Differential Display Technique Research Articles

AraC transcriptional regulator, aspartate semialdehyde dehydrogenase and acyltransferase: Three putative genes in phenol catabolic pathway of Acinetobacter sp. Strain DF4

The objective of this study was to identify genes associated with the biodegradation of phenol by Acinetobacter sp. strain DF4 through the use of differential display (DD) methodology. The bacteria were grown in YEPG medium, and total RNA was extracted and analyzed using labeled primers to detect gene expression differences. Three distinctively expressed cDNA bands (ph1, ph2, and ph3) were identified, cloned, and sequenced. DNA analysis involved searching for open reading frames (ORFs), verifying results with the NCBI database, predicting promoter regions, and constructing phylogenetic trees using bioinformatics tools. The ph1 gene displayed a 97% identity with the AraC transcriptional regulator, suggesting its potential role in regulating the ortho-catabolic pathway of phenol. The ph2 gene showed a 98% identity with aspartate semialdehyde dehydrogenase, which is involved in phenol degradation. The ph3 gene had a 93% identity with acetyltransferase. Essential transcription factors, such as TATA, GTGTGT, CACA, and CTTTT, were detected, and the three genes promoter regions were predicted. This study successfully identified functional genes involved in the metabolism of cyclic chemicals, particularly phenol, using the DD technique. These findings provide insights into the biodegradation pathways of phenol by Acinetobacter sp. Strain DF4 and may contribute to the development of more efficient bioremediation strategies for phenol-contaminated environments.

Identification and Characterization of Genes Responsible for Drought Tolerance in Rice Mediated by Pseudomonas fluorescens

Drought is one of the major abiotic stresses which adversely affect crop plants limiting growth and yield potential. Structural and functional characterization of drought stress-induced genes has contributed to a better understanding of how plants respond and adapt to the drought stress. In the present study, differential display technique was employed to study the gene expression of rice plants at the reproductive stage that were subjected to drought stress by withholding water, Pseudomonas fluorescens strain (Pf1) treated plants subjected for drought stress by withholding water and control (well-watered). Differentially expressed cDNAs of six genes (COX1, PKDP, bZIP1, AP2-EREBP, Hsp20 and COC1) were identified, cloned and sequenced. Real-time qPCR analysis showed that all the six genes were upregulated in drought-stressed plants treated with Pf1. This revealed that the remarkable influence of Pf1 colonization leads to drought tolerance at the reproductive stage. These results showed that high levels of gene expression in plants lacking adequate water can be remarkably influenced by Pf1 colonization, which might be a key element for induced systemic tolerance by microbes.

MECHANISMS OF TOLERANCE TO SALINITY IN BANANA:PHYSIOLOGICAL, BIOCHEMICAL,AND MOLECULAR ASPECTS

ABSTRACT In the northeastern region of Brazil, saline soils are constraints to banana production, becoming necessary to understand the mechanisms of salt tolerance. Two bananas genotypes, Tap Maeo, tolerant, and Berlin, sensitive, were subjected to treatment with 50 mol m-3 NaCl or without salt. This study evaluated the effects of salt on the following physiological aspects: leaf area, content and distribution of Na+, membrane integrity, proton AT Pase activity. Besides, a search for differentially expressed genes was performed using the Differential Display technique. Tap Maeo genotype showed the smallest reduction in leaf area, smaller accumulation of Na+ and malondialdehyde (MDA), and higher activity of proton AT Pase activity. Two sequences differentially expressed in the tolerant genotype, (Musa 07, Musa 23), shared a high degree of identity with the amino acid sequences of the genes SOS1 and SOS2, respectively. The clone Musa 10 was highly similar to amino acid sequence of the ascorbate peroxidase gene, and Musa 26, encodes the enzyme betaine aldehyde dehydrogenase. These significant biological markers indicate that salinity tolerance in banana involves at least two simultaneous mechanisms: the activation of the SOS pathway, increasing the extrusion of Na+, and the activation of antioxidative system, increasing the synthesis of APX and betaine aldehyde dehydrogenase enzyme.

Differential expression analysis in Egyptian redbelly tilapia (Tilapia Zillii) exposed to cold stress

Fish can adapt to a wide range of temperature changes, however, the molecular mechanisms that underlie these adaptations are not fully understood. In this study, fluorescently labeled differential display (FDD) technique was used to detect mRNAs that are differentially expressed in hepatic tissues of Tilapia zillii fish subjected to a temperature reduction regime that combined both accelerated and stationary cold stresses. The two arbitrary primers (DD1 and DD2) with the Oligo d(T)23-C generated a total of 18 and 37 unknown messenger RNA (UMR) bands, respectively. Both DD1 & DD2 arbitrary primers with oligo d(T)23-C scored four stress-related unidentified fragments after refining. Using the two arbitrary primers with the Oligo d(T)23-G, a total of 371 and 372 UmRs were yielded, respectively. Both arbitrary primers scored nine stress-related unidentified fragments after refining. All UmR were tested for its correlation to time and temperature factors scaled for the current experiment, only four were found to be correlated for both factors. Thus, T. zillii responded to temperature reduction by adjusting the expression of a set number of genes that may be required for their adaptation and tolerance to low environmental temperature.

Identification of Differentially Expressed Genes between “Honeycrisp” and “Golden Delicious” Apple Fruit Tissues Reveal Candidates for Crop Improvement

Cultivars of the same species exhibit a large degree of variation in fruit quality traits, which can be directly influenced by differences in gene expression due to allelic variations and interactions with the environment. For Malus × domestica Borkh. (apple), fruit quality traits, including color, texture, aroma, flavor profile, and shelf life, are of utmost economic importance. In order to identify genes potentially influencing these traits, a direct comparative transcriptome profiling approach, based on the differential display technique, was performed using “Golden Delicious” and “Honeycrisp” apple endocarp and peel tissues. A total of 45 differentially expressed sequence tags were identified between the two apple varieties. Reanalysis of a previously published fruit developmental microarray expression experiment revealed that only one of the 45 sequence tags was represented on the array. Differential expression of 31 sequence tags from the peel tissue was validated using quantitative reverse transcription PCR, confirming the robustness of the differential display approach to quickly identify differentially expressed sequence tags. Among these were genes annotated to be involved in ripening, phytohormone signaling, transcription factors, and fruit texture. This work demonstrates yet again the utility of the differential display technique to rapidly identify genes related to desirable traits.

Effect in dedicator of cytokinesis 6 (DOCK6) on steroid production in theca cells of follicular cysts

Ovarian follicular cysts are one of the most common causes of reproductive failure in mammals. A comparative gene expression approach may aid in elucidating the causes of ovarian cyst disease. In the present study, the differential display technique was used to identify mRNA sequences that accumulate preferentially in theca cells of bovine cystic follicles. Dedicator of cytokinesis 6 (Dock6) expression was observed in the theca cells of cystic follicles. Small interfering RNA (siRNA) knockdown of Dock6 increased progesterone (P4) production and StAR expression in theca cells of high-estrogen follicular cysts, but did not affect androstenedione (A4) production. We propose that Dock6 may be a marker associated with the development of follicular cysts. Additionally, Dock6 may be involved in the development of cystic follicles by suppressing P4 production rather than increasing A4 production in theca cells.

DDRT-PCR approaches applied for preeminent results in the isolation of DETs from fish brain tissues.

Differential Display (DD) is a technique widely used in studies of differential expression. Most of these analyses, especially those involving fish species, are restricted to species from North America and Europe or to commercial species, as salmonids. Studies related to South American fish species are underexplored. Thus, the present work aimed to describe DD technique modifications in order to improve outcomes related to the isolation of DETs (Differentially Expressed Transcripts), using Leporinus macrocephalus, a large commercially exploited South American species, as a fish design. Different DDRT-PCR approaches were applied to brain samples and the products of the reactions were analyzed on 6% polyacrylamide gels stained with 0.17% Silver Nitrate (AgNO3). The use of PCR reactions under high stringency conditions and longer oligonucleotides based on VNTR (Variable Number of Tandem Repeats) core sequences led to better results when compared to low stringency PCR conditions and the use of decamer oligonucleotides. The improved approach led to the isolation of differentially expressed transcripts on adult males and females of L. macrocephalus. This study indicates that some modifications on the DDRT-PCR method can ensure isolation of DETs from different fish tissues and the development of robust data related to this approach.

A putative miR172-targeted CeAPETALA2-like gene is involved in floral patterning regulation of the orchid Cymbidium ensifolium.

APETALA2 plays critical roles in establishing meristem and organ identity during plant floral development. In this study, we obtained a CeAP2-like gene by using the mRNA differential display technique to analyze the wild type and a multitepal mutant of the orchid Cymbidium ensifolium. The full-length cDNA encoding the CeAP2-like transcription factor shows significant similarity to the cDNA of AP2 from Erycina pusilla and contains nucleotides complementary to miR172. Using a transient gene expression system of Arabidopsis protoplasts, we found that the accumulation of CeAP2-like protein and transcripts was negatively regulated by miR172, indicating this gene as a putative target of miR172. Northern blotting revealed that CeAP2-like is dominantly expressed in the sepals and petals of the wild-type flower, and shows low expression in the gynostemium. In contrast, the accumulation of CeAP2-like transcripts decreased significantly, especially in the central part of the mutant flower, corresponding to its abnormal petals and the absence of the gynostemium. Furthermore, we found an antagonistic expression pattern between CeAP2-like and AGAMOUS in the wild type, representing A- and C-class genes that specify floral organ fate. However, this antagonistic distribution was modified in the multitepal mutant, and both genes showed lower expression than that in the wild type. This result suggested that the balance between CeAP2-like and AGAMOUS activity was important for the regulation of floral patterning in C. ensifolium. This study represents the first report on a class A gene and its regulatory role for floral development in the orchid C. ensifolium.

Protective effect of oregano water extract against Carbon tetrachloride induced Hepatotoxicity in female rats

Carbon tetrachloride (CCL4) is one of the most toxic chemicals causes oxidative stress in the living organisms. Our experiment started with twenty female rats that were divided into 4 groups; The 1 st group G1 served as control group, 2 nd group G2 injected twice a week with CCL4 diluted in olive oil (1:1 v/v), 3 rd group G3 were fed daily for 14 days with 1ml of Oregano water extract and the 4 th group G4 were injected with CCL4 plus daily feeding with 1ml of Oregano water extract. The biochemical results indicated a significant elevation in the liver and kidney functions including ALT, AST, creatinine, uric acid and urea for the 2 nd group due to the toxicity induced by the CCL4. The levels of ALT, AST, creatinine, urea and uric acid were significantly decreased after the administration of oregano water extract in the 4 th group. Lipid profile was evaluated in the four groups by measuring the levels of total cholesterol, HDL-C and triglycerides. The variation noticed by the over and down expression of the liver genes due to CCl4 was explored. These up and down regulated genes were tracked using differential display technique.

Molecular defense response of mycorrhizal bean plants infected with Rhizoctonia solani

A time course study was conducted to investigate disease development and molecular defense response in common bean (Phaseolus vulgaris L.) plants colonized by a mixture of five arbuscular mycorrhizal (AM) fungi, namely, Glomus mosseae, G. intraradices, G. clarum, Gigaspora gigantea, and Gigaspora margarita, and post-infected with the soil-borne pathogen Rhizoctonia solani. Results showed that pre-colonization of bean plants by AM fungi significantly reduced disease severity and disease incidence. DNA fingerprinting using the differential display technique revealed a genetic polymorphism (86.8 %) in bean plants that resulted from the colonization by AM fungi. Two genetic mechanisms were recorded: (1) switching on of new genes and (2) induction of other active genes, including the defense genes chitinase and β-1,3-glucanase, to a highly expressed state.

Primarily screening and analyzing ESTs differentially expressed in rats' primary liver cancer.

To screen and analyze key express sequence tags (ESTs) which were differentially displayed in every period of SD rats' primary hepatic carcinoma and reveal the molecular mechanism of carcinogenesis. Using diethylnitrosamine (DENA) as a cancerigenic agent, animal models with different phases of primary hepatic cancer were constructed in SD rats. Rats were respectively sacrificed at d 14, d 28, d 56, d 77, d 105 and d 112 after the rats received DENA by gavage, then the livers were harvested. One part of the livers was classified according to their pathological changes, while the other was reserved for molecular mechanism studies on hepatocarcinogenesis. The differentially expressed genes were isolated from both normal and morbid tissues by mRNA differential display technique (DDRT-PCR). After the fragments were sequenced, bioinformatics were used to analyze the results. Twelve differentially expressed cDNA fragments were obtained. Nine fragments had the homology with known cDNA clones, especially EST-7 was similar to BN/SsNHsdMCW mitochondrion gene and the identity was 100% which suggested EST-7 may be the part of BN/SsNHsdMCW mitochondrion gene. In contrast, other three fragments (EST-1, EST-3 and EST-5) had extremely low identity to any genes registered in GENBANK databases. BN/SsNHsdMCW mitochondrion gene was expressed in different periods of hepatocarcinogenesis. Moreover, EST-1, EST-3 and EST-5 were suggested to contribute to the development of rat hepatocarcinogenesis, and thus may be candidates of new targets of oncogenes or cancer suppressor genes.

Altered Gene Expression: Induction/Suppression in Leek Elicited by Iris Yellow Spot Virus Infection (IYSV) Egyptian Isolate

ABSTRACTLeek (Allium porrum) has become one of the major leafy vegetable crops in Egypt and all over the world. Iris yellow spot virus (IYSV) was observed on leek plants in eight different governorates in Egypt, in fields near to onion fields. The symptoms in infected leek plants included yellowish or tan diamond-shaped or irregularly shaped lesions on leaves and flower stalks. Necrotic areas developed on leaves, and some plants had elongated brown lesions or brown flecks resembling thrips injury. Moreover, thrips larvae and adults feed with a punch-and-suck behavior that removes leaf chlorophyll, causing white to silver patches and streaks, which were observed on all infected plants. The plant samples were collected and subjected to ELISA test using IYSV polyantiserum. The results revealed that about 90% of the collected samples with symptoms were positive. For further confirmation, the ELISA positive samples were subjected to PCR amplification using nuclear coat protein specific primers. The PCR results were in agreement with the results obtained by ELISA. Thrip tabaci adults were reared on the infected plant and biological transfer was performed onto new healthy plants. After 15 days to 4 weeks post inoculation, symptoms were observed on the plant. Biologically infected plant samples were collected at different times after thrips-inoculation and the extracted RNA was subjected to Real Time PCR using the coat protein gene primers. The results showed that the expression of the coat protein fluctuated but reached its peak on day five (264%). Differential display technique was performed on the newly infected plant tissues to identify changes in gene expression in leek elicited by IYSV that causes a symptomatic phenotype. Both up- and down- regulated genes were observed in infected plants conjugated with the healthy ones. Sequence analysis of the up-regulated genes was performed and the encoding sequence analysis showed that the obtained genes include: MFS family protein, Pathogenesis- related protein, Mitogen-activated protein kinase, N-acetylserotonin O-methyltransferase-like protein, Serine/threonine-protein kinase, and Putative Retrosat2 Ty3-Gypsy retroelement. On the other hand, only one down-regulated gene was identified, alpha- tubulin suppressor-like protein. Most of the identified genes are suppress defensin genes (innate/adaptive). It can be concluded that viral infection is capable of inducing a huge number of genes which are important in the plant immune system.

Induction of New Defensin Genes in Tomato Plants via Pathogens-Biocontrol Agent Interaction

Defensins and defensin-like peptides are functionally diverse and commonly presented as an immune reaction between plant and pathogen. Trichoderma viride and Bacillus subtilis as biological control agents, were inoculated into the soil, to suppress the activity of the pathogenic fungi Fusarium oxysporum and Rhizoctonia solani on tomato. The up- and down–regulated genes were examined in both treated and non-treated plants, using differential display technique. In treated plants, many up-regulated genes (21) with different molecular sizes, ranging from 50 to 7000 bp, were observed. Only four up-regulated genes were isolated from plants treated with B. subtilis+R. solani. The sequence analysis revealed that the identified genes were defensin genes; Amino Acid/Auxin Permease Family, Endopolygalacturonase PG1, Fructose-1,6-bisphosphatase and glycosyl transferase. Moreover, chitinase gene, defensin genes (DF1 and DF2) were quantitatively determined using RT-PCR. The comparative expression level of the three induced genes was exponentially increased as a function of time, after the application of the biological control agents. However, while the expression levels of DF1 and DF2 were high in plants infected with either F. oxysporum or R. solani in the beginning of the experiment, the highest expression level of these genes was attained in the tomato plant treated with either T. viride or B. subtilis, after 24 hour post inoculation.

Transcriptomic profiling during the post-harvest of heat-treated Dixiland Prunus persica fruits: common and distinct response to heat and cold.

Cold storage is extensively used to slow the rapid deterioration of peach (Prunus persica L. Batsch) fruit after harvest. However, peach fruit subjected to long periods of cold storage develop chilling injury (CI) symptoms. Post-harvest heat treatment (HT) of peach fruit prior to cold storage is effective in reducing some CI symptoms, maintaining fruit quality, preventing softening and controlling post-harvest diseases. To identify the molecular changes induced by HT, which may be associated to CI protection, the differential transcriptome of peach fruit subjected to HT was characterized by the differential display technique. A total of 127 differentially expressed unigenes (DEUs), with a presence-absence pattern, were identified comparing peach fruit ripening at 20°C with those exposed to a 39°C-HT for 3 days. The 127 DEUs were divided into four expression profile clusters, among which the heat-induced (47%) and heat-repressed (36%) groups resulted the most represented, including genes with unknown function, or involved in protein modification, transcription or RNA metabolism. Considering the CI-protection induced by HT, 23-heat-responsive genes were selected and analyzed during and after short-term cold storage of peach fruit. More than 90% of the genes selected resulted modified by cold, from which nearly 60% followed the same and nearly 40% opposite response to heat and cold. Moreover, by using available Arabidopsis microarray data, it was found that nearly 70% of the peach-heat responsive genes also respond to cold in Arabidopsis, either following the same trend or showing an opposite response. Overall, the high number of common responsive genes to heat and cold identified in the present work indicates that HT of peach fruit after harvest induces a cold response involving complex cellular processes; identifying genes that are involved in the better preparation of peach fruit for cold-storage and unraveling the basis for the CI protection induced by HT.

Xyloglucan endotransglycosylase/hydrolase genes from a susceptible and resistant jute species show opposite expression pattern following Macrophomina phaseolina infection

Two of the most widely and intensively cultivated jute species, Corchorus capsularis and Corchorus olitorius, suffer severely from a stem rot disease caused by the fungus Macrophomina phaseolina. Wild jute species, C. trilocularis, shows resistance to this pathogenic fungus. In this study, the technique of differential display was applied to identify genes which are differentially expressed, under both infected and un-infected conditions, between C. trilocularis and C. olitorius var O-72. Two xyloglucan endotransglycosylase/hydrolase (XTH) genes designated CoXTH1 (from Corchorus olitorius) and CtXTH1 (from C.trilocularis) were identified from each of the two species which show different expression patterns upon fungal infection. A steady rise in the expression of CtXTH1 in response to infection was observed by quantitative real time PCR whereas the expression of CoXTH1 was found to be downregulated. Full length sequences of these two genes were determined using primer based gene walking and RACE PCR. This study confirms the involvement of XTH in molecular interactions between M. phaseolina and jute. However, it remains to be explored whether XTH is an essential component of the signaling pathway involved in plant-fungal interaction.

Testis-Specific Cell Adhesion Molecule, CEACAM6-L, Forms Homophilic Interaction at the Cell Adhesion Site in Vitro

Ceacam6-L (carcinoembryonic antigen-related cell adhesion molecule 6-long) has recently been isolated from rat testis by differential display technique followed by RT-PCR and DNA sequence analysis. CEACAM6-L is a member of the immunoglobulin (Ig) superfamily and composed of an Ig-like domain, three IgCAM domains, a transmembrane region, and a short intracellular domain. We previously reported that CEACAM6-L was localized at the interface between Sertoli cells and elongating/elongated spermatids and might be an adhesion molecule contributing to apical ectoplasmic specialization in testis. In this study, we investigate the adhesive capacities and the complex structures of CEACAM6-L, using COS-7 cells as a study model. Transfection and immunoprecipitation experiments showed that CEACAM6-L expressed in the cells was distributed to the plasma membrane and interacted homophilically between transfected COS-7 cells. A chemical cross-linking experiment and a binding assay with recombinant CEACAM6-L proteins suggested that CEACAM6-L could form trans-tetra complexes, constructed by cis-homodimers at the adhesion site between COS-7 cells expressing CEACAM6-L. We hypothesized that CEACAM6-L expressed in both germ cells and Sertoli cells forms homophilic trans-tetra complexes between these cells in the seminiferous epithelium.

Gene expression indicates a zone of heterocyst differentiation within the thallus of the cyanolichen Pseudocyphellaria crocata

Development of the symbiotic association in the bipartite lichen Pseudocyphellaria crocata was investigated by characterizing two regions of the thallus. Thallus organization was examined using microscopy. A HIP1-based differential display technique was modified for use on Nostoc strains, including lichenized strains. Northern hybridization and quantitative real-time polymerase chain reaction were used to confirm differential display results, and determine expression levels of key cyanobacterial genes. Photosystem II yield across the thallus was measured using pulse-amplitude modulated fluorescence. Microscopy revealed structural differences in the thallus margins compared with the centre and identified putative heterocysts in both regions. Differential display identified altered transcript levels in both Nostoc punctiforme and a lichenized Nostoc strain. Transcript abundance of cox2, atpA, and ribA was increased in the thallus margin compared with the centre. Expression of cox2 is heterocyst specific and expression of other heterocyst-specific genes (hetR and nifK) was elevated in the margin, whereas, expression of psbB and PSII yield were not. Structural organization of the thallus margin differed from the centre. Both regions contained putative heterocysts but gene expression data indicated increased heterocyst differentiation in the margins where photosystem II yield was decreased. This is consistent with a zone of heterocyst differentiation within the thallus margin.

Molecular characterization of the hexose transporter gene in benznidazole resistant and susceptible populations of Trypanosoma cruzi.

BackgroundHexose transporters (HT) are membrane proteins involved in the uptake of energy-supplying glucose and other hexoses into the cell. Previous studies employing the Differential Display technique have shown that the transcription level of the HT gene from T. cruzi (TcrHT) is higher in an in vitro-induced benznidazole (BZ)-resistant population of the parasite (17 LER) than in its susceptible counterpart (17 WTS).MethodsIn the present study, TcrHT has been characterized in populations and strains of T. cruzi that are resistant or susceptible to BZ. We investigated the copy number and chromosomal location of the gene, the levels of TcrHT mRNA and of TcrHT activity, and the phylogenetic relationship between TcrHT and HTs from other organisms.ResultsIn silico analyses revealed that 15 sequences of the TcrHT gene are present in the T. cruzi genome, considering both CL Brener haplotypes. Southern blot analyses confirmed that the gene is present as a multicopy tandem array and indicated a nucleotide sequence polymorphism associated to T. cruzi group I or II. Karyotype analyses revealed that TcrHT is located in two chromosomal bands varying in size from 1.85 to 2.6 Mb depending on the strain of T. cruzi. The sequence of amino acids in the HT from T. cruzi is closely related to the HT sequences of Leishmania species according to phylogenetic analysis. Northern blot and quantitative real-time reverse transcriptase polymerase chain reaction analyses revealed that TcrHT transcripts are 2.6-fold higher in the resistant 17 LER population than in the susceptible 17 WTS. Interestingly, the hexose transporter activity was 40% lower in the 17 LER population than in all other T. cruzi samples analyzed. This phenotype was detected only in the in vitro-induced BZ resistant population, but not in the in vivo-selected or naturally BZ resistant T. cruzi samples. Sequencing analysis revealed that the amino acid sequences of the TcrHT from 17WTS and 17LER populations are identical. This result suggests that the difference in glucose transport between 17WTS and 17LER populations is not due to point mutations, but probably due to lower protein expression level.ConclusionThe BZ resistant population 17 LER presents a decrease in glucose uptake in response to drug pressure.

Screening and cloning of differentially expressed genes in Dendrobium nobile induced by orchid mycorrhizal fungus promoting the growth

Appropriate mycorrhizal fungi could effectively promote plant growth and development. Our previous research results showed that the growth of Dendrobium nobile was obviously promoted under inoculating one orchid mycorrhizal fungi, Epulorhiza sp. AR-18. To understand the growth-promoting molecular mechanisms, differential displayed real time polymerase chain reaction (DDRT-PCR), reverse Northern blot and Southern blot were used to isolate and identify genes whose transcription were altered in cultured D. nobile plants that were treated with Epulorhiza sp. AR-18. Amplified by 8 primer combinations from one anchor primers and 8 random primers, a total of 14 complementary DNA (cDNA) fragments including 12 differentially expressed cDNA bands were isolated. Reverse northern blot analysis showed that only 2 genes were differentially displayed cDNA bands. One band was an especially expressed fragment, expressed in the treated group but not in the control; while another was a differentially expressed fragment, weak in the treated and strength in the control. Southern blot analysis demonstrated that the two gene fragments were from the plant and not from the fungus. Sequence analysis and database searches revealed no significant homology to any known sequences. The results suggested that the usefulness of messenger RAN (mRNA) differential display technique for the detection of differentially expressed genes in D. nobile whose growth could be promoted by mycorrhizal fungi. Keywords: Dendrobium nobile , differential displayed real time polymerase chain reaction (DDRT-PCR), orchid mycorrhizal fungus, Epulorhiza sp., reverse northern blot

Regulatory T cells control tolerogenic versus autoimmune response to sperm in vasectomy

The mammalian uterus is normally not receptive to embryo implantation except during the very limited ‘window of implantation’. To identify genes that may be responsible for this phenomenon the technique of RNA differential display (DD-PCR) was applied to implantation and inter-implantation sites on day 4.5 of pregnancy in the mouse, the time at which the blastocyst becomes attached to the endometrium. Three of these genes were identified as splicing factor SC35, calbindin-D9k and monoclonal non-specific suppressor factorβ (MNSFβ). Expression of SC35 mRNA, which is responsible for removal of introns from pre-mRNA, is much higher in implantation than in interimplantation sites during pregnancy. Expression of alternatively spliced mRNAs for SC35 is differentially regulated by early pregnancy and steroid hormones. By contrast, calbindin-D9k, a regulator of calcium, is upregulated by progesterone and its mRNA increases in the uterus during early pregnancy compared with during the cycle, although it is significantly lower in implantation sites than in interimplantation sites on days 4.5–5.5 of pregnancy, but subsequently becomes barely detectable in both sites. The mRNA for calbindin-D9k is predominantly in endometrial luminal epithelium. MNSFβ, a cytokine involved in regulation of the immune system, showed lower expression at implantation sites than interimplantation sites on day 4.5 of pregnancy, when embryos first attach to the uterus and initiate implantation, and on day 5.5 when implantation has advanced. Immunohistochemically, the protein was localized to endometrial stromal cells in the non-pregnant uterus, but disappeared as decidualization progressed. The precise roles of these three proteins in the process of embryo implantation remains to be determined. Homologues of the proteins may contribute to the development of the ‘window of implantation’ in the human and hence be appropriate targets for new post-coital contraceptives or may be manipulated to improve fertility.