Coordinate accumulation of five transcripts in the primary mesenchyme during skeletogenesis in the sea urchin embryo

The sea urchin larval skeleton is produced by the primary mesenchyme (PM), a group of 32 cells descended from the four micromeres of the 16-cell embryo. The development of this lineage proceeds normally in isolated cultures of micromeres. A complementary DNA (cDNA) library was generated from cytoplasmic polyadenylated RNA isolated from differentiated micromere cultures of Strongylocentrotus purpuratus. Five clones were selected on the basis of their enrichment in differentiated PM cell RNA as compared to the polyribosomal RNAs of other embryonic cell types and other developmental stages. Each cloned cDNA hybridized to a distinct RNA that was abundant in the polyribosomes of differentiated PM cells, but absent from larval ectoderm and from 16-cell embryos. These RNAs were encoded by single or low copy genes. In situ hybridization analysis of the most abundant of these RNAs (SpLM 18) demonstrated that it was specifically limited to the skeletogenic PM of intact embryos. During the development of the PM, all five RNAs exhibited the same schedule of accumulation, appearing de novo, or increasing abruptly just before PM ingression, and remaining at relatively high levels thereafter. This pattern of RNA accumulation closely paralleled the pattern of synthesis of PM-specific proteins in general (Harkey and Whiteley, 1983) and of the SpLM 18-encoded protein specifically (Leaf et al., 1987). These results indicate that at least five distinct genes in the sea urchin, each of which encodes a PM-enriched or PM-specific mRNA, are expressed with tight coordination during development of the larval skeleton. They also demonstrate that expression of these genes in the PM is regulated primarily at the level of RNA abundance rather than RNA utilization.     

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 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.     

Differences among the polyadenylated RNA sequences of human leucocyte populations: an approach to the objective classification of human leukaemias

We have constructed a complementary DNA (cDNA) library representing expressed sequences of the white blood cells from a patient with chronic granulocytic leukaemia. The library was screened by colony hybridization of 32P-labelled cDNAs synthesized from the polyadenylated RNAs of the white blood cells from patients with chronic granulocytic or chronic lymphocytic leukaemia. The autoradiographic patterns were compared and 70 recombinants were selected to comprise a panel which distinguished between these two types of leukaemia. Hybridization of this panel with complementary DNAs transcribed from the polyadenylated RNAs of a variety of normal and neoplastic leucocyte populations showed that the RNA sequences in high abundance in leucocytes from chronic granulocytic leukaemias differ quite radically from those in other leucocytes. The patterns of hybridization seen when this panel was challenged with cDNAs representing the RNAs of normal and leukaemic leucocyte populations were sufficiently different to distinguish clearly the peripheral blood leucocytes of chronic granulocytic leukaemias from other populations of white blood cells, both normal and leukaemic. We suggest that this approach might provide additional markers useful in the classification of the acute leukaemias, especially the undifferentiated leukaemias whose identification by conventional methods is uncertain.     

mRNA usage during Drosophila melanogaster embryonic development. Analysis of nine cloned DNA segments

A set of nine phage lambda clones containing inserts from Drosophila melanogaster which are complementary to cDNA made from oocyte poly(A)+ RNA were selected from a larger group. These cloned elements code for a range of middle abundant RNA sequences which show no appreciable change in abundance during Drosophila embryogenesis. Seven of the nine clones are complementary to two oocyte RNAs, one to three RNAs and one to four RNAs. This study describes the changes that occur in these RNAs during embryonic development in the polysomal and non-polysomal fraction, and in the poly(A)+ RNA and poly(A)- RNA fraction. In all nine of these clones, greater than 70% of the complementary RNA is found in the polysomal region of a sucrose gradient. This proportion increases somewhat during development. Specific changes have been found during development in the proportion of RNA that is poly(A)+. Depending to the cloned sequence, this proportion may increase, decrease, or remain unchanged. For those clones that show a change, most of this change occurs between 8 and 19 h of development. Our data suggest, furthermore, the presence of a class of non-adenylated RNA being utilized during embryogenesis.     

Cellular titers and subcellular distributions of abundant polyadenylate-containing ribonucleic acid species during early development in the frog Xenopus laevis.

The distribution of cytoplasmic messenger ribonucleic acids (RNAs) in translationally active polysomes and inactive ribonucleoprotein particles changes during early development. Cellular levels and subcellular distributions have been determined for most messenger RNAs, but little is known about how individual sequences change. In this study, we used hybridization techniques with cloned sequences to measure the titers of 23 mitochondrial and non-mitochondrial polyadenylate-containing [poly(A)+]RNA species during early development in the frog Xenopus laevis. These RNA species were some of the most abundant cellular poly(A)+ RNA species in early embryos. The concentrations of most of the non-mitochondrial (cytoplasmic) RNAs remained constant in embryos during the first 10 h of development, although the concentrations of a few species increased. During neurulation, we detected several new poly(A)+ RNA sequences in polysomes, and with one possible exception the accumulation of these sequences was largely the result of new synthesis or de novo polyadenylation and not due to the recruitment of nonpolysomal (free ribonucleoprotein) poly(A)+ RNA. We measured the subcellular distributions of these RNA species in polysomes and free ribonucleoproteins during early development. In gastrulae, non-mitochondrial RNAs were distributed differentially between the two cell fractions; some RNA species were represented more in free ribonucleoproteins, and others were represented less. By the neurula stage this differential distribution in polysomes and free ribonucleoproteins was less pronounced, and we found species almost entirely in polysomes. Some poly(A)+ RNA species transcribed from the mitochondrial genome were localized within the mitochondria and were mapped to discrete fragments of the mitochondrial genome. Much of this poly(A)+ RNA was transcribed from the ribosomal locus. Nonribosomal mitochondrial poly(A)+ RNA species became enriched in polysome-like structures after fertilization, with time courses similar to the time course of mobilization of cytoplasmic poly(A)+ RNA.     

Identification and Characterization of Neuron-Specific and Developmentally Regulated Gene Transcripts in the Chick Embryo Spinal Cord

Clones corresponding to neuron-specific and developmentally regulated messenger RNA species in the chick have been isolated from a complementary DNA library prepared using polyadenylated RNA from 7-day embryonic spinal cord. The library was initially screened by differential complementary DNA hybridization procedures for clones identifying polyadenylated RNAs present in embryonic spinal cord but absent from or at low abundance in liver tissue. A high proportion of selected recombinant plasmids were found to identify different RNA species which, although present in 14-day embryonic spinal cord, could not be detected in a corresponding region of the developing chick CNS that is devoid of neuronal cell bodies, the optic nerve. The neuron-specific assignment of these mRNAs within the developing neuroectoderm was confirmed using bulk-isolated neuronal and glial-enriched cell fractions from 7-day embryonic spinal cord. In addition, several distinctive patterns of developmentally regulated expression of neuron-specific messenger RNA species have been observed in the chick spinal cord. The studies lay a foundation for detailed examination of the regional and temporal distribution and control of neuronal gene expression in the chick spinal cord during embryogenesis.     

Changes in the frequency and diversity of messenger RNA populations in the course of myogenic differentiation.

Complementary DNAs (cDNAs) were synthesized from polyadenylated RNAs of myoblasts and myotubes and used to analyze changes in the sequence complexity and frequency distribution of messenger RNAs during myogenesis in vitro. cDNA . polyadenylated-RNA hybridization kinetics show the presence of messenger RNA sequences specific for myotubes in fully differentiated muscle cultures. These sequences are accumulated just prior to fusion, as was shown by hybridizations of myotube cDNA and total cytoplasmic RNAs from cells at different stages of differentiation. The myotube cDNA can be enriched 10-fold in myotube-specific RNA species by a hybridization with cytoplasmic RNAs from myoblasts and subsequent removal of these hybridized sequences by hydroxyapatite.     

Changes induced in rat liver polysomal polyadenylated RNAs by depletion of circulating glucocorticoids.

The effects of adrenalectomy on the complexity and the relative abundances of rat liver polyadenylated mRNAs have been investigated. The qualitative and quantitative changes induced by adrenalectomy have been measured by hybridisation of polysomal polyadenylated RNAs from the livers of normal and adrenalectomised rats with total cDNAs, fractionated cDNAs, cDNA representing RNAs specific to normal liver, total unique-sequence DNA and unique-sequence DNA complementary to normal liver polysomal RNA. These analyses indicated that, by 14 days after adrenalectomy, the equivalent of about 7000 sequences of average length 2000 nucleotides can no longer be detected in liver polysomes. Many other sequences are decreased in abundance as compared to normal liver, but some abundant sequences become more abundant. Administration of a glucocorticoid hormone (dexamethasone) very rapidly reverses these changes.     

mRNA abundance changes during flagellar regeneration in Chlamydomonas reinhardtii.

Flagellar amputation in Chlamydomonas reinhardtii induces the accumulation of a specific set of RNAs, many of which encode flagellar proteins. We prepared a cDNA clone bank from RNA isolated from cells undergoing flagellar regeneration. From this bank, we selected clones that contain RNA sequences that display several different patterns of abundance regulation. Based on quantitation of the relative amounts of labeled, cloned cDNAs hybridizing to dots of RNA on nitrocellulose filters, the cloned sequences were divided into five regulatory classes: class I RNAs remain at constant abundance during flagellar regeneration; classes II, III, and IV begin to increase in abundance within a few minutes after deflagellation, reach maximal abundance at successively later times during regeneration, and return to control cell levels within 2 to 3 h; and class V RNA abundance decreases during flagellar regeneration. Alpha- and beta-tubulin mRNAs are included in regulatory class IV. The abundance kinetics of alpha-tubulin mRNAs differ slightly from those of beta-tubulin mRNAs. The availability of these clones makes possible studies on the mechanisms controlling the abundance of a wide variety of different RNA species during flagellar regeneration in Chlamydomonas.     

Polyadenylated RNA sequences which are reduced in concentration following auxin treatment of soybean hypocotyls

Previous work has shown that any effect of exogenous auxin on gene expression in soybean hypocotyl tissue must be restricted to a relatively small fraction of the polyadenylated RNA. However, kinetic hybridization analysis with cDNA probes revealed that a minor abundant class of sequences is markedly reduced in concentrations in the auxin-treated polyadenylated RNA. Recombinant plasmids containing copies of polyadenylated RNA species were constructed using the G-C tailing procedure and clones of auxin-regulated sequences were detected by differential in situ hybridization with cDNA of polyadenylated RNA from auxin-treated or untreated hypocotyls. Although the 12 clones which were selected all contained different size inserts, and were therefore independent, 11 of these apparently hybridized to just two different RNA species. The rate constant of the auxin-sensitive abundant component of the untreated polyadenylated RNA/DNA hybridization was similar to that of the reaction between the two major groups of clones and untreated polyadenylated RNA. This indicates that these cloned sequences are homologous with that cDNA fraction. The twelfth clone is thought to be representative of a group of less abundnt auxin-regulated polyadenylated mRNA species which had been detected in an earlier analysis of the in vitro translation products of soybean hypocotyl RNA. Both the timing and the extent of the influence of auxin on the relative concentration of these cloned sequences are quite consistent with a close relationship between growth regulation by auxin and its effects on gene expression.     

Differential expression of keratin genes during mouse development

Suprabasal layers of the newborn mouse epidermis contain two mRNAs of 2.0 and 2.4 kb which are translated into keratins of 59 and 67 kDa, respectively. To study their expression during development, cDNA sequences corresponding to the 2.0- and the 2.4-kb mRNAs were cloned, characterized by hybridization selection assay, and used as probes to detect keratin sequences in polyadenylated RNA from Day 11, 13, 15, and 17 embryos. In RNA from Day 11 of gestation, two RNAs of 2.8 and 1.8 kb were identified. They were found to have homologies with both epidermal RNAs, suggesting that they are coding for proteins of the keratin family. These two sequences were not detected in sample of later stages. RNAs comigrating with the two epidermal keratin RNAs were identified only in Day 15 and 17 embryos indicating that their expression was induced between Day 13 and 15. Finally, the localization of the 59-kDa keratin mRNA was examined by in situ hybridization. The spinous and granulous cell layers were found to be heavily covered with grains while other regions of the tissue sections were unlabeled. All these results support the hypothesis of a sequential expression of keratins during differentiation of epidermal cells and suggest that proteins related to the keratins expressed specifically in keratinizing cells are expressed earlier during development.     

Estimates of mRNA abundance in the mouse blastocyst based on cDNA library analysis

Studies of gene expression during blastocyst formation in mouse preimplantation development have been limited by the amount of RNA available per embryo. Our present approach to this problem has been to construct a large, representative, blastocyst cDNA library in lambda gt11. Random hexadeoxynucleotides were used as primers with total blastocyst RNA serving as template. RNA collected from 4,100 32-64 cell embryos was used to generate a library with an initial size of 30 X 10(6) recombinants. By using clone frequency as a measure of relative mRNA abundance, our data support previous work on the relative and absolute amounts of actin, histone H2a, and intracisternal A particle. Furthermore, we provide estimates for the abundance of cytokeratin endo A, cytokeratin endo B, and beta-tubulin from clone frequency data. Insert sizes for isolated clones range from 200 bp to 3.6 kb with full-length or near-full-length insert sizes for selected clones, indicating that random primer methods generate cDNAs which can represent a significant portion of the mRNA. We have so far characterized products whose abundance is equal to or greater than 0.002% of total RNA. This library offers the potential for the analyses of presumptive regulatory gene products in the mouse preimplantation embryo which are represented as low abundance (less than 1% of mRNA) RNAs.     

Molecular cloning and characterisation of cDNAs complementary to mRNAs from wounded potato (Solanum tuberosum) tuber tissue

Five cDNA clones complementary to mRNAs representing different abundances and responses to wounding have been isolated from a library of Sau 3A fragments in the bacteriophage M13 mp8. These were characterised by hybrid-release translation and hybridisation to RNA blots. The levels of RNA complementary to two of the clones show a marked increase during the 24h after wounding, one shows a small increase and two show no appreciable changes except that caused by a general increase in the total amount of polyadenylated RNA per microgram of total RNA which increases 2.5-fold during the same period. The would-induced RNAs are not induced in diluted suspension-culture cells, but RNA complementary to each clone is present in varying levels in stems, leaves and roots of intact potato plants.Abbreviations cDNA complementary DNA - poly(A) polyadenosine - poly(A)⁺ RNA polyadenylated RNA - poly(U) polyuridine