scholarly journals Production of transgenic carnation with antisense ACS (1-aminocyclopropane44-carboxy late synthase) gene

2000 ◽  
Vol 6 (4) ◽  
Author(s):  
E. Kiss ◽  
A. Veres ◽  
Zs. Galli ◽  
N. Nagy ◽  
E. Tóth ◽  
...  
Keyword(s):  
Ethylene Production ◽  
Mrna Level ◽  
Dianthus Caryophyllus ◽  
Pcr Analysis ◽  
Vase Life ◽  
Rt Pcr ◽  
Transgene Integration ◽  
Putative Transformants ◽  
Antisense Orientation ◽  
Npt Ii

Dianthus chinensis and Dianthus caryophyllus varieties were tested for shoot regeneration from leaf and petal explants and transformed with Agrobacterium tuniefaciens strains (EHA 105 and LBA 4404) harbouring an apple derived ACS cDNA in antisense orientation in order to reduce ethylene production and influence the ethylene dependant traits in carnation. After transformation regenerating shoots were selected on MS medium containing 50-75-100-125-150 mg/1 kanamycin and supplemented with 1 mg/1 BA, 0.2 mg/1 NAA. Transgene integration was proved by PCR analysis with npt II spcific primers followed by Southern hybridisation of DNA isolated from green shoots on medium containing 150 mg/1 kanamycin. Several putative transformants were subjected to RT-PCR in order to examine the npt 11 expression at mRNA level. Both the transformant and the non-transformant plants were potted into glasshouse to observe the effect of changed ethylene production on flowering time, petal senescence and vase life.  

scholarly journals Down-regulation of ethylene production in carnation (Dianthus Caryphyllus L.) by an apple derived ACC-cDNA

2005 ◽  
Vol 11 (1) ◽  
Author(s):  
A. Veres ◽  
E. Kis ◽  
E. Tóth ◽  
Á. Tóth ◽  
L. Heszky
Keyword(s):  
Genetic Modification ◽  
Ethylene Production ◽  
Plant Material ◽  
Normal Growth ◽  
Acc Synthase ◽  
Vase Life ◽  
Down Regulation ◽  
Transgene Integration ◽  
Relative Value ◽  
Opening Stage

Transgenic carnations were produced with an apple derived antisense ACC-synthase cDNA. Transgenic carnation regenerants were potted in glasshouse. All transformed plants showed normal growth and were true-to-type. Ethylene production — measured at full opening stage — lowered by 30-60 %, no plant with 100 % decrease was identified. The vase-life has been observed for 5 years. 38 % of the transformant carnations showed a higher a relative value in days by more than 2 days to 6 days. Twenty six plants were found exhibiting the most marked alterations in the tested trait. In these plants ethylene production decreased by 37-67 %, they have longer vase-life (by 4 days or more). Since the fragrance variety 'Bíbor' was the plant material for genetic modification of vase-life, this trait has been conserved after transformation in spite of the fact that the position of transgene integration cannot be directed.

scholarly journals Physiological and Molecular Characterization of the Response to Ethylene during Senescence of Carnation Genotypic Variants

1995 ◽  
Author(s):  
William Woodson ◽  
Shimon Mayak ◽  
Haim Rabinowitch
Keyword(s):  
Ethylene Production ◽  
Dianthus Caryophyllus ◽  
Acc Oxidase ◽  
Acc Synthase ◽  
Early Response ◽  
Vase Life ◽  
Petal Senescence ◽  
Ethylene Treatment ◽  
Novel Proteins ◽  
Exogenous Ethylene

The senescence of carnation (Dianthus caryophyllus L.) flowers is associated with increased production of the phytohormone ethylene, which in turn serves to initiate and regulate the processes involved in programmed petal death. We investigated the regulation of ethylene production and petal senescence in carnation. Several carnation genotypes were identified that exhibited extended vase-life in comparison to flowers from typical commercial cultivars. The capacity of these genotypes to produce ethylene during postharvest vase-life and to respond to exogenous ethylene was investigated. Several genotypes, represented by 'Sandrosa' and 87-37G produced little ethylene durig their postharvest vase-life and as a result failed to exhibit the symptoms (in-rolling and wilting) typical of flowers producing elevated levels of ethylene. These genotypes were further separated by their capacity to respond to exogenous ethylene by both increased ethylene synthesis and premature petal senescence. In one case a genotype (799) was identified that was not capable of responding to exogenous ethylene by either increased ethylene production or premature petal senescence. The regulation of ethylene production during petal senescence was investigated both at the enzyme and gene levels. A full length cDNA was identified for the petal senescence-related ACC synthase gene. Utilizing this, and other ethylene biosynthetic pathway cDNA probes, an increase in both ACC synthase and ACC oxidase mRNAs were detected following ethylene treatment. An increase in ACC oxidase mRNA and enzyme activity was detected within 2-3 h following ethylene treatment, indicating the expression of this gene is an early response to ethylene. An investigation into the expression of novel proteins during petal senescence revealed a number of polypeptides increased in abundance and possibly play a role in the regulation or biochemical processes of senescence. One polypeptide of 70 kDa was identified as being encoded by the previously characterized gene SR12 and possibly represents a b-galactosidase involved in the remobilization of carbohydrates during senescence.

scholarly journals Insertion of an SVA Element in MSH2 as a Novel Cause of Lynch Syndrome

2020 ◽  
Author(s):  
Ciyu Yang ◽  
Yirong Li ◽  
Magan Trottier ◽  
Michael Farrell ◽  
Vikas Rai ◽  
...  
Keyword(s):  
Lynch Syndrome ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Dna Mismatch ◽  
Mmr Genes ◽  
Antisense Orientation ◽  
The Family ◽  
Long Range Pcr ◽  
Early Onset Colorectal Cancer ◽  
Generation Sequencing

Germline mutations in the DNA mismatch repair (MMR) genes cause Lynch syndrome (LS). Insertions of retrotransposons in MMR genes have been reported as a rare cause of LS. Here, we present a novel SINE-VNTR-Alu (SVA) insertion in exon 12 of MSH2 in an individual with early-onset colorectal cancer and strong LS family history. RT-PCR analysis indicated a larger aberrant MSH2 transcript in one of the family members. MSK-IMPACT next-generation sequencing testing and long-range PCR revealed an insertion in MSH2 exon 12 at the c.1972 position in an antisense orientation. The insertion was further characterized as an SVA element approximately 3 kb in length, belonging to the SVA_F1 family of retrotransposons.

scholarly journals Rapid competitive PCR determination of relative gene expression in limiting tissue samples

1996 ◽  
Vol 42 (2) ◽  
pp. 227-231 ◽  
Author(s):  
Y H Jiang ◽  
L A Davidson ◽  
J R Lupton ◽  
R S Chapkin
Keyword(s):  
Gene Expression ◽  
Mrna Level ◽  
Internal Standard ◽  
Pcr Amplification ◽  
Pcr Analysis ◽  
Relative Gene Expression ◽  
Competitive Pcr ◽  
Rt Pcr ◽  
Tissue Samples ◽  
Relative Gene

Abstract Reverse transcriptase (RT)-PCR is widely used to study gene transcription in many biological systems. Despite the development of a variety of procedures, quantification of RT-PCR products remains difficult, particularly when processing a large number of samples. Therefore, we developed a novel alternative PCR technique that we term "rapid competitive PCR" (RC-PCR), designed to study the relative expression of specific genes in a large number of small tissue biopsies. RC-PCR is characterized by measuring relative gene expression at the mRNA level of two or more samples with a nonradioactive assay based on competitive PCR amplification between identical sequences of internal standard and target cDNA. Only a single reaction tube per sample is used in this technique, and it was validated by comparing RC-PCR of protein kinase C zeta and alpha expression in rat colonic mucosa samples with competitive RT-PCR analysis (requiring 6-8 reaction tubes per sample). We conclude that RC-PCR is a simple, rapid, highly sensitive technique that is capable of detecting less than twofold differences in mRNA expression.

Endothelial Cells Are Not Derived from Hematopoietic Precursor Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1815-1815
Author(s):  
Frank Timmermans ◽  
Magda De Smedt ◽  
Robrecht Raedt ◽  
Jean Plum ◽  
Bart Vandekerckhove
Keyword(s):  
Bone Marrow ◽  
Endothelial Cells ◽  
Cord Blood ◽  
Mrna Level ◽  
Cell Fraction ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Endothelial Progenitor ◽  
Hematopoietic Stem

Abstract Endothelial outgrowth cells (EOC) can be generated from mononuclear blood cells. Based on proliferative and functional characteristics, EOC were claimed to derive from an immature endothelial progenitor cell or angioblast. Several investigators have claimed that these cells constitute a subpopulation of CD34+ hematopoietic stem cells(HSC). However, the EOC-precursor is not well defined and its nature remains elusive. Methods and results: Umbilical cord blood CD34+ cells were sorted into a small (< 1 %) CD34+CD45− non-hematopoietic cell fraction (purity > 99.5%) and CD34+CD45+ HSC (purity > 99.2 %) (n=5). The cell fractions were cultured separately in EBM2/EGM2 medium (Cambrex, Verviers, Belgium) onto gelatine coated 24 wells. EOC were exclusively derived from the CD34+CD45− cell fraction and not from the CD45+ HSC. We further analysed the CD34+CD45− cell fraction for expression of endothelial progenitor genes. Analysis showed the presence of VEGFR2, VE-Cadherine and CD146 on the CD34+CD45− precursor population whereas CD45+ HSC were consistantly negative for these markers. CD133, which was claimed to be a marker for endothelial progenitors was negative on the CD34+CD45− cells. No VEGFR2+ CD133+ cells could be detected either by flowcytometry or at the mRNA level. In adult bone marrow, EOC only derived from CD45− CD31+ cells, and not from the CD45+ HSC or CD45− CD31− mesenchymal cells. CD34+CD45+ HSC or CD14+ CD45+ monocytes generated under the same conditions large flat adherent cells positive for CD31, LDL uptake and the lectin UEA-1. On RT-PCR and real time RT-PCR analysis, cells were positive for VEGFRII, CD146 and VE cadherin. However, membrane staining was consistently negative for VE-cadherin on flowcytometric analysis and positive for monocytic markers such as CD14 and CD45. In functional assays, the majority of the cells were shown to be phagocytic and were unable to form vascular tubes in the matrigel angiogenesis assay. These data demonstrate that monocytes may acquire a phenotype in vitro which is difficult to discriminate from endothelial cells. Conclusion : Endothelial cell generated in vitro from cord blood or bone marrow derive from a CD45− nonhematopoietic precursor.

scholarly journals Effects of Anti-Ethylene Treatments on Ethylene Production and Antioxidant Activities in Cut Spray Carnation

2012 ◽  
Vol 20 (2) ◽  
pp. 173-182
Author(s):  
Mahnaz Karimi ◽  
Moazzam Hassanpour Asil ◽  
Ghorbanali Nematzadeh ◽  
Hedayat Zakizadeh
Keyword(s):  
Antioxidant Enzymes ◽  
Ethylene Production ◽  
Enzyme Activities ◽  
Antioxidant Activities ◽  
Dianthus Caryophyllus ◽  
Vase Life ◽  
Cut Flowers ◽  
Acc Content ◽  
Oxyacetic Acid

Abstract The present investigation was aimed to study changes in ethylene production, ACC content and antioxidant enzymes of cut spray carnation (Dianthus caryophyllus L.) flowers that had been treated with amino-oxyacetic acid (AOA), benzyladenine (BA) and 1-methylcyclopropene (1-MCP). Maximum vase life in ‘Optima’ spray carnations was obtained with 0.6 mg l-1 1-MCP. Ethylene production was significantly decreased by AOA at concentrations over 100 mg l-1, BA at 30 mg l-1, and 1-MCP at all concentrations, compared with the control. A significant increase in ACC content was observed in 1-MCP treated cut flowers compared with the control. However, the decline in ACC content was observed after using 100 or 150 mg l-1 AOA. A significant increase in SOD, CAT and POX enzyme activities was observed in the treatment with 0.6 mg l-1 1-MCP.

scholarly journals RT-PCR Analysis of TopBP1 Gene Expression in Hereditary Breast Cancer

2010 ◽  
Vol 6 ◽  
pp. 49-59
Author(s):  
Ewa Forma ◽  
Magdalena Bernaciak ◽  
Hanna Romanowicz-Makowska ◽  
Magdalena Bryś
Keyword(s):  
Breast Cancer ◽  
Hereditary Breast Cancer ◽  
Mrna Level ◽  
Pcr Analysis ◽  
Loss Of Function ◽  
Rt Pcr ◽  
Brca1 And Brca2 ◽  
Hereditary Predisposition ◽  
Checkpoint Response ◽  
System P

Hereditary predisposition to breast cancer determined in large part by loss of function mutations in one of two genes BRCA1 and BRCA2. Besides BRCA1 and BRCA2 other genes are also likely to be involved in hereditary predisposition to breast cancer. TopBP1 protein is involved in DNA replication, DNA damage checkpoint response and transcriptional regulation. Expression of TopBP1 gene at the mRNA level was analyzed by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) in 94 samples of hereditary breast cancer. Analysis of TopBP1 mRNA level showed that expression of TopBP1 is significantly downregulated in poorly differentiated breast cancer (grade III according Bloom-Richardson system (P<0.05).

scholarly journals Prolonging the Vase Life of Snapdragons and Carnations with a Natural Lipid, Lysophosphatidylethanolamine (LPE)

HortScience ◽  
1996 ◽  
Vol 31 (4) ◽  
pp. 636f-637 ◽  
Author(s):  
Navjot Kaur ◽  
Jiwan P. Palta
Keyword(s):  
Ethylene Production ◽  
Early Stage ◽  
Membrane Integrity ◽  
Dianthus Caryophyllus ◽  
Stage Iv ◽  
Deionized Water ◽  
Vase Life ◽  
Ion Leakage ◽  
Fresh Weight ◽  
Flower Opening

Freshly cut snapdragon (Antirrhinum majus L) spikes or carnation (Dianthus caryophyllus L cv. White Sim) stems were put in LPE (10 ppm for carnation, 25 ppm for snapdragon) solution for 24 hours and then transferred to deionized water. Parallel controls were kept continuously in deionized water. Snapdragon spikes were harvested when they had one-third of the florets opened which is a standard commercial practice. The carnations used in the experiment were harvested at three different stages of flower development starting from open brush bud stage (Stage IV) to fully opened (Stage VII, petals 45(to the stem) flower. LPE treatment delayed senescence in snapdragon by four days. Furthermore, it enhanced the opening of floral buds and opened all the florets on every spike. LPE treatment also significantly delayed loss in fresh weight of spikes associated with senescence, lowered the endogenous ethylene production and reduced ion leakage from florets. LPE had a similar effect on fresh weight and ion leakage from carnations if it was applied at an early stage of flower opening. Older carnations (Stage VII) were found unresponsive to LPE. In conclusion, LPE has the potential in enhancing the vase life of snapdragons and carnations. Carnations must be harvested at the open brush bud stage for effective LPE application. Our results suggest that LPE is prolonging vase life of cut flowers by reducing ethylene production and maintaining membrane integrity.

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