Production of root hair deformation factors by Rhizobium meliloti nodulation genes in Escherichia coli: HsnD (NodH) is involved in the plant host-specific modification of the NodABC factor

The role of the hsnD (nodH) gene in the determination of the host-specific nodulation ability of Rhizobium meliloti was studied by expressing the common nodulation genes (nodABC) with or without the hsnD gene in Escherichia coli and testing for biological activity on various leguminous plants. In this way, four categories of plants were established. Upon infection with E. coli carrying the nodABC construct, root hair deformation (Had) was detected on clovers while the hsnD gene was additionally needed for the elicitation of the same response on alfalfa and sweet clover. A weak root hair deformation was seen on siratro by inoculation with E. coli harbouring the nodABC genes and was highly increased when hsnD was also introduced. Cowpea and Desmodium did not respond to any of the E. coli strains constructed. Exudates or cytosolicfractions of the respective E. coli derivatives elicited the same root hair deformation as the intact bacteria. These data indicate that not only the nodABC gene products but also the hsnD product are involved in the synthesis of Had factors. Subclones expressing only the nodA, nodB, or nodC genes or the same genes in pairs (nodAB, nodBC, nodAC) did not provide a compound with activity comparable to the NodABC factor, suggesting that all three genes are required for the production of the Had factor which is active on clover. Coinoculation of alfalfa plants with two strains of E. coli, one carrying the nodABC genes and the other expressing only hsnD, or combining exudates or cytosolic fractions from these strains did not result in root hair deformation on alfalfa. These data indicate that the HsnD protein itself or its product is not an additional alfalfa-specific extracellular signal but more likely is enzymatically involved in the modification of the basic compound determined by the nodABC genes.     

Methylation Analyses Reveal Promoter Hypermethylation as a Rare Cause of “Second Hit” in Germline BRCA1-Associated Pancreatic Ductal Adenocarcinoma

Molecular Diagnosis & Therapy - Pancreatic ductal adenocarcinoma (PDAC) is characterized by the occurrence of pathogenic variants in BRCA1/2 in 5–6% of patients. Biallelic loss of BRCA1/2...     

A pseudomolecule‐scale genome assembly of the liverwort Marchantia polymorpha

Marchantia polymorpha has recently become a prime model for cellular, evo‐devo, synthetic biological, and evolutionary investigations. We present a pseudomolecule‐scale assembly of the M. polymorpha genome, making comparative genome structure analysis and classical genetic mapping approaches feasible. We anchored 88% of the M. polymorpha draft genome to a high‐density linkage map resulting in eight pseudomolecules. We found that the overall genome structure of M. polymorpha is in some respects different from that of the model moss Physcomitrella patens. Specifically, genome collinearity between the two bryophyte genomes and vascular plants is limited, suggesting extensive rearrangements since divergence. Furthermore, recombination rates are greatest in the middle of the chromosome arms in M. polymorpha like in most vascular plant genomes, which is in contrast with P. patens where recombination rates are evenly distributed along the chromosomes. Nevertheless, some other properties of the genome are shared with P. patens. As in P. patens, DNA methylation in M. polymorpha is spread evenly along the chromosomes, which is in stark contrast with the angiosperm model Arabidopsis thaliana, where DNA methylation is strongly enriched at the centromeres. Nevertheless, DNA methylation and recombination rate are anticorrelated in all three species. Finally, M. polymorpha and P. patens centromeres are of similar structure and marked by high abundance of retroelements unlike in vascular plants. Taken together, the highly contiguous genome assembly we present opens unexplored avenues for M. polymorpha research by linking the physical and genetic maps, making novel genomic and genetic analyses, including map‐based cloning, feasible.     

Tracing the history of plant traits under domestication in cranberries: potential consequences on anti-herbivore defences

The process of selecting certain desirable traits for plant breeding may compromise other potentially important traits, such as defences against pests; however, specific phenotypic changes occurring over the course of domestication are unknown for most domesticated plants. Cranberry (Vaccinium macrocarpon) offers a unique opportunity to study such changes: its domestication occurred recently, and we have access to the wild ancestors and intermediate varieties used in past crosses. In order to investigate whether breeding for increased yield and fruit quality traits may indirectly affect anti-herbivore defences, the chemical defences have been examined of five related cranberry varieties that span the history of domestication against a common folivore, the gypsy moth (Lymantria dispar). Direct defences were assessed by measuring the performance of gypsy moth caterpillars and levels of phenolic compounds in leaves, and indirect defences by assaying induced leaf volatile emissions. Our results suggest that breeding in cranberry has compromised plant defences: caterpillars performed best on the derived NJS98-23 (the highest-yielding variety) and its parent Ben Lear. Moreover, NJS98-23 showed reduced induction of volatile sesquiterpenes, and had lower concentrations of the defence-related hormone cis-jasmonic acid (JA) than ancestral varieties. However, induced direct defences were not obviously affected by breeding, as exogenous JA applications reduced caterpillar growth and increased the amounts of phenolics independent of variety. Our results suggest that compromised chemical defences in high-yielding cranberry varieties may lead to greater herbivore damage which, in turn, may require more intensive pesticide control measures. This finding should inform the direction of future breeding programmes.     

Validation of prostate cancer risk-related loci identified from genome-wide association studies using family-based association analysis: evidence from the International Consortium for Prostate Cancer Genetics (ICPCG)

Multiple prostate cancer (PCa) risk-related loci have been discovered by genome-wide association studies (GWAS) based on case-control designs. However, GWAS findings may be confounded by population stratification if cases and controls are inadvertently drawn from different genetic backgrounds. In addition, since these loci were identified in cases with predominantly sporadic disease, little is known about their relationships with hereditary prostate cancer (HPC). The association between seventeen reported PCa susceptibility loci was evaluated with a family-based association test using 1,979 hereditary PCa families of European descent collected by members of the International Consortium for Prostate Cancer Genetics, with a total of 5,730 affected men. The risk alleles for 8 of the 17 loci were significantly over-transmitted from parents to affected offspring, including SNPs residing in 8q24 (regions 1, 2 and 3), 10q11, 11q13, 17q12 (region 1), 17q24 and Xp11. In subgroup analyses, three loci, at 8q24 (regions 1 and 2) plus 17q12, were significantly over-transmitted in hereditary PCa families with five or more affected members, while loci at 3p12, 8q24 (region 2), 11q13, 17q12 (region 1), 17q24 and Xp11 were significantly over-transmitted in HPC families with an average age of diagnosis at 65?years or less. Our results indicate that at least a subset of PCa risk-related loci identified by case-control GWAS are also associated with disease risk in HPC families.     

Genetic polymorphisms of NOD1 and IL-8, but not polymorphisms of TLR4 genes, are associated with Helicobacter pylori-induced duodenal ulcer and gastritis

BACKGROUND: Intracellular pathogen receptor NOD1 is involved in the epithelial cell sensing Helicobacter pylori, which results in a considerable interleukin (IL)-8 production. The aim of this study was to evaluate the relationship between NOD1 and IL-8 genetic polymorphisms and the development of H. pylori-induced gastritis and duodenal ulcer (DU), as compared with TLR4 polymorphisms. MATERIALS AND METHODS: Eighty-five patients with DU and 135 patients with gastritis were enrolled in the study. Seventy-five serologically H. pylori-positive subjects without gastric or duodenal symptoms served as controls. The G796A (E266K) NOD1 polymorphism was determined by restriction fragment length polymorphism, and the -251 IL-8 polymorphism by amplification refractory mutation system method. The TLR4 (ASP/299/Gly and Thr/399/Ile) gene polymorphisms were examined by melting point analysis. RESULTS: AA homozygote mutant variants of NOD1 were detected in 20% of the H. pylori-positive patients with DU versus 7% of H. pylori-positive patients with gastritis and versus 6% of the H. pylori-positive healthy controls. The IL-8 heterozygote mutant variant was detected with a significantly higher frequency among the DU patients and those with gastritis than among the H. pylori-positive controls. However, no significant correlation concerning the frequency of the TLR4 gene polymorphism could be revealed between any group of patients and the controls. CONCLUSION: E266K CARD4/NOD1, but not the TLR4 gene polymorphism increases the risk of peptic ulceration in H. pylori-positive patients. The -251 IL-8 polymorphism was significantly associated with either gastritis or DU in H. pylori-infected subjects. Host factors including intracellular pathogen receptors and IL-8 production play an important role in H. pylori-induced gastric mucosal damage.     

Live cell imaging with protein domains capable of recognizing phosphatidylinositol 4,5-bisphosphate; a comparative study

Background  

Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P ₂] is a critically important regulatory phospholipid found in the plasma membrane of all eukaryotic cells. In addition to being a precursor of important second messengers, PtdIns(4,5)P ₂ also regulates ion channels and transporters and serves the endocytic machinery by recruiting clathrin adaptor proteins. Visualization of the localization and dynamic changes in PtdIns(4,5)P ₂ levels in living cells is critical to understanding the biology of PtdIns(4,5)P ₂. This has been mostly achieved with the use of the pleckstrin homology (PH) domain of PLCδ1 fused to GFP. Here we report on a comparative analysis of several recently-described yeast PH domains as well as the mammalian Tubby domain to evaluate their usefulness as PtdIns(4,5)P ₂ imaging tools.     

Special Sm Core Complex Functions in Assembly of the U2 Small Nuclear Ribonucleoprotein of Trypanosoma brucei

The processing of polycistronic pre-mRNAs in trypanosomes requires the spliceosomal small ribonucleoprotein complexes (snRNPs) U1, U2, U4/U6, U5, and SL, each of which contains a core of seven Sm proteins. Recently we reported the first evidence for a core variation in spliceosomal snRNPs; specifically, in the trypanosome U2 snRNP, two of the canonical Sm proteins, SmB and SmD3, are replaced by two U2-specific Sm proteins, Sm15K and Sm16.5K. Here we identify the U2-specific, nuclear-localized U2B″ protein from Trypanosoma brucei. U2B″ interacts with a second U2 snRNP protein, U2-40K (U2A′), which in turn contacts the U2-specific Sm16.5K/15K subcomplex. Together they form a high-affinity, U2-specific binding complex. This trypanosome-specific assembly differs from the mammalian system and provides a functional role for the Sm core variation found in the trypanosomal U2 snRNP.In trypanosomes, trans-splicing is an essential step in the expression of all protein-coding genes. The resulting mRNAs always carry a noncoding spliced leader (SL) sequence of 39 nucleotides at their 5′ ends, which is derived from the SL RNA. In addition to the SL RNA, the small nuclear RNAs (snRNAs) U2, U4, U5, and U6 are essential cofactors during trans-splicing (reviewed in reference 14).In previous studies, we characterized some of the protein components of the spliceosomal small nuclear ribonucleoproteins (snRNPs) from Trypanosoma brucei. All snRNPs contain a core of seven Sm polypeptides (18). Recently, we reported that the identity of the Sm proteins varies among spliceosomal snRNPs; specifically, two of the canonical Sm proteins, SmB and SmD3, are replaced in the U2 snRNP by two novel, U2 snRNP-specific Sm proteins, Sm15K and Sm16.5K (34). There is a similar case of Sm core variation in the U4 snRNP, where a single Sm protein, SmD3, is replaced by a U4-specific LSm protein (32; N. Jaé and A. Bindereif, unpublished data). Trypanosomal snRNAs also differ significantly from what we know in other systems, reflecting the large evolutionary distance and trypanosome-specific properties. For example, both the U1 and U5 snRNAs from trypanosomes represent the shortest known orthologues (6, 19).In addition to the Sm proteins, some snRNP-specific protein factors were found in trypanosomes. Sequence comparisons identified the U2-40K protein as the trypanosomal homologue of the human U2A′ protein (5), a finding that was unexpected, since no immunological relationship could be detected between these proteins (17). As characterized in other systems, including those of humans, yeasts, and plants (9, 29, 31), the U2 snRNP contains a second specific protein, U2B″, a protein closely related to the U1 snRNP protein U1A. Except for the Saccharomyces cerevisiae orthologue, the known U2B″ proteins are built of two RNA recognition motifs (RRMs), with the N-terminal RRM being responsible for snRNA binding specificity (25). The close relatedness of these two proteins is also reflected in Drosophila melanogaster, where a single protein, SNF/D25, combines the functions of both individual proteins (11, 22). Furthermore, genetic and functional redundancy was demonstrated for the two proteins in Caenorhabditis elegans (24).From previous studies on the mammalian U2 snRNP, we know that the U2- specific proteins U2A′ and U2B″ interact with each other, independently of U2 snRNA; moreover, U2B″ binds directly to loop nucleotides of stem-loop IV, but only with the assistance of interacting U2A′ (23, 25, 26). Analogous to the cis-splicing mechanism, the U2 snRNP is likely to play an important role in early trans-spliceosome assembly. Compared with the other snRNAs, the trypanosomatid U2 snRNA differs in several important aspects from its highly conserved counterparts in other species. First, stem-loop III is precisely deleted. Second, the branch point recognition region located between stem-loops I and IIa is missing; in parallel, there is no stringent consensus of branch points in the 3′-splice site region of the polycistronic pre-mRNA. Third, the Sm protein binding site does not follow the general consensus. Finally, only some of the otherwise highly conserved loop IV nucleotides occur in the trypanosomatid U2 snRNAs (7, 12, 15, 33).Here we report the identification and characterization of the U2-specific protein U2B″ of T. brucei. Sequence analysis revealed that the trypanosomal orthologue contains only a single RRM, in contrast to the mammalian, two-RRM domain structure, and that the homology is restricted to this single RRM. We show that U2-40K (U2A′) binds very efficiently to U2B″ in the absence of U2 snRNA and increases the binding affinity of U2B″ to U2 snRNA. Furthermore U2-40K (U2A′) contacts the two specific components of the U2 Sm core, Sm16.5K and Sm 15K, forming together a high-affinity, U2-specific binding complex. This establishes a specific function of the U2 Sm core variation in mediating U2-specific protein-protein interactions.