Properties of the PriA helicase domain and its role in binding PriA to specific DNA structures

Primosome assembly protein PriA functions in the assembly of the replisome at forked DNA structures. Whereas its N-terminal DNA binding domain (DBD) binds independently to DNA, the affinity of DBD protein for forked structures is relatively weak. Although the PriA helicase domain (HD) is required for high affinity fork binding, HD protein had very low affinity for DNA. It had only low levels of ATPase activity, and it hydrolyzed ATP when DNA was absent whereas PriA did not. HD catalyzed unwinding of a minimal substrate composed of a duplex with a 3' single-stranded tail. Single-strand binding protein (SSB) bound to the tail of this substrate inhibited this reaction by full-length PriA but enhanced the reaction by HD. SSB stabilized binding of PriA but not of DBD or HD to duplexes with a 5' or 3' single-stranded tail. On forked substrates SSB enhanced helicase action on the lagging-strand arm by PriA but not by HD. The results indicate that synergy of the DBD and HD allows stable binding at the interface between duplex and single-stranded DNA bound by SSB. This mode of binding may be analogous to fork binding, which orients the helicase to act on the lagging-strand side of the fork.     

Balanced complex chromosomal rearrangements (BCCR) with at least three chromosomes and three or more breakpoints: report of three new cases

Balanced complex chromosomal rearrangements (BCCR) encompass a heterogeneous group of rare chromosomal aberrations. In this paper, we report three cases of BCCRs. In two the probands were referred for either genetic counseling or prenatal management. One case was ascertained after chromosome analysis performed because of psychiatric manifestations; this was an isolated finding. We also outline the molecular cytogenetic techniques, which were essential in confirming and precisely delineating the BCCRs identified in these patients. In addition the various aspects of genetic counseling for this type of chromosomal rearrangement, highlighting the details particular to each individual case are discussed. We discuss the classification for this type of chromosomal mutation.     

Identification of a trafficking motif involved in the stabilization and polarization of P2X receptors

Extracellular ATP-gated channels (P2X receptors) define the third major family of ionotropic receptors, and they are expressed widely in nerve cells, muscles, and endocrine and exocrine glands. P2X subunits have two membrane-spanning domains, and a receptor is thought to be formed by oligomerization of three subunits. We have identified a conserved motif in the cytoplasmic C termini of P2X subunits that is necessary for their surface expression; mutations in this motif result in a marked reduction of the receptors at the plasma membrane because of a rapid internalization. Transfer of the motif to a reporter protein (CD(4)) enhances the surface expression of the chimera, indicating that this motif is likely involved in the stabilization of P2X receptor at the cell surface. In neurons, mutated P2X(2) subunits showed reduced membrane expression and an altered axodendritic distribution. This motif is also present in intracellular regions of other membrane proteins, such as in the third intracellular loop of some G protein-coupled receptors, suggesting that it might be involve in their cellular stabilization and polarization.     

Seeing is believing? A beginners' guide to practical pitfalls in image acquisition

Imaging can be thought of as the most direct of experiments. You see something; you report what you see. If only things were truly this simple. Modern imaging technology has brought about a revolution in the kinds of questions we can approach, but this comes at the price of increasingly complex equipment. Moreover, in an attempt to market competing systems, the microscopes have often been inappropriately described as easy to use and suitable for near-beginners. Insufficient understanding of the experimental manipulations and equipment set-up leads to the introduction of errors during image acquisition. In this feature, I review some of the most common practical pitfalls faced by researchers during image acquisition, and how they can affect the interpretation of the experimental data.This article is targeted neither to the microscopy gurus who push forward the frontiers of imaging technology nor to my imaging specialist colleagues who may wince at the overly simplistic comments and lack of detail. Instead, this is for beginners who gulp with alarm when they hear the word "confocal pinhole" or sigh as they watch their cells fade and die in front of their very eyes time and time again at the microscope. Take heart, beginners, if microscopes were actually so simple then many people (including myself) would suddenly be out of a job!     

Regioselective enzymatic aminoacylation of lobucavir to give an intermediate for lobucavir prodrug

Synthesis of lobucavir prodrug, L-valine, [(1S,2R,3R)-3-(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)-2-(hydroxymethyl)cyclobutyl]methyl ester monohydrochloride (BMS 233866), requires regioselective coupling of one of the two hydroxyl groups of lobucavir (BMS 180194) with valine. Either hydroxyl group of lobucavir could be selectively aminoacylated with valine by using enzymatic reactions. N-[(Phenylmethoxy)carbonyl]-L-valine, [(1R,2R,4S)-2-(2-amino-6-oxo-1H-purin-9-yl)-4-(hydroxymethyl)cyclobutyl]methyl ester (3, 82.5% yield), was obtained by selective hydrolysis of N,N′-bis[(phenylmethoxy)carbonyl]bis[L-valine], O,O′-[(1S,2R,3R)-3-(2-amino-6-oxo-1H-purin-9-yl)cyclobuta-1,2-diyl]methyl ester (1) with lipase M, and L-valine, [(1R,2R,4S)-2-(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)-4-(hydroxymethyl)cyclobutyl]methyl ester monohydrochloride (4, 87% yield) was obtained by hydrolysis of bis[L-valine], O,O′-[(1S,2R,3R)-3-(2-amino-6-oxo-1H-purin-9-yl)cyclobuta-1,2-diyl]methyl ester, dihydrochloride (2), with lipase from Candida cylindracea. The final intermediate for lobucavir prodrug, N-[(phenylmethoxy)carbonyl]-L-valine, [(1S,2R,4R)-3-(2-amino-6-oxo-1H-purin-9-yl)-2-(hydroxymethyl)cyclobutyl]methyl ester (5), could be obtained by transesterification of lobucavir using ChiroCLEC™ BL (61% yield), or more selectively by using immobilized lipase from Pseudomonas cepacia (84% yield).     

Ultraconserved elements in the human genome: association and transmission analyses of highly constrained single-nucleotide polymorphisms

Ultraconserved elements in the human genome likely harbor important biological functions as they are dosage sensitive and are able to direct tissue-specific expression. Because they are under purifying selection, variants in these elements may have a lower frequency in the population but a higher likelihood of association with complex traits. We tested a set of highly constrained SNPs (hcSNPs) distributed genome-wide among ultraconserved and nearly ultraconserved elements for association with seven traits related to reproductive (age at natural menopause, number of children, age at first child, and age at last child) and overall [longevity, body mass index (BMI), and height] fitness. Using up to 24,047 European-American samples from the National Heart, Lung, and Blood Institute Candidate Gene Association Resource (CARe), we observed an excess of associations with BMI and height. In an independent replication panel the most strongly associated SNPs showed an 8.4-fold enrichment of associations at the nominal level, including three variants in previously identified loci and one in a locus (DENND1A) previously shown to be associated with polycystic ovary syndrome. Finally, using 1430 family trios, we showed that the transmissions from heterozygous parents to offspring of the derived alleles of rare (frequency ≤0.5%) hcSNPs are not biased, particularly after adjusting for the rates of genotype missingness and error in the data. The lack of transmission bias ruled out an immediately and strongly deleterious effect due to the rare derived alleles, consistent with the observation that mice homozygous for the deletion of ultraconserved elements showed no overt phenotype. Our study also illustrated the importance of carefully modeling potential technical confounders when analyzing genotype data of rare variants.     

Epoxycarotenoid cleavage by NCED5 fine-tunes ABA accumulation and affects seed dormancy and drought tolerance with other NCED family members

Carotenoid cleavage, catalyzed by the 9-cis-epoxycarotenoid dioxygenase (NCED) constitutes a key step in the regulation of ABA biosynthesis. In Arabidopsis, this enzyme is encoded by five genes. NCED3 has been shown to play a major role in the regulation of ABA synthesis in response to water deficit, whereas NCED6 and NCED9 have been shown to be essential for the ABA production in the embryo and endosperm that imposes dormancy. Reporter gene analysis was carried out to determine the spatiotemporal pattern of NCED5 and NCED9 gene expression. GUS activity from the NCED5 promoter was detected in both the embryo and endosperm of developing seeds with maximal staining after mid-development. NCED9 expression was found at early stages in the testa outer integument layer 1, and after mid-development in epidermal cells of the embryo, but not in the endosperm. In accordance with its temporal- and tissue-specific expression, the phenotypic analysis of nced5 nced6 nced9 triple mutant showed the involvement of the NCED5 gene, together with NCED6 and NCED9, in the induction of seed dormancy. In contrast to nced6 and nced9, however, nced5 mutation did not affect the gibberellin required for germination. In vegetative tissues, combining nced5 and nced3 mutations reduced vegetative growth, increased water loss upon dehydration, and decreased ABA levels under both normal and stressed conditions, as compared with nced3. NCED5 thus contributes, together with NCED3, to ABA production affecting plant growth and water stress tolerance.     

Small molecule screening identifies targetable zebrafish pigmentation pathways

Small molecules complement genetic mutants and can be used to probe pigment cell biology by inhibiting specific proteins or pathways. Here, we present the results of a screen of active compounds for those that affect the processes of melanocyte and iridophore development in zebrafish and investigate the effects of a few of these compounds in further detail. We identified and confirmed 57 compounds that altered pigment cell patterning, number, survival, or differentiation. Additional tissue targets and toxicity of small molecules are also discussed. Given that the majority of cell types, including pigment cells, are conserved between zebrafish and other vertebrates, we present these chemicals as molecular tools to study developmental processes of pigment cells in living animals and emphasize the value of zebrafish as an in vivo system for testing the on- and off-target activities of clinically active drugs.