Niranjan Das

Uterine preparation for implantation in the mouse is associated with coordinate expression of estrogen-responsive finger protein and estrogen receptor.

Estrogen‐responsive finger protein (Efp) is a member of the RING finger‐containing proteins. It is a putative transcription regulator that is speculated to amplify estrogen actions in the target organs. The present study examined the temporal and cell‐type specific expression to Efp mRNA in the periimplantation mouse uterus (days 1–8) by Northern and in situ hybridization. Consistent with previous observation, a 6.0‐kb transcript was detected in uterine RNA samples. The steady‐state levels of Efp mRNA in whole uterine RNAs exhibited modest fluctuations during the periimplantation period. However, results of in situ hybridization showed cell‐specific distribution of Efp mRNA in the periimplantation uterus in a temporal manner. On days 1–2 of pregnancy, distinct autoradiographic signals for Efp mRNA were detected in uterine luminal and glandular epithelia. However, on days 3 and 4, the accumulation of Efp mRNA occurred in stromal cells, in addition to its presence in the epithelium. After initiation of implantation on day 5, signal intensity was higher in stromal cells immediately surrounding the implantation chamber. However, on days 6–8, Efp mRNA was localized throughout the deciduum. To determine whether ovarian steroids influence the uterine expression of this gene, cell‐specific localization of Efp mRNA was examined in the adult ovariectomized mouse uterus at 12 hr and 24 hr after an injection of estradiol‐17β (E2) or progesterone (P4). An injection of E2 caused a modest increase in Efp mRNA levels in the luminal and glandular epithelia, while stromal cell accumulation occurred after an injection of P4. These results suggest that Efp is involved in P4/E2‐mediated uterine cellular proliferation and/or differentiation. Since previous studies showed that Efp is colocalized with estrogen receptor (ER) in target cells, we also examined the cell‐specific nuclear localization of ER in the periimplantation mouse uterus by immunohistochemistry. On days 1–2 of pregnancy, nuclear staining was distinct in the luminal epithelium and glandular epithelium. In contrast, nuclear staining was noted in stromal cells on days 3–4. However, glandular epithelium showed distinct staining during this period. On day 5, stromal cells surrounding the lumen at the mesometrial site were ER‐positive. On days 6–8, the intensity of nuclear staining was very low, and limited to the cells adjacent to the luminal epithelium. The coordinate expression of Efp and ER in specific uterine cells during the preimplantation period (days 1–4) was consistent with the absolute requirement for estrogen in the preparation of the uterus for implantation. Since the amount of estrogen required for the preparation of the uterus is minuscule as compared to that of P4, these results suggest that the coexpression of Efp with ER is involved in amplifying the estrogen effects required for uterine cell proliferation and/or differentiation during implantation. In contrast, discoordinate expression of Efp and ER during the postimplantation period (days 5–8) suggests primary dependence of the decidualization process on P4, but not estrogen. Mol. Reprod. Dev. 46:499–506, 1997.

Molecular cloning and characterization of granule-bound starch synthase I (GBSSI) alleles from potato and sequence analysis for detection of cis-regulatory motifs

Granule-bound starch synthase (GBSS), an isoform of starch synthase, is responsible for amylose synthesis in all starch containing tissues of potato (Solanum tuberosum L.), and in other plant species. In potato, the importance of GBSSI gene function is realized through a number of published reports on both basic and applied research. Here genomic DNA was isolated from six high-yielding Indian potato cultivars, and the cultivar Desiree. Polymerase chain reactions (PCR) were carried out at varying annealing temperatures using GBSSI gene-specific primers and genomic DNA from the individual potato cultivars. Analysis of PCR amplification products provided an overview of GBSSI allelic composition of these potato cultivars which was hitherto unknown. Two partial GBSSI alleles were isolated from one of the Indian potato cultivars. The distinguishing sequence features between them were shown. Multiple sequence alignment of GBSSI alleles from different potato cultivars provided a clear view for comparison between their different regions. Base composition analysis revealed a notably higher GC rich region immediately upstream of the transcription start site of the GBSSI alleles, unlike other potato genes. Preliminary sequence analysis detected a number of different cis-regulatory sequence motifs in the GBSSI alleles which were not documented in earlier reports. The GBSSI sequences from different potato cultivars reported to date were sorted out into four groups as shown in the phylogenetic tree. A promoter was isolated from one GBSSI allele of this study, and found to be strong and tuber-specific in the potato cultivar under field conditions as examined by histochemical GUS assay.

Simplified extraction of good quality genomic DNA from a variety of plant materials

Depending on the nature and complexity of plant material, proper method needs to be employed for extraction of genomic DNA, along with its performance evaluation by different molecular techniques. Here, we optimized and employed a simple genomic DNA isolation protocol suitable for a variety of plant materials covering in vitro grown tender plantlets to relatively complex plant tissues such as field grown mature potato leaves and tubers. Unlike other methods, no detergent was included in the isolation steps. This protocol, based on Dellaporta’s method as reported earlier, worked efficiently both at small and miniscale during handling large number of plant materials. DNA yield was found to be in the range of 70 to 120 μg per gram of the plant material; sufficient for most of the molecular techniques. Purity of DNA was checked by A 260 /A 280 ratio, and restriction analyses including the isoschizomers HpaII and MspI . The DNA preparations were successfully used in polymerase chain reactions using genespecific primers for cloning of different genes. Prolonged storage did not affect the quality of the DNA samples. Taken together, this method could be a reliable substitute to frequently used chemical cetyl trimethylammonium bromide (CTAB) and commercial kits-based plant DNA isolation protocols. Key words : Plant materials, genomic DNA isolation, restriction analyses, HpaII and MspI isoschizomers, genespecific primers, polymerase chain reaction (PCR), molecular cloning, DNA storage

Selective detection of Mg2+ ions via enhanced fluorescence emission using Au–DNA nanocomposites

The biophysical properties of DNA-modified Au nanoparticles (AuNPs) have attracted a great deal of research interest for various applications in biosensing. AuNPs have strong binding capability to the phosphate and sugar groups in DNA, rendering unique physicochemical properties for detection of metal ions. The formation of Au–DNA nanocomposites is evident from the observed changes in the optical absorption, plasmon band, zeta potential, DLS particle size distribution, as well as TEM and AFM surface morphology analysis. Circular dichroism studies also revealed that DNA-functionalized AuNP binding caused a conformational change in the DNA structure. Due to the size and shape dependent plasmonic interactions of AuNPs (33–78 nm) with DNA, the resultant Au–DNA nanocomposites (NCs) exhibit superior fluorescence emission due to chemical binding with Ca2+, Fe2+ and Mg2+ ions. A significant increase in fluorescence emission (λex = 260 nm) of Au–DNA NCs was observed after selectively binding with Mg2+ ions (20–800 ppm) in an aqueous solution where a minimum of 100 ppm Mg2+ ions was detected based on the linearity of concentration versus fluorescence intensity curve (λem = 400 nm). The effectiveness of Au–DNA nanocomposites was further verified by comparing the known concentration (50–120 ppm) of Mg2+ ions in synthetic tap water and a real life sample of Gelusil (300–360 ppm Mg2+), a widely used antacid medicine. Therefore, this method could be a sensitive tool for the estimation of water hardness after careful preparation of a suitably designed Au–DNA nanostructure.