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The core proteins of 35 S hnRNP complexes

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Abstract

Ribonucleoprotein complexes (hnRNP) containing fragments of heterogeneous nuclear (hn)RNA and sedimenting at 35–40 S were isolated from the nuclei of HeLa S3 cells using the pH 8.0/diffusion technique. These hnRNP complexes are thought to be part of the hnRNA processing apparatus. The major protein components (core proteins) were identified by their constant ratios in native particles and in 35S hnRNP particles reconstituted in vitro. All of the core proteins, with one exception, show an increase in Mr on sodium dodecylsulfate (NaDodSO4)/polyacrylamide gels containing 8 M urea, indicative of secondary structure elements resistant to denaturation by NaDodSO4. The nine core proteins found by us are: A1 [Mr(NaDodSO4) 31 − 103/Mr (urea) 38 − 103, apparent isoelectric point, pIapp 9.3], A2 (32.5 − 103/39 − 103, 8.4), B1a (35.5 − 103/41 − 103, 8.8), B1b (35.5 − 103/44 − 103, 8.3), B1c (35.5 − 103/43 − 103, 5.7) B2 (37 − 103/42 − 103, 9.15), C1 (39 − 103/46 − 103, 9.2). C2 (40.5 − 103/45 − 103, 5.55) and C3 (38.5 − 103/37 − 103, 4.8). Individual proteins were electroeluted from two-dimensional gels and their amino acid composition determined. Difference indices were calculated and show a group of closely related basic proteins (A1, A2, B1a, B1b, B2, C1), two related slightly acidic proteins (B1c, C2) and a distinct acidic member (C3). Two-dimensional analysis of tryptic fragments and one-dimensional separation of peptides after V8 protease treatment support these data. Peptide mapping of the proteins A1 and A2 from bovine and human cells yields identical fragments indicating a high degree of cross-species conservation. An additional protein (D4: 44 − 103/55 − 103, >9.5) was found, which preferentially associates with heavier. oligomeric hnRNP structures. Only traces of actin are present in the 35S hnRNP fraction. All core proteins are modified by charge. A large part of the charge isomers arises by phosphorylation, which has been shown by labeling with 32PO4in vivo and with [γ-32P]ATP in vitro. the phosphate transfer is mediated by an endogenous protein kinase associated with the 35S hnRNP complexes. The major core protein A1 exists in two conformeric forms (A1 and A1x) of which only A1x serves as phosphate acceptor in vivo.

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In this and the accompanying paper we demonstrate that certain prokaryotic mRNAs, when modified at their 5'-termini with a cap structure, are translated in a eukaryotic cell-free protein synthesising system as efficiently as, or more efficiently than, eukaryotic mRNAs. Apparently, the prokaryotic mRNA contains all the information necessary for efficient recognition and initiation by eukaryotic translational components, except for the cap structure.
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The protein complement of heterogenous nuclear RNA · protein particles from human HeLA, mouse L and Chinese hamster (CHO) cells has been analysed by two-dimensional gel electrophoresis using the two techniques described by O'Farrell [J. Biol. Chem. (1975) 250, 4007–4021 and Cell (1977) 72, 1133–1142]. Over a hundred individual spots have been reproducibly detected both in stained gel and in autoradiographs of proteins labeled with L-[35S]methionine. Large similarities, especially in the 25000–40000 Mr cluster of basic protein, were found among these three mammalian species. As far as phosphoproteins are concerned, it was observed that the bands already described by one-dimensional gels [Eur. J. Biochem. (1978) 86, 301–310] with Mr values of 28000, 30000, 37000 and 52000 are resolved into about 15 individual spots, suggesting a corresponding number of distinct states of phosphorylation. It was also clearly demonstrated that phosphoproteins are unrelated to the major basic protein species. Particles of different size classes were analysed with respect to their content of individual proteins, both non-phosphorylated and phosphorylated. The most salient feature observed was that phosphoproteins become progressively more abundant with particles of increasing size. This raises the possibility that at least some of these phosphoproteins might belong to a nuclear structure to which hnRNA is normally bound.
Article
A nuclear protein apparently homologous to the two major proteins of 40S heterogeneous nuclear ribonucleoprotein particles from mammalian cells has been isolated from the lower eukaryote , purified, and found to contain a substantial amount of the unusual amino acid NG, NG-dimethylarginine. The apparent homology is based on similar molecular weights, basic isoelectric points and amino acid compositions including the dimethylarginine and a high content of glycine. The implications of the presence of this protein in and the possible significance of the NG, NG-dimethylarginine are discussed.
Article
Nuclear proteins have been fractionated into five distinct classes according to their extractability from rat liver nuclei at different pH and salt concentrations. The fractions have been analyzed for their amino acid composition which shows the presence of NG, NG-dimethylarginine, in sizable amount, in non-histone nuclear proteins (NHNP). This modification is most prominent in proteins which are found associated with rapidly-labeled heterogeneous RNA (HnRNP proteins).
Article
The proteins involved in protein-RNA and protein-protein interactions to form the core structure of nuclear 40S hnRNP particles in HeLa cells have been identified and characterized. Through complete analysis of nuclear extracts on sucrose density gradients and controlled salt dissociation of particle proteins, six lower molecular weight polypeptides are identified as the protein constituents of the 40S ribonucleoprotein complex which appears in the electron microscope as 210 A spherical particles. 40S hnRNP particles isolated from Chinese hamster lung fibroblasts show a strikingly similar protein composition to the human cells. The proteins are specifically associated with rapidly labeled nonribosomal nuclear RNA. Particle proteins from HeLa cells migrate in polyacrylamide gels as three groups of closely spaced doublets (groups A, B and C) and are present in a simple fixed stoichiometry. The group C proteins (C1 and C2 of 42,000 and 44,000 daltons) interact directly with RNA to form a smaller high salt-resistant RNP complex. The group A proteins (A1 and A2 of 32,000 and 34,000 daltons) are major nuclear proteins and constitute 60% total particle protein mass. These two proteins are basic with isoelectric points near 9.2 and 8.4, respectively, and are characterized by an unusual amino acid composition, including high glycine (25%) and the unusual modified basic residue identified as NG,NG-dimethylarginine. The major particle proteins (A1 and A2) interact electrostatically with nucleic acids and apparently function structurally in the packaging and stabilization of hnRNA in a manner analogous to the histones in chromatin υ bodies. The similarity in protein composition of core RNP particles from different cell types (especially in the basic proteins, A1, A2 and B1) is consistent with a conserved particle structure and function in eucaryotes.
Article
To elucidate the mechanisms which achieve and regulate the transport from the nucleus to the cytoplasm of those sequences of heterogeneous nuclear RNA (HnRNA) destined to become cytoplasmic mRNA, we have started a systematic investigation of the proteins associated with these RNA species in the form of the so-called `RNP particles'. The general occurrence of such ribonucleoprotein complexes in eukaryotic cells is widely recognised although they have only recently been described in HeLa cells1,2. The occurrence of phosphorylated proteins in rat brain nuclear particles3, together with the observation that mRNA release from adenovirus-infected KB cell nuclei in vitro is an ATP-dependent process4, has prompted a search for protein kinase activity in HeLa nuclear RNP particles. The role of protein phosphorylation in the regulation of enzyme activity is now recognised5 and we have observed such an activity which does not require the addition of exogenous substrates like histones. The endogenous protein substrates present in HeLa nuclear RNP particles have also been characterised.
Article
Poly (A) polymerase activity from cytoplasm and nuclei of 12-16-day-old mouse embryos has been partially purified by (NH4)2SO4 fractionation, DEAE-cellulose, phosphocellulose and tRNA-Sepharose affinity chromatography, and their properties have been compared. The nuclear and cytoplasmic enzymes exhibit similar chromatographic elution profiles, and similar biochemical and physical properties. Poly(A) polymerase has an absolute requirement for a divalent cation, ATP and an oligo- or polyribonucleotide primer. With tRNA, the divalent salt concentrations for optimum enzyme activity are 1 mM MnCl2 or 10 mM MgCl2. The enzyme activity with MnCl2 is 10-15-fold higher than that with MgCl2. The molecular weight of the native enzyme is about 65 000 and its sedimentation coefficient is around 4.5 S. The average chain length synthesized by the enzyme is between 10 and 13 nucleotides. The inhibitors of RNA polymerase do not affect poly (A) polymerase activity; however, some synthetic rifamycin SV derivatives are potent inhibitors of this enzyme.
Article
Physical factors leading to the separation of oligopeptides in the molecular weight range of 1,200 to 10,000 daltons by analytical-scale electrophoresis in polyacrylamide gel with sodium dodecyl sulfate are described. Increased acrylamide concentration, cross-linkage, and inclusion of 8 M urea to decrease gel porosity, increased gel length, and buffer ions of low mobility are factors which yield improved separation of such peptides. Electrophoretic mobilities of eleven peptides were linearly related to the logarithm of their molecular weights with a standard deviation of 18% in a system of improved resolution. The intrinsic charge and conformation of peptides were found to be relatively more important determinants of electrophoretic mobilities than for proteins larger than 10,000 daltons. Such determinants were relatively more important with four of the eleven peptides examined, leading to deviations from the log-linear slope greater than 18%. Because of the importance of intrinsic charge and conformation, the system, although allowing a first approximation in molecular weight determination, may also be applicable to peptide “mapping,” particularly for “insoluble” peptide mixtures with prominent hydrophobic association, such as encountered in cellular membranes, viruses, and proteolytic digests.
Article
IT is sometimes desirable to study the possible relationship of two proteins when sequence data are unavailable. Few methods are useful for such an analysis: molecular size, functional criteria, genetic localization, end group analysis, tryptic fingerprints all have serious shortcomings. Immunological criteria may be useful when cross-reactivity is found, but such cross-reactivity is frequently one of the earliest common features to be lost. A possible useful criterion is amino-acid composition. Despite considerable changes in protein structure brought about by a limited number of sequence changes, the overall composition of evolutionary related proteins can be expected to remain quite similar. We wish to present a simple method for assessing and describing such compositional relatedness.
Article
Three different methods for hydrolysis and determination of amino acid composition of peptides and proteins were compared. We found, that the method of Matsubara and Sasaki (using 6N HCl and thioglycolic acid) gives comparatively low recoveries for tryptophan, while Liu and Chang's method, using p-toluenesulfonic acid and tryptamine, is more suitable. To eliminate the difficulties of the latter method, we used mercaptoethane-sulfonic acid, which, in the concentration used, results in total hydrolysis of peptide bonds within 22 hr and gives very high tryptophan recoveries. Both sulfonic acid methods were used for hydrolysis of the pentapeptide “pentagastrine” as well as of the proteins lysozyme, cytochrome c, and chymotrypsine. Their amino acid composition was determined using an automatic amino acid analyzer. Similarly to the p-toluenesulfonic acid method, the results of our method are totally reliable only for pure peptides and proteins, though the results obtained with our method using samples containing carbohydrates are better than those of all earlier methods.
Article
A comparison of the amino acid compositions of 30S ribosomal proteins was made using data already published. Similar comparisons were made for a variety of different cytochrome C's as well as for a group of thirty-five functionally unrelated proteins. On the basis of these comparisons it is concluded that some of the 30S ribosomal proteins are likely to possess considerable similarities in amino acid sequence.
Article
Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products. Four major components of the head are cleaved during the process of assembly, apparently after the precursor proteins have assembled into some large intermediate structure.
Article
In previous reports it was shown that the main part of nuclear DNA-like RNA (D-RNA) can be found in homogeneous 30 s particles which are complexes of specific protein particles with D-RNA strands bound to them.It has now been established that 30 s particles are monomers of a more complex polysome-like structure. Such a structure is formed by a long D-RNA strand and a number of globular protein particles bound to it and tightly packed along the D-RNA strand.These complexes occupy a wide zone in sucrose gradients between 30 and 200 s. In the electron microscope, one finds the 30 8 peak contains only single particles; the 45 s peak, dimers; the 55 to 60 s peak, trimers, and so on. In heavier zones long chains of particles (n = 8 to 12) were observed.Treatment with small amounts of RNase quantitatively converts large polyparticles into the 30 s particles.The buoyant density and the protein composition of mono- and poly-particles are the same. They are very different from those of ribosomes. The sedimentation coefficient of RNA isolated from particles is related to the number of monomers in the complex.It is calculated that one protein particle is bound to an RNA strand having a molecular weight of about 2 × 105.The structure and function of the nuclear complexes containing D-RNA are discussed. The name “informofer” is suggested for monomeric protein particles.
Article
A method of preparing a nuclear fraction from HeLa cells is described. The fraction does not contain an appreciable amount of the 16 s RNA component of ribosomes and thus appears to be free of cytoplasmic contamination. All of the early precursors to cytoplasmic ribosomal RNA are contained in the nuclear fraction.Sedimentation analysis of the RNA extracted from the nuclear fraction shows a peak in optical density at 45 s and a larger peak that sediments about 30 s. The RNA in the 30 s region is shown to be polydisperse and contains 35 a and 28 s RNA.Previous work has shown that the initial event in the formation of ribosomal RNA is the synthesis of 45 s RNA. It is shown here that the next step is the cleavage of 45 s RNA form 16 s RNA, which emerges immediately into the cytoplasm, and 35 s RNA which remains in the nucleus. The 35 s RNA undergoes a transition to 28 s and then enters the cytoplasm. The cleavage of 45 s to 35 s and 16 s RNA proceeds in the presence of actinomycin but the subsequent behavior of the 35 s RNA is abnormal.Experiments in which radioactive label is chased shows that there is no high molecular weight RNA permanently associated with the nucleus.
Article
Using a whole cell extract of HeLa cells, we synthesized unspliced RNAs containing the first two leaders and the first intervening sequence of the adenovirus 2 major late transcription unit. Upon incubation of these pre-mRNAs in reaction mixtures containing a nuclear extract and a postnuclear fraction (S100), removal of the first intervening sequence and concomitant joining of the first leader to the second leader was observed. This splicing reaction requires proteins, Mg2+ ions, and ATP. The S100 fraction alone has no splicing activity but stimulates splicing when added to the nuclear extract. Upon fractionation of the postnuclear S100 by chromatography on ion exchange and gel filtration columns, the stimulatory activity copurifies with small ribonucleoprotein particles.
Article
Vaccinia virus RNA guanylyltransferase catalyzes the transfer of GMP from GTP to the 5'-triphosphate or diphosphate terminus of RNA to generate the cap structure G(5')ppp(5')N-. The guanylylation reaction consists of a series of at least two partial reactions: (i) GTP + E in equilibrium E-pG + PPi, (ii) E-pG + (p)ppNpNpN- leads to GpppNpNpN- + E. Inthe first of these, GTP reacts with capping enzyme in the absence of an RNA acceptor to form a covalent enzyme-guanylate intermediate. The GMP is linked to the Mr 95,000 subunit of the capping enzyme via a phosphoamide bond, as judged by the acid-labile, alkali-stable nature of the bond and by the susceptibility of the linkage to cleavage by hydroxylamine at pH 4.75. The isolated enzyme-guanylate complex is able to transfer the guanylate moiety to triphosphate-terminated poly(A) to yield the 5' cap structure GpppA or to pyrophosphate to regenerate GTP. Both partial reactions of transguanylylation require a divalent cation.
Article
Mouse erythroleukemia cells were pulse-labeled with [3H]uridine and irradiated with 254-nm light to produce covalent crosslinks between RNA and proteins in close proximity to one another in vivo. Nuclear ribonucleoprotein particles containing heterogeneous nuclear RNA were isolated and digested with nucleases, and the resulting proteins were subjected to gel electrophoresis. Proteins carrying covalently crosslinked [3H]uridine nucleotides were identified by fluorography. The results demonstrate that heterogeneous nuclear RNA is complexed in vivo with a set of six major proteins having molecular weights between 32,500 and 41,500. Analysis of chromatin fractions indicates that nascent heterogeneous nuclear RNA chains assemble with these six proteins as a very early post-transcriptional event. These data, and other results [Nevins, J. R. & Darnell, J. E. (1981) Cell 15, 1477-1493], lead us to propose the usual order of post-transcriptional events to be: heterogeneous nuclear RNA-ribonucleoprotein particle assembly leads to poly(A) addition leads to splicing.
Article
ADP-ribosylation of nonhistone proteins during the HeLa cell cycle was investigated. Proteins were radiolabeled by incubating interphase nuclei and mitotic cells with the specific precursor, [32P]NAD. Autoradiograms of two-dimensional gels of total nuclear nonhistone proteins showed a large number of modified species (more than 140). A complex pattern was also found for interphase chromatin. Nuclear scaffolds showed a simpler pattern of four major groups of modified species, which appeared to be the lamins and poly(ADP-ribose) polymerase. The labeling pattern for nonhistones of metaphase chromosomes was fundamentally different than with interphase nuclei. Autoradiograms were dominated by the incorporation of label into poly(ADP-ribose) polymerase.
Article
Ribonucleoprotein complexes (hnRNP) sedimenting at 30-40 S and containing fragments of heterogeneous nuclear RNA (hnRNA) have been extracted from HeLa cell nuclei. Besides hnRNA fragments (8-12 S), the complexes contain eight mostly basic core proteins of Mr 31 000-41 000 as shown by two-dimensional gel electrophoresis. Other proteins (mostly of higher molecular weight) seem to be peripherally associated since they are lost after pelleting and recentrifugation of the hnRNP complexes. The particle dissociates into its protein components after digestion of the endogenous hnRNA fragments by micrococcal nuclease. After inactivation of the nuclease and addition of a wide variety of exogenous RNAs [MS2 phage RNA, poly(U), poly(C), poly(A), and poly(A,U)], a RNP particle is re-formed which resembles the native hnRNP complex according to its sedimentation value (35 S), its appearance in the electron microscope, its density in metrizamide, and its protein composition. No particles are formed on double-stranded RNA [poly(A) . poly(U)] or native DNA whereas denatured DNA allows complex formation. On MS2 RNA (3569 nucleotides), the formation of tri- and tetrameric complexes is observed. This indicates the presence of 900-1200 nucleotides per particle. In vivo, 40S hnRNP particles are a unit component of larger RNP structures. Hence, we conclude from our results that the hnRNP core proteins have the intrinsic capability to associate with nascent single-stranded hnRNA regions to form these RNP complexes. Because of the lack of any sequence specificity, the complexes may function in packaging of the hnRNA and in connection with other nuclear components may provide a scaffold for subsequent processing reactions.
Article
We have studied the ribonucleoprotein morphology of nascent hnRNA using chromatin spreading methods. This approach allows visualization of multiple transcripts of the same DNA sequence. Thus the RNP structure of hnRNA molecules with the same nucleotide sequence can be compared. We find that RNP particles averaging 240 A in diameter occur on the majority of hnRNA transcription units, but that RNA fibrils are generally not completely covered with particles. Furthermore, the RNP particle location is nonrandom for the transcripts of a given gene. Particle number and arrangement on RNP fibrils vary from one transcription unit to the next, and are not obviously related to transcription unit size or activity. Analysis of particle location with respect to the 5' RNA terminus allows RNP particle localization to homologous sequences of less than 500 ribonucleotides on the different fibrils of a single transcription unit. RNP particles assemble very soon after synthesis of the particular sequence. Sites of particle formation are distinct from sites of double-stranded hairpin loops on the RNA fibrils. A differential, or differentially stable, RNP structure with respect to RNA sequence may bear on mechanisms of specific hnRNA processing.
Article
Early region 2 of the adenovirus 2 genome (map position 61-75) specifies two poly(A)+ nuclear RNAs (28S and 23S) that appear to be precursors of the 20S cytoplasmic mRNA [Goldenberg, C. J. & Raskas, H. J. (1979) Cell 16, 131-138]. Splicing of these nuclear RNAs in vitro has been obtained with a whole cell extract prepared from MOPC-315 mouse myeloma cells. The in vitro reaction excises sequences from two introns and attaches 5' sequences to the mRNA body. The splicing reaction was demonstrated by two procedures: (i) hybridization of pulse-labeled RNA fractionated by size and (ii) annealing of RNAs with radioactive DNA probes followed by nuclease digestion. The first procedure provided evidence that sequences from the large 2300-nucleotide intron (74.6-68.8) were excised and 5' transcripts were spliced to the mRNA body. Utilizing both S1 and Exo VII nucleases, the second procedure demonstrated excision of sequences from the smaller 720-nucleotide intron (68.5-66.3), the splicing of sequences from the second leader (68.8) to the mRNA body, and the formation of an mRNA body of 1700 nucleotides, the size found in vivo. These findings provide evidence that an in vitro system that splices viral RNAs to yield products comparable to those found in vivo is now available.
Article
Mouse erythroleukemia cell nuclei obtained by three different methods were spread for electron microscopy under low ionic conditions. It was found that this procedure allows the observation of free large ribonucleoprotein (RNP) complexes released from the nuclei during the centrifugation. The morphology of these complexes was readily affected by the conditions of cell treatment and spreading. Two extreme forms of free nuclear RNP structures were obtained, both consisting of spherical particles with diameters of approximately 17-20 nm. The first type was of loosened complexes of irregularly assembled particles interconnected with RNA fibrils. The second represented tightly packed particles forming mostly branched structures. The latter structures appeared to be closer to the native form of the nuclear RNP particles, differing from polyribosomes by their characteristic branching and stability in EDTA solutions.
Article
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 Å. 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 Å fibrous network is a 200–300 Å 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.
Article
The proteins of rat liver cytoplasm, nuclear washes, matrix, membrane, heterogeneous nuclear (hn)RNA proteins and chromatin were examined by two-dimensional gel electrophoresis. The inclusion in the gels of six common protein standards of carefully selected molecular weight and isoelectric point allowed us to clearly follow the distribution of specific proteins during nuclear extraction. In the nuclear washes and chromatin, we observed five classes of proteins: (a) Exclusively cytoplasmic proteins, present in the first saline-EDTA wash but rapidly disappearing from subsequent washes; (b) ubiquitous proteins of 75,000, 68,000, 57,000, and 43,000 mol wt, the latter being actin, found in the cytoplasm, all nuclear washes and the final chromatin pellet; (c) proteins of 94,000, 25,000, and 20,500 mol wt specific to the nuclear washes; (d) proteins present in the nuclear washes and final chromatin, represented by species at 62,000, 55,000, 54,000, and 48,000 mol wt, primarily derived from the nuclear matrix; and (e) two proteins of 68,000 mol wt present only in the final chromatin. The major 65,000-75,000-mol wt proteins seen by one-dimensional gel electrophoresis of nuclear matrix were very heterogeneous and contained a major acidic, an intermediate, and a basic group. A single 68,000-mol wt polypeptide constituted the majority of the membrane-lamina fraction, consistent with immunological studies indicating that a distinct subset of matrix proteins occurs, associated with heterochromatin, at the periphery of the nucleus. Actin was the second major nuclear membrane-lamina protein. Two polypeptides at 36,000 and 34,000 mol wt constituted 60% of the hnRNP. Approximately 80% of the mass of the nonhistone chromosomal proteins (NHP) from unwashed nuclei is contributed by nuclear matrix and hnRNPs, and essentially the same patterns were seen with chromatin NHP. The concept of NHP being a distinct set of DNA-bound proteins is unnecessarily limiting. Many are derived from the nuclear matrix or hnRNp particles and vary in the degree to which they share different intracellular compartments.
Article
A simple and rapid method is presented for the preparation of I/sup 131/- labeled human growth hormone of high specific radioactivity (240-300 mu C/ mu g). Low amounts of carrierfree I/sup 131/ iodide (2 mC) are allowed to react, without prior treatment, with small quantities of protein (5 mu g) in a highyield reaction (approx. 70% transfer of I/sup 131/ to protein). The degree of chemical substitution is minimized (0.5- 1.0 atom of iodine/molecule of protein) by the use of carrier-free I/sup 131/ iodide. The I/sup 131/-labeled hormone (up to 300 mu C/ mu g) contains no detectable degradation products and is immunologically identical with the unlabeled hormone. The loss of immunological reactivity at high specific radioactivities or at high levels of chemical substitution with STAI/sup 127/!iodine is demonstrated. (auth)
  • C Brunel
  • M N Lelay
Brunel, C. & Lelay, M. N. (1979) Eur. J. Biochem. 99,273-283.