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A simple technique to enhance the humoral immune response to intracellular protein antigens in genetic immunizations is demonstrated in mice. In this approach, the intracellular protein is intentionally secreted from expressing cells as a chimeric protein, comprising an N-terminal secreted protein fused to the intracellular protein antigen. Using the Leishmania chagasi Ldccys1 cysteine protease (411CP) as an example of an intracellular protein antigen and both human and murine granulocyte colony stimulating factor (GMCSF) as examples of N-terminal secretion competent fusion partners in chimeric proteins, a humoral response in plasmid DNA immunized mice could only be detected by Western blotting when the expressed 411CP was secreted.

In this study, we tested the protective efficacy of recombinant Leishmania donovani iron superoxide dismutase B1 (SODB1) against Leishmania major infection in BALB/c mice. Mice were challenged with L. major 3 weeks after the second boost immunization with rSODB1 alone or in the presence of adjuvants. Injection of BALB/c mice with rSODB1 alone elicited both humoral and cellular immune responses. Administration of rSODB1 with CpG ODN or GLA-SE (a synthetic toll-like receptor 4 agonist) adjuvant resulted in the induction of anti-SODB1 IgG1, and more importantly of significantly high levels of IgG2a isotype. Immunization of mice with rSODB1 alone or with adjuvant induced the production of IFN-γ by splenocytes in response to stimulation with L. major soluble leishmanial antigens (SLA). Moreover, immunization protocols involving rSODB1 resulted in a significant decrease in IL-10 as compared to controls. The presence of CpG ODN or GLA-SE adjuvant in the immunization protocols resulted in a relative increase in IFN-γ in response to stimulation with rSODB1 in comparison to immunization with rSODB1 alone. Mice immunized with rSODB1 plus CpG ODN or GLA-SE, were able to partially control their Leishmania infections, as indicated by the reduction in footpad swelling and parasite numbers, compared to controls. These results suggest that immunization with recombinant SODB1 protein together with CpG ODN or GLA-SE can be potential vaccine candidate against leishmaniasis.

Cutaneous leishmaniasis (CL) is an increasing public health problem in Ethiopia. There is a concern that it is spreading with increased incidence. In this study, we used isoenzyme electrophoresis and internal transcribed spacer one (ITS1) PCR-RFLP techniques to identify Leishmania species from CL patients in Ethiopia. We obtained isolates from 55 localized cutaneous leishmaniasis (LCL), 3 diffused cutaneous leishmaniasis (DCL) and 36 biopsy samples from 34 LCL and 2 DCL cases from All Africa Leprosy and Tuberculosis Rehabilitation and Training Center (ALERT) and clinically diagnosed CL cases from Ochollo village. Both isoenzyme and ITS1 PCR-RFLP techniques showed that Leishmania aethiopica (L. aethiopica) was the aetiologic agent in all cases. Our study also showed that ITS1 PCR-RFLP could identify Leishmania species from biopsy samples and suggests the method could be used for epidemiological surveillance of leishmaniasis in Ethiopia and for species-specific diagnosis.

There is limited information on the biology and pathogenesis of Leishmania aethiopica, causative agent of cutaneous leishmaniasis (CL) in Ethiopia. In this study we have identified and characterized two cathepsin L-like cysteine protease genes, Laecpa and Laecpb, from L. aethiopica. The predicted amino acid sequence of Laecpa and Laecpb is more than 75% identical with homologous cathepsin L-like cysteine protease genes of other Leishmania species and less than 50% identical with human cathepsin L. Laecpa is expressed predominantly in the stationary, and to a lower level, during the amastigote stage while Laecpb is specifically expressed in the stationary stage of L. aethiopica development. Phylogenetic analysis showed that the two genes are grouped into separate clades which are the result of gene duplication. The isolation of these genes will be useful in developing Leishmania species specific diagnostics for molecular epidemiological studies and serves as a first step to study the role of cysteine proteases in L. aethiopica pathogenesis.

Cutaneous leishmaniasis (CL) is a painful, disfiguring and debilitating disease prevalent in Ethiopia and other countries around the world. In Ethiopia, CL is primarily caused by Leishmania aethiopica and less often by L. tropica and L. major. The intracellular survival mechanisms of Leishmania parasites are still not well understood. Recently a new family of antioxidant enzymes called peroxidoxins have been identified that play an important role in parasite survival. In this study, we have identified two distinct peroxidoxin genes (Pxn1 and Pxn2) that are part of a multi-gene family in L. aethiopica. Protein sequence analysis showed that Pxn1 and Pxn2 are highly homologous to peroxidoxins from other Leishmania species. We have found that L. aethiopica Pxn1 is predominantly expressed in amastigotes and stationary phase promastigotes, whereas Pxn2 is constitutively expressed in the different stages of the parasite. This pattern of RNA expression is consistent with patterns seen in some Leishmania species, but not all. Data from this study will be helpful in enhancing vaccine strategies and drug studies targeted towards peroxidoxins.

This study was performed to characterize the genes that code for superoxide dismutase (SOD) in Leishmania aethiopica. It involved three main steps: specimen collection and parasite isolation, species identification, and molecular characterization of the SOD genes. Out of 20 skin slit specimens cultured and processed from suspected cutaneous leishmaniasis patients enrolled in the study, five (25%) were found to be positive for motile promastigotes. Isoenzyme electrophoresis and PCR-RFLP results confirmed that the isolates were L. aethiopica. Superoxide dismutase-B (SODB) genes were identified from L. aethiopica for the first time. Iron superoxide dismutase-B genes amplified from promastigotes of L. aethiopica (LaeFeSODB) were similar in size to the SODB genes of other Leishmania species. Nucleotide sequences of LaeFeSODB1 showed 95.4, 93.5, and 97.3% identity with L. donovani SODB1 (LdFeSODB1) L. major SODB1 (LmFeSODB1) and L. tropica SODB1 (LtrFeSODB1), respectively. Similarly, LaeFeSODB2 showed 95.9 and 94.1 and 97.6% identity with LdFeSODB2 and LmFeSODB2 and LtrFeSODB2, respectively. On the other hand, predicted amino acid sequence comparison indicated that LaeFeSODB1 had 91.3, 89.8, and 93.9% identity with LdFeSODB1, LmFeSODB1, and LtrFeSODB1, respectively. The difference in nucleic acid sequence of LaeFeSODB from that of LmFeSODB and LtrFeSODB can be utilized to develop specific molecular methods that help differentiate these species in places where there is an overlap in the distribution of these species. In addition, the data provide information about the situation of L. aethiopica with respect to SODB genes.

The mechanisms by which Leishmaniaparasites survive exposure to highly reactive oxygen (ROS) and nitrogen (RNS) species within phagosomes of macrophages are not well known. Recently it has been shown that RNS alone is sufficient and necessary to control Leishmania donovani infection in mice (Murray, H. W., and Nathan, C. F. (1999) J. Exp. Med. 189, 741–746). No enzymatic defense against RNS has been discovered inLeishmania to date. We have previously isolated two peroxidoxins (LcPxn1 and LcPxn2) from Leishmania chagasiand showed that recombinant LcPxn1 protein was capable of detoxifying hydrogen peroxide, hydroperoxide, and hydroxyl radicals (Barr, S. D., and Gedamu, L. (2001) J. Biol. Chem. 276, 34279–34287). In further characterizing the physiological role of peroxidoxins in Leishmania survival, we show here that recombinant LcPxn1 protein can detoxify RNS in addition to ROS, whereas recombinant LcPxn2 protein can only detoxify hydrogen peroxide. LcPxn1 and LcPxn2 are localized to the cytoplasm, and overexpression of LcPxn1 in L. chagasi parasites enhanced survival when exposed to exogenous ROS and RNS and enhanced survival within U937 macrophage cells. Site-directed mutagenesis studies revealed that the conserved Cys-52 residue is essential for detoxifying hydrogen peroxide, t-butyl hydroperoxide, and hydroxyl radicals, whereas the conserved Cys-173 residue is essential for detoxifying t-butyl hydroperoxide and peroxynitrite. This is the first report of an enzymatic defense against RNS in Leishmania.

For the first time, we have reported the establishment and serial propagation of an axenic culture of Leishmania chagasi amastigote-like forms. Parasites were characterized by microscopic evaluation and by the expression of two stage-specific genes, A2 and Ldccys2 amastigote-specific cysteine protease. The differentiated amastigote-like forms were maintained by serial cultivation.

Cathepsin B-like genes from Leishmania donovani and Leishmania chagasi have been isolated and characterized. It is a single gene, which is constitutively expressed in all the life cycle stages of the parasite. Studies using cathepsin B-specific inhibitor treatment suggested that cathepsin B does not seem to play a role in the promastigote stages of the parasite, however it aids in the parasite survival within the host macrophages. Antisense mRNA inhibition of cathepsin B gene also revealed that it plays an important role in the parasite survival within the host macrophages. Furthermore, for the first time, we have shown that Leishmania whole cell lysates as well as the recombinant cathepsin B protein cleaved human recombinant latent transforming growth factor (TGF)-β1 into a mature peptide releasing the latency associated protein, in a cell-free incubation system. Mink lung epithelial cell growth inhibition assay revealed that the cleaved TGF-β1 was biologically active, suggesting thatLeishmania cathepsin B can cleave latent TGF-β1 into mature and active form. These results suggest that cathepsin B plays an important role in Leishmania survival within the host macrophages by activating latent TGF-β1.

Antioxidants have been implicated in protecting cells from oxygen radicals produced as a result of aerobic metabolism and in response to foreign pathogens by phagocytic cells. The mechanisms allowing pathogens to withstand the toxic prooxidant environment within the phagolysosome are poorly understood. We have cloned and characterized three antioxidant genes belonging to the 2-Cys family of peroxidoxins from Leishmania chagasi that may prove to provide these parasites with an enhanced defense mechanism against toxic oxidants. The 5′-untranslated regions and coding regions of each gene are highly conserved, whereas the 3′-untranslated regions have diverged significantly. L. chagasi peroxidoxin 1 (LcPxn1) is predominantly expressed in the amastigote stage, whereas LcPxn2 and LcPxn3 are expressed mainly in the promastigote stage, with LcPxn3 being far less abundant than LcPxn2. LcPxn2 and LcPxn3 possess a nine-amino acid extension at the carboxyl terminus, which LcPxn1 lacks. LcPxn1 appears to exist as high molecular weight multimers in vivo, and recombinant LcPxn1 was shown to detoxify hydrogen peroxide and alkyl hydroperoxides. We also present strong evidence that recombinant LcPxn1 can enzymatically detoxify hydroxyl radicals, an activity never before clearly demonstrated for a protein.

We have isolated and characterized two superoxide dismutase (SOD) cDNAs from a Leishmania chagasi promastigote cDNA library using degenerate primers derived from conserved amino acid residues of previously isolated manganese and iron SODs. Comparison of these two L. chagasi SOD deduced amino acid sequences with previously isolated MnSOD and FeSOD amino acid sequences revealed that they have higher homology to, and complete conservation of, invariant residues found in iron-containing SODs. Southern blot analysis showed that one gene, L.c.FeSODA, is a single copy gene, whereas the other gene, L.c.FeSODB, belongs to a multi-gene family. Transcript levels and enzyme activities of L.c.FeSODA and L.c.FeSODB show differential stage expression, with higher levels present in the amastigote stage of the parasite compared to the promastigote stage. Expression of the L.c.FeSODs in an E. coli SOD null strain protected the bacteria against free radical generating agents. Overexpression of these FeSODs in L. chagasi parasites also showed enhanced protection against the free radical generating agents, paraquat and nitroprusside. The cloning, characterization and overexpression of the L.c.FeSODA and L.c.FeSODB genes and proteins demonstrates the possible role of SODs in Leishmania pathogenesis.

We have cloned and characterized two distinct cysteine protease cDNAs from Leishmania donovani chagasi. One of the cDNAs, Ldccy2, was isolated from a cDNA library prepared from total promastigote RNA while the other cDNA, Ldccys1, was isolated from a cDNA library prepared from total amastigote RNA. Ldccys2 has an open reading frame of 471 amino acids and Ldccys1 has an open reading frame of 447 amino acids. Comparison of the predicted protein sequences of the two distinct cysteine proteases with those of cysteine proteases from Leishmania pifanoi, a member of the L. mexicana complex, showed that the cysteine proteases from the two species of Leishmania are similar in their protein sequences. Each of the two cDNAs is distinct in genomic arrangement and chromosome location. Ldccys1 belongs to a family of cysteine proteases encoded by tandemly organized genes located on chromosome 7 while Ldccys2 appears to be a single cysteine protease gene located on chromosome 10. The organization of the two families of cysteine protease genes in L. donovani donovani was also found to be similar. In this species, the Lddcys1 genes are located on chromosome 5 while the Lddcys2 gene is located on chromosome 8. The Ldccys1 genes are expressed abundantly in the amastigotes recovered from infected hamsters, but at a very low level in the promastigote stage of development. On the other hand, the Ldccys2 gene is expressed both in the promastigote and amastigote stages. We have overexpressed the two cDNAs of cysteine proteases in Leishmania cells and the over-produced cysteine proteases are biologically active and are inhibited by cysteine protease inhibitors. Furthermore, the over-produced and indigenous amastigote specific cysteine protease, Ldccys1, reacted with polyclonal antibodies raised against this protein.

We have analyzed the human (h) metallothionein (MT)-IG proximal promoter region (−174 to +5) using a TATA box mutation (TATCA) and four trinucleotide mutants of the proximal MREa. Transient transfection of HepG2 cells was complemented by in vitro transcription with rat liver nuclear extracts. In both systems, mutations of the TATA box and conserved core of metal responsive element (MRE)a were detrimental to hMT-IG promoter activity suggesting that both elements make significant contributions to hMT-IG transcription. Although MRE binding factors were active in vitro, further metal activation of MT promoter activity was accomplished only by in vivo metal treatment rather than addition of zinc in vitro. Southwestern blotting identified nuclear proteins in rat liver and HepG2 cells which physically interact with MREa in a zinc-dependent manner and could be responsible for MREa function in each system. In addition, the functional effects of the TATCA mutation correlate with altered physical interaction with TATA box-binding protein as observed using DNase I protection.

In this study, the contributions of the two metal-responsive elements (MREs) of the rainbow trout (Salmo gairdnerii) metallothionein (tMT)-B gene promoter (-137 to +5) were analyzed. The effect of MRE mutations on the basal and zinc-induced activities of tMT-B promoter-reporter gene fusions were determined by transfection of a rainbow trout hepatoma (RTH-149) cell line. Together, MREa and MREb cooperate to elicit a significant response to zinc but exhibit differential basal and metal-induced activity. The MREa sequence (-62 to -51) is important for basal promoter activity and can function independently, whereas the more distal MREb (-89 to -100) mainly contributes to metal induction through cooperative interactions with MREa. The degree of basal character of the MREs is partially determined by nucleotide differences at the flexible position N of the MRE consensus TGC(G/A)CNC. In mouse L and HepG2 cells, MREa activity is conserved, but the contributions of the MREb region differ, including reduced cooperativity with MREa. There are also differences in the apparent molecular masses of the rainbow trout and mammalian nuclear factors that bind to the tMT-B promoter and MREa sequence.

In this report, we have measured the cadmium (Cd2+)-induced expression of all known metallothionein I (MT-I) mRNAs in a human hepatoma cell line, Hep G2. Among the human MT-I gene family promoters, marked sequence conservation exists; despite this, the mRNA accumulation level for each species was found to be quite unique. This differential Cd2+ induction of MT-I family members provides an ideal opportunity to assess whether the characteristic response results from subtle isoform-specific variations in promoter structure. Accordingly, we have examined the mechanism for differential expression of two isoforms, MT-IG and MT-IF, by transient transfection into Hep G2 cells. In the presence of Cd2+, MT-IG promoter activity and endogenous mRNA level were, respectively, 4.7- and 3-fold greater than those of MT-IF. This close correlation between promoter activity and mRNA accumulation strongly suggests that differential expression occurs at the level of transcription. The difference in Cd(2+)-stimulated activity was found to be conferred by 240- and 243-base pair promoter fragments spanning nucleotides -174 to +66 and -172 to +71 of the MT-IG and MT-IF genes, respectively. One of the most striking nonhomologies between the promoters is a single A (TATAAA) to C (TATCAA) transversion in the TATA motifs of MT-IG and MT-IF genes, respectively. To determine whether such a subtle change in the TATA motif could account for the marked differences in promoter function, we constructed MT-IG-TATCA and MT-IF-TATAA promoters and measured their activities in transient transfection and cell-free transcription assays. Results of both assays showed a profound difference between the two motifs that paralleled the difference in Cd(2+)-stimulated MT-IG and MT-IF mRNA levels. In summary, we have shown that differential regulation of two MT-I promoters is primarily due to a single base alteration in their TATA motifs.

The sequences responsible for heavy metal-inducible expression are situated within the proximal 437 and 160 base pairs (bp) of MT-IF and MT-IG 5'-flanking sequence, respectively. Only 105 bp of proximal MT-IG 5'-flanking sequence containing a TATA box, two metal responsive elements (MREs), and three GC motifs and 147 bp of proximal MT-IF 5'-flanking sequence containing a TATCA box, four MREs, and two GC motifs were required for heavy metal-inducible expression. However, the proximal 111 bp of MT-IF 5'-flanking sequences (a TATCA box, two MREs, and two GC motifs) was not responsive to heavy metals and competes less efficiently than the 105-bp MT-IG fragment in a competition transfection analysis. The MT-IF promoter fragment containing MREc and MREd is substantially stronger and a more efficient competitor than the MT-IG promoter fragment containing MREc and MREd. Furthermore, the proximal 160 bp of MT-IG 5'-flanking sequence functions as a strong metal-inducible promoter but not as a metal-inducible enhancer. Mobility shift analysis of MT-IF and MT-IG promoter subregions suggests a correlation between protein binding to MRE sequences and MT gene expression. These data illustrate that the overall structural and functional organization of the MT-IF and MT-IG promoters are very different and that the molecular mechanisms governing differential expression levels of human MT genes are quite complex.

In this study we have demonstrated the ability of 5-azacytidine to elevate the basal level expression of the metallothionein (MT)-IF and MT-IG genes and increase the basal level expression of the MT-IIA gene in Hep G2 cells, a cell line which exhibits heavy metal inducible MT gene expression. Atomic absorption analysis of 5-azacytidine treated Hep G2 cells detected a 2-fold increase in the total cellular copper content. Pretreatment of 5-azacytidine exposed cells within hydroxyurea and cycloheximide indicated that the increase in total cellular copper content was a direct response to 5-azacytidine treatment. S1 nuclease analysis illustrated that pretreatment of Hep G2 cells with KCN, a copper specific chelator and uptake inhibitor, suppressed 5-azacytidine- and copper-inducible MT-IG gene expression. Thus, the increase in MT gene expression in response to 5-azacytidine treatment can be correlated to an increase in the total cellular copper content. Possible mechanisms on how 5-azacytidine could alter the influx/efflux of copper in Hep G2 cells are discussed.

Exposure of a trout gonadal fibroblast (RTG-2) cell line to ZnCl2, CdCl2 and CuCl2 resulted in differential levels of accumulation of metallothionein (MT) mRNA. ZnCl2 being the most effective agent induced MT mRNA in 3 h, with 172-fold induction after 48 h and continued accumulation up to 144 h. Following CdCl2 treatment, mRNA could be detected after 24 h, reaching peak levels at 72 h. Furthermore, trout MT mRNA could be detected up to 8 days after withdrawal of extraneous ZnCl2. Using a novel technique of primer extension and DNA sequencing with total RNA as template, specificity of the trout MTa and MTb gene-specific primers was established. Primer extension studies revealed a higher response of MTa to ZnCl2 and CdCl2 compared to MTb. Insensitivity of MT mRNA induction to cycloheximide suggested that induction by the metals was independent of de novo protein synthesis. However, simultaneous exposure of cells to actinomycin D and metals completely inhibited MT mRNA synthesis implying control at the transcriptional level.

The expression of three human metallothionein genes, MT-IIA, MT-IF, and MT-IG was studied in the human hepatoblastoma (HepG2), the hepatocarcinoma (Hep3B2), the embryonic kidney (Hek 293), and the lymphoblastoid-derived (Wi-L2) cell lines. The pattern of expression of each specific MT gene in response to various heavy metals was different among the four cell lines studied indicating differential regulation of MT gene expression. The MT-IF or MT-IG and the MT-IIA genes were regulated in a cell-type specific manner in response to heavy metals and dexamethasone, respectively. DNA methylation was shown to be correlated to cell-type specific regulation of MT gene expression since 5-azacytidine treatment resulted in the expression of the MT-IF and MT-IG genes in response to cadmium and zinc in Wi-L2 cells, of the MT-IIA gene in response to dexamethasone in Wi-L2 cells, and of the MT-IG in response to zinc and copper in Hek 293 cells. Furthermore, transfection studies indicated that all the trans-acting factors necessary for the expression of these genes were present and functional in Wi-L2 and Hek 293 cells. The differential level of expression of the MT-IF and MT-IG genes in response to heavy metals in the Hek 293 cell line was shown to be correlated to their chromatin structure.

Two intronless pseudogenes, corresponding to the human metallothionein MT-I and MT-II processed genes, have been isolated from a human genomic library. MT-I processed gene has accumulated a number of mutations including a nonsense mutation giving rise to a termination codon at amino acid position 21, and a single base deletion at amino acid position 47 causing a shift in the reading frame. MT-II processed gene is a full-length perfect copy of its corresponding mRNA except for a few mutations. Most of the mutations in MT-II processed gene are silent except that the amino acid glycine (GGT) at position 10 is changed to serine (AGT) due to a transition. Both MT-I and MT-II processed genes possess poly(A) sequences of 21 and 17 nucleotides, respectively, 3' to the consensus AATAAA sequence. While these genes are quite similar in their sequences at the 3'-untranslated region, they show less than 50% homology in the 5'-untranslated sequences. Two direct repeats of 16 and 18 nucleotides in length define the limits of the MT-I and MT-II processed genes, respectively, and have been confirmed by S 1 nuclease mapping analysis. In both MT-I and MT-II processed genes these direct repeats towards the 5' end of the gene start with an AhaIII (TTTAAA) restriction site. Our studies suggest that these direct repeats are the results of the insertion site duplication.

At least two classes of protamine mRNA are present in both trout testis polysomal RNA and RNA from the postribosomal supernatant fraction of trout testis hormogenate both of which direct the synthesis of protamine in a Krebs II ascites S-30. One contains poly(A) tracts and the other is devoid of poly(A). Sucrose gradient analyses showed that the poly(A) containing protamine mRNA (poly(A) (+)) sedimented IN THE 6 S region with a shoulder in the 4 S region while the protamine mRNA devoid of poly(A) (poly(A) (-)) appeared to sediment at about 4 S and could not be resolved from tRNA. Analysis of the poly(A) (+) protamine mRNA by boundary sedimentation in an analytical ultracentrifuge showed a sedimentation coefficient of 5.7 S, a value which gives rise to an estimate of 165 to 170 nucleotides per molecule. The poly(A) (+) protamine mRNA migrated as a single species in formamide-containing polyacrylamide gels and its mobility in relation to markers of tRNA (4 S) and 5 S RNA was consistent with its sedimentation velocity of 6 S. The RNA present in the major band on an aqueous polyacrylamide gel was extracted and shown to code for protamine in a wheat germ cell-free system.

We have analyzed the pattern of expression of three human metallothionein (MT) genes (MTI-F, MTI-G, and MTII-A) in the hepatoblastoma cell line, HepG2, in response to the metal ion inducers cadmium, copper, and zinc. The absolute number of transcripts of each of the three genes were measured, and the data clearly suggest differential regulation of these members of the MT gene family by the different inducers both in terms of the rate and the extent of transcript accumulation. The lowest levels of transcript accumulation was observed for MTI-F gene (maximum 4,000 molecules of mRNA per cell) and copper was shown to be its poorest inducer (up to 2,000 molecules per cell). Cadmium is the poorest inducer of MTI-G gene even though transcripts of this gene accumulated at comparatively higher levels than those of MTI-F. Copper- and zinc-induced MTI-G transcript accumulation was up to 12,000 molecules per cell whereas the corresponding value for cadmium was only 4,500. MTII-A was the only gene expressed in the absence of any externally added inducer. Also, in contrast to the MTI genes, the MTII-A gene was equally responsive to all the metal ions tested and the induced levels of accumulation were much higher (up to 75,000 molecules of MTII-A mRNA per cell).

Elevation of the incubation temperature of Chinook salmon embryo cells from 20 to 24 degrees C or exposure to heavy metals such as CdCl2 (5 microM) or ZnCl2 (100 to 500 microM) induces the reversible expression of a set of heat shock or stress proteins. Continuous exposure of the cells to either metal ions or heat shock results in recovery of protein synthesis to a control-like pattern. Treatment of these cells with either ZnCl2 or CdCl2 also induces the protein metallothionein. Heat shock, however, does not induce metallothionein, suggesting that it does not belong to the common group of heat shock or stress proteins. The induction of these stress proteins can be inhibited by pretreatment with actinomycin D, suggesting that their expression is regulated at the transcriptional level. The major stress proteins are detectable in the products of an in vitro translation system programmed with RNA isolated from heat shock- or metal ion-treated cells. A recombinant DNA probe complementary to Drosophila mRNA coding for the 70,000-dalton heat shock protein was found to hybridize to RNA isolated from heat shock-or metal ion-treated cells but not from control cells. The fish mRNA coding for the heat shock protein with a molecular weight of 70,000 appears to be of similar size to the corresponding Drosophila mRNA.

Endogenous levels of metallothionein (MT) mRNA were detected by RNA probes in several somatic and germ-line tissues of rainbow trout, such as eggs, ovaries and immature testis. These levels may be related to metal-ion homeostasis in the observed tissues. The induction kinetics of trout MT isoform B (MT-B) mRNA were studied after single intraperitoneal injections of CdCl1, CuCl2 and ZnCl2. MT-B mRNA was induced within 12 h in liver, kidney, spleen and gills. However, over the 48-h experimental period, the kinetics of MT-B mRNA accumulation differed in response to the three metal salts, possibly due to differential handling of the salts by these tissues. Multiple metal-salt injections induced high levels of MT-B mRNA in the four tissues studied. In the rainbow trout hepatoma cell line, ZnCl2 was a better inducer of the MT-B gene, as compared to CdCl2 and CuCl2. The expression of the exogenous trout MT-B promoter in Chinook salmon embryonic cell line indicates the presence of MT regulatory factors. In contrast, the endogenous MT genes in these cells are quiescent, possibly due to the methylation of their promoter region.

We have investigated the regulation of metallothionein gene expression in two fish cell lines. Rainbow trout hepatoma (RTH) cells synthesized metallothionein in response to heavy metal exposure. The maximum level of metallothionein synthesis detected during zinc exposure was much greater than during cadmium exposure. The time-courses of metallothionein synthesis were different for the different metal inducers, suggesting that metallothionein may be differentially regulated by cadmium and zinc in these cells. The metal-induced synthesis of metallothionein was correlated with increased translational activity and accumulation of metallothionein-mRNA, suggesting that metallothionein may be regulated at the transcriptional and post-transcriptional levels in RTH cells. Chinook salmon embryo (CHSE) cells, unlike RTH cells, did not synthesize metallothionein or metallothionein-mRNA in response to heavy metal exposure. However, when these cells were treated with 5-azacytidine prior to heavy metal exposure, the synthesis of metallothionein was induced, suggesting that DNA methylation may play a role in metallothionein gene expression in fish.

The human metallothionein (MT)-IG gene (hMT-IG) is tandemly linked in a head-to-head fashion with the hMT-IF gene. The hMT-IG gene encodes a MT-I polypeptide and has a tripartite structure. The 5'-flanking region of the hMT-IG gene has a TATAA box, four GC motifs, and at least four metal responsive elements. The 3'-untranslated region has a variation of the polyadenylation signal, AATTAA, and the 3'-flanking region a YGTGTTYY RNA processing signal. This gene is expressed in hepatoma-derived cell lines (Hep G2 and Hep3B2) in response to the heavy metals (cadmium, copper, and zinc) but not to the glucocorticoid analogue dexamethasone. In contrast, the lymphoblastoid cell line (Wi-L2) does not express the hMT-IG gene. These results suggest that the hMT-IG gene is regulated differentially and in a cell type-specific manner. Transient expression studies of the chloramphenicol acetyltransferase (CAT) gene under the transcriptional control of either the hMT-IG or hMT-IF promoter in Hep G2 cells has demonstrated that both promoters contain all the necessary cis-acting elements to elicit a similar pattern of heavy metal inducibility. However, the hMT-IG promoter in all instances is five times more active than the hMT-IF promoter. The differences in promoter activity of these genes could possibly be due to inherent differences in their basal level regulatory sequences. The expression of MT-IGcat in transfected Wi-L2 cells demonstrates that the hMT-IG promoter is not cell type-specific.