Anthony A. Infante

Heterogeneous ribonucleoprotein particles in the cytoplasm of sea urchin embryos.

Abstract Particles containing newly synthesized DNA-like RNA have been described in the cytoplasmic material of sea urchin embryos, sedimenting more slowly than the 74 s monoribosomes. In order to determine their general structure, we compared the physical properties of these particles with those of subribosomal particles occurring abundantly. The buoyant densities of the bulk cytoplasmic particles were measured, after they were fixed with formaldehyde, by banding in preformed CsCl density gradients. These were: monoribosomes, ϱ = 1.57; native 35 s ribosomal subunits, ϱ = 1.55; native 56 s ribosomal subunits, ϱ = 1.61; a 22 s glycoprotein, ϱ = 1.38; soluble protein of less than 15 s, ϱ = 1.30 to 1.35. The subribosomal particles containing newly synthesized RNA sedimented in a range from 15 to 65 s and displayed a range of buoyant densities from ϱ = 1.50 to 1.75. The RNA bound in these particles could be distinguished operationally from free RNA by virtue of its ability to adhere to membrane filters. The sedimentation behavior of the ribonucleoprotein particles was a function of the size of the DNA-like RNA they contained. The DNA-like RNA derived from the totality of subribosomal ribonucleoprotein had a range of sedimentation of approximately 10 to 40 s and a mean sedimentation coefficient of approximately 20 s. The ribonucleoprotein particles could not be distinguished immediately from complexes of RNA and ribosomal subunits, since artificially prepared complexes of DNA-like RNA and 35 s subunits displayed buoyant densities in a range from ϱ = 1.56 to 1.80. Partial deproteinization with pronase caused the particles to shift markedly into a heavier range of buoyant densities, and extended digestion with pronase released DNA-like RNA from these particles. In order to distinguish between simple complexes of (a) DNA-like RNA and ribosomal subunits and (b) DNA-like RNA and protein per se , the particles were disassembled nucleolytically. The products of very mild digestion with ribonuclease were predominantly of buoyant densities less than that of the subunit. Under these conditions the buoyant densities of the subunits were not affected. Therefore, the subribosomal DNA-like RNA could not be bound simply to ribosomal subunits, but could very likely be bound to protein per se , forming unique ribonucleoprotein particles.

Polyubiquitin RNA characteristics and conditional induction in sea urchin embryos

A cDNA of the sea urchin Strongylocentrotus purpuratus was identified as encoding polyubiquitin and used to detect a single gene with transcripts containing multiple ubiquitin coding units. Polyubiquitin transcripts exist as a 3.2-kb RNA in polyribosomes and as three higher molecular weight RNAs in purified nuclei. The amount of polyubiquitin RNA is essentially constant at 10(4) -10(5) transcripts per embryo during the egg-to-blastula period and then declines during further development. Heat shock elicits a transient increase in the level of polyubiquitin RNA, while Zn(II) ions induce a sustained accumulation, that is influenced by developmental parameters: One round of Zn(II) induction elicits the accumulation of the nuclear 7.6- and 5.6-kb RNAs, as well as the 3.2-kb polysomal RNA; however, a second round of induction yields only the 5.6- and 3.2-kb RNAs, suggestive of a change in pre-mRNA size or processing. Polyubiquitin RNA is expressed equally in ectodermal and mesoendodermal tissues and is induced in both tissue fractions by treatment of pluteus larvae with Zn(II). However, in isolated and cultured tissue fractions, polyubiquitin RNA is not inducible by Zn(II), in contrast to the full inducibility of metallothionein mRNAs. Polyubiquitin RNA induction thus appears to be conditioned by the integrity of the embryo, as well as by previous exposure to inducer.

Dissociation of ribosomes induced by centrifugation: Evidence for doubting conformational changes in ribosomes

Abstract 1. The dissociation of sea urchin ribosomes in response to monovalent cations was investigated. Sea urchin egg ribosomes readily dissociate in 0.50 M KCl at o°, and the subunits that are formed are active in translating poly(U) and readily reassociate when the ionic strength is reduced. 2. Subunits are also produced during sucrose gradient sedimentation in moderate concentrations (0.15–0.40 M) of KCl. These subunits display apparent increases in s value, which are not due to conformational changes, since subunits isolated from these gradients resediment only at 35 S and 56 S in a wide range of ionic strength. If prepared in 0.24 M KCl their ability to translate poly(U) is about three times greater than subunits produced in high (0.50 M) KCl. 3. Evidence is presented which supports the concept that ribosomes dissociate into subunits during centrifugation and that the process occurs at an apparently discrete point which is dependent upon: the ionic conditions, the gravitational field and the concentration of ribosomes. We have termed subunits produced during sedimentation as “sedimentation-derived subunits”. 4. The effect of the gravitational field on the sedimentation patterns of ribosomes is particularly striking. When all other conditions are kept constant and the gravitational force imposed on free monoribosomes is varied, totally different sedimentation patterns (hence, s values of the subunits) can be obtained. Furthermore, if the duration of centrifugation in moderate ionic strength is insufficient to resolve the subunits, a single band is observed which can be easily misinterpreted as a reduction in the apparent sedimentation velocity of the monoribosome due to a conformational alteration. These results are discussed in terms of reported alterations in ribosome conformation.

Ribosomal ribonucleic acid of the sea urchin egg and its fate during embryogenesis

Abstract The sedimentation properties of the ribosomal RNA of eggs and that synthesized by late-stage embryos are identical in three species of sea urchin. However, uniquely in the eggs of Strongylocentrotus purpuratus , the 18 s ribosomal RNA can be converted to approximately 13 s (presumably two fragments) by heating briefly at 60 °C. This fragility is peculiar to the RNA of the egg and is not a property of ribosomal RNA synthesized by late-stage embryos. The specific concentration of this 13 s RNA decreased during embryonic development, but not until the 20-hour mesenchyme blastula stage had been reached. The initiation at this stage of substantial ribosomal RNA synthesis, previously demonstrated, suggested the hypothesis that the decline in 13 s RNA content was due to a replacement of egg ribosomes. Hence, we propose that during development, from the 20-hour mesenchyme blastula stage, one-half the original egg ribosomes are replaced in 30 hours.

Collagen polysomes: Site of hydroxylation of proline residues☆

Abstract The biosynthesis of collagen on polysomes has been studied by using a newly devised method for obtaining polysomes in high yield from stationary-phase mouse fibroblast (line 3T6; Goldberg &, Green, 1967). These polysomes were completely disaggregated to monosomes by brief exposure to ribonuclease and they lost most of their radioactivity to the top of the sucrose gradients as a result of a 30-minute chase with unlabeled proline. After a ten-minute pulse with [3H]proline, nascent collagen peptides could be identified in these polysomes on sucrose gradients. Most of the proline residues susceptible to hydroxylation by collagen proline hydroxylase were found, in most cases, to be already hydroxylated in these nascent peptides. The nascent nature of these peptides was confirmed by the observation that treatment of the polysomes with RNase transferred the radioactive collagen peptides to the monosome area and these peptides could subsequently be removed to the soluble material at the top of the gradient upon treatment with puromycin. These findings therefore, show clearly that the hydroxylation of proline residues is occurring, in vivo under normal conditions, on nascent collagen chains. In no case was the degree of hydroxylation of the released collagen chains higher than that on the nascent collagen peptides. It seems likely, therefore, that the major site of proline hydroxylation is the nascent collagen peptide.

Characterization of cytosolic malic enzyme in human tumor cells.

Cytosolic NADP+‐dependent malic enzyme (ME) from human tumor cells was characterized in detail and compared to ME from normal human tissues produced in recombinant E. coli. Kinetic properties, size as seen in SDS gels, and HPLC elution profiles of tryptic digests of human ‘normal cell’ ME and NADP+‐ME from tumor cells were identical. Thus, NADP+‐ME found in tumor cells does not constitute a tumor‐specific isoform as suggested by other studies but is identical to the ‘housekeeping protein’ predominantly expressed in human liver and white adipose tissue.

Relationship between the mRNA of polysomes and free ribonucleoprotein particles in the early sea urchin embryo.

Abstract The cytoplasm of early sea urchin embryos contains nonribosomal, high molecular weight RNA both associated with ribosomes in polysomes and free of ribosomes in particles termed free RNP. In a 1-hr labeling period, 50% of the newly synthesized RNA enters the pool of ribosome-free RNP particles during the cleavage stages, and this percentage decreases until less than 20% of the new RNA in the mesenchyme blastula stage is found in the free RNP. mRNA from both polysomes and free RNP contain poly(A)(+) and poly(A)(−) species. During the cleavage stages only 8–10% of the RNA from each fraction is polyadenylated; however, in the blastula, 40–50% of the nonhistone polysomal RNA is polyadenylated while only 22–30% of the free RNP RNA is polyadenylated. At any developmental stage, the poly(A)(+)RNA from the free RNA and polysomes have identical sedimentation profiles; this is also the case for the poly(A)(−)RNA except for the absence of the 9 S histone mRNA from the free RNP. Changes in poly(A)(+)RNA content and sedimentation profiles during development occur simultaneously in the free RNP and the polysomes. Kinetic studies of these two RNP populations as well as nuclear RNP show that the bulk of the free RNP are not unusually stable cytoplasmic components. The free RNP decay with a half-life of about 40 min while nuclear RNA and polysomal RNA display half-lives of about 12 and 65 min, respectively. Further, the rate of synthesis of the free RNP is not consistent with their being the only precursors for polysomes. Our estimates of the rates of synthesis for nuclear RNA, polysomes, and free RNP are, respectively, 1.1 × 10 −15 , 2.2 × 10 −16 , and 5.0 × 15 −17 g/min/nucleus. The data on free RNP is discussed in terms of translational regulation of protein synthesis in the developing sea urchin.

Stability of free ribosomes, derived ribosomes and polysomes of the sea urchin

Abstract 1. The relative stabilities of free ribosomes and polysomes from various developmental stages of two species of sea urchin were examined on sucrose gradients in a wide range of ionic strengths. With increasing concentrations of KCl there is an increased tendency for free ribosomes to dissociate, and this response to KCl was used to assay any differences in subunit association during development. In moderate and high concentrations of KCl, the free ribosomes from unfertilized eggs dissociate into subunits as readily as free ribosomes from developing embryos. 2. The identical dissociation characteristics of free ribosomes from egg and embryo preparations is also observed during dialysis against Mg 2+ -free buffer, when care is taken in purifying the ribosomes prior to dialysis. Taken together, these results eliminate the possibility, as proposed by others, of a very tight association of ribosomal subunits in the egg which is reduced after fertilization. 3. In contrast to free ribosomes, the ribosomes derived from polysomes through brief nuclease digestion are extremely resistant to dissociation even in high KCl. They can be made susceptible to both moderate and high concentrations of salt by prior treatment with puromycin which causes release of the nascent polypeptides. 4. Both the extraction and integrity of polysomes are impervious to KCl concentrations from 0.05 to 0.6 M when ribonuclease activity in the embryo extract is inhibited. These results are discussed in terms of the methods used in the preparation of sea urchin polysomes and the variations in the sedimentation patterns of polysomes during early embryogenesis.

Pool sizes of the deoxynucleoside triphosphates in the sea urchin egg and developing embryo

Abstract The four deoxynucleoside triphosphate pools in unfertilized eggs of L. pictus and S. purpuratus were measured and found to be very large, ranging from 10 −3 to 10 −2 pmoles per egg. The high levels of the individual dNTP pools are sufficient for one to eight rounds of DNA synthesis. During the first division cycle these pools fluctuate with the highest levels being attained prior to DNA synthesis. The pools then decrease just preceding or during the S period. There is a large reduction in the total cellular dNTP in later stages of development when DNA synthesis is reduced relative to the cleavage stages.

RNA synthesis in unfertilized sea urchin eggs

Abstract We have examined the synthesis of messenger-like RNA in unfertilized sea urchin eggs. Most of the RNA synthesized is restricted to the nucleus and sediments from 16 to 30S. A small fraction can be isolated from the postmitochondrial supernatant and displays a sedimentation profile typical of embryonic mRNA with peaks at 9 and 18S. This cytoplasmic RNA is largely present as free RNPs and we estimate that less than 20% of the RNA is in polysomes. The RNA made in the egg is unstable and reaches a steady state with a half-time of about 30 min. We have examined the accumulation of RNA in the egg and have calculated a rate of synthesis of 1.4 × 10 −14 g of RNA/min/egg which is similar, on a per-nucleus basis, to that found in the just-fertilized egg and very early embryo. It is approximately 10 times greater than the rate of RNA synthesis in the blastula nucleus. We estimate that the RNA synthesized by the unfertilized egg amounts to a maximum of 3 × 10 −13 g of potential mRNA at the time of fertilization, or 10–15% of its immediate needs. This RNA cannot account for the increase in protein synthesis that occurs after fertilization, which must be the result of the translation of another population of more stable egg or oogenic mRNA that is kinetically distinct from the RNA we have measured. The steady-state level of labeled RNA present in the egg does not change upon fertilization until after the first cleavage, at about 2.5 hr after fertilization. Thus the RNA synthesis that occurs in the just-fertilized zygote appears to be merely a continuation (at least quantitatively) of the RNA synthesis taking place in the egg.