Guide Metals and Genetics

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The second type of involvement is catalytic, where the metal is required for the activity of an enzyme associated with gene expression. The third class involves specific regulation, where metal occupancy of a transacting protein modulates transcription of a specific gene.

This type of involvement is different from the first in that it is much more specific, being more interactive than structural in function. Since the catalytic role appears to be relatively unalterable in humans except, perhaps, in extreme deficiency situations during development, this chapter will concentrate on the structural and regulatory aspects of metals in gene expression.

The best examples of the regulation of gene expression by metals are iron and zinc. In the case of iron, metal occupancy decreases the binding of a metal-regulatory binding protein to ferritin mRNA, allowing the translation of ferritin mRNA to increase while simultaneously increasing binding to transferrin receptor mRNA, which increases the degradation of mRNA O'Halloran, Because iron exhibits oxidation-reduction redox chemistry, rapid control of ferritin synthesis at the level of translation is necessary to provide rapid control of free iron levels within cells.

Far more is known about the involvement of zinc in gene expression than that of other elements. The intracellular binding affinity is greater for zinc than for virtually all other metals found in cells, with the exception of copper. However, unlike iron, zinc does not exhibit redox chemistry but has the properties of a Lewis acid and exhibits fast ligand exchange, which is important for its catalytic role da Silva and Williams, Zinc also plays a structural role in the zinc-finger motif of proteins that are involved in DNA binding, as is discussed below.

Finally, as an activator of trans-acting factors, 2 zinc is responsible for regulating the expression of specific genes. The latter is discussed below. Zinc ions serve an important structural function by tetrahedrally coordinating to cysteine or histidine residues of certain proteins to stabilize the structure of a small functional domain. The most prominent role of proteins with zinc-finger motifs is sequence-specific binding to DNA during transcription, and this is one of the most common eukaryotic DNA-binding motifs Klug and Schwabe, Transcription factors that use zinc fingers as DNA-binding domains range from basal transcription components such as Sp1, to tissue type-specific factors such as GATA-1, to inducible factors such as glucocorticoid receptors Lewin, In addition, some zinc fingers have been shown to mediate protein-protein interactions.

The degree of influence of the level of dietary zinc on zinc interaction with finger proteins is not known. Four major but distinct families of zinc-finger motifs have been identified.

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A single, independent motif contains 1 zinc ion coordinated to 2 cysteine and 2 histidine residues, with a loop or finger of 12 amino acids between the 2 pairs of coordinating residues. This DNA-binding structure often functions as a multimer in which the finger motif is repeated in tandem one to nine times, with the spacing between the coordinating residues being highly conserved. This primary structure has served as the starting point for identifying other zinc-finger motifs based on DNA-and amino acid-sequence homology.

The second family of zinc-finger proteins contains a "zinc twist" Vallee et al. This motif consists of two tandem zinc fingers with only cysteines as the zinc coordinating residues. Unlike the classic zinc finger, these two fingers twist around each other to form a single functional unit with two faces. The zinc-twist motif is characteristic of the DNA-binding domain of the steroid hormone receptor super family.

These receptors frequently function as dimers Lewin, The third zinc-finger type motif to be recognized is referred to as a "zinc cluster. The LIM domain is the fourth type of zinc-finger motif. It is composed of two tandem finger-like zinc-binding sites Dawid et al.

The first zinc is coordinated by three cysteines and a histidine, while the second zinc is coordinated by four cysteines. In contrast, for LIM-only proteins, there is mounting evidence that the LIM motif is involved in protein-protein interactions Schmeichel and Beckerle, With such a wide distribution among proteins involved in different cellular processes, zinc fingers are an important focus of research for therapeutic applications. For example, with their high sequence specificity, they are being evaluated as alternatives to anti-sense DNA approaches to modulating gene expression, and new DNA specificities for zinc fingers are being developed by mutagenesis and phage display library screening Rebar and Pabo, ; Wu et al.

In this way, engineered zinc-finger proteins could be selected based on their ability to bind a specific sequence of interest in order to target specific genes or points of regulation. The mechanisms of transcriptional regulation operate through protein interactions with specific sequences of DNA known as response elements. These response elements provide the specificity for protein factors to interact with each other on the promoter and ultimately result in the unique regulation of different genes.

One widely studied eukaryotic response element that confers increased transcription by metals was first identified in the promoter of the metallothionein I gene Stuart et al. This metal response element, or MRE, is a base pair bp consensus sequence found in multiple copies that potentiates a large increase in gene expression when zinc or cadmium is present Cousins, MRE consensus sequences are generally found in the first several hundred base pairs of the promoter and are often near or overlapping with response elements for other transcriptional factors, such as AP1 Lewin, In addition, MREs are orientation independent and confer metal responsiveness when placed in heterologous promoters.

Recently a protein factor that binds to MREs, and is essential for metal responsiveness, has been cloned and characterized from the mouse and human sources. It is not yet clear, however, whether MTF-1 binds or interacts directly with zinc or cadmium to activate transcription or whether another metalloregulatory protein binds zinc or cadmium and then interacts with MTF The fact that MRE elements function independently of other regulatory elements makes them valuable in the construction of chimeric genes for transgenic animals Palmiter et al.

One or several MREs can be incorporated into the promoter of a chimeric gene and allow the expression of the gene to be controlled in vivo.

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In this way, dietary zinc acting through MREs might eventually be coupled to gene therapy to provide some degree of control for therapeutic gene expression, as discussed below. As international genome mapping projects progress, it has become an increasing priority in biology to identify the genes contained in these vast sequences in order to characterize the function of each gene product. Consequently, detection of genes regulated by nutritional status or altered physiological situations has become increasingly important. Most of the techniques for analyzing regulation by nutrition and other factors, however, require information about the gene as a prerequisite, and it is estimated that current international databases have only identified approximately 30 percent of the total genes in the human genome Orr et al.

A recently developed polymerase chain reaction PCR technique, mRNA differential display, can detect genes that are regulated under different physiological states with no prior information about the gene Liang et al. This method is currently being used to detect genes regulated by dietary micronutrients such as zinc and selenium Blanchard and Cousins, ; Kendall and Christensen, The technique of mRNA differential display begins with the isolation of RNA from animals, tissues, or cells exposed to different physiological conditions.

The RNA is reverse transcribed using an oligo d T primer that has an additional two non-T bases at its 3' end in order to anchor the start of the cDNA synthesis to the junction of the 3' untranslated region and the poly A tail. A total of four anchor primers are needed. The resulting cDNAs are then subjected to the polymerase chain reaction using the oligo d T primer and a decanucleotide primer of arbitrary sequence that will only amplify cDNAs representing a small subset of mRNAs from the original sample and incorporate a radioactive label.

Gel electrophoresis and autoradiography are used to display the resulting PCR products.


Any mRNA that contains the arbitrary decanucleotide sequence within approximately bp of poly A tail will have that portion of its 3' end amplified by the primers in the PCR reaction, and this will produce a band of a specific size on the autoradiograph. In order to screen the entire population of mRNA in a given sample effectively, it is necessary to use a battery of at least 26 different arbitrary decamers, as well as the 4 oligo d T primers for all possible combinations at the 3' end.

The intensity of the autoradiographic image is proportional to the amount of a particular mRNA in the samples. Therefore, cDNA bands of the same size that vary in intensity between experimental conditions represent an mRNA that is differentially expressed under those conditions. In addition, since the samples are displayed adjacent to each other on a gel with many lanes, more than two physiological conditions can be evaluated at the same time, which increases the versatility of this technique.

The cDNA for each differentially expressed mRNA is recovered from the electrophoresis gel and cloned into a plasmid to maintain a stable copy of cDNA for further analysis. The cDNA is first used as a probe for a Northern blot analysis of the original RNA samples to confirm the differential expression in the actual RNA and to quantify the levels of expression. Northern blot confirmation of mRNA differential display is important to eliminate false positives from further evaluation.

The sequence generally contains the 3' untranslated region of the mRNA and a small portion of the carboxyl terminal of the protein coding region.

The Genetics of Essential Metal Homeostasis During Development

To round out the collection of covers, on the outside back cover we have an image from Ulrich Bierbach from Wake Forest University , US, and colleagues who have been studying lung cancer cells, and looking at DNA damage produced by a platinum—acridine antitumor agent. These cover papers will be free to read for 6 weeks. Mail will not be published required. Metallomics Impact Factor rises to 3.

Metallomics Issue 7 online: Acidic mine environment, Acidophilic prokaryotes, Metal resistance. Environments regarded as extreme for humans are the hubs of many specialist microorganisms, viz. Low p H regions, occurring either naturally or created by anthropogenic activities such as mining, are colonized by acidophiles, the organisms growing optimally below p H 4 ref. All extremophiles including acidophiles are valuable resources of genes for novel biochemical activities that can be exploited in specific biotechnological processes 5.

Current trends emphasize genetic engineering of ore-leaching bacteria to achieve further success in this area 13,17, Genetic engineering needs a potential vector such as plasmids that carries genes of interest into a cell and a selectable marker phenotype for identification of the engineered cells from the wild types.

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In case of acidophilic bio-mining bacteria, metal resistance is considered as the most suitable phenotypic trait for selection of genetically modified cells 13,17, Living cells are in constant touch with various forms of metals since their genesis on this planet. Thus, the interactions between metals and living organisms are as ancient as the existence of life. This age-old association has made a bunch of metals viz. Co, Cu, Fe, Ni, Zn etc.

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Besides their vital functions in cellular activities, most of the essential as well as non-essential heavy metals may generate toxic reactions mainly due to the strong coordinating abilities in living systems if their levels rise above a threshold concentration in the cell 23, They exhibit toxicity in a wide range of organisms at concentrations of few m M or less in the medium or environment. Albeit toxicity of some metals and metalloids was known to the mankind from the dawn of civilization, the debut of metal toxicity due to industry-related environmental pollution was reported only in when mercury poisoning was proposed as the cause of Minamata disease Interactions between metals and microorganisms are much deeper and widespread in nature 21,22, Almost in every niche on the earth including extremes of p H, salinity and other severe environmental conditions, microorganisms not only thrive but also take part in various geochemical processes, such as lithification, sedimentation, as well as formation, diagenesis and dissolution of minerals Since the geochemistry of sulphide mines and surroundings is quite different from normal soils, leaching of minerals in these mines due to weathering and microbial activities releases acid and sulphates of heavy metals 4, During these natural events, colonizing microorganisms get continuous or intermittent exposition to heavy metals, and may become tolerant or resistant to one or more of them.

Different types of acidophilic prokaryotes, both autotrophs and heterotrophs, colonize acidic sulphidic mines, industrial leaching operations and natural bioleaching sites 13, Another autotrophic species, Acidithiobacillus caldus formerly Thiobacillus caldus , has attracted attention because majority strains of the species are moderately thermophilic Besides autotrophs, heterotrophs are present in sufficient populations in the bioleaching environment 13,29,30,32,33,36 , very often establishing a close symbiotic relationship with an autotroph.

This type of symbiosis with acidophilic heterotrophs has also been demonstrated in the laboratory cultures of Acidithiobacillus At. Many acidophilic heterotrophic bacteria of bioleaching sites have been characterized to the genus and species level 29,30,33,36,37, Among these bacteria, At.

Importance of the other chemolithoautotrophic and heterotrophic bacteria in bioleaching is being well realised 13,30,36, Microorganisms of acidic mine environment are exposed to high levels of heavy metals at acidic p H, and because of continuous contact with high concentrations of heavy metals these bacteria are expected to tolerate higher levels of metals in comparison to normal soil bacteria. However, natural tolerance to single or more heavy metals occurs in many soil bacteria at sufficiently high rates even in the absence of frequent exposure to any known source of metals that may help selection of resistant cells in a population Because of strong influence of various chemical physiological and environmental factors on bacterial tolerance to metals, consensus on the threshold level of maximum tolerable concentration of a specific heavy metal by bacteria could not be reached.

In a study, the tolerable limits of metal concentrations in a complex agar-based medium p H 6. Usually, plasmids carry the metal resistance conferring genes, although chromosomal inheritance of this character occurs 51, Majority of the mesophilic, acidophilic bacteria harbour one or more plasmids. Characterization of these plasmids has attracted attention due to their importance in developing genetic technology for these bacteria.

This article, however, exclusively summarizes the physiological, genetic and molecular aspects of metal resistance in acidophilic prokaryotes inhabiting acidic mine environment; the potential importance of the genetic elements like plasmids is also discussed in this review. Thrust has been paid on the mesophiles.

In recent review, metal resistance mechanisms in acidophilic microorganisms of sulphide mineral environments have been compared with the same in neutrophiles The review also describes the results of preliminary in silico studies on a few metal resistance systems in the sequenced acidophilic genomes. Although this review covers the publications in this area till date, I apologize beforehand for any important omission.

Genetics and molecular biology of metal tolerance and associated properties in At. The systematic laboratory study on metal tolerance of At. It reveals that under iron-oxidizing condition at p H Interestingly, with thiosulphate in the medium p H 4. In a study on arsenic toxicity, it was observed that growth of both At. Tolerance of an At. Vanadium IV did not inhibit sulphur oxidation up to the tested concentration of 0. Sodium tungstate inhibited the growth of iron-oxidizing environmental bacterial isolates strongly at 0.

Among these, an At. Tungsten-binding by the resting cells was specifically inhibited by molybdate but not by vanadate and other divalent metal cations. The cells bound 12 m g of tungsten per mg cell protein at p H 3, and almost half the amount at p H 6 The metal was found mostly on the cell wall and membrane fractions of the treated cells Mercury ion exhibited very high toxicity to the majority of iron-oxidizing strains tested; out of such strains, At.

That the metabolic activity of At. Since copper is the major metal recovered by biohydrometallurgical operations, tolerance of At. Although the adapted strains leached copper from the concentrate more efficiently, the resistance trait was found to be transient depending on the stress, and not acquired by the strains permanently that would reflect mutations under selective conditions 70, It has been suggested that rapid adaptation of this bacterium to adverse conditions might be due to the presence of mobile repeated sequences Other observations demonstrated that on ferrous iron i majority of the At.

Recently, uranium IV tolerance in three eco-types of At.

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While type I and III strains were resistant to 8 and 9 m M of uranium, respectively, the type II strains could not tolerate more than 2 m M of uranium. Chromium resistance in At. This conclusion was later supported by respirometric experiment Inhibition of iron oxidation in an At. It was observed that no growth of copper-treated cells occurred in presence of Cd, Ni and Zn even after hr although untreated cells could grow under identical condition, but in presence of copper, the treated cells grew better in comparison to untreated cells In general, the level and type of metal ion present in the medium affect the growth pattern of At.

Changes in surface properties was noted in an At. The adapted cells showed a higher pI, more hydrophobicity and enhanced attachment to pyrite mineral. Treatment of the adapted cells with proteinase-K resulted in complete loss of tolerance to copper, reduction in copper adsorption and surface hydrophobicity suggesting a role of surface components in copper tolerance and bioleaching of sulphide minerals Gene expression in At.

Among these, the highest induced gene showed similarity with At.

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Products of unknown similarities were also found. In presence of Cu, the level of protein phosphorylation increased; newly synthesized induced proteins were found more in the membrane fractions than in the cytosol of this strain. This observation was in agreement with the hypothesis that membrane proteins sense environmental changes, and transmit the information to cytosolic proteins through phosphorylation events. Presence of these extrachromosomal genetic elements in At.

The first report on plasmid occurrence in this bacterium approved in In the extended study, one to five plasmids of approximate size 7. Interestingly, Shiratori et al. The study suggested that a few homologous plasmids were ubiquitous, and unique plasmid showing no significant homology with others may be present. Presence of one or more plasmids in At. Causal relationship between a plasmid and resistance to a specific metal ion has been reported in many At.


For example, presence of a kb plasmid in all the uranium-resistant and its absence in the uranium-sensitive isolates of a uranium mine indicated involvement of this plasmid in uranium resistance Further, concomitant loss of uranium resistance occurred with the disappearance of this plasmid in one of the resistant strain In several instances, environmental changes-especially with respect to presence or absence of a metal, influenced the plasmid profile in this bacterium.