# Patent

## Title
Sulfotransferase 1A3 sequence variants

## Abstract

Isolated sulfotransferase 1A3 nucleic acid molecules and polypeptides that include nucleotide sequence variants and amino acid sequence variants are described. Methods for determining predisposition to particular clinical conditions and methods for determining sulfonator status also are described.

## Background
STATEMENT AS TO FEDERALLY SPONSORED RESEARCH
This invention was made with government support under grant no. GM61388 awarded by the National Institutes of Health. The government has certain rights in the invention.


TECHNICAL FIELD
This invention relates to sulfotransferase 1A3 nucleic acid and amino acid sequence variants.
BACKGROUND OF THE INVENTION
Sulfate conjugation is an important pathway in the biotransformation of many neurotransmitters, hormones, drugs and other xenobiotics, and is catalyzed by cytosolic sulfotransferase enzymes designated “SULT.” SULT enzymes are encoded by a gene superfamily that, in mammals, is divided into two families: SULT1 or phenol SULTs, and SULT2 or hydroxysteroid SULTs. The SULT1 and SULT2 families share at least 45% amino acid sequence identity, while members of subfamilies within each family share at least 60% amino acid sequence identity. SULT1 subfamilies include the phenol (1A), thyroid hormone (1B), hydroxyarylamine (1C), and estrogen (1E) SULTs. SULT2 subfamilies include two hydroxysteroid SULTs, 2A1 and 2B1.
Members of the SULT1A subfamily, including SULT1A3, catalyze the sulfate conjugation of substrates such as dopamine, serotonin and other catchol monoamines, and Troglitazone. SULT1A3 is expressed in liver, brain, jejunum, kidney, and blood platelets. SULT1A3 also is referred to as “STM,” “M-PST,” or “TL-PST.”
SUMMARY OF THE INVENTION
The invention is based on the discovery of sequence variants that occur in both coding and non-coding regions of SULT1A3 nucleic acids. Certain SULT1A3 nucleotide sequence variants encode SULT1A3 enzymes that are associated with individual differences in enzymatic activity. Other SULT1A3 nucleotide sequence variants in non-coding regions of the SULT1A3 nucleic acid may alter regulation of transcription and/or splicing of the SULT1A3 nucleic acid. Discovery of these sequence variants allows individual differences in the sulfate conjugation of dopamine and similar molecules 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. Identification of SULT1A3 nucleotide sequence variants also allows assessment of an individual's predisposition to conditions such as schizophrenia, affective disorders, and renal disease.
The invention features an isolated nucleic acid molecule containing a SULT1A3 nucleic acid sequence, wherein the nucleic acid molecule is at least ten nucleotides in length, and wherein the SULT1A3 nucleic acid sequence contains a nucleotide sequence variant relative to SEQ ID NO:1. The nucleotide sequence variant can be at a position selected from the group consisting of: a) position 702 relative to the adenine of the SULT1A3 translation initiation codon; b) position 96 relative to the guanine in the splice donor site of intron 1C; c) position 1325 relative to the guanine in the splice donor site of intron 1A; and d) position 1369 relative to the guanine in the splice donor site of intron 4. The nucleotide sequence variant can be a nucleotide substitution. The nucleotide sequence variant can be a thymine substitution for guanine at position 702 relative to the adenine of the SULT1A3 translation initiation codon. The nucleotide sequence variant at position 96 relative to the guanine in the splice donor site of intron 1C can be a thymine substitution for cytosine. The nucleotide sequence variant at position 1325 relative to the guanine in the splice donor site of intron 1A can be a guanine substitution for adenine. The nucleotide sequence variant at position 1369 relative to the guanine in the splice donor site of intron 4 can be an adenine substitution for guanine.
In another aspect, the invention features an isolated nucleic acid encoding a SULT1A3 polypeptide, wherein the polypeptide contains a SULT1A3 amino acid sequence variant relative to the amino acid sequence of SEQ ID NO:4, wherein the amino acid sequence variant is at residue 234.
The invention also features an isolated SULT1A3 polypeptide, wherein the polypeptide contains a SULT1A3 amino acid sequence variant relative to the amino acid sequence of SEQ ID NO:4, wherein the amino acid sequence variant is at residue 234. The amino acid sequence variant at residue 234 can be asparagine.
In another aspect, the invention features an article of manufacture containing a substrate, wherein the substrate contains a population of isolated SULT1A3 nucleic acid molecules of claim 1. The substrate can have a plurality of discrete regions, wherein each region contains a different population of isolated SULT1A3 nucleic acid molecules. Each population of molecules can contain a different SULT1A3 nucleotide sequence variant.
In yet another aspect, the invention features a method for determining if a mammal is predisposed to schizophrenia. The method can include: a) obtaining a biological sample from the mammal, and b) detecting the presence or absence of a SULT1A3 nucleotide sequence variant in the sample, wherein predisposition to the schizophrenia is determined based on the presence or absence of the variant. The method can further include detecting the presence or absence of a plurality of the SULT1A3 nucleotide sequence variants in the sample to obtain a variant profile of the mammal, wherein predisposition to the schizophrenia is determined based on the variant profile.
The invention also features a method for assisting a medical or research professional. The method can include: a) obtaining a biological sample from a mammal, and b) detecting the presence or absence of a plurality of SULT1A3 nucleotide sequence variants in the sample to obtain a variant profile of the mammal. The method can further include communicating the profile to the medical or research professional.
In another aspect, the invention features an isolated nucleic acid molecule containing a SULT1A3 nucleic acid sequence, wherein the nucleic acid molecule is at least ten nucleotides in length, and wherein the SULT1A3 nucleic acid sequence has at least 99% sequence identity to a region of SEQ ID NO:3. Nucleotide 702 relative to the adenine of the SULT1A3 translation initiation codon can be a thymine, and the region can include nucleotides 650 to 750 of SEQ ID NO:3 relative to the adenine of the SULT1A3 translation initiation codon.
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 from the claims.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is the nucleotide sequence of the reference SULT1A3 (SEQ ID NO:1) and its complement (SEQ ID NO:2). Exons are depicted in bold type. Single nucleotide polymorphisms (SNPs) are italicized and in bold type. Primers are underlined. Translation start and stop codons are double-underlined.
FIG. 2A is an mRNA sequence (SEQ ID NO:3) containing the cDNA sequence of the reference SULT1A3 (nucleotides 139 to 885).
FIG. 2B is the amino acid sequence (SEQ ID NO:4) of the reference SULT1A3.
FIG. 3 is a schematic of the location of polymorphisms within the SULT1A3 amino acid sequence in Caucasian Americans (CA) and African Americans (AA).



DETAILED DESCRIPTION
The invention features SULT1A3 nucleotide and SULT1A3 amino acid sequence variants. SULT1A3 catalyzes the transfer of inorganic sulfate to molecules such as dopamine, serotonin and other catechol monoamines, and Troglitazone, and uses 3′-phosphoadenosine-5′-phosphosulfate (PAPS) as the sulfate donor. Sulfation typically detoxifies compounds, as the resulting ionized, organic sulfates are more readily excreted than the unsulfated compounds. Furthermore, functional groups that may interact with biological macromolecules such as nucleic acids or proteins can be masked by the sulfate moiety. Genetically based variations in SULT1A3 activity may affect the metabolism of molecules such as dopamine. For example, a subject with decreased SULT1A3 activity may have higher circulating levels of dopamine, which could lead to schizophrenia or related disorders. Thus, detecting sulfotransferase nucleic acid and amino acid sequence variants can facilitate prediction of therapeutic efficacy and toxicity of drugs on an individual basis, as well as prediction of an individual's ability to biotransform molecules such as dopamine.
Nucleic Acid Molecules
The invention features isolated nucleic acids that include a SULT1A3 nucleic acid sequence. The SULT1A3 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-SULT1A3 proteins). The term “isolated” as used herein with respect to nucleic acids also includes any non-naturally-occurring nucleic acid sequence, since 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 or both of the nucleic acid sequences normally found immediately flanking that DNA molecule in a naturally-occurring genome (one at the 5′ end and one at the 3′ end) 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 recombinant 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 of the invention are at least about 8 nucleotides in length. For example, a 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 of the invention can be in a sense or an antisense orientation, can be complementary to the SULT1A3 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 a nucleic acid. Modifications at the base moiety include deoxyuridine for deoxythymidine, and 5-methyl-2′-deoxycytidine and 5-bromo-2′-deoxycytidine for deoxycytidine. Modifications of the sugar moiety can 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, for example, 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 the SULT1A3 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). Variants include single nucleotide substitutions, deletions of one or more nucleotides, and insertions of one or more nucleotides. The reference SULT1A3 genomic nucleic acid sequence is provided in FIG. 1 (SEQ ID NO:1 and SEQ ID NO:2) and in GenBank (Accession No. U20499). The reference SULT1A3 mRNA including the SULT1A3 cDNA is provided in FIG. 2A (SEQ ID NO:3) and the corresponding amino acid sequence is provided in FIG. 2B (SEQ ID NO:4). Both the mRNA and the amino acid sequences also can be found in GenBank (Accession No. L19956). Transcripts of the SULT1A3 gene are subject to alternative splicing, but this does not result in multiple mRNAs since the alternative splicing takes place in the 5′ untranslated region and affects only exons 1C, 1B, and 1A. The SULT1A3 cDNA is encoded by exons 2-7 and the 5′ end of exon 8.
The nucleic acid and amino acid reference sequences also are referred to herein as “wild type” (WT). As used herein, “untranslated sequence” includes 5′ and 3′ flanking regions that are outside of the 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 are designated as “−X” relative to the “A” in the initiation codon; positions of nucleotide sequence variants in the coding sequence and 3′ untranslated sequence are designated as “+X” or “−X” relative to the “A” in the initiation codon. Nucleotide sequence variants that occur in introns are designated as “+X” or “X” relative to “G” in the splice donor site (GT) or as “−X” relative to the “G” in the splice acceptor site (AG).
In some embodiments, a SULT1A3 nucleotide sequence variant encodes a SULT1A3 polypeptide containing a SULT1A3 amino acid sequence variant. 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, 200-300 residues, or a full-length SULT1A3 polypeptide). SULT1A3 polypeptides may or may not have sulfotransferase catalytic activity, or may have activity that is altered relative to the reference SULT1A3 polypeptide. Polypeptides that do not have activity or that have altered activity can be useful for diagnostic purposes (e.g., for producing antibodies having specific binding affinity for variant sulfotransferase polypeptides).
Corresponding SULT1A3 polypeptides, irrespective of length, that differ in amino acid sequence are herein referred to as allozymes. For example, a SULT1A3 nucleic acid sequence that includes a thymine at nucleotide 702 encodes a SULT1A3 polypeptide having an asparagine at amino acid residue 234. This polypeptide (Lys234Asn) would be considered an allozyme with respect to the reference SULT1A3 polypeptide that contains a lysine at amino acid residue 234.
SULT1A3 allozymes as described above are encoded by a series of sulfotransferase alleles. These alleles represent SULT1A3 nucleic acid sequences containing nucleotide sequence variants, typically multiple nucleotide sequence variants, within coding and non-coding sequences. Representative examples of single nucleotide sequence variants are disclosed in Table 2. Alleles encoding Lys234Asn are commonly observed in African Americans (allele frequencies≧1%). The relatively large number of alleles and allozymes for SULT1A3 indicates the potential complexity of SULT pharmacogenetics. Such complexity emphasizes the need for determining single nucleotide sequence variants, (i.e., single nucleotide polymorphisms, SNPs) as well as complete haplotypes (i.e., the set of alleles on one chromosome or a part of a chromosome) of patients.
Certain SULT1A3 nucleotide sequence variants do not alter the amino acid sequence. Such variants, however, could alter regulation of transcription as well as mRNA stability. SULT1A3 nucleotide sequence variants can occur in intron sequences, for example, within introns 1A, 1B, 1C, 2, 3, 4, 5, 6, or 7. In particular, the nucleotide sequence variant can include a thymine at nucleotide 96 of intron 1C, a cytosine at nucleotide 99 of intron 1C, or a thymine at nucleotide 105 of intron 1C. The nucleotide sequence variant can include a guanine at nucleotide 1325 of intron 1A. Intron 4 variants can include an adenine at nucleotide 1369, and intron 7 variants can include a thymine at nucleotide 113.
SULT1A3 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 SULT1A3 variant can include a guanine at nucleotide 105 relative to the adenine in the translation initiation site, or an adenine at nucleotide 843 relative to the adenine in the translation initiation site.
In some embodiments, nucleic acid molecules of the invention can have at least 98% (e.g., 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:3 that includes one or more variants described herein. The region of SEQ ID NO:1 or SEQ ID NO:3 is at least 15 nucleotides in length (e.g., 50, 60, 70, 75, 100, 150 or more nucleotides in length). For example, a nucleic acid molecule can have at least 99% identity with a region of SEQ ID NO: 1 containing nucleotides −150 to 1000, −250 to −155, −150 to −75, −75 to −30, −25 to 50, 55 to 150, 115 to 200, 205 to 275, 300 to 375, 375 to 475, 455 to 525, 530 to 630, 650 to 750, 755 to 850, 800 to 900, 855 to 950, 955 to 1050, 1005 to 1100, or 1050 to 1150 relative to the adenine of the SULT1A3 translation initiation codon, nucleotides 50 to 150 of SEQ ID NO:1 relative to the guanine in the splice donor site of SULT1A3 intron 1C, nucleotides 1275 to 1375 of SEQ ID NO:1 relative to the guanine in the splice donor site of SULT1A3 intron 1A, nucleotides 1300 to 1400 of SEQ ID NO:1 relative to the guanine in the splice donor site of intron 4, or nucleotides 75 to 160 of SEQ ID NO:1 relative to the guanine in the splice donor site of SULT1A3 intron 7, where the nucleotide sequence of SEQ ID NO:1 includes one or more (e.g., one, two, three, four, five, or more than five) of the variants described herein. For example, the nucleotide sequence of SEQ ID NO:1 can have a guanine at nucleotide 105 relative to the adenine of the SULT1A3 translation initiation codon, a thymine at nucleotide 702 relative to the adenine of the SULT1A3 translation initiation codon, an adenine at nucleotide 843 relative to the adenine of the SULT1A3 translation initiation codon, a thymine at nucleotide 96 relative to the guanine in the splice donor site of intron 1C, a cytosine at nucleotide 99 relative to the guanine in the splice donor site of intron 1C, a thymine at nucleotide 105 relative to the guanine in the splice donor site of intron 1C, a guanine at nucleotide 1325 relative to the guanine in the splice donor site of intron 1A, an adenine at nucleotide 1369 relative to the guanine in the splice donor site of intron 4, or a thymine at nucleotide 113 relative to the guanine in the splice donor site of intron 7, and combinations thereof. The nucleotide sequence of SEQ ID NO:1 also can have an insertion of the sequence 5′ CAGT 3′ between nucleotides 83 and 84 relative to the guanine in the splice donor site of intron 3, an adenine at nucleotide 69 relative to the guanine in the splice donor site of intron 4, or an adenine at nucleotide 69 relative to the guanine in the splice donor site of intron 6. Alternatively, a nucleic acid molecule can have at least 99% identity with a region of SEQ ID NO:1 containing nucleotides 650 to 750, where the nucleotide sequence of SEQ ID NO:3 includes one or more (e.g., one, two, three, four, five, or more than five) of the variants described herein. For example, the nucleotide sequence of SEQ ID NO:3 can have a thymine at nucleotide 702 relative to the adenine of the SULT1A3 translation initiation codon. In some embodiments, the nucleotide sequence can contain at least two (e.g., two, three, four, five, or more than five) of the variants described herein.
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 (Bl2seq) 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 from Fish & Richardson's web site (www.fr.com/blast) or the U.S. government's National Center for Biotechnology Information web site (www.ncbi.nlm.nih.gov). Instructions explaining how to use the Bl2seq program can be found in the readme file accompanying BLASTZ.
Bl2seq 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:3, (2) the Bl2seq program presents 850 nucleotides from the target sequence aligned with a region of the sequence set forth in SEQ ID NO:3 where the first and last nucleotides of that 850 nucleotide region are matches, and (3) the number of matches over those 850 aligned nucleotides is 750, then the 1000 nucleotide target sequence contains a length of 850 and a percent identity over that length of 88 (i.e., 750÷850×100=88).
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 of the invention can be produced by 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 SULT1A3 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 typically are 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 a complementary DNA (cDNA) strand. 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 Genetic Engineering News 12(9):1 (1992); Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA, 87:1874-1878; and Weiss (1991) Science 254:1292.
Isolated nucleic acids of the invention 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 of the invention also can be obtained by mutagenesis. For example, the reference sequence depicted in FIG. 1 or 2A can be mutated using standard techniques such as, for example, oligonucleotide-directed mutagenesis and/or site-directed mutagenesis through PCR. See, Short Protocols in Molecular Biology, Chapter 8, Green Publishing Associates and John Wiley & Sons, Edited by Ausubel et al., 1992. Examples of positions that can be modified include those described above.
Vectors and Host Cells
The invention also provides vectors containing nucleic acids such as those described above. As used herein, a “vector” is a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment. The vectors of the invention can be expression vectors. An “expression vector” is a vector that includes one or more expression control sequences, and an “expression control sequence” is a DNA sequence that controls and regulates the transcription and/or translation of another DNA sequence.
In the expression vectors of the invention, the nucleic acid is operably linked to one or more expression control sequences. As used herein, “operably linked” means incorporated into a genetic construct so that expression control sequences effectively control expression of a coding sequence of interest. Examples of expression control sequences include promoters, enhancers, and transcription terminating regions. A promoter is an expression control sequence composed of a region of a DNA molecule, typically within 100 nucleotides upstream of the point at which transcription starts (generally near the initiation site for RNA polymerase II). To bring a coding sequence under the control of a promoter, it is necessary to position the translation initiation site of the translational reading frame of the polypeptide between one and about fifty nucleotides downstream of the promoter. Enhancers provide expression specificity in terms of time, location, and level. Unlike promoters, enhancers can function when located at various distances from the transcription site. An enhancer also can be located downstream from the transcription initiation site. A coding sequence is “operably linked” and “under the control” of expression control sequences in a cell when RNA polymerase is able to transcribe the coding sequence into mRNA, which then can be translated into the protein encoded by the coding sequence.
Suitable expression vectors include, without limitation, plasmids and viral vectors derived from, for example, bacteriophage, baculoviruses, tobacco mosaic virus, herpes viruses, cytomegalovirus, retroviruses, vaccinia viruses, adenoviruses, and adeno-associated viruses. Numerous vectors and expression systems are commercially available from such corporations as Novagen (Madison, Wis.), Clontech (Palo Alto, Calif.), Stratagene (La Jolla, Calif.), and Invitrogen/Life Technologies (Carlsbad, Calif.).
An expression vector can include a tag sequence designed to facilitate subsequent manipulation of the expressed nucleic acid sequence (e.g., purification or localization). Tag sequences, such as green fluorescent protein (GFP), glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, or FlagTM tag (Kodak, New Haven, Conn.) sequences typically are expressed as a fusion with the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide including at either the carboxyl or amino terminus.
The invention also provides host cells containing vectors of the invention. The term “host cell” is intended to include prokaryotic and eukaryotic cells into which a recombinant expression vector can be introduced. As used herein, “transformed” and “transfected” encompass the introduction of a nucleic acid molecule (e.g., a vector) into a cell by one of a number of techniques. Although not limited to a particular technique, a number of these techniques are well established within the art. Prokaryotic cells can be transformed with nucleic acids by, for example, electroporation or calcium chloride mediated transformation. Nucleic acids can be transfected into mammalian cells by techniques including, for example, calcium phosphate co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, or microinjection. Suitable methods for transforming and transfecting host cells are found in Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd edition), Cold Spring Harbor Laboratory, New York (1989), and reagents for transformation and/or transfection are commercially available (e.g., Lipofectin (Invitrogen/Life Technologies); Fugene (Roche, Indianapolis, Ind.); and SuperFect (Qiagen, Valencia, Calif.)).
SULT1A3 Polypeptides
Isolated SULT1A3 polypeptides of the invention include an amino acid sequence variant relative to the reference SULT1A3 polypeptide (FIG. 2B, GenBank Accession No. L19956). The term “isolated” with respect to a SULT1A3 polypeptide refers to a polypeptide that has been separated from cellular components by which it is naturally accompanied. Typically, a SULT1A3 polypeptide is isolated when it is at least 60% (e.g., 65%, 70%, 75%, 80%, 90%, 95%, or 99%), by weight, free from proteins and naturally occurring organic molecules with which it is naturally associated. In general, an isolated polypeptide will yield a single major band on a non-reducing polyacrylamide gel.
SULT1A3 polypeptides of the invention include variants at residue 234. In particular, an asparagine residue can be substituted at position 234.
In some embodiments, the activity of a SULT1A3 polypeptide is altered relative to a SULT1A3 polypeptide having the reference amino acid sequence set forth in SEQ ID NO:4. Certain SULT1A3 allozymes can have reduced activity (e.g., Lys234Asn), while other allozymes can have activity that is comparable to the reference SULT1A3 polypeptide. Other allozymes can have increased activity relative to the reference SULT1A3 polypeptide. Activity of SULT1A3 polypeptides can be assessed in vitro using a sulfate acceptor substrate such as dopamine and a donor sulfate molecule such as PAPS. In general, recombinant SULT1A3 polypeptides can be incubated at 37° C. for approximately 60 minutes with 14 μM 35S-PAPS and 40 μM dopamine in a 3-[N-tris-(hydroxymethyl)methylamino]propanesulfonic acid (Taps) buffer (50 mM, pH 8.25). Reactions can be stopped by heating at 100° C. for 3 minutes. After centrifugation of the heated reaction, radioactivity in the supernatant can be assessed. SULT1A3 activity is expressed as nmoles of sulfate conjugated product formed per hour of incubation.
Other biochemical properties of allozymes, such as apparent Km values, also can be altered relative to the reference SULT1A3 polypeptide. Apparent Km values can be calculated, for example, by using 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. The apparent Km values for dopamine can vary between different allozymes.
Isolated polypeptides of the invention can be obtained by, for example, extraction from a natural source (e.g., liver tissue), chemical synthesis, or by recombinant production in a host cell. To recombinantly produce SULT1A3 polypeptides, a nucleic acid sequence containing a SULT1A3 nucleotide sequence variant can be ligated into an expression vector that can be used to transform a bacterial or eukaryotic host cell (e.g., insect, yeast, or mammalian cells). In general, nucleic acid constructs include a regulatory sequence operably linked to a SULT1A3 nucleic acid sequence. Regulatory sequences do not typically encode a gene product, but instead affect the expression of the nucleic acid sequence. In bacterial systems, a strain of Escherichia coli such as BL-21 can be used. Suitable E. coli vectors include the pGEX series of vectors (Amersham Biosciences Corp., Piscataway, N.J.) that produce fusion polypeptides with glutathione S-transferase (GST). Transformed E. coli typically are grown exponentially and then stimulated with isopropylthiogalactopyranoside (IPTG) to induce expression of the fusion polypeptide. In general, such fusion polypeptides 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 cloned target gene product can be released from the GST moiety.
In eukaryotic host cells, a number of viral-based expression systems can be utilized to express SULT1A3 variant polypeptides. A nucleic acid encoding a polypeptide of the invention can be cloned into, for example, a baculoviral vector such as pBlueBac (Invitrogen, Carlsbad, Calif.) and then used to co-transfect insect cells such as Spodoptera frugiperda (Sf9) cells with wild type DNA from Autographa californica multiply enveloped nuclear polyhedrosis virus (AcMNPV). Recombinant viruses producing polypeptides of the invention can be identified by standard methodology. Alternatively, a nucleic acid encoding a polypeptide of the invention can be introduced into a SV40, retroviral, or vaccinia based viral vector and used to infect suitable host cells.
Mammalian cell lines that stably express SULT1A3 variant polypeptides can be produced using expression vectors with the appropriate control elements and a selectable marker. For example, the eukaryotic expression vectors pCR3.1 (Invitrogen) and p91023(B) (see Wong et al. (1985) Science 228:810-815) are suitable for expression of sulfotransferase 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 can be selected, e.g., by antibiotic resistance to G418, kanamycin, or hygromycin. Alternatively, amplified sequences can be ligated into a mammalian expression vector such as pcDNA3 (Invitrogen) and then transcribed and translated in vitro using wheat germ extract or rabbit reticulocyte lysate.
SULT1A3 variant polypeptides can be purified by known chromatographic methods including DEAE ion exchange, gel filtration, and hydroxylapatite chromatography. See, e.g., Van Loon and Weinshilboum (1990) Drug Metab. Dispos. 18:632-638; and Van Loon et al. (1992) Biochem. Pharmacol. 44:775-785. SULT1A3 polypeptides can be “engineered” to contain an amino acid sequence that allows the polypeptide to be captured onto an affinity matrix. For example, a tag such as c-myc, hemagglutinin, polyhistidine, or FlagTM (Kodak) can be used to aid polypeptide purification. Such tags can be inserted anywhere within the polypeptide including at either the carboxyl or amino terminus. Other fusions that can be useful include enzymes that aid in the detection of the polypeptide, such as alkaline phosphatase. Immunoaffinity chromatography also can be used to purify SULT1A3 polypeptides.
Non-Human Mammals
The invention features non-human mammals that include SULT1A3 nucleic acids of the invention, 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 of the invention can express a SULT1A3 nucleotide sequence variant in addition to an endogenous SULT1A3 nucleic acid (e.g., a transgenic non-human mammal that includes a SULT1A3 nucleic acid molecule randomly integrated into its genome). Alternatively, an endogenous SULT1A3 nucleic acid can be replaced by a SULT1A3 nucleic acid molecule containing a SULT1A3 nucleotide sequence variant through 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 SULT1A3 nucleic acid (i.e., a knockout), and then a SULT1A3 variant nucleic acid of the invention 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 generally is 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 can 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 since 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 typically 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 generally is 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 SULT1A3 nucleic acid. That is, cultured ES cells can be transformed with a knockout construct and cells selected in which the SULT1A3 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, Sections 9.37-9.52 of Sambrook et al., Molecular Cloning, A Laboratory Manual, second edition, Cold Spring Harbor Press, Plainview; NY, 1989.
To generate a knockout animal, ES cells having at least one inactivated SULT1A3 allele can be 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 SULT1A3 allele. If the original ES cell was heterozygous for the inactivated SULT1A3 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 generate an animal from a cultured cell. 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 SULT1A3 gene is interrupted by a coding region that produces a detectable (e.g., fluorescent) gene product, then the injected eggs can be 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 of the invention. For example, fetal fibroblasts can be genetically modified such that they contain an inactivated endogenous SULT1A3 gene and express a SULT1A3 nucleic acid of the invention, 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, T. et al., 1998, supra.
Non-human mammals of the invention such as mice can be used to screen, for example, toxicity of compounds that are substrates for SULT1A3 polypeptides, drugs that alter SULT1A3 polypeptide activity, or for carcinogenesis. For example, SULT1A3 polypeptide activity or toxicity can be assessed in a first group of such non-human mammals in the presence of a particular compound, and compared with SULT1A3 polypeptides 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 and parenterally. For example, a test compound can be administered parenterally through inhalation, or by intranasal, intravascular, intramuscular, or subcutaneous administration. Enteral routes include sublingual and oral administration. Compounds can be prepared for parenteral administration in the form of liquid solutions or suspensions; for oral administration in the form of tablets or capsules; or for intranasal administration in the form of powders, nasal drops, or aerosols. Compounds can be prepared for other routes of administration using standard techniques. Test compounds can be mixed with non-toxic excipients or carriers before administration. Inhalation formulations can include aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate, or deoxycholate. Other formulations may contain sterile water or saline, or polyalkylene glycols such as polyethylene glycol.
Detecting SULT1A3 Sequence Variants
The invention also provides methods for detecting SULT1A3 sequence variants. SULT1A3 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), or 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 SULT1A3 nucleotide sequence variants. Genomic DNA typically is extracted from a biological sample such as a peripheral blood sample, but also 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). Standard 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, Valencia, Calif.), Wizard® Genomic DNA purification kit (Promega, Madison, Wis.) and the A.S.A.P.TM 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 SULT1A3 gene can be amplified and then directly sequenced. Dye primer sequencing can be used to increase the accuracy of detecting heterozygous samples.
Allele specific hybridization also can be used to detect SULT1A3 nucleotide sequence variants, including complete haplotypes of a mammal. 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 can be selected such that a nucleic acid probe can specifically bind to the sequence of interest, e.g., the SULT1A3 nucleic acid molecule containing a particular SULT1A3 nucleotide sequence variant. Such hybridizations typically are performed under high stringency, as some nucleotide sequence variants include only a single nucleotide difference. High stringency conditions can include, for example, 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 (0.3 M NaCl/0.03 M sodium citrate/0.1% sodium dodecyl sulfate (SDS) and washed in 0.1×SSC (0.015 M 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 SULT1A3 nucleotide sequence variants that introduce a restriction site, restriction digest with the particular restriction enzyme can differentiate the alleles. For SULT1A3 nucleotide 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 a SULT1A3 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, can 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 a SULT1A3 nucleic acid can be amplified using a primer set from either side of the variant. One of the primers typically is 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 fluoroscein 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, SULT1A3 allozymes can be detected by antibodies that have specific binding affinity for the particular allozymes. SULT1A3 allozymes 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 particular SULT1A3 allozyme. 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 heterogenous 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 SULT1A3 allozyme 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; Cole 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. The hybridoma producing the monoclonal antibodies of the invention can be cultivated in vitro and in vivo.
Antibody fragments that have specific binding affinity for a SULT1A3 allozyme can be generated by 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 SULT1A3 allozymes 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.
Methods of the Invention
As a result of the present invention, it is possible to determine sulfonator status of a mammal (e.g., a human subject) as well as to determine if particular SNPs are linked to a particular disease or clinical condition. In some embodiments, it is possible to determine whether a mammal is predisposed (i.e., has a relative greater risk) to, for example schizophrenia, affective disorders, or renal disease. “Sulfonator status” refers to the ability of a mammal to transfer a sulfate group to a substrate (e.g., dopamine). “Predisposition” refers to a relative greater risk for a condition such as schizophrenia. Additional risk factors including, for example, family history of schizophrenia and other genetic factors can be considered when determining risk. Predisposition to a condition such as schizophrenia can be determined based on the presence or absence of a single sulfotransferase sequence variant or based on a variant profile. “Variant profile” refers to the presence or absence of a plurality (e.g., two or more) of SULT1A3 nucleotide sequence variants or SULT1A3 amino acid sequence variants. For example, a variant profile can include the complete SULT1A3 haplotype of the mammal or can include the presence or absence of a set of common non-synonymous SNPs (e.g., single nucleotide substitutions that alter the amino acid sequence of a SULT1A3 polypeptide). In one embodiment, a variant profile includes the presence or absence of two or more non-synonymous SNPs (e.g., two, three, four, or more than four non-synonymous SNPs) described herein. For example, a variant profile can include the presence of variants at nucleotides 99 and 105 relative to the guanine in the splice donor site of intron 1C, or at nucleotide 99 relative to the guanine in the splice donor site of intron 1C and nucleotide 843 relative to the adenine in the SULT1A3 translation initiation site. 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 subjects, while other variants were detected only in Caucasian subjects. In addition, the variant profile can include the presence or absence of any type of SULT1A3 SNP together with any other SULT1A3 SNP (e.g., a polymorphism pair or a group of polymorphism pairs). Such polymorphism pairs include, without limitation, those pairs described in Table 4.
Methods to determine whether a mammal is predisposed to a condition such as schizophrenia, an affective disorder, or renal disease can include providing a biological sample from a mammal and detecting the presence or absence of a SULT1A3 nucleotide sequence variant in the sample. In another embodiment, a method can involve providing a biological sample from a mammal and detecting the presence or absence of a plurality of SULT1A3 nucleotide sequence variants in the sample to obtain a variant profile of the mammal. Such methods also can include communicating the variant profile or the presence or absence of a SULT1A3 nucleotide sequence variant to a medical or research professional, to assist that professional in determining whether the mammal is predisposed to a condition such as schizophrenia, an affective disorder, or renal disease.
Articles of Manufacture
The invention also provides articles of manufacture that include populations of isolated SULT1A3 nucleic acid molecules or SULT1A3 polypeptides immobilized on a substrate. Suitable substrates can 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 SULT1A3 nucleotide or SULT1A3 amino acid sequence variant. Such articles of manufacture can include two or more nucleotide or amino acid sequence variants, or can include all of the sequence variants known for SULT1A3. Furthermore, nucleic acid molecules containing sequence variants for other sulfotransferases, such as SULT1A1, SULT1A2, SULT1B1, and SULT2B1, can be included on the substrate. See, WO 99/64630 and WO 00/20605 for a description of other SULT sequence variants.
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 typically are 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 any 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.
EXAMPLES
Example 1
Methods and Materials: PCR Amplification and DNA Sequencing
Genomic DNA from 60 African American blood donors and 60 Caucasian blood donors was obtained from Coriell Cell Repositories (Camden, N.J.). The DNA was used as a PCR template with SULT1A3-specific primers. The three non-coding and seven coding exons in the SULT1A3 gene were amplified from each of the 120 DNA samples using primers that flanked the exons and that would produce amplification products 400-500 bp in length. Amplification of the entire gene required seven separate reactions for each DNA sample. The hybridization location of each primer was chosen to avoid repetitive sequence and to ensure amplification specificity. All forward primers contained the M13 forward sequence, and all reverse primers contained the M13 reverse sequence for use in dye primer DNA sequencing. 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).
Following amplification, the products from each reaction were sequenced using dye primer DNA sequencing chemistry to identify heterozygous bases. DNA sequencing was performed in the Mayo Clinic Molecular Biology Core Facility with an Applied Biosystems Model 377 DNA sequencers and BigDyeTM (Perkin Elmer, Foster City, Calif.) dye primer sequencing chemistry. In all cases, both DNA strands were sequenced.
DNA sequence analysis: The seven separate SULT1A3 PCR amplifications performed for each of the 120 individual human genomic DNA samples described above generated a total of approximately 900,000 bp of sequence. The DNA chromatograms for this sequence were analyzed both visually and using PolyPhred 3.0, Consed 8.0, and GCG 10.0 software. All sequences were compared to the SULT1A3 gene sequences of GenBank accession number U20499.
COS-1 cell expression: Two different SULT1A3 expression constructs were made using the pCR3.1 expression vector. One of the constructs was designed to express the SULT1A3 Lys234Asn allozyme, while the remaining construct was designed to express a wild type SULT1A3 polypeptide. All SULT1A3 cDNA sequences containing SULT1A3 nucleotide sequence variants used to create the expression constructs were created by site directed mutagenesis using the method described by Ho et al. (1989) Gene 77:51-59. Each SULT1A3 cDNA was amplified by PCR and subcloned into the eukaryotic expression vector pCR3.1 (Promega, Madison, Wis.). After subcloning, all inserts were sequenced to assure that no spurious nucleotide point mutations had been introduced during the PCR amplifications. COS-1 cells were transfected with these expression constructs by the TransFastTM reagent (Promega, Madison, Wis.) as suggested by the manufacturer (i.e., using a 1:1 charge ratio). As a control, a transfection also was performed with “empty” pCR3.1, i.e., vector lacking an insert, to make it possible to correct for endogenous COS-1 cell SULT activity. The control plasmid pSV-β-galactosidase (Promega) was cotransfected with each SULT1A3 construct to make it possible to correct for transfection efficiency. Two independent transfections, each consisting of three separate plates, were performed with each of the expression constructs. After 48 hours in culture, the transfected cells were harvested and high speed supernatant (HSS) cytosol preparations were prepared as described by Wood et al. (1994) Biochem. Biophys. Res. Commun. 198:1119-1127. Aliquots of these cytosol preparations were stored at −80° C. prior to assay.
Enzyme Assays: β-galactosidase activity in each of the COS-1 HSS preparations was measured with the β-galactosidase Enzyme Assay System (Promega, Madison, Wis.). These HSS preparations of recombinant SULT1A3 allozyme were used for the activity studies without any further purification. The protein concentration of each recombinant protein preparation was determined by the dye-binding method of Bradford with bovine serum albumin (BSA) as a standard.
SULT1A3 enzyme activity was measured with an assay that involves sulfate conjugation of a sulfate acceptor substrate, dopamine (40 μM), in the presence of the sulfate donor 3′-phosphoadenosine-5′-phosphosulfate (PAPS). Blank samples did not contain dopamine. Cytosol from COS-1 cells transfected with empty pCR3.1 were used to correct for endogenous SULT activity. Because SULTs display profound substrate inhibition, a range of dopamine concentrations were tested to ensure that the assays were performed at a dopamine concentration that yielded maximal activity for the Lys234Asn allozyme. Enzyme activity was expressed as nanomoles (nmoles) of sulfate conjugated product formed per hour of incubation. Apparent Km values for PAPS were determined in the presence of dopamine, with PAPS concentrations that varied from 0.035 μM to 0.5 μM and dopamine concentrations that varied from 2.5 μM to 80 μM.
Data Analysis: Apparent Km values were calculated by using the method of Wilkinson with a computer program written by Cleland. Wilkinson, Biochem. J., 80:324-332 (1961); and Cleland, Nature, 198:463-365 (1963). Statistical comparisons of data were performed by ANOVA with the StatView program, version 4.5 (Abacus Concepts, Inc., Berkeley, Calif.). Linkage analysis was performed after all DNA samples had been genotyped at each of the 6 polymorphic sites observed. D′ values, a quantitative method for reporting linkage data that is independent of allele frequency (Hartl and Clark, Principles of Population Genetics, 3rd ed. (1997) Sinauer Associates, Inc., (Sunderland, Mass.), pp. 96-106; and Hedrick, Genetics of Populations, 2nd ed. (2000) Jones and Bartlett (Sudbury, Mass.), pp. 396-405), then 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; and Excoffier and Slatkin (1995) Mol. Biol. Evol. 12:921-927). Unambiguous haplotype assignment also was possible on the basis of genotype for samples that contained no more than one heterozygous polymorphism.
Western blot analysis: Quantitative Western blot analysis was performed with recombinant SULT1A3 protein. The quantity of cytosol loaded on the gel for each allozyme was adjusted so that each lane contained an equal quantity of β-galactosidase activity and thus gel loading was corrected for variation in transfection efficiency. Properties of the antibody used to detect the SULT1A3 protein have been described elsewhere. Bound antibody was detected using the ECL system (Amersham Biosciences). The Ambis densitometric system was used to quantitate immunoreactive protein in each lane, and those data were expressed as a percent of the intensity of the control wild type SULT1A3 protein band on that gel.







TABLE 1







PCR primers used for resequencing SULT1A3










Primer Name
Primer Location
Primer Sequence
SEQ ID NO:













UF(−4118)M13
5′-FR
TGTAAAACGACGGCCAGTTAATGGACGATGGGTGCCTTGTACT
5


UR(−3699)M13
5′-FR
CAGGAAACAGCTATGACCATGGGCCCTTAGCAGG
6


UF(−3833)M13
5′-FR
TGTAAAACGACGGCCAGTTGTAATGCCCGCAACAGTGC
7


UR(−3532)M13
5′-FR
CAGGAAACAGCTATGACCTAGGAGGGGGTAAGGACACTGAGGATCA
8


UF(−1705)M13
5′-FR
TGTAAAACGACGGCCAGTCAATACCAATGTTGGCCCCTTTTG
9


UR(−1212)M13
5′-FR
CAGGAAACAGCTATGACCCACCCTGTCTCAAAAATACACAAAGG
10


I1F(−391)M13
Intron 1
TGTAAAACGACGGCCAGTAGCAAAAACTCTGCAAAGGGGC
11


I2R52M13
Intron 2
CAGGAAACAGCTATGACCACCAAGGTGGGGACTGCCG
12


I2F135M13
Exon 2
TGTAAAACGACGGCCAGTACACCTACCCCAAGTCT
13


I4R120M13
Intron 4
CAGGAAACAGCTATGACCTGGGATGTCATCTCTACAGCA
14


I4F(−151)M13
Intron 4
TGTAAAACGACGGCCAGTCGAGCAGGGTTCAGATCCCAG
15


I6R116M13
Intron 6
CAGGAAACAGCTATGACCGAATCCGGGCTTGCTGTGGGAG
16


I6F(−34)M13
Intron 6
TGTAAAACGACGGCCAGTGTTTTGCTCCACTGAGGAGCCC
17


DR1048M13
Exon 8 (3′-UTR)
CAGGAAACAGCTATGACCTGAAATTCGTGGCCTATT
18





Underlined nucleotides indicate M13 tag






Example 2
SULT1A3 Polymorphisms
Sequencing of the 5′ and 3′ untranslated sequences, exons, and introns of the SULT1A3 nucleic acid revealed nine SNPs (Table 2). Polymorphisms in exons, untranslated regions (UTR), and flanking regions (FR) are numbered relative to the adenine in the SULT1A3 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). One of the nine SNPs altered the encoded amino acid (i.e., a non-synonymous SNP), resulting in two different SULT1A3 allozymes. This variant appeared to be “common” (frequency≧1%, Table 2) among the 60 African American samples. The same variant was not detected among the 60 Caucasian samples.
The average number of polymorphisms present in the gene overall, within the ORF, and outside the ORF was 2.2, 2.3, and 2.1 per kb sequenced, respectively, in the African American samples, and 1.3, 0.8, and 1.7 per kb sequenced, respectively, in the Caucasian samples (Table 3). For purposes of comparison, Table 3 also includes data from a large study of polymorphism frequencies in 74 human genes (Halushka et al. (1999) Nat. Genet. 22:239-247). Because Halushka et al. studied a slightly smaller number of samples (74 versus the 120 described), low frequency polymorphisms that would not have been detected by Halushka et al. have been eliminated because of their lower sample number. The genetic variation present within the SULT1A3 sequence was very similar to average values observed in the 74 genes sequenced by Halushka et al. The data in Table 3 also are presented by gene region, with “UTR” representing both exons encoding cDNA untranslated regions and 5′- and 3′-flanking regions.







TABLE 2







Human SULT1A3 sequence variants










Polymorphism
Location
WT Sequence
Variant Sequence












Position
In Gene
Nucleotide
Nucleotide
AA
CA





I1C(96)
5′-FR
C
T
0.000
0.008


I1C(99)
5′-FR
G
C
0.660
0.500


I1C(105)
5′-FR
C
T
0.017
0.050


I1A(1325)
5′-FR
A
G
0.075
0.000


105
Exon 2
A
G
0.033
0.108


I4(1369)
Intron 4
G
A
0.008
0.000


702
Exon 7
G
T
0.042
0.000


I7(113)
Intron 7
C
T
0.008
0.042


843
Exon 8
G
A
0.083
0.000
















TABLE 3







SULT1A3 polymorphism frequencies









Polymorphisms per kb










SULT1A3












African American
Caucasian
74 Human Genes














Gene(s)
1
1
74


Samples
60
60
75


Min. Allele Freq.
0.8
0.8
0.68%


Overall
2.2
1.3
4.6


Coding
2.3
0.8
4.4


Noncoding
2.1
1.7
5.9


UTRs
1.5
1.5
4.4


Introns
2.6
1.3
6.0









Example 3
Linkage Disequilibrium and Haplotype Analysis
Linkage disequilibrium analysis was performed after all of the DNA samples had been genotyped at each of the nine polymorphic sites. Pairwise combinations of these polymorphisms were tested for linkage disequilibrium using the EH program developed by Terwilliger and Ott, Handbook of Human Genetic Linkage, The Johns Hopkins University Press, Baltimore, pp. 188-193 (1994). The output of this program was used to calculate d′ values, a method for reporting linkage data that is independent of sample size. All pairwise combinations with a linkage disequilibrium greater than or equal to 0.75% are shown in Table 4.
Two unequivocal haplotypes were identified by these studies. As shown in Table 5, the haplotype analysis accounted for 8.4% and 5% of all samples based on these unequivocal haplotypes for DNA samples from African-American and Caucasian-American subjects, respectively. The unequivocal haplotypes included one that was common to both ethnic groups and another that was ethnic-specific for African-American subjects.







TABLE 4





SULT1A3 linkage disequilibrium analysis



















Polymorphism Pair African American
d′ Value
X2 Value
















I1C99
I1A(1325)
−1
0.031074



I1C99
843
0.776
0.017388



702
I7113
1
0.022897















Polymorphism Pair Caucasian American
d′ Value
P Value
















I1C99
105
−1
0.002786

















TABLE 5







SULT1A3 haplotype analysis











African
Caucasian





American
American


Frequency
Frequency
I1C99
I1C105
843





0.067
0.000
V
WT
V


0.017
0.050
V
V
WT









Example 4
Activity and Immunoreactivity of Lys234Asn Allozyme
Cell homogenate preparations containing recombinant SULT1A3 Lys234Asn allozyme, prepared as described in Example 1, were used to assess catalytic activity. The resulting activities were adjusted to a percentage of the WT SULT1A3 enzyme activity. Lys234Asn exhibited a 73% reduction in enzyme activity, displaying only 27% of WT activity. These cell preparations contained similar levels of β-galactosidase activity, indicating that the enzyme activity results were not the result of low transfection efficiency.
To determine whether the decreased activity of the Lys234Asn SULT1A3 variant might be related to quantity of immunoreactive enzyme protein, quantitative Western blot analyses were conducted. The decrease in enzyme activity for the Lys234Asn allozyme was paralleled by a similar reduction in the level of immunoreactive protein.
Example 5
Substrate Kinetic Studies
Alterations in amino acid sequence can alter enzyme substrate affinity and/or catalytic efficiency. Substrate kinetic studies were conducted to determine whether the Lys234Asn allozyme differed from the WT SULT1A3 protein in these aspects. A series of dopamine and PAPS concentrations were used to estimate apparent Km values for recombinant wild type SULT1A3 and for the Lys234Asn variant allozyme. These studies revealed no significant difference in apparent Km values for either dopamine (10.4 μM vs. 9.8 μM for WT and Lys234Asn, respectively) or PAPS (0.112 μM vs. 0.107 μM for WT and Lys234Asn, respectively). These results raise the possibility of ethnic-specific genetic alterations of catecholamine sulfation in humans.
Other Embodiments
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.

## Claims

1. An isolated nucleic acid molecule consisting of:
(a) fifteen to 100 contiguous nucleotides of SEQ ID No. 1, wherein said nucleic acid molecule includes nucleotide 7244 of SEQ ID no. 1, with the proviso that the nucleotide at position 7244 is thymine; or
(b) the complement of (a).

2. An article of manufacture comprising a substrate, wherein said substrate comprises the isolated nucleic acid molecule of claim 1.

3. The isolated nucleic acid molecule of claim 1, wherein said isolated nucleic acid molecule is from 20 to 50 nucleotides in length.

4. A vector comprising the isolated nucleic acid molecule of claim 1.

5. The vector of claim 4, wherein said isolated nucleic acid molecule is from 20 to 50 nucleotides in length.

6. An isolated nucleic acid molecule consisting of:
(a) fifteen to 100 contiguous nucleotides of SEQ ID No. 1, wherein said nucleic acid molecule includes nucleotide 7244 of SEQ ID no. 1, with the proviso that the nucleotide at position 7244 is thymine; or
(b) the complement of (a), and with respect to (a) or (b), a label.

7. The isolated nucleic acid molecule of claim 6, wherein said label is a fluorescent moiety.

8. The isolated nucleic acid molecule of claim 6, wherein said label is biotin.

9. An isolated nucleic acid molecule consisting of:
(a) twenty to 100 contiguous nucleotides of SEQ ID No. 1, wherein said nucleic acid molecule includes nucleotide 7244 of SEQ ID no. 1, with the proviso that the nucleotide at position 7244 is thymine; or
(b) the complement of (a).