Distribution of root mRNA species in other vegetative organs of pea (Pisum sativum L.)

Summary A cDNA library was prepared from, poly(A)⁺ RNA from roots of pea (Pisum sativum L.). Twenty five clones were selected by use of random numbers and used as probes on Northern blots to analyse the distribution of their corresponding mRNA species in other vegetative pea organs: leaf, stem and developing cotyledon. Fifteen cDNA inserts hybridised to single mRNA species, five hybridised to two mRNA species and one hybridised to five homologous mRNAs. Four cDNA clones (16% of those selected) gave no hybridization signals, indicating that the steady state levels of mRNAs were below the detection limit (i.e.less than 2.5 x 10^-5% of poly(A)⁺ RNA). Most of the root mRNAs were represented in all four pea organs as sequences of low and medium abundance. All but two cDNAs encoded mRNA species enhanced in root. However, cDNA clones appeared not to encode mRNA species expressed in a strictly organ-specific manner, as no mRNA unique to root was found. Thus, if organ-unique mRNA species are present, they are only present at a very low level of abundance in the poly(A)⁺RNA population.     

Comparison of polysomal polyadenylated RNA from embryonal carcinoma and committed myogenic and erythropoietic cell lines

Using the technique of mRNA-cDNA hybridization, we have examined the polysomal poly(A)⁺ mRNA base-sequence complexity in three different mouse cell lines: mouse embryonal carcinoma cells, myoblast cells and Friend erythroleukemic cells. These cells express 7700, 13,200 and 6200 mRNA sequences, respectively, distributed in three frequency classes. Reciprocal heterologous hybridization experiments revealed that there is a large degree of homology, a subset of 6000 common sequences being present on the polysomes of all three cell types. Myoblast mRNA is capable of hybridizing all reactive embryonal carcinoma cell cDNA, with kinetics close to the homologous embryonal carcinoma cell curve, thus indicating that all embryonal carcinoma cell sequences are present on myoblast polysomes, the majority at similar abundance. Conversely, embryonal carcinoma cell mRNA fails to hybridize 12% of myoblast cDNA, apparently arising primarily from the complex frequency class. This was confirmed by using myoblast fractions partially enriched in abundant and rare sequences. As a proportion of the rare class, this 12% fraction represents about 4500 sequences close to the difference in base-sequence complexity between myoblast and embryonal carcinoma cells.Homologous and heterologous hybridization with total and fractionated Friend cell cDNA probes revealed that all Friend cell polysomal poly(A)⁺ RNA sequences are common to embryonal carcinoma cell polysomes—apart from a small group of sequences drawn from the abundant class, corresponding to about 10% of Friend cell cDNA. This represents about 12 sequences from the abundant class. In addition, certain common sequences in the abundant Friend cell frequency class are present at lower frequency in embryonal carcinoma cell polysomes. Friend cell polysomal poly(A)⁺ RNA fails to hybridize 7–10% embryonal carcinoma cell cDNA apparently derived from the rare frequency class. As a fraction of the rare class, this corresponds approximately to the difference (about 1500 sequences) in complexity between the Friend and embryonal carcinoma cell lines.     

Characterization and Complexity of Wheat Developing Endosperm mRNAs

Free and membrane-bound (MB) polysomes and the corresponding polyadenylated RNAs (polyA⁺ RNAs) have been isolated from developing wheat endosperm (Triticum aestivum L.) Free and MB poly(A)⁺ RNAs, analyzed on isokinetic sucrose gradient with [³H]polyuridylic acid [poly(U)] hybridization detection, appear to be 11S to 12S in size with a 7% poly(A) tail for MB RNAs. cDNAs synthesized using both of these mRNA populations in presence of a potent RNase inhibitor (RNasin), have been used for hybridization kinetics experiments. The mean square fitting analysis of the hybridization kinetics between MB cDNA and its template reveals the presence of two abundance classes representing roughly ⅔ and ⅓ of the MB poly(A)⁺ RNAs and containing the information for approximately 75 superabundant species (21,000 copies per cell) and 750 intermediate species (530 copies per cell), respectively. The mRNA population extracted from free polysomes is divided into three abundance classes. The first one is composed of superabundant sequences which would correspond to the MB superabundant mRNAs. The free mRNAs consist of about 11,000 diverse sequences, most of them being rare sequences. Heterologous hybridizations of MB cDNAs to free mRNAs have shown that some mRNAs are common to both populations. This could be explained either by a partial contamination or by free polysomes en route to their membrane destination. Contrary to the low number of diverse mRNAs corresponding to the legume seed storage proteins, the wheat endosperm superabundant mRNAs consist of about 75 different sequences which would encode most of the seed storage proteins, especially gliadins.     

Poly(A+)mRNA sequences in free mRNP and polysomes of mouse ascites cells

The poly(A⁺)RNA of the free mRNP of mouse Taper ascites cell contains a very reduced number of different mRNA sequences compared to the polysome poly(A⁺)RNA. By the technique of mRNA:cDNA hybridization we have determined that the free mRNP contains approximately 400 different mRNA sequences while the polysomes contain about 9000 different mRNAs. The free mRNP poly(A⁺)RNA sequences are present in two abundance classes, the abundant free mRNP class containing 15 different mRNA sequences and the less abundant free mRNP class containing 400 different mRNAs. The polysome poly(A⁺)RNA consists of three abundance classes of 25, 500 and 8500 different mRNA sequences.Despite its intracellular location in RNP structures not directly involved in protein synthesis the poly(A⁺)RNA purified from the free RNP of these cells was a very effective template for protein synthesis in cell-free systems. Cell-free translation products of free mRNP and polysome poly(A⁺)RNAs were analyzed by two-dimensional gel electrophoresis. This analysis confirmed the hybridization result that the free mRNP poly(A⁺)RNA contained fewer sequences than polysomal poly(A⁺)RNA. The abundant free RNP-mRNA directed protein products were a subset of the polysome mRNA-directed protein products. The numbers of more abundant products of cell-free protein synthesis directed by the free RNP-mRNA and polysomal mRNA were in general agreement with the hybridization estimates of the number of sequences in the abundant classes of these two mRNA populations.     

HeLa cell cytoplasmic mRNA contains three classes of sequences: predominantly poly(A)-free, predominantly poly(A)-containing and bimorphic

The mRNA species which exist in the HeLa cell polyribisomes in a form devoid of A sequences longer than 8 nucleotides constitute the poly(A)-free class of mRNA. The rapidly labelled component of this mRNA class shares no measurable sequence homology with poly(A)-containing RNA. If poly(A)-free mRNA larger than 12 S labelled for 2 h in vivo is hybridized with total cellular DNA, it hybridizes primarily with single-copy DNA. When a large excess of steady poly(A)-containing RNA is added before hybridization of labelled poly(A)-free RNA, no inhibition of hybridization occurs. This indicates the existence of a class of poly(A)-free mRNA with no poly(A)-containing counterpart. Some mRNA species can exist solely as poly(A)-containing mRNAs. These mRNAs in HeLa cells are found almost exclusively in the mRNA species present only a few times per cell (scarce sequences). Some mRNA species can exist in two forms, poly(A)containing and lacking, as evidenced by the translation data in vitro of Kaufmann et al. [Proc. Natl Acad. Sci. U.S.A. 74, 4801--4805 (1977)]. In addition, if cDNA to total poly(A)-containing mRNA is fractionated into abundant and scarce classes, 47% of the scarce class cDNA can be readily hybridized with poly(A)-free mRNA. 10% of the abundant cDNA to poly(A)-containing mRNA will hybridize with poly(A)-free sequences very rapidly while the other 90% hybridize 160 times more slowly, indicating two very different frequency distributions. The cytoplasmic metabolism of these three distinct mRNA classes is discussed.     

Stabilization of mRNA following serum-induction of quiescent 3T3 cells

The stability of mRNA has been measured in 3T3 cells in the resting and the growing states, and also during the transition from the resting to the growing state. Pulse labeled poly(A)⁺ mRNA chased with uridine and cytidine supplemented growth medium decayed with a half-life of 6.5 hr in the resting state, 26 hr during the transition from the resting to the growing condition, and 18 hr during serum-stimulated growth. The half-life of poly(A)⁺ mRNA determined by steady state labeling yielded similar results in resting and serum-stimulated 3T3 cells. Thus during the transition from resting to serum-stimulated growth in 3T3 cells poly(A)⁺ mRNA becomes more stable.     

Sequence complexity of cDNA transcribed from a diverse mRNA population

Mouse liver poly(A)+mRNA was reverse transcribed using oligo-p(dT) or random oligonucleotides as primers to yield cDNA about equal to the mass of the template RNA. The size profile of the oligo-p(dT)-primedd cDNA was similar to that of the template RNA. RNA or cDNA driven saturation annealing of labeled single copy genomic DNA (scDNA) showed that 2% of the scDNA was complementary in either case indicating the sequence complexity of cDNA was equivalent to that of the template mRNA. These results establish for the first time that cDNA represents essentially all of the sequence complexity of a diverse template RNA population in which individual mRNA species are present in vastly different concentrations. RNA driven hydridization of the cDNA showed that about 40% of the cDNA mass represents most of the sequence complexity of the template RNA. Also, kinetics of this hybridization indicate a complexity of 58,000 kb for the template RNA, a value similar to that obtained by scDNA hybridization. We conclude that appropriately characterized cDNA probes can be used to make valid qualitative and quantitative comparisons of the complex, infrequent class mRNAs of different cells and tissues.     

cDNA cloning of mRNAs induced in resistant barley during infection by Erysiphe graminis f.sp. Hordei

Summary Near-isogenic cultivars of Hordeum vulgare which differ for the Mlp gene for resistance to Erysiphe graminis f.sp. hordei were inoculated with race 3 of this pathogen and in vitro translation products of mRNA populations compared by 2-dimensional gel electrophoresis and fluorography. This revealed the presence of new mRNA species in infected leaves compared to non-inoculated controls. These new mRNA species were more abundant in resistant leaves than susceptible leaves. A cDNA library was prepared from poly(A)⁺RNA isolated from infected leaves carrying the Mlp gene for resistance (cvMlp). The library was screened by differential hybridization using [³²P]-labelled cDNA prepared from poly(A)⁺RNA of both control and infected leaves. Six cDNA clones showing greater hybridization to cDNA prepared from infected leaves were selected. These six cDNA clones hybridized to DNA isolated from barley leaves but not to DNA from conidia of the fungus. In Northern blot analysis of RNA from infected leaves the six cDNA clones each hybridized to mRNA species of different size. Translation products for three of the cDNA clones corresponded to infection-related translation products identified on 2-dimensional fluorograms. The cDNA clones were used to study the kinetics of host mRNA induction during infection of the near-isogenic cultivars of barley. The host mRNA species corresponding to the cDNA clones were induced prior to 24 h after inoculation during the primary penetration processes. In addition the mRNAs corresponding to four of the cDNA clones increased to greater amounts in cvMlp than in the near-isogenic susceptible cultivar (cvmlp) over a 2-d period following inoculation. These results suggest that the Mlp gene has a regulatory role in host gene expression resulting in enhanced expression of several host mRNA species following infection by the powdery mildew fungus.     

Three mRNA populations differing in turnover and processing in mouse liver

Using kinetics of approach to steady state labeling, we have found that liver poly(A)⁺ mRNAs fall into three populations, differing in stability and probably in processing, as reflected in their dissimilar delays in reaching polyribosomes and turnover times. There are mRNA-1 (delay 10 min, half-life 1 hr); mRNA-2 (delay 3 hr, half-life 2 hr); and mRNA-3 (delay 40 min, half-life 2.6 hr). The first two species function on free polyribosomes while the third one is operating on bound polyribosomes. The populations listed contribute 10, 20 and 70%, respectively, to the total steady state labeled poly(A)⁺ mRNAs.Abbreviations Poly(A)⁺ and poly(A)^- mRNAs mRNAs with and without poly(A) segments on the 3 end - HnRNA heterogeneous nuclear RNA     

The metabolism of a poly(A) minus mRNA fraction in HeLa cells

About 30% of HeLa cell mRNA lacks poly(A) when labeled in the presence of different rRNA inhibitors. Our method of RNA fractionation precludes contamination of the poly(A)^? mRNA with large amounts of poly(A)⁺ sequences. The poly(A)^? species is associated with polyribosomes, has an average sedimentation value equal to or greater than poly(A)⁺ mRNA, and behaves like the poly(A)⁺ mRNA in its sensitivity to EDTA and puromycin release from polyribosomes. There is very little, if any, hybridization at Rot values characteristic of abundant RNA sequences between the poly(A)^? RNA fractions from total cytoplasm or from polyribosomes and ³H-cDNA made to poly(A)⁺ RNA. This indicates that poly(A)^? mRNA does not arise from poly(A)⁺ mRNA by nonadenylation, deadenylation, or degradation of random abundant mRNA sequences. The rate of accumulation of poly(A)^? mRNA larger than 9S in the cytoplasm parallels the accumulation of poly(A)^? mRNA. The poly(A)^? mRNA is maintained as approximately 30% of the total labeled mRNA in a short (90 min) and in a long (20 hr) time period. These data indicate that poly(A)^? mRNA is not short-lived nuclear or cytoplasmic heterogeneous RNA contamination, and that the half-life of the poly(A)^? mRNA may parallel that of the poly(A)⁺ mRNA. Cordycepin appears to almost completely (95%) inhibit poly(A)⁺ mRNA while only partially (60%) inhibiting the poly(A)^? mRNA. The origin of the cordycepin-insensitive mRNA has not been ascertained.     

Regulation of a group of abundant mRNA sequences during Friend cell differentiation

The changes occurring in the pattern of genes expressed at the polysomal level during induction of Friend cell differentiation with 1.5% dimethylsulfoxide (DMSO) have been examined in two ways. First, homologous and heterologous hybridization experiments between cDNA and polysomal poly(A)⁺ mRNA from differentiated and undifferentiated cells show that about 8000 mRNAs are expressed at both stages of differentiation, the major change being the accumulation of α+β-globin mRNA after DMSO treatment. The vast majority of the mRNA sequences do not change qualitatively, remaining homologous between the undifferentiated and differentiated state. However, in addition to the accumulation of α+β-globin mRNA there is a decrease, after DMSO treatment, in the concentration of abundant and semiabundant sequences found in undifferentiated cells. From control studies with Friend cell variants and fractionated cDNA probes enriched in these sequences, it is shown that the decrease in the abundance of these mRNAs is related to the process of differentiation and not an artefact of DMSO treatment. Comparison of the polysomal poly(A)⁺ mRNAs in differentiated cells to those in pluripotential embryonal carcinoma (EC) cells shows that the vast majority of the sequences are homologous and hence not erythropoiesis specific. Second, comparison of these mRNA populations by in vitro translation and analysis of the protein products on two-dimensional gels also shows that among the more abundant proteins very few qualitatively new proteins appear after differentiation and that the majority are the same as those translated in EC mRNA. There are several proteins prominent in undifferentiated cells which diminish after DMSO treatment, in agreement with the findings from the cDNA studies.     

The disease resistance response in pea is associated with increased levels of specific mRNAs

A cDNA library was constructed using poly(A)⁺RNA fromPisum sativum which had been treated for 8 h with the fungusFusarium solani f. sp.phaseoli. Two thousand four hundred recombinant colonies were screened by differential colony hybridization using³²P-labelled cDNAs prepared from RNA extracted from either noninoculated or inoculated pea tissue. cDNA clones were then selected, which showed greater hybridization with cDNA prepared from pea RNA 8 h post-inoculation than with a cDNA probe from 0 h. Seven distinct hybridization classes were chosen for further study. Northern blot analyses of total cellular RNAs inoculated for 16 h with eitherF. solani phaseoli or water demonstrated that each cDNA clone selected represents an mRNA species which increases substantially in abundance during infection. Results of³H-uridine pulse-labelling experiments suggested that enhanced synthesis is at least partially responsible for the accumulation of the fungus-inducible mRNAs which hybridized with the clones.     

Kinetic study of protein unfolding and refolding using urea gradient electrophoresis

When total cytoplasmic RNA from mouse Friend cells is fractionated using oligo(dT)-cellulose or poly(U)-Sepharose chromatography, approximately 20% of the messenger RNA activity (as measured in the reticulocyte lysate cell-free system) remains in the unbound fraction, even though this contains < 0.5% of the poly(A) (as measured by titration with poly(U)). This RNA, operationally defined as poly(A)^?, is found almost entirely in polysome structures in vivo. Its major translation products, as shown by one-dimensional sodium dodecyl sulphate-containing gels, are the histones and actin. Two-dimensional gels (isoelectric focusing: sodium dodecyl sulphate/gel electrophoresis) show that, with the exception of the mRNAs coding for histones, poly(A)^? mRNA encodes similar proteins to poly(A)⁺ mRNA, though in very different abundances. This is directly confirmed by the arrest of the translation of the abundant poly(A)^? mRNAs after hybridization with a complementary DNA transcribed from poly(A)⁺ RNA.RNA sequences which are rare in the poly(A)⁺ RNA are also found in poly(A)^? RNA, as shown by hybridizing a cDNA transcribed from poly(A)⁺ RNA to total and poly(A)^? polysomal RNA. That this does not simply represent a flow-through of poly(A)⁺ RNA is indicated by (i) the lack of poly(A) by hybridizing to poly(U) in this fraction, (ii) the fact that further passage through poly(U)-Sepharose does not remove the hybridizing sequences, (iii) the very different quantitative distribution of proteins encoded by poly(A)⁺ and poly(A)^? RNAs. We also think that it does not result from removal of poly(A) from polyadenylated RNAs during extraction because RNAs prepared using the minimum of manipulations give similar results. The distribution of both total mRNA and α and β globin mRNAs between poly(A)⁺ and poly(A)^? RNA does not change significantly during the dimethyl sulphoxide-induced differentiation of Friend cells.     

Complex population of nonpolyadenylated messenger RNA in mouse brain

The complexity of nonadenylated mRNA [poly(A)-mRNA] has been determined by hybridization with single-copy DNA (scDNA) and cDNA. Our results show that poly(A)- and poly(A)+ mRNA are essentially nonoverlapping (nonhomologous) sequence populations of similar complexity. The sum of the complexities of poly(A)+ mRNA and poly(A)- mRNA is equal to that of total polysomal RNA or total mRNA, or the equivalent of approximately 1.7 x 10(5) different sequences 1.5 kb in length. Poly(A)- mRNA, isolated from polysomal RNA by benzoylated cellulose chromatography, hybridized with 3.6% of the scDNA, corresponding to a complexity of 7.8 x 10(4) different 1.5 kb sequences. The equivalent of only one adenosine tract of approximately 20 nucleotides per 100 poly(A)- mRNA molecules 1.5 kb in size was observed by hybridization with poly(U). cDNA was transcribed from poly(A)- mRNA using random oligonucleotides as primers. Only 1-2% of the single-copy fraction of this cDNA was hybridized using poly(A)+ mRNA as a driver. These results show that poly(A)- mRNA shares few sequences with poly(A)+ mRNA and thus constitutes a separate, complex class of messenger RNA. These measurements preclude the presence of a complex class of bimorphic mRNAs [that is, species present in both poly(A)+ and poly(A)- forms] in brain polysomes.     

Sequences coding for proteins expressed in liver and for globin in poly(A)+ and poly(A)− RNA fractions from nuclei and cytoplasm of chicken immature red blood cells

By hybridization with [³H]labeled globin cDNA the contents of globin coding sequences in total nuclear RNA, poly(A)⁺nuclear RNA, poly(A)^--nuclear RNA and polysomal RNA of chicken immature red blood cells was determined to be 0.86%, 20%, 0.42% and 1% respectively. As the poly(A)⁺-fraction comprises only about 2% of total nuclear RNA, globin coding sequences are distributed with 49% in the poly(A)⁺-fraction and with 51% in the poly(A)^--fraction.Part of the mRNA sequences which are found in liver are also transcribed in immature red blood cells. These sequences are enriched in poly(A)⁺-nuclear RNA as the globin coding sequences but their total amount in the poly(A)⁺-fraction is much smaller than in the poly(A)^--fraction.When nuclear RNA from immature red blood cells was translated in an ascites tumor cell-free system, 20% of the newly synthesized proteins were globin chains. The percentage of globin chains in the newly synthesized proteins increased to over 70% when poly(A)⁺-nuclear RNA was translated. Only about 7.5% of globin chains were found in proteins coded by poly(A)^--nuclear RNA.