Overexpression of the Tn5 transposase in Escherichia coli results in filamentation, aberrant nucleoid segregation, and cell death: analysis of E. coli and transposase suppressor mutations.

Overexpression of the Tn5 transposase (Tnp) was found to be lethal to Escherichia coli. This killing was not caused by transposition or dependent on the transpositional or DNA binding competence of Tnp. Instead, it was strictly correlated with the presence of a wild-type N terminus. Deletions removing just two N-terminal amino acids of Tnp resulted in partial suppression of this effect, and deletions of Tnp removing 3 or 11 N-terminal amino acids abolished the killing effect. This cytotoxic effect of Tnp overexpression is accompanied by extensive filament formation (i.e., a defect in cell division) and aberrant nucleoid segregation. Four E. coli mutants were isolated which allow survival upon Tnp overexpression, and the mutations are located at four discrete loci. These suppressor mutations map near essential genes involved in cell division and DNA segregation. One of these mutations maps to a 4.5-kb HindIII region containing the ftsYEX (cell division) locus at 76 min. A simple proposition which accounts for all of these observations is that Tnp interacts with an essential E. coli factor affecting cell division and/or chromosome segregation and that overexpression of Tnp titrates this factor below a level required for viability of the cell. Furthermore, the N terminus of Tnp is necessary for this interaction. The possible significance of this phenomenon for the transposition process is discussed.     

Escherichia coli DNA Topoisomerase I and Suppression of Killing by Tn5 Transposase Overproduction: Topoisomerase I Modulates Tn5 Transposition

Tn5 transposase (Tnp) overproduction is lethal to Escherichia coli. The overproduction causes cell filamentation and abnormal chromosome segregation. Here we present three lines of evidence strongly suggesting that Tnp overproduction killing is due to titration of topoisomerase I. First, a suppressor mutation of transposase overproduction killing, stkD10, is localized in topA (the gene for topoisomerase I). The stkD10 mutant has the following characteristics: first, it has an increased abundance of topoisomerase I protein, the topoisomerase I is defective for the DNA relaxation activity, and DNA gyrase activity is reduced; second, the suppressor phenotype of a second mutation localized in rpoH, stkA14 (H. Yigit and W. S. Reznikoff, J. Bacteriol. 179:1704–1713, 1997), can be explained by an increase in topA expression; and third, overexpression of wild-type topA partially suppresses the killing. Finally, topoisomerase I was found to enhance Tn5 transposition up to 30-fold in vivo.     

Migration of Myeloid Cells during Inflammation Is Differentially Regulated by the Cell Surface Receptors Slamf1 and Slamf8

Previous studies have demonstrated that the cell surface receptor Slamf1 (CD150) is requisite for optimal NADPH-oxidase (Nox2) dependent reactive oxygen species (ROS) production by phagocytes in response to Gram- bacteria. By contrast, Slamf8 (CD353) is a negative regulator of ROS in response to Gram+ and Gram- bacteria. Employing in vivo migration after skin sensitization, induction of peritonitis, and repopulation of the small intestine demonstrates that in vivo migration of Slamf1^-/- dendritic cells and macrophages is reduced, as compared to wt mice. By contrast, in vivo migration of Slamf8^-/- dendritic cells, macrophages and neutrophils is accelerated. These opposing effects of Slamf1 and Slamf8 are cell-intrinsic as judged by in vitro migration in transwell chambers in response to CCL19, CCL21 or CSF-1. Importantly, inhibiting ROS production of Slamf8^-/- macrophages by diphenyleneiodonium chloride blocks this in vitro migration. We conclude that Slamf1 and Slamf8 govern ROS–dependent innate immune responses of myeloid cells, thus modulating migration of these cells during inflammation in an opposing manner.     

Temtamy preaxial brachydactyly syndrome is caused by loss-of-function mutations in chondroitin synthase 1, a potential target of BMP signaling

Altered Bone Morphogenetic Protein (BMP) signaling leads to multiple developmental defects, including brachydactyly and deafness. Here we identify chondroitin synthase 1 (CHSY1) as a potential mediator of BMP effects. We show that loss of human CHSY1 function causes autosomal-recessive Temtamy preaxial brachydactyly syndrome (TPBS), mainly characterized by limb malformations, short stature, and hearing loss. After mapping the TPBS locus to chromosome 15q26-qterm, we identified causative mutations in five consanguineous TPBS families. In zebrafish, antisense-mediated chsy1 knockdown causes defects in multiple developmental processes, some of which are likely to also be causative in the etiology of TPBS. In the inner ears of zebrafish larvae, chsy1 is expressed similarly to the BMP inhibitor dan and in a complementary fashion to bmp2b. Furthermore, unrestricted Bmp2b signaling or loss of Dan activity leads to reduced chsy1 expression and, during epithelial morphogenesis, defects similar to those that occur upon Chsy1 inactivation, indicating that Bmp signaling affects inner-ear development by repressing chsy1. In addition, we obtained strikingly similar zebrafish phenotypes after chsy1 overexpression, which might explain why, in humans, brachydactyly can be caused by mutations leading either to loss or to gain of BMP signaling.     

Histological changes in the uterus of the hens after embryonic exposure to bisphenol A and diethylstilbestrol

Many employed chemicals in industries have estrogenic hormone effects on organisms, and these are called as environmental estrogens. Environmental estrogens have adverse effects on development and function of reproductive organs of the birds. Bisphenol A (BPA) is one of the best known environmental estrogens widely found in plastic products. In this study, we injected BPA and the synthetic estrogen diethylstilbestrol (DES) in ovo and then examined and compared the effects of those on the uteri (shell gland) of the adult hens by histological methods. Five groups have been designed in the current study. Only vehicle substance was given in ovo to the control group and BPA (67 or 134 μg/g egg) and DES (0.02 or 0.2 μg/g egg) were administered in the experimental groups. Tissue specimens were taken from uteri of hens at 21 weeks of age, prior to the laying period. Estrogen receptor alpha (ERα) was immunohistochemically stained. It was observed that the hatching proportion in BPA (67 μg and 134 μg/g) was lesser than the other groups (P?<?0.01). Uterine tubular glandular density and thickness of tunica mucosa were found to have reduced (P?<?0.01) in BPA (134 μg/g) and DES (0.2 μg/g) groups, in comparison with those of the control and the other experimental groups. Uterine gland epithelium revealed positive immunoreaction for ERα. These findings suggested that administration of BPA and DES at high doses affected embryonic development in a negative way, and this adverse effect was seen less in adult period.     

Ala-9Val polymorphism of Mn-SOD gene in sickle cell anemia

Oxidative stress may be contributory to the pathophysiology of the abnormalities that underlie the clinical course of sickle cell anemia. We looked for a possible genetic association between the functional polymorphism Ala-9Val in the human Mn-SOD gene and sickle cell anemia. One hundred and twenty-seven patients with sickle cell anemia and 127 healthy controls were recruited into the study. Alanine versus valine polymorphism in the signal peptide of the Mn-SOD gene was evaluated using a primer pair to amplify a 107-bp fragment followed by digestion with the restriction enzyme NgoMIV. In the sickle cell anemia patients, the frequency of Val/Val genotype was approximately 1.4-fold lower and that of Ala/Val was 1.3-fold higher compared to the controls. No significant difference in genotype frequencies was found between patients and controls (χ(2) = 4.561, d.f. = 2, P = 0.101). The Val-9 was the most common allele in patient and healthy subjects. No significant difference in allele frequencies was found between patients and controls (χ(2) = 1.496, d.f. = 1, P = 0.221). We conclude that the Mn-SOD gene polymorphism is not associated with sickle cell anemia.     

Analysis of common MDR1 (ABCB1) gene C1236T and C3435T polymorphisms in Turkish patients with familial Mediterranean fever

The multidrug resistance (MDR1) gene encodes a P-glycoprotein that plays a key role in drug bioavailability and response to drugs in different human populations. More than 50 SNPs have been described for the MDR1 gene. Familial Mediterranean fever (FMF) is considered an autosomal recessive hereditary disease, associated with a single gene named the Mediterranean fever gene (MEFV). However, about one-third of FMF patients have only one mutated allele, suggesting that this disease is expressed as an autosomal dominant trait with partial penetration or an additional gene might be responsible for the disease. We made genotype and haplotype analyses of the MDR1 gene in 142 FMF patients and 130 unrelated Turkish subjects; two MDR-1 genetic markers (C1236T and C3435T) were analyzed by PCR-RFLP analysis. FMF patients had a significantly higher frequency of the 3435 CT genotype compared with the control group (59.9% in FMF patients versus 44.6% in controls; odds ratio [OR] = 1.85; 95% confidence interval [CI] = 1.14-3.00). Based on haplotype analysis, the T-C shift was significantly more frequent in controls (14.4% versus 7.1% in FMF patients). This haplotype could be protective for FMF disease (OR = 0.45; 95%CI = 0.25-0.84). The frequency of CC-CT (1236-3435) binary genotype was significantly higher in FMF patients (14.79% versus 4.61% in controls; OR = 3.59; 95%CI = 1.40-9.20).     

Molecular phylogeny of the Cricetinae subfamily based on the mitochondrial cytochrome b and 12S rRNA genes and the nuclear vWF gene

Despite some popularity of hamsters as pets and laboratory animals there is no reliable phylogeny of the subfamily Cricetinae available so far. Contradicting views exist not only about the actual number of species but also concerning the validity of several genera. We used partial DNA sequences of two mitochondrial (cytochrome b, 12S rRNA) and one partial nuclear gene (von Willebrand Factor exon 28) to provide a first gene tree of the Cricetinae based on 15 taxa comprising six genera. According to our data, Palaearctic hamsters fall into three distinct phylogenetic groups: Phodopus, Mesocricetus, and Cricetus-related species which evolved during the late Miocene about 7-12MY ago. Surprisingly, the genus Phodopus, which was previously thought to have appeared during the Pleistocene, forms the oldest clade. The largest number of extant hamster genera is found in a group of Cricetus-related hamsters. The genus Cricetulus itself proved to be not truly monophyletic with Cricetulus migratorius appearing more closely related to Tscherskia, Cricetus, and Allocricetulus. We propose to place the species within a new monotypic genus. Molecular clock calculations are not always in line with the dating of fossil records. DNA based divergence time estimates as well as taxonomic relationships demand a reevaluation of morphological characters previously used to identify fossils and extant hamsters.