Dynamics of changes in cadmium levels in environmental objects and its impact on the bio-elemental composition of living organisms.

As a result of intensive anthropogenic impact in the biosphere there is a rapid process of accumulation of heavy metal salts. They have led to the aggravation of problems associated with the pollution of ecosystems and basic food products of plant and animal origin. Environmental pollution by these compounds is caused by their persistence in environmental objects, migration ability, accumulation by plants. This contributes to their accumulation in the human environment. A number of studies have shown that heavy metals have mutagenic, toxic effects and affect the intensity of biochemical processes. Therefore, the presence of heavy metals in the environment is extremely undesirable. Moreover, the ecological state of the environment is directly related to changes in the human internal environment. Deficiency or excess of certain bioelements in soils and drinking water or non-compliance with its stable chemical composition causes the development of dysmicroelementosis. The ecological situation of the Carpathian region is closely related to the state of soils and water resources. In this regard, it is advisable to study and control the level of cadmium compounds in the environment of the region. The study of the effect of cadmium intoxication on the macro- and microelement composition of the brain and myocardium of experimental animals is also worthwhile. Materials and methods. Soils and drinking water of the plain, foothill and mountainous zones of the region, as well as organs and tissues of experimental animals served as the object of research. Cadmium levels in drinking water and myocardial tissues and brain of experimental animals have been measured by atomic absorption spectroscopy. Results and discussion. The study of soils in the Prykarpattia region has revealed an increase in the toxic element cadmium. Its content is 1.1-1.5 times higher than background levels. The analysis of drinking water allowed to establish that a significant number of people living in the plain and foothill zone of the region consume water with a high content of cadmium. The main stages of cadmium intake and accumulation in plants have been analyzed. Significant disorders in the body of experimental animals under conditions of excessive intake of cadmium compounds have been revealed. It was accompanied by the accumulation of cadmium in the myocardium and brain, on the background of redistribution of vital macronutrients calcium and magnesium along with micronutrients copper and zinc. Thus, excessive intake of cadmium salts causes the development of dysmicroelementosis, which is accompanied by a violation of the homeostasis of a living organism. It is suggested to conduct continuous monitoring of the level of toxicants in the ecosystem as an integral component of environmental monitoring.

TAF-Containing and TAF-independent forms of transcriptionally active TBP in vivo.

Transcriptional activity in yeast strongly correlates with promoter occupancy by general factors such as TATA binding protein (TBP), TFIIA, and TFIIB, but not with occupancy by TBP-associated factors (TAFs). Thus, TBP exists in at least two transcriptionally active forms in vivo. The TAF-containing form corresponds to the TFIID complex, whereas the form lacking TAFs corresponds to TBP itself or to some other TBP complex. Heat shock treatment altered the relative utilization of these TBP forms, with TFIID being favored. Promoter-specific variations in the association of these distinct forms of TBP may explain why only some yeast genes require TFIID for transcriptional activity in vivo.

Binding of TBP to promoters in vivo is stimulated by activators and requires Pol II holoenzyme.

In eukaryotes, transcriptional activators have been proposed to function by recruiting the RNA polymerase II (Pol II) machinery, by altering the conformation of this machinery, or by affecting steps after initiation, but the evidence is not definitive. Genomic footprinting of yeast TATA-box elements reveals activator-dependent alterations of chromatin structure and activator-independent protection, but little is known about the association of specific components of the Pol II machinery with promoters in vivo. Here we analyse TATA-box-binding-protein (TBP) occupancy of 30 yeast promoters in vivo. We find that TBP association with promoters is stimulated by activators and inhibited by the Cyc8-Tup1 repressor, and that transcriptional activity correlates strongly with the degree of TBP occupancy. In a small subset of promoters, TBP occupancy is higher than expected when gene activity is low, and the activator-dependent increase is modest. TBP association depends on the Pol II holoenzyme component Srb4, but not on the Kin28 subunit of the transcription factor TFIIH, even though both proteins are generally required for transcription. Thus in yeast cells, TBP association with promoters occurs in concert with the Pol II holoenzyme, activator-dependent recruitment of the Pol II machinery occurs at the vast majority of promoters, and Kin28 acts after the initial recruitment.

Cdc53 is a scaffold protein for multiple Cdc34/Skp1/F-box proteincomplexes that regulate cell division and methionine biosynthesis in yeast.

In budding yeast, ubiquitination of the cyclin-dependent kinase (Cdk) inhibitor Sic1 is catalyzed by the E2 ubiquitin conjugating enzyme Cdc34 in conjunction with an E3 ubiquitin ligase complex composed of Skp1, Cdc53 and the F-box protein, Cdc4 (the SCFCdc4 complex). Skp1 binds a motif called the F-box and in turn F-box proteins appear to recruit specific substrates for ubiquitination. We find that Skp1 interacts with Cdc53 in vivo, and that Skp1 bridges Cdc53 to three different F-box proteins, Cdc4, Met30, and Grr1. Cdc53 contains independent binding sites for Cdc34 and Skp1 suggesting it functions as a scaffold protein within an E2/E3 core complex. F-box proteins show remarkable functional specificity in vivo: Cdc4 is specific for degradation of Sic1, Grr1 is specific for degradation of the G1 cyclin Cln2, and Met30 is specific for repression of methionine biosynthesis genes. In contrast, the Cdc34-Cdc53-Skp1 E2/E3 core complex is required for all three functions. Combinatorial control of SCF complexes may provide a basis for the regulation of diverse cellular processes.

Assembly of a bZIP-bHLH transcription activation complex: formation of the yeast Cbf1-Met4-Met28 complex is regulated through Met28 stimulation of Cbf1 DNA binding.

Transcriptional activation of sulfur amino acid metabolism in yeast is dependent on a multi-functional factor, the centromere-binding factor 1 (Cbf1) and on two specific transcription factors, Met4 and Met28. Cbf1 belongs to the basic helix-loop-helix DNA-binding protein family while Met4 and Met28 are two basic leucine zipper (bZIP) factors. We have shown previously that in cell extracts, the three factors are found in a high molecular weight complex. By using mobility shift assays, we report here that the in vitro reconstitution of the Cbf1-Met4-Met28 complex on MET16UAS can be obtained with purified recombinant proteins. DNase I protection experiments confirm that the Cbf1-Met4-Met28 complex is formed over the TCACGTG sequence. The experiments also show that both Met4 and Met28 bind to DNA only in the presence of Cbf1. Moreover, Met28 is shown to enhance the DNA-binding activity of Cbf1. Analysis of MET28 gene regulation reveals that its expression requires Met4. Thus the biochemical activity of Met28 allows the establishment of a positive regulatory loop. The results thus provide evidence of a new functional relationship between bHLH and bZIP proteins and demonstrate that the association of such factors may serve to discriminate between the different TCACGTG sequences found in the chromosomes.

A heteromeric complex containing the centromere binding factor 1 and two basic leucine zipper factors, Met4 and Met28, mediates the transcription activation of yeast sulfur metabolism.

Transcription activation of sulfur metabolism in yeast is dependent on two DNA binding factors, the centromere binding factor 1 (Cbf1) and Met4. While the role of Met4 was clearly established by showing that it acts as a transcription activator, the precise function in transcription of the multi-functional factor Cbf1 remains more elusive. We report here the identification of a new transcription factor Met28 which participates in the regulation of sulfur metabolism. Cloning and sequencing of MET28 revealed that it encodes a new member of the basic leucine zipper DNA binding factor family. We also demonstrate that Met28 possesses no intrinsic transcription activation capabilities. Studies of the DNA binding characteristics of Met28 led us to identify in gel mobility assays a heteromeric complex containing Cbf1, Met4 and Met28. We further demonstrated that the presence of Cbf1 and Met4 stimulates the binding of Met28 to DNA. 'Two-hybrid' studies allowed us to carry out preliminary investigations on the binary protein-protein interactions involved in the formation of the Cbf1-Met4-Met28 complex. Our results give evidence that the leucine zippers of Met4 and Met28, along with the basic helix-loop-helix domain of Cbf1, provide the protein surfaces mediating these interactions. All these results suggest that the multi-functional factor Cbf1 functions in transcription activation by tethering specific activating factors to the DNA.

Met30p, a yeast transcriptional inhibitor that responds to S-adenosylmethionine, is an essential protein with WD40 repeats.

A specific repression mechanism regulates the biosynthesis of sulfur amino acids in Saccharomyces cerevisiae. When the intracellular S-adenosylmethionine (AdoMet) concentration increases, transcription of the sulfur genes is repressed. Using a specific reporter system, we have isolated mutations impairing the AdoMet-mediated transcriptional regulation of the sulfur network. These mutations identified a new gene, MET30, and were shown to also affect the regulation of the methyl cycle. The MET30 gene was isolated and sequenced. Sequence analysis reveals that Met30p contains five copies of the WD40 motif within its carboxy-terminal part, like the yeast transcriptional repressors Hir1p and Tup1p. We identified one target of Met30p as Met4p, a transcriptional activator regulating the sulfate assimilation pathway. By the two-hybrid method, we showed that Met30p interacts with Met4p and identified a region of Met4p involved in this interaction. Further analysis reveals that expression of Met30p is essential for cell viability.

Identification of the yeast methionine biosynthetic genes that require the centromere binding factor 1 for their transcriptional activation.

The yeast Centromere binding factor I (Cbf1) belongs to the family of the DNA binding factors that recognize the consensus sequence CACGTG. Phenotypic studies of cells lacking Cbf1 revealed that this factor is actually involved in two cellular processes; the fidelity of the chromosomal segregation and the metabolism of sulfur amino acids. However, the function of Cbf1 in the regulation of the sulfur amino acid metabolism is now a matter of controversy in literature with conflicting reports about its binding to the CACGTG sequences found upstream to the methionine biosynthetic genes. To provide a reliable basis for the functional analysis of Cbf1, we present an analysis of the transcription of the methionine biosynthesic genes in cells lacking Cbf1. Our results prove that Cbf1 is indeed involved in the transcriptional regulation of the sulfur amino acid metabolism.

Functional analysis of Met4, a yeast transcriptional activator responsive to S-adenosylmethionine.

Transcription of the genes necessary for sulfur amino acid biosynthesis in Saccharomyces cerevisiae is dependent on Met4, a transcriptional activator that belongs to the basic region-leucine zipper protein family. In this report, we show that one mechanism permitting the repression of the sulfur network by S-adenosylmethionine (AdoMet) involves inhibition of the transcriptional activation function of Met4. Using a wide array of deleted LexA-Met4 fusion proteins as well as various Gal4-Met4 hybrids, we identify the functional domains of Met4 and characterize their relationship. Met4 appears to contain only one activation domain, located in its N-terminal part. We demonstrate that this activation domain functions in a constitutive manner and that AdoMet responsiveness requires a distinct region of Met4. Furthermore, we show that when fused to a heterologous activation domain, this inhibitory region confers inhibition by AdoMet. Met4 contains another distinct functional domain that appears to function as an antagonist of the inhibitory region when intracellular AdoMet is low. On the basis of the presented results, a model for intramolecular regulation of Met4 is proposed.