Development and Functional Analysis of Novel Genetic Promoters Using DNA Shuffling, Hybridization and a Combination Thereof

BACKGROUND: Development of novel synthetic promoters with enhanced regulatory activity is of great value for a diverse range of plant biotechnology applications. METHODOLOGY: Using the Figwort mosaic virus full-length transcript promoter (F) and the sub-genomic transcript promoter (FS) sequences, we generated two single shuffled promoter libraries (LssF and LssFS), two multiple shuffled promoter libraries (LmsFS-F and LmsF-FS), two hybrid promoters (FuasFScp and FSuasFcp) and two hybrid-shuffled promoter libraries (LhsFuasFScp and LhsFSuasFcp). Transient expression activities of approximately 50 shuffled promoter clones from each of these libraries were assayed in tobacco (Nicotiana tabacum cv. Xanthi) protoplasts. It was observed that most of the shuffled promoters showed reduced activity compared to the two parent promoters (F and FS) and the CaMV35S promoter. In silico studies (computer simulated analyses) revealed that the reduced promoter activities of the shuffled promoters could be due to their higher helical stability. On the contrary, the hybrid promoters FuasFScp and FSuasFcp showed enhanced activities compared to F, FS and CaMV 35S in both transient and transgenic Nicotiana tabacum and Arabidopsis plants. Northern-blot and qRT-PCR data revealed a positive correlation between transcription and enzymatic activity in transgenic tobacco plants expressing hybrid promoters. Histochemical/X-gluc staining of whole transgenic seedlings/tissue-sections and fluorescence images of ImaGene Green™ treated roots and stems expressing the GUS reporter gene under the control of the FuasFScp and FSuasFcp promoters also support the above findings. Furthermore, protein extracts made from protoplasts expressing the human defensin (HNP-1) gene driven by hybrid promoters showed enhanced antibacterial activity compared to the CaMV35S promoter. SIGNIFICANCE/CONCLUSION: Both shuffled and hybrid promoters developed in the present study can be used as molecular tools to study the regulation of ectopic gene expression in plants

Development of Useful Recombinant Promoter and Its Expression Analysis in Different Plant Cells Using Confocal Laser Scanning Microscopy

BACKGROUND: Designing functionally efficient recombinant promoters having reduced sequence homology and enhanced promoter activity will be an important step toward successful stacking or pyramiding of genes in a plant cell for developing transgenic plants expressing desired traits(s). Also basic knowledge regarding plant cell specific expression of a transgene under control of a promoter is crucial to assess the promoter's efficacy. METHODOLOGY/PRINCIPAL FINDINGS: We have constructed a set of 10 recombinant promoters incorporating different up-stream activation sequences (UAS) of Mirabilis mosaic virus sub-genomic transcript (MS8, -306 to +27) and TATA containing core domains of Figwort mosaic virus sub-genomic transcript promoter (FS3, -271 to +31). Efficacies of recombinant promoters coupled to GUS and GFP reporter genes were tested in tobacco protoplasts. Among these, a 369-bp long hybrid sub-genomic transcript promoter (MSgt-FSgt) showed the highest activity in both transient and transgenic systems. In a transient system, MSgt-FSgt was 10.31, 2.86 and 2.18 times more active compared to the CaMV35S, MS8 and FS3 promoters, respectively. In transgenic tobacco (Nicotiana tabaccum, var. Samsun NN) and Arabidopsis plants, the MSgt-FSgt hybrid promoter showed 14.22 and 7.16 times stronger activity compared to CaMV35S promoter respectively. The correlation between GUS activity and uidA-mRNA levels in transgenic tobacco plants were identified by qRT-PCR. Both CaMV35S and MSgt-FSgt promoters caused gene silencing but the degree of silencing are less in the case of the MSgt-FSgt promoter compared to CaMV35S. Quantification of GUS activity in individual plant cells driven by the MSgt-FSgt and the CaMV35S promoter were estimated using confocal laser scanning microscopy and compared. CONCLUSION AND SIGNIFICANCE: We propose strong recombinant promoter MSgt-FSgt, developed in this study, could be very useful for high-level constitutive expression of transgenes in a wide variety of plant cells

EVALUATION OF ROLE OF HER2 EXPRESSION BY IHC IN PREMALIGNANT AND MALIGNANT LESIONS OF THE UTERINE CERVIX – AN INSTITUTIONAL BASED PROSPECTIVE STUDY.: Her2 expression by IHC in cervical malignant and premalignant lesions.

Background: The most frequent malignancy in Indian women is uterine cervix cancer. According to the clinical stage of the tumour and the surgical findings, surgery is the primary mode of treatment, coupled with radiation and chemotherapy. Despite the use of cisplatin-based combination treatment, patients with advanced or recurring illness have a poor prognosis. In order to replace or supplement the present therapy for these patients, it is becoming more and more important to identify novel therapeutic targets. The goal of the current investigation was to determine whether Her-2/neu expression was present. Methods: In this investigation, 81 cervical specimens were used. These cases included squamous cell carcinoma and adenocarcinoma diagnoses. BioGenex monoclonal mouse anti-human HER-2/neu Receptor IgG1 antibody was used for HER-2/neu immunostaining. Results: Malignant lesions showed higher expression of HER-2/neu compared to premalignant lesions. Additionally, there was a correlation between staining intensity and lymph node metastasis, clinical stage, and malignant tumour grade. Conclusion: Oncoprotein HER-2/neu overexpression has been linked to aggressive biological behaviour, a poor prognosis, and the propensity for metastatic spread

EVALUATION OF ALTERED PTEN EXPRESSION BY IHC AS A DIAGNOSTIC MARKER IN HYPERPLASTIC AND NEOPLASTIC ENDOMETRIUM OBSERVATIONAL DESCRIPTIVE STUDY.: Altered PTEN expression by IHC in endometrial disorders

Introduction: Endometrial carcinoma is a common cancer affecting women. Different molecular alterations have been described in endometrioid endometrial carcinoma (EC). Among them the most frequently altered is the loss of the PTEN protein, a tumor suppressor gene, leading to microsatellite instability. The purpose of this study was to evaluate the expression pattern of PTEN gene in normal proliferative, hyperplastic, and neoplastic endometrium. Objective: To observe the altered expression of PTEN in proliferative endometrium, endometrial hyperplasia, and endometrial carcinoma. Methods: This study was an observational descriptive study conducted in the Department of Pathology, M.K.C.G. Medical College, Brahmpur,Odhisha. over a two-year period in which immuno-histochemical evaluation of PTEN expression was done in 146 patients. Results: PTEN immunoreactivity was present in all normal proliferative endometrium, all simple hyperplasia, 50% of atypical hyperplasia, and in none of EC (P < 0.001). The intensity of PTEN reaction was significantly higher in a group with proliferative endometrium than in atypical hyperplastic endometrium and EC (P < 0.001).  Conclusion: PTEN expression was significantly higher in cyclical endometrium than in atypical hyperplasia and endometrioid carcinoma and PTEN immunostaining may be a new and effective tool for screening malignant and premalignant endometrial lesion

Histochemical localization of GUS activity in transgenic tobacco and Arabidopsis seedlings generated for the respective promoter-<i>GUS</i> constructs.

<p>(a) Histochemical staining of transgenic tobacco seedlings expressing GUS under the control of respective promoter constructs. Photographs were taken using Leica DM LS2 microscope (at 10× magnification) attached to a CCD camera. (b) Histochemical staining of transgenic tobacco stem cross sections expressing GUS under the control of respective promoter constructs. Photographs were taken using Leica DM LS2 microscope (at 10× magnification) attached to a CCD camera. (c) Histochemical staining of transgenic tobacco leaf petiole cross sections expressing GUS under the control of respective promoter constructs. Photographs were taken using Leica DM LS2 microscope (at 10× maginification) attached to a CCD camera. (d) Histochemical staining of transgenic <i>Arabidopsis</i> seedling expressing GUS under the control of respective promoter constructs. Photographs were taken using Leica DM LS2 microscope (at 10× maginification) attached to a CCD camera.</p

Comparative expression analysis (in Agro-infiltration assay) of selected shuffled promoters screened in protoplast transient assay.

<p>In histogram shown, twenty six shuffled promoters giving good activity in transient tobacco protoplast assay selected from six libraries: 4 from LssF, 3 from LssFS, 2 from LmsFFS, 6 from LmsFSF, 4 from LhsFSuasFScp and 7 from LhsFuasFScp; were taken for further comparative expression analysis along with the CaMV35S promoter (35S), parent promoters (F and FS) and hybrid promoters (FuasFScp and FSuasFcp) in Agro-infiltration experiment using whole tobacco plants (<i>Nicotiana tabacum</i> samsun NN) as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031931#s2" target="_blank">Materials and Methods</a>”. The average GUS activity (n mole MU/min/mg protein ± SD) of three replicates of each construct was presented in the histogram. Error bar shows the 95% confidence intervals of the mean. Statistical (one-way analysis of variance, ANOVA) analysis showed an extremely significant <i>P</i> value of <0.001. Empty vector with no GUS gene was treated as ‘Control’.</p

Comparative expression analysis of promoters and promoter fragments fused with reporter genes (GFP and GUS) using CLSM in tobacco transient protoplast assay.

<p>(a) GFP constructs of CaMV35S promoter, parent promoters (F and FS), hybrid promoters (FuasFScp and FSuasFcp), promoter fragments (FScp, Fcp, FSuas and Fuas) were created as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031931#s2" target="_blank">Materials and Methods</a>”. The GFP fluorescence intensity (in gray scale unit) was measured in protoplast transient assay using CLSM as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031931#s2" target="_blank">Materials and Methods</a>”. The average GFP intensity ± SD of two replicates of each construct was presented in the histogram. Error bar shows the 95% confidence intervals of the mean. Statistical (one-way analysis of variance, ANOVA) analysis showed an extremely significant <i>P</i> value of <0.01. Empty vector ‘Control’ with no GFP gene was shown. (b) GUS constructs of CaMV35S promoter, parent promoters (F and FS), hybrid promoters (FuasFScp and FSuasFcp), and promoter fragments (FScp, Fcp, FSuas and Fuas) were generated as described in“<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031931#s2" target="_blank">Materials and Methods</a>”. The GUS activity (n mole MU/min/mg protein) was measured in protoplast transient assay using CLSM as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031931#s2" target="_blank">Materials and Methods</a>”. The average GUS activity ± SD of two replicates of each construct was presented in the histogram. Error bar shows the 95% confidence intervals of the means. Statistical (one-way analysis of variance, ANOVA) analysis showed an extremely significant <i>P</i> value of <0.02. Empty vector ‘Control’ with no GUS gene was shown.</p

Comparative expression analysis of hybrid promoters (FuasFScp and FSuasFcp), and CaMV 35S promoter fused with human alpha defensin-1 (HNP-1) gene in tobacco protoplasts.

<p>The human alpha defensin-1 (HNP-1) gene was expressed in protoplasts under the control of CaMV 35S, FuasFScp and FSuasFcp promoters as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031931#s2" target="_blank">Materials and Methods</a>”. Antibacterial assay of human alpha defensin-1 (HNP-1) protein extracted from tobacco protoplasts was performed using (a) <i>Escherichia coli</i> cell and (b) <i>Staphylococcus aureus</i> cell as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031931#s2" target="_blank">Materials and Methods</a>” section. The data were presented as a mean of two independent experiments with respective SD.</p

CLSM based analysis of localized GUS expression in transgenic tobacco expressing respective promoter-<i>GUS</i> constructs.

<p>(a) Bright field confocal images of transverse sections of transgenic tobacco stem expressing GUS under the control respective promoter constructs. (b) Fluorescence images of transverse sections of transgenic tobacco stem expressing GUS under the control of respective promoter constructs. (c) Superimposed (bright field and fluorescent) images of transverse sections of tobacco stem expressing GUS under the control of respective promoter constructs. (d) Bright field confocal images of transgenic tobacco root expressing GUS under the control of respective promoter constructs. (e) Fluorescence images of tobacco root expressing GUS under the control of respective promoter constructs. (f) Superimposed (bright field and fluorescent) images of tobacco root expressing GUS under the control of respective promoter constructs. Images were captured using CLSM as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031931#s2" target="_blank">Materials and Methods</a>”. Plant samples used were grown aseptically under tissue culture conditions. All figures under row a, b and c were presented in 500 micrometer (µm) scale while figures under row d, e and f were presented in 250 micrometer (µm) scale except figures obtained from promoter FSuasFcp [presented using 100 micrometer (µm) scale].</p