Incorporation of a range of higher concentrations of CuSO 4 _ 5H 2 O in MS medium significantly enhanced direct shoot bud induction and proliferation from cultured leaf and nodal explants taken from mature plants of Stevia rebaudiana Bertoni . Shoot bud induction medium was supplemented with BAP (2.2 µ M) and NAA (2.8 µ M). When the concentration of CuSO 4 _ 5H 2 O in the induction medium was raised to 0.5 µ M (five times the MS level, i.e. 0.1 µ M) there was significant increase in percentage response along with increase in shoot bud number per explant. The shoots were healthy, well developed with dark green broader leaves. There was remarkable increase in total biomass at increased (0.5 µ M) copper level in the medium. During proliferation stage also presence of high copper levels in the medium favoured increase in shoots bud number per explant.
The adenovirus IVa2 gene, which is expressed at an intermediate time in the viral infectious cycle, is separated from the adenovirus major late promoter (MLP) 5' start site by 210 base pairs and is transcribed from the opposite strand. In contrast to the MLP, the IVa2 gene does not contain a "TATA" box upstream from its 5' start sites. By using a series of deletion mutants, two upstream control regions that are rich in cytidine residues, one proximal to the cap site at nucleotide positions -39 to -48 and a distal domain between nucleotide positions -152 and -242 have been identified as essential for IVa2 transcription (IVa2 cap site is nucleotide position + 1). Transcription efficiency is decreased by 70-90% after the deletion of a proximal C-rich domain when either linear or supercoiled DNAs were used as template. However, distal sequences functioned as transcriptional control domains only with covalently closed DNA templates. The deletion of both the proximal and distal regions from covalently closed DNA templates reduces the levels of IVa2 transcription by a factor of 100-150. When the plasmid pAd242 that contains the 5' start sites of adenovirus MLP and IVa2 is transcribed, there is essentially a complete suppression of transcription of the adenovirus IVa2 gene. The transcription efficiency of IVa2 is increased 10-fold after deletion of the MLP cap site. A model based on a shared entry site for RNA polymerase II and competition between major late and IVa2 promoters is proposed to explain the in vitro transcriptional results.
Linear DNAs, containing a copy of the adenovirus serotype 2 (Ad2) inverted terminal repeat sequence at each end, replicate in 293 cells when cotransfected with Ad2 DNA (Hay et al., J. Mol. Biol. 175:493-510, 1984). We have linked either the Ad2 IVa2 promoter (IVa2) or major late promoter (MLP) to the chloramphenicol acetyltransferase gene and inserted this DNA into such a plasmid (pARKR) between its two inverted terminal repeats. These recombinant plasmids were linearized and then used to transfect 293 cells in the presence or absence of Ad2 helper DNA. Synthesis of IVa2 and MLP RNAs, and production of chloramphenicol acetyltransferase was increased dramatically when the Ad2 DNA was included. However, unlike the patterns of temporal regulation which are seen during a cycle of virus replication when these genes are contained within the virion, there was no obvious difference in the timing of RNA synthesis from plasmid IVa2 or MLP after cotransfection. When linearized plasmids containing IVa2 and MLP sequences but lacking inverted terminal repeats at their ends (replication deficient plasmids) were used for transfection, an increase in RNA synthesis from IVa2 or MLP was also observed and similarly required cotransfection with Ad2 DNA. When HeLa cells, which do not constitutively express the adenovirus E1a gene, were cotransfected with linearized plasmids and adenovirus DNA that lacks the E1a region (H5dl312), a stimulation of transcription was also observed, although it was less than the level observed with wild-type DNA. The results of the present study demonstrate that an early gene product(s) besides E1a functions in trans to regulate transcription.
Abstract A mixture of cis ‐9‐[1‐ 14 C] octadecenol and [1‐ 14 C] docosanol was injected into the brains of 19‐day‐old rats, and incorporation of radioactivity into brain lipids was determined after 3, 12, and 24 hr. Both alcohols were metabolized by the brain but at different rates; each was oxidized to the corresponding fatty acid, but oleic acid was more radily incorporated into polar lipids. Substantial amounts of radioactivity were incorporated into 18∶1 alkyl and alk‐1‐enyl moieties of the ethanolamine phosphoglycerides and into 18∶1 alkyl moieties of the choline phosphoglycerides. Even after the disappearance of the 18∶1 alcohol from the substrate mixture (12 hr), the 22∶0 alcohol was not used to any measurable extent for alkyl and alk‐1‐enyl glycerol formation.
Experiments were conducted for the standardization of in vitro culture technique for the mass propagation of Stevia rebaudiana, a medicinally important, zero-calorie value, sweet tasted and an antidiabetic herb. Shoot tip, nodal segment and leaf were used as explants and they were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations of BA, Kn and IAA both in individual and in combined form for shoot inductions and the best results were obtained from MS medium supplemented with BA+ IAA at the concentrations of 1.0 mg/l and 0.5 mg/l respectively. Among the explants used, shoot tip stood first in inducing shoot development. Best root formation of in vitro developed shoots could be achieved on half-strength Nitsch (N6) medium supplemented with IAA at concentration 1.0 mg/l. The in vitro developed plantlets were transferred to pot and they were grown in greenhouse for hardening and finally they were planted in the open filed. Around 82% of plants were successfully established in natural field condition.
Abstract Rat sciatic nerve contains a membrane‐bound phospholipase D that catalyzes the hydrolysis of exogenous phosphatidylcholine (PC) to phosphatidic acid (PA) and choline. The enzyme is associated with a particulate fraction consisting primarily of microsomes and myelin. This fraction also contains phosphatidate phosphohydrolase activity leading to the production of diacylglycerols (DAG). The phosphohydrolase activity can be completely inhibited by NaF. Hydrolysis of exogenous PC requires detergent and is linear up to about 40 μ g of protein at a pH optimum of 6.5. In the absence of NaF, the sum of PA and DAG increases linearly for 40 min, whereas in its presence, PA production is linear for only 15 min. At optimum conditions, PC hydrolysis proceeds at 15 nmol/h/mg ot protein. Addition of increasing amounts of ethanol to the incubation system leads to the generation of increasing amounts of phosphatidylethanol, indicating transphosphatidylation activity. At an ethanol concentration of 0.4 M , phosphatidylethanol represents about one‐half of the reaction products generated at approximately the same rate of enzymic activity observed in the absence of ethanol. Higher ethanol concentrations are inhibitory.
Abstract The role of the adenovirus major late upstream transcription factor (MLTF) in transcription from the adenovirus major late and the IVa2 promoters was studied. The transcription initiation site of the IVa2 promoter is located 210 nucleotides upstream from the CAP site of the major late promoter. Transcription from these two promoters occurs on different DNA strands. Thus, this divergent transcription suggests that the same factor could simultaneously regulate the expression of two different genes. This was investigated utilizing a reconstituted transcription system in vitro. The addition of MLTF to reaction mixtures containing the purified general transcription factors and the major late promoter resulted in a 10-12-fold stimulation of transcription. This stimulation was because of an increase of the stability of the preinitiation complex. MLTF allowed DNA template molecules to undergo multiple rounds of transcription. MLTF also stimulated transcription from the adenovirus-encoded IVa2 promoter. Surprisingly, reconstitution experiments indicated that transcription from the IVa2 promoter which does not have a TATA sequence required all the previously described general transcription factors, including TFIID, the TATA binding protein. The requirement for TFIID was demonstrated by reconstitution experiments as well as by oligonucleotide competition experiments. The implications of this observation are discussed.
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