Molecular characterization of a conserved archaeal copper resistance (cop) gene cluster and its copper-responsive regulator in Sulfolobus solfataricus P2

Using a comparative genomics approach, a copper resistance gene cluster has been identified in multiple archaeal genomes. The cop cluster is predicted to encode a metallochaperone (CopM), a P-type copper-exporting ATPase (CopA) and a novel, archaea-specific transcriptional regulator (CopT) which might control the expression of the cop genes. Sequence analysis revealed that CopT has an N-terminal DNA-binding helix-turn-helix domain and a C-terminal TRASH domain; TRASH is a novel domain which has recently been proposed to be uniquely involved in metal-binding in sensors, transporters and trafficking proteins in prokaryotes. The present study describes the molecular characterization of the cop gene cluster in the thermoacidophilic crenarchaeon Sulfolobus solfataricus. The polycistronic copMA transcript was found to accumulate in response to growth-inhibiting copper concentrations, whereas copT transcript abundance appeared to be constitutive. DNA-binding assays revealed that CopT binds to the copMA promoter at multiple sites, both upstream and downstream of the predicted TATA-BRE site. Copper was found to specifically modulate the affinity of DNA binding by CopT. This study describes a copper-responsive operon in archaea, a new family of archaeal DNA-binding proteins, and supports the idea that this domain plays a prominent role in the archaeal copper response. A model is proposed for copper-responsive transcriptional regulation of the copMA gene cluste

Split transition in ferromagnetic superconductors

The split superconducting transition of up-spin and down-spin electrons on the background of ferromagnetism is studied within the framework of a recent model that describes the coexistence of ferromagnetism and superconductivity induced by magnetic fluctuations. It is shown that one generically expects the two transitions to be close to one another. This conclusion is discussed in relation to experimental results on URhGe. It is also shown that the magnetic Goldstone modes acquire an interesting structure in the superconducting phase, which can be used as an experimental tool to probe the origin of the superconductivity.Comment: REVTeX4, 15 pp, 7 eps fig

Instability, Intermixing and Electronic Structure at the Epitaxial LaAlO3/SrTiO3(001) Heterojunction

The question of stability against diffusional mixing at the prototypical LaAlO3/SrTiO3(001) interface is explored using a multi-faceted experimental and theoretical approach. We combine analytical methods with a range of sensitivities to elemental concentrations and spatial separations to investigate interfaces grown using on-axis pulsed laser deposition. We also employ computational modeling based on the density function theory as well as classical force fields to explore the energetic stability of a wide variety of intermixed atomic configurations relative to the idealized, atomically abrupt model. Statistical analysis of the calculated energies for the various configurations is used to elucidate the relative thermodynamic stability of intermixed and abrupt configurations. We find that on both experimental and theoretical fronts, the tendency toward intermixing is very strong. We have also measured and calculated key electronic properties such as the presence of electric fields and the value of the valence band discontinuity at the interface. We find no measurable electric field in either the LaAlO3 or SrTiO3, and that the valence band offset is near zero, partitioning the band discontinuity almost entirely to the conduction band edge. Moreover, we find that it is not possible to account for these electronic properties theoretically without including extensive intermixing in our physical model of the interface. The atomic configurations which give the greatest electrostatic stability are those that eliminate the interface dipole by intermixing, calling into question the conventional explanation for conductivity at this interface - electronic reconstruction. Rather, evidence is presented for La indiffusion and doping of the SrTiO3 below the interface as being the cause of the observed conductivity

Comprehensive evaluation of methods to assess overall and cell-specific immune infiltrates in breast cancer

Background: Breast cancer (BC) immune infiltrates play a critical role in tumor progression and response to treatment. Besides stromal tumor infiltrating lymphocytes (sTILs) which have recently reached level 1B evidence as a prognostic marker in triple negative BC, a plethora of methods to assess immune infiltration exists, and it is unclear how these compare to each other and if they can be used interchangeably. Methods: Two experienced pathologists scored sTIL, intra-tumoral TIL (itTIL), and 6 immune cell types (CD3+, CD4+, CD8+, CD20+, CD68+, FOXP3+) in the International Cancer Genomics Consortium breast cancer cohort using hematoxylin and eosin-stained (n = 243) and immunohistochemistry-stained tissue microarrays (n = 254) and whole slides (n = 82). The same traits were evaluated using transcriptomic- and methylomic-based deconvolution methods or signatures. Results: The concordance correlation coefficient (CCC) between pathologists for sTIL was very good (0.84) and for cell-specific immune infiltrates slightly lower (0.63-0.66). Comparison between tissue microarray and whole slide pathology scores revealed systematically higher values in whole slides (ratio 2.60-5.98). The Spearman correlations between microscopic sTIL and transcriptomic- or methylomic-based assessment of immune infilt

A somatic-mutational process recurrently duplicates germline susceptibility loci and tissue-specific super-enhancers in breast cancers

Somatic rearrangements contribute to the mutagenized landscape of cancer genomes. Here, we systematically interrogated rearrangements in 560 breast cancers by using a piecewise constant fitting approach. We identified 33 hotspots of large (>100 kb) tandem duplications, a mutational signature associated with homologous-recombination-repair deficiency. Notably, these tandem-duplication hotspots were enriched in breast cancer germline susceptibility loci (odds ratio (OR) = 4.28) and breast-specific 'super-enhancer' regulatory elements (OR = 3.54). These hotspots may b

Regulation of transcription in hyperthermophilic archaea

The aim of the research presented here was to insight in the mechanisms by which transcription in hyperthermophilic archaea is regulated. To accomplish this, we have aimed (I) to identify transcriptional regulatory proteins from hyperthermophilic archaea, (II) to characterize these proteins, and (III) to determine how these proteins modulate the process of transcription initiation.Chapter 1 describes the characteristics of the archaeal transcription machinery, and compiles transcription-related data that was obtained in the past two decades. The archaeal transcription machinery appears to be a simplified version of the eukaryal RNA polymerase II system, lacking various general transcription factors that are essential for eukaryal transcription initiation. However, archaeal genomes encode TFE, a homologue eukaryal TFIIEatranscription factor. Its stimulatory role in transcription is described in Chapter 2 . Although the archaeal transcription machinery is eukaryal-like, many genes encoding members of bacterial regulatory protein families can be found within archaeal genomes. Members of the Lrp family are most abundantly present in archaea and Chapter 3 describes the properties of Lrp-like proteins. When this research project was started, fully sequenced archaeal genomes just became available . Only the gene encoding LrpA from P. furiosus had previously been identified in our laboratory and by others , and our initial strategy included the characterization of this protein, which is described in Chapter 4 . LrpA was shown to negatively autoregulate its own transcription in a ligand-independent manner. The efficient production and purification of recombinant LrpA enabled crystallization of the protein and Chapter 5 describes its resolved three-dimensional structure, which is the first structure of a member of the Lrp family. Subsequently, during the participation of our laboratory in the S.solfataricus P2 genome sequencing project, we were able to identify candidate regulatory genes in a more directed, bioinformatics-based approach, resulting in the identification and characterization of LysM and ChoR. LysM is another example of an archaeal Lrp-like protein, and in Chapter 6 we have used the genomic context of LysM in the S.solfataricus genome to experimentally identify its physiological target and ligand. This study indicates for the first time that an Lrp-like protein may activate archaeal transcriptional.Besides bacterial-like regulators, archaeal genomes encode unique archaeal-specific regulators that can be identified on the basis of a present DNA-binding domain. A putative regulator for c opper ho meostasis (ChoR) was identified in the S.solfataricus genome on the basis of a predicted HTH DNA-binding domain and a metal-binding domain. In Chapter 7 it is demonstrated that ChoR is indeed a metal-responsive DNA-binding protein that is most likely involved in the repression of a heavy metal-efflux system.Chapter 8 summarizes the data presented in this thesis, and adds some concluding remarks with respect to the implications of the work

The Lrp family of transcriptional regulators

Genome analysis has revealed that members of the Lrp family of transcriptional regulators are widely distributed among prokaryotes, both bacteria and archaea. The archetype Leucine-responsive Regulatory Protein from Escherichia coli is a global regulator involved in modulating a variety of metabolic functions, including the catabolism and anabolism of amino acids as well as pili synthesis. Most Lrp homologues, however, appear to act as specific regulators of amino acid metabolism-related genes. Like most prokaryotic transcriptional regulators, Lrp-like regulators consist of a DNA-binding domain and a ligand-binding domain. The crystal structure of the Pyrococcus furiosus LrpA revealed an N-terminal domain with a common helixturnhelix fold, and a C-terminal domain with a typical -sandwich fold. The latter regulatory domain constitutes a novel ligand-binding site and has been designated RAM. Database analysis reveals that the RAM domain is present in many prokaryotic genomes, potentially encoding (1) Lrp-homologues, when fused to a DNA-binding domain (2) enzymes, when fused as a potential regulatory domain to a catalytic domain, and (3) stand-alone RAM modules with unknown function. The architecture of Lrp regulators with two distinct domains that harbour the regulatory (effector-binding) site and the active (DNA-binding) site, and their separation by a flexible hinge region, suggests a general allosteric switch of Lrp-like regulators

The Lrp family of transcriptional regulators

Genome analysis has revealed that members of the Lrp family of transcriptional regulators are widely distributed among prokaryotes, both bacteria and archaea. The archetype Leucine-responsive Regulatory Protein from Escherichia coli is a global regulator involved in modulating a variety of metabolic functions, including the catabolism and anabolism of amino acids as well as pili synthesis. Most Lrp homologues, however, appear to act as specific regulators of amino acid metabolism-related genes. Like most prokaryotic transcriptional regulators, Lrp-like regulators consist of a DNA-binding domain and a ligand-binding domain. The crystal structure of the Pyrococcus furiosus LrpA revealed an N-terminal domain with a common helixturnhelix fold, and a C-terminal domain with a typical -sandwich fold. The latter regulatory domain constitutes a novel ligand-binding site and has been designated RAM. Database analysis reveals that the RAM domain is present in many prokaryotic genomes, potentially encoding (1) Lrp-homologues, when fused to a DNA-binding domain (2) enzymes, when fused as a potential regulatory domain to a catalytic domain, and (3) stand-alone RAM modules with unknown function. The architecture of Lrp regulators with two distinct domains that harbour the regulatory (effector-binding) site and the active (DNA-binding) site, and their separation by a flexible hinge region, suggests a general allosteric switch of Lrp-like regulators