Cloning and analysis of unique human glutaminase isoforms generated by tissue-specific alternative splicing

KHALED M. ELGADI, ROBERT A. MEGUID, MING QIAN, WILEY W. SOUBA and STEVE F. ABCOUWER

Surgical Oncology Research Laboratories, Massachusetts General Hospital; and Department of Surgery, Harvard Medical School, Boston, Massachusetts 02114


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Elgadi, Khaled M., Robert A. Meguid, Ming Qian, Wiley W. Souba, and Steve F. Abcouwer. Cloning and analysis of unique human glutaminase isoforms generated by tissue-specific alternative splicing. Physiol. Genomics 1: 51–62, 1999.—Three human glutaminase (hGA) isoforms were identified, two of which represent isoforms previously unidentified in any species. One isoform contains an open reading frame with high homology with the rat kidney-type glutaminase, suggesting that this isoform represents the human kidney-type glutaminase, hKGA. A second isoform, termed hGAC, contains an open reading frame that matches hKGA except for a unique COOH-terminal amino acid sequence. In addition, a third human glutaminase isoform was identified from a computer search and on further analysis was found to represent an additional unique isoform, hGAM. hKGA is expressed predominantly in brain and kidney but not in liver, hGAC is expressed principally in cardiac muscle and pancreas but not in liver or brain, and hGAM is expressed solely in cardiac and skeletal muscle. hGAC is the predominant isoform expressed by a human breast cancer cell line that exhibits a high rate of glutamine utilization and glutaminase activity. Genomic Southern analysis as well as isolation and analysis of five glutaminase genomic clones suggested that all three hGA isoforms originate from the same locus and therefore represent mRNA species that are produced by tissue-specific alternative splicing of a single pre-mRNA. Furthermore, an RT-PCR assay was developed that can be used to easily differentiate between hKGA and hGAC mRNA species.

glutamine metabolism; mitochondrial proteins; gene expression; mRNA processing; CAG repeat polymorphisms; brain; kidney; muscle; mammary adenocarcinoma


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE MITOCHONDRIAL ENZYME glutaminase catalyzes the hydrolysis of glutamine (Gln) to glutamate and ammonia (5). Through this reaction and the subsequent conversion to {alpha}-ketoglutarate, glutamine serves as a major source of tricarboxylic acid cycle intermediates and ultimately provides a large fraction of cellular energy and reducing equivalents. In culture, most mammalian cells depend on Gln for their survival and proliferation (6), and tumor cells have been identified as particularly avid consumers of Gln. How and why Gln influences cell survival and proliferation and the determinants of Gln utilization rates are not precisely known (2). However, it is believed that the majority of Gln utilization by tumor cells is driven by its enzymatic hydrolysis via glutaminase.

To date, two isoforms of mitochondrial glutaminase have been characterized, liver type (LGA) and kidney type (KGA) (5). LGA is expressed only in periportal hepatocytes of the postnatal liver (9), whereas KGA has been reported to be abundant in the kidney, brain, intestine, fetal liver, lymphocytes, and tumors (5). The two isoenzymes have different structural and kinetic properties and are thought to be the products of different, but related, genes (5). KGA helps to maintain the acid-base balance of the host via ammoniagenesis in kidney tissue (4, 12). In brain tissue, KGA is instrumental in the production of glutamate and {gamma}-aminobutyric acid (GABA) for neurotransmission (32). In intestinal epithelium, KGA is thought to initiate the catabolism of Gln, which serves as a major respiratory fuel source (31).

The present work was initiated to obtain a cDNA of the human KGA, in order to study its expression and its relevance to the growth and Gln utilization of human cancer cells. At the inception of this project, a nearly full-length cDNA and partial cDNA sequences of the KGA had been cloned only from rat and pig libraries, respectively (18, 23). [Recently, however, a full-length human KGA homolog was identified by random cloning as part of a large-scale human cDNA-sequencing project (15).] We used a rat KGA (rKGA) cDNA probe to screen a human cDNA library and obtain a partial cDNA clone of human KGA (hKGA). In addition, a partial cDNA clone containing an open reading frame corresponding to the rKGA with a different COOH-terminal sequence was identified and termed hGAC. Analysis of another cDNA clone (HSAAD20) isolated by Imbert and colleagues (8) revealed that this represents a third human glutaminase homolog, hGAM. These isoforms exhibited unique tissue-specific patterns of expression. Evidence is presented suggesting that these three hGA isoforms originate from a common gene. Therefore, expression of these human glutaminase isoforms is presumably controlled by tissue-specific alternative splicing of a common pre-mRNA. Furthermore, hGAC was found to be the predominant glutaminase splice form expressed by a human breast cancer cell line with an extraordinarily high rate of Gln utilization and glutaminase activity.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Reagents.
[{alpha}-32P]dCTP (sp act 3,000 Ci/mmol) was purchased from Du Pont-New England Nuclear (Boston, MA). A Megaprime DNA labeling kit was purchased from Amersham Life Science (Arlington Heights, IL). A GeneAmp RNA PCR kit was purchased from Perkin Elmer Cetus (Norwalk, CT). Sequenase version 2.0 was obtained from U.S. Biochemical (Cleveland, OH). Human kidney poly(A)+ RNA and human tissue poly(A)+ RNA blot were purchased from Clontech (Palo Alto, CA). Cryopreserved human renal proximal tubule epithelial cells (RPTEC 2601–4) were obtained from Clonetics (San Diego, CA). Nylon and nitrocellulose membranes were obtained from Micron Separations (Westborough, MA). DNA purification kits were purchased from Qiagen (Santa Clarita, CA). E. coli RNase H was purchased from Promega (Madison, WI). Tissue culture plasticware was purchased from Falcon (Becton Dickinson Labware, Franklin Lakes, NJ). Oligomeric DNA primers were synthesized by Genosys (Woodlands, TX). The TSE cell line was established from a primary ductal breast carcinoma and kindly provided by Dr. Simon Powell (Radiation Therapy, Massachusetts General Hospital, Boston, MA). A plasmid construct, pGA104, containing a 2.8-kb insert representing 1.6 kb of the coding sequence and 1.2 kb of the 3'-untranslated region (3'-UTR) of the rat KGA cDNA (23) was kindly provided by Dr. Norman P. Curthoys (Dept. of Biochemistry and Molecular Biology, Colorado State Univ., Fort Collins, CO). dbEST247733 was purchased from Genome Systems (St. Louis, MO). HSAAD20 was kindly provided by Dr. Gaël Yvert (Institut de Génétique et de Biologie Moléculaire et Cellulaire, Strasbourg, France). Cloning vectors, competent cells, and restriction enzymes were purchased from Stratagene (La Jolla, CA), Promega, and New England Biolabs (Beverly, MA). Chemicals were obtained from Sigma Chemical (St. Louis, MO) and Fisher Scientific (Pittsburgh, PA). Fetal bovine serum (FBS) and tissue culture media and reagents were from GIBCO (Grand Island, NY).

Cloning and Analysis
A cDNA library in LambdaGEM-2 vector (Promega) constructed with poly(A)+ RNA isolated from the human colon carcinoma cell line HT-29 cultured on 2.5 mM inosine and harvested during log phase (30) was kindly provided by Dr. Burton Wice (Washington Univ. School of Medicine, St. Louis, MO) via Dr. Daniel K. Podolsky (Dept. of Gastroenterology, Massachusetts General Hospital). An rKGA probe, termed rGA, was generated by random priming of a 1.3-kb template isolated from pGA104 (see Materials) by Cla I-Acc I digestion. After screening the library, positive clones were plaque-purified, and cDNA inserts were separated from the phage fragments by Spe I or partial EcoR I-Xba I digestion and subcloned in pBluescript II-SK+/- (Stratagene). Subcloned inserts were sequenced in both directions by the dideoxynucleotide chain termination reaction method (20) using single-stranded DNA, specific primers, and the Sequenase version 2.0 sequencing kit (U.S. Biological, Swampscott, MA) according to the vendor's protocol. Single-stranded phagmid DNA was obtained by infecting a single bacterial colony carrying plasmid of interest with a helper phage VCS-M13 (Stratagene) and isolating single-stranded DNA from the phage particles according to the vendor's protocol. The sequence data were assembled with assistance from MacVector software (Oxford Molecular Group, Campbell, CA). cDNA sequence information was used to perform a computer-aided comparison to sequences deposited in GenBank, using BLASTN (DNA database comparisons).

Probe templates for analysis of hGA isoform expression were generated as follows: probes 11–1RR (0.5 kb) and 10–1RR (1.4 kb) were generated by EcoR I digestion of hGA11–1 cDNA and hGA10–1 cDNA, respectively; probe 11–1XX (0.5 kb) was generated by Xba I digestion of hGA11–1 cDNA; and probe 10–1NX (1.2 kb) was generated by Hinc II-Xba I digestion of hGA10–1 cDNA. Probe AD20HS (0.6 kb) was generated by Hind III-Sca I digestion of HSAAD20. Probe AD20EB (0.3 kb) was generated by EcoR I-BstE II digestion of HSAAD20.

Cloning of the 5' end of hGAC
The 5' end of hGAC was PCR cloned using 5',3'-rapid amplification of cDNA ends (RACE)-ready Marathon human heart cDNA library (Clontech, San Francisco, CA) and Advantage-GC cDNA PCR kit (Clontech). PCR was performed using hGAC-specific backward primer (hGA MP3) (5'-CGGGACTGAATTTGGCCAGTTGAGG-3') and forward adapter primer (AP1) (5'-CCATCCTAATACGACTCACTATAGGGC-3'). PCR was performed using 5 µl of human heart cDNA (0.5 ng), 1 µl of each backward and forward primer (10 µM each), 1 M GC-Melt (Clontech), 0.2 mM of each dNTP, and 1 µl of Advantage-GC cDNA polymerase mixture in a final volume of 50 µl. PCR was performed as follows: 95°C for 1 min, followed by 35 cycles of 94°C for 30 s and 66°C for 3 min, followed by a final extension step at 66°C for 5 min. A nested PCR reaction was performed using backward primer hGA MP2 (5'-ACCTTTCCTCCAGACTGCTTTTTAGC-3'), forward primer AP1, and 5 µl of a 1:50 dilution of the initial PCR reaction product in 10 mM Tricine-KOH (pH 9.2), 0.1 mM EDTA. PCR products ~1 kb in size from both the original and the nested reactions were cut with restriction enzymes Hinc II and Not I. These DNA restriction fragments were gel-purified and cloned into the pBluescript KS+ vector (Promega). Positive clones were isolated and sequenced.

Northern Blot Analysis
Northern blot analysis of human kidney RNA was performed as described previously (1), using 2 µg of poly(A) RNA from human kidney tissue (Clontech). cDNA fragments representing the homologous and nonhomologous regions of the cloned fragments were used as templates to generate 32P-labeled probes by random hexamer priming. To analyze the tissue-specific expression of hGA mRNA isoforms, a human tissue poly(A) RNA blot (Clontech) was prehybridized with ExpressHyb hybridization solution (Clontech) for 1 h at 65°C and then incubated with 32P-radiolabeled, random hexamer-primed cDNA probes for 2 h at 65°C. After each hybridization, membranes were washed at high stringency (0.1x SSPE, 1% SDS, 65°C, where 1x SSPE is 0.15 M NaCl, 0.01 M Na2HPO4, and 0.001 M EDTA) and exposed to X-ray film (Fuji Medical Systems, Stamford, CT) at -80°C for 8–24 h.

RT-PCR Analysis
Poly(A)+ RNA from human kidney tissue (Clontech) was used as substrate for RT-PCR reactions performed using a GeneAmp RNA PCR Kit (Perkin Elmer Cetus, Branchburg, NJ) according to the manufacturer's protocol. Reverse transcription reactions (10 µl) were prepared by mixing 0.025 µg of RNA, 25 pmol of oligo(dT), 1 µl of 10x PCR buffer (500 mM KCl, 100 mM Tris-HCl), 2 µl of 25 mM MgCl2, 1 µl of 10 mM dNTPs, 10 U of RNase inhibitor, diethyl pyrocarbonate-treated water, and 25 U of Moloney Murine leukemia virus reverse transcriptase. This mixture was incubated at room temperature for 10 min, at 42°C for 15 min, and at 99°C for 5 min and then put on ice. This reaction was used as a substrate for 30 cycles of PCR (2 min at 95°C followed by 30 cycles of 45 s at 95°C and 45 s at 55°C and a final extension step of 7 min at 72°C) in a 50-µl reaction with 1.25 U of Taq polymerase in 1x PCR buffer containing 0.2 mM dNTPs, 2 mM MgCl2, and 0.3 µM of each primer. Primers used for PCR were common forward primer F1 (5'-TGATGGCTGCGACACTGGCTAATG-3'), common nested forward primer F2 (5'-GGTCTCCTCCTCTGGATAAGATGG-3'), hGA11–1-specific backward primer B1 (5'-CCCGTTGTCAGAATCTCCTTGAGG-3'), and hGA10–1-specific backward primer B2 (5'-GATGTCCTCATTTGACTCAGGTGAC-3'). The PCR products were separated on 1% agarose gels.

RNase H Analysis
TSE cells were grown overnight to a subconfluent stage in 150-mm culture plates at 37°C under a humidified atmosphere of 5% CO2-95% air. Cells were maintained in DMEM supplemented with 4 mM L-Gln, 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml amphotericin B, and 10 µg/ml bovine insulin. Total RNA was extracted using RNA Stat-60 (Tel-Test, Friendswood, TX) as described previously (1). Ten micrograms of total RNA was preincubated with 0.5 µg of antisense primer (B2) at 70°C for 5 min in a mixture containing 4 µl of 2.5x Universal RiboClone second-strand buffer [Promega, 100 mM Tris-HCl, pH 7.2, 225 mM KCl, 7.5 mM MgCl2, 7.5 mM dithiothreitol, 0.125 mg/ml bovine serum albumin (BSA)] and diethyl pyrocarbonate-treated water (total vol 9 µl). RNase H (1.5 U; Promega) was added to the mixture, which was then incubated at 37°C for 20 min and put on ice. The reaction was analyzed by Northern blotting as described above.

Southern Analysis of Genomic DNA and Genomic Glutaminase Clones
Human genomic DNA was extracted from a culture of human renal proximal tubule epithelial cells (RPTEC). Briefly, cells were grown to near confluence, harvested and separated by centrifugation, and resuspended in 4.5 ml of DNA extraction buffer (10 mM Tris, pH 8.0, 100 mM EDTA) with 250 µl 10% SDS and 100 µl of 10 mg/ml proteinase K. The mixture was incubated at 55°C for 16 h. DNA was separated by phenol-chloroform extraction and ethanol precipitation. The pellet was then resuspended in TE (10 mM Tris, pH 8.0, 1 mM EDTA) overnight and treated with DNase-free RNase (Boeringer-Mannheim, 500 µg/ml) at 37°C for 1 h, phenol-chloroform extracted, and ethanol precipitated. After resuspension in TE, 5-µg samples of genomic DNA were digested overnight with 50 U of restriction endonuclease (Hind III, BamH I, EcoR I, Pst I, or Not I) in a total volume of 50 µl of appropriate restriction buffer, fractionated in a 0.8% agarose gel, and transferred to nylon membrane.

Five human glutaminase gene (gls) clones were identified and isolated from a bacterial artificial chromosome (BAC) human genomic DNA library by a commercial screening service (Genome Systems, St. Louis, MO) using probe 10–1RR. For Southern analysis, 5 µg of each clone was digested with EcoR I, fractionated in a 0.8% agarose gel, and transferred to nylon membrane. Southern blots were hybridized with differential probes, washed at high stringency (0.1x SSPE, 1% SDS, 65°C) and exposed to X-ray film, as described above for Northern blotting.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Isolation and Analysis of Two Human Glutaminase Homologs
A human colon carcinoma cell line cDNA library was screened with a random-primed probe generated using a 1.3-kb template containing coding sequence from the rKGA cDNA. Screening of ~7 x 105 plaques resulted in the identification of four clones that exhibited sequence similarity to the rKGA cDNA. The two largest of the four rKGA homologous clones, hGA11–1 (1.3 kb) and hGA10–1 (3.2 kb), were sequenced and analyzed in detail.

The 1,285-base sequence of hGA11–1 is presented in Fig. 1. The first 28 bases of this clone are not homologous to rKGA. After the first 28 bases, clone hGA11–1 exhibits 91% sequence homology with a portion of the coding sequence of rKGA [bases 1110–2080 of rKGA (23)] and then an 82% homology with a portion of the 3'-UTR of rKGA [bases 2081–2346 of rKGA (23)]. hGA11–1 contains a 22-base poly(A) tail beginning at base 1264. This tail is not substantially longer than the oligo(dT) primer used in library construction. However, a polyadenylation signal (AATAAA) that is absent in rKGA is located at bases 1180–1185 of hGA11–1. Clone hGA11–1 contained a 999-bp open reading frame (ORF). The translation of the ORF of hGA11–1 has complete homology with the 323 COOH-terminal amino acids of rKGA, excluding the first 9 NH2-terminal amino acids encoded by hGA11–1 (NVFMSLIFL) and three 3 acids within the hGA11–1 ORF (2 of which are not specified because of 2 unidentified nucleic acids). Thus hGA11–1 was deemed a partial cDNA clone of the human kidney-type glutaminase isoform, hKGA. This was confirmed by the isolation and sequencing of a full-length hKGA cDNA by Nagase and co-workers (15). Bases 1287–2522 of this 4,198-bp hKGA cDNA (clone hk03864, GenBank accession no. AB020645) share complete homology with bases 29–1269 of hGA11–1, except for the two unidentified nucleic acids in the hGA11–1 sequence.



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Fig. 1. Nucleic acid base sequence of a partial human kidney-type glutaminase (hKGA) cDNA clone. The sequence of cDNA clone hGA11–1 (GenBank accession no. AF097494) is shown. Nucleotides are numbered at right. Nucleic acids in the open reading frame (ORF) and the noncoding regions are presented in uppercase and lowercase letters, respectively. A polyadenylation signal sequence (aataaa) at bases 1180–1185 is underlined.

 
Comparison of the major ORF of clone hk03864 (bases 251–2257) with the coding sequence of rKGA (23) revealed an 89% nucleotide homology, resulting in a 98% amino acid homology between the two proteins (see Fig. 3). The homology would be nearly 99% if not for the presence of nine Gln repeats in rKGA protein that are not present in the translation of hk03864. The 5'-UTRs of these cDNAs are not homologous except for bases 199–229 of hk03864, which are 84% homologous to bases 16–46 of rKGA. The 3'-UTR of hk03864 is 50% homologous to the corresponding sequences of rKGA. Thus hk03864 undoubtedly represents the human homolog of rKGA. Moreover, kidney glutaminase is highly conserved between the rat and human species.



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Fig. 3. Comparison of glutaminase isoforms. Alignment of the amino acid sequences deduced from rKGA, hKGA, hGAC, and hGAM cDNA sequences is shown. The amino acid sequences that are nonhomologous to the rat KGA amino acid sequence are underlined. The amino acid sequence of hKGA was derived from the nucleotide sequence of cDNA clone hk03864 (GenBank accession no. AB020645).

 
The initial 29 bases of hGA11–1 do not share homology with either rKGA or hk03864. These bases are not the result of a cloning artifact, as evidenced by the existance of an expressed sequence tag (EST) clone obtained from a human endometrial adenocarcinoma cDNA library [dbEST2380672, GenBank accession no. AI564468; National Cancer Institute, Cancer Genome Anatomy Project (CGAP), Tumor Gene Index, 1997, unpublished], which exhibits complete homology with the first 147 nucleotides of the 5' end of hGA11–1.

The complete sequence of hGA10–1 is presented in Fig. 2 (bases 347–3549 of hGAC). hGA10–1 has a very high homology with the 5' end of the rKGA but diverges completely at the 3' end. The 5' end of the mRNA corresponding to hGA10–1 was PCR cloned from a human heart 5'-,3'-RACE-ready cDNA library using hGA10–1-specific primers (complementary to bases 995–1019 and 996–981 in hGAC; Fig. 2). An 879-bp cDNA fragment was cloned and found to overlap with the 5' 533 bases of hGA10–1. The 3' portion of hGA10–1 sequence, which is not homologous to rKGA, was subjected to a BLASTN DNA database search, which identified an EST clone, dbEST247733 (GenBank accession no. R70875). This EST clone was purified from a library constructed from poly(A)+ RNA isolated from full-term placental tissue (Soare's placenta Nb2HP; The WashU-Merck EST Project, L. Hillier et al., 1995, unpublished). dbEST247733 was obtained and fully sequenced. The 5' 938 bases of this 1,847-bp cDNA overlap the 3' end of hGA10–1 with near-perfect homology (1 base deletion compared to hGA10–1). The combined sequence (4,438 bases) of these three clones is presented in Fig. 2. The hGA mRNA species represented by these three partial cDNA clones was termed hGAC.



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Fig. 2. Nucleic acid base sequence of hGAC cDNA. The compiled sequence of hGAC (GenBank accession no. AF158555) is shown. Nucleotides are numbered at right. Bases 1–879 correspond to 5'-rapid amplification of cDNA ends (RACE)-PCR clone, bases 347–3529 correspond to cDNA clone hGA10–1, and bases 2591–4438 correspond to cDNA clone dbEST247733. Nucleic acids in the ORF and the noncoding regions are presented in uppercase and lowercase letters, respectively. A polyadenylation signal sequence (aataaa) at bases 4400–4405 is underlined.

 
The initial 1,894 bases of hGAC exhibit 99.8% homology with the hKGA clone hk03684. This excludes a difference in CAG repeat lengths found in the 5'-UTRs of the two cDNAs. Clone hk03684 contains 15 CAG repeats from bases 40–84, whereas hGAC contains 7 CAG repeats from bases 58–78. After base 1894, hGAC shares virtually no homology with hKGA or rKGA sequences. hGAC contains an ORF that encodes an amino acid sequence identical to that of hKGA up to the point at which these cDNAs lose homology. At amino acid 550 the inferred protein sequence of hGAC diverges from that of hKGA and rKGA (Fig. 3). Consequently, the putative hGAC protein contains a 48-amino acid-long COOH terminus that differs from the 113-amino acid-long COOH termini of hKGA and rKGA proteins. This suggests that hGAC mRNA encodes an isoenzyme of glutaminase different from the kidney type. Clone dbEST247733 contains a poly(A) tail preceded by a polyadenylation signal located at bases 1809–1814 (corresponding to bases 4400–4405 of hGAC). Although this tail is not longer than the oligo(dT) primer used in library construction, the presence of the polyadenylation signal 14 bases upstream suggests that this clone represents the complete 3' end of hGAC. The deduced NH2-terminal sequence of 16 amino acids of hGAC closely matches the putative mitochondrial signal sequence found in the rat kidney isoform with 14 identical and 16 similar amino acids (Fig. 3), suggesting that hGAC also encodes a mitochondrial protein.

Imbert and co-workers (8) reported the isolation and partial sequence of a cDNA clone, HSAAD20, which exhibited partial homology to the rKGA (EMBL accession no. Y08264). This cDNA was one of several clones containing CAG repeats isolated from a spinocerebellar ataxia patient lymphoblastoid cell line library (8). The 2,010 bases of HSAAD20 were fully sequenced (GenBank accession no. AF097495) and compared with hKGA and hGAC. Bases 1–696 are completely homologous to hGAC except for the presence of 14 CAG repeats in HSAAD20 (nucleotides 5–46). At this position, the 5'-UTR of hGAC (obtained from the RACE-ready human heart cDNA) contains only seven GAC repeats. Beyond base 696, HSAAD20 contains a stretch of 356 nucleotides that shares no homology with either hGAC, hKGA, or rKGA. A BLASTN DNA database search confirmed that this stretch is a unique sequence. Bases 1053–1173 of HSAAD20 are again completely homologous to hGAC, but the final 836 bases of HSAAD20 share no homology with hGAC or hKGA. Thus HSAAD20 may represent an hGA mRNA species that shares a 5' end with hGAC but contains a unique internal exon and a unique 3' end. HSAAD20 does not contain a complete 3' end, for this clone was isolated from a random hexamer-primed cDNA library (8). HSAAD20 contains two polyadenylation signals, but these are distal to the 3' end (nucleotides 741–746 and 973–978). Thus HSAAD20 was considered a partial clone of another human glutaminase isoform and termed hGAM. hGAM contains an ORF from base 213 to 719. This ORF encodes a 169-amino acid peptide identical to that encoded by hGAC up to amino acid 161, at which point the hGAM peptide diverges and encodes a unique eight-amino acid COOH terminus (Fig. 3). As the deduced NH2-terminal amino acids of hGAM are the same as those encoded by hGAC, this protein also contains a potential mitochondrial signal peptide.

Figure 4 schematically represents the relationships between rKGA, hKGA, hGAC, and hGAM cDNAs, as well as the probe templates and oligonucleotides used to further analyze these species.



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Fig. 4. Comparison of glutaminase cDNAs: a schematic diagram aligning hKGA, hGAC, and hGAM cDNA sequences to that of rKGA cDNA. The coding regions are presented as thick bars, and the 5'- and 3'-untranslated regions (UTRs) are presented as thin bars. Areas of homology are indicated by the same patterns of shading. Dashed lines indicate alignment of shifted homologous regions. Solid bars represent the different templates used for library screening, Northern, and Southern analysis. Horizontal arrowheads indicate the positions of oligonucleotide primers used for RT-PCR and RNase H analysis.

 
Expression of hGA Isoforms in Human Kidney Tissue
The presence of additional hGA mRNA isoforms was unexpected because previous blotting results demonstrated only two hGA mRNA species of 4.8- and 3.5-kb sizes in both rat kidney (5) and human cell lines (Abcouwer, unpublished observation). Northern blotting analysis was used to investigate the presence and size of mRNA transcripts corresponding to the hKGA and hGAC isoforms in poly(A)-enriched RNA isolated from human kidney. Probe templates that represented common and unique sequences of hGA11–1 (hKGA) and hGA10–1 (hGAC) were prepared (see Fig. 4). 11–1RR represents a homologous region; 11–1XX and 10–1NX represent nonhomologous regions of hGA11–1 and hGA10–1, respectively. Probes 11–1RR, 11–1XX, and 10–1NX each hybridized with two mRNA species ~4.8 kb and 3.5 kb in size (Fig. 5A). The image of 10–1NX hybridization shown was obtained from a fresh, previously unprobed blot, and therefore the signal was not a residual resulting from previous probes. This result suggested that hGAC species are the same size as hKGA mRNA species. In addition, when this blot was hybridized with probe AD20HS, no signal was obtained (data not shown), indicating that hGAM is not appreciably expressed in the kidney.




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Fig. 5. Expression of both hKGA and hGAC mRNA in human kidney. A: Northern blot analysis of human kidney poly(A) RNA. Blots of poly(A) RNA (2 µg) from human kidney were hybridized with 32P randomly labeled differential cDNA probes corresponding to common (11–1RR) and unique (11–1XX and 10–1NX) regions of hKGA and hGAC mRNA isoforms, respectively. The positions of these probes are indicated in Fig. 4. B: RT-PCR analysis using poly(A) RNA from human kidney tissue and endonuclease digestion of the PCR products to confirm presence of internal restriction sites. PCR primer pairs used in each reaction are indicated and include F1 (common forward primer), F2 (common forward nested primer), B1 (hGA 11–1-specific backward primer,) and B2 (hGAC-specific backward primer). Locations hybridized by these primers indicated in Fig. 4. F1/B1-EcoR I and F1/B2-EcoR I lanes represent EcoR I restriction digest of F1/B1 and F1/B2 PCR reactions. PCR and restriction products were separated on 1% agarose gels, ethidium bromide stained, and visualized by ultraviolet light.

 
RT-PCR analysis was used to confirm the presence of hGAC mRNA in poly(A)-enriched RNA isolated from human kidney. Oligonucleotide primers were designed to flank the regions where hGA11–1 and hGA10–1 lose homology (see Fig. 4). After reverse transcription, the cDNAs were amplified using four sets of primers. F1/B1 and F2/B1 primer pairs are specific for hKGA and were expected to produce fragments 680 bp and 464 bp in length, respectively. F1/B2 and F2/B2 primer pairs are specific for hGAC and were expected to produce fragments 464 bp and 248 bp in length, respectively. All four reactions produced DNA fragments of the expected sizes (Fig. 5B). PCR products were not obtained from control reactions to which reverse transcriptase was not added. The authenticity of the PCR products of F1/B1 and F1/B2 was verified by restriction analysis (Fig. 5C); by performing nested PCR using F2/B1 and F2/B2 primer pairs, respectively (Fig. 5B); and by probing Southern blots of the RT-PCR products with probe 11–1RR (data not shown). These results confirmed the existence of both hKGA and hGAC isoforms in kidney tissue.

Human Tissue Distribution of hGA Isoforms
The tissue distribution of the hKGA, hGAC, and hGAM mRNA transcripts was determined by probing a human tissue poly(A)-enriched RNA blot with differential cDNA probes. As shown in Fig. 6, 11–1RR (containing sequences common to hKGA and hGAC) detected a 4.8-kb band in all but liver tissue and a 3.5-kb band in kidney tissue. The intensity of the 4.8-kb band was relatively high in brain, heart, kidney, and pancreas. 11–1XX (specific for hKGA) also detected a 4.8-kb band in all but liver tissue, with a high intensity in brain and kidney and a weak signal in other tissues. The tissue distribution observed with probe 11–1XX is nearly identical to that determined by Nagase and co-workers (15) by RT-PCR with oligonucleotide primers specific to clone hk03864. Probe 11–1XX also detected a 3.5-kb band in kidney and a slightly smaller species (~3.4 kb) in pancreatic tissue. Probe 10–1NX (specific to hGAC) clearly detected a 4.8-kb band in all but brain and liver tissue. This signal was strongest in heart and pancreas, followed by placenta, kidney, and lung. Thus, in contrast to 11–1XX, 10–1NX did not detect the 4.8-kb mRNA species present in brain and produced a relatively weak signal with the kidney sample. Probe 10–1NX also weakly detected a 3.5-kb mRNA species in kidney tissue. By probing a fresh blot, it was determined that this band was not a ghost left from hybridization with probe 11–1NX. Unlike both hKGA- and hGAC-specific probes, AD20HS (specific for hGAM) detected mRNA species in cardiac and skeletal muscle only, which were single bands of 2.6-kb size. These results suggest that three nonhepatic isoforms of hGA exist and are expressed in unique tissue-specific fashions. hKGA is expressed predominantly in brain and kidney. hGAC is expressed primarily in cardiac muscle and pancreas but also appreciably in placenta, kidney, and lung. hGAM is expressed exclusively in cardiac and skeletal muscle tissues.



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Fig. 6. Tissue-specific expression of glutaminase mRNA isoforms. Autoradiograph of human multiple tissue poly(A) RNA blot hybridized with a probe common to hKGA and hGAC (11–1RR), a probe specific to hKGA (11–1XX), a probe specific to hGAC (10–1NX), and a probe specific to hGAM (AD20HS).

 
Theoretically, the absolute amount of mRNA detected with the 11–1XX probe plus that detected with the 10–1NX probe should equal the amount of mRNA detected with the 11–1RR probe. Indeed, this is roughly true for the 4.8-kb mRNA bands. However, this does not seem to be the case for the 3.5-kb and 3.4-kb mRNAs detected in kidney and pancreas. These mRNAs are detected by probe 11–1RR with much less intensity than would be expected from the result obtained with probe 11–1XX. In fact, the 3.4-kb band detected in pancreas by probe 11–1XX is not visible in the 11–1RR blot. At this time, this phenomenon cannot be explained except to hypothesize that there are mRNAs in the kidney and pancreas, of sizes 3.5 and 3.4 kb, respectively, that contain 11–1XX sequences but not 11–1RR sequences.

Analysis of hGA Isoform Expression in a Human Breast Carcinoma Cell Line
The human breast carcinoma cell line TSE was previously found to exhibit a high rate of Gln utilization and an extraordinarily high glutaminase activity (2). It was previously observed that this cell line exhibits a relatively high hGA mRNA content of exclusively 4.8-kb size (Abcouwer, unpublished observation). In light of the identification of multiple isoforms of hGA, it was of interest to determine which isoform was expressed by this cell line. Oligonucleotide-targeted RNAse H digestion of total RNA from subconfluent TSE cells was utilized to determine definitively which isoform of hGA was expressed by these tumor cells. To differentiate the two possible 4.8-kb mRNA species, total RNA was treated with RNase H in the presence of an antisense oligonucleotide (B2) specific for the nonhomologous coding region of hGAC. RNase H has endoribonuclease activity specific for RNA-DNA duplexes and therefore should cleave only that RNA hybridized to B2. The result of this targeted digestion was analyzed by Northern blotting using differential probes. The mRNA band corresponding to hGAC was expected to shift to a lower molecular weight, separating it from the hKGA band. The Northern blots were hybridized with the differential cDNA probes for hGA11–1 and hGAC (Fig. 7). Ethidium bromide staining showed intact ribosomal RNA bands, indicating the absence of any gross nonspecific digestion of RNA. Both 10–1RR and 10–1NX probes detected one 4.8-kb band in the control sample, whereas the same probes detected only 2-kb and 3-kb bands, respectively, in the digested sample. No signal was obtained when this blot was probed with the hKGA-specific probe, 11–1XX (data not shown). This result confirms the existence of an mRNA transcript corresponding to hGAC and also suggests that this is the predominant isoform expressed by TSE cell line. However, the RT-PCR assay (as described in Expression of hGA Isoform in Human Kidney Tissue) established that an amplifiable amount of hKGA mRNA was present in TSE RNA (data not shown).



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Fig. 7. RNase H analysis of hGAC expression in a human breast carcinoma cell line. RNase H digestion and Northern blotting of total RNA from TSE breast carcinoma cells. RNA was incubated with B2 oligonucleotide and RNase H. A control reaction was performed that lacked the oligonucleotide. Reaction products were analyzed by hybridization with 10–1RR and 10–1NX probes corresponding to homologous and nonhomologous regions of hGAC, respectively. EtBr, ethidium bromide.

 
Analysis of the Genomic Origins of hGA mRNA Isoforms
To determine whether the three hGA mRNA isoforms identified originated from a single gene, human genomic DNA was digested with five different restriction enzymes and hybridized with hGA mRNA isoform-specific probes (Fig. 8A). Probe 10–1RR, containing sequences common to hKGA, hGAC, and hGAM, detected single bands in both BamH I-digested and Not I-digested genomic DNA of 14 kb and 16.5 kb, respectively. This suggested that this common region is contained in a single gene locus. In addition, specific probes 10–1NX, 11–1XX, and AD20HS detected the same size bands in these samples. Thus all three probes seemed to hybridize to sequences within the same BamH I and Not I restriction fragments. This result suggests that the three hGA isoforms and four mRNA species identified herein derive from alternative splicing of a primary transcript originating from a common gene.




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Fig. 8. Southern blot analysis of the genomic origins of hGA mRNA isoforms. A: human genomic DNA was digested by 5 separate restriction enzymes and hybridized with probes common to hKGA, hGAC, and hGAM (10–1RR); specific for hKGA (11–1XX); specific for hGAC (10–1NX); and specific for hGAM (AD20HS). B: 5 µg of each glutaminase bacterial artificial chromosome (BAC) clone DNA was digested with EcoR I and hybridized with probes common to hKGA, hGAC, and hGAM (10–1RR); specific for hKGA (11–1XX); specific for hGAC (10–1NX); and corresponding to the 5' end of hGAM and hGAC (AD20EB). Lanes 1–5 refer to BAC clones 21495–21499, respectively. Estimated sizes of the hybridized bands are indicated at right.

 
To confirm this hypothesis, the 10–1RR probe (see Fig. 4) was used to screen a human leukemic cell genomic BAC library (24), and five clones were identified and isolated. Southern analysis of these clones was performed by digestion with EcoR I and hybridization with hGA mRNA isoform-specific probes. As shown in Fig. 8B, probe AD20EB (specific to 5' end of hGAM and hGAC) and probe 10–1RR (common to all isoforms) hybridized to restriction fragments of all five clones, probe 10–1NX (specific to hGAC) hybridized to restriction fragments of four clones (21496–9), and probe 11–1XX (specific to hKGA) hybridized to restriction fragments of three clones (21497–9). In addition, probe AD20HS (specific for the 3' end of hGAM) hybridized to restriction fragments in all five clones and resulted in a pattern of fragment hybridization that was similar to that obtained with probe AD20EB (data not shown). These data suggest that the five BAC clones contain overlapping regions of a common genetic locus that contains sequences of all three hGA mRNA isoforms. Common and 5'-UTR hGA sequences as well as sequences from the 3' end of hGAM are contained in all five clones, sequences from the 3' end of hGAC are contained in four clones, and sequences from the 3' end of hKGA are contained in only three clones. From this analysis it is apparent that the exon(s) corresponding to the 3' end of hGAM lies 5' to the exon(s) corresponding to the 3' end of hGAC, which in turn lies 5' to the exon(s) corresponding to the 3' end of hKGA. This finding further confirms the hypothesis that all three glutaminase isoforms and all five mRNA species examined herein derive from alternative splicing of a primary transcript originating from a common gls gene.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
At the inception of this study, only two isoforms of glutaminase mRNA—KGA and LGA—had been identified. These two isoforms represent the products of separate genes (5). LGA is expressed only in liver, and KGA seemed to represent the only isoform of glutaminase expressed in nonhepatic tissues. Previous cloning and analysis of rat and pig KGA resulted in the conclusion that only two nonhepatic glutaminase mRNA species existed and were produced by alternative polyadenylation of a common pre-mRNA (5). Evidence that other glutaminase mRNA isoforms existed was obtained by Porter and co-workers (18), who detected mRNA species approximately 5.0, 4.5, 3.5, and 2.5 kb in size in RNA isolated from the pig renal cell line, LLC-PK1-F+, using rat and pig KGA cDNA probes. However, these rat and pig KGA cDNA probes detected only the 5.0-kb species and a small amount of the 4.5-kb species in RNA isolated from porcine kidney tissue (18). The data presented here show that three nonhepatic isoforms of human glutaminase—hKGA, hGAC, and hGAM—exist and exhibit different tissue-specific expression patterns. Five human glutaminase mRNA species were identified, including two hKGA species 4.8 kb and 3.5 kb in size, two hGAC species 4.8 kb and 3.5 kb in size, and an hGAM species 2.6 kb in size. Only by obtaining cDNA clones containing divergent regions was the existence of hGAC and hGAM made apparent. The reason, if any, for the coincidental sizes for hKGA and hGAC mRNAs is not known. hGAC does contain a polyadenylation site at nucleotide 3453, which could be utilized to generate the 3.5-kb mRNA species. However, the closest polyadenylation site in hKGA is located at nucleotide 2438 and, therefore, could not be utilized to generate the smaller mRNA corresponding to that isoform. The fact that the 5' 29 bases of both hGA11–1 and dbEST2380672 differ from the sequence of hk03964 suggests there may exist hKGA mRNAs with alternative 5' ends.

Using Southern blotting analysis of human x hamster somatic cell hybrids, the gls gene was previously mapped to human chromosome 2 (13, 15). Human gls was further mapped to the region 2q32–2q34 by in situ hybridization (14). These studies utilized cDNA probes that corresponded to hKGA or rKGA. In the present study, Southern analysis with differential probes indicated that hKGA, hGAC, and hGAM isoforms originate from a single genetic locus contained within 14-kb BamH I and 16.5-kb Not I restriction fragments. In addition, these three hGA mRNA isoforms share appreciable regions of exact sequence homology, suggesting that they contain common exons. Thus it is most likely that these isoforms are produced by alternative splicing of a common pre-mRNA species. This was confirmed by cloning and Southern analysis of the gls BAC clones, which again indicated that exons of the three hGA mRNA isoforms are present in the same genomic locus. However, these BAC clones have not yet been mapped to a chromosomal location. Nonetheless, we propose that human glutaminase represents an example of regulated expression by tissue-specific alternative splicing. This splicing results in the generation of coding sequences with different COOH-terminal amino acid sequences. The implications of these differing COOH termini on the activity and function of these glutaminase enzymes are not yet known.

The 5'-UTRs of hKGA, hGAC, and hGAM contain CAG repeats at the same location but of different lengths (15 repeats in hKGA, 7 repeats in hGAC, and 14 repeats in hGAM). Assuming that these sequences originate from the same exon, these three cDNAs demonstrate a trimorphism in the length of this CAG repeat. CAG repeat-length polymorphisms are associated with several genetic diseases, including myotonic dystrophy and Huntington's (25). Whether the CAG repeat found in the 5'-UTRs of hGA isoforms has an impact on hGA expression is yet to be evaluated. There is an apparent interspecies difference in the location of CAG repeats in GA mRNAs. A CAG repeat is present within the coding sequence of rKGA (nucleotides 329–355), and a BLASTN search of mouse dbEST database identified a cDNA clone similar to rKGA (dbEST2110859, GenBank accession no. AI327299; The WashU-HHMI Mouse EST Project, M. Marra et al., 1996, unpublished), which has a CAG repeat at the same location as the rat clone.

The NH2-terminal amino acid sequence of the rKGA contains a 16-residue mitochondrial signaling sequence that directs the proenzyme to the mitochondria (23, 26, 27). The NH2 terminus of the deduced amino acid sequence of hKGA, hGAC, and hGAM contains a putative mitochondrial signaling sequence that nearly matches that of the rat kidney isoform, suggesting that these human isoforms also encode mitochondrial proteins.

The expression of hGA mRNA isoforms was analyzed in a TSE human breast carcinoma, which was chosen because it exhibits a high rate of Gln consumption (2) and expresses an extraordinarily large amount of glutaminase mRNA and activity (2). In fact, the glutaminase activity measure in these cells (44.8 µmol · mg protein-1 · h-1) is ~4 times higher than that reported in human intestinal epithelial cells(22), 18 times higher than human fibroblasts (21), 19 times higher than rat brain (16), and 12 times higher than rat kidney (3). Moreover, hGAC was found to be the predominant isoform expressed by TSE cells. The fact that these cells exhibit a high glutaminase activity in the absence of detectable hKGA expression strongly suggests that hGAC encodes a functional enzyme. Expression of hGAC by TSE cells should not be interpreted to indicate that this isoform is exclusively associated with neoplastic transformation or with breast tissue, for Northern analysis of several human breast cell lines demonstrated that hGAC expression varied greatly (S. F. Abcouwer, unpublished observation).

In contrast, no data regarding functionality of hGAM can be offered at this time. It is probable that the small peptide encoded by hGAM does not retain glutaminase activity. hGAM encodes only 169 amino acids, 72 of which represent a putative targeting sequence removed during mitochondrial processing. The predicted size of the remaining peptide (<11 kDa) is smaller than any known glutaminase, including the glutaminase subunit of bacterial amidotransferase (33). Because heart and muscle tissue contain relatively low glutaminase activity, it is conceivable that splicing of the primary glutaminase transcript to form hGAM mRNA represents a means to reduce expression of functional glutaminase activity in these tissues. Such a mechanism has been hypothesized to explain the discrepancy between glucokinase mRNA expression and lack of glucokinase activity in the pituitary (11).

By comparison of brain and kidney hybridization signals using probes 11–1RR and 11–1XX, it was estimated that hKGA is approximately six times more abundant than hGAC in kidney (data not shown). Similarly, it was estimated that hGAC is slightly more abundant than hKGA in placenta, lung, cardiac, and skeletal muscle. Expression of these isoforms in normal intestine has not yet been tested. The levels of both hKGA and hGAC mRNA in the HT-29 human colon carcinoma cell line (from which the cDNA library was obtained) were found to be very low (data not shown). Although mRNA quantity is not a direct indicator of protein expression or activity, the results suggest that the expression of the kidney-type glutaminase isoform is indeed dominant in kidney as well as brain. However, it is now apparent that the expression of glutaminase-type C may be considerable in several other tissues. Differential antisera are needed to test this presumption. No information regarding the differential functions of these glutaminase isoforms has yet been obtained, and very little data on the properties of glutaminase enzyme in tissues other than kidney, brain, and liver are available. Therefore, the reason for tissue-specific expression of different glutaminase isoforms remains to be determined.

No analysis of the developmental, hormonal, or environmental regulation of hKGA, hGAC, or hGAM has been presented. In the rat, glutaminase expression is upregulated in kidney in response to acidosis (28). This response has been attributed to an increase in rKGA mRNA stability under acidic conditions that is orchestrated by several AU-rich, pH-responsive instability elements in its 3'-UTR (7). The 3'-UTR of hKGA does not contain any of these elements. The 3'-UTR of hGAC does contain two eight-base sequences (UUUAAAUA) that match the first half of the pH-responsive instability element and two other eight-base sequences (UUAAAAUA) that match the second half of this element (10). Although the results of Laterza and colleagues (10) suggested that an intact 16-base element is needed for full function of this element, the presence of these half-elements raises the possibility that the stability of hGAC mRNA is regulated at the posttranscriptional level by a similar pH-dependent mechanism. However, neither hKGA nor hGAC mRNA levels responded when RPTEC or TSE cells were subjected to acidotic conditions (K. M. Elgadi, unpublished results).

A recent report by Roberg and colleagues (19) claimed that distinct soluble and membrane-bound forms of phosphate-activated glutaminase are present in rat and pig kidney. It is conceivable that these isozymes are the products of KGA and GAC mRNAs. However, reports from the same laboratory, as well as others, also described the presence of distinct soluble and membrane-bound forms of phosphate-activated glutaminase in pig brain (17, 29). If, like human brain tissue, pig brain does not express glutaminase type C, then the origin of these distinct forms of glutaminase enzyme cannot be attributed to the two mRNA isoforms. Instead, soluble and membrane-bound forms of glutaminase may be due to altered mitochondrial processing or represent monomeric and polymeric forms of the same protein (5).

In summary, this report describes a cDNA clone representing the human kidney-type glutaminase isoform, hKGA, and two novel human glutaminase isoforms, hGAC and hGAM. The existence of hGAC mRNA was not suspected because it shares common sizes with the kidney-type mRNA species. This seems to be true for both human and rat isoforms (data for the rat not shown). The existence of hGAM mRNA was not suspected because glutaminase mRNA expression in muscle has not been previously published. Differential probes and a differential RT-PCR assay were used to confirm the existence of the additional isoforms and examine their expression. The RT-PCR assay used represents a particularly convenient method for analysis of hKGA and hGAC mRNA expression. The three nonhepatic glutaminase mRNA isoforms exhibit unique tissue-specific expression patterns. The functionality of one of these novel isoforms, type-C glutaminase, was evidenced by its predominant expression in TSE cells. However, comparison of the enzymatic properties and subcellular localization of the separate isoforms will require further expression studies and the development of isozyme-specific antibodies. It would be of interest to determine whether expression of certain glutaminase isoforms occurs during normal development or is associated with pathological conditions, such as malignant transformation.


    ACKNOWLEDGMENTS
 
The authors thank S. Chandrasekhar for valuable critique of this manuscript and Julianne S. Wattles and Debra Armstrong for editorial assistance.

This work was supported by National Cancer Institute Grant R29-CA-72772 to S. F. Abcouwer and by the Clinical Nutrition Research Center at Harvard University (Pilot Feasibility Grant P30-DK-40561 to S. F. Abcouwer).

The complete nucleotide sequences of hGAC, hGA10–1, hGA11–1, dbEST247733, and hGAM have been deposited in the GenBank database under accession nos. AF158555, AF097492, AF097493, AF097494, and AF097495, respectively.

Address for reprint requests and other correspondence (as of Oct 1, 1999): S. F. Abcouwer, Dept. of Biochemistry and Molecular Biology, The Univ. of New Mexico Health Science Ctr., School of Medicine, Basic Medical Sciences Bldg.-249, Albuquerque, NM 87131-5221 (E-mail: SFAbcouwer{at}salud.unm.edu).


    FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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