Differential Display Reverse Transcription Polymerase Chain Reaction Research Articles

Differential Regulation of Defense-Related Gene Expression in Response to Red Rot Pathogen Colletotrichum falcatum Infection in Sugarcane

Red rot is a serious disease of sugarcane caused by the fungus Colletotrichum falcatum imposing a considerable economic loss annually in all sugarcane-producing countries. In this study, we analyzed the early resistance response of sugarcane to red rot fungus by comparing the differences between control and inoculated stalk tissues. Differential display reverse transcription polymerase chain reaction (DD-RT-PCR) was employed to identify altered expression of genes in disease-resistant cv Co 93009, in response to pathogen infection. DD-RT-PCR identified 300 differentially expressed transcripts of which 112 were selected for further analysis. Cloning and sequence analysis of the isolated cDNA fragments resulted in functional categorization of these clones into five categories, of which the defense/stress/signaling group was the largest, with clones homologous to genes known to be actively involved in various pathogenesis-related functions in plant species. This group showed overexpression of several transcripts related to ethylene-mediated and jasmonic acid pathway of plant defense mechanisms. Of the 112 expressed sequence tags, validation of expression was carried out for five important genes whose role in plant defense mechanisms is well established. This is the first report of Colletotrichum-mediated gene regulation in sugarcane which has provided a set of candidate genes for detailed molecular dissection of signaling and defense responses in tropical sugarcane during the onset of red rot resistance.

Exequibilidade do DDRT-PCR para análise da expressão gênica diferencial em células de medula óssea murina

Modelo do estudo: Estudo Experimental. Introdução: Atualmente a pesquisa com células-tronco temgerado grande interesse devido a sua aplicabilidade no campo na medicina regenerativa. A medulaóssea é considerada a maior fonte de células-tronco adultas e o estabelecimento de novos métodospara a análise da expressão gênica torna-se estritamente necessário. Desse modo, o "DifferentialDisplay Reverse Transcription Polymerase Chain Reaction (DDRT-PCR)", pode ser uma ferramentaacessível para a investigação de pequenas diferenças no nível de expressão gênica em diferentes tiposcelulares, sob distintas condições.Objetivo: Neste presente trabalho nós investigamos a exequibilidade do DDRT-PCR na identificação dediferenças no nível de expressão gênica global em células da medula óssea de camundongos sobduas condições. Métodos: Primeiramente, a medula óssea foi isolada frescamente e uma secundaparte foi cultivada por uma semana sem troca de meio. Posteriormente, as células da medula (fresca ecultivada) foram submetidas a análise da expressão gênica, seguindo a metodologia de DDRT-PCR.Resultados: Inicialmente, foi possível identificar em células da medula óssea com uma semana decultivo, pequenas alterações morfológicas (células ovais para fibroblastóides) e no perfil de proteínas,por meio da visualização de bandas em SDS-Page gel. Finalmente, a análise da expressão gênica porDDRT-PCR, mostrou uma expressão diferencial com a presença de três bandas (1300, 1000 and 225pb) exclusivamente expressas na medula óssea fresca e mais duas bandas (400 and 300 pb) presentes somente nas células de medula cultivadas.Conclusões: Em suma, a metodologia de DDRT-PCR mostrou-se eficiente para a identificação depequenas diferenças no nível de expressão gênica em células da medula óssea sob duas definidascondições. Portanto, nós acreditamos que o DDRT-PCR possa ser designado de forma rápida e eficiente para a análise diferencial de expressão gênica em diferentes tipos de células-tronco, sob diferentescondições.

English

The gene differential expression of Phytophthora sojae during early stage (0 to 7 h) of sporangium formation was studied by differential display reverse transcription polymerase chain reaction (DDRT-PCR) in the present study. Thirty out of 78 primer combinations were selected and 90 stable differential bands were obtained. Thirty-nine positive differential fragments were found in the experiments by using the reverse northern blot, in which 36 sequences of differential fragments were obtained after sequencing. Homology search and analysis were tested by BLASTX in the P. sojae genome database. The results showed that 23 fragments had significant similarities with proteins in P. sojaegenome database, 15 of which matched with the known functional proteins involved in metabolism, cellular processes, and signal transduction processes. Data mining and bioinformatics analysis on 4 fragments which redundancy was greater than 5 showed that sequence AR15-258 had a very high expression level in Phytophthora mycelium, which may be related to mycelium infection or hunger; AR10-263 and GR1-304 were only found in Phytophthora, which might be unique expression in this genus. The analysis and research on important regulatory genes controlling sporangium formation of P. sojae was a foundation for studying of key genes controlling growth and development of P. sojae. Key words: Phytophthora sojae, sporangium formation, mRNA differential display, differential fragments.

Mt-COX1, mt-ND1 and CREBP are indicators of intramuscular fat content in Hanwoo (Korean cattle)

We identified differentially expressed genes associated with intramuscular fat content in Hanwoo Korean cattle. Pre-screening with differential display reverse transcription polymerase chain reaction (DDRT-PCR) identified 15 differentially expressed genes (DEGs) in the muscle of 12- and 27-month-old steers. Eleven genes were validated by qRT-PCR. These 11 DEGs were mainly involved in energy metabolism or were muscle structural or ribosomal proteins. To determine whether these DEGs are associated with adipocyte development, we examined the gene expressions of the 15 genes during preadipocyte differentiation. Two genes associated with energy metabolism, mitochondrial cytochrome c oxidase 1 (mt-COX1) and NADH dehydrogenase I (mt-ND1), and one gene, cAMP response element binding protein (CREBP), showed increased gene expression during adipocyte differentiation. Finally, to determine whether the three genes are associated with intramuscular fat content, we analyzed gene expression in muscles with divergent marbling phenotypes (7.5% vs. 23.5%). The three genes (mt-COX1, mt-ND1 and CREBP) were significantly expressed in highly marbled muscle. Thus, mt-COX1, mt-ND1 and CREBP could be relevant indicators of intramuscular fat content in Hanwoo.

Screening for differentially expressed genes in Anoectochilus roxburghii (Orchidaceae) during symbiosis with the mycorrhizal fungus Epulorhiza sp.

Mycorrhizal fungi promote the growth and development of plants, including medicinal plants. The mechanisms by which this growth promotion occurs are of theoretical interest and practical importance to agriculture. Here, an endophytic fungus (AR-18) was isolated from roots of the orchid Anoectochilus roxburghii growing in the wild, and identified as Epulorhiza sp. Tissue-cultured seedlings of A. roxburghii were inoculated with AR-18 and co-cultured for 60 d. Endotrophic mycorrhiza formed and the growth of A. roxburghii was markedly promoted by the fungus. To identify genes in A. roxburghii that were differentially expressed during the symbiosis with AR-18, we used the differential display reverse transcription polymerase chain reaction (DDRT-PCR) method to compare the transcriptomes between seedlings inoculated with the fungus and control seedlings. We amplified 52 DDRT-PCR bands using 15 primer combinations of three anchor primers and five arbitrary primers, and nine bands were re-amplified by double primers. Reverse Northern blot analyses were used to further screen the bands. Five clones were up-regulated in the symbiotic interaction, including genes encoding a uracil phosphoribosyltransferase (UPRTs; EC 2.4.2.9) and a hypothetical protein. One gene encoding an amino acid transmembrane transporter was down-regulated, and one gene encoding a tRNA-Lys (trnK) and a maturase K (matK) pseudogene were expressed only in the inoculated seedlings. The possible roles of the above genes, especially the UPRTs and matK genes, are discussed in relation to the fungal interaction. This study is the first of its type in A. roxburghii.

Characterization of Edwardsiella tarda rpoN: roles in σ70 family regulation, growth, stress adaption and virulence toward fish

Edwardsiella tarda EIB202, a Gram-negative pathogen with strong virulence, is an opportunistic pathogen capable of causing edwardsiellosis with high mortality to fish. Alternative sigma factor 54 (RpoN) is an important regulator of virulence and stress resistance genes in many bacterial species and mainly responsible for transcription of genes in nitrogen utilization. In this study, the in-frame rpoN deletion mutant was constructed to analyze the function of RpoN in Edwardsiella tarda firstly. Compared to the wild-type and complemented strain rpoN (+), the ΔrpoN was impaired in terms of the ability to survive under oxidative stress, osmotic stress and acid resistance, as well as the growth in Luria-Bertani medium, demonstrating essential roles of RpoN in stress resistance and nitrogen utilization. In addition, the ΔrpoN displayed markedly decreased biofilm formation and chondroitinase activity and was attenuated in virulence reflected in the increased median lethal dose value and extended infection cycle. Real-time polymerase chain reaction revealed that the expression levels of σ(70) class changed in varying degrees in the rpoN mutant. Especially, the expression levels of rpoS and fliA were down-regulated 4.1-fold and 7.9-fold in stationary phase in comparison with the wild type, respectively. Furthermore, two differential expression genes, znuA and flhC, were detected in the wild type and ΔrpoN using the method of differential display reverse transcription PCR.

Identification of Wood Decay Related Genes from Piptoporus Betulinus (Bull. Fr.) Karsten Using Differential Display Reverse Transcription PCR (DDRT-PCR)

ABSTRACTPiptoporus betulinus (Bull. Fr.) Karsten causes brown rot on the wood of weakened birch species. Little work has been published on the molecular mechanism of its wood decaying. In this study differential display reverse transcription PCR (DDRT-PCR) was used to isolate wood decay genes from P. betulinus, which were induced in the wood samples of white birch. We found that 29 differentially expressed cDNA bands (∼4.7%) were especially specific to cDNA samples from the mycelia of P. betulinus inoculated wood samples. Additionly, 12 transcripts from P. betulinus were induced during wood decay. These 12 clones were isolated, cloned and sequenced. Seven of them were highly homologous to known genes including pre-mRNA splicing factor syf2, arabinose 5-phosphate isomerase, alpha/beta hydrolase, lytic transglycosylase, polysaccharide deacetylase, inositol monophosphatase and glucoamylase-like protein. These results suggested that the process of decaying birch wood by P. betulinus was intricate, involving enzyme or protein changes.

Study on differential display gene expression of Eimeria tenella multiple resistance strain isolated from Tangshan in chicken

Total RNA of Eimeria tenella drug-resistant strain from Tangshan was extracted with Trizol. Differential-display reverse transcription- polymerase chain reaction (DDRT-PCR) was established by 3 anchored primers and 20 arbitrary primers. The products ofpolymerase chain reaction (PCR) were analyzed on the denaturing polyacrylamide gels by silver-straining. Ten differential bands were excised from the gels and reampilfied with the same sets of primers. The products were purified and ligated with PMDTM18-T Vector, and then the dot-blot hybridization, sequence analysis and homology comparison. The results showed that through comparison of the nucleotide acid sequence, the similarity was 99% among the sequence S116 from mRNA of Tangshan multiple-resistant strain with the sequence 882 bp lengths in the first chromosome of E.tenella in Genebank and Sanger, which was an unknown protein. This study paved the way for cloning the full-length cDNAs (Complementary Deoxyribonucleic acid) and finding the molecular mechanism about the drug-resistance of E.tenella. Key words: Chicken, E. tenella, mRNA differential display PCR, denaturing polyacrylamide gel, gene cloning.

Differential Gene and Protein Expression in Soybean at Early Stages of Incompatible Interaction with Phytophthora sojae

Soybean root and stem rot caused by Phytophthora sojae is a destructive disease worldwide. Using genetic resistance is an important and major component in the integrated pest management of this disease. To understand molecular mechanisms of root and stem rot resistance in soybeans, the gene and protein expression in hypocotyls and stems of variety Suinong 10 carrying resistance genes Rps1a and Rps2 was investigated by using mRNA differential display reverse transcription PCR and two-dimensional electrophoresis at 0, 0.5, 1, 2, and 4 h after inoculation with P. sojae race 1. The results of the comparison of gene and protein expression showed that at least eight differential fragments at the transcriptional level were related to metabolic pathway, phytoalexin, and signal transduction in defense responses. Sequence analyses indicated that these fragments represented cinnamic acid 4-hydroxylase gene, ATP β gene coding ATP synthase β subunit and ubiquitin-conjugating enzyme gene which upregulated at 0.5 h post inoculation, blue copper protein gene and UDP-N-acetyl-α-D-galactosamine gene which upregulated at 2 h post inoculation, TGA-type basic leucine zipper protein TGA1.1 gene, cyclophilin gene, and 14-3-3 protein gene which upregulated at 4 h post inoculation. Three resistance-related proteins, α-subunit and β-subunit of ATP synthase, and cytochrome P450-like protein, were upregulated at 2 h post inoculation. The results suggested that resistance-related multiple proteins and genes were expressed in the recognition between soybean and P. sojae during zoospore germination, penetration and mycelium growth of P. sojae in soybean.

Low-level expression of cholesterol 7α-hydroxylase is associated with the formation of goose fatty liver

Cholesterol 7α-hydroxylase (Cyp7a1) plays a crucial role in the synthesis of cholic acid and cholesterol catabolism. In this study, Cyp7a1 expression in goose liver was identified for the first time using differential display reverse transcription PCR. This study used real-time PCR and quantified the transcript levels of the cyp7a1 gene under different feeding conditions. In overfeeding, cyp7a1 expression by the liver was lower than that seen for normal feeding and the same result was observed for the Cyp7a1 protein level. The cholesterol level in serum and liver was higher in the overfed group. This study suggests that low-level expression of Cyp7a1 is associated with the formation of goose fatty liver.

Identification and isolation of gene differentially expressed on scrotal circumference in crossbreed bulls

Differential display reverse transcription polymerase chain reaction (DDRT-PCR), reverse northern blot analysis and real-time polymerase chain reaction (RT-PCR) were used to identify tissue-specific expression genes in scrotum of differential scrotal circumference bulls and the function of these specific genes on the development of the bull’s scrotum was analyzed in this study. The results indicate that 19 positive fragments were screened out by DDRT-PCR and reverse northern blot analysis. Results of BLAST with GenBank show that three genes or expressed sequence tag (ESTs) were unknown, and there were eight sequences highly identified to be Bos taurus mRNA for proline-rich protein P-B and other sequences were B. taurus ebd-P2 pseudogene, B. taurus similar to F-box only protein 21 isoform 2, B. taurus similar to Kinesin heavy chain isoform 5C, B. taurus similar to ankyrin repeat domain protein 15 isoform and B. taurus similar to galactosidase, beta 1-like. The result of real-time PCR analysis testified more that B. taurus was similar to galactosidase, beta 1-like, kinesin heavy chain isoform 5C and ankyrin repeat domain protein 15 isoform. B. taurus ebd-P2 pseudogene was highly expressed in bulls which had bigger scrotal circumference. They may be involved with sperm maturation in the epididymis, sperm protection and prevention of the ascent of microorganisms into the adjacent testes and also responsible for converting immature sperm into competent functional cells, as well as movement of spermatozoa. Key words: Bulls, scrotal circumference, differential display reverse transcription polymerase chain reaction (DDRT-PCR), real time polymerase chain reaction (RT-PCR).

Granzyme Gis expressed in the two-cell stage mouse embryo and is required for the maternal-zygotic transition

BackgroundDetailed knowledge of the molecular and cellular mechanisms that direct spatial and temporal gene expression in pre-implantation embryos is critical for understanding the control of the maternal-zygotic transition and cell differentiation in early embryonic development. In this study, twenty-three clones, expressed at different stages of early mouse development, were identified using differential display reverse transcription polymerase chain reaction (DDRT-PCR). One of these clones, which is expressed in 2-cell stage embryos at 48 hr post-hCG injection, shows a perfect sequence homology to the gene encoding the granzyme G protein. The granzyme family members are serine proteases that are present in the secretory granules of cytolytic T lymphocytes. However, the pattern of granzyme G expression and its function in early mouse embryos are entirely unknown.ResultsUpon the introduction of an antisense morpholino (2 mM) against granzyme G to knock-down endogenous gene function, all embryos were arrested at the 2- to 4-cell stages of egg cleavage, and the de novo synthesis of zygotic RNAs was decreased. The embryonic survival rate was dramatically decreased at the late 2-cell stage when serine protease-specific inhibitors, 0.1 mM 3,4-dichloroisocoumarin (3,4-DCI), and 2 mM phenyl methanesulphonyl fluoride (PMSF), were added to the in vitro embryonic culture medium. Survival was not affected by the addition of 0.5 mM EDTA, a metalloproteinase inhibitor.ConclusionWe characterized for the first time the expression and function of granzyme G during early stage embryogenesis. Our data suggest that granzyme G is an important factor in early mouse embryonic development and may play a novel role in the elimination of maternal proteins and the triggering of zygotic gene expression during the maternal-zygotic transition.

Cell homeostasis in a Leishmania major mutant overexpressing the spliced leader RNA is maintained by an increased proteolytic activity

Although several stage-specific genes have been identified in Leishmania, the molecular mechanisms governing developmental gene regulation in this organism are still not well understood. We have previously reported an attenuation of virulence in Leishmania major and L. braziliensis carrying extra-copies of the spliced leader RNA gene. Here, we surveyed the major differences in proteome and transcript expression profiles between the spliced leader RNA overexpressor and control lines using two-dimensional gel electrophoresis and differential display reverse transcription PCR, respectively. Thirty-nine genes related to stress response, cytoskeleton, proteolysis, cell cycle control and proliferation, energy generation, gene transcription, RNA processing and post-transcriptional regulation have abnormal patterns of expression in the spliced leader RNA overexpressor line. The evaluation of proteolytic pathways in the mutant revealed a selective increase of cysteine protease activity and an exacerbated ubiquitin-labeled protein population. Polysome profile analysis and measurement of cellular protein aggregates showed that protein translation in the spliced leader RNA overexpressor line is increased when compared to the control line. We found that L. major promastigotes maintain homeostasis in culture when challenged with a metabolic imbalance generated by spliced leader RNA surplus through modulation of intracellular proteolysis. However, this might interfere with a fine-tuned gene expression control necessary for the amastigote multiplication in the mammalian host.

Differential expression of genes in co-cultured versus separately cultured fibroblasts and epithelial cells from human benign hyperplastic prostate tissues

The prostate is composed mainly of epithelial and stromal cells, whose dynamic interaction is vital to a broad array of cellular processes, including proliferation, differentiation, growth, and apoptosis. To understand intercellular communication in the development and progression of prostatic diseases, we examined gene expression in tissues from five patients diagnosed with benign prostatic hyperplasia (BPH). Fibroblasts and epithelial cells derived from these tissues were grown in a primary co-culture system that retains many characteristics of the intact human prostate. The mRNA levels of expressed genes as assessed by differential-display reverse transcription-PCR revealed that 110 genes were differentially expressed in co-cultured fibroblasts and epithelial cells, compared with expression in separately cultured cells. Eighty-four of these were confirmed by reverse Northern blotting, and 68 were successfully sequenced. Of the sequenced genes, 43 were differentially expressed in epithelial cells (37 upregulated, 6 downregulated), and 25 were differentially expressed in fibroblasts (6 upregulated, 19 downregulated) in co-cultures versus separate cultures. Semi-quantitative RT-PCR analysis of 12 genes with known functions showed that five of these were differentially expressed in co-cultured cells. Human kallikrein gene 7 (KLK7) was markedly upregulated in co-cultured compared with separately cultured epithelial cells (P<0.001), whereas S100 calcium binding protein A11, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, cyclin I, and latexin were significantly downregulated in co-cultured fibroblasts (P<0.05). Quantitative real-time RT-PCR and Western blot analysis confirmed KLK7 up-regulation at both the mRNA and protein levels, respectively. Thus, epithelial-stromal cell interaction and communication are likely to be important in BPH. Epithelial cells and fibroblasts may interplay coordinately or collaboratively to influence cellular growth and death through dynamically differential gene expression in response to physiological and pathophysiological changes.

Molecular Cloning of Cecropin B Responsive Endonucleases in Yersinia ruckeri

We have previously demonstrated that Yersinia ruckeri resists cecropin B in an inducible manner. In this study, we sought to identify the molecular changes responsible for the inducible cecropin B resistance of Y. ruckeri. Differences in gene expression associated with the inducible resistance were investigated. Cultures of Y. ruckeri were exposed to a sublethal concentration of cecropin B and resultant changes in the messenger RNA population of the bacteria were assayed using the differential display reverse transcription polymerase chain reaction (DD-RT-PCR). A single band was consistently increased in intensity in all repeats of the experiment. The band was excised, cloned, sequenced, and used to screen a Y. ruckeri genomic DNA library. The DD-RT-PCR fragment shared 100% identity to the cDNA sequence of an ATP-dependent endonuclease of the overcome lysogenization defect (OLD) family of Y. ruckeri 29473. The genomic clone that was recovered was not identical to the DD-RT-PCR clone, but harbored a gene for a secreted endonuclease 1 (nucM) homologue. It was determined that transcription of the gene was upregulated following exposure to cecropin B via RT-PCR. Furthermore, an increase in the nuclease activity of culture supernatants of Y. ruckeri following exposure to cecropin B was demonstrated. These findings demonstrate that cecropin B exposure increases the expression of at least two endonucleases in Y. ruckeri. The production and secretion of an endonuclease by Y. ruckeri in response to an antimicrobial peptide indicates the involvement of both intracellular and extracellular DNA in the toxic effects of cecropin B.

Identification of differentially expressed genes in the developmental stages from olive flounder Paralichthys olivaceus using an annealing control primer system

We employed a new and improved differential display reverse transcription–polymerase chain reaction (DDRT-PCR) method, which involves annealing control primers (ACPs), to identify the genes that are specifically or prominently expressed in olive flounder (Paralichthys olivaceus) juveniles (35 days post-hatch; dph) compared to larval-stage (dph 21) flounder. Using 60 ACPs, we identified eight differentially expressed genes (DEGs) and basic local alignment search tool (BLAST) searches revealed eight known genes. Gene expression levels were confirmed by RT-PCR. Phosphoglucose isomerase (PGI) was highly expressed at 21 dph, while nephrosin, myosin light chain (MLC), myosin heavy chain (MHC), carboxypeptidase A, chymotrypsin B, fish-egg protein, and matrix protein were expressed at 35 dph. PGI, MLC, and MHC expression was further analyzed by RT-PCR. The differentially expressed genes identified in this study may provide insights into the molecular basis of development in olive flounder.

Identification of Candidate Genes Potentially Relevant to Chamber-Specific Remodeling in Postnatal Ventricular Myocardium

Molecular predisposition of postnatal ventricular myocardium to chamber-dependent (concentric or eccentric) remodeling remains largely elusive. To this end, we compared gene expression in the left (LV) versus right ventricle (RV) in newborn piglets, using a differential display reverse transcription-PCR (DDRT-PCR) technique. Out of more than 5600 DDRT-PCR bands, a total of 153 bands were identified as being differentially displayed. Of these, 96 bands were enriched in the LV, whereas the remaining 57 bands were predominant in the RV. The transcripts, displaying over twofold LV-RV expression differences, were sequenced and identified by BLAST comparison to known mRNA sequences. Among the genes, whose expression was not previously recognized as being chamber-dependent, we identified a small cohort of key regulators of muscle cell growth/proliferation (MAP3K7IP2, MSTN, PHB2, APOBEC3F) and gene expression (PTPLAD1, JMJD1C, CEP290), which may be relevant to the chamber-dependent predisposition of ventricular myocardium to respond differentially to pressure (LV) and volume (RV) overloads after birth. In addition, our data demonstrate chamber-dependent alterations in expression of as yet uncharacterized novel genes, which may also be suitable candidates for association studies in animal models of LV/RV hypertrophy.

Initial Analysis of Gene Expressions in Response to Dengue-2 Virus Infection in Aedes albopictus (Diptera: Culicidae)

In order to understand host responses to viral infection at the transcriptional level, differential display reverse-transcription PCR (DDRT-PCR) was used to identify molecular genetic changes in virus-infected Aedes albopictus (Diptera: Culicidae) following infection with dengue 2 virus (DENV-2). Seven differentially expressed cDNA fragments were identified. Five were up-regulated in virus-infected mosquitoes and two were up-regulated in refractory individuals. Sequence comparisons at the nucleotide level in GenBank showed that clone AS09A had 100% homology to Ae. albopictus ribosomal S5 protein mRNA, 98% homology to Ae. aegypti clone AE-198A ribosomal protein S5 mRNA and the 5-prime end of clone KWOAAA2YJ20AAMI from strain Liverpool of Ae. aegypti, and 91% homology to Drosophila melanogaster ribosomal protein S5a mRNA sequence. The deduced amino acidic sequence of clone AS09A showed 100% identity or conservation to the above sequence. AS12C clone showed significant homology (95%) to Ae. aegypti strain Liverpool. Other clones did not show homology with previously reported gene sequences. The fragment of AS09A may be the ribosomal protein S5 gene of Ae. albopictus that becomes differentially expressed as a result of infection. These results may be helpful in understanding the mechanism of differential susceptibility of Ae. albopictus following dengue virus infection.

Increased ATP generation in the host cell is required for efficient vaccinia virus production

To search for cellular genes up-regulated by vaccinia virus (VV) infection, differential display-reverse transcription-polymerase chain reaction (ddRT-PCR) assays were used to examine the expression of mRNAs from mock-infected and VV-infected HeLa cells. Two mitochondrial genes for proteins that are part of the electron transport chain that generates ATP, ND4 and CO II, were up-regulated after VV infection. Up-regulation of ND4 level by VV infection was confirmed by Western blotting analysis. Up-regulation of ND4 was reduced by the MAPK inhibitor, apigenin, which has been demonstrated elsewhere to inhibit VV replication. The induction of ND4 expression occurred after viral DNA replication since ara C, an inhibitor of poxviral DNA replication, could block this induction. ATP production was increased in the host cells after VV infection. Moreover, 4.5 μM oligomycin, an inhibitor of ATP production, reduced the ATP level 13 hr after virus infection to that of mock-infected cells and inhibited viral protein expression and virus production, suggesting that increased ATP production is required for efficient VV production. Our results further suggest that induction of ND4 expression is through a Bcl-2 independent pathway.

Effect of Hormones on Tumor Formation in Tobacco Hybrids

Genetic tumors can develop on certain tobacco hybrid plant spontaneously. In this study, exogenous hormones were applied on the tumorous tobacco hybrid seedlings (N. glauca × N. langsdorffii), and the data showed that N6-benzyladenine (BA) enhanced the tumor formation under both tissue culture and field conditions, and NAA and ABA inhibited tumor formation under tissue culture condition, whereas ABA accelerated the tumor formation under field condition. Furthermore, Northern analysis indicated that tumor tissue exhibited high level of CycD expression compared with normal tissues, implying abnormal cell cycle of tumor cells. The differential display reverse transcription PCR (DDRT PCR) results indicated some genes may be involved in tumor formation.