Auxin- and ethylene-induced changes in the population of translatable messenger RNA in Basal sections and intact soybean hypocotyl

In vitro translation products of polyadenylated RNA from untreated and auxin-treated basal sections of soybean (Glycine max var. Wayne) hypocotyl were analyzed by two-dimensional polyacrylamide gel electrophoresis. Within one hour of 2,4-dichlorophenoxyacetic acid treatment, the translatable messenger RNAs for at least twelve in vitro translation products are modulated upward. In vitro translation products of polyadenylated RNA from untreated, auxin-treated and Ethephon-treated intact soybean hypocotyl were also analyzed. Within two hours of treatment with either 2,4-dichlorophenoxyacetic acid or Ethephon, the translatable messenger RNAs for a group of high molecular weight in vitro translation products are modulated upward. There is a particular set of translatable messenger RNA, encoding in vitro translation products in the 24,000 to 32,000 molecular weight range, that is specifically modulated upward by auxin treatment in intact soybean hypocotyl and in hypocotyl sections.     

Auxin-induced changes in the population of translatable messenger RNA in elongating sections of soybean hypocotyl

In vitro translation products of polyadenylated RNA from untreated and auxin-treated elongating sections of soybean (Glycine max var. Wayne) hypocotyl were analyzed by two-dimensional polyacrylamide gel electrophoresis. The levels of translatable messenger RNA for at least ten in vitro translation products are increased by auxin treatment. The induction by auxin occurs rapidly (within 15 minutes), and the amounts of the induced in vitro translation products increase with time of auxin treatment. Indoleacetic acid has the same effect on the population of translatable messenger RNA as 2,4-dichlorophenoxyacetic acid. The auxin-induced in vitro translation products disappear rapidly when Actinomycin D is present during the last two hours of a three-hour auxin treatment.     

Kinesin mRNA Is Present in the Squid Giant Axon

Abstract: Recently, we reported the construction of a cDNA library encoding a heterogeneous population of polyadenylated mRNAs present in the squid giant axon. The nucleic acid sequencing of several randomly selected clones led to the identification of cDNAs encoding β-actin and β-tubulin, two relatively abundant axonal mRNA species. To continue characterization of this unique mRNA population, the axonal cDNA library was screened with a cDNA probe encoding the carboxy terminus of the squid kinesin heavy chain. The sequencing of several positive clones unambiguously identified axonal kinesin cDNA clones. The axonal localization of kinesin mRNA was subsequently verified by in situ hybridization histochemistry. In addition, the presence of kinesin RNA sequences in the axoplasmic polyribosome fraction was demonstrated using PCR methodology. In contrast to these findings, mRNA encoding the squid sodium channel was not detected in axoplasmic RNA, although these sequences were relatively abundant in the giant fiber lobe. Taken together, these findings demonstrate that kinesin mRNA is a component of a select group of mRNAs present in the squid giant axon, and suggest that kinesin may be synthesized locally in this model invertebrate motor neuron.     

Comparative analysis of polyadenylated RNA complexity in soybean hypocotyl tissue and cultured suspension cells

Growth parameters of suspension culture cells of soybean (Glycine max L.) were compared between cells grown in medium with (+) auxin and without (−) auxin. Growth rates were greater for (+) auxin cells. Cells transferred to (−) auxin medium primarily expanded in size while (+) auxin cells initially divided and then expanded. Two methods were used to estimate polyadenylated RNA sequence complexity. Kinetic analysis gave a sum of component complexity values of 36,000 and 64,000 diverse poly(A) RNA sequences of about 1,400 nucleotides in (+) and (−) auxin grown cells, respectively. The most striking difference between these cell populations was the increase in the poly(A) RNA sequence complexity in cells grown in medium without auxin. RNA complexities were also determined by the saturation of `single' copy DNA by poly(A) RNAs from (+) and (−) auxin suspension cells. These saturation studies estimated the total complexity of (+) and (−) auxin suspension cells as 41,000 and 57,000 diverse sequences, respectively. Suspension cells in auxin-depleted medium produced about 20,000 more diverse sequences than (+) auxin cells. Comparisons of poly(A) complexities were also made among auxin-treated and untreated hypocotyl cells from the intact plant relative to suspension culture cells. Mixed populations of poly(A) RNA from these tissues and cells allowed the determination of shared sequences among them. When all combinations of poly(A) RNA were mixed, the percentage of `single' copy DNA that saturated was equivalent to diverse sequence complexity estimates of about 60,000. When mixed poly(A) RNA from suspension cells from (+) and (−) auxin medium were compared, they shared about 40,000 sequences and (−) auxin cells contained an additional 20,000. Both (+) and (−) tissue culture cells shared a subset of about 20,000 sequences with cells from (+) and (−) auxin treated hypocotyl. A third subset of about 20,000 sequences was shared by (−) auxin suspension cells and hypocotyl treated with or without auxin, a subset most of which were not shared by (+) auxin suspension cells. Kinetic and saturation data estimates of poly(A) RNA complexity compared favorably and indicated that exogenous auxin treatment can dramatically alter the complexity of all classes of poly(A) RNAs in cultured cells.     

Differences in abundance of individual RNAs in normal and animalized sea urchin embryos

A library of cDNA clones was constructed representing polysomal polyadenylated RNA of mesenchyme blastulae of Strongylocentrotus purpuratus. Using this library, we determined whether or not individual RNA species are associated with animalization of embryos by zinc ions. Clones corresponding to the most actively synthesized RNAs during the period just prior to the mesenchyme blastula stage were selected by screening colonies with in vivo-labeled RNA. The most abundant of these were chosen for further study. Individual RNA abundance was measured as percent of mass of total polyadenylated RNA by hybridizing cDNA exhaustively with cloned DNA on filters. The RNAs in the selected, cloned sequences were present in abundances of 0.01 to 1% of the mass of polyadenylated RNA. Changes in abundance of individual RNA species occurred during normal development and departures from these developmental changes occurred in the zinc-animalized embryos. Two RNA species, which normally increase 10-fold in abundance, are drastically repressed and at least one RNA species increases in abundance dramatically in the animalized embryos. These departures from the normal program of presumptive gene expression may furnish insights into changes in the normal processes of development.     

Accumulation of muscle-specific RNA sequences during myogenesis

DNA complementary to rat skeletal muscle polyadenylated RNA was enriched for sequences specific for terminal differentiation by hybridization to RNA extracted from cloned mononucleated myogenic cells and subsequent removal of the hybridized cDNA. The remaining cDNA (musclespecific cDNA) was hybridized to RNA extracted from primary skeletal muscle cultures harvested at short time intervals during differentiation. The experiments indicate that sequences specific for terminal differentiation accumulate close to the time of cell fusion, possibly a few hours prior to it. DNA complementary to polyadenylated muscle RNA was fractionated by hybridization to its template at a low R₀t and separation of the hybridized (abundant) and nonhybridized (rare) cDNA. Hybridization of these fractions to RNA extracted from cultures harvested prior to or after cell fusion showed that the abundant cDNA is very much enriched for sequences specific for terminal differentiation.     

Molecular Cloning of α-Amylase Genes from DROSOPHILA MELANOGASTER. I. Clone Isolation by Use of a Mouse Probe

A cloned alpha-amylase cDNA sequence from the mouse is homologous to a small set of DNA sequences from Drosophila melanogaster under appropriate conditions of hybridization. A number of recombinant lambda phage that carry homologous Drosophila genomic DNA sequences were isolated using the mouse clone as a hybridization probe. Putative amylase clones hybridized in situ to one or the other of two distinct sites in polytene chromosome 2R and were assigned to one of two classes, A and B. Clone lambda Dm32, representing class A, hybridizes within chromosome section 53CD. Clone lambda Dm65 of class B hybridizes within section 54A1-B1. Clone lambda Dm65 is homologous to a 1450- to 1500-nucleotide RNA species, which is sufficiently long to code for alpha-amylase. No RNA homologous to lambda Dm32 was detected. We suggest that the class B clone, lambda Dm65, contains the functional Amy structural gene(s) and that class A clones contain an amylase pseudogene.     

Molecular cloning of genes related to aflatoxin biosynthesis by differential screening.

A differential hybridization strategy was used to clone genes associated with aflatoxin biosynthesis. A genomic library, formed between nuclear DNA and the pUC19 plasmid, was screened with three different cDNA probes by the colony hybridization procedure. Nineteen clones were selected; all were positively correlated with and presumably enriched with genes associated with aflatoxin production. Some of these clones were further characterized by using them as probes in Northern (RNA blot) hybridizations. Five clones hybridized strongly with some polyadenylated RNAs formed during the transition to or during idiophase when aflatoxin was produced. However, little or no corresponding hybridization occurred with polyadenylated RNAs formed in early and mid-log growth phase. Two of the clones were further used as probes to hybridize with polyadenylated RNAs formed under aflatoxin-permissive and nonpermissive temperatures. Hybridization occurred with RNA species formed under the permissive temperature only.     

Molecular cloning of cDNAs for the nerve-cell specific phosphoprotein, synapsin I.

To provide access to synapsin I-specific DNA sequences, we have constructed cDNA clones complementary to synapsin I mRNA isolated from rat brain. Synapsin I mRNA was specifically enriched by immunoadsorption of polysomes prepared from the brains of 10-14 day old rats. Employing this enriched mRNA, a cDNA library was constructed in pBR322 and screened by differential colony hybridization with single-stranded cDNA probes made from synapsin I mRNA and total polysomal poly(A)+ RNA. This screening procedure proved to be highly selective. Five independent recombinant plasmids which exhibited distinctly stronger hybridization with the synapsin I probe were characterized further by restriction mapping. All of the cDNA inserts gave restriction enzyme digestion patterns which could be aligned. In addition, some of the cDNA inserts were shown to contain poly(dA) sequences. Final identification of synapsin I cDNA clones relied on the ability of the cDNA inserts to hybridize specifically to synapsin I mRNA. Several plasmids were tested by positive hybridization selection. They specifically selected synapsin I mRNA which was identified by in vitro translation and immunoprecipitation of the translation products. The established cDNA clones were used for a blot-hybridization analysis of synapsin I mRNA. A fragment (1600 bases) from the longest cDNA clone hybridized with two discrete RNA species 5800 and 4500 bases long, in polyadenylated RNA from rat brain and PC12 cells. No hybridization was detected to RNA from rat liver, skeletal muscle or cardiac muscle.     

Molecular cloning of genes related to aflatoxin biosynthesis by differential screening.

A differential hybridization strategy was used to clone genes associated with aflatoxin biosynthesis. A genomic library, formed between nuclear DNA and the pUC19 plasmid, was screened with three different cDNA probes by the colony hybridization procedure. Nineteen clones were selected; all were positively correlated with and presumably enriched with genes associated with aflatoxin production. Some of these clones were further characterized by using them as probes in Northern (RNA blot) hybridizations. Five clones hybridized strongly with some polyadenylated RNAs formed during the transition to or during idiophase when aflatoxin was produced. However, little or no corresponding hybridization occurred with polyadenylated RNAs formed in early and mid-log growth phase. Two of the clones were further used as probes to hybridize with polyadenylated RNAs formed under aflatoxin-permissive and nonpermissive temperatures. Hybridization occurred with RNA species formed under the permissive temperature only.     

Cloning and identification of lupin nodule specific cDNA sequences

Yellow lupin nodule specific sequences were selected by screening of cDNA library prepared from lupin nodule poly(A)+RNA. From about 3,000 clones containing fragments of lupin DNA 150-1,500 base pair long, 7% of clones carrying nodule specific sequences were identified. Among them the most abundant sequence species, represented by 32% clones, encodes leghemoglobin. Another abundant species designated pLN13 is represented by 13% clones. The Northern blot analysis of lupin mRNA confirmed nodule specificity of the cloned sequences. The nucleotide sequence of one clone, pLN281 of 225 bp, is presented.     

Cloning, characterization, and tissue distribution of messenger RNA species expressed at moderate and scarce frequencies within the lactating guinea pig mammary gland

In the lactating guinea pig mammary gland, the most abundant mRNA species encoding the major milk proteins, alpha-lactalbumin and caseins A, B, and C, have been extensively studied. Here we describe the isolation and characterization of cloned cDNA sequences representative of moderately abundant and scarce mammary gland mRNA species present at estimated concentrations of 1,400 (pgpO5), 540 (pgpKE6), 36 (pgpK1), and 2 (pgpJF4) copies per sequence per cell. RNA blotting showed these to represent mRNA species of 1,150, 1,900, 1,250, and 3,300 nucleotides in size, respectively. Hybrid selection cell-free synthesis showed that the mRNAs encoded proteins of Mr 33,000 (pgpO5), 58,000 (pgpKE6), and 36,000 (pgpK1). Studies on the tissue distribution of mammary gland mRNAs showed that the mRNA species of lower abundance, but not milk protein mRNAs, were expressed in other tissues but at concentrations differing from those in the mammary gland. None were expressed in all tissues, and so were not typical "housekeeping" proteins. We have used these cloned cDNA species to reinvestigate the apparent differential accumulation of moderately abundant poly(A)-containing mRNA species in polyadenylated and nonpolyadenylated cytoplasmic RNA populations of the mammary gland. Unlike previous observations, based on RNA excess hybridization using fractionated cDNA probes, the use of sequence-specific cloned cDNA probes showed that little intact mRNA was present in the nonpolyadenylated fraction. Thus previous observations were a reflection of the preferential accumulation of fragments of moderately abundant mRNA species, possibly a result of enhanced turnover. The significance of our results in terms of future investigations into factors which determine mRNA accumulation and tissue-specific expression is discussed.     

Comparison of complexity and diversity of polyadenylated polysomal and informosomal messenger ribonucleic acid from Chinese hamster cells.

The sequence complexity and relative abundance of cytoplasmic polyadenylated polysomal (ribosome-bound) mRNA and cytoplasmic polyadenylated informosomal (ribosome-free) mRNA were analyzed in exponentially growing Chinese hamster cells (line CHO) using the technique of cDNA hybridization to excess poly(A)+ mRNA. Polysomal and informosomal mRNAs had similar complexities ( approximately 8300 mRNA species), but both the fraction of mRNA and the number of sequences comprising the mRNA abundance classes were different. Heterologous annealing reactions showed that all of the mRNA sequences detected were shared by the polysomal and informosomal mRNAs. However, the most abundant informosomal mRNA component was considerably different from the most abundant polysomal mRNA component. For a more detailed analysis, cDNA complementary to the most abundant informosomal and polysomal mRNAs was isolated. By use of the fractionated cDNA, it could be demonstrated that the most abundant informosomal mRNA sequences were distributed in the polysomal mRNA with an approximately fivefold reduction in relative frequency. These results are not compatible with models postulating translational control of gene expression by the complete sequestering of some mRNA sequences in an untranslatable form in the cytoplasm. The data are, however, consistent with models encompassing differential rates of initiation on the polysome and/or preferential affinity of some mRNAs for initiation factors.     

Characterization of a class of small auxin-inducible soybean polyadenylated RNAs

Four new auxin-responsive RNAs from soybean (Glycine max (L.) Merr., var. Wayne) are described. The RNAs were identified by hybridization to three cDNA probes obtained from a library enriched for sequences which increase in abundance within 60 min after 2,4-D (2,4-dichlorophenoxyacetic acid) treatment. These RNAs appear to define a new class of small (i.e. approximately 550 nucleotides) RNAs that respond extremely rapidly to application of exogenous auxin. In excised elongating hypocotyl sections, an increase in the abundance of these RNAs can be detected 2 to 5 min after treatment with 50 M 2,4-D. This response is half maximal after 10 min and reaches steady state in 60 min. RNA blot analysis shows that these RNAs are expressed differentially in various parts of the seedling. The degree of inducibility by auxin is also organ-specific, with the elongating hypocotyl being the most responsive of the organs tested. The RNAs display identical response specificities with one exception. Accumulation of one RNA, designated 10A, is completely abolished by simultaneous addition of cycloheximide and 2,4-D. This RNA also displays a different 2,4-D dose response than other RNAs examined. These results suggest that more than one mechanism is involved in rapid modulation of gene expression by auxin.