Impairing methylations at ribosome RNA, a point mutation-dependent strategy for aminoglycoside resistance: The rsmG case / Mutaciones en genes modificadores de ARN ribosómico y la resistencia a aminoglucósidos: el caso del gen rsmG

Introduction: Aminoglycosides like streptomycin are well-known for binding at specific regions of ribosome RNA and then acting as translation inhibitors. Nowadays, several pathogens have been detected to acquire an undefined strategy involving mutation at non structural ribosome genes like those acting as RNA methylases. rsmG is one of those genes which encodes an AdoMet-dependent methyltransferase responsible for the synthesis of m 7 G527 in the 530 loop of bacterial 16S rRNA. This loop is universally conserved, plays a key role in ribosomal accuracy, and is a target for streptomycin binding. Loss of the m 7 G527 modification confers low-level streptomycin resistance and may affect ribosomal functioning. Objectives: After taking into account genetic information indicating that some clinical isolates of human pathogens show streptomycin resistance associated with mutations at rsmG , we decided to explore new hot spots for mutation capable of impairing the RsmG in vivo function and of promoting low-level streptomycin resistance. Materials and methods: To gain insights into the molecular and genetic mechanism of acquiring this aminoglycoside resistance phenotype and the emergence of high-level streptomycin resistance in rsmG mutants, we mutated Escherichia coli rsmG and also performed a genotyping study on rpsL from several isolates showing the ability to grow at higher streptomycin concentrations than parental strains. Results: We found that the mutations at rpsL were preferentially present in these mutants, and we observed a clear synergy between rsmG and rpsL genes to induce streptomycin resistance. Conclusion: We contribute to understand a common mechanism that is probably transferable to other ribosome RNA methylase genes responsible for modifications at central sites for ribosome function.

Introducción. Los aminoglucósidos son moléculas antibióticas capaces de inhibir la síntesis de proteínas bacterianas tras su unión al ribosoma procariota. La resistencia a aminoglucósidos está clásicamente asociada a mutaciones en genes estructurales del ribosoma bacteriano; sin embargo, varios estudios recientes han demostrado, de forma recurrente, la presencia de un nuevo mecanismo dependiente de mutación que no involucra genes estructurales. El gen rsmG es uno de ellos y se caracteriza por codificar una metiltransferasa que sintetiza el nucleósido m 7 G527 localizado en el loop 530 del ribosoma bacteriano, este último caracterizado como sitio preferencial al cual se une la estreptomicina. Objetivo. Partiendo de las recientes asociaciones clínicas entre las mutaciones en el gen rsmG y la resistencia a estreptomicina, este estudio se propuso la caracterización de nuevos puntos calientes de mutación en este gen que puedan causar resistencia a estreptomicina usando Escherichia coli como modelo de estudio. Materiales y métodos. Se indagó sobre el mecanismo genético y molecular por el cual se adquiere la resistencia a estreptomicina y su transición a la resistencia a altas dosis mediante mutagénesis dirigida del gen rsmG y genotipificación del gen rpsL . Resultados. Se encontró que la mutación N39A en rsmG inactiva la proteína y se reportó un nuevo conjunto de mutaciones en rpsL que confieren resistencia a altas dosis de estreptomicina. Conclusiones. Aunque los mecanismos genéticos subyacentes permanecen sin esclarecer, se concluyó que dichos patrones secuenciales de mutación podrían tener lugar en otros genes modificadores del ARN bacteriano debido a la conservación evolutiva y al papel crítico que juegan tales modificaciones en la síntesis de proteínas.

Galectin-3 increases the motility of mouse melanoma cells by regulating matrix metalloproteinase-1 expression

Although mounting evidence indicates the involvement of galectin-3 in cancer progression and metastasis, the underlying molecular mechanisms remain largely unknown. In this study, we investigated the effect and possible mechanism of galectin-3 on the migration and invasion of B16F10, a metastatic melanoma cell line, in which galectin-3 and matrix metalloproteinase-1 (MMP-1) were both found to be highly expressed. Knockdown of galectin-3 with specific siRNA reduced migration and invasion, which was associated with reduced expression of MMP-1. To further investigate the underlying mechanism, we examined the effect of galectin-3 knockdown on the activity of AP-1, a transcriptional factor regulating MMP-1 expression. We found that galectin-3 directly interacted with AP-1 and facilitated the binding of this complex to the MMP-1 promoter that drives MMP-1 transcription. Moreover, silencing of galectin-3 inhibited binding of fra-1 and c-Jun to promoter sites of MMP-1 gene. Consistent with these in vitro findings, our in vivo study demonstrated that galectin-3 shRNA treatment significantly reduced the total number of mouse lung metastatic nodules. Taken together, galectin-3 facilitates cell migration and invasion in melanoma in vitro and can induce metastasis in vivo, in part through, regulating the transcription activity of AP-1 and thereby up-regulating MMP-1 expression.

Polymorphism of a COLIA1 Gene Sp1 Binding Site in Korean Women with Pelvic Organ Prolapse

PURPOSE: To evaluate the possible influence of G-->T substitution at the Sp1-binding site of the COLIA1 gene on the risk of pelvic organ prolapse (POP). MATERIALS AND METHODS: The study group consisted of 15 women with advanced stage POP. Fifteen control subjects with uterine myomas among the postmenopausal women were matched for age and parity. DNA was obtained from peripheral blood leukocytes. The fragments of the first intron of the COLIA1 gene were amplified by real time polymerase chain reaction. The polymorphism was identified using LightCycler Technology with hybridization probes. Sequencing reactions were performed on each template using commercial primer. RESULTS: Two groups had no significant difference in medical history, surgical, and smoking history. The homozygous peaks in two groups were noted at 57degrees C on melting curve analysis. Sequencing reactions confirmed the G/G alleles in the 30 specimens tested. We could not find any polymorphism at the Sp1-binding site in COLIA1 gene with advanced stage POP. Statistical significance was considered to be p < .05. CONCLUSION: The polymorphism of the Sp1-binding site in the COLIA1 gene did not contribute to the development of POP in Korea.

Characterization of the long-terminal repeat single-strand tail-binding site of Moloney-MuLV integrase by crosslinking

Processing of viral DNA by retroviral integrase leaves a dinucleotide single-strand overhang in the unprocessed strand. Previous studies have stressed the importance of the 5' single-stranded (ss) tail in the integration process. To characterize the ss-tail binding site on M-MuLV integrase, we carried out crosslinking studies utilizing a disintegration substrate that mimics the covalent intermediate formed during integration. This substrate carried reactive groups at the 5' ss tail. A bromoacetyl derivative with a side chain of 6 A was crosslinked to the mutant IN 106-404, which lacks the N-terminal domain, yielding a crosslinked complex of 50 kDa. Treatment of IN 106-404 with N-ethylmaleimide (NEM) prevented crosslinking, suggesting that Cys209 was involved in the reaction. The reactivity of Cys209 was confirmed by crosslinking of a more specific derivative carrying maleimide groups that spans 8A approximately. In contrast, WT IN was not reactive, suggesting that the N-terminal domain modifies the reactivity of the Cys209 or the positioning of the crosslinker side chain. A similar oligonucleotide-carrying iodouridine at the 5'ss tail reacted with both IN 106-404 and WT IN upon UV irradiation. This reaction was also prevented by NEM, suggesting that the ss-tail positions near a peptide region that includes Cys209.

Mutation analysis of PAH gene and characterization of a recurrent deletion mutation in Korean patients with phenylketonuria

Phenylketonuria (PKU; MIM 261600) is an autosomal recessive metabolic disorder caused by a deficiency of phenylalanine hydroxylase (PAH; EC 1.14.16.1). Point mutations in the PAH gene are known to cause PKU in various ethnic groups, and large deletions or duplications account for up to 3% of the PAH mutations in some ethnic groups. However, a previous study could not identify ~14% of the mutant alleles by sequence analysis in Korean patients with PKU, which suggests that large deletions or duplication might be frequent causes of PKU in Koreans. To test this hypothesis, we performed multiplex ligation-dependent probe amplification (MLPA) for the identification of uncharacterized mutant alleles after PAH sequence analysis of 33 unrelated Korean patients with PKU. Bi-directional sequencing of the PAH exons and flanking intronic regions revealed 27 different mutations, including four novel mutations (two missense and two deletion mutations), comprising 57/66 (86%) mutant alleles. MLPA identified a large deletion that encompassed exons 5 and 6 in four patients, another large deletion that extended from exon 4 to exon 7 in one patient, and a duplication of exon 4 in one patient. Chromosomal walking characterized the deletion breakpoint of the most common large deletion that involved exons 5 and 6 (c.456_706+138del). The present study shows that the allelic frequency of exon deletion or duplication is 9% (6/66) in Korean PKU patients, which suggests that these mutations may be frequent causes of PKU in Korean subjects.

Mechanism of DNA-binding loss upon single-point mutation in p53.

Over 50% of all human cancers involve p53 mutations,which occur mostly in the sequence-specific DNA-binding central domain (p53c), yielding little/non-detectable af?nity to the DNA consensus site.Despite our current understanding of protein-DNA recognition,the mechanism(s) underlying the loss in protein-DNA binding afnity/ specificity upon single-point mutation are not well understood. Our goal is to identify the common factors governing the DNA-binding loss of p53c upon substitution of Arg 273 to His or Cys,which are abundant in human tumours. By computing the free energies of wild-type and mutant p53c binding to DNA and decomposing them into contributions from individual residues, the DNA-binding loss upon charge/noncharge -conserving mutation of Arg 273 was attributed not only to the loss of DNA phosphate contacts, but also to longer-range structural changes caused by the loss of the Asp 281 salt-bridge. The results herein and in previous works suggest that Asp 281 plays a critical role in the sequence-specific DNA-binding function of p53c by (i)orienting Arg 273 and Arg 280 in an optimal position to interact with the phosphate and base groups of the consensus DNA, respectively, and (ii) helping to maintain the proper DNA-binding protein conformation.

Type 2 diabetes mellitus: phylogenetic motifs for predicting protein functional sites.

Diabetes mellitus, commonly referred to as diabetes, is a medical condition associated with abnormally high levels of glucose (or sugar) in the blood. Keeping this view, we demonstrate the phylogenetic motifs (PMs) identification in type 2 diabetes mellitus very likely corresponding to protein functional sites. In this article, we have identified PMs for all the candidate genes for type 2 diabetes mellitus. Glycine 310 remains conserved for glucokinase and potassium channel KCNJ11. Isoleucine 137 was conserved for insulin receptor and regulatory subunit of a phosphorylating enzyme. Whereas residues valine, leucine, methionine were highly conserved for insulin receptor.Occurrence of proline was very high for calpain 10 gene and glucose transporter.

Incorporating evolution of transcription factor binding sites into annotated alignments.

Identifying transcription factor binding sites (TFBSs) is essential to elucidate putative regulatory mechanisms. A common strategy is to combine cross-species conservation with single sequence TFBS annotation to yield "conserved TFBSs". Most current methods in this field adopt a multi-step approach that segregates the two aspects. Again, it is widely accepted that the evolutionary dynamics of binding sites differ from those of the surrounding sequence. Hence, it is desirable to have an approach that explicitly takes this factor into account. Although a plethora of approaches have been proposed for the prediction of conserved TFBSs, very few explicitly model TFBS evolutionary properties, while additionally being multi-step. Recently, we introduced a novel approach to simultaneously align and annotate conserved TFBSs in a pair of sequences. Building upon the standard Smith-Waterman algorithm for local alignments, SimAnn introduces additional states for profiles to output extended alignments or annotated alignments. That is, alignments with parts annotated as gaplessly aligned TFBSs (pair-profile hits)are generated. Moreover,the pair- profile related parameters are derived in a sound statistical framework. In this article, we extend this approach to explicitly incorporate evolution of binding sites in the SimAnn framework. We demonstrate the extension in the theoretical derivations through two position-specific evolutionary models, previously used for modelling TFBS evolution. In a simulated setting, we provide a proof of concept that the approach works given the underlying assumptions,as compared to the original work. Finally, using a real dataset of experimentally verified binding sites in human-mouse sequence pairs,we compare the new approach (eSimAnn) to an existing multi-step tool that also considers TFBS evolution. Although it is widely accepted that binding sites evolve differently from the surrounding sequences, most comparative TFBS identification methods do not explicitly consider this.Additionally, prediction of conserved binding sites is carried out in a multi-step approach that segregates alignment from TFBS annotation. In this paper, we demonstrate how the simultaneous alignment and annotation approach of SimAnn can be further extended to incorporate TFBS evolutionary relationships.We study how alignments and binding site predictions interplay at varying evolutionary distances and for various profile qualities.

The role of tonicity responsive enhancer sites in the transcriptional regulation of human hsp70-2 in response to hypertonic stress

The inducible 70 kDa heat shock proteins (Hsp70) in mice are encoded by two almost identical genes, hsp70.1 and hsp70.3. Studies have found that only hsp70.1 is induced by hypertonic stress while both hsp70.1 and hsp70.3 genes are expressed in response to heat shock stress. It is unclear if the human counterparts, hsp70-2 and hsp70-1, are differentially regulated by heat shock and osmotic stress. This study found that only hsp70-2 was induced by hypertonic stress in human embryonic kidney epithelial cells and fibroblasts, while heat shock stress induced both hsp70-1 and hsp70-2. The human hsp70-2 promoter region contains three TonE (tonicity-responsive enhancer) sites, which were reported to play an important role in the response to hypertonicity. When the reporter plasmids containing different parts of the 5' flanking region of hsp70-2 were transfected into human embryonic kidney epithelial cells or fibroblasts, one TonE site at -135 was found to play a key role in the response to hypertonicity. The inactivation of the TonE site using site-directed mutagenesis led to the complete loss of induction by hypertonicity, which demonstrates the essential role of the TonE site. This suggests that the TonE site and the TonEBP (TonE binding protein) are the major regulators for the cellular response against high osmolarity in human kidney tissue.

A genomic-scale search for regulatory binding sites in the integration host factor regulon of Escherichia coli K12

We examined general aspects of the DNA-protein interaction between the integration host factor (IHF) global regulator and its regulatory binding sites in the Escherichia coli K12 genome. Two models were developed with distinct weight matrices for the regulatory binding sites recognized by IHF. Using these matrices we performed a genome scale scan and built a set of computationally predicted binding sites for each of the models. The sites found by the model associated with repetitive sequences had a higher score in the sequence to matrix alignment. They were also more rare than the other sites. The sites not associated with repeats rapidly tended to become undistinguishable from the background as statistical stringency was relaxed. We compared our results to the known sites documented in RegulonDB and found new members of the IHF Regulon. The two models exhibit clearly distinct affinity patterns (scores in the sequence to matrix alignments and in the number of regulatory sites), as we vary the stringency of the statistical confidence parameters. We suggest that these differences may play an important role in the dynamics of the network. We concluded that IHF may regulate two genes encoding ATP-dependent RNA helicases. This interaction is not described in RegulonDB, even as a computational prediction. IHF may also regulate RNA modification processes.

Transcriptional regulation of Zic3 by heterodimeric AP-1(c-Jun/c-Fos) during Xenopus development

The heterodimeric c-Jun/c-Fos, an activator protein-1 (AP-1) has been implicated in mesoderm induction (Dong et al., 1996; Kim et al., 1998) whereas the homodimer of c-Jun was reported to be involved in neural inhibition during the early development of Xenopus embryos. During the early vertebrate development AP-1 involvement in the neural induction is still not clearly understood. We report here that AP-1 has a role in Zic3 expression, a critical proneural gene and a primary regulator of neural and neural crest development (Nakata et al., 1997; Nakata et al., 1998). AP-1 was able to induce the Zic3 gene in a dose dependent manner but other homo- or hetero-dimeric proteins, such as c-Jun/c-Jun, JunD/FosB or JunD/Fra-1 were not. The inhibition of AP-1 activity using morpholino antisenses of c-jun mRNAs blocked the Zic3 expression induced by activin. In addition, co-injection of c-jun mRNA rescued the down-regulated Zic3 expression. The promoter region of isolated Zic3 genomic DNA was found to possess several consensus-binding site of AP-1. Thus, in the functional assays, AP-1 could increase promoter activity of Zic3 gene. These findings suggest that proneural gene, Zic3 may be regulated by heterodimeric AP-1(c-Jun/c-Fos) and it may have a role in activin signaling for the regulation of neural specific gene, Zic3.

Weakening of the repressive YY-1 site on the thrombospondin-1 promoter via c-Jun/YY-1 interaction

Thrombospondin-1 (TSP-1) level is tightly regulated at the transcriptional level. To determine the detailed molecular mechanisms of TSP-1 expression, nine serial 5'-deletion constructs of the human genomic tsp-1 promoter (nucleotides -2,220 to +756) were prepared, inserted into luciferase reporter plasmids, and transiently transfected into the Hep3B human hepatocarcinoma cell. Among the nine 5'-deletion constructs, pTSP-Luc-4 (-767~+756) had consistently decreased luciferase activity with or without PMA stimulation, whereas a further truncated construct [pTSP-Luc-4' (-407~+756)] had increased levels of expression. By searching the nucleotides from -767 to -407, a consensus binding sequence (5'-CCATTTT-3') for the repressor Yin Yang-1 (YY-1) at nucleotide -440 was identified. The suppression induced by this site was weakened in the presence of the region upstream of nucleotide -767 (pTSP-Luc-1 and -2). Nuclear protein directly bound to an oligonucleotide containing the repressive YY-1 sequence but the binding capacity of the sequence was decreased by the increased c-Jun levels. Moreover, proteins immunoprecipitated with anti-YY-1 revealed an interaction between c-Jun and YY-1 factor. These data suggest that the repressive YY-1 site of the tsp-1 promoter could not be functional via activating positive cis-elements on the upstream from this site and weakened via c-Jun/YY-1 interactions.

Ets oncogene family.

First member of Ets gene family was discovered a decade ago by studying avian erythroblastosis virus, E26. This virus encodes a tripartite protein gag-myb-ets with a molecular weight of 135 kDa. Subsequently, a series of cellular Ets genes were isolated (Ets-1, Ets-2, Erg, Elk-1, Sap-1, PEA-3, PU.1, Fli-1, Pok/Yan, Etv-1 etc.). These genes share sequence homology to E26 Ets gene (v-ets or viral ets). Ets genes are highly conserved in phylogenetically divergent species from Drosophila to man. Mammalian Ets genes are located on different chromosomes. Ets gene products are transcriptionally active sequence-specific DNA binding proteins and are differentially regulated. Ets genes are involved in certain chromosomal translocations leading to the formation of chimeric fusion proteins that are associated with certain leukemias and soft tissue cancers. Ets genes also have a role in T-cell development and molecular and genetic analysis of Down Syndrome patients have implicated the human Ets-2 and Erg genes in the disease. Down Syndrome afflicted patients have immunologic and thymic disorders as well as a greater risk for leukemic disease. Thus, Ets genes having homology to viral oncogenes, may be instrumental in regulating cellular growth and differentiation, as well as organismal development. Alteration of these genes and their products may cause deregulation of normal cell growth and differentiation and result in a disease.

Interaction of SH3 domain of Hck tyrosine kinase with cellular proteins containing proline-rich regions: evidence for modulation by unique domain.

The Hck tyrosine kinase, a member of Src family, is predominantly expressed in myeloid cells. In this report we have analyzed interaction of cellular proteins with Src homology 3 (SH3) domain of Hck. For this purpose we used various GST-Hck fusion proteins comprising a part of unique region, complete unique region and/or complete SH3 domain of Hck, and glutathione S-transferase (GST). When these fusion proteins (or GST), immobilized on glutathione-agarose beads were incubated with [35S] methionine labelled cell extracts, multiple proteins which interact specifically with SH3 domain of Hck were detected by SDS-PAGE followed by autoradiography. The Hck interacting proteins could also be detected by a tandem blot binding assay in which the blot was incubated with purified fusion protein (or GST) and then the interacting proteins were identified by using antibody against GST. When a part of or complete unique domain was present along with SH3 domain, the interaction of some specific proteins was reduced several fold. These results raise the possibility of unique domain altering the properties of SH3 domain, thus modulating or restricting the interaction of SH3 domain with specific cellular proteins. This modulatory effect of unique domain was localized to 28 amino acids upstream of SH3 domain. SH3 interacting proteins were associated with serine/threonine and tyrosine kinase activities towards exogenous substrates. Most of the SH3 binding proteins were soluble in Triton X-100. Differentiation of promyelocytic leukemia cell line HL-60 into macrophage like cells resulted in appearance of novel SH3 binding proteins. Hck was detected in the eluate of WGA-Sepharose column, suggesting that it interacts with WGA binding glycoprotein (s). A rat spleen cDNA library was screened for the SH3 binding proteins by protein interaction cloning. Sequence analysis of the clones showed the presence of proline rich regions containing PPXP motifs.

Different arrangement of human papillomavirus E2 binding sites distinguishes cutaneous types from those associated with mucosal lesions

High-risk-type human papillomavirus DNA sequences are found in a high percentage of carcinomas from the uterine-cervix, with the viral E1-E2 gene region usually disrupted and the E6 and E7 oncoproteins consistently expressed. The E2 protein is known to repress early transcription from genital HPV promoters having a proximal E2 binding site (2BS) close to the TATA box. On the contrary, the E2 protein activates cutaneous early promotes, the E2 protein activates cutaneous early promoters having a longer distance between these sites. Using an in vivo approach we analyzed the regulation, by the BPV-1 E2 protein, of a natural HPV-18 promoter where proximal E2BS were placed at variable positions relative to the TATA box, and of heterologous promoters where E2BS was placed upstream of any other known DNA-binding elements. Our results confirm that the E2 protein represses or activates HPV early gene transcription depending on the distance between the TATA box and the proximal E2BS

Mutagenic bioassay of certain pharmacological drugs: II. Mebendazole (MBZ)

El Mebendazole (MBZ), o 5-benzoyl- 1H-benzimidazole-2- il ácido metil éster carbámico fue estudiado con tres ensayos de corto plazo. Se probó su capacidad para inducir micronúcleos "in vivo" en eritrocitos policromáticos de médula ósea de ratones CFW. Se analizaron 3 dosis, 25, 50 y 100 mg/kg de peso corporal inyectados intraperitonealmente. Las 3 dieron incremento significativo (p < 0.01). Las pruebas "in vitro" se realizaron en células CHO. Se analizó la capacidad para inducir aberraciones cromosómicas y figuras anormales por prueba de anafase-telofase. Se detectó una frecuencia aumentada en la formación de dicéntricos, gaps, C-mitosis, puentes y cromosomas rezagados. La acción genotóxica observada puede asociarse con la estructura química de un æester carbámico derivado que sugeriría un papel de mutágeno directo