Isolation and Characterization of Messenger RNAs for Seed Lectin and Kunitz Trypsin Inhibitor in Soybeans

The mRNAs for seed lectin and Kunitz trypsin inhibitor of soybean have been highly enriched by immunoadsorption of the polysomes synthesizing these proteins. Polysomes isolated from developing seed of variety Williams were incubated with monospecific rabbit antibodies produced against lectin subunits or trypsin inhibitor protein. The polysomal mixture was passed over a column containing goat anti-rabbit antibodies bound to Sepharose. Bound polysomes were eluted and the mRNA was selected by passage over oligo(dT)-cellulose. Lectin complementary DNA hybridized to an 1150-nucleotide message and trypsin inhibitor complementary DNA hybridized to a 770-nucleotide message in blotting experiments using total poly(A) RNA. Translation of soybean lectin mRNA using a rabbit reticulocyte lysate yielded a major polypeptide of 32,300 whereas the molecular weight for purified lectin subunits was 30,000. Trypsin inhibitor mRNA directed the synthesis of a 23,800-dalton polypeptide as compared to 21,500 daltons for trypsin inhibitor marker protein. Lectin specific polysomes could not be obtained from a soybean variety which lacks detectable lectin protein whereas trypsin inhibitor-specific polysomes were bound by immunoselection. These results confirmed the specificity of the immunoadsorption procedure and strongly indicated that the lectinless variety was deficient or substantially reduced in functional lectin mRNA.     

Mobilisation of NADPH-glutamate dehydrogenase mRNA in regreening cultures of Euglena gracilis

Exposure of dark-grown resting Euglena gracilis Klebs strain Z Pringsheim to light results in a transient increase in the specific activity of NADPH-glutamate dehydrogenase. NADPH-glutamate dehydrogenase antibody was used to detect NADPH glutamate dehydrogenase resulting from the translation of total polyadenylated RNA and polysomal RNA from Euglena in a cell-free rabbit reticulocyte lysate system. NADPH-glutamate dehydrogenase mRNA was present in cells at all stages of development and present on polysomes from dark-grown and regreening cells but not on polysomes from dark-grown resting cells. These results indicate that the light-induced increase in NADPH-glutamate dehydrogenase in dark-grown resting cells represent an increase in the rate enzyme synthesis resulting from the mobilisation of NADPH-glutamate dehydrogenase mRNA onto polysomes.     

Control of tyrosinase gene expression and its relationship to neural crest induction in Rana pipiens. I. Isolation and characterization of amphibian tyrosinase mRNA

Rana pipiens tyrosinase mRNA was isolated from Stage 22 (tailfin circulation) embryos by indirect immunoprecipitation of embryonic polysomes using highly specific rabbit anti-tyrosinase and goat-(anti-rabbit) immunoglobulins. Analysis on sucrose gradients indicated that anti-tyrosinase bound specifically to embryonic polysomes of the 300-350 S class coincident with the location of nascent tyrosinase enzyme activity and tyrosinase mRNA. These same anti-tyrosinase-bound polysomes were fully immunoprecipitated by the addition of goat-(anti-rabbit) IgG. Poly(A+) RNA was obtained from phenol-extracted antibody. polysome complexes by sequential passage over oligo(dT)-cellulose. The final purification of tyrosinase mRNA was achieved by preparative sucrose gradient fractionation. Tyrosinase mRNA sedimented as a single 13 S peak in 5-30% sucrose gradients and tracked on sodium dodecyl sulfate-polyacrylamide gels as a single band of 4.5 X 10(5) Da (1275 nucleotides). When assayed in a cell-free translation system, this mRNA directed the synthesis of a single 35,000-Da protein which co-migrated with native tyrosinase on sodium dodecyl sulfate-polyacrylamide gels and which was greater than 98% immunoprecipitable by anti-tyrosinase immunoglobulin. Final purification was 4103-fold over the starting polysomal RNA.     

In Vitro Synthesis of Polypeptides of Moderately Large Size by Poly(A)-Containing Messenger RNA from Postmortem Human Brain and Mouse Brain

Studies were undertaken to optimize the conditions for isolation and in vitro translation of poly(A)-containing mRNA from human postmortem brain. The comparison of several methods for preparation of mRNA from frozen mouse brain indicated that although the yield of mRNA was increased using polysomes prepared in the presence of ribonucleoside vanadyl complexes and subsequently extracted with guanidinium thiocyanate, the translation products were indistinguishable from those synthesized by total cellular RNA directly extracted from tissue with guanidinium thiocyanate. The oligo d(T)-cellulose-purified poly(A)-containing mRNA preparations were translated in vitro in a rabbit reticulocyte lysate in the presence of L-[35S]methionine. Messenger RNA from frozen mouse brain stimulated protein synthesis from 9- to 20-fold over endogenous mRNA. Over 450 polypeptides were reproducibly synthesized and separated by two-dimensional polyacrylamide gel electrophoresis (PAGE); size classes up to 130,000 daltons were present. Direct extraction of RNA from frozen human cerebral cortex and cerebellum with guanidinium thiocyanate followed by oligo d(T)-cellulose chromatography yielded 1.8 micrograms/g and 2.0 micrograms/g, respectively, of poly(A)-containing mRNA; this represents a two- to fourfold increase over our earlier results. In the rabbit reticulocyte translation system human brain mRNA stimulated protein synthesis nearly threefold over endogenous mRNA. Compared with earlier studies, the number of newly synthesized polypeptides was increased by 30%. Over 300 species were separated by two-dimensional PAGE, and size classes up to 130,000 daltons were present, as compared to 70,000 in an earlier report. The polypeptides synthesized by human cerebral cortex and cerebellum were indistinguishable. However, several appeared to be uniquely human when compared with the products synthesized by mouse brain mRNA. The method described for the preparation of postmortem human brain mRNA eliminates the need to prepare polysomes, which are recovered in variable and low yield from the postmortem human brain. The procedure appears applicable to studies on the synthesis of moderately large human brain polypeptides and for investigations of brain protein polymorphism when relatively large numbers of products are required for analysis.     

Purification of messenger RNA coding for glycine methyltransferase from rat liver

Rat liver messenger RNA coding for glycine methyltransferase was associated preferentially with free polysomes. The mRNA was purified about 1000-fold over the total poly(A)-containing RNA by specific immunoadsorption of polysomes to protein A-Sepharose followed by oligo(dT)-cellulose column chromatography. Sodium dodecyl sulfate-gel electrophoresis of the in vitro translation products in a rabbit reticulocyte lysate system revealed only one major band which migrated to the position of the purified glycine methyltransferase subunit. The result shows that the mRNA isolated is nearly homogeneous and suggests that no precursor form of the enzyme existed. The mRNA sedimented at the position slightly smaller than 18 S rRNA in a sucrose density-gradient centrifugation and was shown to contain about 1,300 nucleotides by the Northern blot hybridization analysis with a cDNA probe.     

Acquisition of ability to utilize Xylitol: disadvantages of a constitutive catabolic pathway in Escherichia coli.

Ribitol+ strains of Escherichia coli acquire the ability to utilize xylitol by mutating to constitutive production of the coordinately controlled ribitol catabolic enzymes ribitol dehydrogenase (RDH) and D-ribulokinase (DRK). Such strains concomitantly acquire toxicity to galacitol and L-arabitol, and to D-arabitol if they are unable to utilize it for growth. Strains selected for resistance to these polyols have DRK structural gene mutations or other mutations that eliminate the constitutive production of DRK, consistent with the view that DRK phosphorylates those polyols to toxic substances. Ribitol+ strains selected for growth on 8 mM xylitol fail to grow on 30 mM xylitol. A product of ribitol and xylitol catabolism represses synthesis of RDH, an enzyme required for growth on xylitol. At 30 mM xylitol, greater than 99% of RDH synthesis is repressed. Strains that grow on 8 mM xylitol can mutate to grow on 30 mM xylitol. Such mutants, relieved of this repression, overproduce RDH, resulting in good growth on the poor substrate, xylitol, but poor growth on the normal substrate, ribitol.     

Specific Polysome Immunoadsorption to Purify an Ammonium-Inducible Glutamate Dehydrogenase mRNA from Chlorella sorokiniana and Synthesis of Full Length Double-Stranded cDNA from the Purified mRNA

A specific polysome immunoadsorption procedure, employing soluble rabbit anti-NADP-GDH IgG and sheep anti-rabbit IgG covalently-linked to an insoluble cellulose matrix, was used to immunoselect polysomes translating mRNA for a chloroplastic ammonium-inducible NADP-GDH in fully induced cells of Chlorella sorokiniana. The immunoselected polysomes were dissociated, and the NADP-GDH mRNA was recovered by oligo (dT)cellulose chromatography. The translatable NADP-GDH mRNA was estimated to be 0.07 and 90% of the total polysomal poly(A)⁺RNA before and after immunoselection of the polysomes, respectively. The immunoadsorption procedure resulted in an 83% recovery and 1,291-fold purification of translatable NADP-GDH mRNA. In vitro translation of the immunoselected poly(A)⁺RNA yielded a single radioactive protein (on sodium dodecyl sufate polyacrylamide gels) with a molecular weight of 58,500, i.e. size of the putative precursor-protein of the NADP-GDH subunit in the holoenzyme in fully induced cells. The purified NADP-GDH mRNA was used for synthesis of a high proportion of nearly full-length single-stranded cDNA and double-stranded cDNA molecules.     

Characterization of polysomes from Xenopus liver synthesizing vitellogenin and translation of vitellogenin and albumin messenger RNA's in vitro.

1. Conditions are described for the isolation of polysomes from the liver of Xenopus laevis. The method involves homogenization of liver in 0.2 M Tris-HCl pH 8.5, treatment with 2% Triton X-100 and subsequent sucrose density gradient fractionation of polysomes from a 10000 X g supernatant. 2. Vitellogenin synthesis was induced in male Xenopus liver by oestradiol treatment. Polysomes were isolated and vitellogenin-synthesizing polysomes characterized by their association with monospecific 125 I-labelled rabbit anti-vitellogenin antibody and by reaction with rabbit anti-vitellogenin immunoglobulins followed by indirect immunoprecipitation with goat anti-rabbit antibody. 3. Changes in liver polysome content following oestrogen treatment of male Xenopus are correlated with the appearance of vitellogenin synthesis using an organ culture assay. 4. RNA extracted from livers of oestradiol-treated male Xenopus and from purified polysomes is shown to code for the synthesis of vitellogenin-specific immunoprecipitable polypeptides in a rabbit reticulocyte cell-free protein-synthesizing system, a major component having a molecular weight of 210000. Xanopus liver RNA is also shown to code for the synthesis of an albumin-specific immunoprecipitable polypeptide of 74000 molecular-weight which coelectrophoresed with Xenopus albumin.     

Role of glucocorticoids and progesterone in the development of rough endoplasmic reticulum involved in casein biosynthesis.

Hydrocortisone acetate injected into pseudopregnant rabbits induced casein synthesis and a parallel accumulation of casein mRNA. These effects were not accompanied by any enrichment of total RNA in the mammary cell. Hydrocortisone acetate did not favour the attachment of polysomes to endoplasmic reticulum. Casein mRNA concentration was enhanced in free and membrane-bound polysomes. After long treatments, the concentration of casein mRNA reached a plateau in membrane bound polysomes whereas it continued to be accumulated in free polysomes, suggesting that a substantial part of casein synthesis is then carried out by free polysomes. Progesterone injected with high doses of prolactin was unable to prevent the stimulatory action of prolactin on the synthesis of casein, the accumulation of casein mRNA and mammary gland growth, as judged by DNA content. By contrast, the increase in the total RNA content of mammary gland was still significantly reduced by progesterone. In addition, progesterone inhibited almost completely the formation of membrane-bound polysomes and the anchorage of casein mRNA to endoplasmic reticulum. From these data, it was concluded that the formation of the endoplasmic reticulum is not a prerequisite for the initiation of casein synthesis. Glucocorticoids do not play a major role in the formation of the endoplasmic reticulum and the Golai apparatus and in the binding of casein synthesizing polysomes to membranes. Progesteronne is capable of inhibiting preferentially and gradually the stimulation of cellular functions requiring the most potent prolactin stimulation.     

Identification of globin mRNA in 10s RNA of rabbit reticulocytes

Electrophoresis on 6% polyacrylamide gels splits 10s RNA of detergenttreated polysomes from rabbit reticulocytes into two major bands. After these two RNAs are isolated separately, the first 10s RNA¹ directs the synthesis of both α and β chains in the Krebs II ascites cell-free system. In contrast, the second 10s RNA is inactive in directing globin synthesis. This result is further documented by separation of the two 10s RNAs by oligo dT-cellulose chromatography and by isolation of globin mRNA after EDTA-treatment of reticulocyte polysomes. Therefore, globin mRNA containing both α- and β-chain synthetic capacity moves as a single RNA species on electrophoresis in polyacrylamide gels.     

A measurement of the sequence complexity of polysomal messenger RNA in sea urchin embryos

The first measurement has been made of the number of diverse mRNA sequences (mRNA sequence complexity) in the total polysomes of a eucaryotic system, the sea urchin gastrula. mRNA was purified of nuclear RNA and any other heterogeneous RNA contaminants by release from polysomes with puromycin. Trace quantities of labeled nonrepetitive DNA fragments were hybridized with an excess of mRNA. The hybridization reaction followed ideal first order kinetics in mRNA concentration. At completion of the hybridization reaction, 1.35% of the nonrepetitive DNA was present as mRNA-DNA hybrid. The hybridized DNA was extracted and was at least 70% hybridizable with mRNA, demonstrating a 50-fold purification of the expressed sequences. This purified DNA fraction reassociated with excess unfractionated sea urchin DNA at a rate identical to that of the total nonrepetitive DNA tracer. The mRNA had therefore been hybridized to nonrepetitive DNA sequence, and the amount of hybrid could be used as a direct measure of the mRNA sequence complexity.The complexity of the gastrula mRNA can be calculated as about 17 million nucleotides, sufficient to comprise some 14,000 distinct structural genes. This result also provides an estimate of the number of diverse proteins being translated in the gastrula. From the rate of mRNA-DNA hybrid formation, we estimate that about 8% of the mRNA belongs to this complex class, and that less than 500 copies of each species of message in this class exist per embryo. Most of the mRNA population consists of a relatively small number of diverse species represented a much larger number of times.     

A Messenger RNA for Tobacco Mosaic Virus Coat Protein in Infected Tobacco Mesophyll Protoplasts

The low molecular weight tobacco mosaic virus (TMV)-specific RNA component (LMC) was demonstrated in tobacco mesophyll protoplasts by polyacrylamide gel electrophoresis of ¹⁴C-uridine-labelled RNA from infected protoplasts. Free and membrane-bound polysomes were isolated from infected protoplasts, and RNA extracted from them was analyzed. TMV-specific RNA species including full-length viral RNA, its replicative intermediate, and LMC were found in both free and membrane-bound polysomes, but were present in free polysomes in much larger amounts. In particular, as much as 37 % of total LMC in protoplasts was found in free polysomes. Fractionation of polysomes by sedimentation in sucrose gradients showed that LMC is associated with small-sized polysomes (mono- to tetrasomes). Polysomes of this size class produced viral coat protein in a cell-free protein synthesizing system from rabbit reticulocytes. On the other hand, full-length TMV-RNA was associated predominantly with larger polysomes which produced in the cell-free system TMV-specific high molecular weight polypeptides but no coat protein. These results indicated that LMC, a subgenomic RNA of TMV, in fact functions in vivo as messenger RNA for viral coat protein, as has been postulated on the basis of in vitro studies.     

The cytoplasmic distribution and characterization of poly(A)+RNA in oocytes and embryos of Drosophilia.

Using the presence of poly(A) tracts as a marker for mRNA, we have examined the distribution of this class of RNA between polysomes and free RNP particles. This has been done in mature oocytes and in embryos aged for various times from fertilization through to hatching of a larva. The proportion of ribosomes that are in polysomes to those that are not has been calculated. In mature oocytes, 58% of the poly(A)⁺ RNA and 72% of the ribosomes are not in polysomes. By 1 hr, this drops to 51% of the poly(A)⁺ RNA and 48% of the ribosomes. By 7 hr, a plateau is reached: 30% of each are not in polysomes. The poly(A)⁺ RNA in the cytoplasm of oocytes and 1-hr embryos is found in particles with an average size of 50S and a range of 30–70S. The poly(A)⁺ RNA ranges in size from 7 to 40S, with an average size of 22S. The polyA from this RNA is 50–200 nucleotides long with an average of 115 nucleotides. These data have allowed us to calculate that 1–2% of the total RNA is poly(A)⁺ RNA.     

Immobilized metal ion affinity chromatography in enzyme fractionation.

Ribitol dehydrogenase from Mycobacterium butyricum and alpha-mannosidase from Lupinus luteus seedlings were fractionated by the immobilized metal ion (Cu2+ or Zn2+) affinity chromatography (IMAC) on iminodiacetic acid coupled to Sepharose 6B. In a single step, ribitol dehydrogenase was purified 10-12 fold with the recovery above 80% when using Zn(2+)-Sepharose 6B as the sorbent and decreasing linear gradient of pH from 7 to 4. In the same conditions purification of alpha-mannosidase was less effective (2-3 fold, recovery 60-70%).     

Purification of immunuglobulin light chain messenger RNA by immunoprecipitation from mouse myeloma tumor, MOPC-31C.

Polysomes producing IgGl(kappa) myeloma protein were specifically selected by an immunoprecipitation method, and immunoglobulin light chain mRNA was purified from the precipitated polysomes. The purified mRNA migrated predominantly as a single band and the molecular weight of this mRNA was calculated to be 410.000 by polyacrylamide gel electrophoresis in 98% formamide. A protein possessing a molecular weight of 25,000, which is the size of the light chain precursor, was synthesized as a major product of translation in a wheat germ cell-free system. DNA complementary to the mRNA (cDNA) was prepared with avian myeloblastosis virus RNA-dependent DNA polymerase. This cDNA had an average size of 8.3S as determined by sedimentation through an alkaline sucrose gradient. Using this cDNA, Crt 1/2 values of template RNA and RNA from various preparations were calculated from the results of molecular hybridization. The relative content of the mRNA increased 4,4-fold during the immunoprecipitation of polysomes.     

Effects of phenobarbital on protein synthesis and polysome levels in rat liver.

Administration of phenobarbital to rats increases the rate of synthesis of certain microsomal drug-metabolizing enzymes in a selective manner and promotes proliferation of smooth endoplasmic reticulum in the liver. Phenobarbital increased a number of factors by which protein synthesis could be enhanced in the liver. It produced a 30% increase in the amount of ribosomes and mRNA per cell. The proportion of ribosomes associated with polysomes was increased by 5-10% over normal liver. There was a 10-30% increase in the rate of ploypeptide elongation and a small increase or no change in polysome size, indicating that the rate of polypeptide initiation was increased proportionately. The product of these effects accounts for the 1.5-fold increase in the rate of total protein synthesis previously reported. The average polysome size, and the size of free polysomes in particular, was maintained when actinomycin D was administered to phenobarbital-pretreated rats, suggesting that the rate of mRNA degradation was decreased selectively. Phenobarbital did not, however, affect the distribution of ribosomes between the free and membrane-bound states or the activity of ribonucleases associated with isolated free and bound polysomes. Thus, we conclude that phenobarbital stimulates protein synthesis by expanding the mRNA pool, at least partially through effects on mRNA degradation, and by augmenting the rate of mRNA translation.     

CHARACTERIZATION OF POLY(A) SEQUENCES IN BRAIN RNA

—Nuclear and polysomal brain RNA from the rabbit bind to Millipore filters and oligo(dT)-cellulose suggesting the presence of poly(A) sequences. The residual polynucleotide produced after RNase digestion of ³²P pulse-labelled brain RNA is 95% adenylic acid and 200-250 nucleotides in length. After longer isotope pulses the polysomal poly(A) sequence appears heterodisperse in size and shorter than the nuclear poly (A). Poly(A) sequences of brain RNA are located at the 3′-OH termini as determined by the periodate-[³H]NaBH₄ labelling technique. Cordycepin interferes with the processing of brain mRNA as it inhibits in vivo poly(A) synthesis by about 80% and decreases the appearance of rapidly labelled RNA in polysomes by about 45%. A small poly(A) molecule 10-30 nucleotides in length is present in rapidly labelled RNA. It appears to be less sensitive to cordycepin than the larger poly(A) and is not found in polysomal RNA.