Asg7p-Ste3p inhibition of pheromone signaling: regulation of the zygotic transition to vegetative growth.

The inappropriate expression of the a-factor pheromone receptor (Ste3p) in the MATa cell leads to a striking inhibition of the yeast pheromone response, the result of a functional interaction between Ste3p and some MATa-specific protein. The present work identifies this protein as Asg7p. Normally, expression of Ste3p and Asg7p is limited to distinct haploid mating types, Ste3p to MATalpha cells and Asg7p to MATa cells. Artificial coexpression of the two in the same cell, either a or alpha, leads to dramatic inhibition of the pheromone response. Ste3p-Asg7p coexpression also perturbs the membrane trafficking of Ste3p: Ste3p turnover is slowed, a result of an Asg7p-mediated retardation of the secretory delivery of the newly synthesized receptor to the plasma membrane. However, in the absence of ectopic Ste3p expression, the asg7Delta mutation is without consequence either for pheromone signaling or overall mating efficiency of a cells. Indeed, the sole phenotype that can be assigned to MATa asg7Delta cells is observed following zygotic fusion to its alpha mating partner. Though formed at wild-type efficiency, zygotes from these pairings are morphologically abnormal. The pattern of growth is deranged: emergence of the first mitotic bud is delayed, and, in its place, growth is apparently diverted into a novel structure superficially resembling the polarized mating projection characteristic of haploid cells responding to pheromone. Together these results suggest a mechanism in which, following the zygotic fusion event, Ste3p and Asg7p gain access to one another and together act to repress the pheromone response, promoting the transition of the new diploid cell to vegetative growth.

Recycling of the yeast a-factor receptor.

The yeast a-factor receptor (Ste3p) is subject to two mechanistically distinct modes of endocytosis: a constitutive, ligand-independent pathway and a ligand-dependent uptake pathway. Whereas the constitutive pathway leads to degradation of the receptor in the vacuole, the present work finds that receptor internalized via the ligand-dependent pathway recycles. With the a-factor ligand continuously present in the culture medium, trafficking of the receptor achieves an equilibrium in which continuing uptake to endosomal compartments is balanced by its recycling return to the plasma membrane. Withdrawal of ligand from the medium leads to a net return of the internalized receptor back to the plasma membrane. Although recycling is demonstrated for receptors that lack the signal for constitutive endocytosis, evidence is provided indicating a participation of recycling in wild-type Ste3p trafficking as well: a-factor treatment both slows wild-type receptor turnover and results in receptor redistribution to intracellular endosomal compartments. Apparently, a-factor acts as a switch, diverting receptor from vacuole-directed endocytosis and degradation, to recycling. A model is presented for how the two Ste3p endocytic modes may collaborate to generate the polarized receptor distribution characteristic of mating cells.

Akr1p and the type I casein kinases act prior to the ubiquitination step of yeast endocytosis: Akr1p is required for kinase localization to the plasma membrane.

Ubiquitination of the plasma membrane-localized yeast a-factor receptor (Ste3p) triggers a rapid, ligand-independent endocytosis leading to its vacuolar degradation. This report identifies two mutants that block uptake by blocking ubiquitination, these being mutant either for the ankyrin repeat protein Akr1p or for the redundant type I casein kinases Yck1p and Yck2p. While no obvious defect was seen for wild-type Ste3p phosphorylation in akr1 or yck mutant backgrounds, examination of the Delta320-413 Ste3p deletion mutant phosphorylation did reveal a clear defect in both mutants. The Delta320-413 deletion removes 18 Ser-Thr residues (possible YCK-independent phosphorylation sites) yet retains the 15 Ser-Thr residues of the Ste3p PEST-like ubiquitination-endocytosis signal. Two other phenotypes link akr1 and yck mutants: both are defective in phosphorylation of wild-type alpha-factor receptor, and while both are defective for Ste3p constitutive internalization, both remain partially competent for the Ste3p ligand-dependent uptake mode. Yck1p-Yck2p may be the function responsible in phosphorylation of the PEST-like ubiquitination-endocytosis signal. Akr1p appears to function in localizing Yck1p-Yck2p to the plasma membrane, a localization that depends on prenylation of C-terminal dicysteinyl motifs. In akr1Delta cells, Yck2p is mislocalized, showing a diffuse cytoplasmic localization identical to that seen for a Yck2p mutant that lacks the C-terminal Cys-Cys, indicating a likely Akr1p requirement for the lipid modification of Yck2p, for prenylation, or possibly for palmitoylation.

Ubiquitination of the PEST-like endocytosis signal of the yeast a-factor receptor.

A 58-residue-long, PEST-like sequence within the yeast a-factor receptor (Ste3p) specifies the ubiquitination, endocytosis, and consequent vacuolar degradation of the receptor protein (Roth, A. F., Sullivan, D. M., and Davis, N. G. (1998) J. Cell Biol. 142, 949-961). The present work investigates three lysyl residues that map within this sequence as the potential ubiquitin acceptor sites. Lys --> Arg substitution mutants were tested for effects on both ubiquitination and endocytosis. Results indicate that the three lysines function redundantly; a severe blockade to both ubiquitination and endocytosis is seen only for receptors having all three lysines replaced. Of the three, Lys(432) plays the predominant role; ubiquitination and turnover are significantly impaired for receptors having just the K432R mutation. CNBr fragmentation of the receptor protein, used for the physical mapping of the ubiquitin attachment sites, showed PEST-like sequence lysines to be modified both with single ubiquitin moieties as well with short multi-ubiquitin chains, two or three ubiquitins long. Thus, in addition to being the signal for ubiquitination, the Ste3p PEST-like sequence also provides the site for ubiquitin attachment. To test if this endocytosis signal functions solely for ubiquitination, we have asked if the requirement for the PEST-like sequence in endocytosis might be bypassed through pre-attachment of ubiquitin to the receptor protein. Indeed, Ste3-ubiquitin translational fusions that have a ubiquitin moiety fused to the receptor in place of the PEST-like signal do undergo rapid endocytosis and vacuolar turnover. We conclude that ubiquitin alone, with no required contribution from receptor sequences, provides the sufficient signal for initiating uptake. In addition, our results confirm conclusions originally drawn from studies with the alpha-factor receptor (Terrell, J., Shih, S., Dunn, R., and Hicke, L. (1998) Mol. Cell 1, 193-202), namely that mono-ubiquitin, and not multi-ubiquitin chains provide the primary recognition determinant for uptake. Although mono-ubiquitination suffices, our results indicate that multi-ubiquitination serves to augment the rate of uptake.

Feedback phosphorylation of the yeast a-factor receptor requires activation of the downstream signaling pathway from G protein through mitogen-activated protein kinase.

The two yeast pheromone receptors, the a and alpha-factor receptors, share many functional similarities: both G protein-coupled receptors couple to the same downstream signal transduction pathway, and both receptors undergo feedback regulation involving increased phosphorylation on their C-terminal domains in response to ligand challenge. The present work, which focuses on the signaling mechanism controlling this feedback phosphorylation, indicates one striking difference. While the alpha-factor-induced phosphorylation of the alpha-factor receptor does not require activation of the downstream G protein-directed signaling pathway (B. Zanolari, S. Raths, B. Singer-Kruger, and H. Riezman, Cell 71:755-763, 1992), the a-factor-induced phosphorylation of the a-factor receptor (Ste3p) clearly does. Induced Ste3p phosphorylation was blocked in cells with disruptions of various components of the pheromone response pathway, indicating a requirement of pathway components extending from the G protein down through the mitogen-activated protein kinase (MAPK). Furthermore, Ste3p phosphorylation can be induced in the absence of the a-factor ligand when the signaling pathway is artificially activated, indicating that the liganded receptor is not required as a substrate for induced phosphorylation. While the activation of signaling is critical for the feedback phosphorylation of Ste3p, pheromone-induced gene transcription, one of the major outcomes of pheromone signaling, appears not to be required. This conclusion is indicated by three results. First, ste12Delta cells differ from cells with disruptions of the upstream signaling elements (e.g., ste4Delta, ste20Delta, ste5Delta, ste11Delta, ste7Delta, or fus3Delta kss1Delta cells) in that they clearly retain some capacity for inducing Ste3p phosphorylation. Second, while activated alleles of STE11 and STE12 induce a strong transcriptional response, they fail to induce a-factor receptor phosphorylation. Third, blocking of new pheromone-induced protein synthesis with cycloheximide fails to block phosphorylation. These findings are discussed within the context of a recently proposed model for pheromone signaling (P. M. Pryciak and F. A. Huntress, Genes Dev. 12:2684-2697, 1998): a key step of this model is the activation of the MAPK Fus3p through the G(betagamma)-dependent relocalization of the Ste5p-MAPK cascade to the plasma membrane. Ste3p phosphorylation may involve activated MAPK Fus3p feeding back upon plasma membrane targets.

A large PEST-like sequence directs the ubiquitination, endocytosis, and vacuolar degradation of the yeast a-factor receptor.

The yeast a-factor receptor (encoded by STE3) is subject to two modes of endocytosis, a ligand-dependent endocytosis as well as a constitutive, ligand-independent mode. Both modes are associated with receptor ubiquitination (Roth, A.F., and N.G. Davis. 1996. J. Cell Biol. 134:661-674) and both depend on sequence elements within the receptor's regulatory, cytoplasmically disposed, COOH-terminal domain (CTD). Here, we concentrate on the Ste3p sequences required for constitutive endocytosis. Constitutive endocytosis is rapid. Receptor is synthesized, delivered to the cell surface, endocytosed, and then delivered to the vacuole where it is degraded, all with a t1/2 of 15 min. Deletion analysis has defined a 36-residue-long sequence mapping near the COOH-terminal end of the Ste3p CTD that is the minimal sequence required for this rapid turnover. Deletions intruding into this interval block or severely slow the rate of endocytic turnover. Moreover, the same 36-residue sequence directs receptor ubiquitination. Mutants deleted for this sequence show undetectable levels of ubiquitination, and mutants having intermediate endocytosis defects show a correlated reduced level of ubiquitination. Not only necessary for ubiquitination and endocytosis, this sequence also is sufficient. When transplanted to a stable cell surface protein, the plasma membrane ATPase Pma1p, the 36-residue STE3 signal directs both ubiquitination of the PMA1-STE3 fusion protein as well as its endocytosis and consequent vacuolar degradation. Alanine scanning mutagenesis across the 36-residue-long interval highlights its overall complexity-no singular sequence motif or signal is found, instead required sequence elements distribute throughout the entire interval. The high proportion of acidic and hydroxylated amino acid residues in this interval suggests a similarity to PEST sequences-a broad class of sequences which have been shown to direct the ubiquitination and subsequent proteosomal degradation of short-lived nuclear and cytoplasmic proteins. A likely possibility, therefore, is that this sequence, responsible for both endocytosis and ubiquitination, may be first and foremost a ubiquitination signal. Finally, we present evidence suggesting that the true signal in the wild-type receptor extends beyond the 36-residue-long sequence defined as a minimal signal to include contiguous PEST-like sequences which extend another 21 residues to the COOH terminus of Ste3p. Together with sequences identified in two other yeast plasma membrane proteins, the STE3 sequence defines a new class of ubiquitination/endocytosis signal.

Congenital orbital teratoma.

BACKGROUND: The occurrence of proptosis at birth is unusual and may be associated with a variety of tumours and structural abnormalities. Congenital orbital teratoma is a rare cause of congenital proptosis. METHODS: A case report is presented of a female infant with gross right proptosis. RESULTS: Computed tomography demonstrated a characteristic multicystic structure with no intracranial involvement. Histological examination found tissues derived from all three germ cell layers, consistent with a congenita orbital teratoma. The tumour was successfully removed preserving the globe and vision. CONCLUSIONS: This report is an example of how early surgical intervention may allow preservation of the globe and vision in some patients with congenital orbital teratoma.

Ubiquitination of the yeast a-factor receptor.

The a-factor receptor (Ste3p) is one of two pheromone receptors in the yeast Saccharomyces cerevisiae that enable the cell-cell communication of mating. In this report, we show that this receptor is subject to two distinct covalent modifications-phosphorylation and ubiquitination. Phosphorylation, evident on the unstimulated receptor, increases upon challenge by the receptor's ligand, a-factor. We suggest that this phosphorylation likely functions in the adaptive, negative regulation of receptor activity. Removal of phosphorylation by phosphatase treatment uncovered two phosphatase-resistant modifications identified as ubiquitination using a myc-epitope-tagged ubiquitin construct. Ste3p undergoes rapid, ligand-independent turnover that depends on vacuolar proteases and also on transport of the receptor from surface to vacuole (i.e., endocytosis) (Davis, N.G., J.L.Horecka, and G.F. Sprague, Jr., 1993 J. Cell Biol. 122:53-65). An end4 mutation, isolated for its defect in the endocytic uptake of alpha-factor pheromone (Raths, S., J. Rohrer, F. Crausaz, and H. Riezman. 1993. J. Cell Biol. 120:55-65), blocks constitutive endocytosis of the a-factor receptor, yet fails to block ubiquitination of the receptor. In fact, both phosphorylation and ubiquitination of the surfacebound receptor were found to increase, suggesting that these modifications may occur normally while the receptor is at the cell surface. In a mutant strain constructed to allow for depletion of ubiquitin, the level of receptor ubiquitination was found to be substantially decreased. Correlated with this was an impairment of receptor degradative turnover-receptor half-life that is normally approximately 20 min at 30 degrees C was increased to approximately 2 h under these ubiquitin-depletion conditions. Furthermore, surface residency, normally of short duration in wild-type cells (terminated by endocytosis to the vacuole), was found to be prolonged; the majority of the receptor protein remained surface localized fully 2 h after biosynthesis. Thus, the rates of a-factor receptor endocytosis and consequent vacuolar turnover depend on the available level of ubiquitin in the cell. In cells mutant for two E2 activities, i.e., ubc4 delta ubc5 delta cells, the receptor was found to be substantially less ubiquitinated, and in addition, receptor turnover was slowed, suggesting that Ubc4p and Ubc5p may play a role in the recognition of the receptor protein as substrate for the ubiquitin system. In addition to ligand-independent uptake, the a-factor receptor also undergoes a ligand-dependent form of endocytosis (Davis, N.G., J.L. Horecka, and G.F. Sprague, Jr. 1993. J. Cell. Biol. 122:53-65). Concurrent with ligand-dependent uptake, we now show that the receptor undergoes ligand-induced ubiquitination, suggesting that receptor ubiquitination may function in the ligand-dependent endocytosis of the a-factor receptor as well as in its constitutive endocytosis. To account for these findings, we propose a model wherein the covalent attachment of ubiquitin to surface receptor triggers endocytic uptake.

Clathrin facilitates the internalization of seven transmembrane segment receptors for mating pheromones in yeast.

The role of clathrin in endocytosis of the yeast phermone receptors was examined using strains expressing a temperature-sensitive clathrin heavy chain. The yeast phermone receptors belong to the family of seven transmembrane segment, G-protein-coupled receptors. A rapid and reversible defect in uptake of radiolabeled alpha-factor pheromone occurred when the cells were transferred to the nonpermissive temperature. Constitutive, pheromone-independent internalization of newly synthesized a-factor phermone receptor was also rapidly inhibited in mutant strains at the nonpermissive temperature. In both cases residual endocytosis, 30-50% of wild-type levels, was detected in the absence of functional clathrin heavy chain. Once internalized, the a-factor receptor was delivered to the vacuole at comparable rates in chc1-ts and wild-type cells at the nonpermissive temperature. Clathrin heavy chain was also required for maximal uptake of a mutant a-factor receptor which is dependent on pheromone for internalization. In the presence of a-factor, the internalization rate of the mutant receptor in chc1-ts cells at the nonpermissive temperature was 2.5 times slower than the rate observed for endocytosis of the mutant receptor in wild-type cells. These experiments provide in vivo evidence that clathrin plays an important role in the endocytosis of the seven trans-membrane segment pheromone receptors in yeast.

Mutations that alter the third cytoplasmic loop of the a-factor receptor lead to a constitutive and hypersensitive phenotype.

The STE3 gene of Saccharomyces cerevisiae encodes a G protein-coupled receptor that is specific for the mating pheromone a-factor. The ste3L194Q mutation, which leads to the substitution of glutamine for leucine-194 within the third cytoplasmic loop of the receptor, resulted in a 20-fold increase in pheromone sensitivity and also caused partial constitutive activation of the response pathway. Moreover, other amino acid substitutions at the 194 position and several deletion mutations that collectively remove most of the third cytoplasmic loop resulted in hyperactive receptors. Therefore, we suggest that one role of the third cytoplasmic loop is to function as a negative regulatory domain involved in the maintenance of a nonsignaling state of the receptor. The constitutive activity and the pheromone hypersensitivity of ste3L194Q cells were recessive, suggesting that the wild-type receptor can antagonize the signal associated with the activated receptor. The ste3 delta 306 mutation, which results in truncation of most of the C-terminal domain of the receptor, led to a 20-fold increase in pheromone sensitivity, indicating that this domain also mediates negative regulation of the receptor. The ste3L194Q and ste3 delta 306 mutations appear to affect receptor activity independently, because the double mutant was associated with a 400-fold increase in pheromone sensitivity.

Cis- and trans-acting functions required for endocytosis of the yeast pheromone receptors.

The Saccharomyces cerevisiae a-factor receptor (STE3) is subject to two modes of endocytosis: a constitutive process that occurs in the absence of ligand and a regulated process that is triggered by binding of ligand. Both processes result in delivery of the receptor to the vacuole for degradation. Receptor mutants deleted for part of the COOH-terminal cytoplasmic domain are disabled for constitutive, but not ligand-dependent internalization. Trans-acting mutants that impair constitutive endocytosis have been isolated. One of these, ren1-1, is blocked at a late step in the endocytic pathway, as receptor accumulates in a prevacuolar endosome-like compartment. REN1 is identical to VPS2, a gene required for delivery of newly synthesized vacuolar enzymes to the vacuole. Based on this identity, we suggest a model in which the transport pathways to the vacuole--the endocytic pathway and the vacuolar biogenesis pathway--merge at an intermediate endocytic compartment. As receptor also accumulates at the surface of ren1 cells, receptor may recycle from the putative endosome to the surface, or REN1 may also be required to carry out an early step in endocytosis.

A hybrid toxin from bacteriophage f1 attachment protein and colicin E3 has altered cell receptor specificity.

A hybrid protein was constructed in vitro which consists of the first 372 amino acids of the attachment (gene III) protein of filamentous bacteriophage f1 fused, in frame, to the carboxy-terminal catalytic domain of colicin E3. The hybrid toxin killed cells that had the F-pilus receptor for phage f1 but not F- cells. The activity of the hybrid protein was not dependent upon the presence of the colicin E3 receptor, BtuB protein. The killing activity was colicin E3 specific, since F+ cells expressing the colicin E3 immunity gene were not killed. Entry of the hybrid toxin was also shown to depend on the products of tolA, tolQ, and tolR which are required both for phage f1 infection and for entry of E colicins. TolB protein, which is required for killing by colicin E3, but not for infection by phage f1, was also found to be necessary for the killing activity of the hybrid toxin. The gene III protein-colicin E3 hybrid was released from producing cells into the culture medium, although the colicin E3 lysis protein was not present in those cells. The secretion was shown to depend on the 18-amino-acid-long gene III protein signal sequence. Deletion of amino acids 3 to 18 of the gene III moiety of the hybrid protein resulted in active toxin, which remained inside producing cells unless it was mechanically released.

Direct characterization of influenza viral NS1 mRNA and related sequences from infected HeLa cells and a cell-free transcription system.

The NS1 mRNA of the influenza A virus WSN (H0N1) strain was isolated from a cell-free transcription system, and from the cytoplasm of virus-infected HeLa cells. The 32P-labeled NS1 mRNA derived from the infected cell cytoplasm was characterized by the secondary enzymatic analysis of sixteen of its large or distinct RNAase T1-resistant oligonucleotides. Several WSN strain-specific nucleotide differences from the previously-determined sequence of NS1 mRNA from the PR8 (H0N1) strain of influenza A virus, were located within these sequences. The RNAase T1-resistant oligonucleotides were placed within the primary sequence of NS1 mRNA, using the PR8 strain sequence data. The resulting linear map was then used to identify NS2 mRNA isolated from the infected cell cytoplasm, and an NS-related RNA species generated from NS1 mRNA incubated in a HeLa cell-free extract.

The fusion-related hydrophobic domain of Sendai F protein can be moved through the cytoplasmic membrane of Escherichia coli.

Recent work on a prokaryotic membrane protein, gene III protein (pIII) of coliphage f1, showed that polypeptide segments of sufficient hydrophobicity functioned to stop transfer of the polypeptide across the cell membrane: strings of 16 or more hydrophobic amino acids sufficed. A fusion-related hydrophobic domain (FRHD) of Sendai F protein, a sequence of 26 consecutive uncharged residues, has been implicated in the fusion of the viral membrane envelope and the target-cell membrane through a hydrophobic interaction. As it is located on the exterior of the viral membrane, this sequence must be transferred across the host-cell membrane during synthesis. We have inserted either the FRHD or the F protein membrane anchor (the COOH-terminal region of the F protein) into an internal site of a secreted pIII, which lacks its natural membrane anchor. These two hydrophobic sequences behave in the bacteria just as they do in their natural eukaryotic cell host. The F protein membrane anchor functions to stop transfer, conferring a membrane-spanning topology to the F-pIII hybrid protein; however, the FRHD is moved through the cytoplasmic membrane and derivatives carrying this sequence are secreted to the periplasm. We discuss how the FRHD is compatible with passage through the membrane and yet is still able to mediate membrane fusion through a presumed hydrophobic interaction.

Subsequent pregnancy following the birth of an extremely low birth-weight infant.

Of 103 mothers who delivered an extremely low birth-weight (ELBW, less than 1,000g) infant, 29% were primiparous; 51% of those who were multiparous had at least one previous miscarriage or perinatal death. The 41 (40%) mothers who decided against subsequent pregnancy were significantly older than the remaining mothers. Mothers were also significantly more likely to decide against subsequent pregnancy if their ELBW infant had survived. The outcome of subsequent pregnancies within 3 years of the ELBW birth was ascertained; 28% ended in miscarriage, 3% in stillbirth, 1% in neonatal death, 21% in a surviving preterm infant and 51% in a survivor born at term. Mothers diagnosed to have cervical incompetence had a significantly higher risk of a subsequent preterm birth. During the study period, 87% of mothers who became pregnant subsequent to their ELBW infant gave birth to at least one surviving child. Of the subsequent livebirths, 36% were less than 2,500g, 11% were less than 1,500g and 5% were less than 1,000g. Significantly more mothers whose ELBW infant had died conceived again within 1 year compared to those whose ELBW infant had survived. The necessary time for recovery from bereavement may be cut short by the subsequent pregnancy. The psychological problems as a result of unresolved mourning which mothers experience and their effects on subsequent children need to be further studied.

An artificial anchor domain: hydrophobicity suffices to stop transfer.

A hydrophobic sequence of 23 contiguous, uncharged residues anchors the coliphage f1 gene III protein (pIII) to the Escherichia coli cytoplasmic membrane; mutations removing this domain allow secretion of the protein to the periplasm. Multiple copies of an oligonucleotide encoding the hydrophobic repeat, Leu-Ala-Leu-Val, were introduced into genes for secreted forms of pIII. Artificial domains of 16 or more hydrophobic residues function to anchor the protein. Pronase protection experiments demonstrate that the new sequences act to halt transfer of the protein across the membrane, thus specifying a transmembrane topology. Relocating the hydrophobic domain within the polypeptide chain predictably alters the resultant protein/membrane topology. Repeats of a polar sequence were inserted with no effect on secretion. Furthermore, an unrelated hydrophobic sequence, uncovered by a gene III frameshift mutation, acts to anchor the protein. We conclude that function simply reflects hydrophobicity and not some more subtle feature of structure or sequence.

Fine structure of a membrane anchor domain.

We describe a detailed deletion analysis of the anchoring domain of a model membrane protein. Removal of the 23 contiguous uncharged amino acids from the carboxy terminus of the bacteriophage fl gene III protein (pIII) converts it from an integral membrane protein to a secreted periplasmic form. Deletions that remove six or fewer residues of the hydrophobic core result in no diminution of the protein's capacity to anchor in the membrane. Longer deletions into this hydrophobic domain gradually destablize the protein-membrane association. pIII derivatives with over half of the hydrophobic core deleted retain substantial residual anchor function. The basic residues, arginine and lysine, which provide a carboxy-terminal boundary for this domain, can be deleted without loss of anchoring capacity.

Messenger RNA processing and nuclear structure: isolation of nuclear ribonucleoprotein particles containing beta-globin messenger RNA precursors.

To explore the relationships between transcription, messenger RNA (mRNA) processing, and nuclear structure, ribonucleoprotein particles containing heterogeneous nuclear RNA (hnRNP) have been purified from globin-producing mouse Friend erythroleukemia cells. These nuclear hnRNP particles sediment at 50S-200S and contain, in addition to high molecular weight hnRNA, a specific set of nuclear proteins predominated by a major component of approximately 38,000 mol wt. The hnRNP particles are free of histones and ribosomal structural proteins, indicating their purification from the two other major nucleoprotein components of the nucleus: chromatin and nucleolar ribosomal precursor RNP particles. Th authenticity of the Friend cell hnRNP particles is demonstrated by the results of reconstruction experiments with deproteinized hnRNA, and by the resistance of the articles to dissociation during isopycnic banding in Cs2SO4 gradients without prior aldehyde fixation. Hybridization analysis with cloned mouse beta-globin DNA demonstrates that hnRNP particles from induced Friend cells contain newly synthesized transcripts of the beta-globin gene. Agarose gel electrophoresis of hnRNP particle-derived RNA denatured in glyoxal followed by "Northern" transfer to diazobenzyloxymethyl paper and hybridization with 32P-labeled cloned mouse beta-globin DNA reveals the presence in hnRNP of two size classes of beta-globin gene transcripts, the larger of which corresponds to the pre-spliced 15S beta-globin mRNA precursor previously identified in whole nuclear RNA, and the smaller of which corresponds to completely processed 9S beta-globin mRNA. These results establish, for the first time, that the nuclear transcripts of a specific, well-defined eukaryotic structural gene can be isolated in an RNP particle form, and that their RNP structure persists throughout mRNA splicing.