Martin Nemer

Co-existence of non-histone messenger RNA species lacking and containing polyadenylic acid in sea urchin embryos.

Abstract The non-histone messenger RNAs of sea urchin embryos have been separated on oligo(T)-cellulose into fractions containing poly(A) and those entirely lacking poly(A). Proof of the existence of the class of mRNA lacking poly(A) is afforded by the following demonstrations: (1) the pulse-labeled RNA analyzed is bound to polyribosomes and has no non-polysomal contamination, (ii) The methods of extraction, as tested by mixing experiments between cytoplasmic extracts of sea urchin embryo and mammalian tissue culture cells, preclude partial degradation of mRNA containing poly(A) to yield artifactual species lacking poly(A), (iii) The non-histone mRNA lacking poly(A) has a mean sedimentation coefficient of 22 S, as measured in a denaturing solvent. It is shown not to consist of molecular aggregates of the putative histone 9 S mRNA, and its base composition differs markedly from those of both 9 S and ribosomal RNA, but resembles closely that of poly(A)-containing mRNA. (iv) Although the non-histone mRNAs lacking and containing poly(A) have similar base compositions and sizes (approximately 22 S), they differ widely in their nucleotide sequences. Complementary DNA, prepared with reverse transcriptase instructed by poly(A)-containing mRNA, hybridized to a negligible extent with RNA lacking poly(A).

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.

Properties of Sea Urchin Embryo Messenger RNA Containing and Lacking Poly(A)

Various properties of nonhistone messenger RNA species containing poly(A), [+A]mRNA, and lacking poly(A), [-A]mRNA, are described: the rates of turnover of these mRNA classes are not significantly different, as indicated by their similar rates of entry into and decay from the cytoplasm; each mRNA class is essentially entirely transcribed from unique DNA sequences; the ratio of [+A] to [-A] nonhistone mRNA increases with increase in size of free polyribosome, although the average molecular weights of these mRNAs are similar in each polysomal size class. These results indicate that the [+A]mRNA species tend to be more fully loaded with ribosomes than the nonhistone [-A]mRNA species.

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.

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.

Polypeptide synthesis in sea urchin embryogenesis: an examination with synthetic polyribonucleotides.

After fertilization of the sea urchin egg the rate of protein synthesis by a cell-free ribosomal system increases markedly. This increase can be attributed to newly synthesized messenger RNA, since (i) the rate of polypeptide synthesis elicited by synthetic messenger polyribonucleotides changes only slightly after fertilization, and (ii) the enzymes for the formation of amino acyl transfer RNAs of phenylalanine and leucine and the polymerization of polypeptide are in excess in the unfertilized egg. Polypeptide synthesis has been characterized in development to the gastrula stage.

Molecular classes of heterogeneous nuclear RNA in sea urchin embryos

Heterogeneous nuclear RNA of sea urchin blastulae has been fractionated into three molecular classes by two separate methods, using oligothymidylate (oligo(dT))-cellulose and polyuridylate (poly(U)) glass fiber filters. These molecular classes, which we have designated α, β and γ HnRNA‡, have distinctly different properties: (i) α HnRNA is not bound by oligo(dT)-cellulose, but is bound by poly(U) filters, and is eluted from the poly(U) by 15% formamide. This class has a mean value (in denaturing solvent) of 37 S, contains an oligoadenylate segment of 25 residues (oligo(A)25) and lacks larger polyadenylate segments (poly(A)). The oligo(A)25 is not present at the 3′ terminus of the molecule, as determined by end-group analysis (ii) β HnRNA is bound by either oligo(dT)-cellulose or poly(U) filters and is eluted from the latter by 40% formamide. This class has a mean value of 31 S, contains a poly(A) segment of approximately 175 residues at the 3′ terminus, but lacks oligo(A)25. In addition, both α and β HnRNAs contain adenylate stretches of approximately 12 residues in length (oligo(A)12). (iii) γ HnRNA is not bound by either oligo(dT) or poly(U). It displays a mean value of 36 S and lacks both poly(A)175 and oligo(A25). Furthermore, our evidence supports the conclusion that these three classes are not artificially derived from HnRNA, but comprise an in vivo population of HnRNA molecules whose mean value is approximately 36 S.

Cytoplasmic Extraction: Polyribosomes and Heterogeneous Ribonucleoproteins without Associated DNA

Two methods are described for preparing cytoplasmic extracts from sea urchin embryos. One method, involving homogenization, yields DNA structures that cosediment with polyribosomes and subribosomal ribonucleoproteins. In addition this method also yields extraneous structures containing RNA that cosediment with polyribosomes. The DNA is not associated with polyribosomes, as shown by buoyant density analysis. Furthermore, this DNA appears to be spurious, because its release into a cytoplasmic extract does not occur when a different method of cell disruption, involving passage of embryos through a hypodermic needle, is used. With this second method, polyribosomes are obtained without extraneous cosedimenting RNA structures and subribosomal ribonucleo-proteins are obtained in the virtual absence of DNA.

Spatial patterns of metallothionein mRNA expression in the sea urchin embryo

Metallothioneins (MTs) are small, cysteine-rich proteins that bind heavy metals which induce their synthesis. Tissue fractionation of embryos at pluteus stage previously demonstrated that in the absence of added zinc, basal expression of MT mRNA is confined to ectoderm, whereas induction by zinc results in increased expression in the endoderm + mesoderm tissue fraction. Using in situ hybridization we now show that expression in the pluteus larva is restricted almost exclusively to the single cell type comprising the aboral ectoderm. Induction by Zn results in a marked accumulation of MT mRNA in gut and oral ectoderm to levels at least as high as that in aboral ectoderm. MT mRNA is also expressed in presumptive aboral ectoderm at earlier stages of normal development. In addition it is transiently expressed at variable levels in oral ectoderm and, to a lesser extent, in presumptive gut.

Transfer of Genetic Information During Embryogenesis

Publisher Summary This chapter describes the transfer of genetic information during embryogenesis. A direct approach for understanding the molecular basis of early embryonic development is to examine the informational macromolecules themselves that are responsible for the transfer of genetic information and the synthesis of specific proteins. Messenger RNA (mRNA) has been postulated as the cytoplasmic representative of the genes, serving as template for the synthesis of specific protein. Nuclear activity in the just-fertilized egg is not needed to supply any of the components of the protein synthesizing system, which in the sea urchin is activated, following fertilization. The role of the egg mRNA, within the animal–vegetal gradient, might be evaluated on the basis of the protein synthesis in the cells derived from these ooplasmic regions. The nature of the dRNA population of the sea urchin egg has been examined, by using it to compete against labeled RNA, from the later stages in hybridization, with DNA.