Novel Interaction of the Z-DNA Binding Domain of Human ADAR1 with the Oncogenic c-Myc Promoter G-Quadruplex

Both G-quadruplex and Z-DNA can be formed in G-rich and repetitive sequences on genome, and their formation and biological functions are controlled by specific proteins. Z-DNA binding proteins, such as human ADAR1, have a highly conserved Z-DNA binding domain having selective affinity to Z-DNA. Here, our study identifies the Z-DNA binding domain of human ADAR1 (hZα_ADAR1) as a novel G-quadruplex binding protein that recognizes c-myc promoter G-quadruplex formed in NHEIII₁ region and represses the gene expression. An electrophoretic migration shift assay shows the binding of hZα_ADAR1 to the intramolecular c-myc promoter G-quadruplex-forming DNA oligomer. To corroborate the binding of hZα_ADAR1 to the G-quadruplex, we conducted CD and NMR chemical shift perturbation analyses. CD results indicate that hZα_ADAR1 stabilizes the parallel-stranded conformation of the c-myc G-quadruplex. The NMR chemical shift perturbation data reveal that the G-quadruplex binding region in hZα_ADAR1 was almost identical with the Z-DNA binding region. Finally, promoter assay and Western blot analysis show that hZα_ADAR1 suppresses the c-myc expression promoted by NHEIII₁ region containing the G-quadruplex-forming sequence. This finding suggests a novel function of Z-DNA binding protein as a regulator of G-quadruplex-mediated gene expression.     

Posttranslational Regulation of Human DNA Polymerase ι

Human DNA polymerases (pols) η and ι are Y-family DNA polymerase paralogs that facilitate translesion synthesis past damaged DNA. Both polη and polι can be monoubiquitinated in vivo. Polη has been shown to be ubiquitinated at one primary site. When this site is unavailable, three nearby lysines may become ubiquitinated. In contrast, mass spectrometry analysis of monoubiquitinated polι revealed that it is ubiquitinated at over 27 unique sites. Many of these sites are localized in different functional domains of the protein, including the catalytic polymerase domain, the proliferating cell nuclear antigen-interacting region, the Rev1-interacting region, and its ubiquitin binding motifs UBM1 and UBM2. Polι monoubiquitination remains unchanged after cells are exposed to DNA-damaging agents such as UV light (generating UV photoproducts), ethyl methanesulfonate (generating alkylation damage), mitomycin C (generating interstrand cross-links), or potassium bromate (generating direct oxidative DNA damage). However, when exposed to naphthoquinones, such as menadione and plumbagin, which cause indirect oxidative damage through mitochondrial dysfunction, polι becomes transiently polyubiquitinated via Lys¹¹- and Lys⁴⁸-linked chains of ubiquitin and subsequently targeted for degradation. Polyubiquitination does not occur as a direct result of the perturbation of the redox cycle as no polyubiquitination was observed after treatment with rotenone or antimycin A, which both inhibit mitochondrial electron transport. Interestingly, polyubiquitination was observed after the inhibition of the lysine acetyltransferase KATB3/p300. We hypothesize that the formation of polyubiquitination chains attached to polι occurs via the interplay between lysine acetylation and ubiquitination of ubiquitin itself at Lys¹¹ and Lys⁴⁸ rather than oxidative damage per se.     

Isolation and characterization of the plasma membrane from the yeast Pichia pastoris

Despite similarities of cellular membranes in all eukaryotes, every compartment displays characteristic and often unique features which are important for the functions of the specific organelles. In the present study, we biochemically characterized the plasma membrane of the methylotrophic yeast Pichia pastoris with emphasis on the lipids which form the matrix of this compartment. Prerequisite for this effort was the design of a standardized and reliable isolation protocol of the plasma membrane at high purity. Analysis of isolated plasma membrane samples from P. pastoris revealed an increase of phosphatidylserine and a decrease of phosphatidylcholine compared to bulk membranes. The amount of saturated fatty acids in the plasma membrane was higher than in total cell extracts. Ergosterol, the final product of the yeast sterol biosynthetic pathway, was found to be enriched in plasma membrane fractions, although markedly lower than in Saccharomyces cerevisiae. A further characteristic feature of the plasma membrane from P. pastoris was the enrichment of inositol phosphorylceramides over neutral sphingolipids, which accumulated in internal membranes. The detailed analysis of the P. pastoris plasma membrane is discussed in the light of cell biological features of this microorganism especially as a microbial cell factory for heterologous protein production.     

Insect clocks: implication in an effective pest management

Abstract

Circadian rhythms are endogenous, and synchronize biological functions at the most suited time of the day. Insects like other organism display a wide array of circadian functions, which are controlled by a central timing system, in coordination with the peripheral clocks found in many tissues. Many insect behaviours including locomotion, courtship, mating, egg laying and photoperiodism, are influenced by the circadian systems. In this mini-review, we briefly describe the involvement of circadian clocks in various physiological processes and correlate their functions in insect life, focusing on lepidopteran pests (major group of Indian crop pests) and discuss their role(s) in the development of effective pest management strategy.     

Sperm number trumps sperm size in mammalian ejaculate evolution

Postcopulatory sexual selection is widely accepted to underlie the extraordinary diversification of sperm morphology. However, why does it favour longer sperm in some taxa but shorter in others? Two recent hypotheses addressing this discrepancy offered contradictory explanations. Under the sperm dilution hypothesis, selection via sperm density in the female reproductive tract favours more but smaller sperm in large, but the reverse in small, species. Conversely, the metabolic constraint hypothesis maintains that ejaculates respond positively to selection in small endothermic animals with high metabolic rates, whereas low metabolic rates constrain their evolution in large species. Here, we resolve this debate by capitalizing on the substantial variation in mammalian body size and reproductive physiology. Evolutionary responses shifted from sperm length to number with increasing mammalian body size, thus supporting the sperm dilution hypothesis. Our findings demonstrate that body-size-mediated trade-offs between sperm size and number can explain the extreme diversification in sperm phenotypes.     

A novel insight into the cost–benefit model for the evolution of botanical carnivory

Background The cost–benefit model for the evolution of botanical carnivory provides a conceptual framework for interpreting a wide range of comparative and experimental studies on carnivorous plants. This model assumes that the modified leaves called traps represent a significant cost for the plant, and this cost is outweighed by the benefits from increased nutrient uptake from prey, in terms of enhancing the rate of photosynthesis per unit leaf mass or area (A_N) in the microsites inhabited by carnivorous plants.Scope This review summarizes results from the classical interpretation of the cost–benefit model for evolution of botanical carnivory and highlights the costs and benefits of active trapping mechanisms, including water pumping, electrical signalling and accumulation of jasmonates. Novel alternative sequestration strategies (utilization of leaf litter and faeces) in carnivorous plants are also discussed in the context of the cost–benefit model.Conclusions Traps of carnivorous plants have lower A_N than leaves, and the leaves have higher A_N after feeding. Prey digestion, water pumping and electrical signalling represent a significant carbon cost (as an increased rate of respiration, R_D) for carnivorous plants. On the other hand, jasmonate accumulation during the digestive period and reprogramming of gene expression from growth and photosynthesis to prey digestion optimizes enzyme production in comparison with constitutive secretion. This inducibility may have evolved as a cost-saving strategy beneficial for carnivorous plants. The similarities between plant defence mechanisms and botanical carnivory are highlighted.     

Root growth dynamics linked to above-ground growth in walnut (Juglans regia)

Background and Aims Examination of plant growth below ground is relatively scant compared with that above ground, and is needed to understand whole-plant responses to the environment. This study examines whether the seasonal timing of fine root growth and the spatial distribution of this growth through the soil profile varies in response to canopy manipulation and soil temperature.Methods Plasticity in the seasonal timing and vertical distribution of root production in response to canopy and soil water manipulation was analysed in field-grown walnut (Juglans regia ‘Chandler’) using minirhizotron techniques.Key Results Root production in walnuts followed a unimodal curve, with one marked flush of root growth starting in mid-May, with a peak in mid-June. Root production declined later in the season, corresponding to increased soil temperature, as well as to the period of major carbohydrate allocation to reproduction. Canopy and soil moisture manipulation did not influence the timing of root production, but did influence the vertical distribution of roots through the soil profile. Water deficit appeared to promote root production in deeper soil layers for mining soil water. Canopy removal appeared to promote shallow root production.Conclusions The findings of this study add to growing evidence that root growth in many ecosystems follows a unimodal curve with one marked flush of root growth in coordination with the initial leaf flush of the season. Root vertical distribution appeared to have greater plasticity than timing of root production in this system, with temperature and/or carbohydrate competition constraining the timing of root growth. Effects on root distribution can have serious impacts on trees, with shallow rooting having negative impacts in years with limited soil water or positive impacts in years with wet springs, and deep rooting having positive impacts on soil water mining from deeper soil layers but negative impacts in years with wet springs.     

Cub growth and maternal care in cheetahs

Using cub growth as an index, I examine the influence of maternalnutrition, litter size, and cub sex on maternal care in cheetahs(Acinonyx jubatus) and compare cub and litter growth rates withthose of other large feilds. Seventy-nine free-living cheetahcubs in 21 litters from 15 mothers were weighed at least oncebetween 6 and 48 days of age. Eleven litters were weighed atthe beginning and end of a 5-day observation of their mothers.The mean cub growth rate varied significantly between litters,due primarily to differences in maternal food intake. Growthdeclined sharply when maternal food intake was less than 1.5kg/ day, but did not increase with greater levels of food intake.Lower limits of growth rates may therefore have been set bythe mother's food intake, whereas upper limits may be set bythe intrinsic physiological ability of cubs to grow. Althoughmale cubs were heavier than female cubs in the same litter whenfirst weighed, major differences in growth rate between thesexes were not apparent at this stage. Both cheetah cubs andlitters grow fast relative to other large felids, and I arguethat this may be an adaptation to the high rate of cheetah juvenilemortality from predation.     

Effects of Peanut Genotypes on Meloidogyne Species Interactions

A 3-year microplot study was conducted to characterize the interaction between Meloidogyne arenaria race 1 (MA1) and M. hapla (MH), as affected by the five peanut genotypes: Florigiant, NC 7, NC 6, NC Ac 18416, and NC Ac 18016. The interactive effects on infection (total parasitic forms per root unit) and reproduction potentials of each nematode species and crop damage were determined. As a single population, MA1 had greater infection capacity and caused more crop damage than did MH, but both species had similar reproduction potentials. In mixed infestations, MA1 was more competitive than MH, as reflected by incidence of infection. Infection and reproduction potentials, and crop-damage capabilities of the mixed populations were similar to those of MA1 alone. All peanut genotypes were susceptible to infection by both nematodes. NC 6 was less susceptible to damage by MA1 and the mixed populations than other genotypes. A nematode treatment x genotype interaction was detected for root infection and crop damage, but not for population density or reproduction. With high preplant nematode levels (Pi), the populations reached their peak by midseason, whereas those with low Pi peaked after midseason. Crop damage in the second and third years was correlated with Pi level.     

Multiple origins of Southern Hemisphere Anemone (Ranunculaceae) based on plastid and nuclear sequence data

 Using two molecular data sets, the plastid atpB-rbcL intergenic spacer region and the nuclear ribosomal internal transcribed spacer regions (ITS), the taxonomic affinities of two newly available Anemone species from the Southern Hemisphere were tested. From previous work based on morphology and geographic distribution, it was assumed that A. tenuicaulis from New Zealand was most closely related to the Tasmanian A. crassifolia, whereas the affinity of A. antucensis from Chile and Argentina was regarded as uncertain. Analyses of molecular sequence data from these and 18 other species of Anemone s.lat. (with Clematis as outgroup) result in trees largely congruent with past analyses based on morphology and plastid restriction site data. They strongly support A. richardsonii and A. canadensis (with boreal distributions in the Northern Hemisphere) as paraphyletic to a well supported Southern Hemisphere clade consisting of A. antucensis and A. tenuicaulis. This group of four species is part of an otherwise predominantly Northern Hemisphere assemblage (subgenus Anemonidium s.lat., chromosome base number x=7), including A. narcissiflora, A. obtusiloba, A. keiskeana and A. (=Hepatica) americana. All other austral species included in the present sampling, A. crassifolia (Tasmania), A. knowltonia (=Knowltonia capensis), and A. caffra (both South African), form a separate clade, sister to A. (=Pulsatilla) occidentalis and other Northern Hemisphere anemones (subgenus Anemone s.lat., x=8). Possible phytogeographical links of the Southern Hemisphere species are discussed. Received April 23, 2001 Accepted October 4, 2001