Isolation and partial characterization of a mitochondrial deoxyribonucleic acid-protein complex from sea urchin embryos

Summary Lysis of mitochondria from sea urchin embryos with Triton X-100 led to a complete conversion of DNA-containing mitochondrial residues into protein-DNA complex with a density higher than 1.22 g/cm³ in sucrose solutions. This complex banded isopycnically in metrizamide gradients at a density of 1..26 g/cm³. Exposure to mixtures of Triton X-100 with Tween 80 resulted in progressively less delipitated and disorganized mitochondria over Tween/Triton weight ratios from 1 to 2, with the retention of the starting buoyant density in sucrose of approximately 1.16 g/cm³ at Tween/Triton ratios above 2.5. The DNA-internal protein complex sedimented with the bulk of the surviving mitochondrial structure under all conditions studied. No free DNA could be detected under any conditions of membrane removal.     

Developmental changes of chromatin nonhistone proteins of sea urchin embryos

Summary The developmental changes in the pattern of chromatin nonhistone proteins have been investigated. The main feature of the observed changes was not the introduction of new or the disappearence of earlier existing species, but the stage-specific alterations in the rate of biosynthesis of the nonhistone protein constituents.     

Biogenesis of the mitochondrial ATPase from sea urchin embryos

The mitochondrial rutamycin-sensitive ATPase from sea urchin eggs was purified to homogeneity. The subunit structure of the enzyme was characterized by SDS-gel electrophoresis. Eight polypeptides were identified with molecular weights of 55,000, 52,000, 39,000, 31,000, 28,000, 23,000, 17,000 and 10,000. Developing sea urchin embryos were incubated with [2H]leucine in the presence of emetine preferentially to label mitochondrially made proteins. Under these conditions sea urchin mitochondria synthesize eight different polypeptides. Two of these proteins, with molecular weights of 31,000 and 23,000, co-purify with the ATPase. Antibody directed against the pure rutamycin-sensitive ATPase precipitated only these two proteins. Therefore, two of the eight sea urchin ATPase subunits appear to be made by mitochondria.     

Heterogenous nuclear and cytoplasmic RNA in Li-treated sea urchin embryos

Ara-CTP differentially inhibits two types of DNA synthetic activity occurring in isolated hepatocyte nuclei in vitro. Ara-CTP inhibits type A synthesis (replication) with a K₁ of 5 × 10^?7 M, whereas type C synthesis (presumed repair) is much less sensitive, the K₁ being 5 × 10^?4 M. Significant inhibition of type C synthesis does not occur until type A synthesis is suppressed by more than 50%.     

Isolation of chromatin bearing nascent RNA from nuclei of sea urchin embryos

Sea urchin embryos were labeled with C¹⁴ thymidine and H³ Uridine, the nuclei isolated, and a chromatin preparation partially deproteinized in salt and detergent. After banding this preparation in a Cs₂SO₄ gradient, the nascent RNA is associated with a small fraction of the chromatin at a density lighter than the bulk chromatin.     

DNA polymerase-beta from the nuclear fraction of sea urchin embryos: characterization of the purified enzyme

Approximately 2,500-fold purifications of DNA polymerase-beta from the nuclear fraction of blastulae of the sea urchin, Hemicentrotus pulcherrimus, was performed. The enzyme preparation, which was devoid of DNase and terminal deoxynucleotidyl transferase as contaminants, showed a sedimentation constant of 3.0 S in a sucrose density gradient, a molecular weight of 50,000 by gel filtration, and an isoelectric point of pH 8.1. The enzyme activity was resistant to sulfhydryl group inhibitors. Its optimal pH was 9.0-9.5 in Tris-maleate buffer and 10.0 in glycine buffer. The optimal NaCl concentration for the activity was 30-60 mM and about half of the activity remained at 0.4 M NaCl. As a template-primer, the enzyme preferred synthetic homopolymers to activated DNA. The order of this preference was as follows; poly (dA)-oligo (dT)12-18 greater than poly (rA)-oligo (dT)12-18 greater than activated DNA. The above results indicate that the enzyme corresponds to DNA polymerase-beta from vertebrate cells.     

Sequence complexity of heterogeneous nuclear RNA in sea urchin embryos.

The sequence complexity of heterogeneous nuclear RNA is sea urchin gastrulas was measured by RNA-driven hybridization reactions with nonrepetitive sea urchin DNA. 28.5% of the sequence complexity of the genome is represented in the nuclear RNA. This amounts to 1.74 X 10(8) nucleotides of diverse sequence, more than 10 times the nucleotide complexity of the polysomal messenger RNA extracted from sea urchin embryos at the same stage. The complex set of nuclear RNA sequences driving this hybridization reaction was shown to be the same as the rapidly labeled hnRNA, using pulse-labeled nuclear RNA as driver.     

Molecular classes of heterogeneous nuclear RNA in sea urchin embryos.

Nucleotide sequences within a phage genome can be detected in individual phage plaques by in situ hybridization with complementary RNA sequences. Results with phage A and a derivative having 10% of its DNA deleted indicate that sequences 500 to 1000 base-pairs long should be detectable with confidence.     

Characterization of two major non-histone protein fractions from chromatin of sea urchin embryos

• 1.1. Chromatin of sea urchin embryos was chromatographed on a hydroxyapatite column, and the two largest fractions of proteins desorbed with 0.05 M and 0.2 M Na-phosphate, pH 6.8, were comparatively characterized.
• 2.2. The mol. wt ranges of these fractions were about 18,000 and 14,000 daltons, respectively, at both molarities of the phosphate buffer.
• 3.3. Both fractions at each elution molarity were found to be acidic, and to differ somewhat in their respective amino acid compositions. No important differences related to the stage of development could be detected in this respect.
• 4.4. Upon re-electrophoresis both large fractions resolved in one major and three minor bands. The amino acid composition of the major band was different for the blastula and gastrula chromatin.
• 5.5. It is likely that these fractions contain glycoprotein subunits.

     

Alkaline phosphatase of sea urchin embryos: Chromatographic and electrophoretic characterization.

A change in the molecular form of alkaline phosphatase in sea urchin embryos accompanies the marked increase in activity that occurs at gastrulation. On the basis of chromatographic and electrophoretic analyses, two major classes of alkaline phosphatase are identified: early enzyme, from unfertilized eggs to mesenchyme blastula, characterized by a major peak of activity, with a K_av of 0.123 on Sephadex G-200 columns, elution from DEAE-Sephadex columns by 0.5 M NaCl, and a migration value of 0.51 (relative to bromophenol blue) after electrophoresis in 7.5% polyacrylamide gels; late enzyme, from gastrula to plutei, characterized by a K_av of 0.137, elution from DEAE-Sephadex by 0.55–0.75 M NaCl, and a migration value of 0.56. By chromatographic and electrophoretic criteria the early enzyme appears to have a slightly greater molecular volume, lower net negative charge, and more heterogeneous composition than the late enzyme. Both enzyme preparations were maximally active at a pH 9.4–9.5. Enzyme from all stages appears to be predominantly associated with cell membranes. Extracting the enzyme by treatment with n-butanol, precipitating the enzyme from the dialyzed aqueous phase with ethyl alcohol, and chromatographing the alcohol preparation on columns of sieving and anion-exchanging media resulted in a substantial purification of the enzyme from all stages.     

Agrobacterium-mediated transformation of sea urchin embryos

Agrobacterium-mediated transformation of higher plants is a well-known and powerful tool for transgene delivery to plant cells. In the present work, we studied whether Agrobacterium can transfer genetic information to animal (sea urchin) embryos. Sea urchin embryos were co-cultivated with A. tumefaciens strains carrying binary vectors containing the nptII marker gene and agrobacterial rolC and rolB oncogenes. Bacterial plasmid T-DNA-sea urchin DNA junction sites were identified in the genome of these embryos, thus indicating successful transformation. The nptII and both rol genes were expressed in the transformed embryos. The processes of transgene integration and transgene expression were suppressed when Agrobacteria contained mutated virA, virB or virG genes, suggesting that Agrobacterium transforms sea urchin cells by a mechanism similar to that which mediates T-DNA transfer to plants. Some of the embryos co-cultivated with Agrobacterium developed teratoma-like structures. The ability of Agrobacterium strains to trigger formation of teratoma-like structures was diminished when they contained the mutated vir genes. In summary, our results demonstrate that Agrobacterium is able to transform animal (sea urchin) embryonic cells, thus indicating a potential of this natural system for gene delivery to animal hosts. We also discuss the possibility of horizontal gene transfer from Agrobacterium to marine invertebrates.