Multi-marker DNA metabarcoding reveals spatial and sexual variation in the diet of a scarce woodland bird.

Avian diet can be affected by site-specific variables, such as habitat, as well as intrinsic factors such as sex. This can lead to dietary niche separation, which reduces competition between individuals, as well as impacting how well avian species can adapt to environmental variation. Estimating dietary niche separation is challenging, due largely to difficulties in accurately identifying food taxa consumed. Consequently, there is limited knowledge of the diets of woodland bird species, many of which are undergoing serious population declines. Here, we show the effectiveness of multi-marker fecal metabarcoding to provide in-depth dietary analysis of a declining passerine in the UK, the Hawfinch (Coccothraustes coccothraustes). We collected fecal samples from (n = 262) UK Hawfinches prior to, and during, the breeding seasons in 2016-2019. We detected 49 and 90 plant and invertebrate taxa, respectively. We found Hawfinch diet varied spatially, as well as between sexes, indicating broad dietary plasticity and the ability of Hawfinches to utilize multiple resources within their foraging environments.

Molecular insights into high-altitude adaption and acclimatisation of Aporrectodea caliginosa.

Here, we explore the high-altitude adaptions and acclimatisation of Aporrectodea caliginosa Population diversity is assessed through mitochondrial barcoding, identifying closely related populations across the island of Pico (Azores). We present the first megabase N50 assembly size (1.2 Mbp) genome for A. caliginosa High- and low-altitude populations were exposed experimentally to a range of oxygen and temperature conditions, simulating altitudinal conditions, and the transcriptomic responses explored. SNP densities are assessed to identify signatures of selective pressure and their link to differentially expressed genes. The high-altitude A. caliginosa population had lower differential expression and fewer co-expressed genes between conditions, indicating a more condition-refined epigenetic response. Genes identified as under adaptive pressure through Fst and nucleotide diversity in the high-altitude population clustered around the differentially expressed an upstream environmental response control gene, HMGB1. The high-altitude population of A. caliginosa indicated adaption and acclimatisation to high-altitude conditions and suggested resilience to extreme weather events. This mechanistic understanding could help offer a strategy in further identifying other species capable of maintaining soil fertility in extreme environments.

Seasonal and ontological variation in diet and age-related differences in prey choice, by an insectivorous songbird.

The diet of an individual animal is subject to change over time, both in response to short-term food fluctuations and over longer time scales as an individual ages and meets different challenges over its life cycle. A metabarcoding approach was used to elucidate the diet of different life stages of a migratory songbird, the Eurasian reed warbler (Acrocephalus scirpaceus) over the 2017 summer breeding season in Somerset, the United Kingdom. The feces of adult, juvenile, and nestling warblers were screened for invertebrate DNA, enabling the identification of prey species. Dietary analysis was coupled with monitoring of Diptera in the field using yellow sticky traps. Seasonal changes in warbler diet were subtle, whereas age class had a greater influence on overall diet composition. Age classes showed high dietary overlap, but significant dietary differences were mediated through the selection of prey; (i) from different taxonomic groups, (ii) with different habitat origins (aquatic vs. terrestrial), and (iii) of different average approximate sizes. Our results highlight the value of metabarcoding data for enhancing ecological studies of insectivores in dynamic environments.

Genome-wide chromatin mapping with size resolution reveals a dynamic sub-nucleosomal landscape in Arabidopsis.

All eukaryotic genomes are packaged as chromatin, with DNA interlaced with both regularly patterned nucleosomes and sub-nucleosomal-sized protein structures such as mobile and labile transcription factors (TF) and initiation complexes, together forming a dynamic chromatin landscape. Whilst details of nucleosome position in Arabidopsis have been previously analysed, there is less understanding of their relationship to more dynamic sub-nucleosomal particles (subNSPs) defined as protected regions shorter than the ~150bp typical of nucleosomes. The genome-wide profile of these subNSPs has not been previously analysed in plants and this study investigates the relationship of dynamic bound particles with transcriptional control. Here we combine differential micrococcal nuclease (MNase) digestion and a modified paired-end sequencing protocol to reveal the chromatin structure landscape of Arabidopsis cells across a wide particle size range. Linking this data to RNAseq expression analysis provides detailed insight into the relationship of identified DNA-bound particles with transcriptional activity. The use of differential digestion reveals sensitive positions, including a labile -1 nucleosome positioned upstream of the transcription start site (TSS) of active genes. We investigated the response of the chromatin landscape to changes in environmental conditions using light and dark growth, given the large transcriptional changes resulting from this simple alteration. The resulting shifts in the suites of expressed and repressed genes show little correspondence to changes in nucleosome positioning, but led to significant alterations in the profile of subNSPs upstream of TSS both globally and locally. We examined previously mapped positions for the TFs PIF3, PIF4 and CCA1, which regulate light responses, and found that changes in subNSPs co-localized with these binding sites. This small particle structure is detected only under low levels of MNase digestion and is lost on more complete digestion of chromatin to nucleosomes. We conclude that wide-spectrum analysis of the Arabidopsis genome by differential MNase digestion allows detection of sensitive features hereto obscured, and the comparisons between genome-wide subNSP profiles reveals dynamic changes in their distribution, particularly at distinct genomic locations (i.e. 5'UTRs). The method here employed allows insight into the complex influence of genetic and extrinsic factors in modifying the sub-nucleosomal landscape in association with transcriptional changes.

Gene dosage effect of WEE1 on growth and morphogenesis from arabidopsis hypocotyl explants.

BACKGROUND AND AIMS: How plant cell-cycle genes interface with development is unclear. Preliminary evidence from our laboratory suggested that over-expression of the cell cycle checkpoint gene, WEE1, repressed growth and development. Here the hypothesis is tested that the level of WEE1 has a dosage effect on growth and development in Arabidospis thaliana. To do this, a comparison was made of the development of gain- and loss-of-function WEE1 arabidopsis lines both in vivo and in vitro. METHODS: Hypocotyl explants from an over-expressing Arath;WEE1 line (WEE1(oe)), two T-DNA insertion lines (wee1-1 and wee1-4) and wild type (WT) were cultured on two-way combinations of kinetin and naphthyl acetic acid. Root growth and meristematic cell size were also examined. KEY RESULTS: Quantitative data indicated a repressive effect in WEE1(oe) and a significant increase in morphogenetic capacity in the two T-DNA insertion lines compared with WT. Compared with WT, WEE1(oe) seedlings exhibited a slower cell-doubling time in the root apical meristem and a shortened primary root, with fewer laterals, whereas there were no consistent differences in the insertion lines compared with WT. However, significantly fewer adventitious roots were recorded for WEE1(oe) and significantly more for the insertion mutant wee1-1. Compared with WT there was a significant increase in meristem cell size in WEE1(oe) for all three ground tissues but for wee1-1 only cortical cell size was reduced. CONCLUSIONS: There is a gene dosage effect of WEE1 on morphogenesis from hypocotyls both in vitro and in vivo.

Phytotracker, an information management system for easy recording and tracking of plants, seeds and plasmids.

BACKGROUND: A large number of different plant lines are produced and maintained in a typical plant research laboratory, both as seed stocks and in active growth. These collections need careful and consistent management to track and maintain them properly, and this is a particularly pressing issue in laboratories undertaking research involving genetic manipulation due to regulatory requirements. Researchers and PIs need to access these data and collections, and therefore an easy-to-use plant-oriented laboratory information management system that implements, maintains and displays the information in a simple and visual format would be of great help in both the daily work in the lab and in ensuring regulatory compliance. RESULTS: Here, we introduce 'Phytotracker', a laboratory management system designed specifically to organise and track plasmids, seeds and growing plants that can be used in mixed platform environments. Phytotracker is designed with simplicity of user operation and ease of installation and management as the major factor, whilst providing tracking tools that cover the full range of activities in molecular genetics labs. It utilises the cross-platform Filemaker relational database, which allows it to be run as a stand-alone or as a server-based networked solution available across all workstations in a lab that can be internet accessible if desired. It can also be readily modified or customised further. Phytotracker provides cataloguing and search functions for plasmids, seed batches, seed stocks and plants growing in pots or trays, and allows tracking of each plant from seed sowing, through harvest to the new seed batch and can print appropriate labels at each stage. The system enters seed information as it is transferred from the previous harvest data, and allows both selfing and hybridization (crossing) to be defined and tracked. Transgenic lines can be linked to their plasmid DNA source. This ease of use and flexibility helps users to reduce their time needed to organise their plants, seeds and plasmids and to maintain laboratory continuity involving multiple workers. CONCLUSION: We have developed and used Phytotracker for over five years and have found it has been an intuitive, powerful and flexible research tool in organising our plasmid, seed and plant collections requiring minimal maintenance and training for users. It has been developed in an Arabidopsis molecular genetics environment, but can be readily adapted for almost any plant laboratory research.

Perturbation of cytokinin and ethylene-signalling pathways explain the strong rooting phenotype exhibited by Arabidopsis expressing the Schizosaccharomyces pombe mitotic inducer, cdc25.

BACKGROUND: Entry into mitosis is regulated by cyclin dependent kinases that in turn are phosphoregulated. In most eukaryotes, phosphoregulation is through WEE1 kinase and CDC25 phosphatase. In higher plants a homologous CDC25 gene is unconfirmed and hence the mitotic inducer Schizosaccharomyces pombe (Sp) cdc25 has been used as a tool in transgenic plants to probe cell cycle function. Expression of Spcdc25 in tobacco BY-2 cells accelerates entry into mitosis and depletes cytokinins; in whole plants it stimulates lateral root production. Here we show, for the first time, that alterations to cytokinin and ethylene signaling explain the rooting phenotype elicited by Spcdc25 expression in Arabidopsis. RESULTS: Expressing Spcdc25 in Arabidopsis results in increased formation of lateral and adventitious roots, a reduction of primary root width and more isodiametric cells in the root apical meristem (RAM) compared with wild type. Furthermore it stimulates root morphogenesis from hypocotyls when cultured on two way grids of increasing auxin and cytokinin concentrations. Microarray analysis of seedling roots expressing Spcdc25 reveals that expression of 167 genes is changed by > 2-fold. As well as genes related to stress responses and defence, these include 19 genes related to transcriptional regulation and signaling. Amongst these was the up-regulation of genes associated with ethylene synthesis and signaling. Seedlings expressing Spcdc25 produced 2-fold more ethylene than WT and exhibited a significant reduction in hypocotyl length both in darkness or when exposed to 10 ppm ethylene. Furthermore in Spcdc25 expressing plants, the cytokinin receptor AHK3 was down-regulated, and endogenous levels of iPA were reduced whereas endogeous IAA concentrations in the roots increased. CONCLUSIONS: We suggest that the reduction in root width and change to a more isodiametric cell phenotype in the RAM in Spcdc25 expressing plants is a response to ethylene over-production. The increased rooting phenotype in Spcdc25 expressing plants is due to an increase in the ratio of endogenous auxin to cytokinin that is known to stimulate an increased rate of lateral root production. Overall, our data reveal important cross talk between cell division and plant growth regulators leading to developmental changes.

Spontaneous and evolutionary changes in the antibiotic resistance of Burkholderia cenocepacia observed by global gene expression analysis.

BACKGROUND: Burkholderia cenocepacia is a member of the Burkholderia cepacia complex group of bacteria that cause infections in individuals with cystic fibrosis. B. cenocepacia isolate J2315 has been genome sequenced and is representative of a virulent, epidemic CF strain (ET12). Its genome encodes multiple antimicrobial resistance pathways and it is not known which of these is important for intrinsic or spontaneous resistance. To map these pathways, transcriptomic analysis was performed on: (i) strain J2315 exposed to sub-inhibitory concentrations of antibiotics and the antibiotic potentiator chlorpromazine, and (ii) on spontaneous mutants derived from J2315 and with increased resistance to the antibiotics amikacin, meropenem and trimethoprim-sulfamethoxazole. Two pan-resistant ET12 outbreak isolates recovered two decades after J2315 were also compared to identify naturally evolved gene expression changes. RESULTS: Spontaneous resistance in B. cenocepacia involved more gene expression changes and different subsets of genes than those provoked by exposure to sub inhibitory concentrations of each antibiotic. The phenotype and altered gene expression in the resistant mutants was also stable irrespective of the presence of the priming antibiotic. Both known and novel genes involved in efflux, antibiotic degradation/modification, membrane function, regulation and unknown functions were mapped. A novel role for the phenylacetic acid (PA) degradation pathway genes was identified in relation to spontaneous resistance to meropenem and glucose was found to repress their expression. Subsequently, 20 mM glucose was found to produce greater that 2-fold reductions in the MIC of multiple antibiotics against B. cenocepacia J2315. Mutation of an RND multidrug efflux pump locus (BCAM0925-27) and squalene-hopene cyclase gene (BCAS0167), both upregulated after chlorpromazine exposure, confirmed their role in resistance. The recently isolated outbreak isolates had altered the expression of multiple genes which mirrored changes seen in the antibiotic resistant mutants, corroborating the strategy used to model resistance. Mutation of an ABC transporter gene (BCAS0081) upregulated in both outbreak strains, confirmed its role in B. cenocepacia resistance. CONCLUSIONS: Global mapping of the genetic pathways which mediate antibiotic resistance in B. cenocepacia has revealed that they are multifactorial, identified potential therapeutic targets and also demonstrated that putative catabolite repression of genes by glucose can improve antibiotic efficacy.

Enacyloxins are products of an unusual hybrid modular polyketide synthase encoded by a cryptic Burkholderia ambifaria Genomic Island.

Gram-negative Burkholderia cepacia complex (Bcc) isolates were screened for antimicrobial activity against cystic fibrosis microbial pathogens, and the ability of B. ambifaria to inhibit B. multivorans was identified. The activity was mapped to a cluster of cryptic, quorum-sensing-regulated modular polyketide synthase (PKS) genes. Enacyloxin IIa and its stereoisomer designated iso-enacyloxin IIa were identified as metabolic products of the gene cluster, which encoded an unusual hybrid modular PKS consisting of multiple proteins with sequence similarity to cis-acyltransferase (cis-AT) PKSs and a single protein with sequence similarity to trans-AT PKSs. The discovery of the potent activity of enacyloxins against drug-resistant bacteria and the gene cluster that directs their production provides an opportunity for engineered biosynthesis of innovative enacyloxin derivatives and highlights the potential of Bcc bacteria as an underexploited resource for antibiotic discovery.

Oxidative stress of Burkholderia cenocepacia induces insertion sequence-mediated genomic rearrangements that interfere with macrorestriction-based genotyping.

Burkholderia cenocepacia can cause serious infections and epidemics in patients with cystic fibrosis (CF). A CF population in the Czech Republic experienced an epidemic outbreak caused by a B. cenocepacia ST-32 strain. The clonality of the isolates was evident by multilocus sequence typing; however, fingerprinting profiles obtained by pulsed-field gel electrophoresis (PFGE) showed substantial band variability. We investigated whether the PFGE pattern diversity resulted from genomic rearrangements mediated by insertion sequences (IS); in addition, we determined whether stressful growth conditions altered the transposition activity of these IS. DNA probes for IS commonly found in B. cenocepacia were designed using the B. cenocepacia J2315 genome. Southern hybridization analysis of ST-32 isolates demonstrated diversity in both the copy number and the insertion site for a homologue of ISBcen20. Movement of the ISBcen20 homologue was detected when the ST-32 isolate CZ1238 was exposed to oxidative stress (growth in the presence of H(2)O(2)). PFGE analysis of CZ1238 derivatives exposed to oxidative stress demonstrated genomic rearrangements. Interestingly, when the closely related B. cenocepacia strain J2315 was exposed to oxidative stress, no movement of ISBcen20 was detected. Since frameshift mutations are present within the transposases of all copies of this IS in J2315, our data suggest that the transposase is inactive. In summary, we have demonstrated for the first time that IS movement can be mediated by oxidative stress and can lead to genomic rearrangements in the CF pathogen B. cenocepacia. These IS movements may alter the PFGE fingerprints of isolates that are clonal by other typing methods.

Use of colony-based bacterial strain typing for tracking the fate of Lactobacillus strains during human consumption.

BACKGROUND: The Lactic Acid Bacteria (LAB) are important components of the healthy gut flora and have been used extensively as probiotics. Understanding the cultivable diversity of LAB before and after probiotic administration, and being able to track the fate of administered probiotic isolates during feeding are important parameters to consider in the design of clinical trials to assess probiotic efficacy. Several methods may be used to identify bacteria at the strain level, however, PCR-based methods such as Random Amplified Polymorphic DNA (RAPD) are particularly suited to rapid analysis. We examined the cultivable diversity of LAB in the human gut before and after feeding with two Lactobacillus strains, and also tracked the fate of these two administered strains using a RAPD technique. RESULTS: A RAPD typing scheme was developed to genetically type LAB isolates from a wide range of species, and optimised for direct application to bacterial colony growth. A high-throughput strategy for fingerprinting the cultivable diversity of human faeces was developed and used to determine: (i) the initial cultivable LAB strain diversity in the human gut, and (ii) the fate of two Lactobacillus strains (Lactobacillus salivarius NCIMB 30211 and Lactobacillus acidophilus NCIMB 30156) contained within a capsule that was administered in a small-scale human feeding study. The L. salivarius strain was not cultivated from the faeces of any of the 12 volunteers prior to capsule administration, but appeared post-feeding in four. Strains matching the L. acidophilus NCIMB 30156 feeding strain were found in the faeces of three volunteers prior to consumption; after taking the Lactobacillus capsule, 10 of the 12 volunteers were culture positive for this strain. The appearance of both Lactobacillus strains during capsule consumption was statistically significant (p < 0.05). CONCLUSION: We have shown that genetic strain typing of the cultivable human gut microbiota can be evaluated using a high throughput RAPD technique based on single bacterial colonies. Validation of this strategy paves the way for future systematic studies on the fate and efficacy of bacterial probiotics during human clinical trials.

Identifying the genetic basis of ecologically and biotechnologically useful functions of the bacterium Burkholderia vietnamiensis.

Signature-tagged mutagenesis (STM) was used to identify genetic determinants of fitness associated with two key ecological processes mediated by bacteria. Burkholderia vietnamiensis strain G4 was used as a model bacterium to investigate: phenol degradation as a model of bioremediation, and pea rhizosphere colonization as a prerequisite to biological control and phytoremediation. A total of 1900 mutants were screened and 196 putative fitness mutants identified; the genetic basis of 137 of these mutations was determined by correlation to the G4 genome. The phenol-STM screen was more successful at identifying phenol degradation mutations (83 mutants; 4.4% hit rate) than a conventional agar-based phenol screen (49 mutants, 5319 screened, 0.92% hit rate). The combination of both screens completely defined the components of the TOM pathway in strain G4 and also identified novel accessory genes not previously implicated in phenol utilization. The rhizosphere-STM screen identified 113 mutants (5.9% hit rate); 107 had reduced tag signals indicative of poor rhizosphere colonization (Rhiz-), while six mutants produced high hybridization signals suggesting increased rhizosphere competence (Rhiz+). Competition assays confirmed that 69% of Rhiz- mutants tested (24/35) were severely compromised in their rhizosphere fitness. Seventy Rhiz- mutations mapped to genes with the following putative functions: amino acid biosynthesis (25; 36%), general metabolism (18; 26%), hypothetical (9; 13%), regulatory genes (4; 5.7%), transport and stress (2 each; 2.8% respectively). One of the most interesting discoveries mediated by the rhizosphere-STM screen was the identification of three Rhiz+ mutants inactivated within a single virulence-associated autotransporter adhesin gene; this mutation consistently produced a hyper-colonization phenotype suggesting a highly novel role for this surface adhesin during plant interactions. Our study has shown that STM can be successfully applied to ecologically important microbial interactions, defining the underlying genetic systems important for biotechnological fitness of environmental bacteria such those from the Burkholderia cepacia complex.

Tobacco BY-2 cells expressing fission yeast cdc25 bypass a G2/M block on the cell cycle.

The mitotic inducer gene from Schizosaccharomyces pombe, Spcdc25, was used as a tool to investigate regulation of G2/M in higher plants using the BY-2 (Nicotiana tabacum) cell line as a model. Spcdc25-expressing BY-2 cells exhibited a reduced mitotic cell size through a shortening of the G2 phase. The cells often formed isodiametric double files both in BY-2 cells and in cell suspensions derived from 35S::Spcdc25 tobacco plants. In Spcdc25-expressing cells, the tobacco cyclin-dependent kinase, NtCDKB1, showed high activity in early S phase, S/G2 and early M phase, whereas in empty vector cells CDKB1 activity was transiently high in early S phase but thereafter remained lower. Spcdc25-expressing cells also bypassed a block on G2/M imposed by the cytokinin biosynthetic inhibitor lovastatin (LVS). Surprisingly, cytokinins were at remarkably low levels in Spcdc25-expressing cells compared with the empty vector, explaining why these cells retained mitotic competence despite the presence of LVS. In conclusion, synchronised Spcdc25-expressing BY-2 cells divided prematurely at a small cell size, and they exhibited premature, but sustained, CDKB1 activity even though endogenous cytokinins were virtually undetectable.

Expression of the fission yeast cell cycle regulator cdc25 induces de novo shoot formation in tobacco: evidence of a cytokinin-like effect by this mitotic activator.

During the last decade, the cell cycle and its control by cyclin-dependent kinases (CDKs) has been extensively studied in eukaryotes. The regulation of CDK activity includes, among others, its activation by Cdc25 phosphatase at G2/M. However, within the plant kingdom studies of this regulation have lagged behind and a plant cdc25 homologue has not been identified yet. Here, we report on the effects of transformation of tobacco (Nicotiana tabacum L., cv. Samsun) with fission yeast (Schizosaccharomyces pombe) cdc25 (Spcdc25) on de novo plant organ formation, a process dependent on rate and orientation of cell division. On shoot-inducing medium (low 1-naphthylacetic acid (NAA), high 6-benzylaminopurine (BAP)) the number of shoots formed on internode segments cultured from transgenic plants was substantially higher than in the non-transformed controls. Anatomical observations indicated that the shoot formation process was accelerated but with no changes in the quality and sequence of shoot development. Surprisingly, and in contrast to the controls, when on root-inducing medium (high NAA, low BAP) cultured segments from transgenic plants failed to initiate hardly any roots. Instead, they continued to form shoots at low frequencies. Moreover, in marked contrast to the controls, stem segments from transgenic plants were able to form shoots even without the addition of exogenous growth regulators to the medium. The results indicate that Spcdc25 expression in culture tobacco stem segments mimicked the developmental effects caused by an exogenous hormone balance shifted towards cytokinins. The observed cytokinin-like effects of Spcdc25 transformation are consistent with the concept of an interaction between cell cycle regulators and phytohormones during plant development.

The Arabidopsis 14-3-3 protein, GF14omega, binds to the Schizosaccharomyces pombe Cdc25 phosphatase and rescues checkpoint defects in the rad24- mutant.

The fission yeast (S. pombe) mitotic inducer gene, Spcdc25, interacts with the plant cell cycle to establish a small cell size phenotype compared with wild-type cells. We have investigated the nature of this interaction by yeast two-hybrid screening using Spcdc25 as bait in a cDNA library prepared from root tips of Arabidopsis thaliana (L.) Heynh. Three 14-3-3 proteins were detected: G-box Factor-like (GF)14kappa, lambda and omega; binding with Spcdc25 was confirmed by an independent immunoprecipitation assay. To test for cell cycle checkpoint function, GF14kappa, lambda and omega were transformed independently, using the strong nmt1+ promoter, into rad24-, a fission yeast mutant deficient in a 14-3-3 checkpoint protein. When exposed to UV irradiation or in the presence of 10 mM hydroxyurea, only cells transformed with GF14omega could fully rescue the defects in the DNA-damage and DNA-replication checkpoints of this mutant. Supporting evidence for a GF14omega cell cycle function was provided by semi-quantitative reverse transcription-polymerase chain reaction indicating that expression of this gene was elevated in regions of the plant that comprise dividing cells whereas GF14kappa and lambda expression was more evenly detected in all tissues examined. The data are consistent with the hypothesis that interaction between Spcdc25 and the plant cell cycle occurs at the level of a 14-3-3 protein with distinct checkpoint properties.

The CUSP DeltaNp63alpha isoform of human p63 is downregulated by solar-simulated ultraviolet radiation.

BACKGROUND: In normal human keratinocytes, a p53-like protein, DeltaNp63alpha, also known as CUSP, is constitutively and abundantly expressed. The significant constitutive expression of DeltaNp63alpha in stratified epithelium has been proposed to maintain the proliferative capacity of basal cells, blocking the consequences of inappropriate p53 activation. OBJECTIVE: To determine the response of keratinocyte DeltaNp63alpha to ultraviolet radiation (UVR), a stimulus for p53 activation. METHODS: Cultured normal human keratinocytes were exposed to graded doses of solar-simulated UVR. The expression of DeltaNp63alpha protein and mRNA were measured with Western and Northern blotting. Normal mouse skin was exposed to UVR, and DeltaNp63alpha expression assessed with immunohistochemistry. RESULTS: Increasing doses of UVR virtually shut off DeltaNp63alpha protein and mRNA expression in cultured normal human keratinocytes and in normal mouse skin in vivo. CONCLUSION: This study supports the hypothesis that in situations where p53 activation is desirable, as with DNA-damaging UVR, DeltaNp63alpha downregulation occurs and may possibly allow for better target gene transcription by p53.

A WEE1 homologue from Arabidopsis thaliana.

Little is known about the genes that regulate cyclinB-Cdc2 complexes at the G2/M transition of the plant cell cycle although in yeast and animals cdc25 and wee1 are central regulators of cdc2. Here we describe the isolation, by reverse transcription polymerase chain reaction (RT-PCR), of a WEE1 cDNA (AtWEE1) in Arabidopsis thaliana (L.) Heynh. Semi-quantitative RT-PCR showed that AtWEE1 expression was confined to actively dividing regions of the plant. The overexpression of AtWEE1 in fission yeast (Schizosaccharomyces pombe) caused cells to arrest, and to grow but not divide, resulting in very elongated cells. Our data provide evidence for a functional WEE1 in A. thaliana.

CUSP/p63 expression in basal cell carcinoma.

Chronic ulcerative stomatitis protein (CUSP), the most abundant cutaneous isoform of p63, is a p53-related gene essential for epithelial development. CUSP lacks the N-terminal transactivation domain found on other p53 family members and has been shown to inhibit p53 function in vitro. In this study, biopsies of normal skin (21 of 21), benign neoplasms [seborrheic keratosis (3 of 3), acrochordon (2 of 3), and verruca plana (3 of 3)], and squamous cell carcinomas (SCC) (4 of 4) displayed strong nuclear CUSP immuno-reactivity in epidermal cells. In contrast few basal cell carcinomas (BCC) (7 of 27) and sebaceous nevi (1 of 2) displayed this pattern of CUSP immunoreactivity. Thus, biopsies of cutaneous conditions characterized by sonic hedgehog (SHH) pathway dysregulation were more than 86 times as likely to lack CUSP/p63 immunofluorescence as were other cutaneous samples. Adjacent normal-appearing skin from patients with basal cell nevus syndrome (BCNS) (2 of 3) also lacked CUSP immuno-staining. Lastly, a BCC arising in a patched heterozygous mouse also lacked CUSP immuno-staining. Because CUSP mRNA and protein were detected via Northern and Western analysis in BCC samples lacking CUSP immuno-staining, we sequenced the coding region of CUSP from two non-staining BCCs but found no mutations. Therefore, CUSP appears to be present, unmutated, and yet frequently undetectable by immunofluorescence in cutaneous lesions in both humans and mice that are associated with SHH pathway dysregulation (BCCs, BCNS, and nevus sebaceous).