Poly A RNA

FIGURE 4.50 FVjrifIcation of mRNA on Toyopearl HW-65F. Column Toyopearl HW-65F. 25 mm X 90 cm. Sample 40 mg of poly(A) RNA/5 ml of formamide, sample from silkworm. Elution 20 mA1 sodium citrate, 5 mM EDTA, 0.5% SDS, 6 M urea, pH 3.5. Flow rate 24 ml/hr. Detection UV at 254, total mRNA activity (—), mRNA activity for major plasma proteins (------------). 

Fig. 2. Time course of accumulation of HSP mRNA. One jUg of poly(A) RNA isolated from soybean hypocotyls after different times of incubation at 42.5 °C (hs) or at additional times after transfer back to 28 °C after 4 h at the elevated temperature (recovery), were electrophoresed in formaldehyde agarose gels. Blots of these gels were hybridised with a mixture of four cDNAs encoding small soybean HSPs ranging from 15 to 23 kDa. From Schoffl Key (1982).

Figure 24.1 Flow chart of RACE-PCR protocol adopted with slight modifications from the instruction manual of the Marathon cDNA amplification kit. Poly A+ RNA is used for the generation of an adaptor-ligated cDNA library. Fragments containing the 5 - and the 3 -end of the cDNA coding for the carrier are amplified from the uncloned library with adaptor primers and gene-specific primers. Afterwards, a full-length clone is generated from the individual RACE products by subcloning or end-to-end PCR.

The issue of whether to use the enriched poly(A) RNA or total RNA was addressed in a later paper by Mahadevappa and Warrington (1999). Using human adenocarcinoma cells, they examined the recovery of detectable transcripts from varying numbers of cells for both protocols. Of the -1800 genes represented on the chip, about 35% were observed to be detectable using either of the two preparahons. 

A similar time course for translatable mRNA has been reported (lA) in barley aleurone layers, in response to glbberel-lic acid. Total poly(A) RNA was found to Increase dramatically in the first 12 hr after hormone application, followed by a rapid decline to 25% of the maximum at 18 hr. On the other hand, the accumulation of ovalbumin mRNA in response to progesterone in chick oviducts (15) is an example that does not appear to behave in this manner. 

Figure 5. Size analysis of Inhibitors I and 11 specific mRNA from levels of 9- and 18-h singly wounded tomato plants and 18-h doubly wounded plants. Poly(A ) RNA was applied to 15-30% linear sucrose gradients and was spun at 25,000 rpm. Twenty-five fractions were collected, the absorbency was measured, and the mRNA was precipitated by cold ethanol. In vitro translations were performed with each fraction in a rabbit reticulocyte system, and isolation of the preinhibitors with preformed antibody precipitates located the position of the two inhibitors. The gradients were calibrated by centrifugation of tomato leaf polyfA)" RNA on an identical gradient. The locations of translatable mRNAs for Inhibitors I and II were identical with RNA obtained from 9- and 18-h singly wounded or 18-h doubly...

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.

A large number of studies (33) support the thesis (59) that the function of GA may be that of a derepressor of gene activity. Giberellic acid has significant effect on the synthesis of all species of RNA s (63, 64), but the formation of < —amylase does not depend on new synthesis of ribosomal and transfer RNA s. Unequivocal proof for GA-induced formation of transcripts was provided by the in vitro synthesis of peptides that are immuno-logically similar to -amylase on poly A+RNA templates that were. isolated from hormone-treated aleurone cells (65, 66, 67). Of particular significance is the finding that detectable levels of -amylase mRNA s were formed within 2 hr of treatment with GA. 

Control of the synthesis of amylase ntRNA s in barley aleurone cells and the synthesis of cellulase mRNAs in pea epicotyl cells are similar in some respects. The control of cellulase activity in pea epicotyl is the only known example of auxin-induced formation of specific mRNA molecules. The formation of cellulase mRNA was demonstrated by the isolation of poly A + RNA s and in vitro synthesis of cellulase (71) using the protein-synthesizing system of wheat germ (72). The formation of cellulase mRNA precedes the increase in cellulase levels by more than 12 hr. Thus, it appears that the increase in rate of synthesis of translatable cellulase mRNA s in the pea epicotyl (71) and that of -amylase mRNA s in barley aleurone cells (65,... 

Bind poly A+ RNA from total RNA and capture poly A+ RNA-oligo(dT) hybrid on streptavidin beads. 

Centrifuge the beads. Wash and elute the poly A+ RNA with water... 

Figure 7.4. Isolation of poly-A+ RNA by biotin-streptavidin affinity matrix. Poly-A+ RNA is captured as a hybrid between poly-A+ RNA and biotinylated oligo(dT) by streptavidin matrix. Most mRNAs carry poly-A+ stretch at their 3 end, and hence poly A-containing RNA can be enriched substantially by this affinity capture method. Poly-A+ RNA can be eluted from the beads by low salt or water. The eluted RNA can be ethanol precipitated.

The effect of JH III on HMG-R expression in I. paraconfusus, I. pini and D. jeffreyi was investigated by determining both a dose-response and time course by standard northern blotting. JH III, dissolved in acetone, was applied topically onto the ventral abdomens of unfed adult insects, while control insects were treated with an equivalent volume of acetone. Total or poly (A)+ RNA was then isolated from intact tissues pooled from up to ten individuals for the Ips studies (Tittiger et al., 1999 Tillman et al., in preparation). For the D. jeffreyi studies, preliminary work localized JH Ill-induced HMG-R expression to the metathorax/... 

Figure 7.3 Regulation of HMG-R expression in male and female /. paraconfusus. A JH Ill-induced HMG-R mRNA levels in females and males following topical application of 5.3 pg JH III. B Northern blot showing the effect of topical JH III application on HMG-R mRNA in males. The doses applied are shown above the image. In both A and B, poly(A)+RNA was isolated 20 h following treatment. The relative levels of HMG-R mRNA (normalized to the actin signal) compared to untreated males is given below each image. Reproduced from Tittiger et al. (1999) with permission.

DOR gene spans 32 kb from transcription initiation sites located between 140 bp and 390 bp upstream of the ATG translation start codon to a polyadeny-lation site located 1.2 kb downstream of the TGA stop codon. RNase protection analysis of the 5 ends of mouse brain poly(A) + RNA and NG108-15 total RNA resulted in identical patterns of multiple protected fragments, suggesting that the DOR gene is transcribed from multiple initiation sites in the TATA-less, 80% G + C-rich sequence between 140 and 390... 

Dworkin, M.B., David, I.B. (1980). Use of a cloned library for the study of abundant poly (A)+ RNA during Xenopus laevis development. Dev. Biol. 76 449-64. 

The genes for the hydrophilic polypeptides of the water-splitting complex have not been isolated, but their location in nuclear DNA is predicted from the synthesis of precursor forms of these polypeptides from poly(A) RNA isolated from spinach [65]. There is no evidence for the location of the genes for the other polypeptides associated with PS II. 

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