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text: No recent wiki edits to this page. OverviewPhase is a single player rhythm and music games developed by Harmonix Music Systems for the 3rd gen iPod Nano, 5th gen iPod video, and the 6th gen iPod classic.GameplayThe game can support virtually any song that can be played on an iPod. The game then translates the songs into levels of play for each difficulty. There are three available difficulties: Easy, Medium, and Hard at the start of the game and two more difficulties (Expert and Insane) can be unlocked later. Just drag and drop whatever songs you want in the game into the Phase playlist and you are ready to go, but there is a 1,000 song limit for the game.The game is similar to Harmonix's other game, Guitar Hero. "Notes", or small circles, travel down the screen towards a series of three holes. When a note reaches a hole, hitting the rewind, center or forward button on the iPod plays that note. Hitting notes gives the player points, and fills up a meter which ranks your gameplay in stars. Unlike Guitar Hero, the game does not have a constant measure of your score. Instead, the game breaks the song into sections, and requires that you gain enough stars in that section to progress to the next one.Preloaded SongsIn addition to the songs you can add yourself, Phase does come with some songs to start off with."Nightlife Commando" by Bang Camaro"Dots and Dashes" by Dealership"Pop Music Is Not a Crime" by Freezepop"Midnight Gamma" by Intersect"Spira Mirabilis" by Kodomo"The Theme of the Awesome" by Speck"Dragonfly Remix" by Universal Hall PassUnlockablesTo unlock the Expert difficulty, beat Marathon mode on Hard.To unlock the Insane difficulty, beat Marathon mode on Expert.


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The largest wildfire in Arizona history is now 89 percent contained.Read More »
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It's a problem for everyone when the well runs dry
Benjamin Franklin probably was not being literal when he wrote, "When the well is dry, we know the worth of water." But the literal interpretation certainly applies to the approximately 25 million Americans who live in Arizona, California, Colorado, Nevada, New Mexico, Utah and Wyoming. People in these states don't need Earth Day or Water Awareness Month celebrations to remind them of the worth of their water. They already know that their well — the Colorado River — is running dry.Read More »
Crews fully contain 1 of 3 major Arizona wildfires
One of three major wildfires burning in Arizona was declared fully contained Sunday, a second was nearly out, and a third was growing only in a rugged area with only a handful of residents.Read More »
After the flames: Advanced ADEQ air monitors find historically high level of 'particularly nasty pollutant'
When it comes to wildfires, size really matters — the bigger the blaze, the greater potential danger to homes, businesses, wildlife, trees, shrubs, the environment, and most important, people.Read More »
Interior Department says federal lands added billions of dollars, thousands of jobs to state
Recreation on federal lands last year supported more than 8,000 jobs in Arizona's rural communities, the fourth-highest in the nation, according to an Interior Department report released Wednesday.Read More »
Crews fared well on key portion of Arizona fire
Crews fighting a wildfire in southern Arizona fared well in setting controlled fires aimed at eliminating forest fuels between the blaze and an upscale neighborhood.Read More »
Feds rely on locals to fight wildfires in national forests
When national forests go up in flames, as they are doing across Arizona right now, the U.S. government turns to local firefighters to help contain the blazes.Read More »
Fight against Arizona fire moves to NM border
Crews battling a massive wildfire in eastern Arizona for two weeks shifted their focus Monday to New Mexico, where they lit fires to stifle flare-ups that skipped along treetops threatening a small mountain town.Read More »
Driggs hauls off memorabilia from family cabin in Greer
Two Arizona senators – one who spends his time in D.C. and one whose office is at the Arizona Capitol – were forced to clear their personal belongings out of family cabins in Eastern Arizona as the Wallow Fire has raged unchecked.Read More »
Arizona National Guard sends truck unit to fire area
An Arizona National Guard cargo truck unit is shifting its summer training site to an armory in eastern Arizona so it can be readily available if needed for missions related with the Wallow fire.Read More »


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In the Klondike settlement of LEGOREDO® Town, young gold diggers can pan gold and have it "melted down" to a shiny gold coin.
LEGOLDMINE®The gold mine at LEGOREDO Town is a classic LEGOLAND® attraction – and an exciting adventure for the whole family. Gold diggers of all ages stand side by side as they pan gold with enthusiastic concentration.
Get a LEGO® gold coin as a souvenirIf you are lucky enough to find some gold dust or perhaps even a gold nugget, you can have it "melted down" into a shiny gold coin to take home as a souvenir.
Worth knowingThere is an extra fee for gold coins.
LEGO, the LEGO logo, the Brick and Knob configurations, the Minifigure, DUPLO, LEGENDS OF CHIMA, NINJAGO and LEGOLAND are trademarks of the LEGO Group. ©2015 The LEGO Group. The LEGO MOVIE © The LEGO Group & Warner Bros Entertainment Inc. © & ™ Lucasfilm Ltd.
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Here is a list of issues that were logged by the community and fixed in this release. This release focused mainly on the support for stored procedures. The following is a breakdown of the complete change log.
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Thank you for submitting this cool story - Trackback from DotNetShoutout
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VB.NET please
Thanks for your feedback, we have been wanting to get feedback on the current templates before implementing VB support. I think we are ready to release VB support in the next version of the templates.
When can we expect the next version for this template ??


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Cottage Grove Recognized in National EMS Research Study
Cottage Grove Department of Public Safety was recognized for their participation in an innovative Emergency Medical Services national research study. In the study, patients experiencing chest pains were given a special study solution—glucose, insulin, and potassium—or a placebo while being transported via ambulance to the hospital.
Cottage Grove Police Paramedic Tim Morning, Police Paramedic Shawn Ebeling, Firefighter Paramedic Kristen Shannon and EMS Captain Brian Bigham, attended the Feb. 15 recognition event where a dozen patients were reunited with their EMS responders. While the research team had regular contact with the patients after they left the hospital, this event was the first time participants could reconnect with their EMS care team.
"The study was a great experience for Cottage Grove EMS as we strive to participate in local and national research projects that help bring better patient care to the citizens we serve," said Wes Halverson, Deputy Fire Chief/EMS.
This same study took place in a total of 13 communities nationwide, including St. Paul. To conduct this study, Regions and Lakeview Hospitals partnered with local paramedics from Cottage Grove, Oakdale, Mahtomedi, Maplewood, White Bear Lake and Lakeview.
Dr. RJ Frascone, medical director for Cottage Grove Public Safety and Regions Hospital EMS, was the local physician in charge of the study. The results of the study showed the group of people who received the study solution had more survivors with smaller areas of damage to their heart.
"At least 250,000 Americans die from heart attacks each year," said Frascone. "Cottage Grove paramedics and EMTs were part of an important investigation that helped find new ways to improve the outcome for people in cardiac arrest across the country. This recognition event was the first of what we hope will be a standard way to share results of studies with the most important stakeholders— our patients."


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Isolated GSTO2 nucleic acid molecules that include a nucleotide sequence variant and nucleotides flanking the sequence variant are described, as well as GSTO2 allozymes. Methods for determining if a subject contains a GSTO2 sequence variant also are described.
1. An isolated nucleic acid molecule consisting of a variant glutathione S-transferase omega 2 (GSTO2) nucleic acid sequence, wherein said variant GSTO2 nucleic acid sequence is selected from the group consisting of:
a) at least fifteen contiguous nucleotides of SEQ ID NO:8, wherein said sequence includes nucleotide 121, 389, or 472 of SEQ ID NO:8, with the proviso that the nucleotide at position 121 is adenine, the nucleotide at position 389 is adenine, or the nucleotide at position 472 is adenine; and
b) the full-length complement of a).
2. The isolated nucleic acid of molecule of
3. The isolated nucleic acid molecule of
4. An article of manufacture comprising a substrate, wherein said substrate comprises a population of the isolated variant GSTO2 nucleic acid molecules of
5. The article of manufacture of
6. An isolated nucleic acid molecule consisting of a variant glutathione S-transferase omega 2 (GSTO2) nucleic acid and a label, wherein the sequence of said variant GSTO2 nucleic acid is selected from the group consisting of:
a) at least fifteen contiguous nucleotides of SEQ ID NO:8, wherein said sequence includes nucleotide 121,389, or 472 of SEQ ID NO:8, with the proviso that the nucleotide at position 121 is adenine, the nucleotide at position 389 is adenine, or the nucleotide at position 472 is adenine; and
b) the full-length complement of a).
7. The isolated nucleic acid of molecule of
8. The isolated nucleic acid molecule of
9. The isolated nucleic acid molecule of
10. The isolated nucleic acid molecule of
This application claims priority to U.S. Provisional Patent Application Ser. No. 60/697,128, filed on Jul. 7, 2005, the entire contents of which are hereby incorporated by reference.
This invention was made with government support under grant nos. GM28157, GM35720, and GM61388, awarded by the National Institutes of Health. The government has certain rights in the invention.
This document relates to GSTO2 nucleic acid and amino acid sequence variants.
The Omega class of glutathione transferases (GSTs) have been identified in organisms including human, mouse, rat, pig, Caenorhabditis elegans, and Drosophila melanogaster. These GSTs, which include glutathione S-transferase omega 2 (GSTO2) have poor activity with common GST substrates, but exhibit novel glutathione-dependent thioltransferase, dehydroascorbate reductase, and monomethylarsonate reductase activities, and also modulate calcium release by ryanodine receptors. The GSTO2 gene contains seven exons, the first of which does not contain GSTO2 coding sequence. The GSTO2 gene is separated from the GSTO1 gene by 7.5 kb on chromosome 10q24.3. GSTO2 is expressed at high levels in the testes, and also is expressed in liver, kidney, skeletal muscle, heart, and lung, with low level expression in the cervix, ovary, and prostate. Substrates for GSTO2 include monomethylarsonic acid (MMV), which is reduced from the +V form to the +III form, as well as dehydroascorbate (DHA) and glutathione (GSH), which can be reacted to give ascorbate and glutathione disulfate.
This document is based on the discovery of sequence variants that occur in both coding and non-coding regions of GSTO2 nucleic acids. Certain GSTO2 nucleotide sequence variants encode GSTO2 enzymes that are associated with individual differences in enzymatic activity. Other GSTO2 sequence variants in non-coding regions of the GSTO2 nucleic acid may alter regulation of transcription and/or splicing of the GSTO2 nucleic acid. Discovery of these sequence variants allows individual differences in the metabolism of drugs and other xenobiotics in humans to be assessed such that particular treatment regimens can be tailored to an individual based on the presence or absence of one or more sequence variants. For example, the presence of absence of a particular variant may indicate an individual's ability to metabolize arsenic, which is a contaminant in drinking water, and which can cause skin, bladder, kidney, liver, and lung cancer in individuals subjected to chronic arsenic exposure. The presence of absence of a particular variant also may indicate an individual's ability to metabolize arsenic trioxide, an agent that can be used to treat acute promyelocytic leukemia. Identification of GSTO2 sequence variants also may allow predisposition to cancer or conditions such as Alzheimer's or Parkinson's disease to be assessed in individuals.
In one aspect, this document features an isolated nucleic acid molecule consisting essentially of a variant GSTO2 nucleic acid sequence, wherein the variant GSTO2 nucleic acid sequence is selected from the group consisting of: a) at least ten contiguous nucleotides of SEQ ID NO:8, wherein said sequence includes nucleotide 121, 389, or 472 of SEQ ID NO:5, with the proviso that the nucleotide at position 121 is adenine, the nucleotide at position 389 is adenine, or the nucleotide at position 472 is adenine; b) at least ten contiguous nucleotides of SEQ ID NO:l, wherein said sequence includes nucleotide 15569, 15811, 15918, 15984, 16006, 16016, 16041, 16403, 16477, 16801, 16851, 16945, or 16948 of SEQ ID NO:1, with the proviso that a guanine is inserted after the nucleotide at position 15569, the nucleotide at position 15811 is cytosine, the nucleotide at position 15918 is thymine, the nucleotide at position 15984 is guanine, the nucleotide at position 16006 is cytosine, the nucleotide at position 16016 is adenine, the nucleotide at position 16041 is thymine, the nucleotide at position 16403 is thymine, the nucleotide at position 16477 is adenine, the nucleotide at position 16801 is cytosine, a thymine is inserted after the nucleotide at position 16851, the nucleotide at position 16945 is thymine, or the nucleotide at position 16948 is guanine; c) at least ten contiguous nucleotides of SEQ ID NO:1, wherein said sequence includes nucleotide 16957, 17056, 17119, 17132, 17158, 17172, or 17348 of SEQ ID NO:1, with the proviso that the nucleotide at position 16957 is guanine, the nucleotide at position 17056 is adenine, the nucleotide at position 17119 is adenine, the nucleotide at position 17132 is cytosine, the nucleotide at position 17158 is adenine, the nucleotide at position 17172 is guanine, or the nucleotide at position 17348 is thymine; d) at least ten contiguous nucleotides of SEQ ID NO:1, wherein said sequence includes nucleotide 17402, 17502, 17659 and 17662, 17683, 17752, 17785, 17827, 19044, 19496, 21635, 22747, or 22754 of SEQ ID NO:1, with the proviso that the nucleotide at position 17402 is guanine, the nucleotide at position 17502 is thymine, the nucleotides at positions 17660 and 17661 are deleted, the nucleotide at position 17683 is cytosine, the nucleotide at position 17752 is thymine, the nucleotide at position 17785 is adenine, a thymine is inserted after the nucleotide at position 17827, the nucleotide at position 19044 is guanine, the nucleotide at position 19496 is guanine, the nucleotide at position 21635 is thymine, the nucleotide at position 22747 is adenine, or the nucleotide at position 22754 is adenine; e) at least ten contiguous nucleotides of SEQ ID NO:1, wherein said sequence includes nucleotide 23291, 26236, 27722, 47154, or 47188 of SEQ ID NO:1, with the proviso that the nucleotide at position 23291 is thymine, the nucleotide at position 26236 is thymine, the nucleotide at position 27722 is cytosine, the nucleotide at position 47154 is guanine, or the nucleotide at position 47188 is thymine; f) at least ten contiguous nucleotides of SEQ ID NO:1, wherein said sequence includes nucleotide 47713, 47876, 48248, 48282, or 48357 of SEQ ID NO:1, with the proviso that the nucleotide at position 47713 is thymine, the nucleotide at position 47876 is guanine, the nucleotide at position 48248 is thymine, the nucleotide at position 48282 is cytosine, or the nucleotide at position 48357 is thymine; and g) the complement of a), b), c), d), e), or f). The isolated nucleic acid can be from 10 to 100 nucleotides in length or from 20 to 50 nucleotides in length.
This document also features a vector comprising the nucleic acid molecule described herein. The nucleic acid molecule can be from 20 to 50 nucleotides in length.
In another aspect, this document features an isolated nucleic acid encoding a GSTO2 polypeptide, wherein the polypeptide contains a GSTO2 amino acid sequence variant relative to the amino acid sequence of SEQ ID NO:9, and wherein the amino acid sequence variant is at a residue selected from the group consisting of 41, 130, and 158. The amino acid sequence variant can be an isoleucine at residue 41, a tyrosine at residue 130, or an isoleucine at residue 158.
In another aspect, this document features an isolated GSTO2 polypeptide, wherein the polypeptide contains a GSTO2 amino acid sequence variant relative to the amino acid sequence of SEQ ID NO:9, and wherein the amino acid sequence variant is at a residue selected from the group consisting of 41, 130, and 158. The amino acid sequence variant can be an isoleucine at residue 41, a tyrosine at residue 130, or an isoleucine at residue 158.
In still another aspect, this document features an article of manufacture comprising a substrate, wherein the substrate comprises a population of isolated variant GSTO2 nucleic acid molecules as disclosed herein. The article of manufacture can comprises a plurality of discrete regions, wherein each region comprises a different population of isolated variant GSTO2 nucleic acid molecules, and wherein each population of molecules comprises a different GSTO2 nucleotide sequence variant.
This document also features a method for determining a GSTO2 genotype. The method can comprise: a) providing a nucleic acid sample from a subject, and b)screening the nucleic acid sample for one or more genetic markers in linkage disequilibrium with a GSTO2 allele. The GSTO2 allele can be selected from the group consisting of a G121A allele, a G389A allele, an A424G allele, or a C472A allele.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims.
This document features GSTO2 nucleotide and amino acid sequence variants. GSTO2 is thought to be a cytoplasmic enzyme that has glutathione transferase and glutathione dehydrogenase activity. For example, GSTO2 can catalyze the transfer of glutathione to an aliphatic, aromatic, or heterocyclic molecule, wherein the linkage is via a thiol group. GSTO2 also can catalyze the dehydrogenation of DHA, which can be reacted with GSH to give ascorbate and glutathione disulfate. Genetically-based variations in GSTO2 activity that lead to altered levels of GSTO2 or altered GSTO2 activity may be important in particular diseases or clinical conditions.
Nucleic Acid Molecules
Provided herein are isolated nucleic acids that include a GSTO2 nucleic acid sequence. The GSTO2 nucleic acid sequence includes a nucleotide sequence variant and nucleotides flanking the sequence variant. As used herein, "isolated nucleic acid" refers to a nucleic acid that is separated from other nucleic acid molecules that are present in a mammalian genome, including nucleic acids that normally flank one or both sides of the nucleic acid in a mammalian genome (e.g., nucleic acids that encode non-GSTO2 proteins). The term "isolated" as used herein with respect to nucleic acids also includes any non-naturally-occurring nucleic acid sequence since such non-naturally-occurring sequences are not found in nature and do not have immediately contiguous sequences in a naturally-occurring genome.
An isolated nucleic acid can be, for example, a DNA molecule, provided one of the nucleic acid sequences normally found immediately flanking that DNA molecule in a naturally-occurring genome is removed or absent. Thus, an isolated nucleic acid includes, without limitation, a DNA molecule that exists as a separate molecule (e.g., a chemically synthesized nucleic acid, or a cDNA or genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences as well as DNA that is incorporated into a vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, lentivirus, adenovirus, or herpes virus), or into the genomic DNA of a prokaryote or eukaryote. In addition, an isolated nucleic acid can include an engineered nucleic acid such as a recombinant DNA molecule that is part of a hybrid or fusion nucleic acid. A nucleic acid existing among hundreds to millions of other nucleic acids within, for example, cDNA libraries or genomic libraries, or gel slices containing a genomic DNA restriction digest, is not to be considered an isolated nucleic acid.
Nucleic acids provided herein are at least about 8 nucleotides in length. For example, the nucleic acid can be about 8, 9, 10-20 (e.g., 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length), 20-50, 50-100 or greater than 100 nucleotides in length (e.g., greater than 150, 200, 250, 300, 350, 400, 450, 500, 750, or 1000 nucleotides in length). Nucleic acids can be in a sense or antisense orientation, can be complementary to the GSTO2 reference sequence, and can be DNA, RNA, or nucleic acid analogs. Nucleic acid analogs can be modified at the base moiety, sugar moiety, or phosphate backbone to improve, for example, stability, hybridization, or solubility of the nucleic acid. Modifications at the base moiety include deoxyuridine for deoxythymidine, and 5-methyl-2′-deoxycytidine or 5-bromo-2′-doxycytidine for deoxycytidine. Modifications of the sugar moiety include modification of the 2′ hydroxyl of the ribose sugar to form 2′-O-methyl or 2′-O-allyl sugars. The deoxyribose phosphate backbone can be modified to produce morpholino nucleic acids, in which each base moiety is linked to a six membered, morpholino ring, or peptide nucleic acids, in which the deoxyphosphate backbone is replaced by a pseudopeptide backbone and the four bases are retained. See, Summerton and Weller (1997) Antisense Nucleic Acid Drug Dev. 7:187-195; and Hyrup et al. (1996) Bioorgan. Med. Chem. 4:5-23. In addition, the deoxyphosphate backbone can be replaced with, for example, a phosphorothioate or phosphorodithioate backbone, a phosphoroamidite, or an alkyl phosphotriester backbone.
As used herein, "nucleotide sequence variant" refers to any alteration in a GSTO2 reference sequence, and includes variations that occur in coding and non-coding regions, including exons, introns, and untranslated sequences. Nucleotides are referred to herein by the standard one-letter designation (A, C, G, or T). Variations include single nucleotide substitutions, deletions of one or more nucleotides, and insertions of one or more nucleotides. The reference GSTO2 nucleic acid sequence is provided in
As used herein, "untranslated sequence" includes 5′ and 3′ flanking regions that are outside of the messenger RNA (mRNA) as well as 5′ and 3′ untranslated regions (5′-UTR or 3′-UTR) that are part of the mRNA, but are not translated. Positions of nucleotide sequence variants in 5′ untranslated sequences can be designated as "−X" relative to the "A" in the translation initiation codon; positions of nucleotide sequence variants in the coding sequence and 3′ untranslated sequence can be designated as "+X" or "X" relative to the "A" in the translation initiation codon. Nucleotide sequence variants that occur in introns can be designated as "+X" or "X" relative to the "G" in the splice donor site (GT) or as "−X" relative to the "G" in the splice acceptor site (AG).
In some embodiments, a GSTO2 nucleotide sequence variant encodes a GSTO2 polypeptide having an altered amino acid sequence. The term "polypeptide" refers to a chain of at least four amino acid residues (e.g., 4-8, 9-12, 13-15, 16-18, 19-21, 22-100, 100-150, 150-200, 200-225 residues, or a full-length GSTO2 polypeptide). GSTO2 polypeptides may or may not have GSTO2 catalytic activity, or may have altered activity relative to the reference GSTO2 polypeptide. Polypeptides that do not have activity or have altered activity can be useful for diagnostic purposes (e.g., for producing antibodies having specific binding affinity for variant GSTO2 polypeptides).
Corresponding GSTO2 polypeptides, irrespective of length, that differ in amino acid sequence are herein referred to as allozymes. For example, a GSTO2 nucleic acid sequence that includes an adenine at position 121 relative to the adenine in the translation initiation codon (i.e., nucleotide 23393 of SEQ ID NO:1 or nucleotide 121 of SEQ ID NO:8) encodes a GSTO2 polypeptide having an isoleucine at amino acid residue 41. This polypeptide (Val4IIle) would be considered an allozyme with respect to the reference GSTO2 polypeptide that contains a valine at amino acid residue 41. Additional non-limiting examples of GSTO2 sequence variants that alter amino acid sequence include variants at nucleotides 389, 424, and 472 relative to the adenine in the translation initiation codon (i.e., positions 27473, 27508, and 45662, respectively, of SEQ ID NO:1, or positions 389, 424, and 472, respectively, of SEQ ID NO:8). For example, a GSTO2 nucleic acid molecule can include an adenine at nucleotide 389 and encode a GSTO2 polypeptide having a tyrosine at amino acid residue 130 in place of a cysteine residue (Cys130Tyr); a guanine at nucleotide 424 and encode a GSTO2 polypeptide having an aspartic acid at amino acid residue 142 in place of an asparagine residue (Asn142Asp), or an adenine at nucleotide 472 and encode a GSTO2 polypeptide having an isoleucine at amino acid 158 in place of a leucine residue (Leu158Ile).
GSTO2 allozymes as described above are encoded by a series of GSTO2 alleles. These alleles represent nucleic acid sequences containing sequence variants, typically multiple sequence variants, within coding and non-coding sequences. Representative examples of single nucleotide variants are described herein. Table 2 sets out a series of GSTO2 alleles that encode GSTO2. Some alleles are commonly observed, i.e., have allele frequencies >1%, such as the allele having a guanine at nucleotide 424 place of an adenine. The relatively large number of alleles and allozymes for GSTO2 indicates the potential complexity of GSTO2 pharmacogenetics. Such complexity emphasizes the need for determining single nucleotide variants, (i.e., single nucleotide polymorphisms, SNPs) as well as complete GSTO2 haplotypes (i.e., the set of alleles on one chromosome or a part of a chromosome) of patients.
Certain GSTO2 nucleotide sequence variants do not alter the amino acid sequence. Such variants, however, could alter regulation of transcription as well as mRNA stability. GSTO2 variants can occur in intron sequences, for example, within introns 1, 2, 4, 5, or 6. For example, nucleotide sequence variants in the first intron can include a guanine substitution at nucleotide 52 (i.e., nucleotide 17402 of SEQ ID NO:1), a thymine substitution at nucleotide 152 (i.e., nucleotide 17502 of SEQ ID NO:1), deletion of cytosine and guanine residues at nucleotides 310 and 311 (i.e., nucleotides 17660 and 17661 of SEQ ID NO:1), an adenine substitution at nucleotide 311 (i.e., nucleotide 17661 of SEQ ID NO:1), a cytosine substitution at nucleotide 333 (i.e., nucleotide 17683 of SEQ ID NO:1), a thymine substitution at nucleotide 402 (i.e., nucleotide 17752 of SEQ ID NO:1), a cytosine substitution at nucleotide 426 (i.e., nucleotide 17776 of SEQ ID NO:1), an adenine substitution at nucleotide 435 (i.e., nucleotide 17785 of SEQ ID NO:1), insertion of a thymine after nucleotide 477 (i.e., nucleotide 17827 of SEQ ID NO:1), a guanine substitution at nucleotide 1694 (i.e., nucleotide 19044 of SEQ ID NO:1), a guanine substitution at nucleotide 2003 (i.e., nucleotide 19353 of SEQ ID NO:1), a guanine substitution at nucleotide 2146 (i.e., nucleotide 19496 of SEQ ID NO:1), an adenine substitution at nucleotide 2168 (i.e., nucleotide 19518 of SEQ ID NO:1), a thymine substitution at nucleotide −1131 (i.e., nucleotide 21635 of SEQ ID NO:1), a guanine substitution at nucleotide −185 (i.e., nucleotide 22581 of SEQ ID NO:1), an adenine substitution at nucleotide −19 (i.e., nucleotide 22747 of SEQ ID NO:1), or an adenine substitution at nucleotide −12 (i.e., nucleotide 22754 of SEQ ID NO:1). Other examples of sequence variants within introns include a thymine substitution at nucleotide −16 of intron 2 (i.e., nucleotide 23291 of SEQ ID NO:1), a cytosine substitution at nucleotide 20 of intron 4 (i.e., nucleotide 26217 of SEQ ID NO:1), a thymine substitution at nucleotide 39 of intron 4 (i.e., nucleotide 26236 of SEQ ID NO:1), a cytosine substitution at nucleotide −38 of intron 4 (i.e., nucleotide 27413 of SEQ ID NO:1), a guanine substitution at nucleotide 74 of intron 5 (i.e., nucleotide 27626 of SEQ ID NO:1), a cytosine substitution at nucleotide 170 of intron 5 (i.e., nucleotide 27722 of SEQ ID NO:1), a guanine substitution at nucleotide −55 of intron 6 (i.e., nucleotide 47154 of SEQ ID NO:1), or a thymine substitution at nucleotide −21 of intron 6 (i.e., nucleotide 47188 of SEQ ID NO:1).
GSTO2 nucleotide sequence variants that do not change the amino acid sequence also can be within an exon or in 5′ or 3′ untranslated sequences. For example, a GSTO2 nucleotide sequence variant within exon 2 can be a guanine substitution at nucleotide −183 relative to the adenine in the translation initiation codon. Exon 7 sequence variants can include, for example, a thymine substitution at nucleotide 591 (i.e., nucleotide 47224 of SEQ ID NO:1 or nucleotide 591 of SEQ ID NO:8), or a thymine substitution at nucleotide 630 (i.e., nucleotide 47263 of SEQ ID NO:1 or nucleotide 630 of SEQ ID NO:8).
GSTO2 variants within the 5′ FR (including the first exon) can be, for example, insertion of a guanine after nucleotide −2012 (i.e., nucleotide 15569 of SEQ ID NO:1) relative to the adenine in the translation initiation codon, a cytosine substitution at nucleotide −1785 (i.e., nucleotide 15797 of SEQ ID NO:1), a cytosine substitution at nucleotide −1771 (i.e., nucleotide 15811 of SEQ ID NO:1), a thymine substitution at nucleotide −1664 (i.e., nucleotide 15918 of SEQ ID NO:1), a guanine substitution at nucleotide −1598 (i.e., nucleotide 15984 of SEQ ID NO:1), a cytosine substitution at nucleotide −1576 (i.e., nucleotide 16006 of SEQ ID NO:1), an adenine substitution at nucleotide −1566 (i.e., nucleotide 16016 of SEQ ID NO:1), a thymine substitution at nucleotide −1541 (i.e., nucleotide 16041 of SEQ ID NO:1), an adenine substitution at nucleotide −1357 (i.e., nucleotide 16225 of SEQ ID NO:1), a cytosine substitution at nucleotide −1189 (i.e., nucleotide 16393 of SEQ ID NO:1), a thymine substitution at nucleotide −1179 (i.e., nucleotide 16403 of SEQ ID NO:1), an adenine substitution at nucleotide −1105 (i.e., nucleotide 16477 of SEQ ID NO:1), a guanine substitution at nucleotide −1102 (i.e., nucleotide 16480 of SEQ ID NO:1), a thymine substitution at nucleotide −1009 (i.e., nucleotide 16573 of SEQ ID NO:1), a cytosine substitution at nucleotide −781 (i.e., nucleotide 16801 of SEQ ID NO:1), insertion of a thymine after nucleotide −730 (i.e., nucleotide 16851 of SEQ ID NO:1), a thymine substitution at nucleotide −637 (i.e., nucleotide 16945 of SEQ ID NO:1), a guanine substitution at nucleotide −634 (i.e., nucleotide 16948 of SEQ ID NO:1), a guanine substitution at nucleotide −625 (i.e., nucleotide 16957 of SEQ ID NO:1), a guanine substitution at nucleotide −530 (i.e., nucleotide 17052 of SEQ ID NO:1), an adenine substitution at nucleotide −526 (i.e., nucleotide 17056 of SEQ ID NO:1), an adenine substitution at nucleotide −463 (i.e., nucleotide 17119 of SEQ ID NO:1), a cytosine substitution at nucleotide −450 (i.e., nucleotide 17132 of SEQ ID NO:1), an adenine substitution at nucleotide −424 (i.e., nucleotide 17158 of SEQ ID NO:1), a guanine substitution at nucleotide −410 (i.e., nucleotide 17172 of SEQ ID NO:1), a thymine substitution at nucleotide −293 (i.e., nucleotide 17289 of SEQ ID NO:1), or a thymine substitution at nucleotide −234 (i.e., nucleotide 17348 of SEQ ID NO:1).
GSTO2 variants within the 3′ untranslated region can be, for example, a thymine substitution at nucleotide 1038 (i.e., nucleotide 47671 of SEQ ID NO:1) relative to the adenine in the translation initiation codon, a thymine substitution at nucleotide 1080 (i.e., nucleotide 47713 of SEQ ID NO:1), a guanine substitution at nucleotide 1243 (i.e., nucleotide 47876 of SEQ ID NO:1), a guanine substitution at nucleotide 1343 (i.e., nucleotide 47976 of SEQ ID NO:1), a thymine substitution at nucleotide 1615 (i.e., nucleotide 48248 of SEQ ID NO:1), a cytosine substitution at nucleotide 1649 (i.e., nucleotide 48282 of SEQ ID NO:1), or a thymine substitution at nucleotide 1724 (i.e., nucleotide 48357 of SEQ ID NO:1).
In some embodiments, a GSTO2 nucleic acid molecule can consist essentially of at least ten (e.g., at least 12, at least 15, at least 18, at least 20, or at least 25) contiguous nucleotides of an GSTO2 reference sequence (e.g., SEQ ID NO:1 or SEQ ID NO:8). Such nucleic acids can contain one or more variant positions, with the proviso that the nucleotides at those positions are variant nucleotides as disclosed herein in Table 2, for example. A GSTO2 nucleic acid "consisting essentially of" a particular sequence has the basic and novel characteristic that it can be used to distinguish, based upon hybridization, a nucleic acid having a sequence that contains a variant from a corresponding nucleic acid having a sequence that does not contain the variant (e.g., a wild type sequence). Such nucleic acid molecules can include additional sequences or labels (e.g., a tag or a fluorescent label as disclosed herein), provided that such additions do not affect the basic and novel characteristic of the nucleic acid molecules.
In some embodiments, a GSTO2 nucleic acid sequence can have at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99.0%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) sequence identity with a region of SEQ ID NO:1 or SEQ ID NO:8. The region of SEQ ID NO:1 or SEQ ID NO:8 is at least ten nucleotides in length (e.g., 10, 15, 20, 50, 60, 70, 75, 100, 150 or more nucleotides in length). For example, a nucleic acid can include a GSTO2 nucleic acid sequence with at least 90% identity to nucleotides 75 to 175, 350 to 450, 375 to 475, 425 to 525, 550 to 650, or 600 to 700 of SEQ ID NO:8. A variant GSTO2 nucleotide sequence can have an adenine at nucleotide 121, an adenine at nucleotide 389, an adenine at nucleotide 424, an adenine at nucleotide 472, a thymine at nucleotide 591, or a thymine at nucleotide 630, and combinations thereof, wherein the numbering of the positions is with respect to the numbering of SEQ ID NO:8.
Percent sequence identity is calculated by determining the number of matched positions in aligned nucleic acid sequences, dividing the number of matched positions by the total number of aligned nucleotides, and multiplying by 100. A matched position refers to a position in which identical nucleotides occur at the same position in aligned nucleic acid sequences. Percent sequence identity also can be determined for any amino acid sequence. To determine percent sequence identity, a target nucleic acid or amino acid sequence is compared to the identified nucleic acid or amino acid sequence using the BLAST 2 Sequences (B12seq) program from the stand-alone version of BLASTZ containing BLASTN version 2.0.14 and BLASTP version 2.0.14. This stand-alone version of BLASTZ can be obtained on the World Wide Web from Fish & Richardson's web site (fr.com/blast) or the U.S. government's National Center for Biotechnology Information web site (ncbi.nlm.nih.gov). Instructions explaining how to use the B12seq program can be found in the readme file accompanying BLASTZ.
B12seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. To compare two nucleic acid sequences, the options are set as follows: −i is set to a file containing the first nucleic acid sequence to be compared (e.g., C:\seq1.txt); −j is set to a file containing the second nucleic acid sequence to be compared (e.g., C:\seq2.txt); −p is set to blastn; −o is set to any desired file name (e.g., C:\output.txt); −q is set to −1; −r is set to 2; and all other options are left at their default setting. The following command will generate an output file containing a comparison between two sequences: C:\B12seq −i c:\seq1.txt −j c:\seq2.txt −p blastn −o c:\output.txt −q −1 −r 2. If the target sequence shares homology with any portion of the identified sequence, then the designated output file will present those regions of homology as aligned sequences. If the target sequence does not share homology with any portion of the identified sequence, then the designated output file will not present aligned sequences.
Once aligned, a length is determined by counting the number of consecutive nucleotides from the target sequence presented in alignment with sequence from the identified sequence starting with any matched position and ending with any other matched position. A matched position is any position where an identical nucleotide is presented in both the target and identified sequence. Gaps presented in the target sequence are not counted since gaps are not nucleotides. Likewise, gaps presented in the identified sequence are not counted since target sequence nucleotides are counted, not nucleotides from the identified sequence.
The percent identity over a particular length is determined by counting the number of matched positions over that length and dividing that number by the length followed by multiplying the resulting value by 100. For example, if (1) a 1000 nucleotide target sequence is compared to the sequence set forth in SEQ ID NO:1, (2) the B12seq program presents 969 nucleotides from the target sequence aligned with a region of the sequence set forth in SEQ ID NO:1 where the first and last nucleotides of that 969 nucleotide region are matches, and (3) the number of matches over those 969 aligned nucleotides is 900, then the 1000 nucleotide target sequence contains a length of 969 and a percent identity over that length of 93 (i.e., 900÷969×100=93).
It will be appreciated that different regions within a single nucleic acid target sequence that aligns with an identified sequence can each have their own percent identity. It is noted that the percent identity value is rounded to the nearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 are rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded up to 78.2. It also is noted that the length value will always be an integer.
Isolated nucleic acid molecules can be produced using standard techniques, including, without limitation, common molecular cloning and chemical nucleic acid synthesis techniques. For example, polymerase chain reaction (PCR) techniques can be used to obtain an isolated nucleic acid containing a GSTO2 nucleotide sequence variant. PCR refers to a procedure or technique in which target nucleic acids are enzymatically amplified. Sequence information from the ends of the region of interest or beyond typically is employed to design oligonucleotide primers that are identical in sequence to opposite strands of the template to be amplified. PCR can be used to amplify specific sequences from DNA as well as RNA, including sequences from total genomic DNA or total cellular RNA. Primers are typically 14 to 40 nucleotides in length, but can range from 10 nucleotides to hundreds of nucleotides in length. General PCR techniques are described, for example in PCR Primer: A Laboratory Manual, ed. by Dieffenbach and Dveksler, Cold Spring Harbor Laboratory Press, 1995. When using RNA as a source of template, reverse transcriptase can be used to synthesize complementary DNA (cDNA) strands. Ligase chain reaction, strand displacement amplification, self-sustained sequence replication, or nucleic acid sequence-based amplification also can be used to obtain isolated nucleic acids. See, for example, Lewis (1992) Genetic Engineering News 12:1; Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878; and Weiss (1991) Science 254:1292.
Isolated nucleic acids also can be chemically synthesized, either as a single nucleic acid molecule (e.g., using automated DNA synthesis in the 3′ to 5′ direction using phosphoramidite technology) or as a series of oligonucleotides. For example, one or more pairs of long oligonucleotides (e.g., >100 nucleotides) can be synthesized that contain the desired sequence, with each pair containing a short segment of complementarity (e.g., about 15 nucleotides) such that a duplex is formed when the oligonucleotide pair is annealed. DNA polymerase is used to extend the oligonucleotides, resulting in a single, double-stranded nucleic acid molecule per oligonucleotide pair, which then can be ligated into a vector.
Isolated nucleic acids also can be obtained by mutagenesis. For example, the reference sequences depicted in
GSTO2 Polypeptides
Isolated GSTO2 polypeptides provided herein include an amino acid sequence variant relative to the reference GSTO2 (SEQ ID NO:9,
GSTO2 polypeptides can include variants at one or more of amino acid residues 41, 130, 142, and 158. In particular, an isoleucine residue can be substituted at position 41, a tyrosine residue at position 130, an aspartic acid residue at position 142, or an isoleucine residue at position 158. In some embodiments, activity of GSTO2 polypeptides is altered relative to the reference GSTO2. Certain GSTO2 allozymes can have reduced activity, while other allozymes can have activity that is comparable to the reference GSTO2. Other allozymes can have increased activity relative to the reference GSTO2. Activity of GSTO2 polypeptides can be assessed in vitro. For example, reduction of DHA catalyzed by bacterially expressed GSTO2 can be detected by monitoring an increase in absorbance at 265 nm. Alternatively, activity of GSTO2 polypeptides can be assessed in vivo (e.g., in COS cells).
Other biochemical properties of allozymes, such as apparent Km values, also can be altered relative to the reference GSTO2. Apparent Km values can be calculated using, for example, the method of Wilkinson with a computer program written by Cleland. Wilkinson (1961) Biochem. J. 80:324-332; and Cleland (1963) Nature 198:463-365.
Isolated polypeptides can be obtained, for example, by extraction from a natural source (e.g., testes, liver, kidney, or skeletal muscle), chemical synthesis, or by recombinant production in a host cell. To recombinantly produce GSTO2 polypeptides, a nucleic acid encoding a GSTO2 nucleotide sequence variant can be ligated into an expression vector and used to transform a prokaryotic (e.g., bacteria) or eukaryotic (e.g., insect, yeast, or mammal) host cell. In general, nucleic acid constructs include a regulatory sequence operably linked to a GSTO2 nucleic acid sequence. Regulatory sequences (e.g., promoters, enhancers, polyadenylation signals, or terminators) do not typically encode a gene product, but instead affect the expression of the nucleic acid sequence. In addition, a construct can include a tag sequence designed to facilitate subsequent manipulations of the expressed nucleic acid sequence (e.g., purification, localization). Tag sequences, such as green fluorescent protein (GFP), glutathione S-transferase (GST), six histidine (His6), c-myc, hemagglutinin, or Flag™ tag (Kodak) sequences are typically expressed as a fusion with the expressed nucleic acid sequence. Such tags can be inserted anywhere within the polypeptide including at either the carboxyl or amino termini. The type and combination of regulatory and tag sequences can vary with each particular host, cloning or expression system, and desired outcome. A variety of cloning and expression vectors containing combinations of regulatory and tag sequences are commercially available. Suitable cloning vectors include, without limitation, pUC18, pUC19, and pBR322 and derivatives thereof (New England Biolabs, Beverly, Mass.), and pGEN (Promega, Madison, Wis.). Additionally, representative prokaryotic expression vectors include pBAD (Invitrogen, Carlsbad, Calif.), the pTYB family of vectors (New England Biolabs), and pGEMEX vectors (Promega); representative mammalian expression vectors include pTet-On/pTet-Off (Clontech, Palo Alto, Calif.), pIND, pVAX1, pCR3.1, pcDNA3.1, pcDNA4, or pUni (Invitrogen), and pCI or pSI (Promega); representative insect expression vectors include pBacPAK8 or pBacPAK9 (Clontech), and p2Bac (Invitrogen); and representative yeast expression vectors include MATCHMAKER (Clontech) and pPICZ A, B, and C (Invitrogen).
In bacterial systems, a strain of Escherichia coli can be used to express GSTO2 variant polypeptides. For example, BL-21 cells can be transformed with a pGEX vector containing a GSTO2 nucleic acid sequence. The transformed bacteria can be grown exponentially and then stimulated with isopropylthiogalactopyranoside (IPTG) prior to harvesting. In general, the GSTO2-GST fusion proteins produced from the pGEX expression vector are soluble and can be purified easily from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the expressed GSTO2 polypeptide can be released from the GST moiety.
In eukaryotic host cells, a number of viral-based expression systems can be utilized to express GSTO2 variant polypeptides. A nucleic acid encoding a polypeptide can be cloned into, for example, a baculoviral vector such as pBlueBac (Invitrogen) and then used to co-transfect insect cells such as Spodoptera frugiperda (Sf9) cells with wild type DNA from Autographa californica multinuclear polyhedrosis virus (AcMNPV). Recombinant viruses producing polypeptides can be identified by standard methodology. Alternatively, a nucleic acid encoding a polypeptide can be introduced into a SV40, retroviral, or vaccinia based viral vector and used to infect suitable host cells.
Eukaryotic cell lines that stably express GSTO2 variant polypeptides can be produced using expression vectors with the appropriate control elements and a selectable marker. For example, the eukaryotic expression vector pCR3.1 (Invitrogen, San Diego, Calif.) and p91023(B) (see Wong et al. (1985) Science 228:810-815) or modified derivatives thereof are suitable for expression of GSTO2 variant polypeptides in, for example, Chinese hamster ovary (CHO) cells, COS-1 cells, human embryonic kidney 293 cells, NIH3T3 cells, BHK21 cells, MDCK cells, and human vascular endothelial cells (HUVEC). Following introduction of the expression vector by electroporation, lipofection, calcium phosphate or calcium chloride co-precipitation, DEAE dextran, or other suitable transfection method, stable cell lines are selected, e.g., by antibiotic resistance to G418, kanamycin, or hygromycin. Alternatively, amplified sequences can be ligated into a eukaryotic expression vector such as pCR3.1, pCDNA3.1 (Invitrogen), or pcDNA4/HisMax TOPO (Promega) and then transcribed and translated in vitro using wheat germ extract or rabbit reticulocyte lysate.
GSTO2 variant polypeptides can be purified by known chromatographic methods including ion exchange and gel filtration chromatography. See, for example, Caine et al. (1996) Protein Expr. Purif: 8:159-166. GSTO2 polypeptides can be "engineered" to contain a tag sequence describe herein that allows the polypeptide to be purified (e.g., captured onto an affinity matrix). Immunoaffinity chromatography also can be used to purify GSTO2 polypeptides.
Non-Human Mammals
Also featured herein are non-human mammals that include GSTO2 nucleic acids as described herein, as well as progeny and cells of such non-human mammals. Non-human mammals include, for example, rodents such as rats, guinea pigs, and mice, and farm animals such as pigs, sheep, goats, horses, and cattle. Non-human mammals can express a GSTO2 variant nucleic acid in addition to an endogenous GSTO2 (e.g., a transgenic non-human that includes a GSTO2 nucleic acid randomly integrated into the genome of the non-human mammal). Alternatively, an endogenous GSTO2 nucleic acid can be replaced with a GSTO2 variant nucleic acid by homologous recombination. See, Shastry (1998) Mol. Cell Biochem. 181:163-179, for a review of gene targeting technology.
In one embodiment, non-human mammals are produced that lack an endogenous GSTO2 nucleic acid (i.e., a knockout), and then a GSTO2 variant nucleic acid as provided herein is introduced into the knockout non-human mammal. Nucleic acid constructs used for producing knockout non-human mammals can include a nucleic acid sequence encoding a selectable marker, which is generally used to interrupt the targeted exon site by homologous recombination. Typically, the selectable marker is flanked by sequences homologous to the sequences flanking the desired insertion site. It is not necessary for the flanking sequences to be immediately adjacent to the desired insertion site. Suitable markers for positive drug selection include, for example, the aminoglycoside 3N phosphotransferase gene that imparts resistance to geneticin (G418, an aminoglycoside antibiotic), and other antibiotic resistance markers, such as the hygromycin-B-phosphotransferase gene that imparts hygromycin resistance. Other selection systems include negative-selection markers such as the thymidine kinase (TK) gene from herpes simplex virus. Constructs utilizing both positive and negative drug selection also can be used. For example, a construct can contain the aminoglycoside phosphotransferase gene and the TK gene. In this system, cells are selected that are resistant to G418 and sensitive to gancyclovir.
To create non-human mammals having a particular gene inactivated in all cells, it is necessary to introduce a knockout construct into the germ cells (sperm or eggs, i.e., the "germ line") of the desired species. Genes or other DNA sequences can be introduced into the pronuclei of fertilized eggs by microinjection. Following pronuclear fusion, the developing embryo may carry the introduced gene in all its somatic and germ cells because the zygote is the mitotic progenitor of all cells in the embryo. Since targeted insertion of a knockout construct is a relatively rare event, it is desirable to generate and screen a large number of animals when employing such an approach. Because of this, it can be advantageous to work with the large cell populations and selection criteria that are characteristic of cultured cell systems. However, for production of knockout animals from an initial population of cultured cells, it is necessary that a cultured cell containing the desired knockout construct be capable of generating a whole animal. This is generally accomplished by placing the cell into a developing embryo environment of some sort.
Cells capable of giving rise to at least several differentiated cell types are "pluripotent." Pluripotent cells capable of giving rise to all cell types of an embryo, including germ cells, are hereinafter termed "totipotent" cells. Totipotent murine cell lines (embryonic stem, or "ES" cells) have been isolated by culture of cells derived from very young embryos (blastocysts). Such cells are capable, upon incorporation into an embryo, of differentiating into all cell types, including germ cells, and can be employed to generate animals lacking an endogenous GSTO2 nucleic acid. That is, cultured ES cells can be transformed with a knockout construct and cells selected in which the GSTO2 gene is inactivated.
Nucleic acid constructs can be introduced into ES cells, for example, by electroporation or other standard technique. Selected cells can be screened for gene targeting events. For example, the polymerase chain reaction (PCR) can be used to confirm the presence of the transgene.
The ES cells further can be characterized to determine the number of targeting events. For example, genomic DNA can be harvested from ES cells and used for Southern analysis. See, for example, Section 9.37-9.52 of Sambrook et al., Molecular Cloning, A Laboratory Manual, second edition, Cold Spring Harbor Press, Plainview; N.Y., 1989.
To generate a knockout animal, ES cells having at least one inactivated GSTO2 allele are incorporated into a developing embryo. This can be accomplished through injection into the blastocyst cavity of a murine blastocyst-stage embryo, by injection into a morula-stage embryo, by co-culture of ES cells with a morula-stage embryo, or through fusion of the ES cell with an enucleated zygote. The resulting embryo is raised to sexual maturity and bred in order to obtain animals, whose cells (including germ cells) carry the inactivated GSTO2 allele. If the original ES cell was heterozygous for the inactivated GSTO2 allele, several of these animals can be bred with each other in order to generate animals homozygous for the inactivated allele.
Alternatively, direct microinjection of DNA into eggs can be used to avoid the manipulations required to turn a cultured cell into an animal. Fertilized eggs are totipotent, i.e., capable of developing into an adult without further substantive manipulation other than implantation into a surrogate mother. To enhance the probability of homologous recombination when eggs are directly injected with knockout constructs, it is useful to incorporate at least about 8 kb of homologous DNA into the targeting construct. In addition, it is also useful to prepare the knockout constructs from isogenic DNA.
Embryos derived from microinjected eggs can be screened for homologous recombination events in several ways. For example, if the GSTO2 gene is interrupted by a coding region that produces a detectable (e.g., fluorescent) gene product, then the injected eggs are cultured to the blastocyst stage and analyzed for presence of the indicator polypeptide. Embryos with fluorescing cells, for example, are then implanted into a surrogate mother and allowed to develop to term. Alternatively, injected eggs are allowed to develop and DNA from the resulting pups analyzed by PCR or RT-PCR for evidence of homologous recombination.
Nuclear transplantation also can be used to generate non-human mammals. For example, fetal fibroblasts can be genetically modified such that they contain an inactivated endogenous GSTO2 gene and express a GSTO2 nucleic acid, and then fused with enucleated oocytes. After activation of the oocytes, the eggs are cultured to the blastocyst stage, and implanted into a recipient. See, Cibelli et al. (1998) Science 280:1256-1258. Adult somatic cells, including, for example, cumulus cells and mammary cells, can be used to produce animals such as mice and sheep, respectively. See, for example, Wakayama et al. (1998) Nature 394:369-374; and Wilmut et al. (1997) Nature 385:810-813. Nuclei can be removed from genetically modified adult somatic cells, and transplanted into enucleated oocytes. After activation, the eggs can be cultured to the 2-8 cell stage, or to the blastocyst stage, and implanted into a suitable recipient. Wakayama et al., supra.
Non-human mammals such as mice can be used, for example, to screen toxicity of compounds that are substrates for GSTO2, drugs that alter GSTO2 activity, or for carcinogenesis. For example, GSTO2 activity or toxicity can be assessed in a first group of such non-human mammals in the presence of a compound, and compared with GSTO2 activity or toxicity in a corresponding control group in the absence of the compound. As used herein, suitable compounds include biological macromolecules such as an oligonucleotide (RNA or DNA), or a polypeptide of any length, a chemical compound, a mixture of chemical compounds, or an extract isolated from bacterial, plant, fungal, or animal matter. The concentration of compound to be tested depends on the type of compound and in vitro test data.
Non-human mammals can be exposed to test compounds by any route of administration, including enterally (e.g., orally) and parenterally (e.g., subcutaneously, intravascularly, intramuscularly, or intranasally). Suitable formulations for oral administration can include tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). Tablets can be coated by methods known in the art. Preparations for oral administration can also be formulated to give controlled release of the compound.
Compounds can be prepared for parenteral administration in liquid form (e.g., solutions, solvents, suspensions, and emulsions) including sterile aqueous or non-aqueous carriers. Aqueous carriers include, without limitation, water, alcohol, saline, and buffered solutions. Examples of non-aqueous carriers include, without limitation, propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters. Preservatives and other additives such as, for example, antimicrobials, anti-oxidants, chelating agents, inert gases, and the like may also be present. Pharmaceutically acceptable carriers for intravenous administration include solutions containing pharmaceutically acceptable salts or sugars. Intranasal preparations can be presented in a liquid form (e.g., nasal drops or aerosols) or as a dry product (e.g., a powder). Both liquid and dry nasal preparations can be administered using a suitable inhalation device. Nebulised aqueous suspensions or solutions can also be prepared with or without a suitable pH and/or tonicity adjustment.
Detecting GSTO2 Sequence Variants
GSTO2 nucleotide sequence variants can be detected, for example, by sequencing exons, introns, 5′ untranslated sequences, or 3′ untranslated sequences, by performing allele-specific hybridization, allele-specific restriction digests, mutation specific polymerase chain reactions (MSPCR), by single-stranded conformational polymorphism (SSCP) detection (Schafer et al. (1995) Nat. Biotechnol. 15:33-39), denaturing high performance liquid chromatography (DHPLC, Underhill et al. (1997) Genome Res. 7:996-1005), infrared matrix-assisted laser desorption/ionization (IR-MALDI) mass spectrometry (WO 99/57318), and combinations of such methods.
Genomic DNA generally is used in the analysis of GSTO2 nucleotide sequence variants, although mRNA also can be used. Genomic DNA is typically extracted from a biological sample such as a peripheral blood sample, but can be extracted from other biological samples, including tissues (e.g., mucosal scrapings of the lining of the mouth or from renal or hepatic tissue). Routine methods can be used to extract genomic DNA from a blood or tissue sample, including, for example, phenol extraction. Alternatively, genomic DNA can be extracted with kits such as the QIAamp® Tissue Kit (Qiagen, Chatsworth, Calif.), Wizard® Genomic DNA purification kit (Promega) and the A.S.A.P.™ Genomic DNA isolation kit (Boehringer Mannheim, Indianapolis, Ind.).
Typically, an amplification step is performed before proceeding with the detection method. For example, exons or introns of the GSTO2 gene can be amplified then directly sequenced. Dye primer sequencing can be used to increase the accuracy of detecting heterozygous samples.
Nucleic acid molecules provided herein can be used to detect variant GSTO2 sequences. For example, allele specific hybridization also can be used to detect sequence variants, including complete haplotypes of a subject (e.g., a mammal such as a human). See, Stoneking et al. (1991) Am. J. Hum. Genet. 48:370-382; and Prince et al. (2001) Genome Res. 11:152-162. In practice, samples of DNA or RNA from one or more mammals can be amplified using pairs of primers and the resulting amplification products can be immobilized on a substrate (e.g., in discrete regions). Hybridization conditions are selected such that a nucleic acid probe can specifically bind to the sequence of interest, e.g., the variant nucleic acid sequence. Such hybridizations typically are performed under high stringency as some sequence variants include only a single nucleotide difference. High stringency conditions can include the use of low ionic strength solutions and high temperatures for washing. For example, nucleic acid molecules can be hybridized at 42° C. in 2×SSC (0.3M NaCl/0.03 M sodium citrate/0.1% sodium dodecyl sulfate (SDS) and washed in 0.1×SSC (0.015M NaCl/0.0015 M sodium citrate), 0.1% SDS at 65° C. Hybridization conditions can be adjusted to account for unique features of the nucleic acid molecule, including length and sequence composition. Probes can be labeled (e.g., fluorescently) to facilitate detection. In some embodiments, one of the primers used in the amplification reaction is biotinylated (e.g., 5′ end of reverse primer) and the resulting biotinylated amplification product is immobilized on an avidin or streptavidin coated substrate.
Allele-specific restriction digests can be performed in the following manner. For nucleotide sequence variants that introduce a restriction site, restriction digest with the particular restriction enzyme can differentiate the alleles. For GSTO2 sequence variants that do not alter a common restriction site, mutagenic primers can be designed that introduce a restriction site when the variant allele is present or when the wild type allele is present. A portion of GSTO2 nucleic acid can be amplified using the mutagenic primer and a wild type primer, followed by digest with the appropriate restriction endonuclease.
Certain variants, such as insertions or deletions of one or more nucleotides, change the size of the DNA fragment encompassing the variant. The insertion or deletion of nucleotides can be assessed by amplifying the region encompassing the variant and determining the size of the amplified products in comparison with size standards. For example, a region of GSTO2 can be amplified using a primer set from either side of the variant. One of the primers is typically labeled, for example, with a fluorescent moiety, to facilitate sizing. The amplified products can be electrophoresed through acrylamide gels with a set of size standards that are labeled with a fluorescent moiety that differs from the primer.
PCR conditions and primers can be developed that amplify a product only when the variant allele is present or only when the wild type allele is present (MSPCR or allele-specific PCR). For example, patient DNA and a control can be amplified separately using either a wild type primer or a primer specific for the variant allele. Each set of reactions is then examined for the presence of amplification products using standard methods to visualize the DNA. For example, the reactions can be electrophoresed through an agarose gel and the DNA visualized by staining with ethidium bromide or other DNA intercalating dye. In DNA samples from heterozygous patients, reaction products would be detected in each reaction. Patient samples containing solely the wild type allele would have amplification products only in the reaction using the wild type primer. Similarly, patient samples containing solely the variant allele would have amplification products only in the reaction using the variant primer. Allele-specific PCR also can be performed using allele-specific primers that introduce priming sites for two universal energy-transfer-labeled primers (e.g., one primer labeled with a green dye such as fluorescein and one primer labeled with a red dye such as sulforhodamine). Amplification products can be analyzed for green and red fluorescence in a plate reader. See, Myakishev et al. (2001) Genome 11:163-169.
Mismatch cleavage methods also can be used to detect differing sequences by PCR amplification, followed by hybridization with the wild type sequence and cleavage at points of mismatch. Chemical reagents, such as carbodiimide or hydroxylamine and osmium tetroxide can be used to modify mismatched nucleotides to facilitate cleavage.
Alternatively, GSTO2 variants can be detected by antibodies that have specific binding affinity for variant GSTO2 polypeptides. Variant GSTO2 polypeptides can be produced in various ways, including recombinantly, as discussed above. Host animals such as rabbits, chickens, mice, guinea pigs, and rats can be immunized by injection of a GSTO2 variant polypeptide. Various adjuvants that can be used to increase the immunological response depend on the host species and include Freund's adjuvant (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. Polyclonal antibodies are heterogeneous populations of antibody molecules that are contained in the sera of the immunized animals. Monoclonal antibodies, which are homogeneous populations of antibodies to a particular antigen, can be prepared using a GSTO2 variant polypeptide and standard hybridoma technology. In particular, monoclonal antibodies can be obtained by any technique that provides for the production of antibody molecules by continuous cell lines in culture such as described by Kohler et al. (1975) Nature 256:495, the human B-cell hybridoma technique (Kosbor et al. (1983) Immunology Today 4:72; Cote et al. (1983) Proc. Natl. Acad. Sci USA 80:2026), and the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96 (1983). Such antibodies can be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof. A hybridoma producing monoclonal antibodies can be cultivated in vitro or in vivo.
Antibody fragments that have specific binding affinity for a GSTO2 variant polypeptide can be generated using known techniques. For example, such fragments include but are not limited to F(ab′)2 fragments that can be produced by pepsin digestion of the antibody molecule, and Fab fragments that can be generated by reducing the disulfide bridges of F(ab′)2 fragments. Alternatively, Fab expression libraries can be constructed. See, for example, Huse et al., Science, 246:1275 (1989). Once produced, antibodies or fragments thereof are tested for recognition of GSTO2 variant polypeptides by standard immunoassay methods including ELISA techniques, radioimmunoassays and Western blotting. See, Short Protocols in Molecular Biology, Chapter 11, Green Publishing Associates and John Wiley & Sons, edited by Ausubel et al., 1992.
As a result of the present disclosure, it is possible to determine GSH transferase status of a subject (e.g., a mammal such as a human). "GSH transferase status" refers to the ability of a subject to transfer a glutathione group to a substrate. GSH transferase status of a subject can be determined by, for example, measuring the level of GSTO2 activity in the subject using, for example, the methods described herein. Alternatively, GSH transferase status can be evaluated by determining whether a GSH transferase nucleic acid sequence (e.g., a GSTO2 nucleic acid sequence) of a subject contains one or more variants (e.g., one or more variants that are correlated with increased or decreased GSH transferase activity). A variant that results in decreased or increased GSTO2 activity, for example, can be said to result in "reduced" or "enhanced" GSH transferase status, respectively. In some embodiments, the variant profile of a subject can be used to determine the GSH transferase status of the subject.
"Variant profile" refers to the presence or absence of a plurality (e.g., two or more) of GSTO2 nucleotide sequence variants or GSTO2 amino acid sequence variants. For example, a variant profile can include the complete GSTO2 haplotype of the mammal (e.g., see Tables 5-9) or can include the presence or absence of a set of particular non-synonymous SNPs (e.g., single nucleotide substitutions that alter the amino acid sequence of a GSTO2 polypeptide). In one embodiment, the variant profile includes detecting the presence or absence of two or more non-synonymous SNPs (e.g., 2, 3, or 4 non-synonymous SNPs) described herein. There may be ethnic-specific pharmacogenetic variation, as certain of the nucleotide and amino acid sequence variants described herein were detected solely in African-American, Caucasian-American, Han Chinese-American, or Mexican-American subjects. In addition, the variant profile can include detecting the presence or absence of any type of GSTO2 SNP together with any other GSTO2 SNP (e.g., a polymorphism pair or a group of polymorphism pairs). Such polymorphism pairs include, without limitation, the pairs described in Table 4. Further, a variant profile can include detecting the presence or absence of any GSTO2 SNP together with any SNP from other GSH transferases.
GSH transferase activity of an enzyme such as GSTO2 can be measured using, for example, in vitro methods such as those described herein. As used herein, the term "reduced GSH transferase status" refers to a decrease (e.g., a 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, or 100% decrease) in GSH transferase activity (e.g., GSTO2 activity) of a subject, as compared to a control level of GSH transferase activity. Similarly, the term "enhanced GSH transferase status" refers to an increase (e.g., a 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, 100%, or more than 100% increase) in GSH transferase activity of a subject, as compared to a control level of GSH transferase activity. A control level of GSH transferase activity can be, for example, an average level of GSH transferase activity in a population of individuals. In one embodiment, the population includes individuals that do not contain particular GSTO2 nucleotide sequence variants or particular GSTO2 amino acid sequence variants (e.g., particular variants that affect GSH transferase status). Alternatively, a control level of GSH transferase activity can refer to the level of GSH transferase activity in a control subject (e.g., a subject that does not contain a GSTO2 nucleic acid containing a variant).
In some embodiments, evaluation of GSH transferase status can be used in diagnostic assays to determine whether a particular therapy may be useful in an individual, or to tailor particular treatment regimens to an individual. For example, the presence of absence of one or more variants may indicate an individual's ability to metabolize arsenic, a drinking water contaminant, or arsenic trioxide, an agent that can be used to treat acute promyelocytic leukemia. In further embodiments, GSH transferase status can be linked to predisposition to a particular disease or condition. For example, identification of GSTO2 sequence variants also may allow predisposition to conditions such as cancer, Alzheimer's, or Parkinson's disease to be assessed in individuals. Additional risk factors including, for example, family history and other genetic factors can be considered when determining risk. Predisposition to such diseases can be determined based on the presence or absence of a single GSTO2 sequence variant or based on a variant profile.
Determination of GSH transferase status and predisposition to conditions such as cancer, Alzheimer's, or Parkinson's disease can include identification of genetic markers (e.g., polymorphisms) in linkage disequilibrium with particular GSTO2 alleles. Although such markers may not be relevant from a functional perspective (i.e., may not directly affect function of GSTO2), their presence can be predictive of functional/clinically relevant polymorphisms. Thus, this document also provides methods for detecting a genotype by screening for genetic markers in linkage disequilibrium with particular GSTO2 alleles. The methods can include providing a nucleic acid sample from a subject (e.g., a human subject), and screening the sample for one or more markers in linkage disequilibrium with a particular GSTO2 allele. For example, a method can include screening for markers in linkage disequilibrium with a SNP at nucleotide 121, 389, 424, or 472 relative to the adenine in the translation initiation codon (nucleotide 23393, 27473, 27508, or 45662, respectively, of SEQ ID NO:1). Methods also can include screening for genetic markers in linkage disequilibrium with any of the other SNPs shown in Table 2.
Articles of Manufacture
Articles of manufacture also are provided herein, and can include populations of isolated GSTO2 nucleic acid molecules or GSTO2 polypeptides immobilized on a substrate. Suitable substrates provide a base for the immobilization of the nucleic acids or polypeptides, and in some embodiments, allow immobilization of nucleic acids or polypeptides into discrete regions. In embodiments in which the substrate includes a plurality of discrete regions, different populations of isolated nucleic acids or polypeptides can be immobilized in each discrete region. Thus, each discrete region of the substrate can include a different GSTO2 nucleic acid or GSTO2 polypeptide sequence variant. Such articles of manufacture can include two or more sequence variants of GSTO2, or can include all of the sequence variants known for GSTO2. For example, the article of manufacture can include two or more of the sequence variants identified herein and one or more other GSTO2 sequence variants, such as nucleic acid variants that occur in the promoter region of the GSTO2 gene. Furthermore, nucleic acid molecules containing sequence variants for other GSH transferases can be included on the substrate.
Suitable substrates can be of any shape or form and can be constructed from, for example, glass, silicon, metal, plastic, cellulose, or a composite. For example, a suitable substrate can include a multiwell plate or membrane, a glass slide, a chip, or polystyrene or magnetic beads. Nucleic acid molecules or polypeptides can be synthesized in situ, immobilized directly on the substrate, or immobilized via a linker, including by covalent, ionic, or physical linkage. Linkers for immobilizing nucleic acids and polypeptides, including reversible or cleavable linkers, are known in the art. See, for example, U.S. Pat. No. 5,451,683 and WO98/20019. Immobilized nucleic acid molecules are typically about 20 nucleotides in length, but can vary from about 10 nucleotides to about 1000 nucleotides in length.
In practice, a sample of DNA or RNA from a subject can be amplified, the amplification product hybridized to an article of manufacture containing populations of isolated nucleic acid molecules in discrete regions, and hybridization can be detected. Typically, the amplified product is labeled to facilitate detection of hybridization. See, for example, Hacia et al. (1996) Nature Genet. 14:441-447; and U.S. Pat. Nos. 5,770,722 and 5,733,729.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
PCR Amplification and DNA Sequencing: DNA samples from 60 Caucasian-American, 60 African-American, 60 Han Chinese-American, and 60 Mexican-American subjects were obtained from the Coriell Institute Cell Repository (Camden, N.J.). These samples had been anonymized, and written informed consent had been obtained from all donors for the use of their DNA for this purpose. All experiments were reviewed and approved by the Mayo Clinic Institutional Review Board. Fourteen PCR reactions were performed with each DNA sample to amplify all GSTO2 exons and splice junctions, as well as a portion of the 5′ flanking region. Primers were designed to hybridize within introns, the 5′-FR, or the 3′-UTR. This approach was used to ensure that a GSTO2 processed pseudogene that maps to chromosome 3 (Whitbread et al. (2003) Pharmacogenet. 13:131-144) would not be amplified. Amplicons were sequenced using dye-primer sequencing chemistry to facilitate the identification of heterozygous bases (Chadwick et al. (1996) Biotechniques 20:676-683). To make that possible, universal M13 sequencing tags were added to the 5′-ends of each forward and reverse primer. Forward primers contained the M13 forward sequence (5′-TGTAAAACGACGGCCAGT-3′; SEQ ID NO:10), and reverse primers contained the M13 reverse sequence (5′-CAGGAAACAGCTATGACC-3′; SEQ ID NO:11). In addition, resequencing of the GSTO2 5′-FR and the intergenic area utilized dye terminator chemistry. The sequences and locations of each primer within the gene are listed in Table 1. "F" represents forward; "R," reverse; "U," upstream; "D," downstream; "I," intron; "FR," flanking region; and "UTR," untranslated region. The locations of primers within the gene were chosen to avoid repetitive sequence.
Amplifications were performed with AmpliTaq Gold DNA polymerase (Perkin Elmer, Foster City, Calif.) using a "hot start" to help ensure amplification specificity. Each 50 μl reaction mixture contained 1 U of DNA polymerase, 5 μl of a 10-fold diluted DNA sample (160-190 ng DNA), 10 pmol of each primer, 0.05 mM dNTP (Boehringer Mannheim, Indianapolis, Ind.), and 5 μl of 10×PCR buffer that contained 15 mM MgCl2 (Perkin Elmer). Amplifications were performed with a Perkin Elmer model 9700 thermal cycler. PCR cycling parameters involved a 12 minutes hot start at 94° C., 30 cycles at 94° C. for 30 seconds, 30 seconds at the annealing temperature, and a final 10 minute extension at 72° C.
Amplicons were sequenced in the Mayo Molecular Biology Core Facility with an ABI 377 DNA sequencer using BigDye™ (Perkin Elmer) dye-primer sequencing chemistry. Both DNA strands were sequenced in all cases. To exclude PCR-induced artifacts, independent amplification followed by DNA sequencing was performed for all samples in which a SNP was only observed once among the samples resequenced, or for samples with ambiguous chromatograms. DNA sequence chromatograms were analyzed using the PolyPhred 3.0 (Nickerson et al. (1997) Nucl. Acids Res. 25:2745-2751) and Consed 8.0 (Gordon et al. (1998) Genome Res. 8:195-202) programs developed by the University of Washington (Seattle, Wash.). The University of Wisconsin GCG software package, Version 10, was also used to analyze nucleotide sequence. GenBank accession numbers for the GSTO2 reference sequences were NT—030059 and NM—183239.
Recombinant GSTO2 Expression Constructs and Allozyme Expression: A WT GSTO2 cDNA open reading frame (ORF) sequence was amplified from a pooled human liver cDNA library (Clontech, Mountain View, Calif.). Expression constructs for the nonsynonymous cSNPs observed during the resequencing studies were generated by site-directed mutagenesis using "circular PCR." Sequences of the site-directed mutagenesis primers are listed in Table 1. Site-directed mutagenesis amplification reactions contained 1 U of Pfu Turbo DNA polymerase (Stratagene, La Jolla, Calif.) in a final volume of 25 μl, 10 pmol of primers, 0.24 mM dNTPs, and 2.5 μl of 10×PCR buffer. The sequences of expression constructs were confirmed by sequencing in both directions after cloning into the eukaryotic expression vector pCR3.1 (Invitrogen, Carlsbad, Calif.).
WT and variant GSTO2 expression constructs were transfected into COS- 1 cells, together with pSV-β-galactosidase DNA (Promega) to make it possible to use β-galactosidase activity to correct for transfection efficiency. Transfections were performed with Lipofectamine 2000 (Invitrogen), and the cells were cultured for 48 hours in Dulbecco's modified Eagles medium (DMEM; Bio Whittaker, Walkersville, Md.) with 10% fetal bovine serum (Clontech). Cells were homogenized with a Polytron homogenizer (Brinkmann Instruments, Westbury, N.Y.). The homogenates were centrifuged at 100,000×g for 1 hour; and supernatant preparations were stored at −80° C.
Western Blot Analysis: Rabbit polyclonal antibodies were generated to GSTO2 (Cocalico Biologicals, Reamstown, Pa.) using a polypeptide that corresponded to amino acids 10 to 30, with an additional cysteine residue at the amino terminus. This synthetic polypeptide was conjugated to keyhole limpet hemocyanin prior to immunization of rabbits. For quantitative Western blot analysis, cytosol preparations of COS-1 cells transfected with the expression constructs were loaded on a 12% polyacrylamide gel on the basis of β-galactosidase activity to correct for possible variation in transfection efficiency. After SDS-PAGE, the proteins were transferred to a nitrocellulose membrane that was probed with rabbit antiserum to GSTO2, diluted 1:2000 with "blocking buffer." Goat anti-rabbit horseradish peroxidase, diluted 1:20,000, was used as the secondary antibody. Bound antibody was detected with the ECL Western blotting system (Amersham Pharmacia Biotech, Piscataway, N.J.). The level of immunoreactive protein was quantitated using IP Gel Lab (Biosystemetica, Plymouth, UK).
In vitro Translation and Degradation: Selected GSTO2 expression constructs were transcribed and translated in vitro in the presence of 35S-methionine (1000 Ci/mmol, 2.5 mCi total activity) (Amersham Pharmacia Biotech) using the TNT coupled rabbit reticulocyte lysate (RRL) system (Promega, Madison, Wis.). Reaction mixtures used to generate radioactively labeled protein were incubated at 30° C. for 90 minutes, and 5 μl aliquots were used to perform SDS-PAGE with 10% gels. Protein degradation studies were performed as described (Wang et al. (2003) Pharmacogenet. 13:555-564). Briefly, 50 μl of an adenosine 5′-triphosphate (ATP) generating system, 50 μl of "untreated" RRL, and 10 μl of 35S-methionine radioactively labeled GSTO2 allozyme were mixed. This mixture was incubated at 37° C., and 10 μl aliquots were removed at various time intervals, followed by SDS-PAGE. After electrophoresis, the gels were dried, exposed to X-ray film and the bands of radioactively labeled protein were quantified.
Data analysis: Statistical comparison of the data was performed by ANOVA using the StatView program, version 4.5 (Abacus Concepts, Inc., Berkeley, Calif.). Values for π, θ, and Tajima's D were calculated as described by Tajima (Tajima (1989) Genet. 123:585-595), followed by correction for length. Linkage analysis was performed after all DNA samples had been genotyped at each of the polymorphic sites observed, using the EH program developed by Terwilliger and Ott, Handbook of Human Genetic Linkage, The Johns Hopkins University Press, Baltimore, pp. 188-193 (1994). D′ values, a quantitative method for reporting linkage data that is independent of allele frequency (Hartl and Clark Principles of Population Genetics, 3rd edition, Sinauer Associates, Inc., (Sunderland, Mass.), pp. 96-106 (1997); and Hedrick Genetics of Populations, 2nd edition, Jones and Bartlett (Sudbury, Mass.), pp. 396-405 (2000)), were calculated. The genotype data also were used to assign inferred haplotypes using a program based on the E-M algorithm (Long et al. (1995) Am. J. Hum. Genet. 56:799-810; Excoffier and Slatkin (1995) Mol. Biol. Evol. 12:921-927; and Schaid et al. (2002) Am. J. Hum. Genet. 70:425-434). Unambiguous haplotype assigunent was possible on the basis of genotype for samples that contained no more than one heterozygous polymorphism. Group mean values were compared using student's t-test. Degradation data were plotted using GraphPad Prism (GraphPad Software, San Diego, Calif.).
GSTO2 Enzyme Activity: GSTO2 activity is measured using a bacterially expressed GSTO2 polypeptide. DHA is combined with DHA and GSH, and GSTO2 activity is measured as increased absorbance at 265 nm, which results from reduction of the DHA. This method also can be used to estimate apparent Km values. Alternatively, GSTO2 enzyme activity is measured in vivo (e.g., in CHO cells).
Quantitative RT-PCR: mRNA is isolated from COS-1 cells cotransfected with a control β-Galactosidase reporter and a GSTO2 WT expression construct or GSTO2 variant expression construct using an RNeasy Mini Kit (Qiagen, Valencia, Calif.), according to the manufacturer's instructions. RT-PCR is performed with primers for both GSTO2 and β-Galactosidase as an internal control.
Confocal microscopy: Fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse immunoglobulin and tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat anti-rabbit immunoglobulin are commercially available (e.g., from Southern Biotech (Birmingham, Ala.)). COS-1 cells are subcultured to 50-70% confluence on coverslips, transfected with expression constructs, and cultured for 48 hours. The cells are washed with phosphate buffered saline (PBS), fixed with 3% paraformaldehyde for 12 minutes at room temperature, washed, and incubated at room temperature for 5 minutes with buffer containing 0.5% Triton X-100. The coverslips are then incubated with primary antibodies—rabbit polyclonal antihuman antibody against calnexin (an endoplasmic reticulum marker) and mouse monoclonal anti-His antibody, followed by FITC-conjugated goat anti-mouse or TRITC-conjugated goat anti-rabbit IgG antibody. The COS-1 cells are viewed using a Zeiss LSM 510 confocal microscope with 488 or 570 nm filters for excitation of the green or red fluorochrome, respectively.
GSTO2 reporter gene constructs and luciferase assay: Luciferase reporter gene constructs are created for the most common human GSTO2 5′-FR haplotypes (e.g., haplotypes having a frequency ≧3% in any of the populations). Specifically, a segment containing about 1000 bp of the GSTO2 5′-FR is amplified from human genomic DNA samples that contained the desired haplotypes. The forward and reverse primers include ACC65I and XhoI restriction sites, respectively, to enable subcloning of the amplicons into pGL-3 Basic (Promega), upstream of the firefly luciferase gene open reading frame (ORF). The insert is sequenced in both directions to ensure that the correct sequence is present.
The luciferase reporter gene constructs are used to transiently transfect primary cells or cell lines (e.g., liver, kidney, skeletal muscle, heart, lung, cervix, ovary, or prostate cells). Cells are transfected with 5 μg purified plasmid DNA with 50 ng pRL-TK (Promega) DNA. The Renilla luciferase activity expressed by pRL-TK is used as a control for transfection efficiency. Cells also are transfected with pGL-3 Basic without insert as a control. Transfection is performed using the TransFast reagent (Promega). After 48 hours, cells are lysed and reporter gene activity is measured using a Promega dual-luciferase assay system. Results are reported as the ratio of firefly luciferase light units to Renilla luciferase light units, and all values are expressed as a percentage of the activity of the pGL3 WT 5′-FR construct. All assays in three cell lines are performed at least in duplicate (e.g., in triplicate).
Fourteen separate PCR amplifications were performed for each of the DNA samples studied. All PCR amplicons were sequenced on both strands, making it possible to verify the presence of polymorphisms using data from the complimentary strand. Approximately 3×106 pg of DNA was sequenced and analyzed, including the "intergenic" area located between the GSTO1 and GSTO2 genes (i.e., between the final exon of GSTO1 and the first exon of GSTO2). A total of 66 polymorphisms were observed (Table 2). Polymorphisms in exons and flanking regions (FR) are numbered relative to the adenine in the GSTO2 translation initiation codon (ATG, adenine is +1). Polymorphisms in introns are numbered separately, either as positive numbers relative to the guanine in the splice donor site (GT, guanine is +1), or as negative numbers relative to the guanine in the splice acceptor site (AG, guanine is −1).
Variant allele frequencies ranged from 0.8% to 78.3%, with differences between the African-American (AA), Caucasian-American (CA), Han Chinese-American (HCA), and Mexican-American (MA) subjects. Fifty-four SNPs were observed in AA subjects, 19 in CA subjects, 17 in HCA subjects, and 36 in MA subjects. Nineteen of these polymorphisms were specific to AA, 5 to CA, and 2 each were specific to HCA and MA subjects.
Six SNPs were observed within the coding-region (cSNPs), and four of those cSNPs—located in exons 2, 4, and 5—were nonsynonymous, resulting in the amino acid alterations Val41Ile, Cys130Tyr, Asn142Asp, and Leu158Ile. The Val41Ile polymorphism had a frequency of 0.9% in African-Americans but was not observed in DNA from Caucasian-American, Han Chinese-American, or Mexican-American subjects. The Cys130Tyr polymorphism had frequencies of 0.8% in African Americans and 1.7% in Han Chinese-Americans, but was not observed in Caucasian-Americans or Mexican-Americans. The Asn142Asp polymorphism had frequencies of 23.3% in African-Americans, 69.2% in Caucasian-Americans, 78.3% in Han Chinese-Americans, and 74.2% in Mexican-Americans. The Leu158Ile polymorphism had a frequency of 0.8% in Caucasian-Americans, but was not observed in African-Americans, Han Chinese-Americans, or Mexican-Americans. To exclude artifacts introduced by PCR-dependent misincorporation, independent amplifications were performed and the amplicons were sequenced in all cases in which a polymorphism was observed only once among the DNA samples studied. All polymorphisms were in Hardy-Weinberg equilibrium (P>0.05).
Nucleotide diversity, a measure of genetic variation that is adjusted for the number of alleles studied, was calculated for the GSTO2 gene and for the intergenic region. Two standard measures of nucleotide diversity are π, average heterozygosity per site, and θ, a population mutation measure that is theoretically equal to the neutral mutation parameter (Fullerton et al. (2000) Am. J. Hum. Genet. 67:881-900). Values for Tajima's D, a test for the neutral mutation hypothesis (Fullerton et al., supra), also were calculated (Table 3). Under conditions of neutrality, Tajima's D should equal 0. For GSTO2, none of the values for Tajima's D were statistically significant. For the intergenic region, Tajima's D value for AA subjects was statistically significant, raising the possibility that this area may be undergoing selection.
Linkage disequilibrium analysis was performed after all of the DNA samples had been genotyped at each of the polymorphic sites. Pairwise combinations of these polymorphisms were tested for linkage disequilibrium using the EH program developed by Terwilliger and Ott, supra. The output of this program was used to calculate d′ values, a method for reporting linkage data that is independent of sample size. Pairwise combinations with a statistically significant linkage disequilibrium (P value <0.001) are shown in Table 4. D′ values greater than 0 indicate a positive association, while d′ values less than 0 indicate a negative association.
The genotype data also were used for haplotype analysis. Haplotypes typically are defined as combinations of alleles on a single chromosome, although they are sometimes referred to in a more restricted definition as all polymorphisms present on a single allele (Altshuler et al. Nature 437:1299-1320 (2005)). Haplotypes can be determined unequivocally if not more than one polymorphism per allele is heterozygous, but haplotypes also can be inferred computationally (Schaid, supra). GSTO2 haplotypes, both observed and inferred, with frequencies ≧0.08%, are listed in Tables 5-8. As shown in the tables, the identified haplotypes accounted for 88.9% of all DNA samples from Caucasian-American subjects, 80.8% of all DNA samples from African-American subjects, 95.0% of all DNA samples from Han Chinese-American subjects, and 90.9% of all DNA samples from Mexican-American subjects. A summary of haplotypes having frequencies ≧1% is presented in Table 9. Haplotypes including one or more cSNPs are included even if their frequency is less than 1%.
As an initial step in the study of the possible functional implications of nonsynonymous cSNPs in the GSTO2 gene, expression constructs were created for all variant allozymes, and those constructs were used to transfect COS-1 cells. This mammalian cell line was transfected to ensure the presence of mammalian post-translational modification and the mammalian machinery for protein degradation. The GSTO2 expression constructs were cotransfected with β-galactosidase to make it possible to correct for variations in transfection efficiency. The antibodies for GSTO2 were directed against peptides that did not include any of the genetically variant amino acids. When GSTO2 constructs were transiently expressed in COS-1 cells, levels of the Cys130Tyr13 and Leu158Ile allozymes, as well as the double variant Asn142Asp/Leu 158Ile allozyme, were strikingly reduced. The Val41Ile and Asn142Asp allozymes were expressed at approximately 80% of the level of the WT allozyme (Table 10). The GSTO2 antibody cross-reacted with a protein in the COS-1 cells with a molecular mass slightly higher than that for recombinant GSTO2.
Attempts were made to complement expression studies with parallel assays of level of enzyme activity, but the assays currently available for GSTO2 activity—assays that have been used to study purified, bacterially expressed protein (Schmuck et al. (2005) Pharmacogenet Genomics 15:493-501)—lacked the sensitivity necessary for application to the cultured mammalian cell cytosol preparations.
To investigate the mechanisms responsible for the striking decrease in expression of the GSTO2 Cys130Tyr and Leu158Ile allozymes, protein degradation studies were performed using RRL. Radioactive WT and variant GSTO2 allozymes were synthesized using "treated" RRL, and the recombinant proteins were incubated for various periods of time with "untreated" RRL and an ATP generating system to follow the time course of protein degradation. The CYS130Tyr, Leu158Ile, and Asn142Asp/Leu158Ile allozymes were degraded much more rapidly than WT GSTO2, with Leu158Ile and Asn142Asp/Leu158Ile being degraded more rapidly than Cys130Tyr (
Cell homogenate preparations containing recombinant GSTO2 allozymes, prepared as described in Example 1, are used to assess catalytic activity. The resulting activities are adjusted to a percentage of the WT GSTO2 enzyme activity. To determine whether alterations in activity of the GSTO2 variants might be related to quantity of immunoreactive enzyme protein, quantitative Western blot analyses are conducted.
Alterations in amino acid sequence can alter enzyme substrate affinity and/or catalytic efficiency. Substrate kinetic studies are conducted to determine whether the allozymes differ from the WT GSTO2 protein in these aspects. A series of DHA concentrations are used to estimate apparent Km values for recombinant wild type GSTO2 and for the variant allozymes.
RT-PCR was used to determine whether differences in protein levels were due to differences in mRNA levels. To evaluate the mRNA level after transient expression of GSTO2 WT and one or more GSTO allozymes, RT-PCR is performed using mRNA isolated from transfected COS-1 cells. β-Galactosidase is used to correct for transfection efficiency and also as an internal control for RT-PCR.
Luciferase reporter assays are conducted to determine whether polymorphisms in the 5′-FR affect transcription of GSTO2 allozymes. Reporter gene constructs including commonly occurring haplotypes (e.g., haplotypes with an allele frequency greater than 3%) are created within the first 1 kb upstream of the translation initiation codon. These constructs are transfected into primary cells or cell lines such as liver, kidney, skeletal muscle, heart, lung, cervix, ovary, or prostate cells. After incubation for 48 hours, cells are harvested and lysed, and luciferase activity is measured using a dual-luciferase assay system.
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
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