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Single Copy (single + copy)
Terms modified by Single Copy Selected AbstractsDetermination of genomic copy number with quantitative microsphere hybridization,,HUMAN MUTATION, Issue 4 2006Heather L. Newkirk Abstract We developed a novel quantitative microsphere suspension hybridization (QMH) assay for determination of genomic copy number by flow cytometry. Single copy (sc) products ranging in length from 62 to 2,304 nucleotides [Rogan et al., 2001; Knoll and Rogan, 2004] from ABL1 (chromosome 9q34), TEKT3 (17p12), PMP22 (17p12), and HOXB1 (17q21) were conjugated to spectrally distinct polystyrene microspheres. These conjugated probes were used in multiplex hybridization to detect homologous target sequences in biotinylated genomic DNA extracted from fixed cell pellets obtained for cytogenetic studies. Hybridized targets were bound to phycoerythrin-labeled streptavidin; then the spectral emissions of both target and conjugated microsphere were codetected by flow cytometry. Prior amplification of locus-specific target DNA was not required because sc probes provide adequate specificity and sensitivity for accurate copy number determination. Copy number differences were distinguishable by comparing the mean fluorescence intensities (MFI) of test probes with a biallelic reference probe in genomic DNA of patient samples and abnormal cell lines. Concerted 5, ABL1 deletions in patient samples with a chromosome 9;22 translocation and chronic myelogenous leukemia were confirmed by comparison of the mean fluorescence intensities of ABL1 test probes with a HOXB1 reference probe. The relative intensities of the ABL1 probes were reduced to 0.59±0.02 &!ndash;fold in three different deletion patients and increased 1.42±0.01 &!ndash;fold in three trisomic 9 cell lines. TEKT3 and PMP22 probes detected proportionate copy number increases in five patients with Charcot-Marie-Tooth Type 1a disease and chromosome 17p12 duplications. Thus, the assay is capable of distinguishing one allele and three alleles from a biallelic reference sequence, regardless of chromosomal context. Hum Mutat 27(4), 376,386, 2006. © 2006 Wiley-Liss, Inc. [source] Chemotaxis in Vibrio choleraeFEMS MICROBIOLOGY LETTERS, Issue 1 2004Markus A. Boin Abstract The ability of motile bacteria to swim toward or away from specific environmental stimuli, such as nutrients, oxygen, or light provides cells with a survival advantage, especially under nutrient-limiting conditions. This behavior, called chemotaxis, is mediated by the bacteria changing direction by briefly reversing the direction of rotation of the flagellar motors. A sophisticated signal transduction system, consisting of signal transducer proteins, a histidine kinase, a response regulator, a coupling protein, and enzymes that mediate sensory adaptation, relates the input signal to the flagellar motor. Chemotaxis has been extensively studied in bacteria such as Escherichia coli and Salmonella enterica serovar Typhimurium, and depends on the activity of single copies of proteins in a linear pathway. However, growing evidence suggests that chemotaxis in other bacteria is more complex with many bacterial species having multiple paralogues of the various chemotaxis genes found in E. coli and, in most cases, the detailed functions of these potentially redundant genes have not been elucidated. Although the completed genome of Vibrio cholerae, the causative agent of cholera, predicted a multitude of genes with homology to known chemotaxis-related genes, little is known about their relative contribution to chemotaxis or other cellular functions. Furthermore, the role of chemotaxis during the environmental or infectious phases of this organism is not yet fully understood. This review will focus on the complex relationship between chemotaxis and virulence in V. cholerae. [source] High-resolution mapping of the 8p23.1 beta-defensin cluster reveals strictly concordant copy number variation of all genes,HUMAN MUTATION, Issue 10 2008Marco Groth Abstract One unexpected feature of the human genome is the high structural variability across individuals. Frequently, large regions of the genome show structural polymorphisms and many vary in their abundance. However, accurate methods for the characterization and typing of such copy number variations (CNV) are needed. The defensin cluster at the human region 8p23.1 is one of the best studied CNV regions due to its potential clinical relevance for innate immunity, inflammation, and cancer. The region can be divided into two subclusters, which harbor predominantly either alpha- or beta-defensin genes. Previous studies assessing individual copy numbers gave different results regarding whether the complete beta-defensin cluster varies or only particular genes therein. We applied multiplex ligation-dependent probe amplification (MLPA) to measure defensin locus copy numbers in 42 samples. The data show strict copy number concordance of all 10 loci typed within the beta-defensin cluster in each individual, while seven loci within the alpha-defensin cluster are consistently found as single copies per chromosome. The exception is DEFA3, which is located within the alpha-defensin cluster and was found to also differ in copy number interindividually. Absolute copy numbers ranged from two to nine for the beta-defensin cluster and zero to four for DEFA3. The CNV-typed individuals, including HapMap samples, are publicly available and may serve as a universal reference for absolute copy number determination. On this basis, MLPA represents a reliable technique for medium- to high-throughput typing of 8p23.1 defensin CNV in association studies for diverse clinical phenotypes. Hum Mutat 0,1,8, 2008. © 2008 Wiley-Liss, Inc. [source] High-throughput single molecule screening of DNA and proteinsTHE CHEMICAL RECORD, Issue 2 2001Edward S. Yeung Abstract We report a novel imaging technology for real time comprehensive analysis of molecular alterations in cells and tissues appropriate for automation and adaptation to high-throughput applications. With these techniques it should eventually be possible to perform simultaneous analysis of the entire contents of individual biological cells with a sensitivity and selectivity sufficient to determine the presence or absence of a single copy of a targeted analyte (e.g., DNA region, RNA region, protein), and to do so at a relatively low cost. The technology is suitable for DNA and RNA through sizing or through fluorescent hybridization probes, and for proteins and small molecules through fluorescence immunoassays. This combination of the lowest possible detection limit and the broadest applicability to biomolecules represents the final frontier in bioanalysis. The general scheme is based on novel concepts for single molecule detection (SMD) and characterization recently demonstrated in our laboratory. Since minimal manipulation is involved, it should be possible to screen large numbers of cells in a short time to facilitate practical applications. This opens up the possibility of finding single copies of DNA or proteins within single biological cells for disease markers without performing polymerase chain reaction or other biological amplification. © 2001 John Wiley & Sons, Inc. and The Japan Chemical Journal Forum Chem Rec 1:123,139, 2001 [source] Local DNA features affect RNA-directed transcriptional gene silencing and DNA methylationTHE PLANT JOURNAL, Issue 1 2008Ute Fischer Summary Transcription of a nopaline synthase promoter (pNOS) inverted repeat provides an RNA signal that can trigger transcriptional gene silencing and methylation of pNOS promoters in trans. The degree of silencing is influenced by the local DNA features close to the target promoter integration sites. Among 26 transgenic Arabidopsis thaliana lines harbouring single copies of a T-DNA including a pNOS- NPTII reporter gene at different chromosomal loci, NPTII RNA levels showed limited variation. When challenged by the silencer transgene providing the pNOS RNA signal, reduction of the NPTII RNA levels in the F1 generation varied by more than 100-fold, ranging from no reduction to reduction to <1% of the non-silenced level. Silencing was generally correlated with proportional DNA methylation in the pNOS region, except for one target transgene showing substantial DNA methylation without adequate silencing. Silencing was progressive through generations. Differences in the degree of silencing among the target transgenes were transmitted at least to the F3 generation, and seemed to be influenced by transgene-flanking sequences. Apparently, close-by repeats promoted, whereas close-by functional genes diminished, the response to the silencing signal. [source] Comprehensive karyotyping of the HT-29 colon adenocarcinoma cell lineGENES, CHROMOSOMES AND CANCER, Issue 1 2002Kanji Kawai The tumor cell line HT-29 was derived from a primary adenocarcinoma of the rectosigmoid colon. HT-29 is hypertriploid (3n+) and has accumulated numerous chromosomal structural aberrations. To identify material involved in chromosome rearrangements, we performed a comprehensive cytogenetic analysis using G-banding, spectral karyotyping (SKY), and fluorescence in situ hybridization (FISH). The combination of molecular cytogenetic techniques enabled us to define the first comprehensive karyotype for HT-29. Seventeen marker chromosomes were found in 75,100% of metaphase cells, generally in a single copy per cell. We confirmed the composition of eight previously described markers, refined the classification of seven others, and identified two novel marker chromosomes. Notable aberrations included a reciprocal translocation between chromosomes 6 and 14 and an unusual, large derivative chromosome 8 composed entirely of 8q material. The telomere status, evaluated by FISH, revealed telomeric signals at the termini of all chromosomes. No interstitial telomeric sequences were observed in any cell. Although numerous chromosomal aberrations are present in HT-29, the cell line appears to have retained a high level of genomic stability during passage in culture since undergoing transformation. The excellent resolving power of SKY, coupled with additional information obtained from molecular cytogenetic analyses, will improve our ability to identify genetic lesions characteristic of cancer. © 2002 Wiley-Liss, Inc. [source] Molecular cloning, characterization, expression pattern and cellular distribution of an ovarian lipophorin receptor in the cockroach, Leucophaea maderaeINSECT MOLECULAR BIOLOGY, Issue 3 2009M. Tufail Abstract A cDNA that encodes a lipophorin receptor (LpR) with a predicted structure similar to that of the low density lipoprotein receptor (LDLR) gene superfamily was cloned from ovaries of the cockroach, Leucophaea maderae (Lem) and characterized. This is the first LpR sequenced from the order Dictyoptera. The cDNA has a length of 3362 bp coding for an 888-residue mature protein with a predicted molecular mass of ~99.14 kDa and a pI value of 4.68. The deduced amino acid sequence showed that the LemLpR harbours eight ligand-binding repeats (LBRs) at the N-terminus similar to the other insect LpRs, and thus resembles vertebrate VLDLRs. In addition to eight tandemly arranged LBRs, the five-domain receptor contains an O -linked sugar region and the classic LDLR internalization signal, FDNPVY. Northern blot analysis revealed the presence of ~4.0 kb ovarian mRNA that was transcribed throughout oogenesis with its peak especially during late previtellogenic and vitellogenic periods (from days 3 to 11). LpR transcript(s) or homologues of LDLRs were also detected in the head, midgut, Malpighian tubules, muscles and in the fat body. RNA in situ hybridization and immunocytochemistry localized the LpR mRNA and protein to germ line-derived cells, the oocytes, and revealed that LpR gene transcription and translation starts very early during oocyte differentiation in the germarium. LpR protein was evenly distributed throughout the cytoplasm during previtellogenic periods of oogenesis. However, during vitellogenic stages, the receptor was accumulated mainly in the cortex of the oocyte. Immunoblot analysis probed an ovarian LpR protein of ~115 and 97 kDa under reducing and nonreducing conditions, respectively. The protein signal appeared on day 2, increased every day and was high during vitellogenic periods from day 4 to day 7. Southern blot analysis suggested the presence of a single copy of the LpR gene in the genome of Le. maderae. [source] Immune activation upregulates lysozyme gene expression in Aedesaegypti mosquito cell cultureINSECT MOLECULAR BIOLOGY, Issue 6 2000Y. Gao Abstract After stimulation with heat-killed bacteria, cultured cells from the mosquito Aedesaegypti (Aag-2 cells) secreted an induced protein with a mass of , 16 kDa that cross-reacted with antibody to chicken egg lysozyme. To investigate whether lysozyme messenger RNA is induced in bacteria-treated cells, we used polymerase chain reaction-based approaches to obtain the complete lysozyme cDNA from Aag-2 cells. The deduced protein contained 148 amino acids, including a 23 amino acid signal sequence. The calculated mass of the precursor protein is 16 965 Da, which is processed to yield a mature lysozyme of 14 471 Da with a calculated pI of 10.1. The lysozyme from Ae. aegypti shared 50% amino acid identity with lysozymes from Anophelesgambiae and Anophelesdarlingi, which in turn shared 70% identity between each other. Northern analysis with the lysozyme cDNA probe showed induction of a 1.3 kb messenger RNA during the first 3 h after treatment of Aag-2 cells with heat-killed bacteria, followed by maximal expression 12,36 h after treatment. Southern analysis suggested that the gene likely occurs as a single copy in the genome of Aag-2 cells. [source] Molecular cloning and functional analysis of Photobacterium damselae subsp. piscicida haem receptor geneJOURNAL OF FISH DISEASES, Issue 2 2005H Naka Abstract A haem receptor gene from Photobacterium damselae subsp. piscicida (formerly known as Pasteurella piscicida) has been cloned, sequenced and analysed for its function. The gene, designated as pph, has an open reading frame consisting of 2154 bp, a predicted 718 amino acid residues and exists as a single copy. It is homologous with the haem receptors of Vibrio anguillarum hupA, V. cholerae hutA, V. mimicus mhuA and V. vulnificus hupA at 32.7, 32.7, 45.6 and 30.9%, respectively, and is highly conserved, consisting of a Phe-Arg-Ala-Pro sequence (FRAP), an iron transport related molecule (TonB) and a Asn-Pron-Asn-Leu sequence (NPNL), binding motifs associated with haem receptors. As a single gene knockout mutant P. damselae subsp. piscicida was able to bind haem in the absence of pph, suggesting that other receptors may be involved in its iron transport system. This study shows that the P. damselae subsp. piscicida pph belongs to the haem receptor family, is conserved and that its iron-binding system may involve more than one receptor. [source] Super-infections of Wolbachia in byturid beetles and evidence for genetic transfer between A and B super-groups of WolbachiaMOLECULAR ECOLOGY, Issue 2 2005G. MALLOCH Abstract Wolbachia are maternally inherited bacteria responsible for altering host reproduction. The two main groups found in insects, A and B, are based on molecular characterization using ribosomal, ftsZ, wsp (Wolbachia surface protein) or groE genes. We have used the wsp and ftsZ genes to study Wolbachia in byturid beetles. Byturus affinis contained a single copy of the ftsZ gene which grouped with A ftsZ sequences and a single copy of the wsp gene which grouped with B wsp sequences. This suggests that genetic exchange between A and B groups has occurred in the Wolbachia of this beetle. FtsZ and wsp sequences that were identical or nearly identical to those of B. affinis were found in B. tomentosus, suggesting that it also contains the same recombinant Wolbachia genotype. Most other byturids had more than one wsp sequence with at least one from the A and B groups, suggesting multiple copies of bacterial genes or multiple infections. B. ochraceus and B. unicolor both had four distinct wsp gene sequences. All the byturids had a closely related A wsp sequence and most a closely related B wsp sequence. Therefore, there appears to be an association between specific A and B wsp types. [source] Single copy nuclear DNA markers characterized for comparative phylogeography in Australian wet tropics rainforest skinksMOLECULAR ECOLOGY RESOURCES, Issue 2 2004G. Dolman Abstract In order to investigate population history and demography in skinks endemic to the wet tropics of Australia, multiple nuclear DNA markers were sought. The utility of 72 primers (including 63 published intron-spanning ,CATS' primers) was tested. Seven loci were characterized and shown to be single copy by single-strand conformation polymorphism analysis. Primers to five nuclear loci were developed, four with utility in skinks and three with utility in frogs. These observations extend the available information on intron-spanning primers for amphibians and reptiles. [source] Genome-wide gene expression profiling and a forward genetic screen show that differential expression of the sodium ion transporter Ena21 contributes to the differential tolerance of Candida albicans and Candida dubliniensis to osmotic stressMOLECULAR MICROBIOLOGY, Issue 1 2009Brice Enjalbert Summary Candida albicans is more pathogenic than Candida dubliniensis. However, this disparity in virulence is surprising given the high level of sequence conservation and the wide range of phenotypic traits shared by these two species. Increased sensitivity to environmental stresses has been suggested to be a possible contributory factor to the lower virulence of C. dubliniensis. In this study, we investigated, in the first comparison of C. albicans and C. dubliniensis by transcriptional profiling, global gene expression in each species when grown under conditions in which the two species exhibit differential stress tolerance. The profiles revealed similar core responses to stresses in both species, but differences in the amplitude of the general transcriptional responses to thermal, salt and oxidative stress. Differences in the regulation of specific stress genes were observed between the two species. In particular, ENA21, encoding a sodium ion transporter, was strongly induced in C. albicans but not in C. dubliniensis. In addition, ENA21 was identified in a forward genetic screen for C. albicans genomic sequences that increase salt tolerance in C. dubliniensis. Introduction of a single copy of CaENA21 was subsequently shown to be sufficient to confer salt tolerance upon C. dubliniensis. [source] Identification and characterization of the immunity repressor (ImmR) that controls the mobile genetic element ICEBs1 of Bacillus subtilisMOLECULAR MICROBIOLOGY, Issue 6 2007Jennifer M. Auchtung Summary ICEBs1 is a mobile genetic element found in the chromosome of Bacillus subtilis. Excision and transfer of ICEBs1 is regulated by the global DNA damage response and intercellular peptide signalling. We identified and characterized a repressor, ImmR (formerly YdcN), encoded by ICEBs1. ImmR represses transcription of genes required for excision and transfer, and both activates and represses its own transcription. ImmR regulates transcription within ICEBs1 by binding to several sites in the region of DNA that contains promoters for both immR and xis (encoding excisionase). In addition, we found that ImmR confers immunity from acquisition of additional copies of ICEBs1. ImmR-mediated regulation serves to keep a single copy of ICEBs1 stably maintained in the absence of induction, allows a rapid response to inducing signals, and helps limit acquisition of additional copies of ICEBs1. [source] Tag-mediated fractionation of yeast ribosome populations proves the monomeric organization of the eukaryotic ribosomal stalk structureMOLECULAR MICROBIOLOGY, Issue 2 2003Esther Guarinos Summary The analysis of the not well understood composition of the stalk, a key ribosomal structure, in eukaryotes having multiple 12 kDa P1/P2 acidic protein components has been approached using these proteins tagged with a histidine tail at the C-terminus. Tagged Saccharomyces cerevisiae ribosomes, which contain two P1 proteins (P1, and P1,) and two P2 proteins (P2, and P2,), were fractionated by affinity chromatography and their stalk composition was determined. Different yeast strains expressing one or two tagged proteins and containing either a complete or a defective stalk were used. No indication of protein dimers was found in the tested strains. The results are only compatible with a stalk structure containing a single copy of each one of the four 12 kDa proteins per ribosome. Ribosomes having an incomplete stalk are found in wild-type cells. When one of the four proteins is missing, the ribosomes do not carry the three remaining proteins simultaneously, containing only two of them distributed in pairs made of one P1 and one P2. Ribosomes can carry two, one or no acidic protein pairs. The P1,/P2, and P1,/P2, pairs are preferentially found in the ribosome, but they are not essential either for stalk assembly or function. [source] A novel mechanism for control of antigenic variation in the haemagglutinin gene family of Mycoplasma synoviaeMOLECULAR MICROBIOLOGY, Issue 4 2000A. H. Noormohammadi High-frequency phase and antigenic variation of homologous lipoprotein haemagglutinins has been seen in both the major avian mycoplasma pathogens, Mycoplasma synoviae and Mycoplasma gallisepticum. The expression and, hence, antigenic variation of the pMGA gene family (encoding these lipoproteins in M. gallisepticum) is controlled by variation in the length of a trinucleotide repeat motif 5, to the promoter of each gene. However, such a mechanism was not detected in preliminary observations on M. synoviae. Thus, the basis for control of variation in the vlhA gene family (which encodes the homologous haemagglutinin in M. synoviae) was investigated to enable comparison with its homologue in M. gallisepticum and with other lipoprotein gene families in mycoplasmas. The start point of transcription was identified 119 bp upstream of the initiation codon, but features associated with control of transcription in other mycoplasma lipoprotein genes were not seen. Comparison of three copies of vlhA revealed considerable sequence divergence at the 3, end of the gene, but conservation of the 5, end. Southern blot analysis of M. synoviae genomic DNA revealed that the promoter region and part of the conserved 5, coding sequence occurred as a single copy, whereas the remainder of the coding sequence occurred as multiple copies. A 9.7 kb fragment of the genome was found to contain eight tandemly repeated regions partially homologous to vlhA, all lacking the putative promoter region and the single-copy 5, end of vlhA, but extending over one of four distinct overlapping regions of the 3, coding sequence. Examination of sequential clones of M. synoviae established that unidirectional recombination occurs between the pseudogenes and the expressed vlhA, with duplication of pseudogene sequence and loss of the corresponding region previously seen in the expressed gene. Expression of the 5, end of two variants of the vlhA gene showed that they differed in their reaction with monoclonal antibodies specific for this region. These data suggest that the control of vlhA antigenic variation in M. synoviae is achieved by multiple gene conversion events using a repertoire of coding sequences to generate a chimeric expressed gene, with the greatest potential for variation generated in the region encoding the haemagglutinin. Thus, completely distinct mechanisms have been adopted to control antigenic variation in homologous gene families. [source] Molecular and Physiological Characterisation of an Insertion Mutant in the ARR21 Putative Response Regulator Gene from Arabidopsis thalianaPLANT BIOLOGY, Issue 3 2003J. Horák Abstract: In our search for insertion mutants of Arabidopsis response regulator (ARR) genes, we identified a candidate for an ARR21 dSpm transposon insertion line in the SLAT collection by searching the SINS sequence database. Molecular characterisation of this line revealed that the transposon is integrated as a single copy 1727 bases downstream of the ATG signal, within the third intron of the ARR21 gene. The transposon insertion segregated in a Mendelian fashion from heterozygous plants that were allowed to self-pollinate. RT-PCR-based expression analysis showed that ARR21 transcript predominantly accumulates in siliques. In contrast, the full-length ARR21 transcript was not detectable in the ARR21 transposon insertion line, indicating that it harbours an insertion of dSpm transposon in the ARR21 gene. The ARR21 insertion mutant (arr21-1) was subjected to several physiological tests for a possible insertion-linked phenotype. However, our results revealed that the insertion in the ARR21 gene did not cause any alterations in viability and fertility, flowering time, sensitivity to ethylene, cytokinin or red light. We discuss these results in the light of recent findings about the function of the other members of the response regulator gene family of Arabidopsis. [source] High-throughput single molecule screening of DNA and proteinsTHE CHEMICAL RECORD, Issue 2 2001Edward S. Yeung Abstract We report a novel imaging technology for real time comprehensive analysis of molecular alterations in cells and tissues appropriate for automation and adaptation to high-throughput applications. With these techniques it should eventually be possible to perform simultaneous analysis of the entire contents of individual biological cells with a sensitivity and selectivity sufficient to determine the presence or absence of a single copy of a targeted analyte (e.g., DNA region, RNA region, protein), and to do so at a relatively low cost. The technology is suitable for DNA and RNA through sizing or through fluorescent hybridization probes, and for proteins and small molecules through fluorescence immunoassays. This combination of the lowest possible detection limit and the broadest applicability to biomolecules represents the final frontier in bioanalysis. The general scheme is based on novel concepts for single molecule detection (SMD) and characterization recently demonstrated in our laboratory. Since minimal manipulation is involved, it should be possible to screen large numbers of cells in a short time to facilitate practical applications. This opens up the possibility of finding single copies of DNA or proteins within single biological cells for disease markers without performing polymerase chain reaction or other biological amplification. © 2001 John Wiley & Sons, Inc. and The Japan Chemical Journal Forum Chem Rec 1:123,139, 2001 [source] Peptide products of the afp-6 gene of the nematode Ascaris suum have different biological actionsTHE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 5 2007Joanne Y. Yew Abstract Matrix-assisted laser desorption/ionization time-of-flight and tandem time-of-flight (MALDI-TOF and MALDI-TOF/TOF) mass spectrometry were used to sequence and localize three novel, related neuropeptides in the nervous system of the nematode Ascaris suum, AMRNALVRFamide (AF21), NGAPQPFVRFamide (AF22), and SGMRNALVRFamide (AF23). The amino acid sequences were used to clone a novel neuropeptide gene (afp-6) that encodes a precursor bearing a single copy of each of the peptides. In situ hybridization and immunocytochemistry revealed that both the transcript and the peptides are expressed in a single cell in the ventral ganglion. Pharmacological studies of intact nematodes injected with these peptides, as well as physiological studies of responses to them in muscle tissue, motor neurons, and the pharynx, reveal that these peptides have potent bioactivity in the locomotory and feeding systems. Further exploration of their effects may contribute to our understanding of neuropeptide modulation of behavior and also to the development of compounds with anthelmintic relevance. J. Comp. Neurol. 502:872,882, 2007. © 2007 Wiley-Liss, Inc. [source] A Transgenic Insertional Inner Ear Mutation on Mouse Chromosome 1,THE LARYNGOSCOPE, Issue 4 2000Rick A. Friedman MD Abstract Objectives/Hypothesis To clone and characterize the integration site of an insertional inner ear mutation, produced in one of fourteen transgenic mouse lines. The insertion of the transgene led to a mutation in a gene(s) necessary for normal development of the vestibular labyrinth. Study Design Molecular genetic analysis of a transgene integration site. Methods Molecular cloning, Southern and northern blotting, DNA sequencing and genetic database searching were the methods employed. Results The integration of the transgene resulted in a dominantly inherited waltzing phenotype and in degeneration of the pars superior. During development, inner ear fluid homeostasis was disrupted. The integration consisted of the insertion of a single copy of the transgene. Flanking DNA was cloned, and mapping indicated that the genomic DNA on either side of the transgene was not contiguous in the wild-type mouse. Localization of unique markers from the two flanks indicated that both were in the proximal region of mouse chromosome 1. However, in the wild-type mouse the markers were separated by 6.3 cM, indicating a sizable rearrangement. Analysis of the mutant DNA indicated that the entire region between the markers was neither deleted nor simply inverted. Conclusions These results are consistent with a complex rearrangement, including at least four breakpoints and spanning at least 6.3 cM, resulting from the integration of the transgene. This genomic rearrangement disrupted the function of one or more genes critical to the maintenance of fluid homeostasis during development and the normal morphogenesis of the pars superior. [source] Methods on erg9 gene deletion in Schizosaccharomyces pombeASIA-PACIFIC JOURNAL OF CHEMICAL ENGINEERING, Issue 5 2009Bing Cheng Abstract Gene modification to key enzymes in biosynthesis pathway becomes familiar technology which can increase efficiency of industrial strains. In the current study, we established a desirable deletion approach based on homologous recombination. It was simple and available for spn5 gene deletion. Also, four kinds of erg9 gene deletion cassettes were constructed in similar manner. The lengths of flanking sequences for homologous integration were from 59 bp to more than 1 Kb, and three selective markers (G418, Zeocin and LEU2) were employed in these cassettes. However, ,erg9 mutant was not obtained in this work. The possible reason is that erg9 gene is an essential gene and a single copy in its chromosome. Deficiency of erg9 gene resulted in nonviability to the cells. In addition, LEU2 marker caused unspecific integration and the difficulty of screening mutants is increased during deletion procedures. This work provides important evidence to modify industrial strains by using deletion technologies. Copyright © 2009 Curtin University of Technology and John Wiley & Sons, Ltd. [source] Cloning, overexpression, purification, crystallization and preliminary X-ray analysis of CheY3, a response regulator that directly interacts with the flagellar `switch complex' in Vibrio choleraeACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2010Susmita Khamrui Vibrio cholerae is the aetiological agent of the severe diarrhoeal disease cholera. This highly motile organism uses the processes of motility and chemotaxis to travel and colonize the intestinal epithelium. Chemotaxis in V. cholerae is far more complex than that in Escherichia coli or Salmonella typhimurium, with multiple paralogues of various chemotaxis genes. In contrast to the single copy of the chemotaxis response-regulator protein CheY in E. coli, V. cholerae contains four CheYs (CheY1,CheY4), of which CheY3 is primarily responsible for interacting with the flagellar motor protein FliM, which is one of the major constituents of the `switch complex' in the flagellar motor. This interaction is the key step that controls flagellar rotation in response to environmental stimuli. CheY3 has been cloned, overexpressed and purified by Ni,NTA affinity chromatography followed by gel filtration. Crystals of CheY3 were grown in space group R3, with a calculated Matthews coefficient of 2.33,Ĺ3,Da,1 (47% solvent content) assuming the presence of one molecule per asymmetric unit. [source] X-chromosome upregulation and inactivation: two sides of the dosage compensation mechanism in mammalsBIOESSAYS, Issue 1 2009Elena V. Dementyeva Abstract Mammals have a very complex, tightly controlled, and developmentally regulated process of dosage compensation. One form of the process equalizes expression of the X-linked genes, present as a single copy in males (XY) and as two copies in females (XX), by inactivation of one of the two X-chromosomes in females. The second form of the process leads to balanced expression between the X-linked and autosomal genes by transcriptional upregulation of the active X in males and females. However, not all X-linked genes are absolutely balanced. This review is focused on the recent advances in studying the dosage compensation phenomenon in mammals. [source] Crystallization and diffraction patterns of the oxy and cyano forms of the Lucina pectinata haemoglobins complexACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 1 2009Carlos R. Ruiz-Martínez The native oxygen-carrier haemoglobins complex (HbII,III) is composed of haemoglobin II (HbII) and haemoglobin III (HbIII), which are found in the ctenidia tissue of the bivalve mollusc Lucina pectinata. This protein complex was isolated and purified from its natural source and crystallized using the vapour-diffusion and capillary counter-diffusion methods. Oxy and cyano derivatives of the complex crystallized using several conditions, but the best crystals in terms of quality and size were obtained from sodium formate pH 5 using the counter-diffusion method in a single capillary. Crystals of the oxy and cyano complexes, which showed a ruby-red colour and nonsingular prismatic shapes, scattered X-rays to resolution limits of 2.15 and 2.20,Ĺ, respectively, using a 0.886,Ĺ synchrotron-radiation source. The crystals belonged to the tetragonal system, space group P42212, with unit-cell parameters a = b = 74.07, c = 152.07 and a = b = 73.83, c = 152.49,Ĺ for the oxy and cyano complexes, respectively. The asymmetric unit of both crystals is composed of a single copy of the heterodimer, with Matthew coefficients (VM) of 3.08 and 3.06,Ĺ3,Da,1 for the oxy and cyano complexes, respectively, which correspond to a solvent content of approximately 60.0% by volume. [source] | |||||||||||||||||||||||||