Single copy DNA and structural gene sequence relationships among four sea urchin species

Measurements of the divergence of single copy DNA sequences among four sea urchin species are presented. At a standard criterion for reassociation (0.12 M phosphate buffer, 60° C, hydroxyapatite binding) we observe the following extents of reaction and reductions in thermal stability for single copy DNA reassociation between Strongylocentrotus purpuratus tracer and heterologous driver DNA: S. dröbachiensis 68% and 2.5°C; S. franciscanus 51% and 3.5° C; Lytechinus pictus 12% and 7.5° C. The implied extents of sequence relatedness are consistent with the phylogenetic relationships of these species. The rate of single copy sequence divergence in the evolutionary lines leading to the Strongylocentrotus species is estimated to be 0.06–0.35% per million years. The rate of divergence of total single copy sequence has been compared to that of structural gene sequences represented in S. purpuratus gastrula polysomal messenger RNA. When closely related species, S. purpuratus and S. franciscanus, are compared, these polysomal sequences are found to diverge at a lower rate than does the total single copy sequence. For two very distantly related species, S. purpuratus and L. pictus, a small fraction of the single copy DNA sequence is probably conserved. These conserved sequences are not enriched in their content of structural gene sequences.Also staff member, Carnegie Institution of Washington, Washington, D.C. 20015     

A comparison of sequence complexity of nuclear and polysomal poly(A)+ RNA from different developmental stages ofXenopus laevis

Summary Nuclear poly(A)⁺ and polysomal poly(A)⁺ RNA were isolated from gastrula and early tadpole stages of the amphibianXenopus laevis. Complementary DNA was synthesized from all RNA preparations. Hybridization reactions revealed that at least all abundant and probably most of the less frequent nuclear and polysomal poly(A)⁺ RNA species present at the gastrula stage are also present at the early tadpole stage. On the other hand, there are nuclear RNA sequences at the latter stage which appear, if at all, only at lower concentrations at the gastrula stage. The polysomal poly(A)⁺ RNA hybridization reactions suggest the existence of polysomal poly(A)⁺ RNA sequences at early tadpole stages which are not present in the corresponding gastrula stage RNA.By cDNA hybridization with poly(A)^– RNA it could be shown that most of the poly(A)⁺ containing RNA sequences transcribed into cDNA were also present within the poly(A)^– RNA. It was estimated, that these sequences are 10 fold more abundant within the poly(A)^– polysomal RNA and 3–6 more abundant within the poly(A)^– nuclear RNA as compared to the poly(A)⁺ RNAs.     

Cloning of cDNA sequences derived from poly(A)+ nuclear RNA ofXenopus laevis at different developmental stages: Evidence for stage specific regulation

Summary Nuclear poly(A)⁺ RNA was isolated from gastrula and early tadpole stages ofXenopus laevis, transcribed into cDNA and integrated as double stranded cDNA by the G-C joining method into the Pst cleavage site of plasmid pBR 322. After cloning inE. coli strain HB 101 the clone libraries were hybridized to³²P labelled cDNA derived from nuclear poly(A)⁺ RNA of the two different developmental stages. About 20% of the clones gave a positive hybridization signal thus representing RNA molecules of high and medium abundance. From these clones, some individual clones were identified containing sequences which are not present at the oocyte and gastrula stages but which are transcribed at the early tadpole stage of embryonic development.     

A gradient of poly(A)+ RNA sequences in Xenopus laevis eggs and embryos

Xenopus laevis eggs and gastrula stage embryos were fractionated into three equal sections normal to the animal-vegetal axis, and poly(A)⁺ RNA was isolated from each section. Hybridization of these poly(A)⁺ RNAs with [³²P]cDNA synthesized using animal or vegetal poly(A)⁺ RNAs showed no detectable differences in the extents or rates of reaction. Thus, the vast majority of poly(A)⁺ RNAs are not segregated along the animal-vegetal axis. To increase the sensitivity of these experiments, [³²P]cDNAs were prepared which had reduced levels of RNA sequences from the animal region of the gastrula stage embryo or spawned unfertilized egg. Hybridization reactions with these probes showed that 3 to 5% of the input cDNA represents poly(A)⁺ RNA sequences enriched 2- to 20-fold in the vegetal region of the egg or gastrula stage embryo.     

Use of a cloned library for the study of abundant poly(A)+RNA during Xenopus laevis development

Over 200 cloned sequences from recombinant DNA libraries prepared from Xenopus laevis embryonic poly(A)⁺RNA have been analyzed by colony hybridization with [³²P]cDNA prepared from poly(A)⁺RNA from several stages of development. The period of early embryogenesis extending through the beginning of gastrulation (stage 10) is marked by the relative constancy of the abundant poly(A)⁺RNA population. Between the gastrula and tailbud stages (stage 24) there is a dramatic change in the pattern of abundant poly(A)⁺RNA species; the new pattern remains fairly constant for at least 2 days of development to the late prefeeding tadpole stages (stage 41). We have also compared nonpolysomal and polysomal poly(A)⁺RNA populations at two different stages. In stage 10 (early gastrula) postribosomal (free ribonucleoprotein) and polysomal poly(A)⁺RNA populations partly overlap; however, many cloned sequences occur in quite different concentrations in one fraction or the other. Among the sequences that are predominantly nonpolysomal at gastrula few become predominantly polysomal at tailbud stages. Thus, we have no evidence for a major recruitment of abundant nonpolysomal RNAs into polysomes with progressing development. We rather observe a general pattern in which a cloned sequence that is nonpolysomal in one stage of development tends to be nonpolysomal (if detectable at all) in other stages as well.     

Long and short repeats of sea urchin DNA and their evolution

Repeated sequences cloned from the DNA of the sea urchin S. purpuratus were used as probes to measure the lengths of individual families of repeats. Some probes reassociated much more rapidly with preparations of long repeats than with short repeats while others reassociated more rapidly with short repeats than with long repeats. In this way two of five cloned repeats were shown to represent families with a great majority of sequences in the long class. One represented a family with similar numbers of long and short class members. Two were members of predominantly short class families. — The cloned repeats representing long class families, formed more precise duplexes than those representing short class families. Thermal stability measurements using S. purpuratus or S. franciscanus driver DNA showed that precise repetitive sequences have as great an interspecies sequence difference as the less precise repeats. Thus the precision of many families may result from recent multiplication rather than from selective pressure on the DNA sequences. Measurements of evolutionary frequency change show a clear correlation between the frequency change and the size of families of repeats in S. purpuratus. Comparison with S. franciscanus indicates that many of the large size families in S. purpuratus are those that have grown in size since these two species diverged.     

Structural gene sets active in embryos and adult tissues of the sea urchin

Structural gene sequences active in a variety of sea urchin adult and embryo tissues are compared. A single-copy ³H-DNA fraction, termed mDNA, was isolated, which contains sequences complementary to the messenger RNA present on gastrula stage polysomes. Gastrula message sequences are 50 fold concentrated in the mDNA compared to total single-copy DNA. mDNA reactions were carried out with excess mRNA from blastula, pluteus, exogastrula, adult ovary, tubefoot, intestine, and coelomocytes, and with excess total mature oocyte RNA. A single-copy ³H-DNA fraction totally devoid of gastrula message sequences, termed null mDNA, was also reacted with these RNAs. Large differences in the extent of both mDNA and null mDNA reaction with the various RNAs were observed, indicating that in each state of differention a distinct set of structural genes is active, generally characterized by several thousand specific sequences. The complexity of gastrula mRNA was shown in previous work to be about 17 × 10⁶ nucleotides. In units of 10⁶ nucleotides, the complexities of the RNA sequence reacting with mDNA and with null mDNA in each tissue are, respectively, as follows: intestine mRNA; 2.1 and 3.7; coelomocyte mRNA: 3.5 and ≤1.4; tubefoot mRNA: 2.7 and ≤0.4; ovary mRNA: 13 and 6.7; oocyte total RNA: 17 and 20; blastula mRNA: 12 and 15; pluteus mRNA: 14 and ≤0.6; exogastrula mRNA: 14 and ≤0.6. The total complexity of each mRNA population is the sum of these values, as verified for several cases by reactions with total single-copy DNA. A relatively small set of mRNAs, the complexity of which is about 2.1 × 10⁶ nucleotides, appears to be shared by several of the tissues studied.     

Molecular cloning of lupin leghemoglobin cDNA

Poly(A)⁺RNA isolated from root nodules of yellow lupin (Lupinus luteus, var. Ventus) has been used as a template for the construction of a cDNA library. The ds cDNA was synthesized and inserted into the Hind III site of plasmid pBR 322 using synthetic Hind III linkers. Clones containing sequences specific for nodules were selected by differential colony hybridization using³²P-labeled cDNA synthesized either from nodule poly(A)⁺RNA or from poly(A)⁺RNA of uninfected root as probes. Among the recombinant plasmids, the cDNA gene for leghemoglobin was identified. The protein structure derived from its nucleotide sequence was consistent with known amino acid sequence of lupin Lb II. The cloned lupin Lb cDNA hybridized to poly(A)⁺RNA from nodules only, which is in accordance with the general concept, that leghemoglobin is expressed exclusively in nodules.     

Evolution of sea urchin non-repetitive DNA

Summary New methods have been applied to the determination of single copy DNA sequence differences between the sea urchin speciesStrongylocentrotus purpuratus, S. franciscanus, S. drobachiensis, andLytechinus pictus. The thermal stability of interspecies DNA duplexes was measured in a solvent (2.4 M tetraethylammonium chloride) that suppresses the effect of base composition on melting temperature. The lengths of duplexes were measured after digestion with S1 nuclease and correction made for the effect of length on thermal stability. The degree of base substitution that has occurred in the single copy DNA during sea urchin evolution is significantly larger than indicated by earlier measurements. We estimate that 19% of the nucleotides of the single copy DNA are different in the genomes of the two sea urchin congeners,S. purpuratus, andS. franciscanus, which apparently diverged only 15 to 20 million years ago.     

Rates of DNA Evolution in Drosophila Depend on Function and Developmental Stage of Expression

DNA-sequence divergence of genes expressed in the embryonic stage was compared with the divergence of genes expressed in adults for 13 species of Drosophila representing various degrees of relatedness. DNA-DNA hybridization experiments were conducted using as tracers complementary DNA (cDNA) reversed transcribed from poly(A)(+) mRNA isolated from different developmental stages. The results indicate: (1) cDNA is less diverged than total single-copy DNA; (2) cDNA sequences are not in the rapidly evolving fraction of the single-copy genome of Drosophila; (3) early in evolutionary divergence embryonic messages are about half as diverged as adult messages; sequence data from some of the species compared indicate this is likely due to differences in rates of silent substitutions in genes expressed at different stages of development; and (4) at greater evolutionary distance, the differences in embryonic and adult messages disappear; this could be due to lineage-specific shifts in codon usage.     

Activation of Balbiani ring genes inChironomus tentans after a pilocarpine-induced depletion of the secretory products from the salivary gland lumen

We have constructed recombinant plasmid libraries containing complementary DNA (cDNA) inserts made to poly(A)⁺ RNA isolated from two stages of Dictyostelium development. The procedure utilized for the cloning allows the excision of the cDNA inserts free of vehicle sequences. The two libraries were screened for inserts complementary to moderately abundant and abundant poly(A)⁺ RNA whose genes are differentially modulated during Dictyostelium development. Several of these plasmids were then further examined by hybridization techniques to determine the reiteration frequencies of their genes, the relative rate of complementary RNA synthesis during development, and the relative accumulation and disappearance of complementary RNA during the Dictyostelium life cycle. RNA complementary to two sequences was found to accumulate from approximately one molecule per cell during vegetative growth to several hundred molecules during preaggregation.     

Repetitive sequences and hairpin-like structures in giant RNA of pigeon erythroid cells

In giant molecules (>45 S) of HnRNA from pigeon bone marrow and peripheral blood erythroid cells a correlation is demonstrated between the amounts of hairpin-like structures and the sequences transcribed from the DNA repetitions. The same correlation is observed in the >45 S poly(A)⁺ and poly(A)^- subfractions.Abbreviations HnRNA heterogeneous nuclear RNA - poly(A)⁺ RNA RNA molecules containing polyadenylic acid sequences - poly(A)^- RNA RNA molecules which do not contain polyadenylic acid sequences - dsRNA double-stranded RNA - SDS sodium dodecylsulphate     

Mitochondrial RNAs are abundant in the poly(A)+RNA population of early frog embryos

Mitochondrial sequences have been identified within a set of cloned complementary DNAs that had been copied from poly(A)⁺RNA of two embryonic stages of Xenopus laevis (Dworkin and Dawid, 1980, Dworkin and Dawid, 1980, Develop. Biol.76, 435–448 and 449–464). Mitochondrial sequences were found to be highly abundant in gastrula stage poly(A)⁺RNA sequences; in tadpole RNA their relative abundance is reduced severalfold. Mitochondrial sequences account for the most abundant poly(A)⁺RNA molecules in the gastrula population. The high abundance of mitochondrial RNA in early stages may be the consequence of the accumulation of large numbers of mitochondria in the egg.     

Expression of two cloned mRNA sequences during development and germination of spores of the sensitive fern,Onoclea sensibilis L.

A library of complementary DNA (cDNA) clones has been prepared from poly(A)⁺RNA of spores of the sensitive fern, Onoclea sensibilis L. By differential hybridization with labeled probes made to poly(A)⁺ RNA of spores, gametophytes and leaves, two spore-specific clones (pOSS68 and pOSS194) were selected and characterized. Northern blot analysis showed that RNA sequences homologous to the two cDNA clones first appear in the post-meiotic spore and increase in abundance during spore maturity. Both RNA sequences decay during photoinduced germination of the spores and do not reappear in the gametophytes. In spores imbibed in the dark under conditions which do not favor germination, no significant decrease in pOSS194-mRNA abundance is noted. In contrast, the decrease in pOSS68 mRNA in dark-imbibed spores parallels that observed in photoinduced spores. The predicted amino-acid sequence of pOSS194 has a striking similarity to the early light-inducible proteins expressed during the greening of etiolated pea and barley seedlings, whereas that of pOSS68 shows some homology to proteins encoded by late-embryogenesis-abundant mRNAs of angiosperm embryos.Abbreviations bp base pairs - cDNA complementary DNA - ds double-stranded - ELIP early light-inducible proteins - LEA late embryogenesis abundant - nt nucleotide - ss single stranded This work was partially supported by a NASA grant (NAGW-901) and by an allocation from the Research Challenge Investigators' Fund of the Ohio State University to V.R. Thanks are due to Mr. Clayton L. Rugh for sequencing our clones and to Dr. Paul A. Fuerst for help in the computer search of sequence alignments.     

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.     

Sequence complexity of messenger RNA in cotyledons of developing pea (Pisum sativum) seeds

The hybridization kinetics of poly(A)⁺-RNA preparations from the cotyledons of developing pea (Pisum sativum seeds to complementary DNAs have shown that the number of distinct sequences in poly(A)⁺ -RNA decreases from ca 20 000 at the early stage of cotyledon development to ca 200 at a late stage of cotyledon development. The decrease in sequences is accounted for entirely by the disappearance of ‘rare’ poly(A)⁺ -RNAs (< 10³ copies/cell) as seed development proceeds. There is an increase (1–6) in very abundant poly(A)⁺-RNA sequences (? 5 × 10⁵ copies/cell) from early- to mid-developmental stages, concomitantly with the increase in the synthesis of seed-specific storage protein polypeptides. In agreement with the continuing synthesis of most of these polypeptides to the end of seed development, the number of very abundant poly(A)⁺-RNAs is maintained to the late cotyledon development stage. Abundant poly(A)⁺-RNA sequences (ca 10⁴ sequences/cell) increase from 80 to 180 during development, possibly corresponding to the polypeptides which are not storage proteins but are known to be accumulated in pea seeds. Hybridization of single-copy pea genomic DNA sequences to poly(A)⁺-RNA from developing seeds showed that ca 5 % of the single-copy sequences were present in mRNA from mid-development cotyledons. In addition, hybridization of cDNA prepared against poly(A)⁺-RNA from nuclei of early development cotyledons to the corresponding cytoplasmic polysomal poly(A)⁺-RNA showed that the cytoplasmic poly(A)⁺-RNA contained ca 50 % of the sequences present in the nuclei. These results are discussed and interpreted in the light of existing results from similar systems.     

Electrophoretic analysis of newly synthesized and stored maternal RNA during oogenesis ofCalliphora erythrocephala (Dipt.)

Summary Ovaries ofC. erythrocephala synthesize large amounts of poly(A)⁺ and poly(A)^– RNA during early and middle stages of oogenesis as shown by labelling with³H-uridine in vivo. After incubation for 1 h, a striking difference in the electrophoretic pattern of newly synthesized labelled poly(A)⁺ RNA and the poly(A)⁺ RNA present in sufficient amounts for optical density measurements (steady state poly(A)⁺ RNA) was observed. During early and mid-oogenesis, in the poly(A)^– RNA fraction, 4S predominantly mature rRNA, 5S RNA and tRNA were labelled. These fractions were no longer synthesized during late oogenesis, whereas poly(A)⁺ RNA was labelled continously During oogenesis stage specific differences in the size distribution of newly synthesized and steady state poly(A)⁺ RNA were not obvious. However, different sizes of labelled poly(A)⁺ RNA species were detected in 0–2h old preblastoderm embryos, after injection of³H-uridine into females either 3–4 days (stage 3–4 of oogenesis) or 24 h before oviposition (stage 5–6 of oogenesis). This difference in RNA synthesis was related to the presence of active nurse cell nuclei. The poly(A)⁺ RNA fraction represents about 2–3% of the total RNA in both ovaries and freshly laid eggs as judged by measurements of optical density and radioactivity bound to oligo(dT). The length of poly(A)-segments in ovarian poly(A)⁺ RNA varied from about 30 to 200 nucleotides.