Native human TATA-binding protein simultaneously binds and bends promoter DNA without a slow isomerization step or TFIIB requirement

The association of TATA-binding protein (TBP) with promoter DNA is central to the initiation and regulation of eukaryotic protein synthesis. Our laboratory has previously conducted detailed investigations of this interaction using yeast TBP and seven consensus and variant TATA sequences. We have now investigated this key interaction using human TBP and the TATA sequence from the adenovirus major late promoter (AdMLP). Recombinant native human protein was used together with fluorescently labeled DNA, allowing real time data acquisition in solution. We find that the wild-type hTBP-DNAAdMLP reaction is characterized by high affinity (Kd < or = 5 nm), simultaneous binding and DNA bending, and rapid formation of a stable human TBP-DNA complex having DNA bent approximately 100 degrees. These data allow, for the first time, a direct comparison of the reactions of the full-length, native human and yeast TBPs with a consensus promoter, studied under identical conditions. The general reaction characteristics are similar for the human and yeast proteins, although the details differ and the hTBPwt-induced bend is more severe. This directly measured hTBPwt-DNAAdMLP interaction differs fundamentally from a recently published hTBPwt-DNAAdMLP model characterized by low affinity (microM) binding and an unstable complex requiring either a 30-min isomerization or TFIIB to achieve DNA bending. Possible sources of these significant differences are discussed.     

Role of protein kinase C alpha for uptake of unopsonized prey and phagosomal maturation in macrophages

Protein kinase C alpha (PKC alpha) participates in F-actin remodeling during phagocytosis and phagosomal maturation in macrophages. Leishmania donovani promastigotes, which inhibit phagosomal maturation, cause accumulation of periphagosomal F-actin instead of the disassembly observed around other prey [Cell. Microbiol. 7 (2001) 439]. This accumulation is induced by promastigote lipophosphoglycan (LPG), which has several effects on macrophages including inhibition of PKC alpha. To investigate a possible connection between PKC alpha and LPG's effects on actin dynamics, we utilized RAW264.7 macrophages overexpressing dominant-negative PCK alpha (DN PKC alpha). We found increased cortical F-actin and decreased phagocytic capacity, as well as defective periphagosomal F-actin breakdown and inhibited phagosomal maturation in the DN PKC alpha-overexpressing cells, effects similar to those seen in controls subjected to LPG-coated prey. The results indicate that PKC alpha is involved in F-actin turnover in macrophages and that PKC alpha-dependent breakdown of periphagosomal F-actin is required for phagosomal maturation, and endorse the hypothesis that intracellular survival of L. donovani involves inhibition of PKC alpha by LPG.     

DNA-protein cross-linking via guanine oxidation: dependence upon protein and photosensitizer

DNA-protein cross-links form when guanine undergoes a 1-electron oxidation in a flash-quench experiment, and the importance of reactive oxygen species, protein, and photosensitizer is examined here. In these experiments, a strong oxidant produced by oxidative quenching of a DNA-bound photosensitizer generates an oxidized guanine base that reacts with protein to form the covalent adduct. These cross-links are cleaved by hot piperidine and are not the result of reactive oxygen species, since neither a hydroxyl radical scavenger (mannitol) nor oxygen affects the yield of DNA-histone cross-linking, as determined via a chloroform extraction assay. The cross-linking yield depends on protein, decreasing as histone > cytochrome c > bovine serum albumin. The yield does not depend on the cytochrome oxidation state, suggesting that reduction of the guanine radical by ferrocytochrome c does not compete effectively with cross-linking. The photosensitizer strongly influences the cross-linking yield, which decreases in the order Ru(phen)(2)dppz(2+) [phen = 1,10-phenanthroline; dppz = dipyridophenazine] > Ru(bpy)(3)(2+) [bpy = 2,2'-bipyridine] > acridine orange > ethidium, in accordance with measured oxidation potentials. A long-lived transient absorption signal for ethidium dication in poly(dG-dC) confirms that guanine oxidation is inefficient for this photosensitizer. From a polyacrylamide sequencing gel of a (32)P-labeled 40-mer, all of these photosensitizers are shown to damage guanines preferentially at the 5' G of 5'-GG-3' steps, consistent with a 1-electron oxidation. Additional examination of ethidium shows that it can generate cross-links between histone and plasmid DNA (pUC19) and that the yield depends on the quencher. Altogether, these results illustrate the versatility of the flash-quench technique as a way to generate physiologically relevant DNA-protein adducts via the oxidation of guanine and expand the scope of such cross-linking reactions to include proteins that may associate only transiently with DNA.     

Viability selection at three early life stages of the tropical tree,Platypodium elegans (Fabaceae,Papilionoideae)

Given the enormous number and high mortality of fertilized ovules in plants, it is possible that selection during the earliest stages of the life cycle plays an important role in shaping the genetic composition of plant populations. Previous research involving selection component analyses found strong evidence for viability selection in annual plant species. Yet despite this evidence, few attempts have been made to identify the magnitude and timing of viability selection as well as the mechanisms responsible for mortality among genotypes. Platypodium elegans, a Neotropical tree with high rates of early fruit mortality, represents an opportunity to study viability selection at a level of discernment not previously possible. Microsatellite markers were used to analyze the genetic composition of aborted embryos, as well as mature seeds and seedlings of the same cohort. While selection resulted in an overall decrease in self-fertilized progeny across each life stage, the greatest change in the genetic composition of progeny occurred between mature seeds and established seedlings. This suggested that inbreeding depression, and not late-acting self-incompatibility, was responsible for early selection. An investigation of the mature seed stage revealed that self-fertilized seeds weigh significantly less than outcrossed seeds. The result of this early selection conceals the mixed-mating system and high levels of inbreeding depression in Platypodium elegans, resulting in an apparently outcrossed adult population that does not differ significantly from Hardy-Weinberg expectations.     

Defective stress kinase and Bak activation in response to ionizing radiation but not cisplatin in a non-small cell lung carcinoma cell line

We have here examined ionizing radiation (IR)-induced apoptotic signaling in one IR-sensitive small cell lung carcinoma (SCLC) and one resistant non-small cell lung carcinoma (NSCLC) cell line, both harboring mutant p53. In the sensitive SCLC cell line, IR induced conformational modulation of Bak and Bax, mitochondrial depolarization, and nuclear fragmentation. These events were not observed in the IR-resistant NSCLC cell line. However, in the same cells, cisplatin, a DNA-damaging drug, induced Bak and Bax modulation, mitochondrial depolarization, and nuclear fragmentation. Pre-mitochondrial signaling events were examined in order to further characterize the differing IR response. In the SCLC cell line, IR-induced apoptotic signaling was found to involve a MEKK1-related pathway and activation of the stress-activated kinases JNK and p38. In comparison, the NSCLC cell line had higher basal levels of activity of JNK and p38, and IR treatment did not further activate these kinases. However, NSCLC cells were sensitive to Bak modulation and apoptosis induced by a kinase-active mutant of MEKK1. Together, the results delineate a mechanism of IR resistance in NSCLC cells and indicate that IR and cisplatin induce Bak modulation and apoptosis via different pathways.     

High-resolution crossover maps for each bivalent of Zea mays using recombination nodules

Recombination nodules (RNs) are closely correlated with crossing over, and, because they are observed by electron microscopy of synaptonemal complexes (SCs) in extended pachytene chromosomes, RNs provide the highest-resolution cytological marker currently available for defining the frequency and distribution of crossovers along the length of chromosomes. Using the maize inbred line KYS, we prepared an SC karyotype in which each SC was identified by relative length and arm ratio and related to the proper linkage group using inversion heterozygotes. We mapped 4267 RNs on 2080 identified SCs to produce high-resolution maps of RN frequency and distribution on each bivalent. RN frequencies are closely correlated with both chiasma frequencies and SC length. The total length of the RN recombination map is about twofold shorter than that of most maize linkage maps, but there is good correspondence between the relative lengths of the different maps when individual bivalents are considered. Each bivalent has a unique distribution of crossing over, but all bivalents share a high frequency of distal RNs and a severe reduction of RNs at and near kinetochores. The frequency of RNs at knobs is either similar to or higher than the average frequency of RNs along the SCs. These RN maps represent an independent measure of crossing over along maize bivalents.     

Effects of biotic and abiotic constraints on the symbiosis between rhizobia and the tropical leguminous trees Acacia and Prosopis

N2-fixing, drought tolerant and multipurpose Acacia and Prosopis species are appropriate trees for reforestation of degraded areas in arid and semiarid regions of the tropics and subtropics. Acacia and Prosopis trees form N2-fixing nodules with a wide range of rhizobia, for example African acacias mainly with Sinorhizobium sp. and Mesorhizobium sp., and Australian acacias with Bradyrhizobium sp. Although dry and hot seasons restrict formation of N2-fixing nodules on Acacia and Prosopis spp., fully grown trees and their symbiotic partners are well adapted to survive in harsh growth conditions. This review on one hand deals with major constraints of arid and semiarid soils, i.e. drought, salinity and high soil temperature, which affect growth of trees and rhizobia, and on the other hand with adaptation mechanisms by which both organisms survive through unfavourable periods. In addition, defects in infection and nodulation processes due to various abiotic and biotic constraints are reviewed. This knowledge is important when Acacia and Prosopis seedlings are used for forestation of degraded areas in arid and semiarid tropics.     

Phosphorylation of p40AUF1 regulates binding to A + U-rich mRNA-destabilizing elements and protein-induced changes in ribonucleoprotein structure

Messenger RNA turnover directed by A + U-rich elements (AREs) involves selected ARE-binding proteins. Whereas several signaling systems may modulate ARE-directed mRNA decay and/or post-translationally modify specific trans-acting factors, it is unclear how these mechanisms are linked. In THP-1 monocytic leukemia cells, phorbol ester-induced stabilization of some mRNAs containing AREs was accompanied by dephosphorylation of Ser83 and Ser87 of polysome-associated p40AUF1. Here, we report that phosphorylation of p40AUF1 influences its ARE-binding affinity as well as the RNA conformational dynamics and global structure of the p40AUF1-ARE ribonucleoprotein complex. Most notably, association of unphosphorylated p40AUF1 induces a condensed RNA conformation upon ARE substrates. By contrast, phosphorylation of p40AUF1 at Ser83 and Ser87 inhibits this RNA structural transition. These data indicate that selective AUF1 phosphorylation may regulate ARE-directed mRNA turnover by remodeling local RNA structures, thus potentially altering the presentation of RNA and/or protein determinants involved in subsequent trans-factor recruitment.     

Underproduction of sigma 70 mimics a stringent response. A proteome approach

When Escherichia coli cells enter stationary phase due to carbon starvation the synthesis of ribosomal proteins is rapidly repressed. In a DeltarelA DeltaspoT mutant, defective in the production of the alarmone guanosine tetraphosphate (ppGpp), this regulation of the levels of the protein synthesizing system is abolished. Using a proteomic approach we demonstrate that the production of the vast majority of detected E. coli proteins are decontrolled during carbon starvation in the DeltarelA DeltaspoT strain and that the starved cells behave as if they were growing exponentially. In addition we show that the inhibition of ribosome synthesis by the stringent response can be qualitatively mimicked by artificially lowering the levels of the housekeeping sigma factor, sigma(70). In other words, genes encoding the protein-synthesizing system are especially sensitive to reduced availability of sigma(70) programmed RNA polymerase. This effect is not dependent on ppGpp since lowering the levels of sigma(70) gives a similar but less pronounced effect in a ppGpp(0) strain. The data is discussed in view of the models advocating for a passive control of gene expression during stringency based on alterations in RNA polymerase availability.