A new method for analyzing gene expression based on frequency analysis of RT-PCR products obtained with degenerate primers

Yams (Dioscorea spp.) are widely grown in China as vegetables and herbal medicine. However, studies on viral diseases on yams are still limited. As a pilot project of a government initiative for improving yam productivity, a small study was conducted in Guangxi, a southern province of China, on viral disease in yams. Incidence of virus-like disease for the three extensively grown D. alata cultivars, GH2, GH5, and GH6, were 12 to 40%, 12 to 29%, and 11 to 25%, respectively, as found in a field survey with a five-plot sampling method in 2010. A total of 112 leaf samples showing mosaic or mottling or leaves without symptoms were collected from the cvs. GH2, GH5, GH6, and seven additional cultivars (D. alata cvs. GY2, GY23, GY47, GY69, GY62, GY72, and D. batatas cv. Tiegun). To determine if the symptoms were caused by Yam mild mosaic virus (YMMV; genus Potyvirus, family Potyviridae), total RNA was extracted from leaves with a commercial RNA purification kit (TIANGEN, Beijing, China), and reverse-transcription (RT)-PCR was conducted with a YMMV-specific primer pair (4) that amplifies the 3′-terminal portion of the viral genome. A PCR product with the predicted size of 262 bp was obtained from samples of GH5 (number testing positive of total number of leaves = 5 of 12), GH6 (24 of 42), and GY72 (1 of 1), but not from asymptomatic leaves. PCR products from a GH5 sample (YMMV-Nanning) and a GH6 sample (YMMV-Luzhai) were cloned and sequenced using an ABI PRISM 3770 DNA Sequencer. The two PCR products were 97% identical at nucleotide (nt) level and with the highest homology (89% identity) to a YMMV isolate (GenBank Accession No. AJ305466). To further characterize the isolates, degenerate primers (2) were used to amplify viral genome sequence corresponding to the C-terminal region of the nuclear inclusion protein b (NIb) and the N-terminal region of the coat protein (CP). These 781-nt fragments were sequenced and a new primer, YMMV For1 (5′-TTCATGTCGCACAAAGCAGTTAAG-3′) corresponding to the NIb region, was designed and used together with primer YMMV UTR 1R to amplify a fragment that covers the complete CP region of YMMV by RT-PCR. These 1,278-nt fragments were sequenced (GenBank Accession Nos. JF357962 and JF357963). CP nucleotide sequences of the YMMV-Nanning and YMMV-Luzhai isolates were 94% similar, while amino acid sequences were 99% similar. BLAST searches revealed a nucleotide identity of 82 to 89% and a similarity of 88 to 97% for amino acids to sequences of YMMV isolates (AF548499 and AF548519 and AAQ12304 and BAA82070, respectively) in GenBank. YMMV is known to be prevalent on D. alata in Africa and the South Pacific, and has recently been identified in the Caribbean (1) and Colombia (3). To our knowledge, this is the first report of the natural occurrence of YMMV in China and it may have implications for yam production and germplasm exchange within China. References: (1) M. Bousalem and S. Dallot. Plant Dis. 84:200, 2000. (2) D. Colinet et al. Phytopathology 84:65, 1994. (3) S. Dallot et al. Plant Dis. 85:803, 2001. (4) R. A. Mumford and S. E. Seal. J. Virol. Methods 69:73, 1997.

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Differential effect of rice husk and xylan on feruloyl esterase activity in Penicillium rubens.

Revista Mvz Córdoba ◽

10.21897/rmvz.1959 ◽

2020 ◽

Vol 26 (1) ◽

pp. e1959

Author(s):

Débora Elizabeth Hernández-Pérez ◽

Carlos Gil-Durán ◽

Renato Chávez-Rosales ◽

Ramon Ovidio Garcia-Rico

Keyword(s):

Gene Expression ◽

Rice Husk ◽

Esterase Activity ◽

Transcriptional Activity ◽

Gene Expression Analysis ◽

Feruloyl Esterase ◽

Rt Pcr ◽

Negative Results ◽

Pcr Products ◽

Positive Effect

Objective. Analyzed the effect of xylan and rice husk as sole sources of carbon on the FAE activity and expression of faeA and faeB genes of Penicillium rubens (Wisconsin 54-1255), in submerged fermentation. Materials and methods. The fermentations were carried out for 24, 48, and 72 h (28°C/250 rpm), in flasks with a modified Sakamoto medium. FAE activity was determined using the synthetic substrate Ethyl 4-hydroxy-3-methoxycinnamate. The transcription of faeA and faeB was analyzed by RT-PCR. The PCR products were resolved by electrophoresis and analyzed by densitometry. Results. The analysis of gene expression showed that the use of xylan had a positive effect on the expression of both genes. The highest transcriptional activity of both faeA and faeB was detected at 48 h. On the other hand, rice husk showed negative results. The data obtained when the FAE activity was determined, supported the results observed in the gene expression analysis. Conclusion. Different from xylan the rice husk did not demonstrate an inducer effect on the feruloyl esterase activity in P. rubens.

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Axl-Gas6 Interaction Counteracts E1A-Mediated Cell Growth Suppression and Proapoptotic Activity

Molecular and Cellular Biology ◽

10.1128/mcb.19.12.8075 ◽

1999 ◽

Vol 19 (12) ◽

pp. 8075-8082 ◽

Cited By ~ 37

Author(s):

Wei-Ping Lee ◽

Yong Liao ◽

Dan Robinson ◽

Hsing-Jien Kung ◽

Edison T. Liu ◽

...

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Kinases ◽

Growth Suppression ◽

Serum Deprivation ◽

Transcriptional Level ◽

Rt Pcr ◽

Degenerate Primers ◽

Pcr Products ◽

Her 2 ◽

Induced Apoptosis

ABSTRACT The adenovirus type 5 early region 1A gene (E1A) has previously been known as an immortalization oncogene because E1A is required for transforming oncogenes, such as ras andE1B, to transform cells in primary cultures. However, E1A has also been shown to downregulate the overexpression of theHer-2/neu oncogene, resulting in suppression of transformation and tumorigenesis induced by that oncogene. In addition, E1A is able to promote apoptosis induced by anticancer drugs, irradiation, and serum deprivation. Many tyrosine kinases, such as the epidermal growth factor receptor, Her-2/Neu, Src, and Axl, are known to play a role in oncogenic signals in transformed cells. To study the mechanism underlying the E1A-mediated tumor-suppressing function, we exploited a modified tyrosine kinase profile assay (D. Robinson, F. Lee, T. Pretlow, and H.-J. Kung, Proc. Natl. Acad. Sci. USA 93:5958–5962, 1996) to identify potential tyrosine kinases regulated by E1A. Reverse transcription (RT)-PCR products were synthesized with two degenerate primers derived from the conserved motifs of various tyrosine kinases. A tyrosine kinase downregulated by E1A was identified by analyzing the AluI-digested RT-PCR products. We isolated the DNA fragment of interest and found that E1A negatively regulated the expression of the transforming receptor tyrosine kinase Axl at the transcriptional level. To study whether downregulation of the Axl receptor is involved in E1A-mediated growth suppression, we transfectedaxl cDNA into E1A-expressing cells (ip1-E1A) to establish cells that overexpressed Axl. The Axl ligand Gas6 triggered a greater mitogenic effect in these ip1-E1A-Axl cells than in ip1-E1A control cells and protected the Axl-expressing cells from serum deprivation-induced apoptosis. These results indicate that downregulation of the Axl receptor by E1A is involved in E1A-mediated growth suppression and E1A-induced apoptosis and thereby contributes to E1A’s antitumor activities.

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Use of Degenerate Primers for Partial Sequencing and RT-PCR-Based Assays of Grapevine Leafroll-Associated Viruses 4 and 5

Phytopathology ◽

10.1094/phyto.1998.88.11.1238 ◽

1998 ◽

Vol 88 (11) ◽

pp. 1238-1243 ◽

Cited By ~ 24

Author(s):

Geoffrey Routh ◽

Yun-Ping Zhang ◽

Pasquale Saldarelli ◽

Adib Rowhani

Keyword(s):

Heat Shock Protein 70 ◽

Plasmid Vector ◽

Pcr Primers ◽

Pcr Detection ◽

Rt Pcr ◽

Chain Reaction ◽

Degenerate Primers ◽

Double Stranded Rna ◽

Pcr Products ◽

Polymerase Chain

Double-stranded RNA (dsRNA) was purified from grapevines infected with grapevine leafroll-associated viruses 4 (GLRaV-4) and 5 (GLRaV-5), two putative closteroviruses. Reverse-transcriptase polymerase chain reaction (RT-PCR) was performed on this dsRNA using degenerate oligonucleotides designed to amplify an approximately 550- to 650-nucleotide fragment from the heat shock protein 70 homolog (HSP70) of the known closteroviruses. RT-PCR products of the appropriate molecular weight were gel-isolated and cloned into the plasmid vector pGEM-T. Clones of RT-PCR products generated by using these primers on dsRNA isolated from a plant infected with GLRaV-4 were sequenced. This sequence was used to develop an immunocapture RT-PCR (IC-RT-PCR) detection protocol capable of detecting GLRaV-4. Similar clones were made from dsRNA isolated from a plant infected with GLRaV-5. These clones were also sequenced. The two sequences were compared, and RT-PCR primers were developed that were able to amplify cDNA from both. These experiments demonstrate that degenerate primers that amplify closterovirus HSP70 sequences can be used to successfully generate sequences useful for IC-RT-PCR detection of these viruses. These data also suggest that it is feasible to use HSP70 sequences to design PCR primers capable of more general PCR detection of multiple GLRaV serotypes. Lastly, the presence of closterovirus-like HSP70 sequences in these putative closteroviruses implies that they are indeed members of this taxonomic group.

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Polyvalent Degenerate Oligonucleotides Reverse Transcription-Polymerase Chain Reaction: A Polyvalent Detection and Characterization Tool for Trichoviruses, Capilloviruses, and Foveaviruses

Phytopathology ◽

10.1094/phyto-95-0617 ◽

2005 ◽

Vol 95 (6) ◽

pp. 617-625 ◽

Cited By ~ 70

Author(s):

Xavier Foissac ◽

Laurence Svanella-Dumas ◽

Pascal Gentit ◽

Marie-Josée Dulucq ◽

Armelle Marais ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Reverse Transcription ◽

Unknown Etiology ◽

Rt Pcr ◽

Chain Reaction ◽

Degenerate Primers ◽

Sequence Comparisons ◽

Pcr Products ◽

Chlorotic Leaf ◽

Polymerase Chain

A polyvalent nested reverse transcription-polymerase chain reaction (RT-PCR) test using degenerate primers containing inosine (polyvalent degenerate oligonucleotides [PDO]) was developed for filamentous fruit tree viruses belonging to the genera Trichovirus, Capillovirus, and Foveavirus. The 362-bp product was amplified from nucleic acid extracts obtained from Prunus and Malus leaf samples. All the viruses targeted were detected, demonstrating the polyvalence of the test. The variability of a collection of Apple chlorotic leaf spot virus isolates was analyzed using the sequence of the PDO RT-PCR amplified cDNAs. The technique was also used to screen stone fruit materials infected with known agents or with virus-like graft-transmissible diseases of unknown etiology. The results obtained further validated the broad specificity of the assay, with positive amplification obtained for uncharacterized or partially characterized viruses associated with cherry and peach disorders. Sequencing the amplified PCR products either directly or after cloning allowed the identification of variants of known agents and the tentative identification of two new agents, a Trichovirus and a Foveavirus. In addition, sequence comparisons demonstrated that the sequence of the targeted region is phylogenetically informative and of predictive taxonomic value.

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Cucurbit leaf crumple virus Identified in Common Bean in Florida

Plant Disease ◽

10.1094/pdis-93-3-0320b ◽

2009 ◽

Vol 93 (3) ◽

pp. 320-320 ◽

Cited By ~ 10

Author(s):

S. Adkins ◽

J. E. Polston ◽

W. W. Turechek

Keyword(s):

Common Bean ◽

Blot Hybridization ◽

Cdna Probe ◽

Hypervariable Region ◽

Rt Pcr ◽

Dot Blot ◽

Fresh Market ◽

Degenerate Primers ◽

Pcr Products ◽

Cucurbit Leaf Crumple Virus

Virus-like symptoms of leaf deformation and rugosity, especially of younger leaves, and a mild mosaic were observed on fresh market common (green) bean (Phaseolus vulgaris L.) plants in Hendry County in southwest Florida in December of 2007 and again in February of 2008. All bean fields were adjacent to watermelon fields in which Cucurbit leaf crumple virus (CuLCrV), Squash vein yellowing virus (SqVYV), and Papaya ringspot virus type W (PRSV-W) infections had previously been confirmed (fall of 2007) by PCR, reverse transcription (RT)-PCR, and/or ELISA. Whiteflies, Bemisia tabaci, were observed on both bean and watermelon plants in December and February. Fifteen samples (eleven with symptoms) were collected in December and two (both with symptoms) in February. Initial ELISA assays using commercially available antisera for potyviruses or Cucumber mosaic virus (Agdia, Elkhart, IN) were negative. Total nucleic acids were extracted and used for PCR testing. All samples tested negative by RT-PCR using specific primers for SqVYV, PRSV-W, and Cucurbit yellow stunting disorder virus, and degenerate primers for potyviruses. Ten of fifteen December samples (ten of eleven symptomatic samples) and both February samples yielded PCR products of the expected size with the degenerate begomovirus primers, PAR1c496/PAL1v1978, which amplify a portion of the begomovirus A component (3). PCR products from three December and both February samples were cloned and sequenced. The 1,159-nt PCR products shared 99% identity with each other and 96% identity with the corresponding region of A component sequences of Arizona and California CuLCrV isolates (GenBank Accession Nos. AF256200 and AF224760, respectively). Additional degenerate begomovirus primers PBL1v2040/PCRc154, which amplify a 381-nt portion of the hypervariable region of the begomovirus B component (3), and AC1048/AV494, which amplify a 533-nt portion of a conserved region of the coat protein gene (4), were used to confirm the identity of CuLCrV in the three December samples. The PBL1v2040/PCRc154 PCR products shared 98 to 99% identity with each other and 94 to 95% identity with the corresponding region of B component sequences of Arizona and California CuLCrV isolates (GenBank Accession Nos. AF327559 and AF224761, respectively), whereas the AC1048/AV494 PCR products shared 99% identity with each other and 97% identity with the corresponding region of A component sequences of Arizona and California CuLCrV isolates. Nucleic acid dot-blot hybridization assays of sap from homogenized leaves of the three December samples (from which the PCR product clones were obtained) with a digoxigenin-labeled CuLCrV cDNA probe also confirmed the presence of CuLCrV. Although CuLCrV has been reported to experimentally infect common bean and tobacco (2), to our knowledge, this is the first report of CuLCrV infecting any noncucurbit host in Florida. This finding suggests that CuLCrV may be more widely distributed than previously known in Florida (1) and that common bean (and potentially other legumes) are potential reservoirs for CuLCrV. References: (1) F. Akad et al. Plant Dis. 92:648, 2008. (2) J. K. Brown et al. Phytopathology 92:734, 2002. (3) M. R. Rojas et al. Plant Dis. 77:340, 1993. (4) S. D. Wyatt and J. K. Brown. Phytopathology 86:1288, 1996.

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First Report of Freesia sneak virus Infecting Lachenalia Cultivars in South Africa

Plant Disease ◽

10.1094/pdis-91-6-0770a ◽

2007 ◽

Vol 91 (6) ◽

pp. 770-770 ◽

Cited By ~ 7

Author(s):

A. M. Vaira ◽

R. Kleynhans ◽

J. Hammond

Keyword(s):

Electron Microscopy ◽

South Africa ◽

The United States ◽

Sequence Information ◽

Rt Pcr ◽

Degenerate Primers ◽

Pcr Products ◽

Leaf Deformation ◽

Complex Hybrid ◽

Commercial Sources

Lachenalia (Lachenalia species, family Hyacinthaceae) is a bulbous ornamental plant endemic to southern Africa. In 1998, several lachenalia lines from ARC-Roodeplaat showing virus-like symptoms, and presumed to be infected with Ornithogalum mosaic virus (OrMV), were sent from South Africa under an APHIS permit for examination in Beltsville, MD. In addition to potyvirus-like particles, fine filamentous particles consistent with those of ophioviruses were observed with electron microscopy in some of the plant samples. Ophiovirus virions are filamentous nucleocapsids approximately 3 nm in diameter forming circularized structures of different lengths and are not easily detectable with electron microscopy. A reverse transcription (RT)-PCR assay using genus-specific degenerate primers that yield a 136-bp fragment from the RdRp gene is currently the best tool for detecting ophioviruses (3). Complementary DNA was produced from lachenalia total RNA extracts using either random hexamers or ophiovirus-specific primer OP1 (3). The ophiovirus diagnostic 136-bp fragment was amplified by PCR from plants of five lines (B12, L. unicolor × L. namaquensis, released in South Africa as cv. Rodelein; B24, L. aloides × L. rubida, cv. Robekkie; B48, a complex hybrid of L. aloides, L. rubida, L. orchioides, and L. bulbifera, cv. Leipoldt; B51, a complex hybrid of L. aloides, L. bulbifera, and L. orchioides, cv. Winsome; and B52, an intraspecies cross of L. aloides, cv. Fransie) of the six lines examined. Electron microscopy revealed ophiovirus particles in three of these five lines. The PCR products from three lachenalia lines were sequenced and found to be identical; the deduced 45 amino acid sequence showed 100% identity with the corresponding sequence obtained from Freesia sneak virus (FreSV), a tentative ophiovirus species referred to in the 8th ICTV report as Freesia ophiovirus (4) (for which the name Freesia sneak virus is now proposed). Currently, available sequence information shows only approximately 50 to 70% similarity between ophiovirus species and almost 100% identity between isolates, suggesting that the lachenalia ophiovirus is an isolate of FreSV. Symptoms associated with ophiovirus-infected lachenalias include fine chlorotic streaking and occasional gray flecking; more prominent chlorotic streaking, necrosis, and/or leaf deformation were observed in plants also infected with OrMV, similar potyviruses, and possibly other viruses. No ophiovirus was detected in five lines of Lachenalia hybrids obtained from U.S. commercial sources showing potyvirus-associated foliar chlorotic streaking, including cv. Fransie. Potyviruses were detected by RT-PCR (1) or ELISA with potyvirus-specific monoclonal antibodies (2) in plants from the United States and South Africa. It is of interest that the known hosts of FreSV, freesia and lachenalia, are both ornamental monocot genera of South African origin. References: (1) J. Chen et al. Arch. Virol. 146:757, 2001. (2) R. L. Jordan and J. Hammond. J. Gen. Virol. 72:25, 1991. (3) A. M. Vaira et al. Arch. Virol. 148:1037, 2003. (4) A. M. Vaira et al. Pages 673–679 in: Virus Taxonomy: 8th Report of the ICTV, 2005.

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Albumin Gene Expression During Mouse Odontogenesis

Advances in Dental Research ◽

10.1177/08959374960100020301 ◽

1996 ◽

Vol 10 (2) ◽

pp. 119-125 ◽

Cited By ~ 7

Author(s):

Z.A. Yuan ◽

K.S. Mcandrew ◽

P.M. Collier ◽

E. Koyama ◽

E. Chen ◽

...

Keyword(s):

Gene Expression ◽

In Situ Hybridization ◽

Primary Source ◽

Periodontal Tissue ◽

Rt Pcr ◽

Albumin Gene ◽

Pcr Products ◽

Old Mice ◽

Albumin Mrna

Albumin protein is present in developing teeth of several species. Oligomer primers and cRNA probes specific for albumin were designed to perform RT-PCR, and for in situ hybridization, respectively. In situ hybridization failed to reveal albumin expression in any tooth cells, however, albumin PCR products were amplified from tissues adhering to the roots of developing teeth from four-week-old mice. It is concluded that this source is not the primary source of albumin protein found in developing enamel, because of the location and level of expression of albumin mRNA in periodontal tissue.

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Differential Expression of Thaumatin-Like Proteins in Sorghum Infested with Greenbugs

Zeitschrift für Naturforschung C ◽

10.1515/znc-2010-3-416 ◽

2010 ◽

Vol 65 (3-4) ◽

pp. 271-276 ◽

Cited By ~ 4

Author(s):

Juan Chou ◽

Yinghua Huang

Keyword(s):

Gene Expression ◽

Real Time Pcr ◽

Time Dependent ◽

Transcriptional Level ◽

Plant Defense Response ◽

Actin Gene ◽

Agarose Gel ◽

Rt Pcr ◽

Susceptible Line ◽

Pcr Products

This study was designed to quantitatively analyze the expression of thaumatin-like protein (TLP) at the transcriptional level in different sorghum lines when they were infested with greenbugs. Three sorghum lines, Tx7000, PI550607, and PI550610, were used. RNAs were isolated from the different sorghum lines that were infested with greenbugs at different infestation times. The resultant mRNA was reverse transcribed into cDNA, and the RT-PCR products were separated by agarose gel. Then, real-time PCR data of the TLP gene expression were analyzed in comparison with the β-actin gene as a reference. The expression levels of the TLP gene were also compared between samples. The results showed that the transcripts of the TLP were induced by greenbug feeding and the increased levels were time-dependent. In the susceptible line, the TLP’s transcripts increased several thousand-fold at 120 hours post infestation, while for the two resistant sorghum lines the TLP expression level increased less than one hundred-fold compared to the controls. This is the first demonstration that thaumatin-like proteins are involved in plant defense response against insects.

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