Post-transcriptional Repair of a Split Heat Shock Protein 90 Gene by mRNA trans-Splicing

Heat shock protein 90 participates in diverse biological processes ranging from protein folding, cell cycle, signal transduction and development to evolution in all eukaryotes. It is also critically involved in regulating growth of protozoa such as Dictyostelium discoideum, Leishmania donovani, Plasmodium falciparum, Trypanosoma cruzi, and Trypanosoma evansi. Selective inhibition of Hsp90 has also been explored as an intervention strategy against important human diseases such as cancer, malaria, or trypanosomiasis. Giardia lamblia, a simple protozoan parasite of humans and animals, is an important cause of diarrheal disease with significant morbidity and some mortality in tropical countries. Here we show that the G. lamblia cytosolic hsp90 ( glhsp90) is split in two similar sized fragments located 777 kb apart on the same scaffold. Intrigued by this unique arrangement, which appears to be specific for the Giardiinae, we have investigated the biosynthesis of GlHsp90. We used genome sequencing to confirm the split nature of the giardial hsp90. However, a specific antibody raised against the peptide detected a product with a mass of about 80 kDa, suggesting a post-transcriptional rescue of the genomic defect. We show evidence for the joining of the two independent Hsp90 transcripts in-trans to one long mature mRNA presumably by RNA splicing. The splicing junction carries hallmarks of classical cis-spliced introns, suggesting that the regular cis-splicing machinery may be sufficient for repair of the open reading frame. A complementary 26-nt sequence in the ``intron'' regions adjacent to the splice sites may assist in positioning the two pre-mRNAs for processing. This is the first example of post-transcriptional rescue of a split gene by trans-splicing.

Photolabeling of the EcoP15 DNA methyltransferase with S-adenosyl-l-methionine

Radioactivity from S-adenosyl-L-[methyl-H-3] methionine ([methyl-H-3]AdoMet) was bound to the EcoP15 DNA methyltransferase (M.EcoP15) following short-wave ultraviolet (UV) irradiation. The labeled protein was subjected to polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE), and detected by fluorography and autoradiography. Labeling was found to be dependent on the concentration of AdoMet and time of UV irradiation. The photolabeling by [methyl-H-3]AdoMet was specific and blocked by S-adenosyl-L-homocysteine (AdoHcy) and sinefungin which are known to function as competitive inhibitors. Limited digestion of the M EcoP15-AdoMet adduct by Staphylococcus aureus protease V8 generated three peptides of approx. 50, 32 and 30 kDa; Interestingly, only the 30-kDa peptide fragment contained radioactivity, as detected by SDS-PAGE, followed by fluorography and autoradiography. Further, sequencing of a few amino acids at the N-terminus of these peptides showed that the 30-kDa fragment was the N-terminal portion of M.EcoP15, These results suggest that photolabeling is at the AdoMet-binding site and that the N-terminal half of M.EcoP15 may be involved in substrate binding.

Biopolymers in nanopores: challenges and opportunities

We review the current status of various aspects of biopolymer translocation through nanopores and the challenges and opportunities it offers. Much of the interest generated by nanopores arises from their potential application to third-generation cheap and fast genome sequencing. Although the ultimate goal of single-nucleotide identification has not yet been reached, great advances have been made both from a fundamental and an applied point of view, particularly in controlling the translocation time, fabricating various kinds of synthetic pores or genetically engineering protein nanopores with tailored properties, and in devising methods (used separately or in combination) aimed at discriminating nucleotides based either on ionic or transverse electron currents, optical readout signatures, or on the capabilities of the cellular machinery. Recently, exciting new applications have emerged, for the detection of specific proteins and toxins (stochastic biosensors), and for the study of protein folding pathways and binding constants of protein-protein and protein-DNA complexes. The combined use of nanopores and advanced micromanipulation techniques involving optical/magnetic tweezers with high spatial resolution offers unique opportunities for improving the basic understanding of the physical behavior of biomolecules in confined geometries, with implications for the control of crucial biological processes such as protein import and protein denaturation. We highlight the key works in these areas along with future prospects. Finally, we review theoretical and simulation studies aimed at improving fundamental understanding of the complex microscopic mechanisms involved in the translocation process. Such understanding is a pre-requisite to fruitful application of nanopore technology in high-throughput devices for molecular biomedical diagnostics.

Germline BRCA1 mutation analysis in Indian breast/ovarian cancer families

Most of the predisposition to hereditary breast and ovarian cancer has been attributed to inherited defects in two tumor suppressor genes BRCA1 and BRCA2. To explore the contribution of BRCA1 mutations to hereditary breast cancer among Indian women, we examined the coding sequence of the BRCA1 gene in 14 breast cancer patients with a positive family history of breast and/or ovarian cancer. Mutation analysis was carried out using conformation sensitive gel electrophoresis (CSGE) followed by sequencing. Three mutations (21%) in the BRCA1 gene were identified. Two of them are novel mutations of which one is a missense mutation in exon 7 near the RING finger domain, while the other is a one base pair deletion in exon 11 which results in protein truncation. The third mutation, 185delAG, has been previously described in Ashkenazi Jewish families. To our knowledge this is the first report of a study of germline BRCA1 mutation analysis in familial breast cancer in India. Our data from 14 different families suggests a lower prevalence but definite involvement of germline mutations in the BRCA1 gene among Indian women with breast cancer and a family history of breast cancer.

The restriction mapping problem revisited

In computational molecular biology, the aim of restriction mapping is to locate the restriction sites of a given enzyme on a DNA molecule. Double digest and partial digest are two well-studied techniques for restriction mapping. While double digest is NP-complete, there is no known polynomial-time algorithm for partial digest. Another disadvantage of the above techniques is that there can be multiple solutions for reconstruction. In this paper, we study a simple technique called labeled partial digest for restriction mapping. We give a fast polynomial time (O(n(2) log n) worst-case) algorithm for finding all the n sites of a DNA molecule using this technique. An important advantage of the algorithm is the unique reconstruction of the DNA molecule from the digest. The technique is also robust in handling errors in fragment lengths which arises in the laboratory. We give a robust O(n(4)) worst-case algorithm that can provably tolerate an absolute error of O(Delta/n) (where Delta is the minimum inter-site distance), while giving a unique reconstruction. We test our theoretical results by simulating the performance of the algorithm on a real DNA molecule. Motivated by the similarity to the labeled partial digest problem, we address a related problem of interest-the de novo peptide sequencing problem (ACM-SIAM Symposium on Discrete Algorithms (SODA), 2000, pp. 389-398), which arises in the reconstruction of the peptide sequence of a protein molecule. We give a simple and efficient algorithm for the problem without using dynamic programming. The algorithm runs in time O(k log k), where k is the number of ions and is an improvement over the algorithm in Chen et al. (C) 2002 Elsevier Science (USA). All rights reserved.

Copper, Zinc-Superoxide Dismutase from Clinically Isolated Escherichia coli: Cloning, Analysis of sodC and Its Possible Role in Pathogenicity

Superoxide dismutase has been discovered within the periplasm of several Gram-negative pathogens. We studied the Cu,Zn-SOD enzyme in Escherichia coli isolated from clinical samples (stool samples) collected from patients suffering from diarrhea. Antibiogram studies of the isolates were carried out to determine the sensitive and resistant strains. The metal co-factor present in the enzyme was confirmed by running samples in native gels and inhibiting with 2 mM potassium cyanide. A 519 bp sodC gene was amplified from resistant and sensitive strains of Escherichia coli. Cloning and sequencing of the sodC gene indicated variation in the protein and amino acid sequences of sensitive and resistant isolates. The presence of sodC in highly resistant Escherichia coli isolates from diarrheal patients indicates that sodC may play role in enhancing the pathogenicity by protecting cells from exogenous sources of superoxide, such as the oxidative burst of phagocytes. The presence of SodC could be one of the factors for bacterial virulence.

A gradient PCR-based screen for use in site-directed mutagenesis

Site-directed mutagenesis is widely used to study protein and nucleic acid structure and function. Despite recent advancements in the efficiency of procedures for site-directed mutagenesis, the fraction of site-directed mutants by most procedures rarely exceeds 50% on a routine basis and is never 100%. Hence it is typically necessary to sequence two or three clones each time a site-directed mutant is constructed. We describe a simple and robust gradient-PCR-based screen for distinguishing site-directed mutants from the starting, unmutated plasmid. The procedure can use either purified plasmid DNA or colony PCR, starting from a single colony. The screen utilizes the primer used for mutagenesis and a common outside primer that can be used for all other mutants constructed with the same template. Over 30 site-specific mutants in a variety of templates were successfully screened and all of the mutations detected were subsequently confirmed by DNA sequencing. A single base pair mismatch could be detected in an oligonucleotide of 36 bases. Detection efficiency was relatively independent of starting template concentration and the nature of the outside primer used. (C) 2003 Elsevier Science (USA). All rights reserved.

The beta-glucoside genes of Klebsiella aerogenes: conservation and divergence in relation to the cryptic bgl genes of Escherichia coli

The ability to metabolize aromatic beta-glucosides such as salicin and arbutin varies among members of the Enterobacteriaceae. The ability of Escherichia coli to degrade salicin and arbutin appears to be cryptic, subject to activation of the bgl genes, whereas many members of the Klebsiella genus can metabolize these sugars. We have examined the genetic basis for beta-glucoside utilization in Klebsiella aerogenes. The Klebsiella equivalents of bglG, bglB and bglR have been cloned using the genome sequence database of Klebsiella pneumoniae. Nucleotide sequencing shows that the K. aerogenes bgl genes show substantial similarities to the E. coli counterparts. The K. aerogenes bgl genes in multiple copies can also complement E. coli mutants deficient in bglG encoding the antiterminator and bglB encoding the phospho-beta-glucosidase, suggesting that they are functional homologues. The regulatory region bglR of K aerogenes shows a high degree of similarity of the sequences involved in BglG-mediated regulation. Interestingly, the regions corresponding to the negative elements present in the E. coli regulatory region show substantial divergence in K aerogenes. The possible evolutionary implications of the results are discussed. (C) 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.v. All rights reserved.

Shape memory alloy based motor

Design and characterization of a new shape memory alloy wire based Poly Phase Motor has been reported in this paper. The motor can be used either in stepping mode or in servo mode of operation. Each phase of the motor consists of an SMA wire with a spring in series. The principle of operation of the poly phase motor is presented. The motor resembles a stepper motor in its functioning though the actuation principles are different and hence has been characterized similar to a stepper motor. The motor can be actuated in either direction with different phase sequencing methods, which are presented in this work. The motor is modelled and simulated and the results of simulations and experiments are presented. The experimental model of the motor is of dimension 150mm square, 20mm thick and uses SMA wire of 0·4mm diameter and 125mm of length in each phase.

Mutations in WDR62, encoding a centrosomal and nuclear protein, in Indian primary microcephaly families with cortical malformations

Primary microcephaly is an autosomal recessive disorder characterized by smaller than normal brain size and mental retardation. It is genetically heterogeneous with seven loci: MCPH1-MCPH7. We have previously reported genetic analysis of 35 families, including the identification of the MCPH7 gene STIL. Of the 35 families, three families showed linkage to the MCPH2 locus. Recent whole-exome sequencing studies have shown that the WDR62 gene, located in the MCPH2 candidate region, is mutated in patients with severe brain malformations. We therefore sequenced the WDR62 gene in our MCPH2 families and identified two novel homozygous protein truncating mutations in two families. Affected individuals in the two families had pachygyria, microlissencephaly, band heterotopias, gyral thickening, and dysplastic cortex. Using immunofluorescence study, we showed that, as with other MCPH proteins, WDR62 localizes to centrosomes in A549, HepG2, and HaCaT cells. In addition, WDR62 was also localized to nucleoli. Bioinformatics analysis predicted two overlapping nuclear localization signals and multiple WD-40 repeats in WDR62. Two other groups have also recently identified WDR62 mutations in MCPH2 families. Our results therefore add further evidence that WDR62 is the MCPH2 gene. The present findings will be helpful in genetic diagnosis of patients linked to the MCPH2 locus.

Combined Electron Transfer Dissociation-Collision-Induced Dissociation Fragmentation in the Mass Spectrometric Distinction of Leucine, Isoleucine, and Hydroxyproline Residues in Peptide Natural Products

Distinctions between isobaric residues have been a major challenge in mass spectrometric peptide sequencing. Here, we propose a methodology for distinction among isobaric leucine, isoleucine, and hydroxyproline, a commonly found post-translationally modified amino acid with a nominal mass of 113 Da, through a combined electron transfer dissociation-collision-induced dissociation approach. While the absence of c and z(center dot) ions, corresponding to the Yyy-Xxx (Xxx = Leu, Ile, or Hyp) segment, is indicative of the presence of hydroxyproline, loss of isopropyl (Delta m = 43 Da) or ethyl radicals (Delta m = 29 Da), through collisional activation of z(center dot) radical ions, are characteristic of leucine or isoleucine, respectively. Radical migration processes permit distinctions even in cases where the specific e ions, corresponding to the Yyy-Leu or -Ile segments, are absent or of low intensity. This tandem mass spectrometric (MSn) method has been successfully implemented in a liquid chromatography MSn platform to determine the identity of 23 different isobaric residues from a mixture of five different peptides. The approach is convenient for distinction of isobaric residues from any crude peptide mixture, typically encountered in natural peptide libraries or proteomic analysis.

Familial Diarrhea Syndrome Caused by an Activating GUCY2C Mutation

BACKGROUND Familial diarrhea disorders are, in most cases, severe and caused by recessive mutations. We describe the cause of a novel dominant disease in 32 members of a Norwegian family. The affected members have chronic diarrhea that is of early onset, is relatively mild, and is associated with increased susceptibility to inflammatory bowel disease, small-bowel obstruction, and esophagitis. METHODS We used linkage analysis, based on arrays with single-nucleotide polymorphisms, to identify a candidate region on chromosome 12 and then sequenced GUCY2C, encoding guanylate cyclase C (GC-C), an intestinal receptor for bacterial heat-stable enterotoxins. We performed exome sequencing of the entire candidate region from three affected family members, to exclude the possibility that mutations in genes other than GUCY2C could cause or contribute to susceptibility to the disease. We carried out functional studies of mutant GC-C using HEK293T cells. RESULTS We identified a heterozygous missense mutation (c.2519G -> T) in GUCY2C in all affected family members and observed no other rare variants in the exons of genes in the candidate region. Exposure of the mutant receptor to its ligands resulted in markedly increased production of cyclic guanosine monophosphate (cGMP). This may cause hyperactivation of the cystic fibrosis transmembrane regulator (CFTR), leading to increased chloride and water secretion from the enterocytes, and may thus explain the chronic diarrhea in the affected family members. CONCLUSIONS Increased GC-C signaling disturbs normal bowel function and appears to have a proinflammatory effect, either through increased chloride secretion or additional effects of elevated cellular cGMP. Further investigation of the relevance of genetic variants affecting the GC-C-CFTR pathway to conditions such as Crohn's disease is warranted. (Funded by Helse Vest Western Norway Regional Health Authority] and the Department of Science and Technology, Government of India.)

A variant peptide of buffalo colostrum beta-lactoglobulin inhibits angiotensin I-converting enzyme activity

beta-lactoglobulin is a rich source of bioactive peptides. The LC-MS separated tryptic peptides of buffalo colostrum beta-lactoglobulin (BLG-col) were computed based on MS-MS fragmentation for de novo sequencing. Among the selected peptides (P1-P8), a variant was detected with methionine at position 74 instead of glutamate. The sequences of two peptides were identical to hypocholesterolemic peptides whereas the remaining peptides were in accordance with buffalo milk beta-lactoglobulin. Comparative sequence analysis of BLG-col to milk beta-lactoglobulin was carried out using CLUSTALW2 and a molecular model for BLG-col was constructed (PMDB ID-PM0076812). The synthesized variant pentapeptide (IIAMK, m/z-576 Da) was found to inhibit angiotensin I-converting enzyme (ACE) with an IC50 of 498 +/- 2 mu M, which was rationalized through docking simulations using Molgrow virtual docker. (C) 2012 Elsevier Masson SAS. All rights reserved.

Genetic analysis of an Indian family with members affected with Waardenburg syndrome and Duchenne muscular dystrophy

Purpose: Waardenburg syndrome (WS) is characterized by sensorineural hearing loss and pigmentation defects of the eye, skin, and hair. It is caused by mutations in one of the following genes: PAX3 (paired box 3), MITF (microphthalmia-associated transcription factor), EDNRB (endothelin receptor type B), EDN3 (endothelin 3), SNAI2 (snail homolog 2, Drosophila) and SOX10 (SRY-box containing gene 10). Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder caused by mutations in the DMD gene. The purpose of this study was to identify the genetic causes of WS and DMD in an Indian family with two patients: one affected with WS and DMD, and another one affected with only WS. Methods: Blood samples were collected from individuals for genomic DNA isolation. To determine the linkage of this family to the eight known WS loci, microsatellite markers were selected from the candidate regions and used to genotype the family. Exon-specific intronic primers for EDN3 were used to amplify and sequence DNA samples from affected individuals to detect mutations. A mutation in DMD was identified by multiplex PCR and multiplex ligation-dependent probe amplification method using exon-specific probes. Results: Pedigree analysis suggested segregation of WS as an autosomal recessive trait in the family. Haplotype analysis suggested linkage of the family to the WS4B (EDN3) locus. DNA sequencing identified a novel missense mutation p.T98M in EDN3. A deletion mutation was identified in DMD. Conclusions: This study reports a novel missense mutation in EDN3 and a deletion mutation in DMD in the same Indian family. The present study will be helpful in genetic diagnosis of this family and increases the mutation spectrum of EDN3.

Distinct and Contrasting Transcription Initiation Patterns at Mycobacterium tuberculosis Promoters

Although sequencing of Mycobacterium tuberculosis genome lead to better understanding of transcription units and gene functions, interactions occurring during transcription initiation between RNA polymerase and promoters is yet to be elucidated. Different stages of transcription initiation include promoter specific binding of RNAP, isomerization, abortive initiation and promoter clearance. We have now analyzed these events with four promoters of M. tuberculosis viz. P-gyrB1, P-gyrR, P-rrnPCL1 and P-metU. The promoters differed from each other in their rates of open complex formation, decay, promoter clearance and abortive transcription. The equilibrium binding and kinetic studies of various steps revealed distinct rate limiting events for each of the promoter, which also differed markedly in their characteristics from the respective promoters of Mycobacterium smegmatis. Surprisingly, the transcription at gyr promoter was enhanced in the presence of initiating nucleotides and decreased in the presence of alarmone, pppGpp, a pattern typically seen with rRNA promoters studied so far. The gyr promoter of M. smegmatis, on the other hand, was not subjected to pppGpp mediated regulation. The marked differences in the transcription initiation pathway seen with rrn and gyr promoters of M. smegmatis and M. tuberculosis suggest that such species specific differences in the regulation of expression of the crucial housekeeping genes could be one of the key determinants contributing to the differences in growth rate and lifestyle of the two organisms. Moreover, the distinct rate limiting steps during transcription initiation of each one of the promoters studied point at variations in their intracellular regulation.