European Ancestry Predominates in Neuromyelitis Optica and Multiple Sclerosis Patients from Brazil

Background: Neuromyelitis optica (NMO) is considered relatively more common in non-Whites, whereas multiple sclerosis (MS) presents a high prevalence rate, particularly in Whites from Western countries populations. However, no study has used ancestry informative markers (AIMs) to estimate the genetic ancestry contribution to NMO patients. Methods: Twelve AIMs were selected based on the large allele frequency differences among European, African, and Amerindian populations, in order to investigate the genetic contribution of each ancestral group in 236 patients with MS and NMO, diagnosed using the McDonald and Wingerchuck criteria, respectively. All 128 MS patients were recruited at the Faculty of Medicine of Ribeirão Preto (MS-RP), Southeastern Brazil, as well as 108 healthy bone marrow donors considered as healthy controls. A total of 108 NMO patients were recruited from five Neurology centers from different Brazilian regions, including Ribeirão Preto (NMO-RP). Principal Findings: European ancestry contribution was higher in MS-RP than in NMO-RP (78.5% vs. 68.7%) patients. In contrast, African ancestry estimates were higher in NMO-RP than in MS-RP (20.5% vs. 12.5%) patients. Moreover, principal component analyses showed that groups of NMO patients from different Brazilian regions were clustered close to the European ancestral population. Conclusions: Our findings demonstrate that European genetic contribution predominates in NMO and MS patients from Brazil. © 2013 Brum et al.

Methicillin resistance in Staphylococcus aureus and coagulase-negative staphylococci: Epidemiological and molecular aspects

Infections caused by the genus Staphylococcus are of great importance for human health. Staphylococcus species are divided into coagulase-positive staphylococci, represented by S. aureus, a pathogen that can cause infections of the skin and other organs in immunocompetent patients, and coagulase-negative staphylococci (CNS) which comprise different species normally involved in infectious processes in immunocompromised patients or patients using catheters. Oxacillin has been one of the main drugs used for the treatment of staphylococcal infections; however, a large number of S. aureus and CNS isolates of nosocomial origin are resistant to this drug. Methicillin resistance is encoded by the mecA gene which is inserted in the SCCmec cassette. This cassette is a mobile genetic element consisting of five different types and several subtypes. Oxacillin-resistant strains are detected by phenotypic and genotypic methods. Epidemiologically, methicillin-resistant S. aureus strains can be divided into five large pandemic clones, called Brazilian, Hungarian, Iberian, New York/Japan and Pediatric. The objective of the present review was to discuss aspects of resistance, epidemiology, genetics and detection of oxacillin resistance in Staphylococcus spp., since these microorganisms are increasingly more frequent in Brazil.

Replacement of the proximal heme thiolate ligand in chloroperoxidase with a histidine residue

Chloroperoxidase is a versatile heme enzyme which can cross over the catalytic boundaries of other oxidative hemoproteins and perform multiple functions. Chloroperoxidase, in addition to catalyzing classical peroxidative reactions, also acts as a P450 cytochrome and a potent catalase. The multiple functions of chloroperoxidase must be derived from its unique active site structure. Chloroperoxidase possesses a proximal cysteine thiolate heme iron ligand analogous to the P450 cytochromes; however, unlike the P450 enzymes, chloroperoxidase possesses a very polar environment distal to its heme prosthetic group and contains a glutamic acid residue in close proximity to the heme iron. The presence of a thiolate ligand in chloroperoxidase has long been thought to play an essential role in its chlorination and epoxidation activities; however, the research reported in this paper proves that hypothesis to be invalid. To explore the role of Cys-29, the amino acid residue supplying the thiolate ligand in chloroperoxidase, Cys-29 has been replaced with a histidine residue. Mutant clones of the chloroperoxidase genome have been expressed in a Caldariomyces fumago expression system by using gene replacement rather than gene insertion technology. C. fumago produces wild-type chloroperoxidase, thus requiring gene replacement of the wild type by the mutant gene. To the best of our knowledge, this is the first time that gene replacement has been reported for this type of fungus. The recombinant histidine mutants retain most of their chlorination, peroxidation, epoxidation, and catalase activities. These results downplay the importance of a thiolate ligand in chloroperoxidase and suggest that the distal environment of the heme active site plays the major role in maintaining the diverse activities of this enzyme.

Proteomic and Metabolomic Analyses of Mitochondrial Complex I-deficient Mouse Model Generated by Spontaneous B2 Short Interspersed Nuclear Element (SINE) Insertion into NADH Dehydrogenase (Ubiquinone) Fe-S Protein 4 (Ndufs4) Gene.

Eukaryotic cells generate energy in the form of ATP, through a network of mitochondrial complexes and electron carriers known as the oxidative phosphorylation system. In mammals, mitochondrial complex I (CI) is the largest component of this system, comprising 45 different subunits encoded by mitochondrial and nuclear DNA. Humans diagnosed with mutations in the gene NDUFS4, encoding a nuclear DNA-encoded subunit of CI (NADH dehydrogenase ubiquinone Fe-S protein 4), typically suffer from Leigh syndrome, a neurodegenerative disease with onset in infancy or early childhood. Mitochondria from NDUFS4 patients usually lack detectable NDUFS4 protein and show a CI stability/assembly defect. Here, we describe a recessive mouse phenotype caused by the insertion of a transposable element into Ndufs4, identified by a novel combined linkage and expression analysis. Designated Ndufs4(fky), the mutation leads to aberrant transcript splicing and absence of NDUFS4 protein in all tissues tested of homozygous mice. Physical and behavioral symptoms displayed by Ndufs4(fky/fky) mice include temporary fur loss, growth retardation, unsteady gait, and abnormal body posture when suspended by the tail. Analysis of CI in Ndufs4(fky/fky) mice using blue native PAGE revealed the presence of a faster migrating crippled complex. This crippled CI was shown to lack subunits of the "N assembly module", which contains the NADH binding site, but contained two assembly factors not present in intact CI. Metabolomic analysis of the blood by tandem mass spectrometry showed increased hydroxyacylcarnitine species, implying that the CI defect leads to an imbalanced NADH/NAD(+) ratio that inhibits mitochondrial fatty acid β-oxidation.

The rulB gene of plasmid pWW0 is a hotspot for the site-specific insertion of integron-like elements found in the chromosomes of environmental Pseudomonas fluorescens group bacteria

The rulAB operon of Pseudomonas spp. confers fitness traits on the host and has been suggested to be a hotspot for insertion of mobile elements that carry avirulence genes. Here, for the first time, we show that rulB on plasmid pWW0 is a hotspot for the active site-specific integration of related integron-like elements (ILEs) found in six environmental pseudomonads (strains FH1–FH6). Integration into rulB on pWW0 occurred at position 6488 generating a 3 bp direct repeat. ILEs from FH1 and FH5 were 9403 bp in length and contained eight open reading frames (ORFs), while the ILE from FH4 was 16 233 bp in length and contained 16 ORFs. In all three ILEs, the first 5.1 kb (containing ORFs 1–4) were structurally conserved and contained three predicted site-specific recombinases/integrases and a tetR homologue. Downstream of these resided ORFs of the ‘variable side’ with structural and sequence similarity to those encoding survival traits on the fitness enhancing plasmid pGRT1 (ILEFH1 and ILEFH5) and the NR-II virulence region of genomic island PAGI-5 (ILEFH4). Collectively, these ILEs share features with the previously described type III protein secretion system effector ILEs and are considered important to host survival and transfer of fitness enhancing and (a)virulence genes between bacteria.

P element insertion-dependent gene activation in the Drosophila eye

Insights into the function of a gene can be gained in multiple ways, including loss-of-function phenotype, sequence similarity, expression pattern, and by the consequences of its misexpression. Analysis of the phenotypes produced by expression of a gene at an abnormal time, place, or level may provide clues to a gene’s function when other approaches are not illuminating. Here we report that an eye-specific, enhancer–promoter present in the P element expression vector pGMR is able to drive high level expression in the eye of genes near the site of P element insertion. Cell fate determination, differentiation, proliferation, and death are essential for normal eye development. Thus the ability to carry out eye-specific misexpression of a significant fraction of genes in the genome, given the dispensability of the eye for viability and fertility of the adult, should provide a powerful approach for identifying regulators of these processes. To test this idea we carried out two overexpression screens for genes that function to regulate cell death. We screened for insertion-dependent dominant phenotypes in a wild-type background, and for dominant modifiers of a reaper overexpression-induced small eye phenotype. Multiple chromosomal loci were identified, including an insertion 5′ to hid, a potent inducer of apoptosis, and insertions 5′ to DIAP1, a cell death suppressor. To facilitate the cloning of genes near the P element insertion new misexpression vectors were created. A screen with one of these vectors identified eagle as a suppressor of a rough eye phenotype associated with overexpression of an activated Ras1 gene.

DNA sequence insertion and evolutionary variation in gene regulation.

Current evidence on the long-term evolutionary effect of insertion of sequence elements into gene regions is reviewed, restricted to cases where a sequence derived from a past insertion participates in the regulation of expression of a useful gene. Ten such examples in eukaryotes demonstrate that segments of repetitive DNA or mobile elements have been inserted in the past in gene regions, have been preserved, sometimes modified by selection, and now affect control of transcription of the adjacent gene. Included are only examples in which transcription control was modified by the insert. Several cases in which merely transcription initiation occurred in the insert were set aside. Two of the examples involved the long terminal repeats of mammalian endogenous retroviruses. Another two examples were control of transcription by repeated sequence inserts in sea urchin genomes. There are now six published examples in which Alu sequences were inserted long ago into human gene regions, were modified, and now are central in control/enhancement of transcription. The number of published examples of Alu sequences affecting gene control has grown threefold in the last year and is likely to continue growing. Taken together, all of these examples show that the insertion of sequence elements in the genome has been a significant source of regulatory variation in evolution.

Targeted gene inactivation in petunia by PCR-based selection of transposon insertion mutants.

Establishment of loss-of-function phenotypes is often a key step in determining the biological function of a gene. We describe a procedure to obtain mutant petunia plants in which a specific gene with known sequence is inactivated by the transposable element dTph1. Leaves are collected from batches of 1000 plants with highly active dTph1 elements, pooled according to a three-dimensional matrix, and screened by PCR using a transposon- and a gene-specific primer. In this way individual plants with a dTph1 insertion can be identified by analysis of about 30 PCRs. We found insertion alleles for various genes at a frequency of about 1 in 1000 plants. The plant population can be preserved by selfing all the plants, so that it can be screened for insertions in many genes over a prolonged period.

Insertion of an E-coli lacZ gene in Acetobacter xylinus for the production of cellulose in whey

A mini-Tn10:lacZ: kan was inserted into a wild-type strain of Acetobacter xylinus by random transposon mutagenesis, generating a lactose-utilising and cellulose-producing mutant strain designated ITz3. Antibiotic selection plate assays and Southern hybridisation revealed that the lacZ gene was inserted once into the chromosome of strain ITz3 and was stably maintained in non-selective medium after more than 60 generations. The modified strain had, on the average, a 28-fold increase in cellulose production and a 160-fold increase in beta-galactosidase activity when grown in lactose medium. beta-Galactosidase activity is present in either lactose or sucrose medium indicating that the gene is constitutively expressed. Cellulose and beta-galactosidase production by the modified strain was also evaluated in pure and enriched whey substrates. Utilisation of lactose in whey substrate by ITz3 reached 17 g l(-1) after 4 days incubation. (C) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

A versatile and reliable two-component system for tissue-specific gene induction in Arabidopsis

Developmental progression and differentiation of distinct cell types depend on the regulation of gene expression in space and time. Tools that allow spatial and temporal control of gene expression are crucial for the accurate elucidation of gene function. Most systems to manipulate gene expression allow control of only one factor, space or time, and currently available systems that control both temporal and spatial expression of genes have their limitations. We have developed a versatile two-component system that overcomes these limitations, providing reliable, conditional gene activation in restricted tissues or cell types. This system allows conditional tissue-specific ectopic gene expression and provides a tool for conditional cell type- or tissue-specific complementation of mutants. The chimeric transcription factor XVE, in conjunction with Gateway recombination cloning technology, was used to generate a tractable system that can efficiently and faithfully activate target genes in a variety of cell types. Six promoters/enhancers, each with different tissue specificities (including vascular tissue, trichomes, root, and reproductive cell types), were used in activation constructs to generate different expression patterns of XVE. Conditional transactivation of reporter genes was achieved in a predictable, tissue-specific pattern of expression, following the insertion of the activator or the responder T-DNA in a wide variety of positions in the genome. Expression patterns were faithfully replicated in independent transgenic plant lines. Results demonstrate that we can also induce mutant phenotypes using conditional ectopic gene expression. One of these mutant phenotypes could not have been identified using noninducible ectopic gene expression approaches.

Association between polymorphisms in the progesterone receptor gene and endometriosis

The progesterone receptor (PR) is a candidate gene for the development of endometriosis, a complex disease with strong hormonal features, common in women of reproductive age. We typed the 306 base pair Alu insertion (AluIns) polymorphism in intron G of PR in 101 individuals, estimated linkage disequilibrium (LD) between five single-nucleotide polymorphisms (SNPs) across the PR locus in 980 Australian triads (endometriosis case and two parents) and used transmission disequilibrium testing (TDT) for association with endometriosis. The five SNPs showed strong pairwise LD, and the AluIns was highly correlated with proximal SNPs rs1042839 (Δ2 = 0.877, D9 = 1.00, P < 0.0001) and rs500760 (Δ2 = 0.438, D9 = 0.942, P < 0.0001). TDT showed weak evidence of allelic association between endometriosis and rs500760 (P = 0.027) but not in the expected direction. We identified a common susceptibility haplotype GGGCA across the five SNPs (P = 0.0167) in the whole sample, but likelihood ratio testing of haplotype transmission and non-transmission of the AluIns and flanking SNPs showed no significant pattern. Further, analysis of our results pooled with those from two previous studies suggested that neither the T2 allele of the AluIns nor the T1/T2 genotype was associated with endometriosis.