Development and evaluation of a Gal4-mediated LUC/GFP/GUS enhancer trap system in Arabidopsis

BACKGROUND: Gal4 enhancer trap systems driving expression of LacZ and GFP reporters have been characterized and widely used in Drosophila. However, a Gal4 enhancer trap system in Arabidopsis has not been described in the primary literature. In Drosophila, the reporters possess a Gal4 upstream activation sequence (UAS) as five repeats (5XUAS) and lines that express Gal4 from tissue specific enhancers have also been used for the ectopic expression of any transgene (driven by a 5XUAS). While Gal4 transactivation has been demonstrated in Arabidopsis, wide use of a trap has not emerged in part because of the lack of detailed analysis, which is the purpose of the present study. RESULTS: A key feature of this study is the use of luciferase (LUC) as the primary reporter and rsGFP-GUS as secondary reporters. Reporters driven by a 5XUAS are better suited in Arabidopsis than those containing a 1X or 2X UAS. A 5XUAS-LUC reporter is expressed at high levels in Arabidopsis lines transformed with Gal4 driven by the full, enhanced 35S promoter. In contrast, a minimum 35S (containing the TATA region) upstream of Gal4 acts as an enhancer trap system. Luciferase expression in trap lines of the T1, T2, and T3 generations are generally stable but by the T4 generation approximately 25% of the lines are significantly silenced. This silencing is reversed by growing plants on media containing 5-aza-2'-deoxycytidine. Quantitative multiplex RT-PCR on the Gal4 and LUC mRNA indicate that this silencing can occur at the level of Gal4 or LUC transcription. Production of a 10,000 event library and observations on screening, along with the potential for a Gal4 driver system in other plant species are discussed. CONCLUSION: The Gal4 trap system described here uses the 5XUAS-LUC and 5XUAS rsGFP-GUS as reporters and allows for in planta quantitative screening, including the rapid monitoring for silencing. We conclude that in about 75% of the cases silencing is at the level of transcription of the Gal4 transgene and is at an acceptable frequency to make the Gal4 trap system in Arabidopsis of value. This system will be useful for the isolation and comprehensive characterization of specific reporter and driver lines

Essential histidine pairs indicate conserved haem binding in epsilonproteobacterial cytochrome c haem lyases

Bacterial cytochrome c maturation occurs at the outside of the cytoplasmic membrane, requires transport of haem b across the membrane, and depends on membrane-bound cytochrome c haem lyase (CCHL), an enzyme that catalyses covalent attachment of haem b to apocytochrome c. Epsilonproteobacteria such as Wolinella succinogenes use the cytochrome c biogenesis system II and contain unusually large CCHL proteins of about 900 amino acid residues that appear to be fusions of the CcsB and CcsA proteins found in other bacteria. CcsBA-type CCHLs have been proposed to act as haem transporters that contain two haem b coordination sites located at different sides of the membrane and formed by histidine pairs. W. succinogenes cells contain three CcsBA-type CCHL isoenzymes (NrfI, CcsA1 and CcsA2) that are known to differ in their specificity for apocytochromes and apparently recognize different haem c binding motifs such as CX2CH (by CcsA2), CX2CK (by NrfI) and CX15CH (by CcsA1). In this study, conserved histidine residues were individually replaced by alanine in each of the W. succinogenes CCHLs. Characterization of NrfI and CcsA1 variants in W. succinogenes demonstrated that a set of four histidines is essential for maturing the dedicated multihaem cytochromes c NrfA and MccA, respectively. The function of W. succinogenes CcsA2 variants produced in Escherichia coli was also found to depend on each of these four conserved histidine residues. The presence of imidazole in the growth medium of both W. succinogenes and E. coli rescued the cytochrome c biogenesis activity of most histidine variants, albeit to different extents, thereby implying the presence of two functionally distinct histidine pairs in each CCHL. The data support a model in which two conserved haem b binding sites are involved in haem transport catalysed by CcsBA-type CCHLs

Looking beyond the exome: a phenotype-first approach to molecular diagnostic resolution in rare and undiagnosed diseases.

PurposeTo describe examples of missed pathogenic variants on whole-exome sequencing (WES) and the importance of deep phenotyping for further diagnostic testing.MethodsGuided by phenotypic information, three children with negative WES underwent targeted single-gene testing.ResultsIndividual 1 had a clinical diagnosis consistent with infantile systemic hyalinosis, although WES and a next-generation sequencing (NGS)-based ANTXR2 test were negative. Sanger sequencing of ANTXR2 revealed a homozygous single base pair insertion, previously missed by the WES variant caller software. Individual 2 had neurodevelopmental regression and cerebellar atrophy, with no diagnosis on WES. New clinical findings prompted Sanger sequencing and copy number testing of PLA2G6. A novel homozygous deletion of the noncoding exon 1 (not included in the WES capture kit) was detected, with extension into the promoter, confirming the clinical suspicion of infantile neuroaxonal dystrophy. Individual 3 had progressive ataxia, spasticity, and magnetic resonance image changes of vanishing white matter leukoencephalopathy. An NGS leukodystrophy gene panel and WES showed a heterozygous pathogenic variant in EIF2B5; no deletions/duplications were detected. Sanger sequencing of EIF2B5 showed a frameshift indel, probably missed owing to failure of alignment.ConclusionThese cases illustrate potential pitfalls of WES/NGS testing and the importance of phenotype-guided molecular testing in yielding diagnoses

Last Men Standing: Chlamydatus Portraits and Public Life in Late Antique Corinth

Notable among the marble sculptures excavated at Corinth are seven portraits of men wearing the long chlamys of Late Antique imperial office. This unusual costume, contemporary portrait heads, and inscribed statue bases all help confirm that new public statuary was created and erected at Corinth during the 4th and 5th centuries. These chlamydatus portraits, published together here for the first time, are likely to represent the Governor of Achaia in his capital city, in the company of local benefactors. Among the last works of the ancient sculptural tradition, they form a valuable source of information on public life in Late Antique Corinth

The CydDC family of transporters

The CydDC family of ABC transporters export the low molecular weight thiols glutathione and cysteine to the periplasm of a variety of bacterial species. The CydDC complex has previously been shown to be important for disulfide folding, motility, respiration, and tolerance to nitric oxide and antibiotics. In addition, CydDC is thus far unique amongst ABC transporters in that it binds a haem cofactor that appears to modulate ATPase activity. CydDC has a diverse impact upon bacterial metabolism, growth, and virulence, and is of interest to those working on membrane transport mechanisms, redox biology, aerobic respiration, and stress sensing/tolerance during infection

Dissecting the Shared Genetic Architecture of Suicide Attempt, Psychiatric Disorders, and Known Risk Factors

Background Suicide is a leading cause of death worldwide, and nonfatal suicide attempts, which occur far more frequently, are a major source of disability and social and economic burden. Both have substantial genetic etiology, which is partially shared and partially distinct from that of related psychiatric disorders. Methods We conducted a genome-wide association study (GWAS) of 29,782 suicide attempt (SA) cases and 519,961 controls in the International Suicide Genetics Consortium (ISGC). The GWAS of SA was conditioned on psychiatric disorders using GWAS summary statistics via multitrait-based conditional and joint analysis, to remove genetic effects on SA mediated by psychiatric disorders. We investigated the shared and divergent genetic architectures of SA, psychiatric disorders, and other known risk factors. Results Two loci reached genome-wide significance for SA: the major histocompatibility complex and an intergenic locus on chromosome 7, the latter of which remained associated with SA after conditioning on psychiatric disorders and replicated in an independent cohort from the Million Veteran Program. This locus has been implicated in risk-taking behavior, smoking, and insomnia. SA showed strong genetic correlation with psychiatric disorders, particularly major depression, and also with smoking, pain, risk-taking behavior, sleep disturbances, lower educational attainment, reproductive traits, lower socioeconomic status, and poorer general health. After conditioning on psychiatric disorders, the genetic correlations between SA and psychiatric disorders decreased, whereas those with nonpsychiatric traits remained largely unchanged. Conclusions Our results identify a risk locus that contributes more strongly to SA than other phenotypes and suggest a shared underlying biology between SA and known risk factors that is not mediated by psychiatric disorders.Peer reviewe

Reciprocal Leaf and Root Expression of AtAmt1.1 and Root Architectural Changes in Response to Nitrogen Starvation

Nitrogen is an essential macronutrient for plant growth and survival. Here, the temporal and spatial sensing of nitrogen starvation is analyzed in Arabidopsis (Arabidopsis thaliana). The promoter for the high-affinity ammonium transporter, AtAmt1.1, is shown to be a valid indicator for nitrogen status in leaves and roots. An AtAmt1.1-Gal4 transgene using three 5× upstream activating sequence-driven reporters (luciferase, green fluorescent protein, and β-glucuronidase) facilitated in vivo profiling at the whole-plant and cellular levels. The effects of nitrogen supply, light duration, light intensity, and carbon on the expression of the AtAmt1.1 gene in the roots and aerial tissues are reported. Under nitrogen starvation, high expression is observed in the roots and, under nitrogen-sufficient conditions, high expression is observed in the leaves. This reciprocal regulation of AtAmt1.1 was confirmed by quantitative reverse transcription-polymerase chain reaction, which was also used to quantitate expression of the five other Amt genes in Arabidopsis. Although some of these show tissue specificity (roots or leaves), none exhibit reciprocal regulation like the AtAmt1.1-encoded high-affinity transporter. This robust reciprocal expression suggests that Arabidopsis undergoes rapid resource reallocation in plants grown under different nitrogen supply regimens. Ultimately, nitrogen starvation-mediated reallocation results in root architectural restructuring. We describe the precise timing and cellular aspects of this nitrogen limitation response