Induction of metallothionein-I mRNA in cultured cells by heavy metals and iodoacetate: evidence for gratuitous inducers.

A mouse hepatocyte cell line selected for growth in 80 microM CdSO4 (Cdr80 cells) was used to test the role of metallothioneins in heavy metal detoxification. The cadmium-resistant (Cdr80) cells have double minute chromosomes carrying amplified copies of the metallothionein-I gene and accumulate ca. 20-fold more metallothionein-I mRNA than unselected cadmium-sensitive (Cds) cells after optimal Cd stimulation. As a consequence, the amount of Cd which inhibits DNA synthesis by 50% is ca. 7.5-fold higher in Cdr80 cells than in Cds cells. Cds and Cdr80 cells were compared in terms of their resistance to other heavy metals. The results indicate that although Zn, Cu, Hg, Ag, Co, Ni, and Bi induce metallothionein-I mRNA accumulation in both Cdr80 and Cds cells, the Cdr80 cells show increased resistance to only a subset of these metals (Zn, Cu, Hg, and Bi). This suggests that not all metals which induce metallothionein mRNA are detoxified by metallothionein and argues against autoregulation of metallothionein genes. Metallothionein-I mRNA is also induced by iodoacetate, suggesting that the regulatory molecule has sensitive sulfhydryl groups.     

Metallothionein mediates gene expression of 3.1 mRNA (PTZ17) related to epileptic seizure

Genes differentially expressed in association with disruption of the metallothionein gene were screened using two hepatic stellate cell lines isolated and established from the livers of normal 129/Sv (IMS/N cells) and transgenic mice deficient in the genes for metallothionein-I and -II (IMS/MT (-) cells). We found one cDNA (tentatively named NM31) that was expressed only in IMS/IN cells. Transfecting IMS/MT (-) cells with the genes for both metallothionein-I and -II resulted in NM31 expression. These results suggest that metallothionein is essential for NM31 gene expression. The nucleotide sequence of NM31 (294 bp) was identical to the 3' region of 3.1 mRNA (PTZ 17), which is abundant in the embryonic mouse brain and is related to chemically induced seizures. The present study indicates that metallothionein mediates the expression of specific genes. This is a novel explanation for some of the functions of metallothionein.     

Cloning, nucleotide sequence and characterization of a New Zealand rabbit metallothionein-I gene

We have isolated a rabbit metallothionein-I gene from a lambda gt10 library. The coding sequence of this gene is interrupted by two introns occurring at amino acid positions 9 1/3 and 30 1/3. Comparison of the promoter sequence of this gene with the promoters of other metallothionein genes identified a number of oligonucleotide sequences which are recognized by trans-acting proteins involved in the regulation of these genes by heavy metals, glucocorticoids and alpha interferon.     

Transient expression of murine interferon-alpha genes in mouse and monkey cells

The coding regions of murine interferon-alpha (IFN-alpha) genes were combined with promoter and 3'-noncoding sequences from other eukaryotic genes. Transient expression of these fusion genes was achieved in monkey COS cells and in a mouse cell line (TOP cells) expressing polyoma virus (Py) large T antigen constitutively. The efficiency of the different expression plasmids was determined by measuring the amount of IFN secreted into the medium. Replacement of the 3'-noncoding region of an IFN-alpha gene by that of the rabbit beta-globin gene resulted in a fourfold higher IFN-alpha production. The SV40 early promoter and the Moloney murine leukemia virus (MoMLV) long terminal repeat (LTR) produced similar amounts of IFN-alpha in COS cells. However, a tandem combination of the SV40 enhancer/early promoter and the mouse metallothionein-I promoter appeared fivefold more active than the SV40 early promoter. In TOP cells the MoMLV LTR was found to be threefold more active than the Py early promoter.     

Dramatic pituitary hyperplasia in transgenic mice expressing a human growth hormone-releasing factor gene

A transgenic animal model system was used to analyze the mitogenic effects of GRF on its target cell, the pituitary somatotroph. We have previously established a strain of mice that express a mouse metallothionein-I/human GRF (hGRF) fusion gene, and that grow to be abnormally large due to GH hypersecretion. We show here that chronic GRF production in these mice leads to the development of enormous pituitary glands. The increase in pituitary size appears to be largely the result of a selective proliferation (hyperplasia) of somatotrophs, the GH-producing cells. This observation provides direct evidence that a neuropeptide may act as a specific trophic factor for its target cell. In addition to this effect on pituitary development, we find that the pituitary is a major site of expression of mouse metallothionein-I/hGRF mRNA, and of hGRF peptide. This tissue specificity was unexpected in that neither component of the fusion gene is highly expressed in the normal pituitary. It suggests that pituitary somatotrophs might produce and respond to GRF in an essentially autocrine fashion in these transgenic animals.     

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.     

Differential regulation of metallothionein-thymidine kinase fusion genes in transgenic mice and their offspring

A fusion plasmid, pMK, containing the mouse metallothionein-I promoter/regulatory region joined to the structural gene of herpesvirus thymidine kinase, was introduced into mice by microinjection into fertilized eggs followed by reinsertion of the eggs into foster mothers. Fifteen percent (10 of 69) of the mice developing from this procedure carried pMK sequences. Seven of these mice expressed high levels of viral thymidine kinase in the liver. This enzyme is inducible by heavy metals, as indicated by assay of thymidine kinase activity following sequential partial hepatectomies with or without cadmium treatment. However, glucocorticoid treatment has been ineffective in all transgenic mice tested. The pMK sequences are extensively methylated at a variety of restriction sites, indicating the existence of a de novo methylation enzyme. We have analyzed the inheritance of pMK sequences and their expression in several pedigrees. These fusion genes are inherited as though they were integrated into a single chromosome; however, their expression may be extinguished, diminished or enhanced in the offspring relative to that of the parent. In some animals there is a correlation between changes in DNA methylation and expression of these fusion genes.     

Identification and characterization of the two isoforms of the vertebrate H2A.Z histone variant

Histone variants play important roles in the epigenetic regulation of genome function. The histone variant H2A.Z is evolutionarily conserved from yeast to vertebrates, and it has been reported to have multiple effects upon gene expression and insulation, and chromosome segregation. Recently two genes encoding H2A.Z were identified in the vertebrate genome. However, it is not yet clear whether the proteins transcribed from these genes are functionally distinct. To address this issue, we knocked out each gene individually in chicken DT40 cells. We found that two distinct proteins, H2A.Z-1 and H2A.Z-2, were produced from these genes, and that these proteins could be separated on a long SDS–PAGE gel. The two isoforms were deposited to a similar extent by the SRCAP chromatin-remodeling complex, suggesting redundancy to their function. However, cells lacking either one of the two isoforms exhibited distinct alterations in cell growth and gene expression, suggesting that the two isoforms have differential effects upon nucleosome stability and chromatin structure. These findings provide insight into the molecular basis of the multiple functions of the H2A.Z gene products.     

Parental methylation patterns of a transgenic locus in adult somatic tissues are imprinted during gametogenesis.

The methylation status of a mouse metallothionein-I/human transthyretin fusion gene was studied during gametogenesis in transgenic mice. In the adult tissues of this mouse line, the promoter region of the transgene on chromosome 11 is methylated when it is maternally inherited and undermethylated when it is paternally inherited. Germ cells from various developmental stages of gametogenesis were isolated, and their DNAs were assayed using methylation-sensitive restriction endonucleases and the polymerase chain reaction. Only low to nonexistent levels of transgene methylation were detected in germ cells from 14.5-day-old male and female fetuses irrespective of the parental origin of the transgene. This undermethylated state persisted in oocytes from newborn females as well as in testicular spermatogenic cells and sperm. By contrast, the transgene promoter was completely methylated in fully grown oocytes arrested at the first meiotic prophase. The endogenous metallothionein-I gene promoter, located on a different chromosome, remained undermethylated at all stages examined, consistent with previous findings reported for a typical CpG island. Taken together, the results suggest that parental-specific adult patterns of transgene methylation are established during gametogenesis.     

Antisense polynucleotides and prospects for their use in fighting viruses

Natural or synthetic anti-sense (as) polynucleotides complementary to distinct functional regions of mRNA (asRNA or asDNA) are able to inhibit the expression of any target gene. If certain viral mRNAs important for virus replication are targeted the inhibition of viral infection by asRNA or asDNA takes place. Inhibitory effects of complementary polynucleotides on gene activity in eukaryotic cells is due to the disturbance of translation of corresponding mRNAs as well as to the impairment of their splicing or transportation from the nuclei to cytoplasm. In prokaryotic cells, obviously, only the first factor is operating. The recombinant genes programming anti-viral asRNA can confer the resistance to the infection by other virus to the transformed cells. The resistance to viral infection observed in transgenic animals, expressing asRNA genes, may be considered as a new unnatural form of informational immunity.     

Pharmacological characterization of cyclic AMP receptors mediating gene regulation in Dictyostelium discoideum.

Extracellular molecules regulate gene expression in eucaryotes. Exogenous cyclic AMP (cAMP) affects the expression of a large number of developmentally regulated genes in Dictyostelium discoideum. Here, we determine the specificity of the receptor(s) which mediates gene expression by using analogs of cAMP. The order of potency with which these analogs affect the expression of specific genes is consistent with the specificity of their binding to a cell surface receptor and is distinct from their affinity for intracellular cAMP-dependent protein kinase. Dose-response curves with cAMP and adenosine 3',5'-monophosphorothioate, a nonhydrolyzable analog, revealed that the requirement for high concentrations of exogenous cAMP for regulating gene expression is due to the rapid degradation of cAMP by phosphodiesterase. The addition of low concentrations of cAMP (100 nM) or analogs in pulses also regulates gene expression. Both the genes that are positively regulated by exogenous cAMP and the discoidin gene, which is negatively regulated, respond to cAMP analogs to the same degree. Genes expressed in prespore or prestalk cells are also similarly regulated. These data suggest that the effects are mediated through the same receptor. The specificity of this receptor is indistinguishable from that of the well-characterized cell surface cAMP receptor.     

Distinct patterns in the regulation and evolution of human cancer genes

Understanding the mechanism of regulation of cancer genes and the constraints on their coding sequences is of fundamental importance in understanding the process of tumour development. Here we test the hypothesis that tumour suppressor genes and proto-oncogenes, due to their involvement in tumourigenesis, have distinct patterns of regulation and coding selective constraints compared to non-cancer genes. Indeed, we found significantly greater conservation in the promoter regions of proto-oncogenes, suggesting that these genes are more tightly regulated, i.e. they are more likely to contain a higher density of cis-regulatory elements. Furthermore, proto-oncogenes appear to be preferentially targeted by microRNAs and have longer 3' UTRs. In addition, proto-oncogene evolution appears to be highly constrained, compared to tumour suppressor genes and non-cancer genes. A number of these trends are confirmed in breast and colon cancer gene sets recently identified by mutational screening.     

The single-minded gene of Drosophila is required for the expression of genes important for the development of CNS midline cells

The single-minded (sim) gene of Drosophila encodes a nuclear protein that plays a critical role in the development of the neurons, glia, and other nonneuronal cells that lie along the midline of the embryonic CNS. Using distinct cell fate markers, we observe that in sim mutant embryos the midline cells fail to differentiate properly into their mature CNS cell types and do not take their appropriate positions within the developing CNS. We further present evidence that sim is required for midline expression of a group of genes including slit, Toll, rhomboid, engrailed, and a gene at 91F; that the sim mutant CNS defect may be largely due to loss of midline slit expression; and that the snail gene is required to repress sim and other midline genes in the presumptive mesoderm.