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With rising demand for effective and sustainable measures in wound healing researchers are compelled to explore diverse methods that combine folk medicine with existing wound care to double the impact. Hemigraphis colorata (Blume) H G Hallier, an exotic plant in the Acanthaceae family, is a perennial herb that has potential therapeutic benefits. In the present investigation, a comparative study was conducted between the effect of tender and mature leaf extracts of H. colorata as well as the differentially expressed genes of both leaves using four upstream primers and one downstream (HT11C) primer. The rate of cell migration was studied by creating a scratch wound in HaCaT monolayer culture. The finding indicated that the application of tender leaf extract resulted in a higher rate of wound closure, whereas the mature leaf extract led to a two-fold increase in the secretion of TGF-β protein, as measured by ELISA. This demonstrates the therapeutic effectiveness of tender leaf extract in promoting wound healing compared to mature leaf extract. Additionally, the results of DDRT-PCR unveiled a diverse range of differentially expressed genes, which were registered as molecular markers (ESTs) and did not show homology with any known gene sequences in the NCBI database. [ABSTRACT FROM AUTHOR]

Viral isolation in cell cultures has been regarded for decades as the “gold standard” for the laboratory diagnosis of influenza viral infections. Not all viral strains could be isolated from clinical samples. This study aimed to quantify the viral load in the samples before isolation to save working time and improve working efficiency. Four hundred samples from patients with influenza-like cases were confirmed pdmH1N1 positive (200 cases) and B Victoria (BV) positive (200 cases) by whole-genome sequencing and analyzed by ddPCR for viral load in samples before isolation, and isolation results were verified by hemagglutination (HA) assay and hemagglutination-inhibition (HI) tests. Probit regression analysis was used to calculate the isolation viral load limit with a 95% probability level by SPSS 19.0 software. The results showed that the isolation limit of viral load was 4.9 × 104 (95% CI: 2.5 × 104–9.0 × 104) copies/mL for pdmH1N1 and 1.9 × 104 (95% CI: 7.8 × 103–3.6 × 104) copies/mL for BV. The isolation rate of clinical samples is positively correlated with the viral load in clinical samples, which can be used for viral culture, providing important guidance for daily work.

Abstract Background In rice, the cortex and outer tissues play a key role in submergence tolerance. The cortex differentiates into aerenchyma, which are air-containing cavities that allow the flow of oxygen from shoots to roots, whereas exodermis suberification and sclerenchyma lignification limit oxygen loss from the mature parts of roots by forming a barrier to root oxygen loss (ROL). The genes and their networks involved in the cellular identity and differentiation of these tissues remain poorly understood. Identification and characterization of key regulators of aerenchyma and ROL barrier formation require determination of the specific expression profiles of these tissues. Results We optimized an approach combining laser microdissection (LM) and droplet digital RT-PCR (ddRT-PCR) for high-throughput identification of tissue-specific expression profiles. The developed protocol enables rapid (within 3 days) extraction of high-quality RNA from root tissues with a low contamination rate. We also demonstrated the possibility of extracting RNAs from paraffin blocks stored at 4 °C without any loss of quality. We included a detailed troubleshooting guide that should allow future users to adapt the proposed protocol to other tissues and/or species. We demonstrated that our protocol, which combines LM with ddRT-PCR, can be used as a complementary tool to in situ hybridization for tissue-specific characterization of gene expression even with a low RNA concentration input. We illustrated the efficiency of the proposed approach by validating three of four potential tissue-specific candidate genes detailed in the RiceXpro database. Conclusion The detailed protocol and the critical steps required to optimize its use for other species will democratize tissue-specific transcriptome approaches combining LM with ddRT-PCR for analyses of plants.

Background: In rice, the cortex and outer tissues play a key role in submergence tolerance. The cortex differentiates into aerenchyma, which are air-containing cavities that allow the flow of oxygen from shoots to roots, whereas exodermis suberification and sclerenchyma lignification limit oxygen loss from the mature parts of roots by forming a barrier to root oxygen loss (ROL). The genes and their networks involved in the cellular identity and differentiation of these tissues remain poorly understood. Identification and characterization of key regulators of aerenchyma and ROL barrier formation require determination of the specific expression profiles of these tissues. Results: We optimized an approach combining laser microdissection (LM) and droplet digital RT-PCR (ddRT-PCR) for high-throughput identification of tissue-specific expression profiles. The developed protocol enables rapid (within 3 days) extraction of high-quality RNA from root tissues with a low contamination rate. We also demonstrated the possibility of extracting RNAs from paraffin blocks stored at 4 °C without any loss of quality. We included a detailed troubleshooting guide that should allow future users to adapt the proposed protocol to other tissues and/or species. We demonstrated that our protocol, which combines LM with ddRT-PCR, can be used as a complementary tool to in situ hybridization for tissue-specific characterization of gene expression even with a low RNA concentration input. We illustrated the efficiency of the proposed approach by validating three of four potential tissue-specific candidate genes detailed in the RiceXpro database. Conclusion: The detailed protocol and the critical steps required to optimize its use for other species will democratize tissue-specific transcriptome approaches combining LM with ddRT-PCR for analyses of plants. [ABSTRACT FROM AUTHOR]

Cadmium (Cd) is one of the hazardous environmental pollutants, and it can be harmful to human health through consumption of food-plants capable of bioaccumulating Cd. Therefore, lowering cadmium accumulation in plants is highly desirable. Here, a rice cultivar 'Qisanzhan' was studied using differential display reverse transcription-polymerase chain reaction (DDRT-PCR). Fifty-six differentially expressed genes were found in the root tips of 4-leaf stage rice seedlings exposed to 4 and 12 h of 50 µmol/L Cd(NO3)2 in a nutrient solution using DDRT-PCR. Further validation using semi-quantitative RT-PCR showed that the expression patterns of 16 genes were consistent with those found in DDRT-PCR. These genes encode receptor-like protein kinase, pleiotropic drug resistance protein, aquaporin protein, plasma membrane ATPase, etc. The differentially genes identified here can be used to obtain a better understanding of the molecular mechanisms of Cd absorption and accumulation in plants. [ABSTRACT FROM AUTHOR]

Differential gene expression profile was studied in Dimocarpus longan Lour. in response to treatments of simulated acid rain with pH 2.5, 3.5, and a control (pH 5.6) using differential display reverse transcription polymerase chain reaction (DDRT-PCR). Results showed that mRNA differential display conditions were optimized to find an expressed sequence tag (EST) related with acid rain stress. The potential encoding products had 80% similarity with a transcription initiation factor IIF of Gossypium raimondii and 81% similarity with a protein product of Theobroma cacao. This fragment is the transcription factor activated by second messenger substances in longan leaves after signal perception of acid rain. [ABSTRACT FROM AUTHOR]

Even though the major benefit of arbuscular mycorrhizae is the increased uptake of phosphate from the soil solution and translocation to the plant, changes in the activity of enzymes involved in nitrogen (N) metabolism have been detected in mycorrhizal roots. Using differential display of reverse-transcripts of tobacco roots not-inoculated or inoculated with Glomus intraradices (Gi), we have cloned two partial cDNAs (NtGi2 and NtGi3). The presence of a conserved CobW/HypB/UreG domain and phylogenetic analyses suggest that NtGi2 and NtGi3 encode isoforms of urease accessory protein G (ureG) highly similar to ureG from fungi. The steady state levels of the putative ureG transcripts were shown to be higher in roots colonized by Gi, as compared to non-mycorrhizal controls. Urease activities were also determined in tobacco roots inoculated with Glomus clarum (Gc) or Gi and grown in substrate containing 50, 100 or 150 mg N kg-1 in the form of ammonium sulfate (N-AMS) or urea (N-URE). Urease activities were shown to be induced in mycorrhizal roots fertilized with 100 mg N-AMS kg-1. In Gc-colonized roots fertilized with N-URE, induction of urease activities was observed at the lowest N concentration. In contrast, at the highest N-URE concentration, suppression of urease activities was observed in Gc and Gi-colonized roots, as compared to non-mycorrhizal controls. Urease activities in roots were modulated by soil N availability and source, and arbuscular mycorrhizal fungal inoculation.Muito embora o maior benefício de micorrizas arbusculares seja o incremento na absorção de fosfato da solução do solo e translocação para a planta, alterações nas atividades de enzimas envolvidas no metabolismo de nitrogênio (N) têm sido detectadas em raízes micorrizadas. Usando "differential display of reverse-transcripts" de raízes de tabaco não-inoculadas ou inoculadas com Glomus intraradices (Gi), dois cDNAs parciais (NtGi2 e NtGi3) foram clonados. A presença de um domínio CobW/HypB/UreG conservado e a análise filogenética sugerem que NtGi2 e NtGi3 codificam isoformas de proteínas acessórias da urease G (ureG) altamente similares a ureG de fungos. Os níveis de transcritos dos genes ureG putativos foram mais elevados em raízes colonizadas por Gi, em relação ao controle não-micorrizado. As atividades de urease foram determinadas em raízes de tabaco inoculadas com Glomus clarum (Gc) ou Gi e cultivadas em substrato contendo 50, 100 ou 150 mg N kg-¹, na forma de sulfato de amônio (N-AMS) ou uréia (N-URE). As atividades de ureases foram induzidas em raízes micorrizadas cultivadas com 100 mg N-AMS kg-¹. Em raízes colonizadas por Gc e cultivadas com N-URE, a indução das atividades de urease foi observada na concentração mais baixa de N. Em contraste, na concentração mais elevada de N-URE, supressão das atividades de urease em raízes colonizadas por Gc e Gi, em relação aos controles não-micorrizados, foi observada. As atividades de urease nas raízes foram moduladas pela disponibilidade e fonte de N no solo, e pela inoculação com fungos micorrízicos arbusculares.

Introduction: The pathogenic bacterium Salmonella enterica serovar Typhimurium elicits a variety of genetic programs to adapt to stress conditions encountered within hostile environments such as host phagocytes and preserved food. Methodology: In this work, differential display (DD) methodology was used to investigate the effect of one month starvation in a salty microcosm (0.5 M NaCl) on transcript profiling in a Salmonella Typhimurium LT2 strain. cDNA fragments resulting from differentially expressed mRNA were eluted from the gel, re-amplified, cloned, and then sequenced. Results: A total of 21 differentially expressed bands were detected by DD reverse transcription-polymerase chain reaction (RT-PCR). However, only 12 of them were successfully identified as upregulated genes in stressed cells. Based on the sequencing data and BLAST analysis, these genes were sopA, ssaD, yhhK, gmK, cspC, uspA, ompR, phoP, stcC, fimA, acrA,and yehZ. As a confirmation of the differential expression, RT-PCR was carried out using a set of specific primers. Remarkably, the expression levels of these genes were significantly increased in starved bacteria compared to standard laboratory conditions. Conclusions: Our results indicate that the starvation of Salmonella Typhimurium over one month in a salty microcosm changes the expression of stress proteins, response regulator in a two-component system, outer membrane proteins, effector proteins translocated by Salmonella pathogenicity island SPI1 and SPI2 type III secretion systems (TTSS), several metabolic enzymes, efflux pumps, and transport proteins. This suggests that the expression of the identified genes is important for the response of this pathogen to starvation in salt. [ABSTRACT FROM AUTHOR]

We have compared gene expression, using the Differential Display Reverse Transcriptase - Polymerase Chain Reaction (DDRT-PCR) technique, by means of mRNA profile in Melipona scutellaris during ontogenetic postembryonic development, in adult worker, and in both Natural and Juvenile Hormone III-induced adult queen. Six, out of the nine ESTs described here, presented differentially expressed in the phases L1 or L2, or even in both of them, suggesting that key mechanisms to the development of Melipona scutellaris are regulated in these stages. The combination HT11G-AP05 revealed in L1 and L2 a product which matches to thioredoxin reductase protein domain in the Clostridium sporogenes, an important protein during cellular oxidoreduction processes. This study represents the first molecular evidence of differential gene expression profiles toward a description of the genetic developmental traits in the genus Melipona.Nesse estudo nós usamos a técnica de Differential Display Reverse Transcriptase - Polymerase Chain Reaction (DDRT-PCR) para comparamos o perfil de mRNA em Melipona scutellaris durante o desenvolvimento ontogenético pós-embrionário e em operárias adultas, rainha natural e induzida pelo Hormônio Juvenil III. Fragmentos diferencialmente expressos foram detectados usando as seguintes combinações de primers: HT11G-AP05; HT11C-AP05; HT11G-OPF12; HT11G-OPA16. Dos 9 ESTs descrito nesse trabalho, 6 tiveram expressão diferencial nas fases de larva L1 e L2, sugerindo serem mecanismos chave no regulação do desenvolvimento larval em Melipona. A combinação HT11G-AP05 revelou em L1 e L2 um produto com similaridade à proteína tioredoxina redutase de Clostridium sporogenes, uma proteína importante durante os processos de oxidoredução. Esse estudo representa as primeiras evidências moleculares do perfil de expressão durante o desenvolvimento ontogenético em abelhas do gênero Melipona.

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The inflammatory response elicited by various stimuli such as microbial products or cytokines is determined by differences in the pattern of cellular gene expression. We have used the differential display RT-PCR (DDRT-PCR) strategy to identify mRNAs that are differentially expressed in various murine cell types stimulated with pro-inflammatory cytokines, microbial products or anti-inflammatory drugs. Mouse embryonic fibroblasts (MEFs) were treated with IFNs, TNF, or sodium salicylate. Also, peritoneal macrophages from C3H/Hej mice were stimulated with T. cruzi-derived GPI-mucin and/or IFN-g. After DDRT-PCR, various cDNA fragments that were differentially represented on the sequencing gel were recovered, cloned and sequenced. Here, we describe a summary of several experiments and show that, when 16 of a total of 28 recovered fragments were tested for differential expression, 5 (31%) were found to represent mRNAs whose steady-state levels are indeed modulated by the original stimuli. Some of the identified cDNAs encode for known proteins that were not previously associated with the inflammatory process triggered by the original stimuli. Other cDNA fragments (8 of 21 sequences, or 38%) showed no significant homology with known sequences and represent new mouse genes whose characterization might contribute to our understanding of inflammation. In conclusion, DDRT-PCR has proven to be a potent technology that will allow us to identify genes that are differentially expressed when cells are subjected to changes in culture conditions or isolated from different organs.

The English grain aphid, Sitobion avenae (F.) (Hemiptera: Aphididae), is a common worldwide pest of wheat ( Triticum aestivum L.). The use of improved resistant cultivars by the farmers is the most effective and environmentally friendly method to control this aphid in the field. The winter wheat genotypes 98-10-35 and Amigo are resistant to S. avenae. To identify genes responsible for resistance to S. avenae in these genotypes, differential-display reverse transcription-polymerase chain reaction was used to identify the corresponding differentially expressed sequences in current study. Two backcross progenies were obtained by crossing the two resistant genotypes with the susceptible genotype 1376. Six potential expected-differential bands were sequenced. Lengths of the expressed sequence tags ranged from 128 to 532 bp. Although these expressed sequences were likely associated with S. avenae resistance, there was one expressed sequence tag located on 7DL chromosome, and its potential function may associate with the ability to maintain photosynthesis in wheat. That serves as an active way for tolerant common wheat with resistant to S. avenae. Cloning the full length of these sequences would help us thoroughly understand the mechanism of wheat resistance to S. avenae and be valuable for breeding cultivars with S. avenae resistance. [ABSTRACT FROM AUTHOR]

Plants are under constant assault by biotic and abiotic agents. When an elicitor is prologued, an immense reprogramming of plant gene expression and defense responses are initiated, which could be a natural source for potential drug development and insertional mutagenesis. In this regard, differential expression analysis of a medicinal plant Moringa oleifera was performed for bioactive genes at seedling stage, using differential display-RT-PCR technique. Infected seedlings with a fungus Fusarium solani collected at different time intervals, showed a massive change in their gene expression profile. The data analysis revealed that at least 150 pathogen-induced and about 60 suppressed genes were differentially expressed at 8-h postinoculation of the biotic stress. Fifty-five selective genes were disunited and reamplified. Sequence analysis of these potential genes illustrated that these genes had properties of some induced peroxidase mRNA, cell proliferation, others were mitogen activated protein kinases, ribosomal protein genes, defense regulating genes, and a few also had structural properties. Further studies about the utility of these genes in plant metabolism could assist to develop improved transgenic breeds with enhanced value of infection tolerance not only of M. oleifera but of other cultivars also. [ABSTRACT FROM AUTHOR]

Zerumbone, a natural cyclic sesquiterpene, has been the focus of recent research as it has been found to exhibit selective toxicity towards cancer cells compared to normal cells. Studies on the cell cycle phase-specific effects of this interesting compound, however, remain sparse. Hence, concentration and time-dependent effects of zerumbone were evaluated employing a suitable model system, the naturally synchronous surface cultures of Physarum polycephalum. Zerumbone treatment in S, early, and late G2 phases resulted in G2 arrest. Early G2 phase exhibited the highest sensitivity ( P < 0.001) to the compound. Protein profiles showed a complete inhibition of cyclin B1 expression following zerumbone treatment. Furthermore, FACS and comet analysis revealed that zerumbone inhibited DNA synthesis ( P < 0.001) without being genotoxic at the concentrations tested. Differential display of mRNA showed distinct zerumbone-induced variations in transcript profiles, an analysis of which suggested a likely link between cellular networks involving stress-related gene expression and G2 arrest in P. polycephalum. [ABSTRACT FROM AUTHOR]

Copyright of Brazilian Journal of Biology is the property of Instituto Internacional de Ecologia and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Modelo do estudo: Estudo Experimental. Introdução: Atualmente a pesquisa com células-tronco tem gerado grande interesse devido a sua aplicabilidade no campo na medicina regenerativa. A medula óssea é considerada a maior fonte de células-tronco adultas e o estabelecimento de novos métodos para a análise da expressão gênica torna-se estritamente necessário. Desse modo, o "Differential Display Reverse Transcription Polymerase Chain Reaction (DDRT-PCR)", pode ser uma ferramenta acessível para a investigação de pequenas diferenças no nível de expressão gênica em diferentes tipos celulares, sob distintas condições. Objetivo: Neste presente trabalho nós investigamos a exequibilidade do DDRT-PCR na identificação de diferenças no nível de expressão gênica global em células da medula óssea de camundongos sob duas condições. Métodos: Primeiramente, a medula óssea foi isolada frescamente e uma secunda parte foi cultivada por uma semana sem troca de meio. Posteriormente, as células da medula (fresca e cultivada) 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 de cultivo, 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 por DDRT-PCR, mostrou uma expressão diferencial com a presença de três bandas (1300, 1000 and 225 pb) 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 de pequenas diferenças no nível de expressão gênica em células da medula óssea sob duas definidas condições. 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 diferentes condições.

Karnal bunt caused by Tilletia indica is a quarantine disease responsible for qualitative and quantitative losses in wheat. The present study aimed to determine the expression of defense-related genes involved in early stages of infection in response to T. indica by using the differential display reverse transcriptase-PCR method (DDRT-PCR) in susceptible (WL 711) and resistant (HD 29) cultivars of wheat. The DDRT-PCR banding profiles generated by the random primers in combination with oligo-dT primers of resistant and susceptible wheat plants were evaluated at 0 h, 12 h, 24 h, and 48 h after inoculation. Out of 9 sets of primers, 7 sets of primers produced remarkable induced transcripts. The highest up-regulated transcripts were observed in the resistant cultivar (HD 29), whereas the down-regulated transcripts were found in the susceptible (WL 711) cultivar. Several sets of primers were designed from the defenserelated gene sequences available in the GenBank database, and of these, 4 primer sets amplified the desired specific bands. Based on the putative functions, 3 defense-related genes, namely puroindoline protein PINB (AT_KB2), ß-1,4-glucanase (AT_KB3), and chitinase (AT_KB7), and 1 housekeeping gene, actin (AT_KB1), were characterized. [ABSTRACT FROM AUTHOR]

Plants respond to stress in part by modulating gene expression either constitutively or in an inducible manner which ultimately leads to the restoration of cellular homeostasis, detoxification of toxins, and recovery of growth. Upon introduction to various elicitors such as pathogen-associated molecular patterns, a massive reprogramming of plant gene expression is initiated. Differential display PCR offers rapid and multiple comparisons of gene expression to various stress durations and intensities. Nigella sativa has acclaimed many medicinal properties in traditional medicine. To explore the underlying molecular mechanisms in response to stress in the plants, Fusarium solani (a fungus) stress was induced at different time intervals ranging from 0 to 48 h. RNA was subjected to complementary DNA (cDNA) synthesis followed by PCR using different sets of anchored primers and arbitrary primers. The expression was visualized after silver staining on urea-PAGE. Out of the 23 upregulated re-amplified cDNA products, ten differential fragments showed significant homologies with domains related to cellular metabolism, signal transduction, and disease resistance. Such genes could be an informative source for developing genetically improved breeds under infectious stress. [ABSTRACT FROM AUTHOR]

The objective of the present study was to determine by differential display reverse transcriptase-polymerase chain reaction (DDRT-PCR) the effects of juvenile hormone (JH) III applied during the late larval 3 (L3) phase on gene expression in Melipona scutellaris. A temporal window of expression of feminizing genes exists during the late L3 and pre-defecating larval phases when these genes can be turned on or off by the action of JH, which is able to mediate the differentiation of female larvae into queens. Combination of the HT11A-AP4 primers revealed differential expression in L3 individuals treated with JH III for 1 h, with weak expression of the transcript, while intense expression was observed for controls and individuals treated for 4 h. Combination of the HT11G-AP4 and HT11G-AP5 primers showed suppression of the gene products for each primer combination in 1-h treated larvae compared to untreated control individuals of the same age and individuals treated for 4 h. Differential gene expression was also observed during development. These results demonstrate that the JH III may suppress or alter gene expression profiles during phase L3 of M. scutellaris.

A técnica Differential Display (DD), descrita primeiramente em 1992, promoveu um grande avanço nos estudos de expressão gênica em eucariotos, permitindo análises comparativas, isolamento e caracterização de novos genes relacionados aos mais variados processos biológicos. A larga aplicação do DD se deve, principalmente, ao fato de ser uma técnica rápida, fácil e necessitar de pequena quantidade de RNA total. Entretanto, o uso de radioisótopos em uma determinada etapa do DD restringe esse método a laboratórios que tenham áreas específicas para manipular esses reagentes e o torna inviável à grande maioria dos centros de pesquisa existentes no país. Assim, esse trabalho busca adaptar a coloração por nitrato de prata, não radioativa, à técnica do DD; utilizando como modelo biológico a espécie Apis mellifera (abelha africanizada). Os resultados têm sido promissores e mostram que a coloração por nitrato de prata é eficiente no DD e pode tornar a técnica mais difundida nos laboratórios nacionais, inclusive aplicada aos estudos de câncer. UNITERMOS: DDRT-PCR, Expressão gênica, Differential Display, método não radioativo.

The adaptive response of bacteria to stress is variable owing to the effects of various environmental factors and the time of exposure to stress conditions. We have studied the morphological and biochemical characters of resuscitated Pseudomonas aeruginosa ATCC 27853 that had been incubated for 14 years in natural sterilized seawater. The resuscitated cells were identified by PCR analysis of the internal transcribed spacer region. The biochemical profile of the resuscitated cells was partially inactive according to the API-20NE system. The cells had lost the ability to reduce nitrate and hydrolyse gelatin but acquired urease activity. In addition, the atomic force micrographs showed a reduction in cell size and an evolution to coccoid shapes. After incubation in a rich medium, the resuscitated cells regained their initial biochemical characters and their original rod shape. The differential display 'DDRT-PCR' technique was used to determine the effects of stress on gene expression in this P. aeruginosa strain. The genes algU and iscR were successfully identified in stressed P. aeruginosa ATCC 27853 as two differentially expressed gene fragments. Our results demonstrate that the DDRT-PCR technique can be a useful tool in gene expression analysis studies looking at the stress response in P. aeruginosa. [ABSTRACT FROM AUTHOR]

Haploid seed-producing semigamy in cotton is controlled by a single incompletely dominant gene ( Se). However, its molecular mechanism is currently poorly understood. In this study, the mRNA differential display (DD) was employed to compare gene expression between non-semigametic Pima S-1 ( Gossypium barbadense) and its naturally occurring semigametic mutant 57-4. Of the 70 DD cDNA fragments isolated from a total of 3,000 cDNA fragments, some exhibited semigamy-specific expression across anthers and/or ovules. Of the 61 cDNA fragments cloned and sequenced, nine showed high homology to genes encoding for α-tubulin, SPK1, Rab11, sucrose synthase, isocitrate lyase, phosphoethanolamine N-methyltransferase, phospholipase D, MADS 7, and WRKY 2. From cDNA-single-strand conformational polymorphism using primers designed from DD sequences, qualitative expression differences in six genes between the semigametic and non-semigametic genotypes were validated. However, a primer pair designed from an α-tubulin gene sequence yielded PCR products with quantitative difference in band intensity, which is linked to the semigamy gene at a recombination frequency of 0.316 in a (57-4 × Pima S-1) × Pima S-1 BC1F1 population. Its expression in the semigametic 57-4 anthers was suppressed by 2-fold as compared to that in Pima S-1 using a quantitative reverse transcription PCR analysis. This study identified several novel genes that were missed in prior microarray analysis, and their potential roles with regards to reproductive biology are discussed. [ABSTRACT FROM AUTHOR]

Identification of ethylene-regulated and ripening-related genes from banana ( Musa acuminata Var. Harichaal) fruits using DDRT-PCR led to the isolation of differentially expressed partial cDNA of pectin methylesterase inhibitor (MaPMEI) gene. Its full-length cDNA sequence consisted of a 567 bp ORF, encoding a protein of 189 aa with deduced molecular mass 19.6 kDa. Expression pattern of MaPMEI gene revealed that upon ethylene treatment, this gene is up-regulated initially giving maximum expression in post-climacteric stage then decreases slightly in later stages of ripening. 1-MCP, a known ethylene perception inhibitor, inhibits both fruit ripening as well as the transcript level of this gene. Also, the transcripts of MaPMEI gene were not detected during the short time ethylene treatment suggesting this gene appears to be not directly induced by ethylene. Interestingly, MaPMEI gene showed fruit specific expression that indicates its possible role in the regulations of PMEs in fruits. In silico analysis revealed a predicted signal peptide sequence necessary for localization of MaPMEI in the cell wall. Furthermore, the four Cys residues involved in disulfide bridges are conserved in MaPMEI similar to other PMEIs and invertase inhibitors. Phylogenetic analysis further suggests that the MaPMEI identified in this study is more closely related to PMEIs than to invertase inhibitors. [ABSTRACT FROM AUTHOR]

D-myo-inositol-3-phosphate synthase (MIPS) catalyzes the reaction from d-glucose 6-phosphate to D-myo-inositol 3-phosphate (MIP), which is the first and rate-limiting step in myo-inositol biosynthesis. In this study, Jatropha curcas MIPS cDNA (JcMIPS) (GenBank accession no. EF 185781) has been isolated using mRNA differential display technology (DDRT) and the rapid amplification of cDNA ends (RACE). The cDNA clone of JcMIPS is comprised of 1,957 bp, encoding 509 amino acids, with a predicted molecular weight of 56.4 kDa. The JcMIPS protein is highly homologous to those from other plant species, ranging from 88.4 to 91.18% homology at the amino acid levels. Real-time quantification polymerase chain reaction (PCR) analysis has revealed that JcMIPS transcripts are highly present in seed and leaf tissues, but are at low levels in stem and flower tissues. Furthermore, the transcription of JcMIPS in leaves is up-regulated by abscisic acid (ABA) (100 μM), drought (30% PEG-6000), NaCl (200 mM), and low-temperature (4°C) treatments. The observed increase of JcMIPS enzyme activity is also detected following treatments with ABA, drought, and NaCl. Interestingly, JcMIPS enzyme activity is only slightly changed following low-temperature treatment. [ABSTRACT FROM AUTHOR]

Genes induced by phosphorus deficiency in leaves and roots of winter rape ( Brassica napus) seedlings were isolated and analyzed. The mRNA differential display technique was used to visualize cDNA fragments derived from the phosphorus-tolerant cv. Zy 821 and the phosphorus-sensitive cv. No. 1182 grown under conditions of phosphorous starvation. Approximately 2000 cDNA bands were visualized by differential display using 78 primer pair combinations. A total of 61 phosphorus-starvation-induced cDNA fragments differentially expressed were isolated. Among these, 40 were derived from roots and only 21 from leaves. Sequence analysis of 16 fragments revealed that they represented distinct cDNAs. A subsample of five cDNAs was analyzed by semi-quantitative RT-PCR, which showed that they were truly different products. Transcripts coding for enzymes involved in photosynthesis (thylakoid membrane phosphoprotein) and root hair initiation (xyloglucan endotransglycosylase), and two regulators (RNA-binding/nucleic acid-binding mRNA and a structural constituent of ribosomal mRNA) were found to be differentially expressed. These results show that the mechanism of cv. Zy 821 adaptation to P starvation is complex. Although its P-tolerance trait is controlled by a major gene, many other genes influencing P acquisition and remobilization, carbon and secondary metabolism, developmental processes, and regulatory pathways are also involved. [ABSTRACT FROM AUTHOR]

Using a differential display (DDRT-PCR) approach to screen genes involved in response to osmotic stress, we isolated six cDNA clones from potato cell cultures. One of these, clone #54, is a member of the gene family encoding the α-subunit of translation elongation factor 1 (EF-1α). After sequencing, this clone showed a deduced amino acid sequence of 447 residues. Southern blot analysis revealed that this EF-1α is a member of a family consisting of at least nine genes. The expression of EF-1α increased upon osmotic stress mediated by polyethylene glycol (PEG) but was unaffected by abscisic acid (ABA) treatment. Salt also increased EF-1α expression but with a different kinetics. These results indicate that we cloned an EF-1α in potato which is ABA responsive, independent of osmotic stress. Abbreviations: DDRT-PCR, differential display reverse transcriptase-polymerase chain reaction; PEG, polyethylene glycol; RACE, rapid amplification of cDNA ends; RT-PCR, reverse transcriptase-polymerase chain reaction [ABSTRACT FROM AUTHOR]

The genus Calanthe includes species of terrestrial orchids that produce attractive flowers with diverse floral traits. Breeding programs have been established to improve the horticultural value of various Calanthe species, but studies to identify the genetic components contributing to the key phenotypic characteristics have not been undertaken. To understand the molecular mechanisms underlying floral development associated with floral morphology, color, and fragrance production, the flower buds of two typical Korean Calanthe species, C. discolor and C. sieboldii, were subjected to gene expression analysis by differential display RT-PCR (DDRT-PCR). A total of 66 non-redundant differentially expressed genes (DEGs) were isolated and sequenced. Of these, 26 and 40 DEGs were found to be highly expressed in C. discolor and C. sieboldii, respectively. Moreover, the expression patterns of a subset of genes presumably implicated in signal transduction, metabolic pathways, and hormonal signaling differed between the two species. The data presented here may improve our understanding of the mechanisms underlying floral development and contribute to advances in orchid biotechnology. [ABSTRACT FROM AUTHOR]

Summary: We report here about the physiological and molecular responses of Populus canadensis (clone Dorskamp) to drought. The stress was applied to young rooted cuttings by PEG 6000 application over 30 days. This stress induces a decrease in predawn leaf water potential. After 10 days of stress, there was a decrease in stomatal conductance and a slight retardation of leaf growth, but the osmotic potential remained constant. Using the differential display technique, we searched for genes differentially expressed in response to drought at this date. Thirty-six differentially expressed leaf cDNAs were detected between stressed and control conditions. Thirty-four cDNAs clones were successfully cloned and 23 were found to share high identity with Arabidopsis thaliana and Populus trichocarpa genes. The transcriptional regulation of 21 genes was examined by reverse RNA dot blot, confirming an increase in expression for 16 of them after 10 days of treatment. Among these 16 genes, most of them are involved in a different cellular metabolic pathway. These differentially expressed genes are also involved and/or regulated by other treatments such as salt, withholding water or auxin application. The maintenance of growth observed during the first 10 days of the stress period could be due to the regulation of these genes and can be a common response between herbaceous plants and trees. [Copyright &y& Elsevier]

Abstract: Cellular senescence is regulated by specific genes in many organisms. The identification and functional analysis of senescence-associated genes could provide valuable insights into the senescence process. Here, we employed a new and improved differential display reverse transcription-polymerase chain reaction (DDRT-PCR) method that involves annealing control primers (ACPs) to identify genes that are differentially expressed in human umbilical endothelial cells during replicative senescence. Using 120 ACPs, we identified 31 differentially expressed genes (DEGs). Basic local alignment search tool (BLAST) search revealed 29 known genes and two unknown genes. Expression levels of the 29 known genes were confirmed by real-time quantitative RT-RCR and by Western blotting for eight of these genes. CD9 antigen, MHC class I chain-related sequence A (MICA) and cell division cycle 37 homolog (CDC37) were up-regulated, and bone morphogenetic protein 4 (BMP4), dickkopf-1 (DKK1), and transcription factor 7-like 1 (TCF7L1) were down-regulated in old cells. Treatment with recombinant human MICA caused a decrease in cell proliferation and an increase in senescence-associated β-galactosidase staining. Further analysis of differentially expressed genes may provide insights into the molecular basis of replicative senescence and vascular diseases associated with cellular senescence. [Copyright &y& Elsevier]

Abstract: This study is focused on the identification of early response genes involved in the resistance mechanism of one of the most important diseases of wheat, yellow rust. The strategy undertaken was to use differential display reverse transcriptase-PCR method (DDRT-PCR) on two of the yellow rust differential lines of wheat, were infected with the virulent and the avirulent Puccinia striiformis f. sp. tritici races together with appropriate control inoculations. Upon infection of the plant materials (resistant and/or susceptible), different time points were evaluated on DDRT-PCR banding profiles. The expression level differences of 33 genes in DDRT-PCR were further analyzed using quantitative real-time PCR (qRT-PCR). Among which only those (14 gene fragments) resulting reproducible induction or repression levels were reported with measured fold changes. Based on the sequence similarity analysis, all but one were absent in NCBI databank, thus they are newly found wheat sequences. Among those, two genes were identified as full ORF, including 5′ and 3′ end untranslated regions (UTR); namely cyclophilin like protein (putative antifungal activity) and ubiquitin-conjugating enzyme (E2) or Rad6. The sequence homology analysis of the cloned gene fragments revealed that the genes detected may have roles in ubiquitinylation, programmed cell death (apoptosis), putative antifungal activities, disease resistance responses, pathogenesis related responses, and including a few with no known function. [Copyright &y& Elsevier]

Summary: We have now entered in the post-genomic era, where we have knowledge of plethora of fungal genomes and cutting edge technology is available to study global mRNA, protein and metabolite profiles. These so-called ‘omic’ technologies (transcriptomics, proteomics and metabolomics) provide the possibility to characterize plant–pathogen interactions and pathogenesis at molecular level. This article provides an overview of transcriptomics and its applications in fungal plant pathology. [Copyright &y& Elsevier]

Vibrio vulnificus is thought to employ a quorum-sensing system to control the expression of a global gene. In this study, proteomes and transcriptomes of a lacZ null mutant, VvSRΔ Z, and a luxS– smcR double mutant, VvSRΔZSR, were compared with the parent strain, VvAR, by means of two-dimensional gel electrophoresis (2D-PAGE) and differentially displayed reverse transcriptase PCR (DDRT-PCR). 2D-PAGE analysis showed that 36 protein spots were differentially expressed, 14 of which have been identified by peptide-mass fingerprinting. The expression of eight cellular proteins was repressed by luxS and smcR mutation: Zn-dependent protease, 6-phosophofructokinase, periplasmic ABC-type Fe3+ transport system, deoxyribose-phosphate aldolase, phosphomannomutase, orotidine-5′-phosphate decarboxylase, uridylate kinase, and an unidentified protein. These proteins are involved in virulence, adaptation to environmental stress, biosynthesis of LPS, and cell multiplication. Phage shock protein A, a chemotaxis signal transduction protein, and an uncharacterized low-complexity protein were activated in the cellular components of the luxS-smcR mutant. However, only three proteins, of unknown function, were identified in the extracellular components of the mutants. Analysis of transcriptomes with DDRT-PCR showed that two genes, phosphoribosylformylglycinamidine synthase and ATP-dependent protease HslVU protease were regulated at the transcriptional level by luxS and smcR gene mutation. The results from this study show conclusively that luxS/smcR quorum sensing endows a global change in gene expression to V. vulnificus. [ABSTRACT FROM AUTHOR]

Researchers hope to increase γ-globin expression by controlling potential trans-acting factors that specifically activate the γ-globin gene in fetuses or silence this gene in adults to potentially treat sickle cell disease and β-thalassemias. To characterize genes encoding such factors, we analyzed the differential expression of mRNAs in erythroid induction cultures of CD34+ cells derived from normal adult bone marrow, umbilical cord blood, and bone marrow from a patient with heterocellular hereditary persistence of fetal hemoglobin. Using differential-display – reverse-transcription PCR analysis, we identified a number of genes with differential expression in the above-mentioned cells. The differential expression of some genes was also confirmed by quantitative real-time PCR. Our data provide important clues for identifying and validating trans-activators that activate the γ-globin gene in fetuses, and trans-acting factors involved in silencing the γ-globin gene in adults. [ABSTRACT FROM AUTHOR]

Summary: Alkali grass (Puccinellia tenuiflora), a monocotyledonous halophyte, can serve as a model of salt tolerance in monocotyledon crops. To elucidate the molecular events associated with salt tolerance in alkali grass, we generated a directional cDNA library from leaves treated with the alkali salt, NaHCO3. Large-scale sequencing of the cDNA library identified 2942 ESTs representing 2366 non-redundant transcripts. These have been deposited in the dbEST division of GenBank. BLASTX evaluation indicated that 1274 of the ESTs were homologous to various known genes/proteins in a broad range of organisms, especially gramineae species. The other 1092 ESTs, which showed little if any homology to known sequences, were considered novel. Based on the encoded proteins, the 1274 identified ESTs fell into 12 functional categories, of which four were abundant, namely metabolism (18.84%), transcription (12.48%), unclassified (11.22%) and cell rescue/defense (9.66%). The 162 unique transcripts corresponding to possible salt-related genes were also identified. This study provides an overview of gene expression in NaHCO3-stressed alkali grass, as well as useful information for further investigation of salt resistance in plants. [Copyright &y& Elsevier]

Abstract: This study was designed to investigate changes in gene expression associated with stage-specific programmed cell death (PCD) in intersegmental muscles (ISMs) of the moth, Manduca sexta. The technique of differential display reverse transcription PCR was applied to compare mRNA levels before and after the onset of PCD in ISMs. Expression of E75B transcription factor was repressed while another factor, βFTZ-F1, stayed at a very low level. However, gene coding for a translation-initiation factor (eIF1A) was upregulated. Expression of these genes had not been previously reported to be altered in dying ISMs. An ecdysteroid agonist, RH-5849, that prevented PCD in ISMs also blocked these changes. [Copyright &y& Elsevier]

The effect of Chinese traditional medicine "Bushen Yinao Pian," a complex prescription used for anti-aging, on the cerebral gene expression of the senescence-accelerated mouse prone 8/Ta (SAMP8/Ta) had been studied with messenger ribonuclear acids reverse transcription differential display polymerase chain reaction (mRNA DDRT-PCR). Eight differential displayed bands had been discerned and sequenced. The sequences of those fragments are matched to adipocyte-specific protein-5; low density lipoprotein receptor-related protein associated protein-1; reticulon-3; cysteine and histidine-rich domain (CHORD)-containing, zinc-binding protein-1; cytochrome c oxidase subunit-2 (Cox-2); cytochrome c gene, MC1; DNA sequence from clone RP23-72M11 on chromosome X, respectively and a novel sequence fragment. Most of these genes are aging-related. It can be proved that the "Bushen Yinao Pian" truly has anti-aging function. [ABSTRACT FROM AUTHOR]

Genes differentially expressed under high irradiance (HI) stress in mature flag leaves of super-hybrid rice Liangyoupeijiu ( Oryza sativa ssp. indica) were studied by the silver-staining mRNA differential display technique. We obtained 167 differentially displayed bands on silver-stained polyacrylamide gels and searched for their genetic origins. Five cDNA fragments, which were upregulated by HI stress, were cloned and sequenced. The clones of G25×320, A31×380, and G29×280 putatively encode a cytosolic monodehydroascorbate reductase (MDAR), a phosphatidylinositol 4-kinase (PI-4-K), and a DEAH-box RNA helicase, respectively. Most differentially expressed genes in hybrid rice were inherited from parents and many of them were related with both parents, although some were derived from one parent only. The differentially expressed cDNA fragments having no distinct genetic origins indicated the involvement of some unclear mechanisms in the inheritance processes from parents to hybrid. [ABSTRACT FROM AUTHOR]

Abstract: The molecular mechanism of the Chinese traditional medicine ‘Bushen Yinao Pian’ (a complex prescription used for clinical anti-ageing in China for over 20 years) is elusive. In this study, the cDNA of low-density lipoprotein related-receptor associated protein-1 (LRPAP-1), an ageing-related gene, which functions as a chaperon or escort protein in the intracellular transport of low-density lipoprotein related-receptor, a transporter of amyloid beta protein (AβP), had been cloned by screening cDNA library based on analyzing the gene expression in cerebral tissue between the test and the control accelerated senescence-prone mouse 8/Ta (SAMP8/Ta). The result shows that this complex prescription increased the expression level of LRPAP-1. It indicated that the Chinese traditional medicine ‘Bushen Yinao Pian’ plays an important role in anti-ageing by increasing LRPAP-1 expression level. [Copyright &y& Elsevier]

Copper and iron act at different levels on gene expression. Due to their chemical reactivity, both metals could play a role in the regulation of the protein machinery involved in their metabolism, and/or of the metabolic function they are involved in. Experimental and clinical evidences raise also the hypothesis of the existence of genes commonly regulated by both metals. Purpose of this work was to find genes modulated by copper and iron in the rat intestine. A panel of 24 animals was randomly divided into three nutritional treatments including a control, a copper-deficient and an iron-deficient diet. The positive regulation of iron responsive element (IRE)-DMT1 gene was found, with different extent, in both experimental groups. A differential display reverse transcription (DDRT)–polymerase chain reaction (PCR) analysis carried out on the rat intestinal mRNAs demonstrated the differential expression of five cDNA fragments. Among these, the Cytochrome c oxidase (COX) subunit II mitochondrial gene resulted to be regulated by both metals, the Serum and Glucocorticoids-regulated Kinase (SGK) gene mainly by iron, and an Ebnerin-like 2 kb mRNA dramatically down-regulated by copper. Two residual clones showed low identity scores with sequences present in data bank. Finally, we observed that both iron and copper are able to modulate the expression of the three characterized genes in some tissues, other than intestine. [Copyright &y& Elsevier]

Paracoccidioides brasiliensis, the etiologic agent of paracoccidioidomycosis, is a dimorphic fungus which is found as mycelia (M) at 26 °C and as yeasts (Y) at 37 °C, or after the invasion of host tissues. Although the dimorphic transition in P. brasiliensis and other dimorphic fungi is an essential step in the establishment of infection, the molecular events regulating this process are yet poorly understood. Since the differential gene expression is a well-known mechanism which plays a central role in the dimorphic transition as well as in other biological process, in this work we describe the identification and characterization of two differentially expressed P. brasiliensis hydrophobin cDNAs (Pbhyd1 and Pbhyd2). Hydrophobins are small hydrophobic proteins related to a variety of important functions in fungal biology, including cell growth, development, infection, and virulence. These two hydrophobin genes are present as single copy in P. brasiliensis genome and Northern blot analysis revealed that both mRNAs are mycelium-specific and highly accumulated during the first 24 h of M to Y transition. [Copyright &y& Elsevier]

In this study, the ageing-specific expression genes of the murine cerebrum were investigated by employing differential-display reverse transcription polymerase chain reaction (DDRT-PCR) in three senescence-accelerated mouse (SAM) strains: SAMP8/Ta, SAMP10/Ta and SAMR1TA. In the comparison of gene expression profile in different strain mice, 16 differential fragments have been detected, 3, 6 and 7 of them belong to SAMP10/Ta, SAMP8/Ta and SAMR1TA, respectively. Sequencing data indicated that those fragments are homologous with heat shock protein cognate protein 70, ATP-dependent mitochondrial RNA helicase, Dleu2 mRNA, Mouse DNA sequence from clone RP23-334C3 on chromosome X, ubiquinol-cytochrome c reductase complex (7.2 kDa), 60S ribosomal protein L21, FIS, phenylalkylamine Ca2+ antagonist (emopamil) binding protein, fucosyltransferase 9, glial cell line derived neurotrophic factor family receptor alpha 1, endonuclease/reverse transcriptase, PER1 interacting protein of the suprachiamatic nucleus homolog, centrosomal protein CG-NAP, ferritin heavy chain gene, NID-2 gene, and prkdc gene for DNA-dependent protein kinase catalytic subunit. [Copyright &y& Elsevier]

Studies comparing in vivo and in vitro functional capacities of leukocytes from non-parturient and periparturient dairy cows have provided substantial evidence that systemic and local mammary immune defenses are deficient around parturition. This evidence has lead to the reasonable hypothesis that immune deficiency underlies the heightened mastitis susceptibility of periparturient cows. Nutrition and vaccine studies substantiate this hypothesis, showing that dietary antioxidant supplementation and rigorous immunization regimes can bolster innate and humoral immunity to the point that mastitis severity and time for return to normal milk production are reduced. However, completely effective resolution of this significant production disease has not been achieved because so little is understood about its complex etiology. In particular, we possess almost no knowledge of how or why immune cells responding to parturient physiology end up with deficient functional capacities. Fluctuations in reproductive steroid hormones and chronic shifts in neuroendocrine hormones with roles in nutrient partitioning and appetite control may affect the expression of critical leukocyte genes in periparturient dairy cows. A thorough understanding of leukocyte biology during periparturition would seem a critical goal for future development of effective mastitis prevention strategies. Recently, our group has begun to use cDNA microarray technology to explore bovine leukocyte RNA for global gene expression changes occurring around parturition. We are working within the context of a hypothesis that the physiology of parturition negatively affects expression of critical genes in blood leukocytes. In the current study we initiated hypothesis testing using leukocyte RNA from a high producing Holstein cow collected at 14 days prepartum and 6 hours postpartum to interrogate a cDNA microarray spotted with >700 cDNAs representing unique bovine leukocyte genes. This analysis revealed 18 genes with ≥1.2-fold higher expression 14 days prepartum than 6 hours postpartum. BLASTN analysis of these genes revealed only one that can be considered a classical immune response gene. All other repressed genes were either unknown or putatively identified as encoding key proteins involved in normal growth and metabolism of cells. Given the critical roles of these repressed genes in normal cell functions, we may have identified good candidates to pursue with respect to periparturient immunosuppression and mastitis susceptibility.

Studies comparing in vivo and in vitro functional capacities of leukocytes from non-parturient and periparturient dairy cows have provided substantial evidence that systemic and local mammary immune defenses are deficient around parturition. This evidence has lead to the reasonable hypothesis that immune deficiency underlies the heightened mastitis susceptibility of periparturient cows. Nutrition and vaccine studies substantiate this hypothesis, showing that dietary antioxidant supplementation and rigorous immunization regimes can bolster innate and humoral immunity to the point that mastitis severity and time for return to normal milk production are reduced. However, completely effective resolution of this significant production disease has not been achieved because so little is understood about its complex etiology. In particular, we possess almost no knowledge of how or why immune cells responding to parturient physiology end up with deficient functional capacities. Fluctuations in reproductive steroid hormones and chronic shifts in neuroendocrine hormones with roles in nutrient partitioning and appetite control may affect the expression of critical leukocyte genes in periparturient dairy cows. A thorough understanding of leukocyte biology during periparturition would seem a critical goal for future development of effective mastitis prevention strategies. Recently, our group has begun to use cDNA microarray technology to explore bovine leukocyte RNA for global gene expression changes occurring around parturition. We are working within the context of a hypothesis that the physiology of parturition negatively affects expression of critical genes in blood leukocytes. In the current study we initiated hypothesis testing using leukocyte RNA from a high producing Holstein cow collected at 14 days prepartum and 6 hours postpartum to interrogate a cDNA microarray spotted with >700 cDNAs representing unique bovine leukocyte genes. This analysis revealed 18 genes with ≥1.2-fold higher expression 14 days prepartum than 6 hours postpartum. BLASTN analysis of these genes revealed only one that can be considered a classical immune response gene. All other repressed genes were either unknown or putatively identified as encoding key proteins involved in normal growth and metabolism of cells. Given the critical roles of these repressed genes in normal cell functions, we may have identified good candidates to pursue with respect to periparturient immunosuppression and mastitis susceptibility.

The expression of mouse two-cell stage specific genes was studied using the modified DDRT-PCR method, which overcame the paucity of (he experimental materials of preimplantation embryos. Embryo tissues equivalent to that of four blastomeres are sufficient for amplification of target genes as visualized using polyacrytamide gel. Sequence analyses and reverse Northern blots indicate that the genes of ATPase 6 and Ywhaz are expressed specifically in two-cell embryos. ATPase 6 is essential for one-cell to two-cell transition and plays an important role in establishment of oxidative phosphorylation, while Ywhaz is related to initiating cellular communication system. [ABSTRACT FROM AUTHOR]

Self-incompatibility is a genetically controlled process used to prevent self-pollination. We report here the characterization of pollen cDNA clones of Lycopersicon peruvianum, and the identification of a genotype-specific pollen factor involved in self-incompatibility. To identity the latter, differential mRNA display RT-PCR was performed on pollen cDNAs from S12Sa and S12Sa genotypes. We isolated lour cDNA fragments expressed preferentially in S12Sapollen, and screened a cDNA library from S12Sa pollen with the lour cDNA fragments to isolate the corresponding full length cDNAs. One of the four isolated cDNAs encoded part of an actin depolymerizing factor protein that we named LpADF. LpADF is highly homologous to actin depolymerizing factors of Arabidopsis thaliana, Lilium longiflorum, and Zea mays. RNA blot analysis revealed that LpADF is only expressed in mature pollen of the S12Sagenotype, and is therefore a candidate pollen factor in the gametophyte self-incompatibility system of L. peruvianum. [ABSTRACT FROM AUTHOR]

A differentially expressed OsIM1 gene was isolated from rice salt-tolerant mutant M-20 by differential display. Sequence analysis revealed that the amino-acid sequence of OsIM1 showed 66% and 62% identity with PTOX from tomato (Capsicum annuum) and AtIM from Arabidopsis, both of which encoded chloroplast-orientated terminal oxidase. Comparison of the nucleotide sequence of the OsIM1 cDNA with its genomic sequence revealed that OsIM1 genomic DNA contained nine exons and eight introns. A pseudo-transcript (OsIM2), which probably resulted from the abnormal splicing of the OsIM1 pre-mRNA, was also identified. Southern-blot analysis showed that there existed only one copy of the OsIM1 gene in the rice genome. RFLP analysis located it on rice chromosome 3. The Northern blot revealed that OsIM1 was up-regulated by NaCl and ABA treatment. RT-PCR analysis indicated that OsIM1 and OsIM2 co-existed in the OsIM transcript pool, and the ratio of OsIM1/OsIM2 was differentially regulated by salt stress in the salt-sensitive variety and the salt-tolerant varieties. [ABSTRACT FROM AUTHOR]

Differential display reverse transcriptase polymerase chain reaction (DDRT-PCR) in combination with semi-quantitative RT-PCR was used to compare differences in gene expression between the solitary and gregarious phase of Schistocerca gregaria. Twenty-six primer combinations were used, which produced 8 differential bands. Two out of the 8 differentials, one typical for the solitary and one for the gregarious phase, were further analyzed by semi-quantitative RT-PCR. The expression level of the solitary phase specific gene (SSG) was 2 times higher in solitary animals as compared to gregarious ones, while the gregarious specific gene (GSG) gave a 4-fold higher expression level in gregarious animals than in solitaries. Sequence analysis demonstrated that SSG does not belong to a known gene family, while the GSG belongs to the SPARC protein family. [Copyright &y& Elsevier]

Differences in mRNA expression between immature adult worms (IAW) and mature adult worms (MAW) of Echinococcus granulosus were determined using polymerase chain reaction-based differential display (DDRT-PCR). Twenty-eight putative differential cDNA fragments were isolated, cloned and sequenced. mRNAs from IAW and MAW were probed with the labelled fragments. Six cDNA fragments (coded as egM12, egM13, egM22, egM26, egM30 and egM34) were putatively determined to be specific to MAW by Northern hybridisation. The stage-specificity of egM12, egM13 and egM34 was confirmed by RT-PCR. RNAs of IAW, MAW, protoscoleces and oncospheres, probed with egM13 and egM30, showed that the mRNAs were expressed exclusively in MAW, which implied involvement in the regulation of egg development. Using the labelled fragments to screen a cDNA library of MAW, 99 clones were identified and analysed. An alignment of selected clones showed that the MAW-specific mRNAs belonged to a family. Examination of the deduced amino acid sequence of three of the corresponding cDNAs (egM4, egM9 and egM123) indicated they were cysteine-rich and contained a 24 amino acid repeat sequence, repeated four to six times. The repeat regions were predominantly alpha helical in nature with interspersed turns, forming alternating zones of positive and negative charge. The functional significance of each of the cDNAs identified is unclear as none had significant sequence similarity to genes of known function. However, polypeptides encoded by egM4 and egM123 were recognised by antibodies in a serum pool from dogs experimentally infected with E. granulosus, suggesting they could prove of value in serodiagnosis of definitive hosts. [Copyright &y& Elsevier]