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P Kille

Publications and source records attributed to P Kille.

36 records · Page 2Linked to original sources
PubMed1997

Functional comparison of the metal-regulated transcriptional control regions of metallothionein genes from cadmium-sensitive and tolerant fish species.

The promoter region of teleost metallothioneins (MTs) contains multiple metal-responsive elements (MREs) organized in proximal and distal clusters which together mediate gene induction by heavy metals. This arrangement of MREs is found both in cadmium-sensitive species, such as the rainbow trout, and in cadmium-tolerant species such as the pike and the stone loach. On comparison of the putative regulatory elements identified within the 5'-flanking region of these genes the major differences are that the number of MREs differ between the different species and that, while both the stone loach and rainbow trout MT genes contain TATA boxes, the pike MT gene has a TTTA box. In order to investigate if the metal sensitivity of a species is correlated to the regulatory potential of the putative MT detoxification system the promoter regions of MT genes from all three species were assessed for their ability to enhance transcription in response to the heavy metals Zn, Cd and Cu. The polymerase chain reaction was used to produce nested deletion sets of each promoter region and these were cloned into the mammalian expression vector pGL-2 upstream of the firefly luciferase gene. The inducibility of the different constructs in response to heavy metal challenge was tested in two cell lines, one fish cell line (homologous to rainbow trout and heterologous to the two other species), the rainbow trout hepatoma, RTH-149, cell line and one cell line that was heterologous to all studied species, the human hepatoblastoma; HepG2, cell line. Maximum inducibility of each gene was achieved with constructs containing both the proximal and the distal MRE clusters. Both the rainbow trout and the stone loach MT genes showed inducibility of comparable amplitude whilst the pike MT gene on the other hand was less inducible, partly due to fewer MREs and partly due to the TTTA box. These data indicate that more than one mechanism is responsible for the differences in cadmium sensitivity of these three teleost species. Although MT is the main heavy-metal detoxifying system in most vertebrates and appears to be contributing to the differences seen between rainbow trout and pike, the present study shows that the relative sensitivity of these species is not primarily due to MT.

Animals↗
PubMed1997

Difference in hepatic metallothionein content in Antarctic red-blooded and haemoglobinless fish: undetectable metallothionein levels in haemoglobinless fish is accompanied by accumulation of untranslated metallothionein mRNA.

Icefish (family Channichthyidae, suborder Nothothenioidei) are a group of Antarctic fish that have evolved unique phenotypes in order to adapt to the environment in which they live. Besides the lack of haemoglobin and the drastic reduction in the number of erythrocyte-like cells, another striking feature of the icefish is that their liver is devoid of metallothionein. These cysteine-rich heavy-metal-binding proteins are usually present in large amounts in a large variety of organisms, from bacteria to mammals. Despite the failure to detect appreciable levels of metallothionein in icefish liver, a cDNA encoding metallothionein was produced from total RNA by reverse transcriptase PCR. The icefish metallothionein showed high percentage identity with metallothionein from Trematomus bernachii, a red-blooded Antarctic fish in which a normal content of hepatic metallothionein was found. Steady-state mRNA levels were assessed in fish liver by high-stringency hybridization of the metallothionein probe with total RNA. The results showed that icefish livers retain large amounts of untranslated metallothionein mRNA. The stability of the icefish transcript might be correlated with the lack of specific motifs in the untranslated 3' ends of mRNA.

Adaptation, Physiological↗
PubMed1997

Cloning and sequencing of a full-length sea bream (Sparus aurata) beta-actin cDNA.

A full-length cDNA clone encoding beta-actin (beta-actin) was isolated from a sea bream (Sparus aurata) liver cDNA library. Sequencing of this clone reveals an open reading frame encoding a 375 amino acid protein that shares a high degree of conservation to other known actins. The sea bream beta-actin sequence showed 98% identity to carp and human beta-actin and 95% and 94% identity to sea squirt and Dictyostelium cytoplasmic actins, respectively.

Actins↗
PubMed1996

Cloning and sequencing a novel metallothionein I isoform expressed in human reticulocytes.

Total RNA from human reticulocytes was purified and reverse-transcribed into cDNA using an oligo-dT primer. This cDNA was used as a template for a polymerase chain reaction (PCR) with a primer specific for the N-terminal sequence of mammalian metallothioneins (MT) and a universal primer. A single amplified fragment was thus generated which when cloned and sequenced revealed two distinct MT cDNAs of almost identical molecular weights. One sequence was identical to that previously reported for human MT II and the other encoded a novel MT I isoform (MT IR). The DNA sequence of MT 1R is distinct from those documented for other MT I isoforms.

Amino Acid Sequence↗
PubMed1996

Effect of stress on mouse and rat brain metallothionein I and III mRNA levels.

The effect of immobilization stress on brain and liver metallothionein (MT) mRNA levels has been studied in mice and rats. Stress increased brain and liver MT-I mRNA levels in mice in a time-dependent manner, in agreement with the MT-I+II protein levels, suggesting an increased gene transcription during stress. In contrast, the brain-specific isoform, MT-III, tended to decrease during stress. In selected brain areas of rats, the overall tendency for both MT-I and MT-III mRNA levels was to be transiently decreased by stress in hippocampus, and increased in hypothalamus, cerebellum and the remaining brain tissues; adrenalectomy significantly affected MT mRNA levels either in basal conditions or during stress, with very different temporal patterns of response depending on the brain area studied. These results suggest that glucocorticoids could be involved in MT-I but also MT-III regulation. In both rats and mice, the subtle response to stress observed in the brain contrasts with the robust response in the liver, suggesting that the factors involved in MT regulation in both tissues differ substantially. In primary cultures enriched in astrocytes or neurons, MT-III mRNA was clearly detected by Northern blotting in both cases, suggesting that it is expressed in both types of cells. Dexamethasone appeared to decrease MT-III mRNA levels in cultured neurons and to increase them in astrocytes, which indicates that glucocorticoids have a different role in MT-III regulation in both cell types.

Animals↗
PubMed1995

Structural and functional analysis of the rainbow trout (Oncorhyncus mykiss) metallothionein-A gene.

In the present study, the distal part of the 5'-flanking region of the rainbow trout metallothionein-A promoter was sequenced in order to identify cis-acting regulatory elements. Analysis of this sequence combined with that previously reported for the 5'-flanking region directly proximal to the start of transcription revealed several putative regulatory sequences. In total, six metal-responsive elements (MREs) were identified; these sequences were organised into two clusters, one containing two copies of MRE and located close to the predicted TATA box sequence, and a second consisting of four MREs and lying 500-700 bp upstream from the start of transcription. In addition, the 5'-flanking region contained sequences sharing high similarity with the activator protein 1 consensus sequence as well as one nuclear-factor-interleukin-6-responsive element. Functional analysis of the promoter was performed by introducing deletion mutants of the 5'-flanking region into the vector pGL-2, directly upstream from the luciferase reporter gene. Both MRE clusters were needed for maximal metal inducibility in both rainbow trout hepatoma (RTH-149) and human hepatoblastoma (Hep G2) cell lines. Furthermore, the distal region was found to be functional in promoting gene transcription following exposure of RTH-149 cells to hydrogen peroxide.

Amino Acid Sequence↗
PubMed1994

Memories of metallothionein.

Metallothionein (MT) has provided nature with a small molecule which exhibits multiple facets. The distinct arrangement of cysteine residues which occurs within the two domains of MT confers predisposed metal specificity upon each domain. Furthermore, subtle changes in primary sequence may be built onto the metal cluster scaffold. These not only bestow immunodistinction but may also potentially allow specific members of this family such as MT-III to fulfill unique biological roles. An understanding of how the structures of MT molecules predetermine their biochemical characteristics may allow the design of novel metal-binding molecules specific for the metal ion of choice. Already, using nature as a blueprint, a semi-specific cadmium-binding molecule has been constructed from a polymer of mammalian C-terminal domains. This novel protein has been used to protect tobacco plants from cadmium toxicity. In addition, modeling of biologically active determinants which are located on the external face of MT-III may facilitate the design of small synthetic molecules which mimic the biological activity of MT-III and prevent the distressing effects of memory and speech loss associated with Alzheimer's disease. Memories of metallothionein may yet be something worth remembering!

Amino Acid Sequence↗
PubMed1993

Analysis of regulatory elements flanking metallothionein genes in Cd-tolerant fish (pike and stone loach).

From genomic libraries constructed for both pike and stone loach, clones were isolated containing the metallothionein genes from these two species of cadmium-tolerant fish. A single copy metallothionein gene was identified in each species by Southern blot analysis. Sequencing revealed that each gene consisted of three exons followed by polyadenylation signals at the 3' end. In the 5' flanking region, putative metal responsive elements were identified both close to the transcription start site and clustered distally approx. 500 bp upstream. Neither gene locus showed any homology with the glucocorticoid or interferon responsive elements that have been identified in mammalian species. The significance of the absence of such responsive elements and their replacement by additional metal responsive elements in the same location of the 5'-flanking region of the MT genes is discussed in relation to the organisation of the MT gene loci in (the Cd-sensitive) rainbow trout and higher mammalian species.

Animals↗
PubMed1993

Cloning and sequencing of a full length cDNA encoding ovine lipoprotein lipase.

A cDNA clone encoding lipoprotein lipase has been isolated from an ovine adipocyte library. Sequencing of this clone has revealed a single open reading frame encoding a 450 amino acid protein. Comparison with known LPL sequences from other species shows a high degree of conservation in the sequence of the protein and in the 5' untranslated region of the DNA sequence.

Adipose Tissue↗
PubMed1992

Sequestration of cadmium and copper by recombinant rainbow trout and human metallothioneins and by chimeric (mermaid and fishman) proteins with interchanged domains.

A family of synthetic genes was constructed encoding a rainbow trout metallothionein (MT), a human MT, and two chimeric molecules which contained respectively (i) the N-terminal (or head) domain of human MT followed by the C-terminal (or tail) domain of a fish MT (termed mermaid MT) and (ii) the head domain of fish MT fused with the tail domain of human MT (denoted fishman MT). These were expressed in Escherichia coli and the four recombinant proteins were purified to homogeneity therefrom. All four were found to bind 7 g atoms of Cd(II) per mol; at pH 7.0, but not at pH 8.6, four Cd(II) ions were sequestered preferentially in the tail (or alpha) domain. Reciprocally, copper was found to bind preferentially in the head (or beta) domain. The human and fishman MTs displayed a stoichiometry of 12 g atoms of Cu(I) per mol, while rainbow trout and mermaid MTs bound only 10. The significance of these findings is discussed in relation to the different positional organization of cysteine residues close to the N and C termini of mammalian and piscine metallothioneins.

Amino Acid Sequence↗
PubMed1991

A plant metallothionein produced in E. coli.

A metallothionein cDNA was generated from pea (Pisum sativum L.) roots, amplified by PCR and inserted into a plasmid for expression in E. coli. Purification of the resultant product generated 3 pools of cadmium-containing material after DEAE-cellulose chromatography. The amino acid composition of each was in excellent agreement with that predicted for pea metallothionein. A cadmium content of approximately 6 g.atoms per mole of protein was estimated. N-terminal sequence analysis revealed that the recombinant molecule had been proteolysed within the extended region linking the 2 cysteine-rich (putative) metal-binding regions. The significance of these findings in terms of the protein folding/targeting of the molecule are considered.

Amino Acid Sequence↗
PubMed1991

Elucidation of cDNA sequences for metallothioneins from rainbow trout, stone loach and pike liver using the polymerase chain reaction.

Metallothionein cDNAs were generated from the livers of three fish species and amplified by PCR. Two distinct coding sequences (A and B) were elucidated for rainbow trout metallothioneins but single isoforms were encoded by genes isolated from the stone loach and pike. Different sized transcripts were observed both with stone loach and pike but these were accounted for by altered lengths of 3' untranslated regions.

Amino Acid Sequence↗
PubMed1990

The expression of a synthetic rainbow trout metallothionein gene in E. coli.

A synthetic gene for rainbow trout metallothionein was constructed and inserted into a dual origin plasmid where expression was induced by a temperature shift in a proteinase-deficient strain of Escherichia coli. The recombinant protein was purified to homogeneity, and a partial amino acid sequence was determined to confirm its identity. Its immunochemical characteristics were similar to those of native metallothionein from rainbow trout. The amounts of recombinant metallothionein produced were quantified in soluble cell extracts by ELISA. Low concentrations were detected when growth was performed either in L-broth or defined (GMM-II) medium. Supplementation of the medium with zinc or copper had no effect on the amount of metallothionein produced. By contrast, when cadmium was included in either L-broth or GMM-II medium, much higher concentrations of the protein within the cells (approx. 13 micrograms/mg soluble cell protein) were detected. This stabilisation of the protein by metal reconstitution in vivo is considered in relation to the selective uptake/exclusion of metals by the cells and its significance for the scavenging of certain precious or toxic heavy metals is discussed.

Amino Acid Sequence↗
PubMedDate not supplied

Developing a new method for soil pollution monitoring using molecular genetic biomarkers.

Physiological responses to environmental stressors may induce changes in gene expression as part of an organism's homeostatic mechanisms. Thus molecular genetic biomarkers have the potential to be used for monitoring sublethal chemical exposure in ecosystems. This paper describes a methodological assessment of the suitability of a protocol to monitor selected biomarkers. The TaqMan real-time quantitative polymerase chain reaction was used to measure gene transcription in earthworms (Lumbricus rubellus) maintained on control or cadmium- or copper-spiked soil. Changes in the expression of two target genes, that encoding metallothionein isoform 2 (MT-2) and that encoding the mitochondrial large ribosomal subunit (MLRS), were quantified against the internal control gene beta-actin. The protocol used produced reliable and reproducible results. Transcript levels displayed qualitative and quantitative differences in the responses to the two metal ions. MLRS gene levels were unaffected by exposure to cadmium but displayed a response to high levels of copper. Conversely, cadmium greatly induced MT-2 gene expression, but copper only altered transcription of this gene at high exposure concentrations. This study demonstrates that it is now technically feasible to use gene expression as an index of pollution exposure in environmentally relevant organisms.

Adaptation, Physiological↗