Home About us Contact
|
Home About us Contact | ||
|
|
||
Southern Hybridization Analysis (southern + hybridization_analysis)
Selected AbstractsDevelopment of a sensitive diagnostic assay for fish nervous necrosis virus based on RT-PCR plus nested PCRJOURNAL OF FISH DISEASES, Issue 5 2000L Dalla Valle A polymerase chain reaction (PCR)-based assay to detect nervous necrosis virus (NNV) in fish was developed by using two sets of primers designed on a highly conserved region of the coat protein gene encoded by RNA2 of NNV. The first pair of primers amplified a fragment of 605 bp by one-step reverse-transcription (RT)-PCR, while the second pair amplified an internal segment of 255 bp by nested PCR. Addition of nested PCR increased the assay sensitivity 100-fold when carried out in a separate tube (two-step assay) and 10-fold when performed in the same tube (one-step assay). The sensitivity of the two-step assay was 104 times higher than that of virus cultivation. Nested PCR served also to confirm the specificity of the first amplification, as verified also by Southern hybridization analysis and direct sequencing. In species known to be susceptible to infection, such as European sea bass, Dicentrarchus labrax, and gilthead seabream, Sparus aurata, NNV was often detectable in brain tissue by RT-PCR alone but only by the two-step assay in blood, sperm, ovarian tissue or larvae. The same was true for sperm and ovarian tissue of shi drum, Umbrina cirrosa. NNV was also detected in the brains of Japanese red seabream, Pagrus major and brown meagre, Sciaena umbra, suggesting that these species can also be infected. No NNV was detected in samples of Artemia salina nauplii and rotifers obtained from a fish farm with an NNV outbreak. The inclusion of nested PCR in the assay appears to be necessary to screen out NNV-positive broodfish by blood sampling and testing of their larval progeny. [source] ,-TUBULIN GENE OF PORPHYRA PURPUREA ( RHODOPHYTA),JOURNAL OF PHYCOLOGY, Issue 5 2002Ron M. MacKay The life cycle of the marine red alga Porphyra purpurea (Roth) C. Agardh includes a shell-boring filamentous sporophyte and a leafy gametophyte. A single intronless gene for the microtubule protein ,-tubulin was discovered by molecular cloning of P. purpurea cDNA and genomic DNA. This gene, named TubB1, encodes a ,-tubulin with a divergent amino acid sequence, showing 74% identity with the conserved ,-tubulin of Chlamydomonas reinhardtii P. A. Dangeard. Southern hybridization analysis of nuclear DNA confirmed that P. purpurea has a single TubB1 gene. Transcripts (1.8 kb) of TubB1 are present in the sporophyte and gametophyte. Codon bias indicates strong expression of TubB1. The divergent nature of the TubB1 genes suggests that the absence of axonemal structures has allowed substantial genetic drift in red algal ,-tubulin genes. [source] Magnaporthe oryzae isolates causing gray leaf spot of perennial ryegrass possess a functional copy of the AVR1-CO39 avirulence geneMOLECULAR PLANT PATHOLOGY, Issue 3 2006REBECCA PEYYALA SUMMARY Gray leaf spot of perennial ryegrass (Lolium perenne) is a severe foliar disease caused by the ascomycete fungus Magnaporthe oryzae (formerly known as Magnaporthe grisea). Control of gray leaf spot is completely dependent on the use of fungicides because currently available perennial ryegrass cultivars lack genetic resistance to this disease. M. oryzae isolates from perennial ryegrass (prg) were unable to cause disease on rice cultivars CO39 and 51583, and instead triggered a hypersensitive response. Southern hybridization analysis of DNA from over 50 gray leaf spot isolates revealed that all of them contain sequences corresponding to AVR1-CO39, a host specificity gene that confers avirulence to rice cultivar CO39, which carries the corresponding resistance gene Pi-CO39(t). There was also an almost complete lack of restriction site polymorphism at the avirulence locus. Cloning and sequencing of the AVR1-CO39 gene (AVR1-CO39Lp) from 16 different gray leaf spot isolates revealed just two point mutations, both of which were located upstream of the predicted open reading frame. When an AVR1-CO39Lp gene copy was transferred into ML33, a rice pathogenic isolate that is highly virulent to rice cultivar CO39, the transformants were unable to cause disease on CO39 but retained their virulence to 51583, a rice cultivar that lacks Pi-CO39(t). These data demonstrate that the AVR1-CO39 gene in the gray leaf spot pathogens is functional, and suggest that interaction of AVR1-CO39Lp and Pi-CO39(t) is responsible, at least in part, for the host specificity expressed on CO39. This indicates that it may be possible to use the Pi-CO39(t) resistance gene as part of a transgenic strategy to complement the current deficiency of gray leaf spot resistance in prg. Furthermore, our data indicate that, if Pi-CO39(t) can function in prg, the resistance provided should be broadly effective against a large proportion of the gray leaf spot pathogen population. [source] Two homologous parasitism-specific proteins encoded in Cotesia plutellae bracovirus and their expression profiles in parasitized Plutella xylostellaARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 4 2008Sunyoung Lee Abstract A wasp, Cotesia plutellae, parasitizes the diamondback moth, Plutella xylostella, and interrupts host physiology for wasp survival and development. Identification of parasitism-specific factors would be helpful to understand the host,parasitoid interaction. This study focused on identification of a 15-kDa protein found only in plasma of the parasitized P. xylostella. Degenerate primers were designed after N-terminal amino acid sequencing of the parasitism-specific protein and used to clone the corresponding gene from the parasitized P. xylostella by a nested reverse transcriptase-polymerase chain reaction (RT-PCR). Two homologous genes were cloned and identified as "CpBV15," and "CpBV15,," respectively, due to the identical size (158 amino acid residues) of the predicted open reading frames, in which they shared amino acid sequences in both terminal regions, but varied in internal sequences. Southern hybridization analysis indicated that both genes were located on C. plutellae bracovirus genome. Real-time quantitative RT-PCR revealed that both genes were mostly expressed at the late parasitization period, which was further confirmed by an immunoblotting assay using CpBV15 antibody. A recombinant CpBV15 protein was produced from Sf9 cells via a baculovirus expression system. The purified CpBV15 protein could enter hemocytes of P. xylostella and were localized in the cytosol. Along with the sequence similarities of CpBV15s with eukaryotic initiation factors, their putative biological role has been discussed in terms of the host translation inhibitory factor. Arch. Insect Biochem. Physiol. 67:157,171, 2008. © 2008 Wiley-Liss, Inc. [source] | ||||||||||