Leu Glu Thr Leu Asn Lys Ser Gly 65 70 75 80 Asn Asn Lys Val Asp Asp
Asp Lys Leu Pro Val Phe Leu Ile Lys Leu 85 90 95 Trp Asn Ile Val
Glu Asp Pro Asn Leu Gln Ser Ile Val His Trp Asp 100 105 110 Asp Ser
Gly Ala Ser Phe His Ile Ser Asp Pro Tyr Leu Phe Gly Arg 115 120 125
Asn Val Leu Pro His Phe Phe Lys His Asn Asn Met Asn Ser Met Val 130
135 140 Arg Gln Leu Asn Met Tyr Gly Phe Arg Lys Met Thr Pro Leu Ser
Gln 145 150 155 160 Gly Gly Leu Thr Arg Thr Glu Ser Asp Gln Asp His
Leu Glu Phe Ser 165 170 175 His Pro Cys Phe Val Gln Gly Arg Pro Glu
Leu Leu Ser Gln Ile Lys 180 185 190 Arg Lys Gln Ser Ala Arg Thr Val
Glu Asp Lys Gln Val Asn Glu Gln 195 200 205 Thr Gln Gln Asn Leu Glu
Val Val Met Ala Glu Met Arg Ala Met Arg 210 215 220 Glu Lys Ala Lys
Asn Met Glu Asp Lys Met Asn Lys Leu Thr Lys Glu 225 230 235 240 Asn
Arg Asp Met Trp Thr Gln Met Gly Ser Met Arg Gln Gln His Ala 245 250
255 Arg Gln Gln Gln Tyr Phe Lys Lys Leu Leu His Phe Leu Val Ser Val
260 265 270 Met Gln Pro Gly Leu Ser Lys Arg Val Ala Lys Arg Gly Val
Leu Glu 275 280 285 Ile Asp Phe Cys Ala Ala Asn Gly Thr Ala Gly Pro
Asn Ser Lys Arg 290 295 300 Ala Arg Met Asn Ser Glu Glu Gly Pro Tyr
Lys Asp Val Cys Asp Leu 305 310 315 320 Leu Glu Ser Leu Gln Arg Glu
Thr Gln Glu Pro Phe Ser Arg Arg Phe 325 330 335 Thr Asn Asn Glu Gly
Pro Leu Ile Ser Glu Val Thr Asp Glu Phe Gly 340 345 350 Asn Ser Pro
Val Gly Arg Gly Ser Ala Gln Asp Leu Phe Gly Asp Thr 355 360 365 Phe
Gly Ala Gln Ser Ser Arg Tyr Ser Asp Gly Gly Ala Thr Ser Ser 370 375
380 Arg Glu Gln Ser Pro His Pro Ile Ile Ser Gln Pro Gln Ser Asn Ser
385 390 395 400 Ala Gly Ala His Gly Ala Asn Glu Gln Lys Pro Asp Asp
Met Tyr Met 405 410 415 Gly Ser Gly Pro Leu Thr His Glu Asn Ile His
Arg Gly Ile Ser Ala 420 425 430 Leu Lys Arg Asp Tyr Gln Gly Ala Ser
Pro Ala Ser Gly Gly Pro Ser 435 440 445 Thr Ser Ser Ser Ala Pro Ser
Gly Ala Gly Ala Gly Ala Arg Met Ala 450 455 460 Gln Lys Arg Ala Ala
Pro Tyr Lys Asn Ala Thr Arg Gln Met Ala Gln 465 470 475 480 Pro Gln
Gln Asp Tyr Ser Gly Gly Phe Val Asn Asn Tyr Ser Gly Phe 485 490 495
Met Pro Ser Asp Pro Ser Met Ile Pro Tyr Gln Pro Ser His Gln Tyr 500
505 510 Leu Gln Pro His Gln Lys Leu Met Ala Ile Glu Asp Gln His His
Pro 515 520 525 Thr Thr Ser Thr Ser Ser Thr Asn Ala Asp Pro His Gln
Asn Leu Tyr 530 535 540 Ser Pro Thr Leu Gly Leu Ser Pro Ser Phe Asp
Arg Gln Leu Ser Gln 545 550 555 560 Glu Leu Gln Glu Tyr Phe Thr Gly
Thr Asp Thr Ser Leu Glu Ser Phe 565 570 575 Arg Asp Leu Val Ser Asn
His Asn Trp Asp Asp Phe Gly Asn Asn Val 580 585 590 Pro Leu Asp Asp
Asp Glu Glu Gly Ser Glu Asp Pro Leu Arg Gln Leu 595 600 605 Ala Leu
Glu Asn Ala Pro Glu Thr Ser Asn Tyr Asp Gly Ala Glu Asp 610 615 620
Leu Leu Phe Asp Asn Glu Gln Gln Tyr Pro Glu Asn Gly Phe Asp Val 625
630 635 640 Pro Asp Pro Asn Tyr Leu Pro Leu Ala Asp Glu Glu Ile Phe
Pro His 645 650 655 Ser Pro Ala Leu Arg Thr Pro Ser Pro Ser Asp Pro
Asn Leu Val 660 665 670 12 200 PRT Artificial Sequence Description
of Artificial Sequencepoly Gly flexible linker 12 Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 1 5 10 15 Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 20 25 30
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 35
40 45 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly 50 55 60 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly 65 70 75 80 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly 85 90 95 Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly 100 105 110 Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly 115 120 125 Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 130 135 140 Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 145 150 155 160
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 165
170 175 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly 180 185 190 Gly Gly Gly Gly Gly Gly Gly Gly 195 200
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