Mosaic Gene Research Articles

Prokaryotic expression of a Thunberg fritillary mosaic virus CP gene and antiserum preparation

Wei, C. B., Wu, Q. Y., Yang, Y. and Liu, C. Y. 2012. Prokaryotic expression of a Thunberg fritillary mosaic virus CP gene and antiserum preparation. Can. J. Plant Sci. 92: 495–500. Fritillaria anhuiensis is a valued traditional Chinese medicinal plant. Thunberg fritillary mosaic virus (TFMV) was found to cause mosaic symptoms. Virus particles of the Lu'an isolate were purified and its RNA genome was extracted. cDNA was synthesized from the RNA and specific PCR primers were used to amplify the virus coat protein (CP) gene. It shared 96.8% nucleotide identity and 97.6% amino acid identity with the sequences of CPs from the other TFMV isolates. The CP gene was inserted into pSBET and expressed in Escherichia coli BL21 (DE3) plys S strain. The expressed protein was first purified by 12% (wt/vol) SDS-PAGE and subsequently by 5–20% (wt/vol) gradient SDS-PAGE. Antiserum against the CP was raised in mouse and its specificity was confirmed by Western blot analysis suggesting that it may be useful for detecting the virus.

Prokaryotic expression of a Thunberg fritillary mosaic virus CP gene and antiserum preparation

Wei, C. B., Wu, Q. Y., Yang, Y. and Liu, C. Y. 2012. Prokaryotic expression of a Thunberg fritillary mosaic virus CP gene and antiserum preparation. Can. J. Plant Sci. 92: 495–500. Fritillaria anhuiensis is a valued traditional Chinese medicinal plant. Thunberg fritillary mosaic virus (TFMV) was found to cause mosaic symptoms. Virus particles of the Lu'an isolate were purified and its RNA genome was extracted. cDNA was synthesized from the RNA and specific PCR primers were used to amplify the virus coat protein (CP) gene. It shared 96.8% nucleotide identity and 97.6% amino acid identity with the sequences of CPs from the other TFMV isolates. The CP gene was inserted into pSBET and expressed in Escherichia coli BL21 (DE3) plys S strain. The expressed protein was first purified by 12% (wt/vol) SDS-PAGE and subsequently by 5–20% (wt/vol) gradient SDS-PAGE. Antiserum against the CP was raised in mouse and its specificity was confirmed by Western blot analysis suggesting that it may be useful for detecting the...

A novel Cre/loxP system for mosaic gene expression in the Drosophila embryo

Mosaic analysis is used to assess gene function and cell autonomy in a subset of cells in an organism, and has been extensively applied in Drosophila studies. However, it is difficult to generate mosaic cells in Drosophila embryonic tissues using existing methods. Therefore, we developed a new method for generating genetic mosaic embryos using a modified Cre/loxP system. In this report, we also characterized the capabilities and limitations of this novel method. We first constructed a novel cassette combining loxP with the Actin 5C enhancer and Gal4 cDNA, and generated a transgenic fly carrying this construct (Aloxg-Gal4). In Aloxg-Gal4, the activation of Gal4 expression is suppressed by the gypsy insulator. Once the gypsy insulator is removed, however, Gal4 is expressed when site-specific recombination between loxP sites is induced by Cre recombinase. This system allowed the mosaic expression of Gal4 in Drosophila embryonic tissues (epidermis, amnioserosa, tracheal system, malpighian tubules, foregut, hindgut, midgut, and neuron), leading to the Gal4-dependent activation of arbitrary genes under the control of the upstream activation sequence (UAS). This practical method can be used to generate mosaic cells in Drosophila embryonic tissues and can be applied to any gene without specialized equipment.

Screening and identification of random amplified polymorphic DNA (RAPD) markers linked to mungbean yellow mosaic virus (MYMV) resistance in mungbean (Vigna radiata (L.) Wilczek)

Bulk segregant analysis (BSA) and random amplified polymorphic DNA (RAPD) techniques were used to analyse the F2 individuals of susceptible VBN (Gg) 2 × resistant KMG 189 to screen and identify the molecular marker linked to mungbean yellow mosaic virus (MYMV) resistant gene in mungbean. Two DNA bulks namely resistant bulks and susceptible bulks were setup by pooling equal amount of DNA from five randomly selected plants of each disease response. A total of 72 random sequence decamer oligonucleotide primers were used for RAPD analysis. Primer OPBB 05 (5′-GGGCCGAACA-3′) generated OPBB 05 260 fragment in resistant parent and their bulks but not in the susceptible parent and their bulks. Co segregation analysis was performed in resistant and susceptible F2 individuals, it confirmed that OPBB 05 260 marker was tightly linked to mungbean yellow mosaic virus resistant gene in mungbean.

The 2011 Garrod Lecture: From penicillin-binding proteins to molecular epidemiology

In this review, based on my Garrod Lecture to the British Society for Antimicrobial Chemotherapy, I have given a brief outline of my career over the past 40 years, starting with research in the 1970s into the properties and functions of penicillin-binding proteins (PBPs), leading to the identification of the high molecular mass PBPs as the physiological targets of penicillin, and subsequent studies showing the emergence of low-affinity PBPs in penicillin-resistant clinical isolates by inter-species recombination and the generation of mosaic PBP genes. The studies of clinical isolates of gonococci, meningococci and pneumococci with PBP-mediated resistance to penicillin led to new interests in molecular epidemiology and the population and evolutionary biology of bacterial pathogens. The development (with colleagues) of multilocus sequence typing provided a method for the unambiguous characterization of bacterial strains that has proved to be very widely used, but the recent remarkable (and ongoing) developments in DNA sequencing technologies have provided the prospect of being able routinely to use whole genome sequences to characterize pathogen isolates. These developments will soon have major implications for diagnostic microbiology, outbreak investigations and our ability to follow the spread of strains of community-acquired and nosocomial pathogens at local, national and international levels. However, there are major barriers to be overcome, particularly with respect to how the avalanche of genome sequence data will be stored so that its transformative potential for molecular epidemiology and international public health are fully realized.

Surveillance of gonococcal antimicrobial susceptibility resulting in early detection of emerging resistance

To undertake laboratory and clinical surveillance of gonococcal antimicrobial susceptibility to various therapeutic agents in Hong Kong, so as to monitor for emerging resistance and to inform on appropriate choice of empirical therapy. Trends in susceptibility of gonococci to ceftriaxone, spectinomycin, ceftibuten and azithromycin were monitored over time. Isolates with reduced susceptibility to oral extended-spectrum cephalosporins were further characterized by detection of the mosaic penA gene and typing by Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST). Correlation with clinical and epidemiological findings was undertaken on isolates positive for the mosaic penA gene. Trends in susceptibility of gonococci to ceftriaxone, spectinomycin and ceftibuten remained stable between 2005 and 2010. In 2010, 30.3% of tested strains were not susceptible to azithromycin. The percentage of gonococcal strains harbouring the mosaic penA gene increased from 1.0% during April to December 2010 to 8.2% during January to September 2011 (P < 0.0001). Review of available clinical records showed that, out of 35 patients infected by strains positive for the mosaic penA gene, 30 had laboratory-documented treatment failure. This study showed that ceftriaxone and spectinomycin remained effective against gonorrhoea in Hong Kong. There was an alarming increase in strains with reduced susceptibility to oral extended-spectrum cephalosporins associated with clinical treatment failure. One-third of gonococcal isolates were non-susceptible to azithromycin. The need to switch to agents other than oral extended-spectrum cephalosporins for empirical treatment is imminent. Continued surveillance with strain characterization is essential to monitor the effectiveness of currently recommended therapy.

Synergistic Effect of MWNTs/CeO2/CHIT Film for Detection of CdSe Nanoparticle Labeled Sequence‐Specific of 35S Promoter of Cauliflower Mosaic Virus Gene

AbstractA porous composite film was fabricated combining the advantages of multiwalled carbon nanotubes, CeO2 and chitosan. The synergistic effect of the film improved the immobilization of probe ssDNA. The loaded probe ssDNA was used for detection of CdSe quantum dots labeled target DNA. The DNA hybridization reaction was detected by differential pulse anodic stripping voltammetry of Cd2+ after the oxidative release of labeled CdSe quantum dots. The established DNA biosensor can discriminate different target sequences associated with 35S promoter of cauliflower mosaic virus gene with relatively wide linear range and low detection limit (2.4×10−13 mol/L).

What makes a chloroplast? Reconstructing the establishment of photosynthetic symbioses

Earth is populated by an extraordinary diversity of photosynthetic eukaryotes. Many eukaryotic lineages contain chloroplasts, obtained through the endosymbiosis of a wide range of photosynthetic prokaryotes or eukaryotes, and a wide variety of otherwise non-photosynthetic species form transient associations with photosynthetic symbionts. Chloroplast lineages are likely to be derived from pre-existing transient symbioses, but it is as yet poorly understood what steps are required for the establishment of permanent chloroplasts from photosynthetic symbionts. In the past decade, several species that contain relatively recently acquired chloroplasts, such as the rhizarian Paulinella chromatophora, and non-photosynthetic taxa that maintain photosynthetic symbionts, such as the sacoglossan sea slug Elysia, the ciliate Myrionecta rubra and the dinoflagellate Dinophysis, have emerged as potential model organisms in the study of chloroplast establishment. In this Commentary, we compare recent molecular insights into the maintenance of chloroplasts and photosynthetic symbionts from these lineages, and others that might represent the early stages of chloroplast establishment. We emphasise the importance in the establishment of chloroplasts of gene transfer events that minimise oxidative stress acting on the symbiont. We conclude by assessing whether chloroplast establishment is facilitated in some lineages by a mosaic of genes, derived from multiple symbiotic associations, encoded in the host nucleus.

Watermelon transformation with Zucchini yellow mosaic virus coat protein gene and comparison with parental cultivar

The objective of this work was to transfer Zucchini yellow mosaic virus coat protein (ZYMV-CP) and neomycin phosphotransferase II (NPT II) genes to the watermelon 'Crimson Sweet'(CS) genome, and to compare the transgenic progenies T1 and T2 with the nontransformed parental cultivar for morphological, pomological, growth and yield characteristics. The ZYMV-CP gene was transferred by Agrobacterium tumefaciens. The presence of the gene in transgenic T0, T1 and T2 plants was determined by polymerase chain reaction, and the results were confirmed by Southern blot. Two experiments were performed, one in the winter-spring and the other in the summer-autumn. In both experiments, the hypocotyl length of transgenic seedlings was significantly higher than that of nontransgenic parental ones. In the second experiment, the differences between transgenic and nontransgenic individuals were significant concerning fruit rind thickness, flesh firmness, fruit peduncle length, size of pistil scar, and a* values for fruit stripe or flesh color. Transferring ZYMV-CP gene to CS genome affected only a few characteristics from the 80 evaluated ones. The changes in rind thickness, flesh firmness and flesh color a* values are favorable, while the increase in the size of pistil scar is undesirable. The transgenic watermelon line having ZYMV-CP gene and the parental cultivar CS are very similar.

Detection of multidrug-resistant gonorrhoea in the Gauteng province

Two cefpodoxime-resistant Neisseria gonorrhoeae isolates were identifed with high minimum inhibitory concentrations (MIC) for oral cephalosporins. The ceftriaxone MICs for both isolates were still in the susceptible range, although raised at 0.064 mg/l. Both isolates were cultured from urethral specimens that were collected from male patients who had presented to their general practitioners with urethral discharge. Patient 1 was treated empirically with azithromycin. Patient 2, who reported having had sex with other men, was given intravenous ceftriaxone as he had recently failed two courses of oral cefxime. Both isolates were also highly resistant to ciprofoxacin, resistant to penicillin (s-lactamase negative) and resistant to tetracycline (and thus doxycycline), and exhibited decreased susceptibility to both azithromycin and gentamicin. On molecular testing, both isolates were found to have mosaic penA genes, which encode for penicillin-binding protein 2. Mosaic penA gene formation is the most common ge...

Tetracycline resistance genes of ureaplasmas

In the medical setting, limited information is available on antimicrobial susceptibilities and resistance development in ureaplasmas. This study addresses tetracycline and doxycycline resistance in clinical isolates of Ureaplasma parvum and Ureaplasma urealyticum. Culture, with species polymerase chain reaction (PCR) confrmatory techniques, was applied to 191 endocervical specimens, collected during the period January-March 2006. Minimum inhibitory concentration (MIC) determinations were performed by microbroth dilution, with tetracycline resistance (tetM), and int-Tn genes were characterised by PCR and sequencing. Sixty-six ureaplasma cultures (35 U. parvum, 9 U. urealyticum, 22 U. parvum and U. urealyticum) were obtained. On screening the ureaplasma cultures, tetM gene regions were demonstrated from tetracycline-susceptible and -resistant ureaplasmas. Seven isolates (six U. parvum and one U. urealyticum) were resistant to tetracycline, with dual doxycycline resistance observed in three strains. Int-Tn gene characterisation of the seven tetracycline-resistant strains revealed that three types were present, indicating that transposons from different origins had integrated into ureaplasma genomes. TetM sequences of fve tetracycline-resistant strains were seen to be highly mosaic in structure. The fnding of transposon and/or tetM regions in all ureaplasma cultures investigated, with, or without, full expression of tetracycline resistance, in conjunction with tetM and int-Tn gene mosaic or diversity, verifes that ureaplasmas undergo extensive genetic exchange of transposon or resistance genes, with concomitant genomic remodelling.

Two loci in sorghum with NB-LRR encoding genes confer resistance to Colletotrichum sublineolum

The aim of this work was to identify plant resistance genes to the sorghum anthracnose fungus Colletotrichum sublineolum. cDNA-AFLP transcript profiling on two contrasting sorghum genotypes inoculated with C. sublineolum generated about 3,000 informative fragments. In a final set of 126 sequenced genes, 15 were identified as biotic stress related. Seven of the plant-derived genes were selected for functional analysis using a Brome mosaic virus-based virus-induced gene silencing (VIGS) system followed by fungal inoculation and quantitative real-time PCR analysis. The candidate set comprised genes encoding resistance proteins (Cs1A, Cs2A), a lipid transfer protein (SbLTP1), a zinc finger-like transcription factor (SbZnTF1), a rice defensin-like homolog (SbDEFL1), a cell death related protein (SbCDL1), and an unknown gene harboring a casein kinase 2-like domain (SbCK2). Our results demonstrate that down-regulation of Cs1A, Cs2A, SbLTP1, SbZnF1 and SbCD1 via VIGS, significantly compromised the resistance response while milder effects were observed with SbDEFL1 and SbCK2. Expanded genome analysis revealed that Cs1A and Cs2A genes are located in two different loci on chromosome 9 closely linked with duplicated genes Cs1B and Cs2B, respectively. The nucleotide binding-leucine rich repeat (NB-LRR) encoding Cs gene sequence information is presently employed in regional breeding programs.

Mapping of VSG similarities in Trypanosoma brucei

The protozoan parasite Trypanosoma brucei switches its variant surface glycoprotein (VSG) to subvert its mammalian hosts’ immune responses. The T. brucei genome contains as many as 1600 VSG genes (VSGs), but most are silent noncoding pseudogenes. Only one functional VSG, located in a telomere-linked expression site, is transcribed at a time. Silent VSGs are copied into a VSG expression site through gene conversion. Truncated gene conversion events can generate new mosaic VSGs with segments of sequence identity to other VSGs. To examine the VSG family sub-structure within which these events occur, we combined the available VSG sequences and annotations with scripted BLAST searches to map the relationships among VSGs in the T. brucei genome. Clusters of related VSGs were visualized in 2- and 3-dimensions for different N- and C-terminal regions. Five types of N-termini (N1–N5) were observed, within which gene recombinational events are likely to occur, often with fully-coding ‘functional’ or ‘atypical’VSGs centrally located between more dissimilar VSGs. Members of types N1, N3 and N4 are most closely related in the middle of the N-terminal region, whereas type N2 members are more similar near the N-terminus. Some preference occurs in pairing between specific N- and C-terminal types. Statistical analyses indicated no overall tendency for more related VSGs to be located closer in the genome than less related VSGs, although exceptions were noted. Many potential mosaic gene formation events within each N-terminal type were identified, contrasted by only one possible mosaic gene formation between N-terminal types (N1 and N2). These data suggest that mosaic gene formation is a major contributor to the overall VSG diversity, even though gene recombinational events between members of different N-terminal types occur only rarely.

Towards an accurate identification of mosaic genes and partial horizontal gene transfers

Many bacteria and viruses adapt to varying environmental conditions through the acquisition of mosaic genes. A mosaic gene is composed of alternating sequence polymorphisms either belonging to the host original allele or derived from the integrated donor DNA. Often, the integrated sequence contains a selectable genetic marker (e.g. marker allowing for antibiotic resistance). An effective identification of mosaic genes and detection of corresponding partial horizontal gene transfers (HGTs) are among the most important challenges posed by evolutionary biology. We developed a method for detecting partial HGT events and related intragenic recombination giving rise to the formation of mosaic genes. A bootstrap procedure incorporated in our method is used to assess the support of each predicted partial gene transfer. The proposed method can be also applied to confirm or discard complete (i.e. traditional) horizontal gene transfers detected by any HGT inferring method. While working on a full-genome scale, the new method can be used to assess the level of mosaicism in the considered genomes as well as the rates of complete and partial HGT underlying their evolution.

Mosaic Analysis of Gene Function in Postnatal Mouse Brain Development by Using Virus-based Cre Recombination

Normal brain function relies not only on embryonic development when major neuronal pathways are established, but also on postnatal development when neural circuits are matured and refined. Misregulation at this stage may lead to neurological and psychiatric disorders such as autism and schizophrenia1,2. Many genes have been studied in the prenatal brain and found crucial to many developmental processes3-5. However, their function in the postnatal brain is largely unknown, partly because their deletion in mice often leads to lethality during neonatal development, and partly because their requirement in early development hampers the postnatal analysis. To overcome these obstacles, floxed alleles of these genes are currently being generated in mice 6. When combined with transgenic alleles that express Cre recombinase in specific cell types, conditional deletion can be achieved to study gene function in the postnatal brain. However, this method requires additional alleles and extra time (3-6 months) to generate the mice with appropriate genotypes, thereby limiting the expansion of the genetic analysis to a large scale in the mouse brain. Here we demonstrate a complementary approach that uses virally-expressed Cre to study these floxed alleles rapidly and systematically in postnatal brain development. By injecting recombinant adeno-associated viruses (rAAVs)7,8 encoding Cre into the neonatal brain, we are able to delete the gene of interest in different regions of the brain. By controlling the viral titer and coexpressing a fluorescent protein marker, we can simultaneously achieve mosaic gene inactivation and sparse neuronal labeling. This method bypasses the requirement of many genes in early development, and allows us to study their cell autonomous function in many critical processes in postnatal brain development, including axonal and dendritic growth, branching, and tiling, as well as synapse formation and refinement. This method has been used successfully in our own lab (unpublished results) and others8,9, and can be extended to other viruses, such as lentivirus 9, as well as to the expression of shRNA or dominant active proteins 10. Furthermore, by combining this technique with electrophysiology as well as recently-developed optical imaging tools 11, this method provides a new strategy to study how genetic pathways influence neural circuit development and function in mice and rats.

Mosaic Analysis of Gene Function in Postnatal Mouse Brain Development by Using Virus-based Cre Recombination

Normal brain function relies not only on embryonic development when major neuronal pathways are established, but also on postnatal development when neural circuits are matured and refined. Misregulation at this stage may lead to neurological and psychiatric disorders such as autism and schizophrenia. Many genes have been studied in the prenatal brain and found crucial to many developmental processes. However, their function in the postnatal brain is largely unknown, partly because their deletion in mice often leads to lethality during neonatal development, and partly because their requirement in early development hampers the postnatal analysis. To overcome these obstacles, floxed alleles of these genes are currently being generated in mice. When combined with transgenic alleles that express Cre recombinase in specific cell types, conditional deletion can be achieved to study gene function in the postnatal brain. However, this method requires additional alleles and extra time (3-6 months) to generate the mice with appropriate genotypes, thereby limiting the expansion of the genetic analysis to a large scale in the mouse brain. Here we demonstrate a complementary approach that uses virally-expressed Cre to study these floxed alleles rapidly and systematically in postnatal brain development. By injecting recombinant adeno-associated viruses (rAAVs) encoding Cre into the neonatal brain, we are able to delete the gene of interest in different regions of the brain. By controlling the viral titer and coexpressing a fluorescent protein marker, we can simultaneously achieve mosaic gene inactivation and sparse neuronal labeling. This method bypasses the requirement of many genes in early development, and allows us to study their cell autonomous function in many critical processes in postnatal brain development, including axonal and dendritic growth, branching, and tiling, as well as synapse formation and refinement. This method has been used successfully in our own lab (unpublished results) and others, and can be extended to other viruses, such as lentivirus, as well as to the expression of shRNA or dominant active proteins. Furthermore, by combining this technique with electrophysiology as well as recently-developed optical imaging tools, this method provides a new strategy to study how genetic pathways influence neural circuit development and function in mice and rats.

P4-S1.05 Polymerase chain reaction-based typing of penA genes exhibiting elevated MIC values to cephalosporins in Isolates of Neisseria gonorrhoeae

ObjectiveDevelop a PCR based assays for rapid analysis of isolates that exhibit elevated MIC values to penicillin and cephalosporins (MICs of 0.015–0.06 ug/ml to ceftriaxone). These primers were designed to...

O1-S03.03 Clonally related Neisseria gonorrhoeae isolates with decreased susceptibility to extended-spectrum cephalosporins in Amsterdam, the Netherlands

BackgroundBetween 2006 and 2008, the prevalence of Neisseria gonorrhoeae (NG) isolates with decreased susceptibility (0.125<MIC<0.5 μg/ml) to the extended-spectrum cephalosporin (ESC) cefotaxime (CTX) among visitors of the STI clinic in...

The Dahlia mosaic virus gene VI product N-terminal region is involved in self-association

The genome of the floriculture pathogen Dahlia mosaic caulimovirus (DMV) encodes six open reading frames. Generally, caulimovirus gene VI products (P6s) are thought to be multifunctional proteins required for viral infection and it is likely that self-association is required for some of these functions. In this study, yeast two-hybrid and maltose binding protein (MBP) pull-down assays indicated that full-length DMV P6 specifically self-associates. Further analyses indicated that only the DMV P6 N-terminal region, consisting of 115 amino acids, interacts with full-length P6 and with itself. This distinguishes the DMV P6 from its Cauliflower mosaic virus counterpart, which contains four regions involved in self-association. Thus, our results suggest that each caulimovirus P6 may possess a unique pattern of protein–protein interactions. Bioinformatic tools identified a putative nuclear exclusion signal located between amino acid residues 10–20, suggesting another possible function for the P6 N-terminal region.

Identification of an amino acid residue required for differential recognition of a viral movement protein by the Tomato mosaic virus resistance gene Tm-22

The Tm-2 gene of tomato and its allelic gene, Tm-2 2 , confer resistance to Tomato mosaic virus (ToMV) and encode a member of the coiled-coil/nucleotide binding-ARC/leucine-rich repeat (LRR) protein class of plant resistance ( R) genes. Despite exhibiting only four amino acid differences between the products of Tm-2 and Tm-2 2 , Tm-2 2 confers resistance to ToMV mutant B7, whereas Tm-2 is broken by ToMV-B7. An Agrobacterium-mediated transient expression system was used to study the mechanism of differential recognition of the movement proteins (MPs), an avirulence factor for ToMV resistance, of ToMV-B7 by Tm-2 and Tm-2 2. Although resistance induced by Tm-2 and Tm-2 2 is not usually accompanied by hypersensitive response (HR), Tm-2 and Tm-2 2 induced HR-like cell death by co-expression with MP of a wild-type ToMV, a strain that causes resistance for these R genes, and Tm-2 2 but not Tm-2 induced cell death with B7-MP in this system. Site-directed amino acid mutagenesis revealed that Tyr-767 in the LRR of Tm-2 2 is required for the specific recognition of the B7-MP. These results suggest that the Tyr residue in LRR contributes to the recognition of B7-MP, and that Tm-2 and Tm-2 2 are involved in HR cell death.