Mouse Metallothionein Gene Research Articles

Generation of an Enhancer-Trapping Vector for Insertional Mutagenesis in Zebrafish.

Enhancer trapping (ET) is a powerful approach to establish tissue- or cell-specific reporters and identify expression patterns of uncharacterized genes. Although a number of enhancer-trapping vectors have been developed and a large library of fish lines with distinct tissue- or cell-specific expression of reporter genes have been generated, the specificity and efficiency of trapping vectors need to be improved because of the bias interaction of minimal promoters with genomic enhancers. Accordingly, we generated an enhancer-trapping vector pTME that contains a minimal mouse metallothionein gene (mMTI) promoter upstream of EGFP reporter. In the first round of screening, twelve zebrafish lines that carry a single copy of ET cassettes were characterized to have tissue- or cell-specific EGFP expression. One of the highly conserved noncoding elements near an insertion site of trapping cassettes was characterized as an enhancer that can specifically regulate the expression of EGFP in cells of the central nervous system. In addition, the pTME vector contains a mutation-cassette that is able to effectively block the transcription of an endogenous gene in an ET line with ubiquitous EGFP expression. Thus, the pTME vector can be used as an alternative tool for both enhancer trapping and mutagenesis across a target genome.

Metallothionein expression in chloroplasts enhances mercury accumulation and phytoremediation capability

Genetic engineering to enhance mercury phytoremediation has been accomplished by expression of the merAB genes that protects the cell by converting Hg[II] into Hg[0] which volatilizes from the cell. A drawback of this approach is that toxic Hg is released back into the environment. A better phytoremediation strategy would be to accumulate mercury inside plants for subsequent retrieval. We report here the development of a transplastomic approach to express the mouse metallothionein gene (mt1) and accumulate mercury in high concentrations within plant cells. Real-time PCR analysis showed that up to 1284 copies of the mt1 gene were found per cell when compared with 1326 copies of the 16S rrn gene, thereby attaining homoplasmy. Past studies in chloroplast transformation used qualitative Southern blots to evaluate indirectly transgene copy number, whereas we used real-time PCR for the first time to establish homoplasmy and estimate transgene copy number and transcript levels. The mt1 transcript levels were very high with 183,000 copies per ng of RNA or 41% the abundance of the 16S rrn transcripts. The transplastomic lines were resistant up to 20 μm mercury and maintained high chlorophyll content and biomass. Although the transgenic plants accumulated high concentrations of mercury in all tissues, leaves accumulated up to 106 ng, indicating active phytoremediation and translocation of mercury. Such accumulation of mercury in plant tissues facilitates proper disposal or recycling. This study reports, for the first time, the use of metallothioneins in plants for mercury phytoremediation. Chloroplast genetic engineering approach is useful to express metal-scavenging proteins for phytoremediation.

Integrative roles of transforming growth factor-α in the cytoprotection mechanisms of gastric mucosal injury

BackgroundTransforming growth factor α (TGFα) protects against gastric mucosal injury and facilitates wound healing. However, its overexpression is known to induce hypertrophic gastropathy resembling Menetrier's disease in transgenic (TG) mice on an FVB background, as one of the authors reported previously. We studied another TGFα-expressing mouse line on a CD1 background, whose gastric mucosa appears normal. Since this TG mouse had a strong resistance to ethanol-induced gastric injury, we considered the long-term effect of TGFα on several gastric protection mechanisms.MethodsTGFα-expressing transgenic (TG) mouse lines bearing human TGFα cDNA under the control of the mouse metallothionein gene I promoter were generated on a CD1 mouse background, and analyzed their ethanol injury-resistant phenotypes produced by TGFα.ResultsIn the TG mucosa, blood flow was well maintained after ethanol injury. Further, neural and inducible types of NO synthases were consistently and widely expressed in the TG mucosa, compared with the limited distribution of neural type NO synthase in the luminal pit region of the wild-type (WT) mucosa. COX-2 and its upstream transcription factor NfkB were constitutively elevated in the TG mucosa even before ethanol administration, whereas they were induced in the same region of the WT mucosa only after ethanol injury. Two anti-apoptotic proteins, HSP70 and Bcl-2, were upregulated in the TG mucosa even before ethanol administration, while they were not expressed in the WT mucosa before the injury. Furthermore, pro-caspase 3 activation was inhibited in the TG mucosa, while it was converted to the active form in the WT mucosa following ethanol administration.ConclusionWe conclude that TGFα maintains the gastric mucosal defense against gastric injury by integrating other cytoprotective mechanisms.

Cadmium-mediated Activation of the Metal Response Element in Human Neuroblastoma Cells Lacking Functional Metal Response Element-binding Transcription Factor-1

Metal response element-binding transcription factor-1 (MTF-1) binds specifically to metal response elements (MREs) and transactivates metallothionein (MT) gene expression in response to zinc and cadmium. This investigation contrasts the mechanism of mouse MT gene (mMT-I) promoter activation by cadmium and zinc in IMR-32 human neuroblastoma cells to determine whether MTF-1 binding to the MRE is necessary for activation by these metals. Cadmium activated a mMT-1 promoter (-150 base pairs) luciferase reporter 20-25-fold through a MRE-dependent mechanism. In contrast, zinc had little effect on the mMT-1 luciferase reporter. IMR-32 cells lacked MRE binding activity, and treatment with zinc in vitro or in vivo did not generate a MTF-1. MRE complex, suggesting that IMR-32 cells lack functional MTF-1. Overexpression of mMTF-1 regenerated a zinc-mediated induction of the MRE without affecting cadmium activation. Because no other transition metals tested activated the MRE, this effect appeared to be cadmium-specific. These data demonstrate that in IMR-32 human neuroblastoma cells, zinc and cadmium can use independent mechanisms for activation of the mMT-I promoter and cadmium-mediated MRE activation is independent of MTF-1 and zinc.

A pair of adjacent glucocorticoid response elements regulate expression of two mouse metallothionein genes.

Synthesis of mouse metallothionein (MT)-I and MT-II is transcriptionally induced by the synthetic glucocorticoid, dexamethasone (DEX) or both in vivo as well as in numerous cell lines. However, the location(s) of a glucocorticoid response element (GRE) has not been described. The observation that a marked MT-I gene, as well as heterologous genes, when placed in the context of 17 kb of flanking sequence from the MT locus, are inducible by DEX and lipopolysaccharide in transgenic mice renewed the search for the GRE. Analysis of a series of deletion constructs from this 17-kb region in cultured cells identified a single 455-bp region that conferred DEX induction on a reporter gene. This 455-bp region contains two GREs that bind to the glucocorticoid receptor as assessed by gel mobility shift. Deletion of this fragment from the 17-kb flanking region eliminates the DEX responsiveness of reporter genes. The two GREs, which are located approximately 1 kb upstream of the MT-II gene and approximately 7 kb upstream of the MT-I gene, are necessary for induction of both genes and can function independently of elements within the proximal promoter region of either gene.

Construction and Characterization of a Recombinant Adenovirus Vector Carrying the Human Preproinsulin Gene under the Control of the Metallothionein Gene Promoter

A new adenovirus vector carrying human-preproinsulin (h-PPI) genomic DNA, which was placed under the control of the mouse metallothionein gene promoter, was constructed. In the recombinant virus-infected cells, h-PPI gene expression increased as a function of ZnSO4concentration. Reversed-phase high-performance liquid chromatography analysis revealed that the recombinant adenovirus-infected cells secreted immature insulin containing proinsulin and incorrectly processed insulin. Tyrosyl phosphorylation of human insulin receptor substrate 1 occurred when HepG2 cells were treated with the cultured medium, indicating that the h-PPI gene product was functionally activein vitro.We also examined the biological activity of the product using diabetic severe combined immunodeficient mice and confirmed that the h-PPI gene product reduced the blood glucose concentrationin vivo.This study suggests that the adenovirus vector can be used to express a foreign gene under the control of an external promoter in various human cells.

Transgenic model for the discovery of novel human secretory non-pancreatic phospholipase A 2 inhibitors

Transgenic mice were created which overexpress human secretory non-pancreatic phospholipase A 2 (sPLA 2) pansomatically as a potential disease and drug-testing model. The mice were produced using a DNA construct in which the inducible mouse metallothionein gene promoter drives expression of a human sPLA 2 minigene. High levels of sPLA 2 were detected in several tissues by immunofluo-rescence localization. Expression in the testes caused hypospermia and male infertility. Circulating catalytically active sPLA 2 could be induced to levels observed in patients undergoing a systemic inflammatory response but had no detectable effect on the mice. Therefore, these results suggest that sPLA 2 hyperphospholipasemia alone may have only limited pathophysiological consequences. We further show that 3-[3-acetamide-1-benzyl-2-ethylindolyl-5-oxy]propane phosphonic acid (LY311727), a potent new inhibitor of phospholipase A 2 catalysis developed by our group, dramatically suppresses the circulating enzyme activity in these animals whereas 3-[3-acetamide-1-benzyl-2-propylindolyl-5-oxy]propane phosphonic acid (LY314024), a substantially less potent LY311727 analog, is without effect. These later results thus motivate the further development of this compound as a potential new therapeutic agent and valuable research tool.

Functional analysis of human tissue kallikrein in transgenic mouse models.

Clinical studies show that an inverse correlation exists between blood pressure and urinary kallikrein levels. It has been postulated that the tissue kallikrein-kinin system contributes to the maintenance of normal blood pressure. To test this hypothesis, we have established transgenic mice that overexpress human tissue kallikrein under the promoter control of the mouse metallothionein gene and a liver-targeted albumin gene. These animals secrete human tissue kallikrein in plasma at levels 10- to 40-fold higher than that found in normal human serum, and they are chronically hypotensive. This hypotensive effect can be reversed by the injection of aprotinin, a potent tissue kallikrein inhibitor, or Hoe 140, a specific bradykinin receptor antagonist. Transgenic mice overexpressing human tissue kallikrein show a sustained reduction in blood pressure throughout their life spans, indicating the lack of sufficient compensatory mechanisms to reverse the hypotensive effect of kallikrein. Somatic gene delivery of rat kallikrein-binding protein by muscle injection increases the blood pressure of the hypotensive transgenic mice to levels comparable with those in normotensive control mice. These results indicate that a direct link exists between kallikrein gene expression and alterations in blood pressure. In addition, we have developed normotensive transgenic mice that harbor the human tissue kallikrein gene containing 801 bp of its native promoter. The tissue distribution pattern of human kallikrein in these transgenic mice is similar to that in human tissues, with the highest level in the pancreas and much lower levels in the kidney and salivary gland. These transgenic mice provide new animal models for investigating the tissue-specific regulation of tissue kallikrein and its role in altering blood pressure.

Activation of the complete mouse metallothionein gene locus in the maternal deciduum

The mouse metallothionein (MT) gene family consists of four known members (MT-I through IV) clustered on chromosome 8. Studies reported herein examine the expression and regulation of the MT-III and MT-IV genes in specific cell types in the maternal reproductive tract, developing embryo, and fetus known to express the MT-I and -II genes. MT-III and MT-IV mRNAs were absent from the visceral yolk sac, placenta, and fetal liver, tissues with high levels of MT-I and MT-II mRNAs. In contrast, MT-III and MT-IV mRNAs were both abundant in the maternal deciduum, and in experimentally induced deciduoma on 7 and 8 days postcoitum (1 dpc = vaginal plug), as are MT-I and -II mRNAs. The abundance of each of these MT mRNAs increased coordinately during development of the deciduum (6-8 dpc), and in situ hybridization localized MT-I, MT-III, and MT-IV mRNAs to the secondary decidual zone of the antimesometrial region on 8 dpc, where in some regions all of the cells were apparently positive. Thus, all of the known mouse MT genes are co-expressed in at least some of the cells in the secondary decidual zone. Electrophoretic analysis of decidual MT suggested that the MT-I, -II, and -III isoforms are abundant proteins in the secondary deciduum. Bacterial endotoxin-lipopolysaccharide (LPS) and Zn are powerful inducers of MT-I and MT-II gene expression in many adult organs, whereas these agents apparently have little effect on MT-III and MT-IV gene expression. Neither of these agents significantly effected levels of decidual MT-III or MT-IV mRNAs in vivo or in primary cultures of decidual cells in vitro, and only modest effects of Zn on MT-I mRNA levels were noted. During 2 days of in vitro culture, decidual cell MT-I and MT-III mRNA levels remained elevated while MT-IV mRNA levels decreased. Thus, expression of the mouse MT gene locus in the deciduum appears to be developmentally regulated, and in this tissue, the MT genes are refractory to induction by Zn or inflammation.

Metallothionein gene expression and metal regulation during preimplantation mouse embryo development (MT mRNA during early development)

In order to provide information concerning gene expression and regulation in the preimplantation mammalian embryo, and to explore the roles of metallothionein (MT) during this period of development, the constitutive and metal-induced MT mRNA levels in mouse ova, preimplantation embryos, and oviducts were determined. These results were correlated with the effects of transient exposure to high levels of metals (zinz (Zn) or cadmium (Cd)) on the continued development of preimplantation embryos into blastocysts in culture. RNA from preimplantation mouse embryos at different stages of development (Days 1 through 4 of gestation; D1 = vaginal plug) was analyzed using the reverse transcriptase-polymerase chain reaction (RT-PCR) to specifically amplify MT-I and MT-II mRNA transcripts. MT-I mRNA in ova, preimplantation embryos, and oviducts was detected using in situ hybridization. This mRNA in the oviduct was also analyzed by Northern blotting. The results establish that the mouse MT genes are coordinately and constitutively expressed at low basal levels in ova and preimplantation mouse embryos. In unfertilized (ova), fertilized (one-cell) eggs, and two-cell embryos, the MT-I gene was not detectably responsive to metal ions, whereas in later cleavage stage embryos (four- and eight-cell) the MT-I gene was detectably responsive to metals in some blastomeres of some of the embryos. In contrast, after the third cleavage this gene was highly metal-inducible in essentially all cells of the embryo (morula/blastocyst). Surprisingly, the appearance of metal responsiveness of the MT genes during development correlated with decreased Zn toxicity and increased Cd toxicity; two-cell embryos were Zn-sensitive and Cd-resistant, whereas eight-cell and older embryos were Zn-resistant and Cd-sensitive. In the oviduct, MT-I mRNA was not abundant in total RNA, but was detected specifically in the epithelial cells of the isthmus region and was elevated in these cells on D3 and D4 of gestation. In the oviduct, only isthmus epithelial cells responded to metals (Zn or Cd) by increased accumulation of this mRNA. These studies suggest that preimplantation mouse embryo develops the capacity to respond to metals in the environmental milieu by induction of MT gene expression at about the third cleavage. Whether the lack of responsiveness of these genes before this stage reflects transcriptional repression or attenuated metal ion influx and/or enhanced efflux remains to be determined. Sensitivity and resistance of preimplantation embryos to acute metal toxicity involve mechanisms other than MT gene expression in preimplantation mouse embryos. The cell-specific expression of MT genes in the isthmus epithelia of the oviduct suggests that these cells play a role in metal ion homeostasis on D3 and D4 of pregnancy, perhaps by creating a local environment rich in the essential metals Zn and copper and/or by protecting from the toxic effects of Cd.

Restriction fragment length variations and chromosome mapping of two mouse metallothionein genes, Mt-1 and Mt-2.

Restriction endonuclease fragment length variations (RFLVs) were found through the use of cDNA probes for metallothionein genes 1 (Mt-1) and 2 (Mt-2) in the mouse. RFLVs were detected in restriction patterns generated by BglII and XbaI in the Mt-1 gene and by PvuII in the Mt-2 gene. All laboratory strains carry the Mt-1a and Mt-2a alleles. Among strains of wild origin, some Western European subspecies (Mus mus domesticus and M. m. brevirostris) also carry the Mt-1a and Mt-2a alleles. In contrast, a European subspecies (M. m. musculus) and the great majority of subspecies from East Asian countries (M. m. molossinus, Chinese mice of wild origin, and M. m. yamashinai) carry the Mt-1b and Mt-2b alleles. A domesticus strain from Bulgaria and two castaneus strains from Thailand and Philippines carry the intermediate combination of Mt-1b and Mt-2a alleles. Using the RFLVs, we mapped the Mt-1 and Mt-2 genes on chromosome 8, and they appear to be very closely linked since no recombination was observed between them in any of the mice examined. Data from three-point cross tests showed that the recombination frequencies are 4.31% between Os and Mt, 15.52% between Mt and Prt-2, and 19.83% between Os and Prt-2. The gene order of Os-Mt-1,Mt-2-Prt-2 has been confirmed.

A cis-acting sequence within the rat ribosomal DNA enhancer region can modulate RNA polymerase II-directed transcription of the metallothionein I gene in vitro.

Plasmids were constructed by inserting a 557-bp or 174-bp spacer fragment of rat ribosomal (r)DNA containing an enhancer element(s) at -148 bp upstream from a cloned mouse metallothionein gene (pMT-I). Transcription of these plasmids in a fractionated nuclear extract from a rat hepatoma resulted in 5 to 20-fold stimulation of MT-I gene transcription. This enhancement occurred independent of orientation of the enhancer or its distance from the metallothionein gene promoter or in the presence of the MT-I gene enhancer, and was sensitive to low levels of alpha-amanitin. Stimulation of MT-I gene transcription under the direction of the rDNA spacer element also occurred in HeLa nuclear extract, albeit to a smaller extent. Prior incubation of the nuclear extract with the 557-bp or 174-bp fragment resulted in as much as 5- to 10-fold stimulation of MT-I gene transcription. No significant effect on MT-I gene transcription was observed following preincubation with other DNAs. Preincubation of the extract with three subfragments of the 174-bp spacer inhibited MT-I gene transcription, which suggests that the majority of the 174-bp domain is required for binding to the negative regulatory factor(s) for MT-I gene transcription and that the subfragments can only interact with the positive core promoter-binding factor. The 37-bp subfragment, which has been shown to interact with a positive rDNA trans-acting factor, could also interact with a positive polymerase II (pol II) trans-acting factor. These studies have demonstrated that the 174-bp rat rDNA spacer element containing the pol I enhancer can also modulate pol II-directed transcription.

Expression of a mouse metallothionein gene in transgenic plant tissues

Three gene constructions based on a mouse metallothionein I gene ( mMT-I) were introduced into tobacco using an Ri plasmid vector system to test the effectiveness of animal gene regulatory signals in plant cells. No transcription from the native mouse gene was observed. In plant cells bearing chimeric mMT-I genes in which transcription was driven by the nopaline synthase promoter, neither polyadenylation nor splicing of mMT-I pre-mRNA was observed. Detailed comparisons of mMT-I sequences with those of known plant genes were carried out; slight differences in regions of known consensus sequences may be at least partly responsible for the non-recognition of mMT-I gene regulatory signals in plant cells, though other as yet unidentified, potentially necessary sequences may also be involved.

Inducible expression and cytogenetic effects of the EcoRI restriction endonuclease in Chinese hamster ovary cells.

The cytogenetic endpoints sister chromatid exchange (SCE) and chromosome aberrations are widely used as indicators of DNA damage induced by mutagenic carcinogens. Chromosome aberrations appear to result directly from DNA double-strand breaks, but the lesion(s) giving rise to SCE formation remains unknown. Most compounds that induce SCEs induce a spectrum of lesions in DNA. To investigate the role of double-strand breakage in SCE formation, we constructed a plasmid that gives rise to one specific lesion, a staggered-end ("cohesive") DNA double-strand break. This plasmid, designated pMENs, contains a selectable marker, neo, which is a bacterial gene for neomycin resistance, and the coding sequence for the bacterial restriction endonuclease EcoRI attached to the mouse metallothionein gene promoter. EcoRI recognizes G decreases AATTC sequences in DNA and makes DNA double-strand breaks with four nucleotides overhanging as staggered ends. Cells transfected with pMENS were resistant to the antibiotic G418 and contained an integrated copy of the EcoRI gene, detectable by DNA filter hybridization. The addition of the heavy metal CdSO4 resulted in the intracellular production of EcoRI, as measured by an anti-EcoRI antibody. Cytogenetic analysis after the addition of CdSO4 indicated a dramatic increase in the frequency of chromosome aberrations but very little effect on SCE frequency. Although there was some intercellular heterogeneity, these results confirm that DNA double-strand breaks do result in chromosome aberrations but that these breaks are not sufficient to give rise to SCE formation.

Inducible expression and cytogenetic effects of the EcoRI restriction endonuclease in Chinese hamster ovary cells.

The cytogenetic endpoints sister chromatid exchange (SCE) and chromosome aberrations are widely used as indicators of DNA damage induced by mutagenic carcinogens. Chromosome aberrations appear to result directly from DNA double-strand breaks, but the lesion(s) giving rise to SCE formation remains unknown. Most compounds that induce SCEs induce a spectrum of lesions in DNA. To investigate the role of double-strand breakage in SCE formation, we constructed a plasmid that gives rise to one specific lesion, a staggered-end ("cohesive") DNA double-strand break. This plasmid, designated pMENs, contains a selectable marker, neo, which is a bacterial gene for neomycin resistance, and the coding sequence for the bacterial restriction endonuclease EcoRI attached to the mouse metallothionein gene promoter. EcoRI recognizes G decreases AATTC sequences in DNA and makes DNA double-strand breaks with four nucleotides overhanging as staggered ends. Cells transfected with pMENS were resistant to the antibiotic G418 and contained an integrated copy of the EcoRI gene, detectable by DNA filter hybridization. The addition of the heavy metal CdSO4 resulted in the intracellular production of EcoRI, as measured by an anti-EcoRI antibody. Cytogenetic analysis after the addition of CdSO4 indicated a dramatic increase in the frequency of chromosome aberrations but very little effect on SCE frequency. Although there was some intercellular heterogeneity, these results confirm that DNA double-strand breaks do result in chromosome aberrations but that these breaks are not sufficient to give rise to SCE formation.

Promoter and enhancer elements from the rat elastase I gene function independently of each other and of heterologous enhancers.

An elastase-human growth hormone (hGH) fusion gene containing 205 base pairs of elastase 5' flanking region is expressed exclusively in pancreatic acinar cells of transgenic mice. This paper shows that the promoter region (-72 to +8) and the enhancer (-205 to -73) function independently of each other. The elastase enhancer can activate the heterologous mouse metallothionein gene and the hGH gene promoters; conversely, enhancers from the thymocyte-specific murine leukemia virus MCF13 and the metal regulatory elements from the metallothionein gene can activate the elastase promoter in a variety of cell types. Combinations of immunoglobulin and elastase enhancers with a heterologous promoter and the hGH gene result in expression in all of the tissues predicted by the sum of each enhancer acting alone. Thus these enhancer elements act independently of each other, suggesting that they do not have silencing activity in cells in which they are normally inactive.

Promoter and enhancer elements from the rat elastase I gene function independently of each other and of heterologous enhancers.

An elastase-human growth hormone (hGH) fusion gene containing 205 base pairs of elastase 5' flanking region is expressed exclusively in pancreatic acinar cells of transgenic mice. This paper shows that the promoter region (-72 to +8) and the enhancer (-205 to -73) function independently of each other. The elastase enhancer can activate the heterologous mouse metallothionein gene and the hGH gene promoters; conversely, enhancers from the thymocyte-specific murine leukemia virus MCF13 and the metal regulatory elements from the metallothionein gene can activate the elastase promoter in a variety of cell types. Combinations of immunoglobulin and elastase enhancers with a heterologous promoter and the hGH gene result in expression in all of the tissues predicted by the sum of each enhancer acting alone. Thus these enhancer elements act independently of each other, suggesting that they do not have silencing activity in cells in which they are normally inactive.

Different types of hypersensitive sites in the mouse metallothionein gene region.

We have examined the chromatin structure of the metallothionein (MT) gene region in MT- S49 mouse lymphoma cells and in derivatives which express MT-I alone, MT-II alone, or both genes. In all lines, these genes are contained in a 16-kilobase pair region between two DNase I sensitive sites: one site located 5.3 kilobase pairs 5' of MT-II (the 5' gene) is present in naked DNA and retained in the chromatin of all lines; the other site located 3.1 kilobase pairs 3' of MT-I is hypersensitive. Hypersensitivity at three other sites is dependent on the expression of MT genes. Two sites 5' of MT-II disappear, and a site 3' of MT-I appears regardless of which gene is activated. The fact that these sites respond when either gene is activated suggests that the regulation of the two genes is interdependent and that the region undergoes a general change in conformation with MT activation. In addition, a single site in the 5' region of MT-II becomes hypersensitive with activation of the gene and may be related directly to expression.

Structure and regulation of the sheep metallothionein-Ia gene.

Screening of a sheep genomic lambda library with a sheep metallothionein-I cDNA clone resulted in the isolation of a 13,200-base-pair fragment containing a metallothionein gene which DNA sequence analysis identified as the gene encoding the cloned cDNA. The two introns occur at identical positions to those in other mammalian metallothioneins but are considerably larger. The first intron contains a DNA element that is present in a related but not identical form in many places in the sheep genome. Comparison of the promoter sequences of this gene (sMT-Ia) with the promoters of metallothionein genes from other species identified a number of conserved regions which may be important in the regulation of this gene by heavy metals, glucocorticoids and alpha-interferon. In sheep fibroblasts, the levels of sMT-Ia mRNA was found to be maximally elevated (95-fold) in the presence of zinc or cadmium and elevated 30-fold in the presence of copper. Dexamethasone had no effect upon mRNA levels. Thus this gene shows a pattern of regulation similar to the human MT-If gene, but distinct from the other human and mouse metallothionein genes so far reported.

Morphological transformation of established rodent cell lines by high-level expression of the adenovirus type 2 E1a gene.

When a strong promoter derived from the mouse metallothionein gene was substituted for the homologous adenovirus type 2 E1a promoter, leading to enhanced levels of E1a RNAs and proteins in cells transfected with the chimeric gene, the E1a gene alone was able to induce in established cell lines alterations in cellular morphology and growth properties similar to those produced by the combined action of E1a and E1b genes. The qualitative effects of E1a gene expression upon cellular properties thus depend on the level of expression of the E1a gene. Furthermore, E1a may be the primary transforming gene of adenoviruses, since it produced many of the characteristics of transformed cells that had previously been attributed to E1b.