Title: LETM1: modulators of cellular proliferation
Abstract: The present invention relates to regulation of cellular proliferation. More particularly, the present invention is directed to nucleic acids encoding LETM1 ("leucine zipper EF hand transmembrane receptor"), which is involved in modulation of cellular proliferation and cell cycle regulation. The invention further relates to methods for identifying and using agents, including small molecule chemical compositions, antibodies, siRNA, antisense nucleic acids, and ribozymes, that modulate cell cycle regulation and cellular proliferation via modulation of LETM1 and LETM1 related signal transduction; as well as to the use of expression profiles and compositions in diagnosis and therapy related to cell cycle regulation and modulation of cellular proliferation.
Patent Number: 7,005,254 Issued on 02/28/2006 to Demo, et al.
| Inventors:
|
Demo; Susan (Sunnyvale, CA);
Hitoshi; Yasumichi (Mountain View, CA);
Pearsall; Denise (Belmont, CA)
|
| Assignee:
|
Rigel Pharmaceuticals, Incorporated (South San Franicsco, CA)
|
| Appl. No.:
|
160663 |
| Filed:
|
May 31, 2002 |
| Current U.S. Class: |
435/4; 435/6; 435/7.1 |
| Current Intern'l Class: |
C12Q 1/00 (20060101); C12Q 1/68 (20060101); G01N 33/53 (20060101) |
| Field of Search: |
435/4,71,6
514/1
|
References Cited [Referenced By]
U.S. Patent Documents
| 2002/0132753 | Sep., 2002 | Rosen et al.
| |
Other References
Endele, et al.; "LETM1, A Novel Gene Encoding a Putative EF-Hand Ca2+-Binding
Protein, Flanks the Wolf-Hirschhorn Syndrome (WHS) Critical Region and Is Deleted
in Most WHS Patents", Genomics 60, 218-225 (1999).
Database GenBank, Accession No. AF061025, Oct. 5, 1999.
|
Primary Examiner: Nickol; Gary B.
Attorney, Agent or Firm: Townsend and Townsend and Crew LLP
Parent Case Text
CROSS-REFERENCES TO RELATED APPLICATION
The present application is related to U.S. Pat. No. 60/296,817, filed Jun. 7,
2001, and U.S. Pat. No. 60/347,970, filed Oct. 19, 2001, herein each incorporated
by referenced in its entirety.
Claims
What is claimed is:
1. A method for identifying a compound that activates or inhibits cellular proliferation,
the method comprising the steps of:
(i) contacting the compound with a LETM1 polypeptide, the polypeptide encoded
by a nucleic acid that hybridizes under stringent conditions to a nucleic acid
encoding a polypeptide having the amino acid sequence of SEQ ID NO:2, wherein the
stringent conditions comprise 50% formamide, 5×SSC, and 1% SDS, incubating
at 42° C., or in 5× SSC, 1% SDS, incubating at 65° C., with wash
in 0.2×SSC, and 0.1% SDS at 65° C.; and
(ii) determining the functional effect of the compound upon the LETM1 polypeptide.
2. The method of claim 1, wherein the functional effect is measured in vitro.
3. The method of claim 2, wherein the functional effect is a physical effect.
4. The method of claim 3, wherein the functional effect is determined by measuring
ligand binding to the polypeptide.
5. The method of claim 2, wherein the functional effect is a chemical effect.
6. The method of claim 1, wherein the polypeptide is expressed in a eukaryotic
host cell or cell membrane.
7. The method of claim 6, wherein the functional effect is a physical effect.
8. The method of claim 7, wherein the functional effect is determined by measuring
ligand binding to the polypeptide.
9. The method of claim 6, wherein the functional effect is a chemical or phenotypic effect.
10. The method of claim 9, wherein the chemical or phenotypic effect is determined
by measuring cellular proliferation.
11. The method of claim 10, wherein the cellular proliferation is measured by
assaying for DNA synthesis or fluorescent marker dilution.
12. The method of claim 11, wherein DNA synthesis is measured by
3H
thymidine incorporation, BrdU incorporation, or Hoechst staining.
13. The method of claim 11, wherein the fluorescent marker is selected from the
group consisting of a cell tracker dye or green fluorescent protein.
14. The method of claim 1, wherein the compound inhibits cancer cell proliferation.
15. The method of claim 6, wherein the host cell is a cancer cell.
16. The method of claim 15, wherein the cancer cell is a breast, prostate, colon,
or lung cancer cell.
17. The method of claim 15, wherein the cancer cell is a transformed cell line.
18. The method of claim 15, wherein the cancer cell is p53 null or mutant.
19. The method of claim 15, wherein the cancer cell is p53 wild-type.
20. The method of claim 1, wherein the polypeptide is recombinant.
21. The method of claim 1, wherein the polypeptide is encoded by a nucleic acid
comprising a sequence of SEQ ID NO: 1.
22. The method of claim 1, wherein the compound is an antibody.
23. The method of claim 1, wherein the compound is an antisense molecule.
24. The method of claim 1, wherein the compound is an siRNA molecule.
25. The method of claim 1, wherein the compound is a small organic molecule.
26. The method of claim 1, wherein the compound is a peptide.
27. The method of claim 1, wherein said LETM1 polypeptide comprises a transmembrane sequence.
28. A method for identifying a compound that inhibits cellular proliferation,
the method comprising the steps of:
(i) contacting the compound with a LETM1 polypeptide, the polypeptide encoded
by a nucleic acid that hybridizes under stringent conditions to a nucleic acid
encoding a polypeptide having the amino acid sequence of SEQ ID NO:2, wherein the
stringent conditions comprise 50% formamide, 5×SSC, and 1% SDS, incubating
at 42° C., or in 5× SSC, 1% SDS, incubating at 65° C., with wash
in 0.2×SSC, and 0.1% SDS at 65° C.; and
(ii) determining the functional effect of the compound upon the LETM1 polypeptide.
29. The method of claim 28, wherein said LETM1 polypeptide causes G
0/G
1
cell cycle arrest in a p53 independent and a Rb dependent manner.
Description
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH
AND DEVELOPMENT
Not applicable.
FIELD OF THE INVENTION
The present invention relates to regulation of cellular proliferation. More particularly,
the present invention is directed to nucleic acids encoding LETM1 ("leucine zipper
EF hand transmembrane receptor"), which is involved in modulation of cellular proliferation
and cell cycle regulation. The invention further relates to methods for identifying
and using agents, including small molecule chemical compositions, antibodies, siRNA,
antisense nucleic acids, and ribozymes, that modulate cell cycle regulation and
cellular proliferation via modulation of LETM1 and LETM1 related signal transduction;
as well as to the use of expression profiles and compositions in diagnosis and
therapy related to cell cycle regulation and modulation of cellular proliferation.
BACKGROUND OF THE INVENTION
Cell cycle regulation plays a critical role in neoplastic disease, as well as
disease caused by non-cancerous, pathologically proliferating cells. Identifying
membrane proteins, their ligands, and downstream signal transduction pathways is
important for developing therapeutic regents to treat cancer and other proliferative diseases.
In recent years, there have been major developments in the understanding of the
cell cycle. Normal cell proliferation is tightly regulated by the activation and
deactivation of a series of proteins that constitute the cell cycle machinery.
The expression and activity of components of the cell cycle can be altered during
the development of a variety of human disease such as cancer, cardiovascular disease,
psoriasis, etc., where aberrant proliferation contributes to the pathology of the
illness. There are genetic screens to isolate critical components for cell cycle
regulation using different organisms such as yeast, worms, flies etc., since cell
cycle regulation is the most common machinery among all eukaryotic cells. However,
there is a need to establish screening for understanding human diseases caused
by disruption of cell cycle regulation and for the development of new therapeutics.
SUMMARY OF THE INVENTION
The present invention therefore provides nucleic acids encoding LETM1, which
is involved in modulation of cell cycle regulation and cellular proliferation.
The invention therefore provides methods of screening for compounds, e.g., small
molecules, antibodies, siRNA, antisense molecules, and ribozyme, that are capable
of modulating cell cycle regulation and cellular proliferation, e.g., either inhibiting
or activating cellular proliferation. Therapeutic and diagnostic methods and reagents
are also provided.
In one aspect of the invention, nucleic acids encoding LETM1 membrane receptors
are provided. In another aspect, the present invention provides nucleic acids,
such as probes, siRNA, antisense oligonucleotides, and ribozymes, that hybridize
to a gene encoding LETM1. In another aspect, the invention provides expression
vectors and host cells comprising LETM1-encoding nucleic acids. In another aspect,
the present invention provides LETM1 protein, and antibodies thereto.
In another aspect, the present invention provides a method for identifying a
compound
that modulates cell cycle regulation and cellular proliferation, the method comprising
the steps of: (i) contacting the compound with a LETM1 polypeptide; and (ii) determining
the functional effect of the compound upon the LETM1 polypeptide.
In one embodiment, the functional effect is a physical effect or a chemical effect.
In one embodiment, the polypeptide is expressed in a eukaryotic host cell. In another
embodiment, the functional effect is determined by measuring receptor or signal
transduction activity, e.g., increases in intracellular calcium or other signaling compounds.
In another aspect, the present invention provides a method of modulating cell
cycle regulation and cellular proliferation 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.
In one embodiment, the compounds identified using the assays of the invention
are used to treat neuromuscular disease, cancer, cardiovascular disease, and psoriasis.
In another aspect, the present invention provides a method of detecting the presence
of LETM1 nucleic acids and polypeptides in human tissue, the method comprising
the steps of: (i) isolating a biological sample; (ii) contacting the biological
sample with a LETM1-specific reagent that selectively associates with LETM1; and,
(iii) detecting the level of LETM1-specific reagent that selectively associates
with the sample.
In one embodiment, the human LETM1-specific reagent is selected from the group
consisting of: human LETM1-specific antibodies, human LETM1 specific oligonucleotide
primers, and human LETM1-nucleic acid probes.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 provide exemplary wild type nucleotide (SEQ ID NO: 1) and amino acid
(SEQ ID NO: 2) sequences for LETM1.
FIG. 2 provides data for cDNAs identified in a screen for inhibitors of cellular proliferation.
FIG. 3 demonstrates DNA transfer of phenotype for clone A4-976 (LETM1).
FIG. 4 shows analysis of LETM1 functional domains.
FIG. 5 provides functional pathway mapping for LETM1.
FIG. 6 provides a schematic pathway showing that LETM1 induces G
0/G
1
arrest through Rb (retinoblastoma protein).
FIG. 7 shows RT-PCR analysis of LETM1 expression in several tumor cell lines
and primary tumor samples.
FIG. 8 shows Taqman analysis of LETM1 expression in several tumor cell lines
and primary tumor samples. Prostate tumor cells (DU145 and PC3) showed increased
LETM1 levels are compared to primary prostate epithelial cells. LETM1 levels are
high in primary breast cells.
FIG. 9 shows that LETM1 overexpression in lung tumor cells relative to associated
normal tissues. LETM1 is downregulated in breast tumors.
DETAILED DESCRIPTION OF THE INVENTION
Introduction
Here we report the identification and molecular characterization of a cell surface
molecule, leucine zipper-EF-hand containing transmembrane protein 1 (LETM1), through
a retroviral cDNA/peptide library-based functional screening using Tet-regulatable
gene expression system. For the first time, LETM1 has been identified as a transmembrane
protein involved in modulation of cell cycle regulation and cellular proliferation.
LETM1 was identified from a functional genetic screen that selects for cells with
cell cycle arrest. Clone A4-976 caused G
0/G
1 arrest and was
found to encode LETM1, a membrane receptor (see, e.g., Endele et al.,
Genomics
60:218-225 (1999)). LETM-1 was originally identified as the gene that flanks
the critical region of the wolf-hirschhorn syndrome (WHS), a chromosomal disorder
characterized by a deletion at 4p16.3 and 4p monosomy resulting in facial dysmorphic
features and neurological manifestations, e.g., severe pre- and post-natal growth
retardation, severe mental retardation, developmental delay with microcephaly,
"Greek warrior helmet," impairment of muscular tone, seizures, and B cell deficiency.
LETM1 is deleted in most WHS patients and is localized to human chromosome 4p16.3.
Translocations t(4;14(p 16.3;q3) are associated with multiple myeloma. The results
provided herein demonstrate LETM-1 causes potent G
0/G
1 arrest
in p53 independent and Rb dependent manor. Cell cycle regulation through LETM-1
also plays an important role in neuromuscular development.
LETM1 contains a leader sequence, RGD, a transmembrane region, a leucine zipper
and two EF hands. Accession numbers AF061025 and NM
—012318 (SEQ
ID NO:1) provide an exemplary nucleotide sequence of wild type LETM1, and Accession
numbers NP
—036450 (SEQ ID NO:2) and AAD13138 provide exemplary
amino acid sequences of wild type LETM1. Expression analysis provided herein shows
that LETM1 is ubiquitously expressed in tumor cells. LETM1 is further over or underexpressed
in tumor cells as compared to normal tissues.
LETM1 is a protein involved in tumor progression that therefore represents
a drug target for compounds that suppress or activate cellular proliferation, or
cause cell cycle arrest, or cause release from cell cycle arrest. Agents identified
in these assays, including small molecule chemical compositions, antibodies, siRNA,
antisense nucleic acids, and ribozymes, that modulate cell cycle regulation and
cellular proliferation via modulation of LETM1 and LETM1 related signal transduction,
can be used to treat diseases related to cellular proliferation. Such modulators
are useful for treating cancers, such as melanoma, breast, ovarian, lung, gastrointestinal
and colon, prostate, and leukemia and lymphomas, e.g., multiple myeloma. LETM1
is also associated with Wolf Hirschhorn syndrome (WHS) and therefore modulators
of LETM1 should be useful for treating WHS. In addition, such modulators are useful
for treating noncancerous disease states caused by pathologically proliferating
cells such as thyroid hyperplasia (Grave's disease), psoriasis, benign prostatic
hypertrophy, neurofibromas, atherosclerosis, restenosis, and other vasoproliferative disease.
Definitions
By "disorder associated with cellular proliferation" or "disease associated with
cellular proliferation" herein is meant a disease state which is marked by either
an excess or a deficit of cellular proliferation. Such disorders associated with
increased cellular proliferation include, but are not limited to, cancer and non-cancerous
pathological proliferation.
The terms "LETM1" or a nucleic acid encoding "LETM1" refer to nucleic acids and
polypeptide polymorphic variants, alleles, mutants, and interspecies homologs that:
(1) have an amino acid sequence that has greater than about 60% amino acid sequence
identity, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98% or 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 an LETM1 nucleic acid (Accession numbers AF061025
and NM
—012318 or amino acid sequence of an LETM1 protein (Accession
numbers NP
—036450 and AAD13138); (2) bind to antibodies, e.g.,
polyclonal antibodies, raised against an immunogen comprising an amino acid sequence
of an LETM1 protein (Accession numbers NP
—036450 and AAD13138),
and conservatively modified variants thereof; (3) specifically hybridize under
stringent hybridization conditions to an anti-sense strand corresponding to a nucleic
acid sequence encoding an LETM1 protein (Accession numbers AF061025 and NM—12318
and Accession numbers NP
—036450 and AAD13138), and conservatively
modified variants thereof; (4) have a nucleic acid sequence that has greater than
about 95%, preferably greater than about 96%, 97%, 98%, 99%, or higher nucleotide
sequence identity, preferably over a region of at least about 25, 50, 100, 200,
500, 1000, or more nucleotides, to an LETM1 nucleic acid (Accession numbers AF061025
and NM
—12318. A polynucleotide or polypeptide sequence is typically
from 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.
"Membrane receptor activity," refers to signal transduction in response
to extracellular stimuli and production of second messengers such as IP3, cAMP,
and Ca2+ via stimulation of enzymes such as phospholipase C and adenylate cyclase.
Such activity can be measured by examining increases in intracellular calcium using
(Offermans & Simon,
J. Biol. Chem. 270:15175-15180 (1995)). Receptor activity
can be effectively measured by recording ligand-induced changes in [Ca
2+]
i
using fluorescent Ca
2+-indicator dyes and fluorometric imaging.
Such receptors have transmembrane, extracellular and cytoplasmic domains that
can be structurally identified using methods known to those of skill in the art,
such as sequence analysis programs that identify hydrophobic and hydrophilic domains
(see, e.g., Kyte & Doolittle,
J. Mol. Biol. 157:105-132 (1982)). Such domains
are useful for making chimeric proteins and for in vitro assays of the invention.
The phrase "functional effects" in the context of assays for testing compounds
that modulate activity of an LETM1 protein includes the determination of a parameter
that is indirectly or directly under the influence of an LETM1, e.g., a functional,
physical, or chemical effect, such as the ability to increase or decrease cellular
proliferation. It includes cell cycle arrest, the ability of cells to proliferate,
and other characteristics of proliferating cells. "Functional effects" include
in vitro, in vivo, and ex vivo activities.
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
an LETM1 protein, e.g., functional, physical and chemical 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; measuring cell surface
marker expression; measurement of changes in protein levels for LETM1-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
2+); identification of downstream
or reporter gene expression (CAT, luciferase, β-gal, GFP and the like), e.g.,
via chemiluminescence, fluorescence, calorimetric reactions, antibody binding,
inducible markers, and ligand binding assays.
"Inhibitors", "activators", and "modulators" of LETM1 polynucleotide
and polypeptide sequences are used to refer to activating, inhibitory, or modulating
molecules identified using in vitro and in vivo assays of LETM1 polynucleotide
and polypeptide sequences. 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 LETM1 proteins, e.g., antagonists.
"Activators" are compounds that increase, open, activate, facilitate, enhance activation,
sensitize, agonize, or up regulate LETM1 protein activity. Inhibitors, activators,
or modulators also include genetically modified versions of LETM1 proteins, e.g.,
versions with altered activity, as well as naturally occurring and synthetic ligands,
antagonists, agonists, antibodies, siRNA, antisense molecules, ribozymes, small
chemical molecules and the like. Such assays for inhibitors and activators include,
e.g., expressing LETM1 protein in vitro, in cells, or cell membranes, applying
putative modulator compounds, and then determining the functional effects on activity,
as described above.
Samples or assays comprising LETM1 proteins 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 activity value
of 100%. Inhibition of LETM1 is achieved when the activity value relative to the
control is about 80%, preferably 50%, more preferably 25-0%. Activation of LETM1
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.
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 cellular proliferation. 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.
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, most preferably a mammal such as a primate e.g., chimpanzee or human;
cow; dog; cat; a rodent, e.g., guinea pig, rat, mouse; rabbit; or a bird; reptile;
or fish.
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 70% identity, preferably 75%, 80%, 85%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region
(e.g., FIG. 1, 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 (see, e.g., NCBI web site). 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.
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.
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)).
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).
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.
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. An example
of potassium channel splice variants is discussed in Leicher, et al.,
J. Biol.
Chem. 273(52):35095-35101 (1998).
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.
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, γ-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 a 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.
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.
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.
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)).
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
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., transmembrane domains, extracellular domains,
and cytoplasmic tail domains. Domains are portions of a polypeptide that form a
compact unit of the polypeptide and are typically 15 to 350 amino acids long. Exemplary
domains include extracellular domains, transmembrane domains, and cytoplasmic domains.
Typical domains are made up of sections of lesser organization such as stretches
of β-sheet and α-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.
An "siRNA" or "RNAi" refers to a nucleic acid that forms a double stranded RNA,
which double stranded RNA has the ability to reduce or inhibit expression of a
gene or target gene when the siRNA expressed in the same cell as the gene or target
gene. "siRNA" thus refers to the double stranded RNA formed by the complementary
strands. The complementary portions of the siRNA that hybridize to form the double
stranded molecule typically have substantial or complete identity. In one embodiment,
an siRNA refers to a nucleic acid that has substantial or complete identity to
a target gene and forms a double stranded siRNA. The sequence of the siRNA can
correspond to the full length target gene, or a subsequence thereof. Typically,
the siRNA is at least about 15-50 nucleotides in length (e.g., each complementary
sequence of the double stranded siRNA is 15-50 nucleotides in length, and the double
stranded siRNA is about 15-50 base pairs in length, preferable about preferably
about 20-30 base nucleotides, preferably about 20-25 nucleotides in length, e.g.,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.
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
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.
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.
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).
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° C. lower than the thermal melting point (T
m) for
the specific sequence at a defined ionic strength pH. The T
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
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.
Exemplary stringent hybridization conditions can be as following: 50% as
formamide, 5×SSC, and 1% SDS, incubating at 42° C., or, 5×SSC, 1%
SDS, incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDS at 65°
C. For PCR, a temperature of about 36° C. is typical for low stringency amplification,
although annealing temperatures may vary between about 32° C. and 48°
C. depending on primer length. For high stringency PCR amplification, a temperature
of about 62° C. is typical, although high stringency annealing temperatures
can range from about 50° C. to about 65° C., depending on the primer
length and specificity. Typical cycle conditions for both high and low stringency
amplifications include a denaturation phase of 90° C.-95° C. for 30 sec-2
min., an annealing phase lasting 30 sec.-2 min., and an extension phase of about
72° 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.).
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° C., and a wash in 1×SSC
at 45° 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
"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.
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
L)
and variable heavy chain (V
H) refer to these light and heavy chains respectively.
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)′
2, a dimer of Fab which itself is a light chain joined
to V
H-C
H1 by a disulfide bond. The F(ab)′
2
may be reduced under mild conditions to break the disulfide linkage in the hinge
region, thereby converting the F(ab)′
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))
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)).
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.
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.
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 LETM1 protein, 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 LETM1proteins 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).
Isolation of Nucleic Acids Encoding LETM1
This invention relies on 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)).
LETM1 nucleic acids, polymorphic variants, orthologs, and alleles that are
substantially identical to an amino acid sequence the Accession numbers provided
herein can be isolated using LETM1 nucleic acid probes and oligonucleotides under
stringent hybridization conditions, by screening libraries. Alternatively, expression
libraries can be used to clone LETM1 protein, polymorphic variants, orthologs,
and alleles by detecting expressed homologs immunologically with antisera or purified
antibodies made against human LETM1 or portions thereof.
To make a cDNA library, one should choose a source that is rich in LETM1 RNA.
The mRNA is then made into cDNA using reverse transcriptase, ligated into a recombinant
vector, and transfected into a recombinant host for propagation, screening and
cloning. Methods for making and screening cDNA libraries are well known (see, e.g.,
Gubler & Hoffinan,
Gene 25:263-269 (1983); Sambrook et al., supra; Ausubel
et al, supra).
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).
An alternative method of isolating LETM1 nucleic acid and its 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 human LETM1 directly from mRNA, from cDNA, from genomic libraries
or cDNA libraries. Degenerate oligonucleotides can be designed to amplify LETM1
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 LETM1 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.
Gene expression of LETM1 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
+ RNA, northern blotting, dot blotting, in situ hybridization,
RNase protection, high density polynucleotide array technology, e.g., and the like.
Nucleic acids encoding LETM1 protein can be used with high density oligonucleotide
array technology (e.g., GeneChip™) to identify LETM1 protein, orthologs,
alleles, conservatively modified variants, and polymorphic variants in this invention.
In the case where the homologs being identified are linked to modulation of cell
cycle regulation, they can be used with GeneChip™ 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).
The gene for LETM1 is 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
To obtain high level expression of a cloned gene, such as those cDNAs encoding
LETM1, one typically subclones LETM1 into an expression vector that contains a
strong promoter to direct transcription, a transcription/translation terminator,
and if for a nucleic acid encoding a protein, 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 the LETM1 protein 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.
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.
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 the LETM1 encoding nucleic acid in host cells. A typical
expression cassette thus contains a promoter operably linked to the nucleic acid
sequence encoding LETM1 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.
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.
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.
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
+, pMTO10/A
+, 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.
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.
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 a LETM1 encoding sequence under the direction of the
polyhedrin promoter or other strong baculovirus promoters.
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.
Standard transfection methods are used t
