Evidence for a recent mutation giving rise to a truncated copy of a cysteine proteinase gene in Leishmania pifanoi

We have previously identified and characterized two amastigote-specific cysteine proteinases of Leishmania pifanoi. The slightly different isoforms of the more abundant proteinase are coded by a gene family of approximately 20 gene copies, that contain a C-terminal extension characteristic of cysteine proteinases of trypanosomatids. In this gene family, we have detected a copy that codes for a truncated form of this proteinase, lacking the C-terminal extension. Interestingly, when the deletion of a nucleotide that creates a stop codon causing this truncation is disregarded, the translated sequence gives rise to a divergent C-terminal extension that has many conserved amino acids when compared to Leishmania and Trypanosome, suggesting that a recent mutation led to the truncation.     

Molecular cloning and expression of a Tetrahymena pyriformis ubiquitin fusion gene coding for a 53-amino-acid extension protein.

Summary The genome of Tetrahymena pyriformis has been shown to contain a ubiquitin multigene family consisting of several polyubiquitin genes and at least one ubiquitin fusion gene. We report here the isolation and characterization of one genomic clone (pTUl1), that encodes a ubiquitin extension protein. A comparison of the predicted amino acid sequence of the ubiquitin extension protein gene of T. pyriformis with those from other organisms indicated a high degree of homology. However, the Tetrahymena ubiquitin extension protein contains 53 and not 52 amino acids. This feature is different from all ubiquitin 52-amino-acid extension protein genes thus far sequenced. Furthermore, we found an array of four cysteine residues similar to those found in nucleic acid binding proteins. Also, the C-terminal sequence possesses a conserved motif which may represent a nuclear translocation signal. The ubiquitin 53-amino-acid extension protein gene encodes the smallest class of ubiquitin mRNAs in T. pyriformis.     

The amino acid composition of the zinc-induced metallothionein isoforms in rat brain

Recent investigation from this laboratory has identified in the rat brain a zinc-inducible and actinomycin D-inhibited metallothionein with an elution volume (Ve/Vo) of 2.08 and a molecular weight of smaller than 10,000 daltons. Furthermore, purification of the zinc-induced metallothionein by ion exchange chromatography on DEAE-Sephadex A-25 columns produced two isoforms, eluting, respectively, at 68 and 130 mM of Tris-acetate buffer, pH 7.5. In this paper, we report that zinc-induced metallothionein produces also two distinct isoforms on reverse phase high performance liquid chromatography that exhibit retention times of 17.23 and 18.53 minutes, respectively. Brain metallothionein was characterized further by studies showing that the zinc-induced metallothionein incorporated a large quantity of [³⁵S]cysteine and that isoforms I and II contain 17 and 18 cysteine residues, respectively, while being devoid of any arginine, histidine, leucine, phenylalanine or tyrosine. The precise functions of the brain metallothionein isoforms, which may be related to the transport and homeostasis of essential elements such as zinc and copper, remain to be elucidated.     

The molecular characteristics of two metallothioneins in Ostrinia furnacalis and expression under conditions of heavy metal stress

Metallothioneins are ubiquitously-expressed metal-binding proteins. Despite their potential ecological relevance, no prior reports have identified any metallothioneins in Ostrinia furnacalis or other Lepidoptera species. A better understanding of the molecular characteristics and regulatory dynamics of metallothionein genes in O. furnacalis under heavy metal stress conditions would enable future studies of the roles played by these proteins in the context of heavy metal detoxification. Herein, we identified and characterized two metallothionein (OfMT) genes in O. furnacalis, including the 147 bp OfMT1 gene encoding a 48 amino acid protein containing 10 cysteine residues, and the 141 bp OfMT2 gene encoding a 46 amino acid protein containing 12 cysteine residues. The expression of OfMT2 was found to be related to Cu and Cd concentrations in a dose-dependent manner but was unaffected by Zn exposure. Overall, these results indicate that OfMT genes likely encode metal-binding proteins consistent with their potential role in the maintenance of heavy metal homeostasis.     

Copper ions and the regulation ofSaccharomyces cerevisiae metallothionein genes under aerobic and anaerobic conditions

We have previously reported that theSaccharomyces cerevisiae CRS5 metallothionein gene is negatively regulated by oxygen. The mechanism of this repression was the focus of the current study. We observed that the aerobic repression ofCRS5 is rapid and occurs within minutes of exposing anaerobic cultures to air. Furthermore, theCUP1 metallothionein gene ofS. cerevisiae was found to be subject to a similar down-regulation of gene expression. We provide evidence that the aerobic repression of yeast metallothioneins involves copper ions and Ace1, the coppertrans-activator ofCUP1 andCRS5 gene expression. A functional Ace1 binding site was found to be necessary for the aerobic repression ofCRS5. Moreover, the aerobic down-regulation of the metallothioneins was abolished when cells were treated with elevated levels of copper. Our studies show that anaerobic cultures accumulate higher levels of copper than do aerobic cells and that this copper is rapidly lost when cells are exposed to air. In fact, the kinetics of this copper loss closely parallels the kinetics ofCUP1 andCRS5 gene repression. The yeast metallothionein genes, therefore, serve as excellent markers for variations in copper accumulation and homeostasis that occur in response to changes in the oxidative status of the cell.     

Isolation and characterization of a metallothionein-1 protein in Chloris virgata Swartz that enhances stress tolerances to oxidative, salinity and carbonate stress in Saccharomyces cerevisiae

Chloris virgata Swartz (C. virgata) is a gramineous wild plant that is found in alkaline soil areas in northeast China and is highly tolerant to carbonate stress. We constructed a cDNA library from C. virgata seedlings treated with NaHCO₃, and isolated a type1 metallothionein (MT1) gene (ChlMT1: AB294238) from the library. The amino acid sequence of ChlMT1 contained 12 cysteine residues that constituted the Cys-X-Cys (X = amino acid except Cys) motifs in the N- and C-terminal regions. Northern hybridization showed that expression of ChlMT1 was induced by several abiotic stresses, from salts (NaCl and NaHCO₃), a ROS inducer (paraquat), and metals (CuSO₄, ZnSO₄, and CoCl₂). ChlMT1 expression in leaf was induced by 200 mM NaCl and 100 mM NaHCO₃. About 5 μM Paraquat, 500 μM Zn²⁺, and 500 μM Co²⁺ also induced expression of ChlMT1 in leaf after 6 h, and 100 μM Cu²⁺ induced it after 24 h. Saccharomyces cerevisiae when transformed with the ChlMT1 gene had dramatically increased tolerances to salts (NaCl and NaHCO₃) and ROS.     

小白菜BcUBCE2基因的克隆及表达分析

采用cDNA-AFLP和RACE技术从小白菜中克隆得到泛素结合酶E2基因(ubiquitin conjugating enzyme E2),命名为BcUBCE2。序列分析表明,BcUBCE2基因cDNA全长830bp,包含1个456bp的开放阅读框,编码152个氨基酸。结构分析发现,该序列包含一个泛素结合酶E2活性位点和一个高度保守的半胱氨酸。进化分析显示,小白菜BcUBCE2蛋白同拟南芥E2蛋白的亲缘关系最近。qRT-PCR分析表明,BcUBCE2基因在小白菜根、茎、叶中均有表达,铜处理10d时BcUBCE2基因的表达量最高。研究认为,BcUBCE2基因可能在铜胁迫响应中发挥重要作用。     

Evidence for a recent mutation giving rise to a truncated copy of a cysteine proteinase gene in Leishmania pifanoi

We have previously identified and characterized two amastigote-specific cysteine proteinases of Leishmania pifanoi. The slightly different isoforms of the more abundant proteinase are coded by a gene family of approximately 20 gene copies, that contain a C-terminal extension characteristic of cysteine proteinases of trypanosomatids. In this gene family, we have detected a copy that codes for a truncated form of this proteinase, lacking the C-terminal extension. Interestingly, when the deletion of a nucleotide that creates a stop codon causing this truncation is disregarded, the translated sequence gives rise to a divergent C-terminal extension that has many conserved amino acids when compared to Leishmania and Trypanosome, suggesting that a recent mutation led to the truncation.     

Identification of a metallothionein gene in honey bee Apis mellifera and its expression profile in response to Cd,Cu and Pb exposure

Metallothioneins are ubiquitous proteins important in metal homeostasis and detoxification. However, they have not previously been identified in honey bees or other Hymenoptera, where metallothioneins could be of ecophysiological and ecotoxicological significance. Better understanding of the molecular responses to stress induced by toxic metals could contribute to honey bee conservation. In addition, honey bee metallothionein could represent a biomarker for monitoring environmental quality. Here we identify and characterize a metallothionein gene in Apis mellifera (AmMT). AmMT is 1,680 bp long and encodes a 48 amino acids protein with 15 cysteines and no aromatic residues. A metal response element upstream of the start codon, coupled with numerous cis‐regulatory elements indicate the functional context of AmMT. Molecular modelling predicts several transition metal binding sites, and comparative phylogenetic analysis revealed five putative metallothionein proteins in three other hymenoptera species. AmMT was characterized by cloning the full‐length coding sequence of the putative metallothionein. Recombinant AmMT was found to increase metal tolerance upon overexpression in Escherichia coli supplemented with Cd, Cu or Pb. Finally, in laboratory tests on honey bees, gene expression profiles showed a dose‐dependant relationship between Cd, Cu and Pb concentrations present in food and AmMT expression, while field experiments showed induction of AmMT in bees from an industrial site compared to those from an urban area. These studies suggest that AmMT has metal binding properties in agreement with a possible role in metal homeostasis. Further functional and structural characterization of metallothionein in honey bees and other Hymenoptera are necessary.     

The Evolution of Codon Preferences in Drosophila: A Maximum-Likelihood Approach to Parameter Estimation and Hypothesis Testing

Synonymous codon usage in related species may differ as a result of variation in mutation biases, differences in the overall strength and efficiency of selection, and shifts in codon preference—the selective hierarchy of codons within and between amino acids. We have developed a maximum-likelihood method to employ explicit population genetic models to analyze the evolution of parameters determining codon usage. The method is applied to twofold degenerate amino acids in 50 orthologous genes from D. melanogaster and D. virilis. We find that D. virilis has significantly reduced selection on codon usage for all amino acids, but the data are incompatible with a simple model in which there is a single difference in the long-term N _e, or overall strength of selection, between the two species, indicating shifts in codon preference. The strength of selection acting on codon usage in D. melanogaster is estimated to be |N _e s|≈ 0.4 for most CT-ending twofold degenerate amino acids, but 1.7 times greater for cysteine and 1.4 times greater for AG-ending codons. In D. virilis, the strength of selection acting on codon usage for most amino acids is only half that acting in D. melanogaster but is considerably greater than half for cysteine, perhaps indicating the dual selection pressures of translational efficiency and accuracy. Selection coefficients in orthologues are highly correlated (ρ= 0.46), but a number of genes deviate significantly from this relationship. Received: 20 December 1998 / Accepted: 17 February 1999     

Degradation of 35S-labeled metallothionein in the liver and the kidney of Brindled mice: Model for Menkes' disease

An amino acid analysis of the renal copper-binding protein of heterozygous Brindled mice indicated that the protein labeled with L-[³⁵S]cystine was metallothionein.The metabolism of ³⁵S-labeled hepatic and renal metallothionein of adult normal (Mo^+/+) and heterozygous (Mo^br/+) Brindled mice was investigated without prior induction with metals. After incorporation of L-[³⁵S] cysteine into hepatic and renal metallothionein, ³⁵S-labeled metallothionein is normally degraded with two half-lives (liver: 11.6 ± 1.3 hours and 3.1 ± 0.3 days; kidney: 8.22 ± 0.08 hours and 3.5 ± 1.2 days). However, ³⁵S-labeled renal metallothionein of the heterozygous Brindled mice is exclusively degraded with a half-life of 3.1 ± 0.2 days.The results imply that the mutation in Brindled mice causes an impaired renal reabsorption of copper (transport of copper from the tubular cells into the blood circulation).     

Spectroscopic characterization of Cicer arietinum metallothionein 1

The plant metallothioneins differ distinctively from other metallothionein families with respect to the cysteine distribution patterns, the presence of aromatic amino acids in most and histidine in some forms, as well as long cysteine-free amino acid stretches between cysteine-rich regions. Although known for more than 25 years, research activity on plant metallothioneins has been low increasing only in the past few years. In the following, we will present the first characterization of Cicer arietinum (chickpea) MT1. In this root-specific protein two cysteine-rich regions with six cysteine residues each are separated by a 42 amino acids long linker region. A synthetic gene encoding MT1 was designed, cloned into a suitable vector, and the protein was over-expressed in Escherichia coli. We could show, that MT1 has the ability to coordinate up to five Zn²⁺ or Cd²⁺ ions and even higher amounts of Hg²⁺. According to titration experiments pH-dependent zinc- and cadmium-thiolate cluster stability in MT1 is considerably lower than in vertebrate metallothioneins. The approximate contribution of secondary structural elements to the overall structure was assessed with circular dichroism and infrared spectroscopy. Hypothetical metal-thiolate cluster structures will be presented.     

Use of Sequence-Specific Antisense Oligodeoxynucleotides to Determine the Protozoan Parasite Antigen Recognized by Nonspecific Cytotoxic Cells

The antigen on the protozoan parasite Tetrahymena pyriformis recognized by catfish nonspecific cytotoxic cells (NCC) is a 46- to 48-kDa protein referred to as NKTag. The complete cDNA-derived amino acid sequence of NKTag has been obtained. The antigenic determinant of NKTag corresponding to the NCC binding site has been determined with synthetic peptides in target cell competition experiments. To more directly characterize the mechanism of parasite:effector cell interaction, we applied NKTag sequence-specific antisense oligodeoxynucleotides to Tetrahymena in vitro. NKTag mRNA translation by Tetrahymena was blocked by specific antisense (AS) oligodeoxynucleotides. 5′-3′ sense (S) oligodeoxynucleotide sequences were synthesized corresponding to the first 17 N-terminal amino acids of NKTag (in addition to −2 untranslated codons plus the start codon). Complimentary AS oligodeoxynucleotides were likewise synthesized. To determine the optimum in vitro conditions for AS treatment, we tested parasites at various phases of their growth cycle for the effects of a single AS treatment. At 9 h post-AS treatment (during the linear phase of the growth curve), maximum reduction in membrane expression of NKTag was observed. Eighty-five percent of Tetrahymena were positive for expression of NKTag at 0 time post-AS treatment versus 13% positive at 9 h. Membrane expression of AS-treated parasites returned to normal levels by 24 h post-treatment. In cold target inhibition experiments, the reduced NKTag expression by Tetrahymena at 9 h AS treatment was confirmed by observing a complete inability (compared with S-treated parasites) to compete with IM-9 cells for binding with NCC. These data demonstrated a unique experimental in vitro system to define the antigen determinant on target cells responsible for recognition by cytotoxic effector cells that participate in innate immune responses. Received: 14 June 1999 / Accepted: 4 September 1999     

Cloning and characterization of cDNA probes for the analysis of metallothionein gene expression in the Mediterranean bivalves: <Emphasis Type="Italic">Ruditapes decussatus</Emphasis> and <Emphasis Type="Italic">Cerastoderma glaucum</Emphasis>

cDNA probes have been developed for subsequent use in monitoring the cadmium exposure of the clam Ruditapes decussatus and the cockle Cerastoderma glaucum using metallothionein (MT) gene expression in different tissues of these species. Two partial MT cDNAs were isolated from Ruditapes decussatus and Cerastoderma glaucum. The identification of the nucleotide sequences showed that the cDNAs consist of 480 bp coding 72 amino acid proteins containing 21 cysteine residues organized in Cys–X–Cys motifs as classically described for MTs. The induction of MT gene expression in CdCl₂ treated bivalves was confirmed by dot blot analysis and suggests a potential specific tissue expression rate.     

Coexpression of thecys E andcys M genes ofSalmonella typhimurium in mammalian cells: a step towards establishing cysteine biosynthesis in sheep by transgenesis

TheSalmonella typhimurium genes for serine acetyltransferase (cys E) and O-acetylserine sulphydrylase B (cys M) were isolated and characterized in order to express these as transgenes in sheep to establish a cysteine biosynthesis pathway and, thereby, to achieve an increased rate of wool growth. Comparison of theS. typhimurium andEscherichia coli genes showed considerable homology, both at the nucleotide and amino acid sequence levels. Thein vitro andin vivo expression studies showed that both genes could be transcribed and translated in eukaryotic cells and that their products could function as active enzymes. Thecys M gene ofS. typhimurium possessed a GUG initiation codon, like itsE. coli counterpart, but translation could be initiated using this codon in eukaryotic cells to give an active enzyme product. Chinese hamster ovary cells, stably transfected with a tandem arrangement of the two genes, showed a capacity to synthesize cysteinein vivo, indicating the establishment of a cysteine biosynthesis pathway in these cells. The measured levels of activity of the gene products suggest that improved wool growth is possible by transgenesis of sheep with these genes.