Gene expression of rat glutathione S-transferases. Evidence for gene conversion in the evolution of the Yb multigene family

We have characterized a cDNA with complete coding sequence for the rat liver glutathione S-transferase subunit 4 (Yb2) isolated from a constructed lambda gt10 cDNA library. Functional expression of the cDNA sequence has resulted in the purification to homogeneity of an enzymatically active anionic glutathione S-transferase. In addition to three previously described Yb-type subunits (Yb1, Yb2, Yb3), we now report characterization of a fourth Yb subunit sequence in the form of a genomic DNA clone lambda GTR15-2. The Yb4 gene has no apparent defect, and the deduced Yb4 polypeptide sequence differs from the other three Ybs by 40 to 53 amino acids. The Yb4 gene organization is similar to that of the Yb2 gene in having a minimum of eight exons. Three out of the seven introns between the two genes are conserved to the extent of more than 88% nucleotide identity. We propose that gene conversion may have played a role in the evolution of these Yb genes.     

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.     

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.     

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.     

Expression of Yb1 glutathione S-transferase using a baculovirus expression system

A full-length cDNA clone was isolated for rat liver Yb1 glutathione S-transferase (EC 2.5.1.18). The coding sequence of Yb1 cDNA was inserted into a baculovirus vector for infection of Spodoptera frugiperda (SF9) cells. The enzymatically active recombinant Yb1 glutathione S-transferase protein has a native molecular weight of 42,000 daltons (by molecular sieve chromatography), a subunit molecular weight of 26,500 daltons (by SDS-polyacrylamide gel electrophoresis), a pI of 8.4 and an extinction coefficient E1%280 of 5.6 +/- 0.4.     

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.     

Selective induction of glutathione S-transferase D in rat testis by phenobarbital

Testis cytosol is shown to contain the Yb2Yb2 -homodimer glutathione S-transferase D in addition to the previously described glutathione S-transferases A ( Yb1Yb1 ) and C ( Yb1Yb2 ). Treatment of rats with phenobarbital induces the level of glutathione S-transferase D in testis with no increase in the activities of glutathione S-transferases A and C. This result indicates a specific induction of the Yb2 subunit in testis, in contrast with the situation in rat liver, where phenobarbital specifically induces the Yb1 subunit.     

水稻中一个谷胱甘肽转移酶基因的克隆、表达和酶活性分析

OsGSTL1是位于水稻3号染色体上的一个λ类谷胱甘肽转移酶基因,由8个内含子和9个外显子组成,编码一个由243个氨基酸组成的多肽链。将OsGSTL1克隆到酵母表达载体pYTV上,转化大肠杆菌,然后再转化酵母菌PEP4。Western印迹分析表明外源OsGSTL1基因在转基因酵母中表达,分析半乳糖诱导表达的酵母粗提液的谷胱甘肽转移酶活性表明:转基因酵母的谷胱甘肽转移酶较非转基因酵母和未诱导的酵母高,说明OsGSTL1在转基因酵母中受半乳糖的诱导表达,具有谷胱甘肽转移酶活性。     

Nucleotide sequence of the gene for the b subunit of human factor XIII

Factor XIII (Mr 320,000) is a blood coagulation factor that stabilizes and strengthens the fibrin clot. It circulates in blood as a tetramer composed of two a subunits (Mr 75,000 each) and two b subunits (Mr 80,000 each). The b subunit consists of 641 amino acids and includes 10 tandem repeats of 60 amino acids known as GP-I structures, short consensus repeats (SCR), or sushi domains. In the present study, the human gene for the b subunit has been isolated from three different genomic libraries prepared in lambda phage. Fifteen independent phage with inserts coding for the entire gene were isolated and characterized by restriction mapping, Southern blotting, and DNA sequencing. The gene was found to be 28 kilobases in length and consisted of 12 exons (I-XII) separated by 11 intervening sequences. The leader sequence was encoded by exon I, while the carbonyl-terminal region of the protein was encoded by exon XII. Exons II-XI each coded for a single sushi domain, suggesting that the gene evolved through exon shuffling and duplication. The 12 exons in the gene ranged in size from 64 to 222 base pairs, while the introns ranged in size from 87 to 9970 nucleotides and made up 92% of the gene. The introns contained four Alu repetitive sequences, one each in introns A, E, I, and J. A fifth Alu repeat was present in the flanking 3' end of the gene. Two partial KpnI repeats were also found in the introns, including one in intron I and one in intron J. The KpnI repeat in intron J was 89% homologous to a sequence of approximately 2200 nucleotides flanking the gene coding for human beta globin and approximately 3800 nucleotides from the L1 insertion present in the gene for human factor VIII. Intron H also contained an "O" family repeat, while two potential regions for Z-DNA were identified within introns G and J. One nucleotide change was found in the coding region of the gene when its sequence was compared to that of the cDNA. This difference, however, did not result in a change in the amino acid sequence of the protein.     

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.     

The human Hb (mu) class glutathione S-transferases are encoded by a dispersed gene family

The human glutathione S-transferases are products of a gene superfamily which consists of at least four gene families. The various glutathione S-transferase genes are located on different human chromosomes, and new gene(s) are still being added to the gene superfamily. We have characterized a cDNA in pGTH4 encoding human glutathione S-transferase subunit 4 (GST mu) and mapped its gene (or a homologous family member) on chromosome 1 at p31 by in situ hybridization. Genomic Southern analysis with the 3' noncoding region of the cDNA revealed at least four human DNA fragments with highly homologous sequences. Using a panel of DNAs from mouse-human somatic cell hybrids in genomic DNA hybridization we show that the Hb (or B) genes of human glutathione S-transferases are on three separate chromosomes: 1, 6, and 13. Therefore, the glutathione S-transferase B gene family, which encodes the Hb (mu) class subunits, is a dispersed gene family. The GST mu (psi) gene, whose expression is polymorphic in the human population, is probably located on chromosome 13. We propose that the GST mu (psi) gene was created by a transposition or recombination event during evolution. The null phenotype may have resulted from a lack of DNA transposition just as much as from the deletion of an inserted gene.     

The Euglena gracilis chloroplast ribulose-1,5-bisphosphate carboxylase gene. I. Complete DNA sequence and analysis of the nine intervening sequences

The nucleotide sequence of 6225 base pairs (bp) of Euglena gracilis chloroplast DNA including the complete DNA sequence of the chloroplast-encoded ribulose-1,5-bisphosphate carboxylase large subunit gene along with the flanking DNA sequences is presented. The gene is greater than 5.5 kilobase pairs in length and is organized as 10 exons coding for 475 amino acids, separated by 9 introns. The exons range in size from 45 to 438 bp, while the introns range in size from 382 to 568 bp. The introns have highly conserved boundary sequences with the consensus, 5'-N GTGTGGATTT...(intron)...TTAATTTTAT N-3'. The introns are 82-85 mol% AT, with a pronounced T greater than A greater than G greater than C base bias in the RNA-like strand. They do not appear to encode any polypeptides. In addition, the introns have a conserved sequence 30-50 bp from their 3'-ends with the consensus, 5'-TACAGTTTGAAAATGA-3'. The 5'-TACA sequence bears some homology to the 5'-end of the TACTAACA sequence found in a similar location in yeast nuclear mRNA introns. The conserved sequences of the Euglena rbcL introns may be indicative of a splicing mechanism similar to that of eucaryotic nuclear mRNA introns and group II mitochondrial introns.     

Structure and evolution of the bovine prothrombin gene

The cloned bovine prothrombin gene has been characterized by partial DNA sequence analysis, including the 5' and 3' flanking sequences and all the intron-exon junctions. The gene is approximately 15.4 x 10(3) base-pairs in length and comprises 14 exons interrupted by 13 introns. The exons coding for the prepro-leader peptide and the gamma-carboxyglutamic acid-containing region are similar in organization to the corresponding exons in the factor IX and protein C genes. This region has probably evolved as a result of recent gene duplication and exon shuffling events. The exons coding for the kringles and the serine protease region of the prothrombin gene are different in organization from the homologous regions in other genes, suggesting that introns have been inserted into these regions after the initial gene duplication events.