An overview of the structures of protein-DNA complexes

On the basis of a structural analysis of 240 protein-DNA complexes contained in the Protein Data Bank (PDB), we have classified the DNA-binding proteins involved into eight different structural/functional groups, which are further classified into 54 structural families. Here we present this classification and review the functions, structures and binding interactions of these protein-DNA complexes.     

Leakage of a liquid 188Re-filled balloon system during intracoronary brachytherapy. A case report

We are reporting the first case of an accidental radioactive 188Re leakage of a liquid-filled balloon system. Different analytical methods estimated that approximately 4 mCi 188Re were released. The radiation burden was reduced considerably by the combined therapy with perchlorate and forced volume diuresis. Estimated exposures to all organs were very low with 1.8 rad. A total body nuclear scintigraphy demonstrated uniform 188Re distribution, without specific organ concentration.     

Filamentous growth of Saccharomyces cerevisiae is regulated by manganese

The Candida albicans INT1 gene is a virulence factor that contributes to both adhesion and filamentous growth of the fungus. Expression of INT1 in the budding yeast Saccharomyces cerevisiae directs both adhesion and filamentous growth. Because Int1p contains two predicted divalent cation-binding motifs, we asked whether divalent cations are important for the role of Int1p in filament formation. In this study, we found that INT1-induced filamentous growth (I-IFG) is sensitive to the divalent cation chelator EDTA and that this EDTA sensitivity can be ameliorated by the addition of Mn(2+), but not Mg(2+) or Ca(2+) ions. The addition of MnCl(2) restored both the proportion of cells forming filaments and the length of filaments formed. Expression of INT1 in S. cerevisiae mutants that reduce the intracellular concentration of Mn(2+) did not affect I-IFG. Interestingly, the Mn(2+) dependence of I-IFG is not dependent upon the presence of the putative divalent cation-binding domains found in INT1. Rather, we found that polarized growth induced by mutations in CDC12 and CLA4 or by expression of excess SWE1 was also sensitive to EDTA treatment and was restored by the addition of MnCl(2) but not by the addition of CaCl(2). Thus, our results suggest that in S. cerevisiae polarized growth is dependent upon the presence of Mn(2+) ions.     

Insertion of telomere repeat sequence decreases plasmid DNA condensation by cobalt (III) hexaammine.

Telomere repeat sequence (TRS) DNA is found at the termini of most eukaryotic chromosomes. The sequences are highly repetitive and G-rich (e.g., [C(1-3)A/TG(1-3)]n for the yeast Saccharomyces cerevisiae) and are packaged into nonnucleosomal protein-DNA structures in vivo. We have used total intensity light scattering and electron microscopy to monitor the effects of yeast TRS inserts on in vitro DNA condensation by cobalt (III) hexaammine. Insertion of 72 bp of TRS into a 3.3-kb plasmid depresses condensation as seen by light scattering and results in a 22% decrease in condensate thickness as measured by electron microscopy. Analysis of toroidal condensate dimensions suggests that the growth stages of condensation are inhibited by the presence of a TRS insert. The depression in total light scattering intensity is greater when the plasmid is linearized with the TRS at an end (39-49%) than when linearized with the TRS in the interior (18-22%). Circular dichroism of a 95-bp fragment containing the TRS insert gives a spectrum that is intermediate between the A-form and B-form, and the anomalous condensation behavior of the TRS suggests a noncanonical DNA structure. We speculate that under conditions in which the plasmid DNA condenses, the telomeric insert assumes a helical geometry that is similar to the A-form and is incompatible with packing into the otherwise B-form lattice of the condensate interior.     

Characterization of SPACR, a sialoprotein associated with cones and rods present in the interphotoreceptor matrix of the human retina: immunological and lectin binding analysis

Rod and cone photoreceptors project from the outer retinal surface into a carbohydrate-rich interphotoreceptor matrix (IPM). Unique IPM glycoconjugates are distributed around rods and cones. Wheat germ agglutinin (WGA) strongly decorates the rod matrix domains and weakly decorates the cone matrix domains. This study characterizes the major WGA-binding glycoprotein in the human IPM, which we refer to as SPACR (sialoprotein associated with cones and rods). SPACR, which has a molecular weight of 147 kDa, was isolated and purified from the IPM by lectin affinity chromatography. A polyclonal antibody to SPACR was prepared that colocalizes in tissue preparations with WGA-binding domains in the IPM. Sequential digestion of SPACR with N- and O- glycosidases results in a systematic increase in electrophorectic mobility, indicating the presence of both N- and O-linked glycoconjugates. Complete deglycosylation results in a reduction in the relative molecular mass of SPACR by about 30%. Analysis of lectin binding allowed us to identify some of the structural characteristics of SPACR glycoconjugates. Treatment with neuraminidase exposes Galbeta1- 3GalNAc disaccharide as indicated by positive peanut agglutinin (PNA) staining, accompanied by the loss of WGA staining. Maackia amurensis agglutinins (MAA-1 and MAA-2), specific for sialic acid in alpha2-3 linkage to Gal, bind SPACR, while Sambucus nigra agglutinin (SNA), specific for alpha2-6 linked sialic acid, does not, indicating that the dominant glycoconjugate determinant on SPACR is the O-linked carbohydrate, NeuAcalpha2-3Galbeta1-3GalNAc. The abundance of sialic acid in SPACR suggests that this glycoprotein may contribute substantially to the polyanionic nature of the IPM. The carbohydrate chains present on SPACR could also provide sites for extensive crosslinking and participate in the formation of the ordered IPM lattice that surrounds the elongate photoreceptors projecting from the outer retinal surface.      

The structure, catalytic mechanism and regulation of adenylyl cyclase

The recent structure determinations of the mammalian effector enzyme adenylyl cyclase reveal the structure of its catalytic core, provide new insights into its catalytic mechanism and suggest how diverse signaling molecules regulate its activity.     

Cloning and Functional Characterization of the Maize (Zea mays L.) Carotenoid Epsilon Hydroxylase Gene

The assignment of functions to genes in the carotenoid biosynthesis pathway is necessary to understand how the pathway is regulated and to obtain the basic information required for metabolic engineering. Few carotenoid ε-hydroxylases have been functionally characterized in plants although this would provide insight into the hydroxylation steps in the pathway. We therefore isolated mRNA from the endosperm of maize (Zea mays L., inbred line B73) and cloned a full-length cDNA encoding CYP97C19, a putative heme-containing carotenoid ε hydroxylase and member of the cytochrome P450 family. The corresponding CYP97C19 genomic locus on chromosome 1 was found to comprise a single-copy gene with nine introns. We expressed CYP97C19 cDNA under the control of the constitutive CaMV 35S promoter in the Arabidopsis thaliana lut1 knockout mutant, which lacks a functional CYP97C1 (LUT1) gene. The analysis of carotenoid levels and composition showed that lutein accumulated to high levels in the rosette leaves of the transgenic lines but not in the untransformed lut1 mutants. These results allowed the unambiguous functional annotation of maize CYP97C19 as an enzyme with strong zeinoxanthin ε-ring hydroxylation activity.     

Cold-hardiness of the phoretic mites Anoetus myrmicarum (Acariformes,Anoetidae) from the ants Formica lemani (Hymenoptera,Formicidae) in the Kolyma Highland

In the sparse larch forests of the upper Kolyma River, hypopi of the mite Anoetus myrmicarum (Scheucher, 1957) were found in several nests of the ant Formica lemani Bondroit, 1917. These mites were not found in hundreds of nests of the other 10 ant species examined in northeastern Asia. A possible ecological and physiological conditionality of the restricted distribution of phoretic mites was analyzed. For this purpose, coldhardiness of mites and their ant hosts, the biotopic distribution and the structure of nests, and the temperature conditions of overwintering were examined. At the stage of hypopus, the mites overwintered on ants in the overcooled stage; their mean supercooling temperatures (SCP) varied from ?25.8 ± 0.3°C to ?27.7 ± 0.4°C (min ?32.2°C, n = 157). These values were by 0.1 to 7.0°C lower than the mean SCP of the ants from 8 tested nests of F. lemani (?20.7 ± 0.5°C to ?25.7 ± 0.8°C). The soil temperatures at the level of winter chambers varied from ?12°C to ?15°C. Scarcity of findings of Anoetus myrmicarum in the Kolyma Highland is not associated with the limited cold-hardiness of the examined stages, but is most probably determined by interrelations between mites and ants.     

Genomic Plasticity of the Human Fungal Pathogen Candida albicans

The genomic plasticity of Candida albicans, a commensal and common opportunistic fungal pathogen, continues to reveal unexpected surprises. Once thought to be asexual, we now know that the organism can generate genetic diversity through several mechanisms, including mating between cells of the opposite or of the same mating type and by a parasexual reduction in chromosome number that can be accompanied by recombination events (2, 12, 14, 53, 77, 115). In addition, dramatic genome changes can appear quite rapidly in mitotic cells propagated in vitro as well as in vivo. The detection of aneuploidy in other fungal pathogens isolated directly from patients (145) and from environmental samples (71) suggests that variations in chromosome organization and copy number are a common mechanism used by pathogenic fungi to rapidly generate diversity in response to stressful growth conditions, including, but not limited to, antifungal drug exposure. Since cancer cells often become polyploid and/or aneuploid, some of the lessons learned from studies of genome plasticity in C. albicans may provide important insights into how these processes occur in higher-eukaryotic cells exposed to stresses such as anticancer drugs.The purpose of this review is to describe the tools used to detect genome changes, to highlight recent advances in our understanding of large-scale chromosome changes that arise in Candida albicans, and to discuss the role of specific stresses in eliciting these genome changes. The types of genomic diversity that have been characterized suggest that C. albicans can undergo extreme genomic changes in order to survive stresses in the human host. We propose that C. albicans and other pathogens may have evolved mechanisms not only to tolerate but also to generate large-scale genetic variation as a means of adaptation.C. albicans is a polymorphic yeast with a 16-Mb (haploid) genome organized in 8 diploid chromosomes (140, 154, 203). The C. albicans genome displays a very high degree of plasticity. This plasticity includes the types of genomic changes frequently observed with cancer cells, including gross chromosomal rearrangements, aneuploidy, and loss of heterozygosity (reviewed in references 100, 117, and 157). Similar to somatic cancer cells, C. albicans reproduces primarily through asexual clonal division (65, 84). Nonetheless, it has retained much of the machinery needed for mating and meiosis (189), yet meiosis has never been observed (13, 120).C. albicans has two mating-type-like (MTL) alleles, MTLa and MTLα (76). The MTL locus is on the left arm of chromosome 5 (Chr5), approximately 80 kbp from the centromere. Most C. albicans isolates are heterozygous for the MTL locus, but approximately 3 to 10% of clinical isolates are naturally homozygous at MTL (104, 108). Mating can occur between strains carrying the opposite MTL locus, and most strains that were found to be naturally MTL homozygous are mating competent (104, 108). MTL-homozygous strains were also constructed from MTL-heterozygous strains by deletion of either the MTLa or MTLα locus (77) or by selection for Chr5 loss on sorbose (87, 115).Mating between these diploid strains of opposite mating type can occur both in vitro (115) and in vivo (77, 97). The products are tetraploid and do not undergo a conventional meiotic reduction in ploidy (12, 120). Rather, they undergo random loss of multiple chromosomes, a process termed “concerted chromosome loss,” until they reach a near-diploid genome content (2, 12, 53, 85). A subset of these cells also undergoes multiple gene conversion events reminiscent of meiotic recombination, and most remain trisomic for one to several chromosomes (53). While mating and concerted chromosome loss have been induced in the laboratory, the role of the parasexual cycle during the host-pathogen interaction and in the response to stresses, such as exposure to antifungal drugs, remains unclear. The prevailing model is that adaptive mutations (such as those that occur with the acquisition of drug resistance) evolve through somatic events, including point mutations, recombination, gene conversion, loss of heterozygosity, and/or aneuploidy (13).