Heat Transport in Herbertsmithite: Can a Quantum Spin Liquid Survive Disorder?

One favorable situation for spins to enter the long-sought quantum spin liquid (QSL) state is when they sit on a kagome lattice. No consensus has been reached in theory regarding the true ground state of this promising platform. The experimental efforts, relying mostly on one archetypal material ZnCu_{3}(OH)_{6}Cl_{2}, have also led to diverse possibilities. Apart from subtle interactions in the Hamiltonian, there is the additional degree of complexity associated with disorder in the real material ZnCu_{3}(OH)_{6}Cl_{2} that haunts most experimental probes. Here we resort to heat transport measurement, a cleaner probe in which instead of contributing directly, the disorder only impacts the signal from the kagome spins. For ZnCu_{3}(OH)_{6}Cl_{2}, we observed no contribution by any spin excitation nor obvious field-induced change to the thermal conductivity. These results impose strong constraints on various scenarios about the ground state of this kagome compound: while certain quantum paramagnetic states other than a QSL may serve as natural candidates, a QSL state, gapless or gapped, must be dramatically modified by the disorder so that the kagome spin excitations are localized.

Rat glutathione S-transferase M4-4: an isoenzyme with unique structural features including a redox-reactive cysteine-115 residue that forms mixed disulphides with glutathione.

Although the existence of the rat glutathione S-transferase (GST) M4 (rGSTM4) gene has been known for some time, the corresponding protein has not as yet been purified from tissue. A recombinant rGSTM4-4 was thus expressed in Escherichia coli from a chemically synthesized rGSTM4 gene. The catalytic efficiency (k(cat)/K(m)) of rGSTM4-4 for the 1-chloro-2,4-dinitrobenzene (CDNB) conjugation reaction was 50-180-fold less than that of the well-characterized homologous rGSTM1-1, and the pH optimum for the same reaction was 8.5 for rGSTM4-4 as opposed to 6.5 for rGSTM1-1. Molecular-modelling studies predict that key substitutions in the helix alpha4 region of rGSTM4-4 account for this pK(a) difference. A notable structural feature of rGSTM4-4 is the Cys-115 residue in place of the Tyr-115 of other Mu-class GSTs. The thiol group of Cys-115 is redox-reactive and readily forms a mixed disulphide even with GSH; the S-glutathiolated form of the enzyme is catalytically active. A mutated rGSTM4-4 (C115Y) had 6-10-fold greater catalytic efficiency than the wild-type rGSTM4-4. Trp-45, a conserved residue among Mu-class GSTs, is essential in rGSTM4-4 for both enzyme activity and binding to glutathione affinity matrices. Antibodies directed against either the unique C-terminal undecapeptide or tridecapeptide of rGSTM4 reacted with rat and mouse liver GSTs to reveal an orthologous mouse GSTM4-4 present at low basal levels but which is inducible in mouse liver. This subclass of rodent Mu GSTs with redox-active Cys-115 residues could have specialized physiological functions in response to oxidative stress.

Diagnosis of congenital hypothyroidism from human anagen scalp hair by infrared microspectroscopy.

A highly efficient Fourier transform infrared (FT-IR) microscopy was used to determine the biophysical structure of anagen scalp hair roots of neonates suffering from congenital hypothyroidism (CH) due to ectopic thyroid. The present results indicate that the lower composition near 1,053 cm(-1) (also assigned to the aromatic iodide stretching band) in the infrared (IR) spectra of the hair roots for CH patients was directly associated with the lower serum level of T4 and fT4, and the elevated TSH levels determined by RIA method. This strongly implies the lower evidence of the aromatic iodide stretching band in the IR spectra of hair roots. These findings suggest that FT-IR microscopy has the potential to become a good diagnostic tool and that hair can be useful as a genetic marker.

Differentiation of hair growth cycle from scalp hair roots for the diagnosis of glucose-6-phosphate dehydrogenase deficiency in neonates.

Hair analysis can be used as a screening tool in the diagnosis of genetic diseases. The scalp hair roots of 67 normal neonates and 39 neonates with glucose-6-phosphate dehydrogenase (G6PD) deficiency were analysed using Fourier transform infrared (FT-IR) microspectroscopy to differentiate the stages of the hair growth cycle and to diagnose the genetic disorder on the basis of spectral differences. We have demonstrated that FT-IR microspectroscopy is a rapid and effective noninvasive diagnostic method to differentiate scalp hair roots of normal neonates into the anagen, catagen or telogen phases of the hair growth cycle, using IR spectral differences within the 3000-2800 cm(-1) region and the IR peak area ratio of 2854 cm(-1)/2873 cm(-1) or 1084 cm(-1)/amide II band (p<0.001). Moreover, G6PD-deficient neonates could be accurately diagnosed from telogen phase hair roots owing to significant differences in IR peak area ratios of 2854 cm(-1)/2873(-1) or 1084 cm(-1)/amide II band compared to normal values in healthy neonates. The result suggests that the application of FT-IR microspectroscopy may be capable not only of differentiating the hair growth cycle into anagen, categen or telogen phases but also of detecting G6PD deficiency. Hair root analysis promises to be a useful complement to serum and urine analysis in the diagnosis of genetic diseases.

Heterologous expression of rat liver microsomal glutathione transferase in simian COS cells and Escherichia coli.

The cDNA coding for rat liver microsomal glutathione transferase was subcloned into the mammalian expression vector pCMV-5 and the construct was transfected into, and transiently expressed in, simian COS cells. This resulted in high expression (0.7% of the microsomal protein). The activity towards 1-chloro-2,4-dinitrobenzene in microsomes was 15-30 nmol/min per mg, which increased upon N-ethylmaleimide treatment to 60-200 nmol/min per mg. Control and antisense-vector-treated cells displayed very low activity (3-6 nmol/min per mg). A DNA fragment coding for rat microsomal glutathione transferase was generated by PCR, cloned into the bacterial expression vector pSP19T7LT and transformed into Escherichia coli strain BL21 (DE3) (which contained the plasmid pLys SL). Isopropyl beta-D-thiogalactopyranoside (IPTG; 1 mM) induced the expression of significant amounts of enzymically active protein (4 mg/l of culture as measured by Western blots). The recombinant protein was purified and characterized and found to be indistinguishable from the rat liver enzyme with regard to enzymic activity, molecular mass and N-terminal amino acid sequence. Human liver cDNA was used to obtain the coding region of human microsomal glutathione transferase by PCR. This PCR product was cloned into pSP19T7LT, which, upon induction with IPTG, yielded significant amounts (9 mg/l of culture) of active enzyme in BL21 (DE3) cells. Thus, for the first time, it is now possible to express both human and rat microsomal glutathione transferase in an enzymically active form in Escherichia coli.

Glutathione S-transferases in tracheobronchial epithelium.

The purpose of this study is to characterize glutathione S-transferase (GST) gene expression in airway epithelium both in vivo and in vitro. Immunohistochemical staining of nonhuman primate lungs of well-controlled healthy animals reveals the presence of alpha- and pi-class GST isoenzymes in ciliated bronchial epithelium. The stain of mu-GST antibody is either very low or absent in some of these monkey lungs. We observed that primary tracheobronchial epithelial (TBE) cells isolated from human and monkey pulmonary tissues maintain a relatively high level of GST enzymatic activity in culture, compared with various immortalized human TBE cell lines and other nonpulmonary cell lines. Northern blot analysis demonstrated the presence of mu-, pi-, and microsomal-GST messages but not the alpha-class message in cultures of primary TBE cells as well as in various human TBE cell lines. The expression of mu- and pi-class GST genes can be further regulated in culture by various environmental factors; however, most of these regulating factors are associated with TBE cell differentiation in culture. For instance, vitamin A treatment, which was shown to enhance mucous cell differentiation in vitro, stimulated the message levels of mu- and pi-class GST. Furthermore, plating cells on collagen gel substrata, which also enhanced mucous cell differentiation in culture, instead of plastic culture surface, enhanced total GST enzymatic activity by eightfold, and this enhancement is related to an increase in the expression of the pi-class GST gene. These results demonstrated that GST genes are differentially expressed and regulated by various environmental factors in primary TBE cells and various cell lines, and the regulation is correlated to the mucous cell differentiation in culture.

Pentobarbital-induced changes in Drosophila glutathione S-transferase D21 mRNA stability.

The Drosophila glutathione S-transferase (gstD) genes are a family of divergently transcribed, intronless genes and pseudogenes. Under control conditions, the steady-state level of gstD1 mRNA is 20-fold higher than that of the gstD21 mRNA despite a lower transcription rate of the gstD1 gene. The GST D1 protein level is four times as abundant as the GST D21 protein. The gstD1 and gstD21 genes responded rapidly to pentobarbital (PB) as changes in mRNA levels were detectable within 30 min of treatment. Maximal induction of gstD1 and gstD21 resulted in 3-fold and 20-fold elevation of their respective mRNA levels. The major mechanism for the increase in gstD1 mRNAs appears to be transcriptional activation. The 2-fold increase in the rate of gstD21 transcription, however, cannot fully account for the 20-fold increase in the steady-state level of gstD21 mRNA. Therefore, post-transcriptional mechanism(s) should also be responsible for the increase of gstD21 mRNA by PB. Because the gstD21 mRNA is relatively unstable under control conditions, induction of the intronless gstD21 mRNA by PB occurs mainly at the level of enhanced mRNA stability. The GST D1 protein level in adult Drosophila was increased approximately 2-fold after PB treatment, whereas the GST D21 level remained relatively the same. Thus, an increase in gstD21 mRNA stability by PB treatment is probably coupled to a regulatory effect at the translational level.

Drosophila glutathione S-transferase D27: functional analysis of two consecutive tyrosines near the N-terminus.

The Drosophila glutathione S-transferase D27 (GST D27) has been purified and characterized after direct expression of the intronless gstD27 gene in E. coli. The GST D27 has both conjugation activity against the common substrate 1-chloro-2,4-dinitrobenzene and peroxidase activity against cumene hydroperoxide. Its pH optimum is 8.5 in 0.125 M bis-tris propane buffer at 22 degrees C. It is more thermal labile than the human GST121. The GST D27 has two tyrosines at positions 3 and 4. Both of them appear to be important but neither of them is essential for the enzyme activity. Thus, other residues may also participate in catalysis. The two tyrosines of GST D27 could also be important in binding to GSH or S-hexyl GSH. Results from in vitro biochemical analyses were confirmed by the in vivo activity-based CDNB growth inhibition analyses. Our results clearly indicate that the Drosophila GST D isozymes are different from any of the known mammalian GSTs.

A molecular genetic approach for the identification of essential residues in human glutathione S-transferase function in Escherichia coli.

The common substrate for glutathione S-transferases (GSTs), 1-chloro-2,4-dinitrobenzene (CDNB), is an inhibitor of Escherichia coli growth. This growth inhibition by CDNB is enhanced when E. coli expresses a functional GST. Cells under growth inhibition have reduced intracellular GSH levels and form filaments when they resume growth. Based on this differential growth inhibition by CDNB we have developed a simple procedure to select for null-mutants of a human GST in E. coli. Null mutations in the human GST gene from hydroxylamine mutagenesis or oligonucleotide-directed mutagenesis can be selected for on agar plates containing CDNB after transformation. The molecular nature of each mutation can be identified by DNA sequence analysis of the mutant GST gene. We have identified three essential amino acid residues in an alpha class human GST at Glu31, Glu96, and Gly97. Single substitution at each of these residues, E31K, E96K, G97D, resulted in mutant GST proteins with loss of CDNB conjugation activity and failure in binding to the S-hexyl GSH affinity matrix. In contrast, a mutant GST (Y8F) resulting from substitution of the conserved tyrosine near the N terminus has much reduced CDNB conjugation activity but was still capable of binding to the S-hexyl GSH-agarose. Additional mutant GSTs with substitutions at position 96 (E96F, E96Y) and 97 (G97P, G97T, G97S) resulted in changes in both Km and kcat to different extents. The in vitro CDNB conjugation activity of the purified mutant enzymes correlate negatively with the plating efficiencies of strains encoding them in the presence of CDNB. Based on the x-ray structure model of human GST 1-1, two of these residues are involved in salt bridges (Arg19-Glu31, Arg68-Glu96) and the third Gly97 is in the middle of the helix alpha 4. Our results provide evidence in vivo that Tyr8, Gly97, and the two salt bridges are important for GST structure-function. This molecular genetic approach for the identification of essential amino acids in GSTs should be applicable to any GSTs with CDNB conjugation activity. It should also complement the x-ray crystallographic approach in understanding the structure and function of GSTs.

Biochemical characterization of Drosophila glutathione S-transferases D1 and D21.

The genomic DNA for the two Drosophila genes, gstD1 and gstD21, were engineered for expression in Escherichia coli by polymerase chain reaction using a pair of specially designed primers. This newly designed expression system produced consistently high yields of the recombinant glutathione S-transferases (GSTs), which were purified to electrophoretic homogeneity by S-hexyl-GSH affinity chromatography. Consistent with their differences in size, GST D1 and GST D21 displayed different mobilities on SDS-polyacrylamide gel electrophoresis. Circular dichroism spectrometry revealed some differences in the protein secondary structural organization between the two GST D isozymes. Polyclonal antibodies against GST D1 and GST D21 revealed that they are immunologically distinct from each other. The GST D1 antiserum cross-reacted weakly with GST D21, but the GST D21 antiserum had no detectable cross-reactivity with GST D1. The amino acid sequences of GST D1 and GST D21 have 70% identity. GST D1 is active toward CDNB with 17% of the catalytic efficiency of the human alpha GST121, whereas CDNB is a poor substrate for GST D21. Both GST D1 and GST D21 have similar levels of GSH peroxidase activity against cumene hydroperoxide. Another major difference in substrate specificities between GST D1 and GST D21 is in the activity of 1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane (DDT) dehydrochlorinase, which exists only in the GST D1 isozyme. This is the first definitive demonstration that DDT dehydrochlorinase activity is an intrinsic property of a Drosophila GST. Our results suggest that GST D1 may play a role in DDT metabolism in Drosophila.

A surface mutant (G82R) of a human alpha-glutathione S-transferase shows decreased thermal stability and a new mode of molecular association in the crystal.

A chimeric enzyme (GST121) of the human alpha-glutathione S-transferases GST1-1 and GST2-2, which has improved catalytic efficiency and thermostability from its wild-type parent proteins, has been crystallized in a space group that is isomorphous with that reported for crystals of GST1-1. However, a single-site (G82R) mutant of GST121, which exhibits a significant reduction both in vitro and in vivo in protein thermostability, forms crystals that are not isomorphous with GST1-1. The mutant protein crystallizes in space group P2(1)2(1)2(1), with cell dimensions a = 49.5, b = 92.9, c = 115.9 A, and one dimer per asymmetric unit. Preliminary crystallographic results show that a mutation of the surface residue Gly 82 from a neutral to a charged residue causes new salt bridges to be formed among the GST dimers, suggesting that the G82R mutant might aggregate more readily than does GST121 in solution, resulting in a change of its solution properties.

Implementation of stereotactic focal radiotherapy for intracranial arteriovenous malformations using a linear accelerator.

A system of stereotactic focal radiotherapy using a linear accelerator has been developed in cooperation with a neurosurgeon. The treatment is delivered using a carefully calibrated 10 MV machine and the Cosman-Roberts-Wells (CRW) system. The precision of the method as well as its quality assurance is described. Eight patients with intracranial arteriovenous malformations (AVM) received irradiation from August 1990 to November 1991. The prescribed dose at the periphery of the AVM was 8 Gy per session, with six patients receiving two sessions and two patients receiving one session. The field size, encompassing the 90% isodose, ranged from 20 mm to 35 mm. In four patients, follow-up angiography was performed one year after the full course of therapy; total obliteration of the AVM was noted in three (75%) with a partial response in the other. In the other four patients, follow-up angiography was not performed; one patient, who had only one session of irradiation, experienced rebleeding six months later and died, and the other three patients had no further episodes of bleeding during their follow-up of 28, 18 and 14 months, respectively. Linear accelerator-based stereotactic focal radiotherapy can attain a precisely defined and reproducible dose distribution. The effects of this treatment may take one to two years to develop. Our preliminary study suggests that it is an effective alternative treatment for surgically inaccessible lesions. Patients with a small cavernous sinus dural AVM appear to have a better and more rapid response.

The glutathione S-transferase D genes. A divergently organized, intronless gene family in Drosophila melanogaster.

We have characterized a cluster of glutathione S-transferase genes located at 87B on the Drosophila polytene chromosome near the heat shock genes, hsp70. These genes, designated gst Ds in the glutathione S-transferase gene superfamily, are closely linked within a approximately 60-kilobase DNA segment. The gene family has a minimum of eight intronless genes organized in divergent orientations. Two of the genes are probably GST pseudogenes in that their open reading frames are shorter than functional GSTs, and no RNAs from them have been detected thus far. The amino acid sequence identity among the functional genes ranges from 53 to 75% in pairwise comparisons. The intergenic regions are much more AT rich (63-73%) than the coding regions (41-52%), consistent with being promoter/regulatory sequences in Drosophila melanogaster. The mRNA size for each gene suggests that these genes are probably expressed individually from separate promoters. This is the first documentation of definitive physical linkage of a functional glutathione S-transferase multigene family. The genes are divergently organized, and a gradation of sequence similarity exists among the encoded GST isozymes. The patterns of sequence similarity in pairwise comparisons of the family members suggest that gene conversion may have played a role in the evolution of this GST multigene family. We propose that the Drosophila gst D genes provide a unique system for studying GST gene regulation, in vivo physiological functions, and evolution of substrate specificities with a global perspective. The gst D genes in other organisms should be intronless and can be isolated directly from genomic DNAs for functional analyses at the gene and protein levels.

Preferential increase of glutathione S-transferase class alpha transcripts in cultured human hepatocytes by phenobarbital, 3-methylcholanthrene, and dithiolethiones.

In rodents, a diversity of compounds are able to protect against acute and chronic toxicities of various xenobiotics including carcinogens, at least in part through induction of drug-metabolizing enzymes including glutathione S-transferase (GST) enzymes. We have posed the question as to whether or not these compounds also induce GSTs in human liver. Primary human hepatocyte cultures were exposed to phenobarbital, 3-methylcholanthrene, and two dithiolethiones [1,2-dithiole-3-thione and its 5-(2-pyrazinyl)-4-methyl derivative, oltipraz], and steady-state mRNA levels of GST classes alpha, mu, and pi were determined by Northern blot analysis. After 3 daily treatments, the two dithiolethiones were the most potent inducers; phenobarbital was also effective but to a lesser extent and 3-methylcholanthrene increased GST mRNA in only 2 of the 6 samples, although it stimulated cytochrome P-450 1A2 mRNA in all cell preparations. Whatever the compound only GSTA1 and/or A2 transcripts were induced. GST M1 mRNAs were not responsive or only slightly responsive, and GST P1 mRNAs, which were mostly undetectable in control cells, were not affected by treatment with any of the four chemicals. Large individual variations were observed in the level of induction of GST A1 and/or A2 mRNAs, and no sex difference could be demonstrated. These results clearly indicate that phenobarbital, 3-methylcholanthrene, and dithiolethiones are able to markedly increase mRNA levels of GST in human hepatocytes and that the GST alpha class is preferentially involved.

[Implementation of stereotactic focal radiotherapy using 10 MV x-ray].

Stereotactic focal irradiation is also called stereotactic radiosurgery by some neurosurgeons. This irradiation is used for the treatment of brain AVM (arterio-venous malformation) and small tumor. Application of stereotactic focal irradiation was developed with CRW (Cosman-Roberts-Wells) stereotactic device and two dimensional (2D) computer treatment planning system using 10 MV x-ray from a linear accelerator. This process of irradiation includes: (a) Identification and localization of a target volume in CRW stereotactic frame by CT scan or angiography. (b) To verify the alignment, a linear accelerator was used as a simulator to take portal films in anterior and lateral views. This was done to ascertain the angles between arc therapy, to encompass the target volume, and to exclude the critical organs such as lens at 0 degrees, 45 degrees, 90 degrees and 315 degrees couch angles. (c) A 2D computer treatment planning system was used to generate an isodose curve distribution for each couch angle. Then this was used to calculate the monitor unit per degree for rotation treatment. (d) 10 MV x-ray was used to implement the stereotactic focal radiotherapy.

[Measurements of dose distribution in small fields of 10 MV X-rays].

Dose distribution measurement in small fields include: 1. Field size factor, 2. Percentage depth dose, 3. Isodose curves charts, 4. Tissue-phantom ratio, and 5. Penumbra width. Field size factor was measured by using different detectors and it was found that the dimension of detector must be less than half of the field size to avoid under-estimation. If a 0.6 cc cylindrical ion chamber was used for small fields measurement, it would under-estimate the field size factor by 19.7% for a 2x2 cm2 field. The percentage depth dose and the tissue-phantom ratio increased with increasing the field size. The penumbra width increased with increasing the field size which was larger for a square field than the corresponding circular field by less than 1 mm.

Expression of human mu or alpha class glutathione S-transferases in stably transfected human MCF-7 breast cancer cells: effect on cellular sensitivity to cytotoxic agents.

Increased expression of certain glutathione S-transferase (GST) isoenzymes has frequently been associated with the development of resistance to alkylating agents and other classes of antineoplastic drugs in drug-selected cell lines. The question arises whether this phenomenon is causal or is a stress-induced response associated with drug resistance in these cell lines. We have constructed mammalian expression vectors containing the human GST mu and GST alpha 2 (Ha2) cDNAs and stably transfected them into the human breast cancer cell line MCF-7. Whereas the parental and pSV2neo-transfected cell lines display low GST activity, three individual transfected clones were identified in each group that expressed either GST mu or GST alpha 2. The range of GST activities was similar to those observed in cells selected for anticancer drug resistance. The GST mu specific activities were 56, 150, and 340 mlU/mg, compared with 10 mlU/mg of endogenous GST mu in control lines. Specific activities in GST alpha 2-transfected clones were 17, 28, and 52 mlU/mg, compared with no detectable alpha class GST in control lines. These clonal lines and the parental and pSV2neo-transfected control lines were tested for sensitivity to antineoplastic agents and other cytotoxic compounds. The clones with the highest activity in each group were 1.7-fold (GST alpha 2) to 2.1-fold (GST mu) resistant to the toxic effects of ethacrynic acid, a known substrate for GSTs. However, the GST-transfected cell lines were not resistant to doxorubicin, L-phenylalanine mustard, bis(2-chloroethyl)-1-nitrosourea, cisplatin, chlorambucil, or the GST substrates 1-chloro-2,4-dinitrobenzene or tert-butyl hydroperoxide. Thus, although L-phenylalanine mustard, bis(2-chloroethyl)-1-nitrosourea, chlorambucil, tert-butyl hydroperoxide, and 1-chloro-2,4-dinitrobenzene are known to be metabolized by glutathione-dependent GST-catalyzed reactions, there was no protection against any of these agents in MCF-7 cell lines overexpressing GST mu or GST alpha 2. We conclude that, at the levels of GST obtained in this transfection model system, overexpression of GST mu or GST alpha 2 is not by itself sufficient to confer resistance to these anticancer agents. These studies do not exclude the possibility that GST may be a marker of drug resistance or that other gene products not expressed in MCF-7 cells might cooperate with GST to confer drug resistance.

Drosophila glutathione S-transferases have sequence homology to the stringent starvation protein of Escherichia coli.

The Drosophila glutathione S-transferase D genes encode a family of isozymes. We have determined the amino acid sequence of a new member of this family by nucleotide sequence analysis of a genomic DNA clone. The open reading frame of this intronless gene should encode an isozyme subunit of 211 amino acids. This sequence has significant homology to the E. coli stringent starvation protein, SSP, which is also a protein of two identical 211 amino acid subunits. The two proteins have very similar overall amino acid composition as well. It is possible that SSP may be a glutathione S-transferase(s) in E. coli or is evolutionarily related to glutathione S-transferases. Because SSP is known to be tightly associated with the RNA polymerase holoenzyme during purification, it is conceivable that Drosophila glutathione S-transferase(s) may potentially interact with the transcription machinery in a fashion similar to SSP's interaction with E. coli RNA polymerase holoenzyme.

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.