Messenger RNA isolation

Messenger RNA isolated from differentiated HL-60 cells is thus a convenient source of transcripts for the receptor, and this mRNA (50-100 ng) can be injected into X. laevis oocytes. These oocytes actively translate foreign mRNA molecules, and often the expressed protein is functional 2-5 days later, functional fMet-Leu-Phe receptors can be detected on the cell surface. The identity of a functional fMet-Leu-Phe receptor was confirmed because ... 

RNA is only a small fraction of total RNA, but it is expressed in high concentrations in tissues that express the target protein. Therefore messenger RNA isolated from enriched sources is likely to contain RNA sequences that encode for the target protein. 

Wallach, M., and Kilejian, A. (1982). The importance of tRNA for the in vitro cell-free translation of messenger RNA isolated from the malaria parasite Plasmodium lophurae. Mol. Biochem. Parasitol. 5, 245-261. 

If Karlson s model is valid, it could be expected that RNA extracted from ecdysone-activated epidermis cells would reproduce the effects of the hormone on these cells. Evidence is not forthcoming on the action of such RNA on intact cells. The problems of cell penetration and degradation make the probability of technical success of these experiments very low. However, the introduction, in the uterine cavity of oophorectomized rat, of messenger RNA isolated from the uterus of oestrogen-treated castrated rats, had clear oestrogenic effects. Evidence of the activity of RNA extracted from activated epidermis cells in the synthesis of DOPA decarboxylase in acellular systems is still not clear. 

Saito H., Mimmack M., Keveme E.B., Kishimoto J. and Emson P. (1998). Isolation of mouse vomeronasal receptor genes and their co-localization with specific G-protein messenger RNAs. Molec Brain Res 60, 215-227. 

Anti-arthritic effect. Oral administration of AJA, a cannabinoid acid devoid of psychoactivity, reduced joint tissue damage in rats with adjuvant arthritis. Peripheral blood monocytes (PBM) and synovial fluid monocytes (SFM) were isolated from healthy subjects and patients with inflammatory arthritis, respectively, treated with AJA (0-30 mM) in vitro, and then stimulated with lipopolysaccharide. Cells were harvested for messenger RNA (mRNA), and supernatants were collected for cytokine assay. Addition of AJA to PBM and SFM in vitro reduced both steady-state levels of interleukin-ly (IL-ly) mRNA and secretion of IL-ly in a concentration-dependent manner. Suppression was maximal (50.4%) at 10 mM AJA (p < 0.05 vs untreated controls, n = 7). AJA did not influence tumor necrosis factor-a (TNF-a) gene expression in or secretion from PBM . 

The wound-induced synthesis and accumulation of proteinase Inhibitors I and II in tomato leaves has provided a model system to study the regulation of proteinase inhibitor genes in plants. The simplicity of the phenomenon has made it possible to Isolate the wound-factor, or hormone, and to study its release, direction and rate of transport in tomato plants. Messenger RNA has been isolated from leaves of wounded plants and contains translatable mRNAs for the two proteinase inhibitors. Studies with these mRNAs have provided a basis for the initiation of a program to clone inhibitor cDNAs for studies of the molecular basis of the wound-Induced process of inhibitor synthesis. 

Probes may also consist of DNA copied from mRNA. This is known as cDNA and is also widely used to determine indirectly the sequences of mRNA molecules. Messenger RNA may be isolated from the total cellular RNA by affinity chromatography on bound poly (dT) or poly (U). These materials selectively hold RNA with the poly (A) tails characteristic of most eukaryotic mRNA (see Chapter 28). Another source of mRNA is polyribosomes (polysomes), which are "reading" mRNA and actively making proteins. 

Messenger RNA is isolated from the tissue source selected to contain the Ab-secreting cells. Spleen, bone marrow, tonsil, and lymph node have all been successfully used as a source Copy DNA (cDNA) is then generated by reverse transcription and Fv or Fab regions amplified by PCR. 

There is also some interplay between the Zn2+ and Mn2+ cations. The RNA polymerase isolated from Euglena gracilis grown on a zinc-deficient medium contains two Zn2+ per molecule but differs from the RNA polymerases isolated from zinc-sufficient cells. The messenger RNA from the zinc-deficient cells had a different base composition, with a two-fold increase in the (G+ C)/(A+U) ratio. Since intracellular [Mn2+] increases in zinc-deficient cells, the effect of [Mn2+] on the RNA polymerase from zinc-deficient cells was investigated. It was shown that increased levels of Mn2+ lead to increased incorporation of GMP relative to UMP.346... 

These observations are consistent with the conclusion that auxin treatment leads to the synthesis of BS cellulase, which then accumulates in smooth ER vesicles. There is direct evidence that the synthesis occurs in rough ER vesicles (11). Cellulase activity was shown (25) to increase in RNA-rich pea microsomes, provided these were isolated from auxin-treated tissue, when the preparations were incubated with ingredients necessary for carrying out protein synthesis in vitro. Messenger RNA (mRNA) from these microsomes has been translated in a different ribo-somal system and shown to synthesize BS cellulase protein (II). Thus, it is legitimate to use the term "induction to apply to the ability of auxin to evoke the appearance of mRNA for BS cellulase. 

Stavnezer, J., Huang, R.C.C., Stavnezer, E., Bishop, J.M. (1974). Isolation of messenger RNA for an immunoglobulin kappa chain and enumeration of the genes for the constant region of kappa chain in the mouse. J. Mol. Biol. 88,43-63. 

Fig. 3. A northern blot of poly(A)+ RNA probed with a psi cDNA clone showed enhanced levels of psi messenger RNA as phosphate starvation became more severe. Poly(A)+ RNA was isolated from cultures at 3, 6 and 9 days after transfer to —Pi or +Pi medium (A. Danon et al., unpublished data in preparation). Equal amounts of RNA (1 pg per treatment) were separated on a denaturing formaldehyde/agarose gel and used for a northern blot. The filter was probed using a psi cDNA clone (identified by —/+ screening using standard methods) as the probe. Enhanced levels of mRNA for this clone are seen as early as 3 days after transfer to —Pi medium although cell growth equivalent to the unstressed control continued until day 8.

All the bases in the synthetic messenger RNA prepared by Nirenberg were U therefore, the codon is UUU. By referring to the codons in Table 27.4, we see that the UUU codes for phenylalanine. A polypeptide in which all the amino acid residues were phenylalanine was isolated in Nirenberg s experiment. 

There are three different types of RNA in cells, namely ribosomal RNA (rRNA), transfer RNA (tRNA) and messenger RNA (mRNA). E. granulosus is the only cestode in which RNA has been investigated in depth (8) and the evidence indicates that the RNA species in this worm and their formation conform with those of other eukaryotes. The rRNA had sedimentation coefficients of 29.4 and 19.6, was rich in guanosine-5 -monophosphate and had a G+C content of around 50%. In addition, a light RNA fraction, with a sedimentation coefficient of 6.3, was isolated... 

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