• Volume 21, Issue 6

      December 1996,   pages  735-860

    • A facile method for the construction of synthetic genes

      Pradhyuman K Singh Bijaya K Sarangi Rakesh Tuli

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      A simple protocol for rapid assembly of chemically synthesized deoxyoligonucleotides into double stranded DNA is described. Several parameters of a ligation-free method were investigated to allow efficient assembly of a large number of oligonucleotides into double stranded DNA by polymerase chain reaction. Synthesis of a 701 bp DNA was carried out in a single reaction by assembling 28 oligonucleotides designed with partial overlaps at complementary ends. An estimate of error rate was made by sequencing several independent clones of the synthesized DNA

    • Interactions of 3′ terminal and 5′ terminal regions of physalis mottle virus genomic RNA with its replication complex

      V Srividhya H S Savithri

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      Physalis mottle virus (PhMV) belongs to the tymogroup of positive-strand RNA viruses with a genome size of 6 kb. Crude membrane preparations from PhMV-infectedNicotiana glutinosa plants catalyzed the synthesis of PhMV genomic RNA from endogenously bound template. Addition of exogenous gnomic RNA enhanced the synthesis which was specifically inhibited by the addition of sense and antisense transcripts corresponding to 3′ terminal 242 nucleotides as well as the 5′ terminal 458 nucleotides of PhMV genomic RNA while yeast tRNA or ribosomal RNA failed to inhibit the synthesis. This specific inhibition suggested that the 5′ and 3′ non-coding regions of PhMV RNA might play an important role in viral replication

    • Mapping of assembled epitopic regions of human chorionic gonadotropin reveals proximity of CTPα to the determinant loop β93–100

      N S Srilatha G S Murthy

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      Three overlapping assembled epitopes of βhCG have been mapped using MAb probes and a single step solid phase radioimmunoassay. These epitopes have been shown to be at receptor binding region comprising of the loop region μ Cys93-Cys100. Importance of disulphide bonds in maintaining integrity of these epitopes is assessed. Two MAbs (INN 58 and INN 22) interact with the μ region as well as the β C-terminal peptide, while the other MAb INN 24 interacts with only the μ region. Cross-reactivity pattern with μhCG and hLH as well as the reported crystal structure of hCG substantiates the epitope identification. The results demonstrate utility of MAbs as probes in investigations on three-dimensional structure of gonadotropins

    • Spatial patterning of the distribution of Ca2+ in Dictyostelium discoideum as assayed in fine glass capillaries

      M Azhar Vidyanand Nanjundiah

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      We have shown previously that the Ca2+-specific fluorescent dyes chlortetracycline (CTC) and indo-1/AM can be used to distinguish between prestalk and prespore cells inDictyostelium discoideum at a very early stage. In the present study, pre-and post-aggregative amoebae ofDictyostelium discoideum were labelled with CTC or indo-1 and their fluorescence monitored after being drawn into a fine glass capillary. The cells rapidly form two zones of Ca2+-CTC or Ca2+-indo-1 fluorescence. Anterior (air side) cells display a high level of fluorescence; the level drops in the middle portion of the capillary and rises again to a lesser extent in the posteriormost cells (oil side). When bounded by air on both sides, the cells display high fluorescence at both ends. When oil is present at both ends of the capillary, there is little fluorescence except for small regions at the ends. These outcomes are evident within a couple of minutes of the start of the experiment and the fluorescence pattern intensifies over the course of time. By using the indicator neutral red, as well as with CTC and indo-1, we show that a band displaying strong fluorescence moves away from the anterior end before stabilizing at the anterior-posterior boundary. We discuss our findings in relation to the role of Ca2+ in cell-type differentiation inDictyostelium discoideum.

    • Spontaneous recombination in males ofDrosophila bipectinata

      B N Singh Rakhee Banerjee

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      Spontaneous recombination in males ofDrosophila bipectinata was tested in five wild type laboratory stocks of different geographic origins by using sepia eye and black body colour double recessive mutant stock. The results indicate thatDrosophila bipectinata exhibits spontaneous male recombination. Further, recombination occurs at low rate and there is interstrain variation with respect to the rate of male crossing-over. This is the first report of spontaneous recombination in males ofDrosophila bipectinata.

    • Changes in the haemocyte profile ofSpilostethus hospes (Fab) (Heteroptera: Lygaeidae) in relation to eclosion, sex and mating

      K P Sanjayan T Ravikumar S Albert

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      Five types of haemocytes have been identified in the haemolymph ofSpilostethus hospes. Their morphology and micrometric measurements have been provided. Changes in the total and differential haemocyte population [total haemocyte count (THC) and differtial haemocyte count (DHC)] as well as in the absolute number of haemocytes in circulation have been assessed in relation to eclosion, sex and mating. The haemogram profile was studied prior to and immediately after eclosion and also prior to and after copulation. Though the THC was not significantly different immediately before and after eclosion, there was a significant increase in total count prior to copulation. Mated females registered an increase in total count but there was no appreciable change in the mated males. Granulocytes were the most abundant of the haemocyte types in both the sexes and mating caused a significant increase in the plasmatocyte count in females. Changes in the blood volume as well as the mitotic activity of the haemocytes is also discussed

    • Chronobiotic effect of melatonin following phase shift of light/dark cycles in the field mouseMus booduga

      M Singaravel V K Sharma R Subbaraj N Gopukumar Nair

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      The objective of this study was to assess whether melatonin accelerates the re-entrainment of locomotor activity after 6 h of advance and delay phase shifts following exposure to LD 12:12 cycle (simulating jet-lag/shift work). An experimental group of adult male field mice Mus booduga were subjected to melatonin (1 mg/kg) through i.p. and the control group were treated with 50 % DMSO. The injections were administered on three consecutive days following 6h of phase advance and delay, at the expected time of “lights off”. The results show that melatonin accelerates the re-entrainment after phase advance (29%) when compared with control mice. In the 6 h phase delay study, the experimental mice (melatonin administered) take more cycles for re-entrainment (51%) than the control. Further, the results suggest that though melatonin may be useful for the treatment of jet-lag caused by eastward flight (phase advance) it may not be useful for westward flight (phase delay) jet-lag

    • Do thyroid and testis modulate the effects of pineal and melatonin on haemopoietic variables inClarias batrachus?

      Maya Shedpure Atanu Kumar Pati

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      Involvement of pineal and its major hormone, melatonin, in the process of erythropoiesis in a freshwater catfish,Clarias batrachus has been investigated. The study was conducted during four phases, namely preparatory phase, spawning phase, postspawning phase and late postspawning phase of its annual reproductive cycle. During each phase a fish received either melatonin injections or subjected to pinealectomy. In addition, each fish in all the groups, received either iopanoic acid or cyproterone acetate or vehicle in the morning or late afternoon. Results clearly indicate that melatonin stimulates the rate of erythropoiesis inClarias batrachus. It appears that the extent of stimulation depends upon the phase of the annual reproductive cycle. However, in general, the pineal-or melatonin-induced modulation of blood variables is gonad dependent and thyroid seems to play a time of the day dependent subtle role

    • Kinetics and mechanism of tolerance induction on acclimation ofVillorita cyprinoides (Hanley) to copper and zinc

      B Sathyanathan

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      The typical euryhaline clamVillorita cyprinoides (Hanley) was acclimated to copper and zinc at salinity 13 × 10−3 and < 1 × 10−3 (fresh water). Acclimation enhanced the lethal tolerance, as denoted by dose-survival curves, which was more pronounced after zinc acclimation. In fresh water copper acclimation sensitized the organisms. The copper accumulation trend was significantly changed consequent to metal acclimation, especially after zinc acclimation, indicating some tissue metal regulatory effect. Acclimation to copper equiped the organism to survive for longer periods with increased body burden of copper, while zinc acclimation supressed the uptake of the more toxic ion copper. The earlier report of increased uptake of zinc by this organism during combined exposure with copper is corelated in the present context. The role of metallothionein like protein in providing protection against metal toxicity, the environmental implication of acclimation phenomena are indicated

    • Fatty acid synthesis by isolated leucoplasts from developingBrassica seeds: Role of nucleoside triphosphates and DHAP-shuttle as the source of energy

      Ranjan Gupta Randhir Singh

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      Fatty acid synthesis in leucoplasts isolated from developing seeds ofBrassica campestris was absolutely dependent on external source of ATP. None of the other nucleoside triphosphates could replace ATP in the reaction mixture. Use of ADP alone also resulted in reduced rates of fatty acid synthesis. However, in combination with inorganic phosphate or inorganic pyrophosphate, it improved the rate of fatty acid synthesis, giving up to 50% of the ATP-control activity. Inorganic phosphate or inorganic pyrophosphate alone again did not serve as an energy source for fatty acid synthesis. AMP, alongwith inorganic pyrophosphate could promote fatty acid synthesis to up to 42% of the activity obtained with ATP. The three components dihydroxy acetone phosphate, oxaloacetic acid, inorganic phosphate of dihydroxy acetone phosphate-shuttle together could restore 50% of the activity obtained with ATP. Omission of any one of the components of this shuttle drastically reduced the rate of fatty acid synthesis to 15–24% of the ATP-control activity. Inclusion of ATP in reaction mixtures containing shuttle components enhanced the rate of synthesis over control. The optimum ratio of shuttle components dihydroxy acetone phosphate, oxaloacetic acid, inorganic phosphate determined was 1:1:2. Maximum rates of fatty acid synthesis were obtained when dihydroxy acetate phosphate was used as the shuttle triose. Glyceraldehyde-3-P, 3-phosphoglycerate, 2-phosphoglycerate and phosphoenolpyruvate as shuttle trioses were around 35–60% as effective as dihydroxy acetone phosphate in promoting fatty acid synthesis. The results presented here indicate that although the isolated leucoplasts readily utilize exogenously supplied ATP for fatty acid synthesis, intraplastidic ATP could also arise from dihydroxy acetone phosphate shuttle components or other appropriate metabolites

    • Embryogenesis in flowering plants: Recent approaches and prospects

      K Sankara Rao

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      The overall architectural pattern of the mature plant is established during embryogenesis. Very little is known about the molecular processes that underlie embryo morphogenesis. Last decade has, nevertheless, seen a burst of information on the subject. The synchronous somatic embryogenesis system of carrot is largely being used as the experimental system. Information on the molecular regulation of embryogenesis obtained with carrot somatic embryos as well as observations on sandalwood embryogenic system developed in our laboratory are summarized in this review. The basic experimental strategy of molecular analysis mostly relied on a comparison between genes and proteins being expressed in embryogenic and non-embryogenic cells as well as in the different stages of embryogenesis. Events such as expression of totipotency of cells and establishment of polarity which are so critical for embryo development have been characterized using the strategy. Several genes have been identified and cloned from the carrot system. These include sequences that encode certain extracellular proteins (EPs) that influence cell proliferation and embryogenesis in specific ways and sequences of the abscisic acid (ABA) inducible late embryogenesis abundant (LEA) proteins which are most abundant and differentially expressed mRNAs in somatic embryos. That LEAs are expressed in the somatic embryos of a tree flora also is evidenced from studies on sandalwood. Several undescribed or novel sequences that are enhanced in embryos were identified. A sequence of this nature exists in sandalwood embryos was demonstrated using aCuscuta haustorial (organ-specific) cDNA probe. Somatic embryogenesis systems have been used to assess the expression of genes isolated from non-embryogenic tissues. Particular attention has been focused on both cell cycle and histone genes

    • Acknowledgements

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