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A site of hypersensitivity to DNase I has been found at the 5'-side of the chicken alpha-globin gene domain, ca. 8 Kbp upstream to the first gene. The presence of this site in different types of chicken cells correlates with the transcriptional status of the domain. A matrix attachment region (MAR) has also been found in the same subfragment, suggesting that it may be involved in the control of transcription of the chicken alpha-globin genes.

The sequence of a DNA fragment about 1 Kbp long located at the 3' boundary of the chicken alpha globin gene domain, including the 3'-side matrix attachment point and the site of transcription termination, was determined. It contains a repetitive DNA element and the AT-rich (easily denaturable) DNA segment conserved at the same position in the duck genome. The repetitive sequence was identified by computer analysis as being a member of the CR1 family. Within the non-repetitive part of the AT-rich DNA fragment, four topoisomerase II recognition sites were found which might be indicative of matrix attachment. Furthermore, two distinct regions were identified, possessing strong homology to a number of noncoding consensus sequences, one of them to a limited part of the LTR of HTLVIII, and the other to the replication origin of Polyoma virus JC. DNA shift experiments showed that the CR1 repeat binds specifically an abundant nuclear protein factor. The binding site for this factor was identified by footprinting and turned out to be closely related to the previously described recognition site for the TGGCA-binding protein, the chicken analog of nuclear factor 1 (NF-1). Finally, the CR1 repeats within the chicken alpha and beta globin gene domains were mapped. All these observations are discussed in terms of the organization of the 5' and 3' boundaries of the functional genomic domains forming a chromatin loop including all avian alpha type globin genes.

Duck erythroblast nuclei, isolated under conditions designed to preserve lysosome integrity, were extracted to yield a bulk population of nuclear ribonucleoprotein (nRNP) particles which, when fractionated on sucrose gradients, was resolved into two classes of RNA-protein complexes containing low-molecular-weight RNA. The classical pre-messenger ribonucleoprotein complex (pre-mRNP), which had been previously characterized in this laboratory and was shown to contain rapidly-labeled pre-messenger RNA (pre-mRNA) sequences, was observed to contain approximately eight discrete species of nuclear RNA between 90 and 230 nucleotides in length (major species of Mr 80000 and 55000, and minor species of Mr 61 000, 58000, 51000, 38000, 36000 and 32000). The size and relative distribution of these RNA sequences indicated that they were the small nuclear RNA species previously described in the literature. In addition, two smaller ribonucleoprotein complexes with sedimentation coefficients of 17 S and 12 S were characterized and shown to be composed of characteristic, although not mutually exclusive, sets of low-Mr RNA species. Comparison of these RNA species in the two smaller nRNP complexes with those found in the 40-S pre-mRNP particles demonstrated that all but three of the major low-Mr RNA bands found in the small nRNP were found in the pre-mRNP. The additional RNA species unique to the small nRNP were major ones of MI 150000 and 110000, minor ones of Mr 48000, 34000 and 27000 and three major bands of Mr approximately 25000 as well. Further comparison revealed that while each particle was composed of a characteristic subset of polypeptides, several common proteins were observed in both small nRNP and pre-mRNP. The common RNA and polypeptide components observed in these two classes of nuclear ribo- nucleoprotein suggested that these small nRNPs may represent either integral parts of intact physiological pre-mRNP or nuclear structures which are closely associated with pre-mRNP in vivo. No difference was observed in either the RNA or protein composition of small nRNP or pre-mRNP when prepared in the presence of the protease inhibitor phenylmethylsulfonyl fluoride, indicating that endogenous protease activity was not a problem in the preparation of nRNP from duck erythroblasts. CsCl gradient analysis of the two fractionated small nRNPs revealed buoyant densities of 1.39 and 1.40 g/ml for the 17-S and 12-S particles respectively. From these densities, an approximate RNA: protein ratio of 1 : 4, similar to that previously determined for the 40-S pre-mRNP complex, was estimated. Isoelectric focusing of the small nRNP on a pH gradient between pH 4 and pH 6 and electron microscopy studies reaffirmed the particulate nature of these smaller ribonucleoprotein complexes. In addition, ultrastructural analysis of small nRNP particles indicated an average diameter of 15 nm, compared with an average diameter of 35 - 40 nm for the bulk of the 40-S pre-mRNP particles. The poly(A)-protein complex of pre-RNA, sedimenting at a position of 15 S on the sucrose gradients, was bound to oligo(dT)-cellulose and subsequently eluted from the column in buffer containing 50 % formamide. Six major proteins were associated with this poly(A) sequence, including the 73 000-Mr poly(A)-binding protein typically seen associated with the poly(A) sequence of mRNA and pre-mRNA. In contrast, neither the 17-S nor the 12-S nRNP was bound to the oligo(dT)- cellulose column indicating that these nRNPs did not contain poly(A) sequences and were, there- fore, nRNP complexes distinctly different from the previously described poly(A)-protein complex of pre-mRNA.

In eukaryotic cells mRNA is present in the form of messenger ribonucleoprotein complexes (mRNP particles). When polyribosomes, from a variety of cell types and tissues, are dissociated with EDTA or puromycin, the released mRNA is found tightly associated with 3 major proteins o fM r ~ 43 000-52 000 and 72 000-78 000 (review [1]). The larger of these proteins was shown to interact with the 3'-polyadenylate sequence of mRNA [2,3] since it may be recovered with poly(A) following digestion of polyribosomes or purified mRNP with ribonucleases A and T1. From density measurements of the poly(A) RNP particle it may be estimated that ~4 5 copies of this protein are associated with the average-sized poly(A) fragment [4]. The affinity of the poly(A)-binding protein for poly(A) is very high since it resists not only 0.5 M KC1 [1] but also centrifugation through Cs2SO4 density gradients [5,6] and affords protection ofpoly(A) against ribonucleases and nucleases [7 10]. Furthermore, this protein binds not only poly(A) but also regions of poly(A) adjacent and non-adjacent to poly(A) [81. A protein of the same M r as the polyribosomal poly(A)-binding protein has been observed to be associated with the 3'-poly(A)region of nuclear pre

We have previously shown that AEV infection of leukosis-free chickens provokes an erythroleukemia in the infected birds, and that the target cell for virus transformation is most likely an early pro-erythroblast comparable to the BFU-E. The virus infection correlates with a block in differentiation which was studied at a specific cellular gene level, namely that of the globin genes. In live birds, the viral transformation has been found to affect the transcriptional activity to two out of three adult globin genes and the phenotypic expression of the third. Indeed, the alpha A gene that is transcribed is silent as well, since its transcription product is fully eliminated in the nucleus following an "abortive processing". Chick bone marrow cells can also be transformed in vitro by AEV in which case the virus recruits, as targets, cells of the erythroid lineage belonging to a wider spectrum and hence in varying degrees of differentiation. In contrast to the results obtained with in vivo transformed cells, molecular hybridization with globin cDNA probes showed that in the erythroblasts transformed in vitro under tissue culture conditions all the three adult globin genes are transcriptionally active. In fact, low but detectable amounts of globin mRNA sequences are present in both the nuclear and cytoplasmic compartment of the cell prior to exposure to any chemical inducers of red cell differentiation. Judging from our present results, we may propose that AEV transformation in vitro does not shut off out only modulates the already functioning erythroid cell differentiation program.

In this report we describe some recent and, in some cases, preliminary results on the characterization of messenger ribonucleoprotein complexes isolated from the nucleus and various subcellular compartments of immature avian red blood cells. Reviewing investigations carried out over the last ten years, we try to draw some general conclusions regarding the composition and significance of the protein associated with pre-mRNA in the nucleus and with translated and untranslated mRNA in the cytoplasm. Furthermore, we propose a flow diagram through the cell of the various consecutive carriers of genetic information. In the case of nuclear RNP the main conclusions for our research are that pre-mRNA is associated with a relatively small group of approximately 20 basic proteins which may be involved in structural functions, and with a larger population estimated to contain at least 60 acidic polypeptides. This latter group has the theoretical characteristics of agents involved in post-transcriptional regulation, analogous to the putative role of non-histone proteins in transcription. The proteins of translated mRNP complexes are associated with specific segments of the mRNA; their function is unclear since they neither inhibit nor stimulate translation of their endogenous mRNA. However, in addition there exists genuinely repressed mRNA in the terminally differentiated erythroblast since the mRNA population in polyribosomes contains ten times fewer messages than the ribosome-free mRNP complexes. Of the order of 25% of the cytoplasmic globin mRNA is found in the untranslated fraction; thisin vivo repressed globin mRNP is also untranslatablein vitro, unless it is fully deproteinized. Since different messages in the untranslated mRNP compartment are associated with different sets of proteins we suppose that specific mRNAs can be recognized by specific sets of proteins in low redundancy, and can be drawn into the repressed state. We conclude that it is probable that RNP proteins play some role in post-transcriptional regulation of gene expression.

This paper represents a first attempt to follow a specific eukaryotic messenger RNA from the moment of its transcription from the nuclear DNA to the site of its expression by protein synthesis in polyribosomes. A direct demonstration that the globin mRNA sequence is transcribed from DNA in form of a giant precursor RNA (pre-mRNA) is presented: The anti-messenger DNA produced by transcription of purified globin mRNA with the RNA-dependent DNA-polymerase hybridizes to nuclear RNA of more than 2.5 × 106 MW (isolated on DMSO gradients) equally well as to its template. This proves that the nascent pre-mRNA is an informative precursor to mRNA. However, there exists no direct evidence demonstrating a physical precursor-product relationship between nascent pre-mRNA and mRNA. Nascent pre-mRNA molecules of 5–20 × 106 MW are cleaved with a halftime of about 30 min into intermediate size (1–5 × 106 MW) pre-mRNA molecules which are considerably more stable. This first processing step is accompanied by the solubilisation of about 50 % of the pre-mRNA. Small size nuclear pre-mRNA 1–10 × 105 MW) is metabolically very stable. However, no peak of nuclear 9S globin mRNA can be observed on polyacrylamide gels. Thus the globin mRNA sequence, processed either by direct cleavage of pre-mRNA or by a process of retranscription, is exported very rapidly to the cytoplasm. Ribosome-free ribonucleoprotein particles containing translatable globin mRNA can be isolated in the cytoplasm. Newly synthesized 9S mRNA rapidly fills up this free pool and the globin messenger is transferred to polyribosomes. The pool of free mRNP particles contains a larger spectrum of messenger molecules than polyribosomes where the 9S mRNA is predominant; some of the other mRNA-types are possibly never translated. Thus we conclude that a pre-translational control must operate, reducing quantitatively and qualitatively the mRNA spectrum in free RNP particles to that translated in polyribosomes. The globin mRNA in polyribosomes is doubly heterogeneous: newly synthesized molecules have molecular weights of about 230 000 whereas the steady-state spectrum of mRNA molecules is represented by a heterogeneous population of molecules with an average molecular weight of about 180 000. On this basis, we assert that processing of mRNA continues right throughout the translation process. Nascent globin pre-mRNA is associated with specific proteins; their major component cannot be observed among the proteins associated with cytoplasmic mRNA. The proteins associated with globin mRNA in the free mRNP particles are different from those associated with the translated globin message. Both protein populations include some phosphorylated species which contain phosphoserine. Thus it is possible that exchange of messenger-associated proteins and their phosphorylation reflect pre-translational and translational controls of globin synthesis. Compared to mRNA from other species, duck globin mRNA is poorly translated in all protein-synthesizing lysate systems tested. However, in a purified and reconstituted ribosome system, it is translated equally well as rabbit 9S mRNA provided that the system is supplemented with a high amount of a particular fraction of heterologous initiation factors. This requirement may be explained by a particular secondary structure in the programming sequence of the duck globin message. It is proposed that the transcriptional unit of gene regulation reflected in the nascent pre-mRNA molecule contains, in addition to one or several messenger sequences and possible no-sense sequences, programming sequences; interacting with specific proteins, such sequences may permit post-transcriptional controls involved in pre-mRNA processing and in messenger formation and translation. A general discussion of the regulation of the highly complex system of mRNA formation and translation in eukaryotic cells is presented with particular reference to the Cascade Regulation Hypothesis.

A method for electrophoretic analysis of RNA under fully denaturing conditions on exponential gradient polyacrylamide gels is described. Full denaturation, and strand separation of DNA - RNA hybrids and double-stranded RNA is obtained in dry formamide only if electrophoresis is carried out at 45 degrees and 55 degrees C, respectively. In such conditions, the effects of secondary structure of RNA, important in aqueous medium, are suppressed and a linear correlation is obtained between the logarithm of the molecular weight of an RNA and its final position in the gel over the entire molecular weight range of 10(4) - 10(7). Based on absolute molecular weight standards, obtained from sequenced rRNA of Escherichia coli and tRNA and extrapolating to higher molecular weights the size of animal cell was reexamined. Precursor tRNA from HeLa cells migrates according to a molecular weight of 4.1 x 10(6). Nascent precursor mRNA has molecular weights of up to 5 x 10(6) in the case of duck erythroblasts and of up to 10(7) in HeLa cells. This seems to represent the largest size of non-viral animal-cell RNA molecules.

Cells transformed by avian erythroblastosis virus were grown in vitro for up to 5 months. After a few days in culture, synthesis of hemoglobin was undetectable and could not be induced by dimethyl sulfoxide. As shown by globin cDNA hybridization to nuclear and cytoplasmic RNA carried to Crot values of 10(5) moles of nucleotide per liter X sec, globin genes in these cells are transcribed into pre-mRNA, but no trace of globin mRNA appears in the cytoplasm. The implications of this observation for schemes of post-transcriptional regulations and viral transformation are discussed.

The “prosomes”, a novel type of ubiquitous ribonucleoprotein particle of extraordinary stability and of defined electron microscopical structure, have been characterized in several cell types and species. Identified as a 19 S sub-component of free mRNA-protein complexes, including globin and other repressed mRNA, in the cytoplasm of duck, mouse and HeLa cells, they were previously found to inhibit protein synthesis in vitro. In all cells studied, electron microscopy shows an identical, seemingly ring-like but rather raspberry-shaped particle of 12 nm diameter, resistant to EDTA and 1% (w/v) Sarkosyl. Two-dimensional electrophoretic analysis of prosomal proteins shows a characteristic pattern in the 19,000 to 35,000 Mr range of pI 4 to 7, with an additional 56,000 Mr component specific to avian species. The prosomes found in globin mRNA-protein complexes contain about 25 protein components, 16 of which have identical molecular weight and pI values in duck and mouse, and which are also found in the prosomes of the heterogeneous free mRNPs of HeLa cells. Seral and monoclonal antibodies raised in mice against the prosomes of duck erythroblasts cross-react with some of the proteins of the mouse and HeLa cell particles. Prosomes isolated from duck and mouse globin mRNP, both contain small cytoplasmic RNAs of 70 to 90 nucleotides, which represent about 15% of the particle mass. The molecular weight and the 3′-terminal oligonucleotide of each one of these small cytoplasmic RNAs are identical in the two animal species; fingerprints of their oligonucleotides generated by RNase T1 show that more than 80% of spots are identical. In contrast, the prosomes of HeLa cells, associated with a large population of repressed mRNA, contain at least 12 small cytoplasmic RNA species. All prosomal RNAs tested so far hybridize to mRNA. The data available indicate that prosomes constitute a novel class of ubiquitous cellular ribonucleoprotein complexes, present in the nucleus and cytoplasm that, in its structural variations shown here, reflects function and species.

The steady-state distribution of globin gene-specific transcripts was analysed in avian erythroblast nuclear RNA with the aim of determining the maximal extent of the corresponding transcriptional units. The experimental approach included gel electrophoresis in pure formamide at high temperature, which allows absolute molecular weight determinations excluding secondary structure effects and intermolecular aggregation. The fraction of molecules in the 2 × 106 to 5 × 106 Mr range, which includes the rapidly turning over bulk primary transcripts, was re-isolated after electrophoresis and shown by Cr0t ‡ analysis with a globin gene-specific probe to contain both α and β globin messenger RNA sequences at a Cr 0 t 1 2 value of 10.2; a heterologeous mRNA added as an internal control was totally absent from this fraction. Quantitative evaluations show that as little as one molecule per cell is present in the Mr range of 3 × 106 to 5 × 106 and about ten molecules per cell with 2 × 106 to 3 × 106 Mr. These numbers, which correlate well with synthesis time and turnover rates of RNA in this molecular weight range, demonstrate the difficulty of detecting these full transcripts by less sensitive techniques. Determination of the steady-state pattern of globin pre-mRNA shows that, in addition to the rare full transcripts, globin pre-mRNA molecules are found with 1 × 106 to 2 × 106 (“28 S”) and 0.2 × 106 to 0.8 × 106 Mr (“15 S”); the latter represent the most abundant class of globin pre-mRNA (about 1000 molecules/cell). We conclude that the transcriptional units for the α and β globin genes may include up to 15,000 base-pairs rather than only the 1600 or so base-pairs corresponding to the 15 S pre-mRNA, the substrate for the final splicing event. The question remains open as to whether the full transcripts we observe are the compulsory primary pre-mRNA, or only facultative transcripts of the globin genes.

DNA fragments containing replication origins (oriDNA) were isolated from a chicken erythroblast cell line by a modified procedure of Zannis-Hadjopoulos et al. and studied in the renaturation reaction driven by either total or nuclear matrix DNA (nmDNA) from the same cells or from mature erythrocytes. We found that the unique sequences of nmDNA from erythroblasts (5 kb long) represented a specific subset of sequences constituting about a quarter of total DNA unique sequences, while the erythrocyte nmDNA 5 kb fragments constitute only about one tenth of total unique DNA and all are recovered among erythroblast nmDNA. Virtually all oriDNA sequences are present in the fraction of erythrocyte nmDNA. Thereafter, the putative positions of replication origins within the alpha-globine gene domain have been mapped by hybridization experiments. They were found to coincide with the previously established positions of permanent sites of DNA attachment to the nuclear matrix.

Two types of in vivo untranslated 'free' mRNA-protein particles (mRNP) were isolated from duck erythroblast cytoplasm and characterised. Both types, namely the highly purified globin mRNA-specific '20S' mRNP and the '35S' mRNP containing a heterogenous non-globin mRNA population, are not translatable in rabbit reticulocyte lysates, but yield active mRNA upon deproteinisation. In vivo, 90% of globin mRNA is translated, but the majority of mRNA types are found in the inactive mRNP fraction, including fully repressed mRNA species. Searching for the factors controlling differential mRNA repression, we characterised and compared the protein composition of globin and '35S' mRNP using two dimensional gel electrophoresis, in vivo labelling with [35S]methionine and in vivo phosphorylation. The major proteins ubiquitously bound to globin or any other mRNA in the polyribosomes (e.g., the 73 K mol. wt. poly(A) binding protein) were not detected in purified inactive mRNP. In the latter some polypeptides appear to be associated with only one of the two inactive mRNA types while some others are common to both mRNPs. Furthermore, different rates of synthesis and phosphorylation characterize the protein populations of the two types of repressed mRNP. The specificity in composition and metabolism of the populations of polypeptides associated with different subpopulations of inactive cytoplasmic mRNA, as shown here, argues in favour of a role of mRNP proteins in mRNA recognition and selective translational repression, possibly in association with the ScRNA previously found as components of the free mRNP and able to inhibit protein synthesis.

Poly(A)-containing mRNA has been prepared from the polyribosomes and post-polyribosomal mRNP fraction of duck reticulocytes. The coding capacity of the respective mRNA populations has been examined by translation in vitro followed by two-dimensional electrophoresis of the 35S-labeled polypeptides. A detailed analysis of these results is given elsewhere (Imaizumi-Scherrer, M.-T., Maundrell, K., Civelli, O., and Scherrer, K. (1982) Dev. Biol. 93, 126-138). Here, we focus on one of these translation products which migrates as a slightly basic protein of 73,000 molecular weight. By two-dimensional electrophoretic analysis and partial peptide mapping, we show that this protein is indistinguishable from the poly(A)-binding protein. We conclude that the majority of the coding sequences for this protein are translationally repressed in the reticulocyte cytoplasm.

Made available in DSpace on 2016-10-10T03:52:20Z (GMT). No. of bitstreams: 5 The poly_A binding protein facilitates in vitro translation of poly_A _ rich mRNA.pdf: 703457 bytes, checksum: cb7f3441f13194c0dd85e8c846d5dc6c (MD5) license_url: 52 bytes, checksum: 2f32edb9c19a57e928372a33fd08dba5 (MD5) license_text: 24372 bytes, checksum: 94b0a37ff5ec51de8c55507bff4a7ff9 (MD5) license_rdf: 24623 bytes, checksum: 378d22d8fe50e084ee2f354be78cbe62 (MD5) license.txt: 1887 bytes, checksum: 445d1980f282ec865917de35a4c622f6 (MD5) Previous issue date: 1988 To investigate the role of the 73-kDa poly(A)-binding protein in protein synthesis, the effect of the addition of homo-polyribonucleotides on the translation of polyadenylated and non-adenylated mRNA was studied in the rabbit reticulocyte lysate. Poly(A) was found to be the most effective polynucleotide in inhibiting duck-globin mRNA translation, whereas it had no effect on the translation of polyribosomal duck-globin mRNP, or on the endogenous synthesis of the rabbit reticulocyte lysate. The translation of poly(A)-free mRNA was not affected by the addition of poly(A). Furthermore, we found that the inhibiting effect of poly(A) can be reversed by addition of purified poly(A)- binding protein. It is thus likely that the 73-kDa poly(A)-binding protein is an essential factor necessary for poly(A)-rich mRNA translation. Sim Publicado

EDTA dissociation of polyribosomes from duck erythroblasts allowed us to isolate the 15-S globin messenger ribonucleoproteins (mRNP) by sucrose gradient centrifugation or affinity chromatography on poly(U)-Sepharose or oligo(dT)-cellulose columns. Their protein composition was compared by one and two-dimensional electrophoresis in sodium dodecyl sulfate to the free 20-S mRNP containing the repressed fraction of globin mRNA [Vincent, A., Civelli, O., Maundrell, K., and Scherrer, K. (1980) Eur. J. Biochem. 112, 617--633]. The protein composition of the 15-S mRNP isolated by these methods in different ionic strength conditions, was characterized by a major 73 000-Mr polypeptide and seven minor polypeptides with Mr ranging from 45 000 to 68 000, all of which are slightly basic, and about five acidic ones in the 80 000--130 000-Mr range. All these are retained in the 15-S mRNP core particle isolated at 0.5 M KCl. At low ionic strength, in addition, a specific group of acidic polypeptides in the Mr range 35 000--105 000 was also found associated with globin mRNA. Oligo(dT)-cellulose chromatography of mRNP digested with ribonucleases A and T1 indicated that the 73 000-Mr major protein is bound to the poly(A) segment; some other proteins resolved as minor components interact with both the poly(A) and non-poly(A) regions of globin mRNA. Characterization of proteins interacting with the poly(A) segment of non-polyribosomal globin mRNA in 20-S free mRNP demonstrated the absence of the polyribosomal 73 000-Mr poly(A)-binding protein. Furthermore, it confirmed that the protein compositions of translatable polyribosomal and repressed free globin mRNP are very different. Indeed, the respective core (0.5 M KCl) particles contain only two possibly common polypeptides. The specificity of proteins associated with globin mRNA in two different functional states shown here supports the hypothesis of a role of mRNP proteins in translational control of mRNA.

A residual protein matrix has been prepared from avian erythroblast nuclei by extensive extraction with salines and detergent and subsequent digestion with high concentrations of RNase and DNase. Ultrastructural examination reveals considerable internal structure, the most prominent feature being the remains of the nucleoli embedded in a network of fibres of fairly uniform diameter of 50 A. The proteins which make up this structure have been examined by two-dimensional electrophoresis and are shown to consist of a characteristic set of about 30, mainly acidic components, including four prominent species of 43 000, 52 000, 66 000 and 68 000 molecular weight (MW). In parallel preparations of the nuclear matrix digested with DNase alone, much of the nuclear RNA is found associated with the residual structure, including globin-coding sequences. These results correlate well with the ultrastructural appearance of DNase-digested matrix preparations which show that superimposed on the 50 A fibrous network is a 200–300 A granular component, the combined fibrillo-granular structure resembling the interchromatin RNP previously identified in situ. However, the proteins of the DNase-digested matrix seen by two-dimensional electrophoresis are indistinguishable from the proteins of matrix preparations digested with both DNase and RNase. Furthermore, two-dimensional comparison between the proteins of the DNase-digested matrix and purified 40S nuclear RNP particles shows that the bulk of the proteins found associated with nuclear RNA in vitro are extracted during matrix preparation, and only two, with MWs of 43 000 and 73 000, remain. The latter species co-migrates with the poly(A)-binding protein.

The genomic domain of about 20 kbp of the chicken alpha-type globin genes, framed by AT-rich linkers (ATRLs; Moreau et al. 1982) and repetitive sequences (Broders et al. 1986), was cut into 13 fragments and subcloned. The in vitro labelled individual restriction fragments were used to test the extent of the transcribed domain by blot-hybridization of nuclear RNA in large excess from normal adult chicken and Avian Erythroblastosis Virus (AEV)-transformed erythroblasts. In both these types of cells, the AT-rich segments situated 6 kbp upstream of the first gene as well as all the domain including the embryonic pi and the adult alpha D and alpha A genes down to the AT-rich segment placed 3 kbp downstream were found to be transcribed. Electrophoresis of nuclear RNA, Northern blotting and hybridization with most of the nick-translated DNA probes revealed in all cases the presence of heterogeneous globin RNA molecules in the 3-12 kb range, as well as some distinct RNA bands. Single-stranded RNA probes of some genomic segments indicated asymmetrical transcription of the minus strand. A 12 kb globin-specific RNA including the pi and alpha A genes but not the intervening alpha D gene was observed in AEV-transformed cells: it includes sequences located far upstream and downstream from the alpha globin genes and might represent a processing product of a full length transcript spanning the whole domain. Reverse transcription by extension of primers placed in the first exon of each of the three globin genes confirmed the presence of continuous transcripts of the domain including the two adult and the embryonic globin genes.

In the circulating blood of anemic ducks, 5% of all erythroid cells synthesize DNA. Immature erythroblasts, at all stages of differentiation, synthesize DNA although to a varying degree, while reticulocytes and erythrocytes do not. In the erythroid cell population labeled in vitro 2 h with 32Pi, half of the labeled DNA sediments as small-molecular-weight molecules, suggesting that these molecules fail to integrate into the high-molecular-weight components. Labeled DNA is found in the cytoplasmic postmitochondrial fractions and it is in a form of deoxyribonucleoproteins which cosediment with ribosomes as well as subribosomal particles in sucrose gradients. However, fixation with HCHO and centrifugation to equilibrium in CsCl gradient of these particles shows that the deoxyribonucleoprotein bands at the density different than the ribosomes and, thus, not physically linked to them. In EDTA-dissociated ribosomes, the deoxyribonucleoprotein particles cosediment with ribosomes as well as subribosomal particles in sucorse gradients. However, fixation with HCHO and centrifugation to equilibrium in CsCl gradient of these particles shows that the deoxyribonucleoprotein bands at the density different than the ribosomes and, thus, not physically linked to them. In EDTA-dissociated ribosomes, the deoxyribonucleoprotein particles cosdeiment with ribosomal subunits in such a way that the larger the particle, the larger the molecular weight of the DNA cosedimenting with it. The specific radioactivity of the cytoplasmic ribosome-derived and postribosomal-particle-derived DNAs and the small molecular-weight nuclear DNA is similar and 10-20-fold higher than that of the bulk nuclear DNA. The former three DNA species sediment between 4-14 S. It is concluded that the cytoplasmic nonmitochondrial DNA species are of the nuclear origin. Less than 0.5% of the total cellular nonmitochondrial DNA can be purified from the nucleus and the cytoplasm as fast-labeled small-molecular-weight components. All of the cellular nonmitochondrial DNA species band at the same mean buoyand density in Cs2SO4/urea gradients. All behave as native structures in hydroxyapatite and contain less than 5% of their length as single-stranded regions.

Duck immature red-cell polyribosomes were dissociated by different concentrations of EDTA At 0.02 μmol EDTA260 unit of ribosomes the dissociation was complete and reversible as shown by the ability of the reassociated ribosomes to synthesize globins. The behaviour of the mRNA during dissociation and reassociation was studied. Possible mechanisms of the reassociation are discussed.

There is evidence that the polyribosomal messenger RNA (mRNA) in animal cells is specifically associated with proteins [l-7] . In the case of cytoplasmic [5,8] or nuclear [9] messenger-like RNA (mlRNA) similar RNA-protein (RNP) complexes have also been described. Contrary to the claim that such associations may be non-specific [ 10, 1 l] , adequate control experiments suggest that they pre-exist in the cell and are not artefacts produced during lysis [5,8, 121. The exact physiological significance of these structures is not known. They may play a variety of roles such as: protection against RNase attack [5,8], RNA transport [ 1,8,9] , cleavage of giant mlRNA and selection of the RNA molecules to be transferred to the cytoplasm [5,9] , or initiation of protein synthesis [7] . In particular, two reports [ 13, 141 claim that the protein bound to the giant nuclear mlRNA is the same as that associated with mRNA in polyribosomes, indicating a possible function in mRNA transport. We have studied these mRNP particles in a highly differentiated system: duck immature red blood cells. In contrast to the hemoglobin producing cells in mammals, duck red cells are nucleated, making possible the investigation of the mland mRNA complexes in the nucleus and in the cytoplasm of a cell where a specific mRNA can be identified.

The size of virus-specific RNA synthesized in cultured mouse kidney cells infected with polyoma virus was estimated by electrophoresis and sedimentation analysis of RNA extracts from whole cells. Newly synthesized “late” polyoma-specific RNA appears as “giant” molecules of heterogeneous size, up to several times larger than a strand of polyoma DNA (1.5 × 10 6 daltons). Treatment with dimethylsulfoxide or urea showed that the large size of these molecules is not due to aggregation. Giant polyoma-specific RNA is strikingly similar in size distribution to “nuclear messenger-like” RNA (“heterogeneous nuclear” RNA) of the host cell. Subsequent to its synthesis, some of the giant polyoma-specific RNA appears to be cleaved to at least three smaller species.

New methods are presented for the isolation of nucleoli and the extraction of intact preribosomes under mild conditions avoiding EDTA and high salt buffers. These nucleolar preribosomes were characterized by an improved technique of polyacrylamide-gel electrophoresis which gives better resolution than sedimentation analysis for both ribonucleoprotein particles and free-RNA molecules. Thus, the 50-S native-ribosomal subunit could be separated from its 55-S precursor containing 32-S pre-RNA, and another nucleolar precursor, a “70-S” particle, could be identified. The separation technique newly developed is disc electrophoresis using exponential polyacrylamide gels. The resolution with these gels is greater than that with uniform gels over a wider range of molecular weights. The final resolution is independent of the volume in which the sample is loaded and the diffusion of molecules during migration is eliminated. Molecular weights of RNA molecules can be estimated in exponential gels as in uniform gels. Electrophoresis in quartz tubes allows the direct scanning of ultraviolet absorbance during and after the run. As all handling is thus avoided, gels of very low concentration can be used.

The RNA metabolism in immature duck erythrocytes has been investigated in order to determine the characteristics of messenger RNA (mRNA) in a highly differentiated animal cell. mRNA-like fractions were obtained from polysomes, on the one hand, and from pulse-labeled total cells or isolated nuclei, on the other, and were characterized by sedimentation, labeling kinetics, base composition, and hybridization to homologous DNA. At the translational level, the pulse-labeled RNA from polysomes consists of a predominant species sedimenting with about 9S and of a class of polydisperse material sedimenting between 6 and 28S. Very little ribosomal RNA (rRNA) is synthesized. The 9S RNA has been purified. Its base composition is relatively high in G + C (but different from rRNA or transfer RNA) – as determined, after alkaline hydrolysis, by 32P distribution or spectrophotometric analysis. The polydisperse RNA has a base composition characterized by relatively high proportions of U and A and is similar in this respect to nuclear RNA. Total polysomal RNA hybridizes to homologous DNA. The biological activity tested in a cell-free protein-synthesizing system of Escherichia coli is highest in the 16 to 18S zone of polysomal RNA. The rapidly labeled RNA synthesized at the transcriptional level in the nuclei sediments predominantly in the 30 to 80S zone. Base-composition analysis of this RNA reveals the presence of a predominant fraction of high-U-type RNA and of a small amount of 45 and 32S rRNA precursors. The former fraction — tentatively termed nascent, messenger-like RNA (nascent mlRNA), with respect to its base composition and capacity of selective hybridization — is metabolically more unstable than the precursor rRNA. Hybridization experiments demonstrate that up to 7% of the DNA is homologous to the nascent RNA fractions. Polysomal RNA hybridizes to a much smaller extent and competes only slightly with the heavy nuclear fractions. The significance of this heavy, nascent mlRNA and its eventual role in the regulation of protein synthesis in animal cells is discussed. We conclude that in a highly differentiated cell many more mRNA species are produced than would be expressed phenotypically through protein synthesis in the polysome. A surprisingly large part of the genome is activated, but an important fraction of the transcription products never reaches the sites of protein synthesis. Thus, the spectrum of functional RNA is not defined through synthesis only, but is restricted during metabolism. Under these conditions, control of differentiation is probably not limited exclusively to the transcription of the genome, but is subject to regulation mechanisms operating at the intermediate or translational level.

We have investigated some properties of a class of cytoplasmic ribonucleoprotein particles in HeLa cells. After 3.5 h of labelling in the presence of 0.04 μg/ml actinomycin D to inhibit rRNA synthesis, about 20% of the newly synthesized cytoplasmic RNA, that sediments faster than 7 S, is contained in two types of particles sedimenting at 45 S and 30 S in sucrose gradients. We have examined this material in more detail with the following results. 1 The fixed particles band as two sharp peaks of density o= 1.440 g/cm3 for the 45-S and density o= 1.460 g/cm3 for the 30-S material. We call them the 45-S-M and 30-S-M particles, respectively. 2 The RNA of the 45-S-M and 30-S-M particles sediment in sucrose gradients as sharp peaks at 16 S and 12 S, respectively. 3 The synthesis of these two RNA species is inhibited by ethidium bromide, but resistant to low concentrations of actinomycin D. Two RNA species with these drug sensitivities and sedimentation characteristics have been found in the mitochondria of HeLa cells [1]. 4 If cell homogenization and fractionation are carried out without detergents, the 45-S-M and 30-S-M particles are quantitatively bound to mitochondria. 5 Under most of the conditions commonly used for cell lysis and fractionation, these particles are extracted into the postmitochondrial-cytoplasmic fraction.

Two RNA · protein complexes were isolated from duck erythroblast postribosomal supernatants. Their nominal sedimentation values on sucrose gradients are 12 S and 20 S, respectively. The 12-S particle contains a 4–6 S RNA, in the 20-S particle a 9-S RNA is found. This 9-S RNA is shown to direct the synthesis of all duck globin chains in a cell-free, messenger RNA-dependent protein-synthesizing system. The protein moiety of these ribosome-free particles is described and compared with the proteins found in the mRNA · protein complex liberated by EDTA from polyribosomes. We show that in the free-cytoplasmic particles no protein can be found which is identical to any of the polypeptides associated with polyribosomal mRNA. Some of these protein are phosphorylated and contain phosphoserine. The electrophoretic patterns of phosphorylated proteins from the two globin mRNA-containing complexes differ, as do those of the unlabelled polypeptides. We conclude that the mRNA-associated protein population is exchanged when the mRNA enters the translation machinery. The possible role of the RNA-associated proteins in the post-transcriptional and translational control of eukaryotic protein synthesis is discussed.

Duck globin messenger RNA purified by three different procedures from duck immature erythrocyte polyribosomes was translated in a mammalian cell-free system which is strongly dependent upon exogenous mRNA. The system consists of purified mouse liver ribosome sub-units, partially purified rabbit reticulocyte initiation factors, and rat liver pH-5 enzymes (containing elongation and termination factors, aminoacyl-tRNA synthetases and tRNA). If offered alone, duck globin mRNA directed the synthesis of all known duck globin chains. The efficiency of duck globin synthesis was similar to that of rabbit globin if the two types were translated separately. If duck and rabbit globin mRNA were offered together each in saturating amounts in this system, rabbit globin synthesis occurred almost exclusively. Differences in the secondary structure between the two types of globin mRNA must be involved in this strongly discriminating translation in the competition experiments. Furthermore, the quantitative requirement of at least one initiation factor was found to be different for duck and rabbit globin mRNA translation. While there is no evidence that this factor is directly involved in mRNA binding, it seems likely that the difference in factor requirement is related to the preferential translation of rabbit globin mRNA in the competition experiments.

Duck erythroblasts prosomes were analysed by small angle neutron scattering (SANS), dynamic light scattering and (cryo-)electron microscopy. A molecular weight of approximately 720,000 +/- 50,000, a radius of gyration of 64 +/- 2 A and a hydrodynamic radius of approximately 86 A were obtained. Electron micrographs show a hollow cylinder-like particle with a diameter of 120 A, a height of 170 A and a diameter of 40 A for the cavity, built of four discs, the two outer ones being more pronounced than those in the center. Results from SANS indicate less then 5% of RNA in the purified prosomes, but nuclease protection assays confirm its presence.

have been isolated under low ionic strength conditions: the actively translatable 15 S mRNP isolated from polyribosomes and the in vivo and in vitro translationally repressed 20 S free cyto- plasmic mRNP [l-4]. A specific group of proteins (possibly equivalent to initiation factor eIF-3) is removed from 15 S polyribosomal mRNP when puri- fied in a 0.5 M KC1 containing buffer. Furthermore, the 20 S free mRNP is split into several sub-particles at this ionic concentration. Two of these still transla- tionally repressed subparticles, namely the 16 S and 13 S mRNPs, contain both (Y- and /3-globin mRNA [I] and differ partially in their protein composition [2]. The protein composition of these two free globin mRNP particles is also clearly different from that of the translatable 15 S polyribosomal globin mRNP, suggesting a relationship between protein composition of mRNP and mRNA function [3,5,6]. We have shown that proteins do not interact at random with mRNA within mRNPparticles, but rather specifically. Indeed, specific segments of the mRNA are protected in the RNP against staphylococcal nuclease action [7,8]. To gain further insight into the structure/function rela- tionship of the active and repressed globin mRNP complexes, we pursued our comparative studies on the structure of cytoplasmic-free and polyribosomai duck globin mRNPs and investigated: (i) Whether in the different forms of mRNP particles, i.e., 16 S, 13 S and 15 S, different proteins inter- act with the same region of the mRNA sequence; We show here that the specific sets of mRNP proteins induce specific structural alterations in the globin mRNA chain which may be related to translational activity or repression. 2. MateriaIs and methods RNase-free sucrose was from Merck. [-Y-~‘P] ATP (3000 Ci/mmol) was from the Radiochemical Center (Amersham).

Recent findings indicate that messenger RNA from a wide variety of cellular and viral sources contains methylated constituents [l-5] . Studies of complex mixtures of mammalian mRNA’s have indicated that the methyl groups occur in internal residues of N6 methyl adenylate and in unusual sequences at the 5’-termini [S-l 11. The 5’-terminal sequences appear to be of the form X5’ ppp” (Nmp) 1-2N~ . . . in which a ‘capping nucleoside’ X (generally m’G), is joined by a 5’-5’ triphosphate linkage to sequences containing either one or two 2’0ribose methylated nucleotides (Nmp). In order to determine whether such components are also characteristic of a well-defined species of eukaryotic mRNA, we investigated the methylated derivatives of mRNA in immature duck erythrocytes. These cells synthesize readily detectable quantities of 9 S globin mRNA in addition to a variety of other mRNA’s [ 121. Our findings indicate that 9 S globin mRNA also contains capped 5’-terminal sequences, but very little, if any, internal base methylated derivatives. Moreover, compared to a mixture of other mRNA species synthesized by these cells, the 9 S mRNA is relatively enriched in the sequences that contain two 2’0methyl nucleotides.

In vivo protein synthesis in duck erythroblasts was compared to in vitro translation of polyribosomal and free cytoplasmic mRNA. The in vivo study showed the absence of de novo synthesis of the Mr 73 000 poly(A)-binding protein found associated with all polyribosomal mRNA. In vitro translation demonstrated that the mRNA for this protein is absent from the polyribosomal mRNA fraction but constitutes a medium frequency messenger among the repressed free mRNA. This result confirms the existence of a qualitative translational control in terminal differentiating duck erythroblasts leading eventually to the arrest of the protein synthesizing machinery.

We have detected a transcriptional enhancer sequence downstream from the adult beta-globin (beta A-globin) genes of chicken and duck. DNA segments from the beta-globin coding and flanking sequences were cloned into expression vectors containing the SV40 promoter linked to either the T antigen gene or the cat gene. The expression of these genes was measured in a chicken erythroid cell line transfected with the recombinant plasmids. We found that segments located about 400 bp downstream from the poly(A) site of both the chicken and duck beta A-globin genes (and about 1.5 kb upstream from the embryonic epsilon-globin gene) stimulate transcription of the test genes about five-fold. In chicken essentially the same segment was also found by others to act as an erythroid cell-specific enhancer [Hesse et al., Proc. Natl. Acad. Sci. USA 83 (1986) 4312-4316; Choi and Engel, Nature 323 (1986) 731-734]. The sequence containing this enhancer is conserved in evolution. A high degree of homology, reaching 84% in a segment 180 bp in length, was found between chicken and duck despite an evolutionary divergence of 70 myr.

Low molecular weight RNAs (1mw RNA) extracted from isolated small nuclear ribonucleoprotein complexes (snRNP) of purified duck erythroblast nuclei were enzymatically labeled invitro at the 5′ terminus using (32P)-ATP and T4 polynucleotide kinase (1). Hybridization of these labeled 1mw RNA species to purified fractions of duck nuclear and cytoplasmic RNA revealed 2 principal RNA species of approximately 27,000 and 58,000 molecular weight which hydrogen-bonded to nuclear pre-messenger RNA (both poly(A)+ and poly(A)−) and to purified cytoplasmic globin messenger RNA.

The study of the interaction between mRNA and proteins in the polyribosomal 15 S duck globin messenger ribonucleoprotein complex showed that proteins protect specific mRNA sequences against digestion by the nonspecific micrococcal nuclease (Nucleic Acids Research 6 (8) 2787, 1979). Here we report the isolation of the poly(A)-protein RNP complex from nuclease digested 15 S mRNP by two different methods: sucrose gradient sedimentation and oligo(dT)-cellulose chromatography. We show by fingerprint analysis, that aprt from the periodically fragmented poly(A) segment, mRNA sequences adjacent and non-adjacent to the poly(A) segment are protected by the poly(A) binding proteins against nuclease digestion. The duck globin poly(A)-protein RNP complex, with a sedimentation coefficient between 7 S and 10 S, shows a characteristic protein composition, with a major 73,000 MW polypeptide and some minor components. The results are discussed in view of a dynamic ribonucleoprotein structure.