Cloning and sequence analysis of a cDNA for a rat liver glutathione S-transferase Yb subunit.

We have isolated a Yb-subunit cDNA clone from a GSH S-transferase (GST) cDNA library made from rat liver polysomal poly(A) RNAs. Sequence analysis of one of these cDNA, pGTR200, revealed an open reading frame of 218 amino acids of Mr = 25,915. The deduced sequence is in agreement with the 19 NH2-terminal residues for GST-A. The sequence of pGTR200 differs from another Yb cDNA, pGTA/C44 by four nucleotides and two amino acids in the coding region, thus revealing sequence microheterogeneity. The cDNA insert in pGTR200 also contains 36 nucleotides in the 5' noncoding region and a complete 3' noncoding region. The Yb subunit cDNA shares very limited homology with those of the Ya or Yc cDNAs, but has relatively higher sequence homology to the placental subunit Yp clone pGP5. The mRNA of pGTR200 is not expressed abundantly in rat hearts and seminal vesicles. Therefore, the GST subunit sequence of pGTR200 probably represents a basic Yb subunit. Genomic DNA hybridization patterns showed a complexity consistent with having a multigene family for Yb subunits. Comparison of the amino acid sequences of the Ya, Yb, Yc, and Yp subunits revealed significant conservation of amino acids (approximately 29%) throughout the coding sequences. These results indicate that the rat GSTs are products of at least four different genes that may constitute a supergene family.     

Subunit composition of rat liver glutathione S-transferases

The plasmid pGTR112 contains partial coding sequences for one of the rat liver glutathione S-transferase subunits. We have used immobilized pGTR112 DNA to select for complementary and homologous liver poly(A)-RNAs under conditions of increasing stringency for hybridization. Each fraction of selected poly(A)-RNAs was assayed by in vitro translation followed by immunoprecipitation. A total of four distinct polypeptides precipitated by antiserum against rat liver glutathione S-transferases were resolved by NaDodSO₄ polyacrylamide gel electrophoresis. They are separated into two pairs according to the sequence homology of their poly(A)-RNAs with the pGTR112 DNA. Purified rat liver glutathione S-transferases can be resolved on gradient NaDodSO₄ polyacrylamide gels into four polypeptides. There should be ten isozymes of different binary combinations from four distinct subunits for the rat liver glutathione S-transferases.     

Cloning and sequence analysis of a cDNA plasmid for one of the rat liver glutathione S-transferase subunits.

We describe the construction and characterization of a cDNA plasmid for one of the rat liver glutathione S-transferase subunits. Poly(A)-RNA isolated from rat livers was enriched for glutathione S-transferase mRNA activity and used as templates to synthesize double stranded cDNA. The double stranded cDNAs were annealed to pBR322 through terminal deoxynucleotidyl transferase generated GC-tails followed by transformation into E. coli. Several candidate clones were selected by colony hybridization using polynucleotide kinase labeled liver and testis poly(A)-RNA probes. These candidate clones were further characterized by hybrid-selected translation of mRNA followed by immunoprecipitation and SDS gel electrophoresis. The positive clone, pGTR112 was mapped with restriction endonuclease analysis and sequenced by the chemical method of Maxam and Gilbert. The largest upen reading frame contains 142 amino acids very rich in Arg and Lys residues. The C-terminal residue phenylalanine of this open reading frame is consistent with what was reported for one of the ligandin subunits by Bhargava et al., (J. Biol. Chem. 253, 4116-4119, 1978). Among the 352 nucleotides covered by both pGTR112 and pGST94 described by Kalinyak and Taylor (J. Biol. Chem. 257, 523-530, 1982), there are only 9 nucleotide differences resulting in four changes of amino acid sequences.     

Characterization of a variant rat glutathione S-transferase by cDNA expression in Escherichia coli

We have isolated a glutathione S-transferase Yb1 subunit cDNA from a lambda gt11 cDNA collection constructed from rat testis poly(A) RNA enriched for glutathione S-transferase mRNA activities. This Yb1 cDNA, designated pGTR201, is identical to our liver Yb1 cDNA clone pGTR200 except for a shorter 5'-untranslated sequence. Active glutathione S-transferase is expressed from this Yb1 cDNA driven by the tac promoter on the plasmid construct pGTR201-KK. The expressed glutathione S-transferase protein begins with the third codon (Met) of the cDNA, and is missing the N-terminal proline of rat liver glutathione S-transferase 3-3. Therefore, our Escherichia coli expressed glutathione S-transferase protein represents a variant form of glutathione S-transferase 3-3 (Yb1Yb1), designated GST 3-3(-1). The expressed Yb1 subunits are assembled into a dimer as purified from sonicated E. coli crude extracts. In the absence of dithiothreitol three active isomers can be resolved by ion-exchange chromatography. The pure protein has an extinction coefficient of 9.21 x 10(4) M-1 cm-1 at 280 nm or E0.1% 280 = 1.78 and a pI at 8.65. It has a substrate specificity pattern similar to that of the authentic glutathione S-transferase 3-3. The GST 3-3(-1) has a KM of 202 microM for reduced GSH and of 36 microM for 1-chloro-2,4-dinitrobenzene. The turnover number for this conjugation reaction is 57 s-1. Results of kinetic studies of this reaction with GST 3-3(-1) are consistent with a sequential substrate binding mechanism. We conclude that the first amino acid proline of glutathione S-transferase 3-3 is not essential for enzyme activities.     

The human liver glutathione S-transferase gene superfamily: expression and chromosome mapping of an Hb subunit cDNA.

We have isolated from a lambda gt10 cDNA library a clone lambda GTH4 which encodes a human liver glutathione S-transferase Hb subunit, designated as subunit 4. Expression of this cDNA in E. coli and subsequent purification and immunoblotting analysis provided a definitive assignment of a structure and function relationship. RNA blot hybridization with human liver poly(A) RNA revealed a single band of approximately 1200 nucleotides, comparable in size to the rat brain Yb3 mRNA. Divergence analysis of amino acid replacement sites in subunit 4 relative to the four rat Yb subunits revealed that it is most closely related to the brain-specific Yb3 subunit. This conclusion is further substantiated by the nucleotide sequence homology between lambda GTH4 and the Yb3 cDNA in their 3' untranslated region. In situ chromosome mapping has located this glutathione S-transferase gene in the region of p31 on chromosome 1. Results from many laboratories, including ours, indicate that the human glutathione S-transferases are encoded by a gene superfamily which is located on at least two different chromosomes.     

Preferential over-expression of the class alpha rat Ya2 glutathione S-transferase subunit in livers bearing aflatoxin-induced pre-neoplastic nodules. Comparison of the primary structures of Ya1 and Ya2 with cloned class alpha glutathione S-transferase cDNA sequences.

Normal rat liver expresses Ya (Mr 25,500), Yc (Mr 27,500) and Yk (Mr 25,000) Class Alpha glutathione S-transferase (GST) subunits. The Ya-type subunit can be resolved into two separate polypeptides, designated Ya1 and Ya2, by reverse-phase h.p.l.c. In rat livers that possess aflatoxin B1-induced pre-neoplastic nodules, a marked increase is observed in the expression of Ya1, Ya2, Yc and Yk; of these subunits, Ya2 exhibited the greatest increase in concentration. The Ya1 and Ya2 subunits isolated from nodule-bearing livers were cleaved with CNBr, and the purified peptides were subjected to automated amino-acid-sequence analysis. Differences in the primary structures of the two Ya GST subunits were found at positions 31, 34, 107 and 117. These data demonstrate that Ya1 and Ya2 are distinct polypeptides and are the products of separate genes. The amino acid sequences obtained from Ya1 and Ya2 were compared with the cloned cDNAs pGTB 38 [Pickett, Telakowski-Hopkins, Ding, Argenbright & Lu (1984) J. Biol. Chem. 259, 4112-4115] and pGTR 261 [Lai, Li, Weiss, Reddy & Tu (1984) J. Biol. Chem. 259, 5182-5188], which encode rat Ya-type subunits. From these comparisons it appears probable that Ya1 represents the GST subunit encoded by pGTR 261, whereas Ya2 represents the subunit encoded by pGTB 38. It is likely that the over-expression of Ya1 and Ya2 in nodule-bearing livers is of major significance in the acquired resistance of nodules to aflatoxin B1, since previous work [Coles, Meyer, Ketterer, Stanton & Garner (1985) Carcinogenesis 6, 693-697] has shown that the Ya-type GST subunit has high activity towards aflatoxin B1 8,9-epoxide.     

Rat glutathione S-transferases supergene family. Characterization of an anionic Yb subunit cDNA clone

High multiplicity of GSH S-transferases (GST) with overlapping substrate specificities may be essential to their multiple roles in xenobiotics metabolism, drug biotransformation, and protection against peroxidative damage. Subunit composition analysis of rat liver GSH S-transferases indicated that heterodimer associations were not random, limiting the generation of GST isozyme multiplicity. We have analyzed a Yb subunit cDNA clone, pGTR187, that may correspond to an anionic Yb subunit sequence. Comparison with other GSH S-transferase cDNA sequences and blot hybridization results indicates that the multiple Yb subunits are encoded by a multigene family. This Yb subunit sequence has very limited homology to Ya and Yc subunit cDNAs, but slightly more sequence homology to the Yp subunit cDNA. More consistent sequence homology is found at the amino acid level with 28% conservation throughout the coding sequences. These results and results published from other laboratories clearly indicate that rat GSH S-transferases are products of at least four different gene families that constitute a supergene family. Conceptually, the supergene family may encode GSH S-transferases of very different structures that are essential to metabolize a multitude of xenobiotics in addition to serving other physiologically important functions.     

Rat liver glutathione S-transferases. Nucleotide sequence analysis of a Yb1 cDNA clone and prediction of the complete amino acid sequence of the Yb1 subunit

We have constructed a nearly full length cDNA clone, pGTA/C44, complementary to the rat liver glutathione S-transferase Yb1 mRNA. The nucleotide sequence of pGTA/C44 has been determined, and the complete amino acid sequence of the Yb1 subunit has been deduced. The cDNA clone contains an open reading frame of 654 nucleotides encoding a polypeptide comprising 218 amino acids with Mr = 25,919. The NH2-terminal sequence deduced from DNA sequence analysis of pGTA/C44 is in agreement with the first 19 amino acids determined for purified glutathione S-transferase A, a Yb1 homodimer, by Frey et al. (Frey, A. B., Friedberg, T., Oesch, F., and Kreibich, G. (1983) J. Biol. Chem. 258, 11321-11325). The DNA sequence of pGTA/C44 shares significant sequence homology with a cDNA clone, pGT55, which is complementary to a mouse liver glutathione S-transferase (Pearson, W. R., Windle, J. J., Morrow, J. F., Benson, A. M., and Talalay, P. (1983) J. Biol. Chem. 258, 2052-2062). We have also determined 37 nucleotides of the 5'-untranslated region and 348 nucleotides of the 3'-untranslated region of the Yb1 mRNA. The Yb1 mRNA and subunit do not share any sequence homology with the rat liver glutathione S-transferase Ya or Yc mRNAs or their corresponding subunits. These data provide the first direct evidence that the Yb1 subunit is derived from a gene or gene family which is distinct from the Ya-Yc gene family.     

Rat glutathione S-transferase. Cloning of double-stranded cDNA and induction of its mRNA

Messenger RNA extracted from the livers of normal, phenobarbital-treated, and trans-stilbene oxide-treated rats was translated in a mRNA-dependent protein-synthesizing system. Immunoprecipitation of the translation products by antibodies against the Ya and Yc subunits of glutathione S-transferase detected two polypeptides of molecular weights 23,500 and 25,000. Subsequently, a clone containing glutathione S-transferase sequences was identified from a rat liver double-stranded cDNA library that had been prepared by homopolymeric tailing and cloning into the Pst I site of pBR322. Confirmation of the identity of the clone was obtained by recloning the 550-bp insert DNA into the phage vector M13 and utilizing the single strand recombinant phage DNA in specific hybrid selection of mRNA followed by translation and immunoprecipitation with antibodies to the Ya and Yc subunits. This recombinant phage, M13GST94, was also utilized in a new technique to synthesize 32P-labeled cDNA specific to the glutathione S-transferase insert DNA that was used subsequently in RNA excess solution hybridization to determine the relative concentration of glutathione S-transferase mRNA. Phenobarbital treatment resulted in a 3.2-fold increase in glutathione S-transferase mRNA over levels found in control rats, while trans-stilbene oxide increased glutathione S-transferase mRNA levels 5.7-fold. The DNA sequence of the clone was determined and utilized to propose a partial amino acid sequence.     

Expression of a cDNA encoding a rat liver glutathione S-transferase Ya subunit in Escherichia coli

A full length cDNA clone, pGTB38 (C. B. Pickett et al. (1984) J. Biol. Chem. 259, 5182-5188), complementary to a rat liver glutathione S-transferase Ya mRNA has been expressed in Escherichia coli. The cDNA insert was isolated from pGTB38 using MaeI endonuclease digestion and was inserted into the expression vector pKK2.7 under the control of the tac promoter. Upon transformation of the expression vector into E. coli, two protein bands with molecular weights lower than the full-length Ya subunit were detected by Western blot analysis in the cell lysate of E. coli. These lower-molecular-weight proteins most likely result from incorrect initiation of translation at internal AUG codons instead of the first AUG codon of the mRNA. In order to eliminate the problem of incorrect initiation, the glutathione S-transferase Ya cDNA was isolated from the expression vector and digested with Bal31 to remove extra nucleotides from the 5' noncoding region. The protein expressed by this expression plasmid, pKK-GTB34, comigrated with the Ya subunit on sodium dodecyl sulfate polyacrylamide gels and was recognized by antibodies against the YaYc heterodimer. The expressed Ya homodimer was purified by S-hexylglutathione affinity and ion-exchange chromatographies. Approximately 50 mg pure protein was obtained from 9 liters of E. coli culture. The expressed Ya homodimer displayed glutathione-conjugating, peroxidase, and isomerase activities, which are identical to those of the native enzyme purified from rat liver cytosol. Protein sequencing indicates that the expressed protein has a serine as the NH2 terminus whereas the NH2 terminus of the glutathione S-transferase Ya homodimer purified from rat liver cytosol is apparently blocked.     

Rat liver glutathione S-transferases. Construction of a cDNA clone complementary to a Yc mRNA and prediction of the complete amino acid sequence of a Yc subunit

Using polysomal immunoselected rat liver glutathione S-transferase mRNAs, we have constructed cDNA clones using DNA polymerase I, RNase H, and Escherichia coli ligase (NAD+)-mediated second strand cDNA synthesis as described by Gubler and Hoffman (Gubler, U., and Hoffman, B. S. (1983) Gene 25, 263-269). Recombinant clone, pGTB42, contained a cDNA insert of 900 base pairs whose 3' end showed specificity for the Yc mRNA in hybrid-select translation experiments. The nucleotide sequence of pGTB42 has been determined, and the complete amino acid sequence of a Yc subunit has been deduced. The cDNA clone contains an open reading frame of 663 nucleotides encoding a polypeptide comprising 221 amino acids with a molecular weight of 25,322. The NH2-terminal sequence deduced from pGTB42 is in agreement with the first 39 amino acids determined for a Ya-Yc heterodimer by conventional protein-sequencing techniques. A comparison of the nucleotide sequence of pGTB42 with the sequence of a Ya clone, pGTB38, described previously by our laboratory (Pickett, C. B., Telakowski-Hopkins, C. A., Ding, G. J.-F., Argenbright, L., and Lu, A.Y.H. (1984) J. Biol. Chem. 259, 5182-5188) reveals a sequence homology of 66% over the same regions of both clones; however, the 5'- and 3'-untranslated regions of the Ya and Yc mRNAs are totally divergent in their sequences. The overall amino acid sequence homology between the Ya and Yc subunits is 68%, however, the NH2-terminal domain is more highly conserved than the middle or carboxyl-terminal domains. Our data suggest that the Ya and Yc subunits of the rat liver glutathione S-transferases are products of two different mRNAs which are derived from two related yet different genes.     

Cloning and the nucleotide sequence of rat glutathione S-transferase P cDNA.

A cDNA library prepared from poly(A)+ RNA of 2-acetylaminofluorene (AAF) induced rat hepatocellular carcinoma was screened by synthetic DNA probes deduced from a partial amino acid sequence of glutathione S-transferase P subunit that had been isolated from the tumor by two-dimensional gel electrophoresis. One of the four clones analyzed contained an mRNA region encoding the total amino acid sequence of this enzyme subunit and the complete 3'-noncoding region. The nucleotide sequence indicates that this enzyme subunit has 209 amino acids (calculated Mr=23,307) distinct from other glutathione S-transferase subunits such as Ya and Yc. Comparison of the amino acid sequences between these proteins indicates that glutathione S-transferase P subunit gene has been evolved from the ancestral gene at an earlier stage than the separation of Ya and Yc and that there are at least three domains having a considerable homology with each other in these enzymes. The very large increase of this mRNA in chemically induced hepatocellular carcinoma suggests a characteristic derepression of this gene during hepatocarcinogenesis.     

Rat liver glutathione S-transferases. Complete nucleotide sequence of a glutathione S-transferase mRNA and the regulation of the Ya, Yb, and Yc mRNAs by 3-methylcholanthrene and phenobarbital

With the use of cDNA probes reverse transcribed from purified glutathione S-transferase mRNA templates, four cDNA clones complementary to transferase mRNAs have been identified and characterized. Two clones, pGTB38 and pGTB34, have cDNA inserts of approximately 950 and 900 base pairs, respectively, and hybridize to a mRNA(s) whose size is approximately 980 nucleotides. In hybrid-select translation experiments, pGTB38 and pGTB34 select mRNAs specific for the Ya and Yc subunits of rat liver glutathione S-transferases. Clone pGTB33, which harbors a truncated cDNA insert, hybrid-selects only the Ya mRNA. All of the clones, pGTB38, pGTB34, and pGTB33, hybrid-select another mRNA which is specific for a polypeptide with an electrophoretic mobility slightly greater than the Ya subunit. The entire nucleotide sequence of the full length clone, pGTB38, has been determined and the complete amino acid sequence of the corresponding polypeptide has been deduced. The mRNA codes for a protein comprising 222 amino acids with Mr = 25,547. We have also identified a cDNA clone complementary to a Yb mRNA of the rat liver glutathione S-transferases. This clone, pGTA/C36, hybrid-selects only Yb mRNA(s) and hybridizes to a mRNA(s) whose size is approximately 1200 nucleotides. Although the Ya, Yb, and Yc mRNAs are elevated coordinately by phenobarbital and 3-methylcholanthrene, the Ya-Yc mRNAs are induced to a much greater extent compared to the Yb mRNA(s). These data suggest that the mRNAs for each transferase isozyme are regulated independently.     

Isolation and characterization of a DNA sequence complementary to rat liver glutathione S-transferase B mRNA

Total rat liver poly(A+)-RNA has been isolated from phenobarbital-treated rats and fractionated on sucrose gradients to enrich for glutathione S-transferase B mRNA. Poly(A+)-RNA fractions were assayed for glutathione S-transferase B mRNA activity by in vitro translation and those fractions enriched in glutathione S-transferase B mRNA were used as a template for cDNA synthesis. The cDNA was cloned into the PstI site of pBR322 by G-C tailing. Bacterial clones harboring inserts complementary to glutathione S-transferase mRNA were identified by colony hybridization using a [32P]cDNA probe reverse transcribed from poly(A+)-RNA enriched significantly in glutathione S-transferase B mRNA and by hybrid-select translation. Two recombinant clones, pGTB6 and pGTB15 hybrid-selected the mRNAs specific for the Ya and Yc subunits, indicating these two mRNAs share significant sequence homology. Radiolabeled pGTB6 was utilized in RNA gel-blot experiments to determine that the size of glutathione S-transferase B mRNA is 980 nucleotides and the degree of induction of the mRNA in response to 3-methylcholanthrene administration is threefold.     

Expression of glutathione S-transferases in rat brains

The tissue-specific expression of glutathione S-transferases (GSTs) in rat brains has been studied by protein purification, in vitro translation of brain poly(A) RNAs, and RNA blot hybridization with cDNA clones of the Ya, Yb, and Yc subunit of rat liver GSTs. Four classes of GST subunits are expressed in rat brains at Mr 28,000 (Yc), Mr 27,000 (Yb), Mr 26,300, and Mr 25,000. The Mr 26,3000 species, or Y beta, has an electrophoretic mobility between that of Ya and Yb, similar to the liver Yn subunit(s) reported by Hayes (Hayes, J. D. (1984) Biochem. J. 224, 839-852). RNA blot hybridization of brain poly(A) RNAs with a liver Yb cDNA probe revealed two RNA species of approximately 1300 and approximately 1100 nucleotides. The band at approximately 1300 nucleotides was absent in liver poly(A) RNAs. The Mr 25,000 species, or Y delta, can be immunoprecipitated by antisera against rat heart and rat testis GSTs, but not by antiserum against rat liver GSTs. Therefore, the Y delta subunit may be related to the "Mr 22,000" subunit reported by Tu et al. (Tu, C.-P.D., Weiss, M.J., Li, N., and Reddy, C. C. (1983) J. Biol. Chem. 258, 4659-4662). The abundant liver GST subunits, Ya, are not expressed in rat brains as demonstrated by electrophoresis of purified brain GSTs and a lack of isomerase activity toward the Ya-specific substrate, delta 5-androstene-3,17-dione. This is apparently because of the absence of Ya mRNA expression prior to RNA processing. The data on the preferential expression of Yc subunits in rat brains, together with the differential phenobarbital inducibility of the Ya subunit(s) in rat liver reported by Pickett et al. (Pickett, C. B., Donohue, A. M., Lu, A. Y. H., and Hales, B. F. (1982) Arch. Biochem. Biophys. 215, 539-543), suggest that the Ya and Yc genes for rat GSTs are two functionally distinct gene families even though they share 68% DNA sequence homology. The expression of multiple GSTs in rat brains suggests that GSTs may be involved in physiological processes other than xenobiotics metabolism.     

Cloning of the C alpha catalytic subunit of the bovine cAMP-dependent protein kinase.

The bovine C alpha type catalytic subunit of the cAMP-dependent protein kinase was cloned. A partial cDNA was isolated from a bovine heart cDNA library. This clone contained 120 bp of the coding sequence and the entire 3' untranslated region of 1431 bp. The complete coding region was cloned by PCR amplification from total bovine heart and skeletal muscle RNA. The sequence of the 3' oligonucleotide was taken from the partial cDNA clone whereas the 5' oligonucleotide was chosen by comparison of sequences of published C alpha subunits from other species. In the deduced amino acid sequence there is one deviation from the published bovine C alpha protein sequence, aspartic acid 286 is exchanged by an asparagine. The C alpha mRNA was found to be expressed differentially in various bovine tissues.     

Molecular cloning, cDNA structure, and regulation of the regulatory subunit of type II cAMP-dependent protein kinase from rat ovarian granulosa cells

One isoform of the regulatory subunit of type II cAMP-dependent protein kinase (R-II51; Mr = 51,000) and its electrophoretic variants (R-II51.5 and R-II52; Mr = 51,500 and 52,000, respectively) are selectively induced by estradiol and follicle-stimulating hormone (cAMP) in rat ovarian granulosa cells. To ascertain the amino acid sequence of R-II51 and to gain insight into the molecular events regulating the intracellular content of ovarian follicular R-II51, we constructed a lambda gt11 cDNA expression library from poly(A)+ RNA of hormone-primed rat granulosa cells. A 1.5-kilobase (kb) cDNA insert, isolated from a plaque-purified R-II antibody positive bacteriophage clone, selectively bound R-II51 mRNA as demonstrated by analysis of the hybrid-selected translation product. Restriction maps and sequence analyses of the 1.5-kb cDNA insert and of the 1.8- and 2.2-kb cDNA inserts from two additional clones showed overlapping sequences which span a region of 3065 nucleotides in size. The 1.5- and 1.8-kb cDNA inserts each contained poly(A) addition signals (1508 and 1761 nucleotides, respectively), terminal poly(A) sequences, and the entire coding region for R-II51 (1204 nucleotides) except for a small number of nucleotides at the 5' end. The 2.2-kb cDNA insert contained 394 nucleotides of the coding region a long 3' untranslated region and two more poly(A) addition signals (3041 and 3059 nucleotides). An amino acid microsequence surrounding the autophosphorylation site of pure rat ovarian R-II51 agreed with the amino acid sequence deduced from the nucleotide sequence of the cDNA. Northern blot analyses demonstrated two major mRNA species (1.8 and 3.2 kb in size) in hormone-primed rat ovaries which were approximately 10- and 50-fold greater than the R-II mRNA content in rat brain and rat heart, respectively. Southern blot analysis of rat liver DNA indicated that a single gene codes for R-II51 mRNA. Structural differences among rat ovarian R-II51, rat heart R-II54, and the known amino acid sequences of bovine R-II and R-I subunits also indicate that the rat ovarian R-II51 subunit is the product of a distinct gene.     

Identification of glutathione S-transferase Yb1 mRNA as the androgen-repressed mRNA by cDNA cloning and sequence analysis

Androgens, while stimulating the growth of the rat ventral prostate, can also repress the levels of a limited number of mRNAs. The cDNA for one of the androgen-repressed mRNAs has been identified by nucleotide sequence analysis as coding for the glutathione S-transferase Yb1 subunit. The prostate cDNA is 1071 nucleotides long, and only 2 or 4 bases of this sequence do not match the two published sequences of the cDNA for the Yb1 subunit of rat liver glutathione S-transferase. The amino acids in the protein encoded by the prostate cDNA matched completely with that for one of the liver cDNAs and differ with the other cDNA only in two of 218 amino acids. The identification of the androgen-repressed mRNA as a glutathione S-transferase subunit may indicate that some of the cellular actions of the enzyme may be important in the control of androgen-dependent growth of the prostate. Since Yb forms of the transferases have been colocalized with uridylic acid-rich small nuclear RNAs at interchromatinic regions of the cell nucleus, autoregulation of prostate growth by androgens may be carried out through the modulation of RNA production or processing in this target organ.