Synthesis and Hybridization Studies of Oligonucleotide Sequences with Modified Fluorescent Nucleoside Analogs

Abstract

Two complementary oligodeoxynucleotide hexamers CATGAA and TTCATG and a pentamer with a fluorescent nucleoside analog viz. 9-N-(2′-deoxy-β-D-ribofuranosyl) carbazole (C*) incorporated into it, TTC*ATG were synthesized and characterised by spectroscopic and chromatographic studies. The comparative fluorescent studies of the two nucleoside analogs viz. 9-N-(2′-deoxy-β-D-ribofuranosyl) acridone and its carbazole analog (C*) have been carried out under different experimental conditions. The effect on fluorescence by incorporation of (C*) into the sequence and its subsequent hybridization with the complementary sequence have been studied.     

Importance of partially unfolded conformations for Mg(2+)-induced folding of RNA tertiary structure: structural models and free energies of Mg2+ interactions

RNA molecules in monovalent salt solutions generally adopt a set of partially folded conformations containing only secondary structure, the intermediate or I state. Addition of Mg2+ strongly stabilizes the native tertiary structure (N state) relative to the I state. In this paper, a combination of experimental and computational approaches is used to estimate the free energy of the interaction of Mg2+ with partially folded I state RNAs and to consider the possibility that Mg2+ favors "compaction" of the I state to a set of conformations with a higher average charge density. A sequence variant with a drastically destabilized tertiary structure was used as a mimic of I state RNA; as measured by small-angle X-ray scattering, it adopted a progressively more compact conformation over a wide Mg2+ concentration range. Average free energies of the interaction of Mg2+ with the I state mimic were obtained by a fluorescence titration method. To interpret these experimental data further, we generated molecular models of the I state and used them in calculations with the nonlinear Poisson-Boltzmann equation to estimate the change in Mg2+-RNA interaction free energy as the average I state dimensions decrease from expanded to compact. The same models were also used to reproduce quantitatively the experimental difference in excess Mg2+ between N and I states. On the basis of these experiments and calculations, I state compaction appears to enhance Mg2+-I state interaction free energies by 10-20%, but this enhancement is at most 5% of the overall Mg2+-associated stabilization free energy for this rRNA fragment.     

Reprogramming of Protein Synthesis from a Developmental to a Germinative Mode Induced by Desiccation of the Axes of Phaseolus vulgaris

Immature seeds of Phaseolus vulgaris cv Taylor's Horticultural removed from the pod at 32 days of development do not germinate unless first subjected to desiccation. Our results show that premature drying not only redirects metabolism from a developmental to a germination program but it does so permanently, thus effecting an irreversible switch. This is shown by in vitro protein synthesis, and analysis of poly(A)⁺ mRNA with a cDNA probe specific for phaseolin message. For example, the pattern of proteins synthesized in vitro by the mRNA fraction from fresh and prematurely dried axes show strong similarities; on the other hand, the mRNA population from rehydrated axes code for a different set of proteins. Also, the message for phaseolin is preserved following the normal maturation process and premature desiccation of seeds. Following rehydration of immature seeds at the desiccation-tolerant stage, this message is no longer detectable in the axes.     

An analysis of avoidance generalization and food preference transitivity in goldfish,Carassius auratus

To examine the effect of novelty in food selection by fishes, goldfish were trained by feeding them on red or green food pellets. Individual fish and fish in shoals of two, three, and five were then given a choice between equal numbers of familiar pellets and novel (yellow) pellets. In experimental groups, the yellow pellets were soaked in 15% quinine hydrochloride to make them unpalatable. Fish were next presented with equal numbers of familiar and a second novel pellet. It was expected that fish would generalize from their experience with the unpalatable pellets and demonstrate avoidance to sampling a second novel food item. However, as groups, neither controls nor experimentals were reluctant to sample the second novel pellets. Also, fish did not eat significantly more familiar than novel pellets when both were palatable. A third set of experiments examined food preference transitivity in which each pellet type was presented alone and in three pair-wise combinations to individual fish. Although red and yellow pellets were preferred over green, they were preferred equally, indicating an absence of transitivity in pellet choice and, perhaps, a “preference” for a mixture of red and yellow pellets.     

Heterogeneity in Composition along the Length of Cuscuta filaments

Considerable variations in the content of free amino acids, elhanol-soluble carbohydrates, starch, protein, chlorophyll, phylic acid, RNA and DNA exist in different regions of the long filaments of Cuscuta reflexa. The distinction is especially pronounced when comparison is made between the hanstoria-bearing curls of the parasite and the apical portions of the overhanging filament.     

Assembly and channel opening in a bacterial drug efflux machine

Drugs and certain proteins are transported across the membranes of Gram-negative bacteria by energy-activated pumps. The outer membrane component of these pumps is a channel that opens from a sealed resting state during the transport process. We describe two crystal structures of the Escherichia coli outer membrane protein TolC in its partially open state. Opening is accompanied by the exposure of three shallow intraprotomer grooves in the TolC trimer, where our mutagenesis data identify a contact point with the periplasmic component of a drug efflux pump, AcrA. We suggest that the assembly of multidrug efflux pumps is accompanied by induced fit of TolC driven mainly by accommodation of the periplasmic component.     

RNase E, an RNA processing enzyme from Escherichia coli.

An activity, RNase E, was purified about 100-fold from Escherichia coli cells, it can process p5 rRNA from a 9 S RNA molecule which accumulates in a mutant of E. coli defective in the maturation of 5 S rRNA. The enzyme requires Na+, K+, or NH4+, and Mg2+ or Mn2+. The molecular weight of the enzyme is about 70,000 and its pH optimum is 7.6 to 8.0. Its temperature optimum is around 30 degrees C, and it can be irreversibly inactivated at 50 degrees C. It has a very high degree of specificity but the reaction can be inhibited by nonspecific RNAs. We interpret its mode of action in producing p5 RNA as being accomplished in two steps, 9 S RNA is first processed to 7 S and 4 S, and subsequently 7 S is further processed to p5.     

Screening and selection of media components for lactic acid production using Plackett–Burman design

Plackett–Burman design was used to efficiently select important media components influencing lactic acid production in a two step screening procedure. A total of 36 screening experiments were conducted for studying the effect of various media components such as carbon and nitrogen (simple and complex) sources, minerals/buffering agents and a specific inducer for the production of lactic acid by Lactobacillus plantarum NCIM 2084. The eleven ingredients chosen after the first screening experiments were further screened by a Plackett-Burman design consisting of 12 experiments. Liquefied starch, wheat bran extract, ammonium nitrate, manganese sulphate and sodium acetate were chosen as promising ingredients for further optimisation studies. The highest yield of 41.9?g/l of lactic acid was obtained at the end of 24 hours of fermentation which corresponded to 90% conversion, on the basis of sugar supplied.