Mitochondrial RNA synthesis in sea urchin embryos

Chamberlain, John P.; Metz, Charles B.

doi:10.1016/0022-2836(72)90085-x

Cited by 43 publications

(6 citation statements)

References 15 publications

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“…The major 1.16-kb HindIII band as well as the minor HindIlI and EcoRI bands must, however, result from hybridization to related sequences in the genomic DNA. The concentrations of the putative mitochondrial RNAs decrease fivefold between the blastula and pluteus stages, agreeing with earlier investigations which indicate that mitochondrial RNAs are a smaller fraction of the total RNA at later stages (6,11).…”

Section: Discussionsupporting

confidence: 91%

Molecular cloning of five individual stage- and tissue-specific mRNA sequences from sea urchin pluteus embryos.

Fregien¹,

Dolecki²,

Mandel³

et al. 1983

Mol. Cell. Biol.

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Five developmentally regulated sea urchin mRNA sequences which increase in abundance between the blastula and pluteus stages of development were isolated by molecular cloning of cDNA. The regulated sequences all appeared in moderately abundant mRNA molecules of pluteus cells and represented 4% of the clones tested. There were no regulated sequences detected in the 40% of the clones which hybridized to the most abundant mRNA, and the screening procedures were inadequate to detect possible regulation in the 20 to 30% of the clones presumably derived from rare-class mRNA. The reaction of 32P[cDNA] from blastula and pluteus mRNA to dots of the cloned DNAs on nitrocellulose filters indicated that the mRNAs complementary to the different cloned pluteus-specific sequences were between 3-and 47-fold more prevalent at the pluteus stage than at the blastula stage. Polyadenylated RNA from different developmental stages was transferred from electrophoretic gels to nitrocellulose filters and reacted to the different cloned sequences. The regulated mRNAs were undetectable in the RNA of 3-h embryos, became evident at the hatching blastula stage, and reached a maximum in abundance by the gastrula or pluteus stage. Certain of the clones reacted to two sizes of mRNA which did not vary coordinately with development. Transfers of RNA isolated from each of the three cell layers of pluteus embryos that were reacted to the cloned sequences revealed that two of the sequences were found in the mRNA of all three layers, two were ectoderm specific, and one was endoderm specific. Four of the regulated sequences were complementary to one or two major bands and one to at least 50 bands on Southern transfers of restriction endonuclease-digested total sea urchin DNA.The sea urchin embryo appears to be one of the most accessible developmental systems for studying the molecular events of cell type determination during early embryogenesis. After fertilization, eggs divide into a number of cells whose developmental fate is fixed somewhat before they differentiate into the various cell types. Most of the cells in the sea urchin embryo contribute to the three layers of the relatively simple pluteus larva: the ectodermal covering, the skeletal-forming mesenchyme, and the gut. These differentiated tissues function during the 1 to several months of larval life. The actual sea urchin emerges at metamorphosis after it has grown from a few cells set aside during embryogenesis for its development.Despite the simplicity of the system and the numerous molecular studies on sea urchin embryos, these larval tissues have not received much attention as specialized cells synthesizing cell type-specific proteins. The major reason has been that there is no evidence of very abundant With the advent of molecular cloning, it has become possible to define, isolate, and study genes coding for proteins produced only in limited abundance, including putative specialized proteins of the three cell layers of the pluteus. Clones of mRNA and genomic sequences which code for sev...

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Section: Discussionsupporting

confidence: 91%

Molecular cloning of five individual stage- and tissue-specific mRNA sequences from sea urchin pluteus embryos.

Fregien¹,

Dolecki²,

Mandel³

et al. 1983

Mol. Cell. Biol.

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“…A similar preferential inhibition of mitochondrial RNA synthesis by ethidium bromide has been observed in HeLa cells (2,84) and sea urchin embryos (17,21) . In addition, ethidium bromide was found to preferentially inhibit mitochondrial DNA synthesis in cultured mammalian cells (57,73) and slime mold (41) and to reversibly inhibit the accumulation of cytochrome oxidase in cultured human fibroblasts (59) and HeLa and L cells (42) .…”

Section: Mitochondrial Differentiation and Embryo Development In The supporting

confidence: 76%

“…In this species mitochondrial RNA-and DNA-synthesis is absent up to the gastrula stage when synthesis of mitochondrial ribosomal RNA (but not 4S RNA) begins; mitochondrial DNA content and total mitochondrial mass begin to increase in the swimming tadpole (19) . In the sea urchin, mitochondrial RNA synthesis occurs both during normal cleavage and in activated nonnucleate fragments (16,17,20,39,71) ; however, mitochondrial DNA synthesis appears to be absent during early development (38,65) .…”

Section: Mitochondrial Differentiation In Control Embryosmentioning

confidence: 99%

“…The role interest for the study of mitochondrial genetic ac-tivity because of the presence of large amounts of mitochondrial DNA in these eggs as shown in frog (22), sea urchin (68), and echiuroid worm (25) . Biochemical evidence indicates that the mitochondrial DNA is transcribed during early development in sea urchins (16,17,20,39,71), but the role of this transcriptive activity is not clear . On the other hand, in frog eggs the mitochondrial genome may be inactive until gastrulation (19) .…”

Section: Introductionmentioning

confidence: 99%

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Role of the Mitochondrial Genome During Early Development in Mice

Pikó

Chase

1973

The Journal of Cell Biology

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The role of the mitochondrial genome in early development and differentiation was studied in mouse embryos cultured in vitro from the two to four cell stage to the blastocyst (about 100 cells) . During this period the mitochondria undergo morphological differentiation : progressive enlargement followed by an increase in matrix density, in number of cristae, and in number of mitochondrial ribosomes . Mitochondrial ribosomal and transfer RNA synthesis occurs from the 8 to 16 cell stage on and contributes to the establishment of a mitochondrial protein-synthesizing system . Inhibition of mitochondrial RNA-and proteinsynthesis by 0 .1 µg/ml of ethidium bromide or 31 .2 ug/ml of chloramphenicol permits essentially normal embryo development and cellular differentiation . Mitochondrial morphogenesis is also nearly normal except for the appearance of dilated and vesicular cristae in blastocyst mitochondria . Such blastocysts are capable of normal postimplantation development when transplanted into the uteri of foster mothers. Higher concentrations of these inhibitors have general toxic effects and arrest embryo development . It is concluded that mitochondrial differentiation in the early mouse embryo occurs through the progressive transformation of the preexisting mitochondria and is largely controlled by the nucleocytoplasmic system . Mitochondrial protein synthesis is required for the normal structural organization of the cristae in blastocyst mitochondria . Embryo development and cellular differentiation up to the blastocyst stage are not dependent on mitochondrial genetic activity .

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“…It will be interesting to find out whether polyadenylation after fertilization occurs by lengthening of the poly(A) sequences present in RNA of unfertilized eggs. Since about half the adenosine incorporated during the first 2 hr after fertilization goes into polyadenylate, and much of the remaining adenosine incorporation is in mitochondrial RNA (14) and turnover of the 3'-OH terminus of transfer RNA (15), it is possible there is very little nuclear transcription during this period of development.…”

Section: Discussionmentioning

confidence: 99%

Polyadenylation of Maternal RNA of Sea Urchin Eggs After Fertilization

Wilt

1973

Proc. Natl. Acad. Sci. U.S.A.

139

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Between fertilization, or parthenogenetic activation, and the two-cell stage, the content of polyadenylic acid in sea urchin eggs doubles, and the increase occurs primarily in the ribosome-polyribosome fraction. The increase is due to polyadenylation of preexisting RNA molecules synthesized during oogenesis. The polyadenylation occurs in activated, enucleated merogons. It is argued that cytoplasmic polyadenylation may play a role in mobilization of maternal messenger RNA for translation and the polyadenylic acid does not subserve an exclusively nuclear function.Fertilization or parthenogenetic activation of sea urchin eggs entrains a profound series of changes in the egg, including dramatic increases in synthesis of nucleic acids and proteins, as well as complete reorganization of the structure and permeability of the cell surface (1). The increase in protein synthesis is not affected by enucleation (2) or inhibition of transcription (3). The increase in protein synthesis reflects the increased capacity of the eggs to synthesize protein, is accompanied by a gradual increase in the number (but not size) of polyribosomes (4), and occurs on mRNA templates synthesized during oogenesis. The mechanism(s) of sequestration or inactivity of untranslated maternal mRNA of unfertilized eggs and its mobilization into active polysomes after fertilization is unknown.I will report experiments that are based on the assumption that most mRNA molecules of metazoa contain sequences of poly(A) (5). First, I will show that during the 2-hr time span from activation to first cleavage (the time during which protein synthesis attains its maximum) the content of poly(A) doubles, and that the increase occurs primarily in the ribosome-polyribosome fraction of the cell. While this work was in progress, papers reporting similar conclusions appeared (6, 7). Second, the increase in poly(A) is primarily due to a polyadenylation after fertilization of RNA molecules transscribed during oogenesis. Third, polyadenylation occurs in enucleated activated merogons; hence, the reaction is cytoplasmic. Sea urchin gametes were obtained, and embryos were raised in Millipore-filtered sea water containing 50,4g/ml of streptomycin by conventional techniques. Parthenogenetic activation was done by a modified butyric acid method (9) followed by exposure for 20 min to sea water containing an extra 30 g/liter of NaCl, after which the eggs were returned to normal sea water.Merogons were obtained by layering 0.2-1.0 ml of a 20%suspension of eggs over a linear gradient constructed from 1.0 M sucrose and a mixture of equal parts of sea water and 1.0 M sucrose. Gradients were centrifuged in either an SW 39 or SW 25 rotor at 5000 X g (middle of tube) for 6-7 min, followed by centrifugation at 26,000 X g (middle of tube) for 6-10 min. The well-separated egg fragments were collected by dripping the gradients through a puncture made in the bottom of the tube and were immediately washed in normal sea water. Eggs and embryos were fractionated by washing twice in 1.5 ...

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Mitochondrial RNA synthesis in sea urchin embryos

Cited by 43 publications

References 15 publications

Molecular cloning of five individual stage- and tissue-specific mRNA sequences from sea urchin pluteus embryos.

Molecular cloning of five individual stage- and tissue-specific mRNA sequences from sea urchin pluteus embryos.

Role of the Mitochondrial Genome During Early Development in Mice

Polyadenylation of Maternal RNA of Sea Urchin Eggs After Fertilization

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