87 research outputs found

    Promoter trapping in tobacco and Arabidopsis.

    Full text link
    A promoter trapping system based on the promoter trap vector pDeltaGUSBin19 was studied for the identification and isolation of developmentally regulated plant genes. pDeltaGUSBin19 contained a promoterless gusA gene and a nos promoter-driven nptII gene. The previously unknown regions of pDeltaGUSBin19 were sequenced. A method of high frequency transformation of Arabidopsis thaliana by Agrobacterium-tumefaciens was developed. The T-DNA of pDeltaGUSBin19 was introduced into tobacco and Arabidopsis to generate a large number of transformed lines. The levels and patterns of gusA gene activation in diverse organs and cell types of transgenic plants were analysed. Line atvt1 exhibited GUS fusion activity in the tapetum and vascular tissues. It contained a single copy of the T-DNA and the gusA gene fusion was transcribed as a fusion transcript. Wild-type genomic and cDNA clones corresponding to the tagged gene were isolated using a molecular probe generated by IPCR of genomic sequence flanking the T-DNA. The native mRNA was approximately 4.4 kb. The foil length cDNA of this gene was cloned, sequenced and analysed. It encoded a putative nucleic acid helicase, designated HVT1 (Helicase in Vascular tissue and Tapetum). Of a predicted 1291 amino acid residues, HVT1 is homologous to the Drosophila MALELESS, human RNA helicase A and bovine nuclear DNA helicase proteins, and represents the first identified member of a new subgroup within the mle group of the DEAH protein family. Low stringency genomic Southern blot analysis indicates there exists another structurally related gene in Arabidopsis. The value of promoter trapping as a complement to other approaches of gene isolation and possible function of the HVT1 protein is discussed

    iTRAQ-Based Proteomics Investigation of Aqueous Humor from Patients with Coats' Disease

    No full text
    <div><p>Background</p><p>Coats' disease is an uncommon form of retinal telangiectasis, and the identification of novel proteins that contribute to the development of Coats' disease is useful for improving treatment efficacy. Proteomic techniques have been used to study many eye diseases; however, few studies have used proteomics to study the development of Coats' disease.</p><p>Methods</p><p>Isobaric tagging for relative and absolute protein quantification (iTRAQ) was employed to screen differentially expressed proteins (DEPs) in the aqueous humor (AH) between stage 3A patients (n = 8), stage 3B patients (n = 14), stage 4 patients (n = 2) and control patients (n = 20). Differentially co-expressed proteins (DCPs) were present in all three stages of Coats' disease and were considered disease-specific proteins. These proteins were further analyzed using Gene Ontology (GO) functional annotations.</p><p>Results</p><p>A total of 819 proteins were identified in the AH, 222 of which were significantly differentially expressed (fold change > 2 and P < 0.05) in the samples from at least one stage of Coats' disease. Of the DEPs, 46 were found among all three stages of Coats' disease and the controls; therefore, they were considered Coats' disease-specific proteins (DCPs). A GO classification analysis indicated that the DCPs were closely related to structural molecule activity, cell adhesion molecule binding and receptor binding. Western blotting confirmed the expression levels of haptoglobin and apolipoprotein C-I were significantly up-regulated in Coats’ disease.</p><p>Conclusions</p><p>The 46 Coats' disease-specific proteins may provide additional insights into the mechanism of Coats' disease and represent potential biomarkers for identifying individuals with Coats' disease.</p></div

    Heat map analysis of the differentially expressed proteins between the CK group and three groups of patients with different stages of Coats' disease.

    No full text
    <p>The color scale shown at the top illustrates the relative protein expression level across all the samples. Red represents an expression level lower than the mean, whereas green represents an expression level above the mean.</p

    Venn diagram indicating the differentially co-expressed proteins (DCPs) from the patients with the three stages of Coats' disease.

    No full text
    <p>The numbers in parentheses indicate the total number of DCPs in stage 3A, stage 3B and stage 4, which are represented by purple, yellow and green, respectively.</p

    DEPs between the patients with stage 3A, 3B and 4 Coats’ disease and the controls (<i>p</i> < 0.05).

    No full text
    <p>DEPs between the patients with stage 3A, 3B and 4 Coats’ disease and the controls (<i>p</i> < 0.05).</p

    Schematic representation of the workflow of the iTRAQ experiment.

    No full text
    <p>Schematic representation of the workflow of the iTRAQ experiment.</p

    Forty-six DCPs, including 29 down-regulated proteins (log FC < -1) and 17 up-regulated proteins (log FC > 1).

    No full text
    <p>Forty-six DCPs, including 29 down-regulated proteins (log FC < -1) and 17 up-regulated proteins (log FC > 1).</p

    GO term classification of the DCPs between the patients and CK with <i>p</i>-values < 0.01.

    No full text
    <p>GO term classification of the DCPs between the patients and CK with <i>p</i>-values < 0.01.</p

    Image_3_Genome deletions to overcome the directed loss of gene function in Leishmania.tiff

    No full text
    With the global reach of the Neglected Tropical Disease leishmaniasis increasing, coupled with a tiny armory of therapeutics which all have problems with resistance, cost, toxicity and/or administration, the validation of new drug targets in the causative insect vector borne protozoa Leishmania spp is more important than ever. Before the introduction of CRISPR Cas9 technology in 2015 genetic validation of new targets was carried out largely by targeted gene knockout through homologous recombination, with the majority of genes targeted (~70%) deemed non-essential. In this study we exploit the ready availability of whole genome sequencing technology to reanalyze one of these historic cell lines, a L. major knockout in the catalytic subunit of serine palmitoyltransferase (LCB2), which causes a complete loss of sphingolipid biosynthesis but remains viable and infective. This revealed a number of Single Nucleotide Polymorphisms, but also the complete loss of several coding regions including a gene encoding a putative ABC3A orthologue, a putative sterol transporter. Hypothesizing that the loss of such a transporter may have facilitated the directed knockout of the catalytic subunit of LCB2 and the complete loss of de novo sphingolipid biosynthesis, we re-examined LCB2 in a L. mexicana line engineered for straightforward CRISPR Cas9 directed manipulation. Strikingly, LCB2 could not be knocked out indicating essentiality. However, simultaneous deletion of LCB2 and the putative ABC3A was possible. This indicated that the loss of the putative ABC3A facilitated the loss of sphingolipid biosynthesis in Leishmania, and suggested that we should re-examine the many other Leishmania knockout lines where genes were deemed non-essential.</p

    Table_3_Genome deletions to overcome the directed loss of gene function in Leishmania.xlsx

    No full text
    With the global reach of the Neglected Tropical Disease leishmaniasis increasing, coupled with a tiny armory of therapeutics which all have problems with resistance, cost, toxicity and/or administration, the validation of new drug targets in the causative insect vector borne protozoa Leishmania spp is more important than ever. Before the introduction of CRISPR Cas9 technology in 2015 genetic validation of new targets was carried out largely by targeted gene knockout through homologous recombination, with the majority of genes targeted (~70%) deemed non-essential. In this study we exploit the ready availability of whole genome sequencing technology to reanalyze one of these historic cell lines, a L. major knockout in the catalytic subunit of serine palmitoyltransferase (LCB2), which causes a complete loss of sphingolipid biosynthesis but remains viable and infective. This revealed a number of Single Nucleotide Polymorphisms, but also the complete loss of several coding regions including a gene encoding a putative ABC3A orthologue, a putative sterol transporter. Hypothesizing that the loss of such a transporter may have facilitated the directed knockout of the catalytic subunit of LCB2 and the complete loss of de novo sphingolipid biosynthesis, we re-examined LCB2 in a L. mexicana line engineered for straightforward CRISPR Cas9 directed manipulation. Strikingly, LCB2 could not be knocked out indicating essentiality. However, simultaneous deletion of LCB2 and the putative ABC3A was possible. This indicated that the loss of the putative ABC3A facilitated the loss of sphingolipid biosynthesis in Leishmania, and suggested that we should re-examine the many other Leishmania knockout lines where genes were deemed non-essential.</p
    • …
    corecore