Barbara R. Hough-Evans

Introduction of Cloned DNA into Sea Urchin Egg Cytoplasm: Replication and Persistence during Embryogenesis

Cloned DNA sequences were introduced into the cytoplasm of unfertilized sea urchin eggs by a simple microinjection technique. Sperm was then added, and development allowed to proceed. If linearized plasmids are injected they form random concatenates, and during the early development of the embryos replicate repeatedly. Eukaryotic sequences are not required for replication of the exogenous DNA. Injected supercoiled DNAs neither ligate nor replicate. Both forms of exogenous DNA persist in the embryo through pluteus stage.

Sea urchin embryo mRNA sequences expressed in the nuclear RNA of adult tissues

The representation of message sequences in nuclear RNA was studied in sea urchin tissues which utilize these messages in their polysomes and in tissues which do not. A ^3H-labeled single-copy DNA tracer highly enriched for sequences complementary to blastula embryo mRNA was prepared. This tracer (mDNA) reacted to 78% with excess blastula mRNA, compared with 2.1% for the starting single-copy DNA. As expected from previous data, the mDNA reacted to only 12% with cytoplasmic RNA of adult intestine, since most embryo mRNA sequences are not detectable in this tissue. The mDNA reacted to the same extent, however, with nuclear RNA from either adult intestine, adult coelomocytes or gastrula embryos as with polysomal mRNA from blastula embryos. Thus virtually all of the blastula mRNA sequences are present in adult tissue nuclear RNAs, although most of these mRNA sequences are absent from cytoplasmic or polysomal RNA in the adult cells. The blastula mRNA sequences are present in heterogeneous nuclear RNAs at the same concentrations as are nuclear transcripts of most single-copy DNA sequences. Calculations based on the steady state concentration of structural gene transcripts in the embryo nuclei suggest that a majority of these molecules do not serve as message precursors.

Appearance and persistence of maternal RNA sequences in sea urchin development

This paper deals with the relationship between the single copy transcripts represented in mature oocytes of the sea urchin and the RNA sequences present in immature oocytes and embryos. We term the oocyte transcripts from single copy DNA the maternal single copy sequence set. A single copy [^3H]DNA fraction ([^3H]oDNA) enriched for sequences complementary to the maternal single copy sequence set was prepared and reacted with the different RNA preparations. The complexity of the mature oocyte RNA is estimated to be 37 × 10^6 nucleotides. At kinetic termination, [3H]oDNA reacted with the polysomal mRNA of 16-cell embryos to 73% of the reaction with mature oocyte RNA, indicating that 27 × 10^6 nucleotides of the maternal sequence set are present. With blastula mRNA the reaction equals about 56%, a complexity of 21 × 10^6 nucleotides; with gastrula mRNA, 53%, a complexity of 19 × 10^6 nucleotides. The relative amount of hybridization of [^3H]oDNA was 100% with cytoplasmic RNA of the 16-cell stage and became progressively less with the cytoplasmic RNAs of later stages. The total RNA of immature oocytes was found to include about 26 × 10^6 nucleotides of the maternal sequence set. Results of these experiments are discussed, and an interpretation of the pattern of utilization of structural genes during oocyte and embryo development is suggested.

Mosaic incorporation and regulated expression of an exogenous gene in the sea urchin embryo

A fusion gene construct in which the bacterial chloramphenicol acetyltransferase (CAT) gene is controlled by CyIIIa actin gene cis-regulatory sequences was injected into unfertilized eggs of the sea urchin Strongylocentrotus purpuratus. The distribution of CAT DNA sequences was measured directly by in situ hybridization in squashed 24-hr blastula preparations derived from these eggs. Earlier studies had shown that stable mosaic incorporation of the exogenous DNA occurs during cleavage, after which the exogenous sequences replicate at approximately the pace of the host cell genomes. The fractions of embryonic cells observed in this study to include CAT DNA sequences imply that their stable incorporation into a replicating nuclear form occurs most often in a single cell at the 3rd or 4th cleavage stages, though it may occur as early as 2nd cleavage, or as late as 7th cleavage. Corroborative measurements were carried out by the same method on squashed preparations of embryos at earlier stages, and by in situ hybridizations of CAT mRNA, both in dissociated embryos and in cytological sections of 72-hr pluteus-stage embryos. Hybridizations to CAT mRNA and to CAT DNA were carried out on alternate sections of several embryos. The results confirm unequivocally that although CAT mRNA appears only in the aboral ectoderm in embryos derived from eggs injected with the CyIIIa.CAT fusion gene, the exogenous sequences are indeed present, though silent, in the various other cell types of the late embryo.

Limited Complexity of the RNA in Micromeres of Sixteen-Cell Sea Urchin Embryos

The sequence complexity of sea urchin embryo micromere RNA is about 75% of that of total 16-cell embryo cytoplasmic RNA, as reported earlier by Rodgers and Gross [Rodgers, W. H., and Gross, P. R. (1978) Cell, 14, 279–288]. In contrast to the rest of the embryo, there are few, if any, complex maternal RNA species in the micromere cytoplasm which are not represented in the polysomes. The micromeres do not contain detectable quantities of high-complexity nuclear RNA, though such RNA exists in other cells of the fourth-cleavage embryo.

Correct cell-type-specific expression of a fusion gene injected into sea urchin eggs☆

A fusion gene construct containing the bacterial chloramphenicol acetyltransferase (CAT) gene under the control of CyIIIa actin gene regulatory sequences was injected into unfertilized eggs of the urchin Strongylocentrotus purpuratus, and early pluteus stage embryos that developed from these eggs were fixed and sectioned for analysis by in situ hybridization. A [3H]RNA antisense probe for CAT mRNA was hybridized to 5-micron embryo sections. Autoradiographic signal denoting the presence of CAT mRNA was detected only over aboral ectoderm cells, in which the CyIIIa gene is normally expressed, and not over any recognizable regions of gut or oral ectoderm included in the same sections.

Proteins of the sea urchin egg vitelline layer

The vitelline layers (VL) of unfertilized sea urchin eggs were isolated, and the diversity of their polypeptide constitutents estimated by two-dimensional polyacrylamide gel electrophoresis. At least 25 components are reproducibly observed. While VL polypeptides are almost certainly synthesized in the growing oocyte, they are not among the more prevalent newly synthesized proteins detected in oocytes that were isolated and labeled in vitro for 4 hr. A set of monoclonal antibodies was raised against VL components and partially characterized. The 31 monoclonals analyzed fell into 11 classes with respect to their avidity for VL proteins solubilized under mild and under strongly denaturing conditions, and to their reactions with surface components of the VLs of living eggs. Fluorescence microscopy showed diverse patterns of surface reactivity when different monoclonal antibodies were compared. Two of the monoclonal antibodies reacted with specific sets of three proteins each on VL protein blots. It is concluded that the VL is a complex structure containing a large number of different polypeptide components, the genes for several of which should now be experimentally accessible.

RNA complexity in developing sea urchin oocytes

Nuclear and cytoplasmic RNAs extracted from previtellogenic and vitellogenic oocytes of the sea urchin Strongylocentrotus purpuratus were characterized by hybridization reactions with radioactively labeled single-copy sea urchin DNA. The complexity of nuclear RNA from previtellogenic oocytes was 1.6 × 10^8 nucleotides. The previtellogenic nuclear RNA sequence set is included in the hnRNA of gastrula stage embryos. The nulcear RNA of vitellogenic oocytes may also contain a class of more prevalent transcripts. A single-copy [^3H]DNA tracer enriched for the sequences of mature egg RNA was reacted with cytoplasmic RNA of previtellogenic oocytes. This experiment showed that less than half of the mature egg RNA sequence set is accumulated before the onset of vitellogenesis. Therefore, a large fraction of the maternal message sequences appears in the egg during the last several weeks of oocyte development.

Genome size and DNA complexity of Plasmodium falciparum

Plasmodium falciparum DNA was prepared from cells cultured in vitro in human erythrocytes. The P. falciparum DNA was mixed with a tritium-labeled Escherichia coli DNA standard, and the kinetics of reassociation were measured using hydroxyapatite chromatography. It was found that the P. falciparum genome size is equal to 3.8 . 10(8) nucleotide pairs, and that a repetitive component is present which contains about 10% of the DNA. The average repetition frequency in this component is 95 copies of each sequence.

Complexity of RNA in developing oocytes of Drosophila melanogaster

RNA was extracted from Drosophila egg chambers which had been separated into four size classes, and in addition from mature ovarian eggs. The RNAs were hybridized with a Drosophila single-copy DNA tracer. Egg chamber RNAs from all stages of oogenesis examined contained single-copy DNA transcripts equal in complexity to the RNA of the mature egg.