Showing papers in "The EMBO Journal in 1985"

Repetitive zinc‐binding domains in the protein transcription factor IIIA from Xenopus oocytes.

Abstract: The 7S particle of Xenopus laevis oocytes contains 5S RNA and a 40-K protein which is required for 5S RNA transcription in vitro. Proteolytic digestion of the protein in the particle yields periodic intermediates spaced at 3-K intervals and a limit digest containing 3-K fragments. The native particle is shown to contain 7-11 zinc atoms. These data suggest that the protein contains repetitive zinc-binding domains. Analysis of the amino acid sequence reveals nine tandem similar units, each consisting of approximately 30 residues and containing two invariant pairs of cysteines and histidines, the most common ligands for zinc. The linear arrangement of these repeated, independently folding domains, each centred on a zinc ion, comprises the major part of the protein. Such a structure explains how this small protein can bind to the long internal control region of the 5S RNA gene, and stay bound during the passage of an RNA polymerase molecule.

Neuronal origin of a cerebral amyloid: neurofibrillary tangles of Alzheimer's disease contain the same protein as the amyloid of plaque cores and blood vessels.

Abstract: The protein component of Alzheimer's disease amyloid [neurofibrillary tangles (NFT), amyloid plaque core and congophilic angiopathy] is an aggregated polypeptide with a subunit mass of 4 kd (the A4 monomer). Based on the degree of N-terminal heterogeneity, the amyloid is first deposited in the neuron, and later in the extracellular space. Using antisera raised against synthetic peptides, we show that the N terminus of A4 (residues 1-11) contains an epitope for neurofibrillary tangles, and the inner region of the molecule (residues 11-23) contains an epitope for plaque cores and vascular amyloid. The non-protein component of the amyloid (aluminum silicate) may form the basis for the deposition or amplification (possible self-replication) of the aggregated amyloid protein. The amyloid of Alzheimer's disease is similar in subunit size, composition but not sequence to the scrapie-associated fibril and its constituent polypeptides. The sequence and composition of NFT are not homologous to those of any of the known components of normal neurofilaments.

Levels of nerve growth factor and its mRNA in the central nervous system of the rat correlate with cholinergic innervation.

Abstract: The levels of nerve growth factor (NGF) and its mRNA in the rat central nervous system were determined by two-site enzyme immunoassay and quantitative Northern blots, respectively. Relatively high NGF levels (0.4-1.4 ng NGF/g wet weight) were found both in the regions innervated by the magnocellular cholinergic neurons of the basal forebrain (hippocampus, olfactory bulb, neocortex) and in the regions containing the cell bodies of these neurons (septum, nucleus of the diagonal band of Broca, nucleus basalis of Meynert). Comparatively low, but significant NGF levels (0.07-0.21 ng NGF/g wet weight) were found in various other brain regions. mRNANGF was found in the hippocampus and cortex but not in the septum. This suggests that magnocellular cholinergic neurons of the basal forebrain are supplied with NGF via retrograde axonal transport from their fields of innervation. These results, taken together with those of previous studies showing that these neurons are responsive to NGF, support the concept that NGF acts as trophic factor for magnocellular cholinergic neurons.

The molecular basis of the specific anti-influenza action of amantadine.

Abstract: Amantadine (1-aminoadamantane hydrochloride) is effective in the prophylaxis and treatment of influenza A infections. In tissue culture this selective, strain-specific antiviral activity occurs at relatively low concentrations (5 microM or less), which inhibit either the initiation of infection or virus assembly. The data reported here demonstrate that the basis of these actions is similar and resides in the virus-coded M2 membrane protein, the product of a spliced transcript of RNA segment 7. Mutations which confer resistance to amantadine are restricted to four amino acids within a hydrophobic sequence, indicating that the drug is targetted against the putative membrane-associated portion of the molecule. The influence of the virus haemagglutinin on the amantadine sensitivity of virus strains implies that the drug may interfere with interactions between these two virus proteins.

Primary structure of human fibronectin: differential splicing may generate at least 10 polypeptides from a single gene.

Abstract: Cellular and plasma fibronectins are heterodimers consisting of similar but not identical polypeptides. The differences between fibronectin subunits are due in part to the variability of internal primary sequences. This results from alternative splicing in at least two regions (ED and IIICS) of the pre-mRNA. The complete primary structure of human fibronectin, including most of the internal variations, has been determined by sequencing a series of overlapping cDNA clones. In total, they covered 7692 nucleotides and represented the mRNA sequence coding from the amino terminus of the mature protein to the poly(A) tail. The deduced amino acid sequence of fibronectin has been analysed in terms of the arrangement of internal homologies and the different binding domains.

Cultivation in a semi-defined medium of animal infective forms of Trypanosoma brucei, T. equiperdum, T. evansi, T. rhodesiense and T. gambiense.

Abstract: A semi-defined medium for the cultivation of bloodstream forms of the African trypanosome brucei subgroup was developed. Out of 14 different strains tested, 10 could be cultured including Trypanosoma brucei, T. equiperdum, T. evansi, T. rhodesiense and T. gambiense. The presence of a reducing agent (2-mercaptoethanol or thioglycerol) was found to be essential for growth. The standard medium consisted of Hepes buffered minimum essential medium with Earle's salts supplemented with 0.2 mM 2-mercaptoethanol, 2 mM pyruvate and 10% inactivated serum either from rabbit (T. brucei, T. equiperdum, T. evansi and T. rhodesiense) or human (T. gambiense). Although a general medium could be defined for the long-term maintenance of trypanosome cultures, the initiation to culture nevertheless required particular conditions for the different strains. The cultured trypanosomes had all the characteristics of the in vivo bloodstream forms including: morphology, infectivity, antigenic variation and glucose metabolism.

The common 90-kd protein component of non-transformed '8S' steroid receptors is a heat-shock protein.

Abstract: Non-transformed steroid receptors have an approximately 8S sedimentation coefficient that corresponds to an oligomeric structure of 250-300 kd which includes a non-hormone binding 90-kd protein. A monoclonal antibody BF4 raised against the purified, molybdate-stabilized, 8S progesterone receptor (8S-PR) from chick oviduct, recognizes 8S forms of all steroid hormone receptors. BF4 was found specific for a 90-kd protein present in great abundance in all chicken tissues, including that present in 8S-forms of steroid receptors. Here, using immunological and biochemical techniques, we demonstrate that this ubiquitous BF4-positive 90-kd protein is in fact the chicken 90 kd heat-shock protein (hsp 90): it increased in heat-shocked chick embryo fibroblasts, and displayed identical migration in two-dimensional gel electrophoresis and the same V8 peptide map as the already described hsp 90. We discuss the possibility that the interaction between hsp 90 and steroid hormone-binding subunits may play a role in keeping the receptor in an inactive form.

Identification of a set of genes expressed during the G0/G1 transition of cultured mouse cells.

Abstract: To identify previously undetected genes that may be involved in the transition from a resting state (G0) to a proliferative state (G1) of mammalian cells, we set out to isolate cDNA clones derived from mRNAs that appear in serum-stimulated cells in the absence of protein synthesis A lambda cDNA library was prepared using poly(A)+ RNA from BALB/c 3T3 cells that had been brought to quiescence and subsequently stimulated with serum in the presence of cycloheximide Approximately 50 000 recombinant phage plaques were screened, and 357 clones were isolated that hybridized to probes derived from stimulated-cell RNA but not to probes from resting-cell RNA Cross hybridization analysis showed that four RNA sequence families account for approximately 90% of these clones One of the clones hybridized to an actin probe; none hybridized to any of 13 oncogene probes tested Five different RNAs that appear to be previously uncharacterized have been further analyzed These RNAs accumulate and decay rapidly following stimulation by serum or purified growth factors, or by a tumor promoter, and they are superinduced by serum in the presence of cycloheximide Three of the RNAs could be enriched by hybridization to cDNAs and translated in vitro, yielding proteins of approximately 43, 40 and 35 kd, respectively

High level expression of introduced chimaeric genes in regenerated transformed plants

Abstract: Promoter DNA sequences from a petunia chlorophyll a/b binding protein gene were fused to octopine synthase DNA sequences and the resulting chimaeric genes were introduced into petunia and tobacco cells. Populations of transformed regenerated petunia plants containing the chimaeric genes were examined so that the expression of any particular construction could be compared between independent transformants. Substantial variation was observed between transformants in the level of chimaeric gene expression. In general, transcriptional fusions in which a linker sequence interrupted the 5'-untranslated region gave rise to less chimaeric mRNA accumulation than a translational fusion. In the most actively expressing transformants the amount of mRNA from the introduced chimaeric genes was half that of the endogenous wild-type gene. Transcription initiated at the same place in the chimaeric and endogenous genes. Construction of the translational cab/ocs fusion caused three amino acid changes in the octopine synthase protein and functional octopine synthase enzyme was absent from plants in which mRNA for the chimaeric gene was abundantly expressed.

New cloning vehicles for transformation of higher plants.

Abstract: We have constructed a set of small vectors based on the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens which allow the transfer of exogenous DNA into plant chromosomes. These vectors contain: (i) a chimeric gene containing the transcriptional control signals from the nopaline synthase gene and the coding sequence for neomycin phosphotransferase; (ii) the ColE1 replicon; (iii) the cos site of bacteriophage λ; (iv) the border sequences from the ends of the T-DNA region of the Ti plasmid; and (v) a wide host range replicon. Due to the small size of these cosmid vectors, DNA fragments up to 35 kbp can be inserted by an in vitro packaging method in Escherichia coli. The ability of these vectors to be stably replicated in both E. coli and A. tumefaciens allows their subsequent transfer to and maintenance in Agrobacterium without intermediate genetic manipulations. We demonstrate that DNA cloned into these vectors in A. tumefaciens can efficiently transform plants when in trans with a wild-type Ti plasmid which donates the functions necessary for DNA transfer and integration. We also show that only the right border of the T-DNA is necessary for DNA transformation.

Transformation of Aspergillus niger by the amdS gene of Aspergillus nidulans.

Abstract: Aspergillus niger grows poorly on acetamide as a nitrogen or carbon source and lacks sequences detectably homologous to the amdS gene encoding the acetamidase of Aspergillus nidulans. We have taken advantage of these observations to develop a transformation system for A. niger using the amdS gene as a dominant heterologous marker for selecting transformants on the basis of acetamide utilization. Transformants varied in their ability to grow on amide media and the number of integrated copies of the amdS plasmid ranged from 1 or 2 to greater than 100. Southern analysis of transformants revealed that the multiple copies were integrated into the chromosome in tandem arrays. This result indicates that transformation of A. niger is more similar to mammalian cells than to yeast. Analysis of enzyme activity levels and RNA levels showed that most of the copies of amdS were expressed. Mitotic stabilities of transformants were found to be high. A transformant containing greater than 100 copies of the amdS gene was impaired in omega-amino acid utilization, a result that has also been found in A. nidulans. Since, in A. nidulans, omega-amino acids induce acetamidase via a characterizied regulatory gene (amdR/intA) this observation implies that titration of an analogous A. niger regulatory gene product by multiple amdS copies has occurred. Additional evidence suggested that the amdS gene is regulated in A. niger. It has also been shown that an unselected plasmid can be co-transformed with the amdS plasmid into A. niger.

A Tn3 lacZ transposon for the random generation of beta-galactosidase gene fusions: application to the analysis of gene expression in Agrobacterium.

Abstract: The construction and use of a Tn3-lac transposon, Tn3-HoHo1, is described. Tn3-HoHo1 can serve as a transposon mutagen and provides a new and useful system for the random generation of both transcriptional and translational lacZ gene fusions. In these fusions the production of beta-galactosidase, the lacZ gene product, is placed under the control of the gene into which Tn3-HoHo1 has inserted. The expression of the gene can thus be analyzed by monitoring beta-galactosidase activity. Tn3-HoHo1 carries a non-functional transposase gene; consequently, it can transpose only if transposase activity is supplied in trans, and is stable in the absence of this activity. A system for the insertion of Tn3-HoHo1 into sequences specifically contained within plasmids is described. The applicability of Tn3-HoHo1 was demonstrated studying three functional regions of the Agrobacterium tumefaciens A6 Ti plasmid. These regions code for octopine catabolism, virulence and plant tumor phenotype. The regulated expression of genes contained within each of these regions was analyzed in Agrobacterium employing Tn3-HoHo1 generated lac fusions.

Complete amino acid sequence of human vitronectin deduced from cDNA. Similarity of cell attachment sites in vitronectin and fibronectin.

Abstract: cDNA clones for vitronectin, a cell adhesion-promoting plasma and tissue protein, were isolated from a lambda gt11 library containing cDNA inserts made from human liver mRNA. The library was screened with anti-vitronectin antibodies and the positive clones were further identified with synthetic oligonucleotide probes deduced from the partial amino acid sequence of vitronectin. Nucleotide sequence analysis showed that the largest insert was 1545 bp long and contained the whole sequence corresponding to plasma vitronectin. It showed that vitronectin contains the entire 44-amino acid somatomedin B peptide at its NH2 terminus and, near its COOH terminus, a 34-amino acid glycosaminoglycan binding site in which half of the amino acids are basic residues. Three potential carbohydrate attachment sites are present in the sequence. An Arg-Gly-Asp sequence, which has previously been shown to be the cell attachment site in fibronectin, was found in vitronectin immediately after the NH2-terminal somatomedin B sequence. No other homologies with fibronectin were found. The Arg-Gly-Asp sequence appears to constitute the cell attachment site of vitronectin, since it is in the region where we have previously localized the cell attachment site, its presence correlate with cell attachment activity among the insert-coded polypeptides, and because previous results have shown that synthetic peptides containing the Arg-Gly-Asp sequence inhibit the cell attachment function of vitronectin. The discovery of an Arg-Gly-Asp cell attachment site in a protein with a known cell attachment function emphasizes the general importance of this sequence in cell recognition.

Erythroid-specific expression of human beta-globin genes in transgenic mice.

Abstract: Transgenic mice carrying human beta-globin genes were produced by microinjecting linear DNA molecules containing cloned beta-globin genes with up to 4300 bp of 5'-flanking sequence and 1700 bp of 3'-flanking sequence. Most (15 of 20) of these transgenic mice expressed the human beta-globin genes in blood cells and the level of expression in some mice was comparable with that obtained from endogenous beta-globin genes. Human beta-globin gene expression appeared to be restricted to cells of the erythroid lineage and was first detected between 11 and 14 days of development, in parallel with mouse beta-globin. Constructs with as little as 48 bp of 5'-flanking sequence also appeared to be expressed appropriately. The mRNA transcripts had correct 5' ends and directed human beta-globin synthesis in reticulocyte lysates. Human beta-globin protein was detectable in mature erythrocytes from progeny of one of these mice. The frequency and extent of expression was severely depressed when the procaryotic vector DNA was not removed prior to microinjection.

Structural details of the binding of guanosine diphosphate to elongation factor Tu from E. coli as studied by X-ray crystallography.

Abstract: Structural details of the guanosine diphosphate binding to a modified form of elongation factor Tu from Escherichia coli, resulting from X-ray crystallographic studies, are reported. The protein elements that take part in the nucleotide binding are located in four loops connecting beta-strands with alpha-helices. These loops correspond to regions in primary sequences which show a high degree of homology when compared with other prokaryotic and eukaryotic elongation factors and initiation factor 2.

Activation of the pp60c-src kinase by middle T antigen binding or by dephosphorylation.

Abstract: The transforming protein of polyoma virus, middle T antigen, associates with the protein tyrosine kinase pp60c-src, and analysis of mutants of middle T suggests that this complex plays an important role in transformation by polyoma. It has recently been reported that pp60c-src from polyoma virus-transformed cells has enhanced tyrosine kinase activity in vitro. The data presented here confirm these findings and show that the enhanced kinase activity of pp60c-src is due to an increase in the Vmax of the enzyme. Sucrose density gradient analysis demonstrates that only the form of pp60c-src which is bound to middle T antigen is activated. The difference in enzyme activity between pp60c-src from normal and middle T-transformed cells is more marked when the enzyme is prepared from lysates containing the phosphotyrosine protein phosphatase inhibitor, sodium orthovanadate. pp60c-src from middle T transformed cells is unaffected, but pp60c-src from normal cells has reduced kinase activity if dephosphorylation is prevented. The kinase activity of pp60c-src thus appears to be regulated by its degree of phosphorylation at tyrosine, and data are presented which support this hypothesis. pp60c-src is the first example of a protein tyrosine kinase whose activity is inhibited by phosphorylation at tyrosine. Middle T antigen may increase the kinase activity of pp60c-src by preventing phosphorylation at this regulatory site.

The human ubiquitin multigene family: some genes contain multiple directly repeated ubiquitin coding sequences.

Abstract: Ubiquitin coding sequences were isolated from a human genomic library and two cDNA libraries. One human ubiquitin gene consists of 2055 nucleotides and codes for a polyprotein consisting of 685 amino acid residues. The polyprotein contains nine direct repeats of the ubiquitin amino acid sequence and the last ubiquitin sequence is extended with an additional valyl residue at the C-terminal end. No spacer sequences separate the ubiquitin repeats and the coding regions are not interrupted by intervening sequences. This particular gene is transcribed since cDNAs corresponding to the genomic sequence have been isolated. At least two more types of ubiquitin genes are encoded in the human genome, one coding for an ubiquitin monomer while another presumably codes for three or four direct repeats of the ubiquitin sequence. Human DNA contains many copies of the ubiquitin sequence. Ubiquitin is therefore encoded in the human genome as a multigene family.

The uncoupling protein from brown fat mitochondria is related to the mitochondrial ADP/ATP carrier. Analysis of sequence homologies and of folding of the protein in the membrane.

Abstract: We report here, for the first time, the primary structure of uncoupling protein as established by amino acid sequencing. Like the ADP/ATP carrier, this protein has a tripartite structure comprising three similar sequences of approximately 100 residues each. These six 'repeats' exhibit striking conservation of several residues, in particular glycine and proline, at possible structurally strategic positions. Although the two proteins differ strongly in their amino acid composition, their sequences are distantly homologous. Three membrane-spanning alpha-helices can be deduced from hydropathy plots. A modified plot accounting for amphiphilic helices indicates 5-6 such alpha-segments. In addition an amphiphilic beta-strand of membrane-spanning length can be discerned. The tripartite sequence structure is also distinctly reflected in the hydropathy distribution. Based on the membrane disposition of the segments of the ADP/ATP carrier, a model for the transmembrane folding path of the polypeptide chain of the uncoupling protein is proposed.

Immobilization and purification of enzymes with staphylococcal protein A gene fusion vectors

Abstract: Two improved plasmid vectors, containing the gene coding for staphylococcal protein A and adapted for gene fusions, have been constructed. These vectors allow fusion of any gene to the protein A moiety, giving fusion proteins which can be purified, in a one-step procedure by IgG affinity chromatography. One vector, pRIT2, is designed for temperature-inducible expression of intracellular fusion proteins in Escherichia coli and the other pRIT5, is a shuttle vector designed for secretion. The latter gives a periplasmatic fusion protein in E. coli and an extracellular protein in Gram-positive hosts such as Staphylococcus aureus. The usefulness of these vectors is exemplified by fusion of the protein A gene and the E. coli genes encoding the enzymes beta-galactosidase and alkaline phosphatase. High amounts of intact fusion protein are produced which can be immobilized on IgG-Sepharose in high yield (95-100%) without loss of enzymatic activity. Efficient secretion in both E. coli and S. aureus, was obtained for the alkaline phosphatase hybrid, in contrast to beta-galactosidase which was only expressed efficiently using the intracellular system. More than 80% of the protein A alkaline-phosphatase hybrid protein can be eluted from IgG affinity columns without loss of enzymatic activity.

Involvement of ATP in the nuclear and nucleolar functions of the 70 kd heat shock protein.

Abstract: The major heat shock protein, hsp70, is an ATP-binding protein which is synthesized in very large amounts in response to stress. In unstressed, or recovered, mammalian cells it is found in both nucleus and cytoplasm. Under these conditions, its interaction with nuclei is weak, and it is readily released from them upon lysis of cells in isotonic buffer. After heat shock, hsp70 binds tightly first to some nuclear component(s) and then to nucleoli. It can be released from these binding sites rapidly and specifically in vitro by as little as 1 microM ATP, but not by non-hydrolysable ATP analogues. Studies of hsp70 deletion mutations show that the ability of mutants to be released by ATP correlates with their ability to migrate to heat-shocked nucleoli and aid their repair in vivo. We propose a model in which ATP-driven cycles of binding and release of hsp70 help to solubilize aggregates of proteins or RNPs that form after heat shock. Cells also contain proteins related to hsp70 that are synthesized in the absence of stress. The most abundant of these shows the same behaviour as hsp70 after heat shock, and thus may perform a related function in both normal and stressed cells.

Epidermal growth factor and oncogenes induce transcription of the same cellular mRNA in rat fibroblasts.

Abstract: We have isolated and sequenced a cloned cDNA corresponding to an mRNA present in significantly higher levels in rat cells transformed by polyoma virus, Rous sarcoma virus, and the cellular oncogene H-ras than in the normal parental cell lines The mRNA transcript is also rapidly induced by the polypeptide growth factor epidermal growth factor, providing a new link between oncogenes and growth factors Both the growth factor and the oncogenes control expression of the corresponding gene at the transcriptional level Our results point to the existence of intracellular mechanisms that are common to the action of both growth factors and oncogenes

Mitochondrial protein synthesis is required for maintenance of intact mitochondrial genomes in Saccharomyces cerevisiae.

Abstract: The genes of Saccharomyces cerevisiae coding for the mitochondrial threonine and tryptophan tRNA synthetases and for a putative mitochondrial ribosomal protein have been cloned. These, and the previously cloned gene for a mitochondrial elongation factor, were used to disrupt or partially delete the wild-type chromosomal copies of the genes in the respiratory-competent strain W303. In each case, inactivation of a gene whose product is required for mitochondrial protein synthesis causes an instability in mitochondrial DNA. Although intact mitochondrial genomes are rapidly and quantitatively eliminated in the protein synthesis defective strains, specific rho- genomes can be maintained stably over many generations. These results indicate that mitochondrial protein synthesis is required for the propagation of wild-type mitochondrial DNA in yeast.

DO beta: a new beta chain gene in HLA-D with a distinct regulation of expression.

Abstract: The HLA-D region of the human major histocompatibility complex encodes the genes for the alpha and beta chains of the DP, DQ and DR class II antigens. A cDNA clone encoding a new class II beta chain (designated DO) was isolated from a library constructed from mRNA of a mutant B-cell line having a single HLA haplotype. Complete cDNA clones encoding the four isotypic beta chains of the DR1, DQw1, DPw2 and putative DO antigens were sequenced. The DO beta gene was mapped in the D region by hybridization with DNA of HLA-deletion mutants. DO beta mRNA expression is low in B-cell lines but remains in mutant lines which have lost expression of other class II genes. Unlike other class II genes DO beta is not induced by gamma-interferon in fibroblast lines. The DO beta gene is distinct from the DP beta, DQ beta and DR beta genes in its pattern of nucleotide divergence. The independent evolution and expression of DO beta suggest that it may be part of a functionally distinct class II molecule.

Laminin-immunoreactive glia distinguish regenerative adult CNS systems from non-regenerative ones.

Abstract: Most regions of the adult mammalian central nervous system (CNS) do not support axonal growth and regeneration. Laminin, expressed by cultured astrocytes and known to promote neurite outgrowth of cultured neurons, is normally present in brain basement membranes, and only transiently induced in adult brain astrocytes by injury. Here I provide three lines of evidence which suggest that the continued expression of laminin by astrocytes may be a prerequisite for axonal growth and regeneration in adult CNS. Firstly, laminin is continuously present in astrocytes of adult rat olfactory bulb apparently in close association with the olfactory nerve axons. Secondly, laminin is continuously expressed by astrocytes in adult frog brain, and sectioning of the optic tract further increases laminin immunoreactivity in astrocytes of the optic tectum during the period of axonal regeneration. Lastly, laminin appears normally in astrocytes of the frog and goldfish optic nerves which regenerate, but not in astrocytes of the rat or chick optic nerves which do not regenerate. The selective association of laminin with axons that undergo growth and regeneration in vivo is consistent with the possibility that astrocytic laminin provides these central nervous systems with their regenerative potential.

Rapid and reversible translocation of the catalytic subunit of cAMP-dependent protein kinase type II from the Golgi complex to the nucleus.

Abstract: In unstimulated interphase bovine epithelial (MDBK) cells, both regulatory (R II) and catalytic (C) subunits of the type II enzyme of cAMP-dependent protein kinase (cAMP-dPK II) are associated with the Golgi complex. However, as demonstrated by indirect immunofluorescence microscopy, within 5 min after stimulation of adenylate cyclase by forskolin, the C subunit dissociates from the Golgi-associated R II and becomes diffusely distributed. With increasing time of forskolin treatment, C subunits accumulate in the nucleus, while R II subunits remain associated with the Golgi complex. The effect of forskolin is rapidly reversible in that C subunits begin to reassociate with the Golgi complex within a few minutes after drug removal. C subunit translocations similar to those produced by forskolin also occur after treatment of MDBK cells with dibutyryl-cAMP, confirming that the observed effects are most likely mediated by elevation of intracellular cAMP levels. These results suggest that nuclear translocation of activated protein kinase subunits may represent an important link between hormonal stimuli and physiological responses.

Nucleotide sequences of STE2 and STE3, cell type-specific sterile genes from Saccharomyces cerevisiae

Abstract: The nucleotide sequences of STE2 and STE3, cell type-specific sterile genes of Saccharomyces cerevisiae, were determined; major open reading frames encode 431 and 470 amino acids, respectively. STE2 and STE3 proteins seem to be folded in a similar fashion and are likely to be membrane-bound. Both consist of seven hydrophobic segments in each NH2-terminal region with a long hydrophilic domain in each COOH-terminal region. However, the two putative gene products do not exhibit extensive sequence homology. The STE2 protein has no obvious hydrophobic signal peptide; the NH2 terminus of the STE3 protein might serve as a signal peptide. The STE2 transcript, 1.7 kb, was detected in MATa strains but not in MATα strains, while the STE3 transcript, also 1.7 kb, was detected only in MATα cells. In STE2, two canonical TATA sequences are located 18 and 27 bp upstream of the mRNA start site, which has been mapped 32 bp before the initiator ATG codon, while STE3 contains a similar sequence (TATAGA), which is preceded by a long AT sequence, 140 bp upstream of the initiator ATG codon. Transcription of STE2 in a cells seems to be enhanced by exogenous α-factor.

Transcription occurs at a nucleoskeleton

Abstract: Native chromatin aggregates under isotonic conditions so it is generally studied using higher or lower salt concentrations. This has led to different interpretations of how transcription might occur. Studies using hypertonically-isolated preparations suggest that DNA functions in close association with a skeletal nuclear substructure, the matrix or cage, but such a structure is not usually seen under hypotonic conditions (e.g., in 'Miller-spreads'). Using a novel method for preparing chromatin under isotonic conditions we have investigated the site of transcription. We find that all three constituents of the transcription complex, nascent transcripts, active RNA polymerase and genes being transcribed are all closely associated with some structure too large to be electroeluted from the nucleus. Hypotonic treatment partly disrupts this association. We suggest a model for transcription that involves the participation of a nucleoskeleton at the active site and reconcile the contradictory results obtained using different salt concentrations.

The use of nuclear-encoded sequences to direct the light-regulated synthesis and transport of a foreign protein into plant chloroplasts.

Abstract: The light-inducible nuclear gene coding for the small subunit of ribulose-1,5-bisphosphate carboxylase (Rubisco), produces a precursor protein with an amino-terminal transit peptide which is transported into the plastids and cleaved by a specific proteinase To test whether the promoter and transit peptide-coding sequences of the small subunit gene can be used to direct the light-inducible synthesis and transport of a foreign protein into chloroplasts, a chimaeric gene was constructed consisting of the promoter, first exon and intron as well as part of the second exon of the small subunit Rubisco gene fused to the amino-terminal end of the neomycin phosphotransferase II gene, (nptII) of Tn5 Tobacco tissue, as well as whole plants, into which this chimaeric gene was introduced, were resistant to kanamycin The transcription of the chimaeric gene as well as the NPTII activity of the resulting fusion protein were shown to be light inducible The fusion protein is processed and located within the chloroplasts of the transformed plants

The 70-kd mammalian heat shock proteins are structurally and functionally related to the uncoating protein that releases clathrin triskelia from coated vesicles.

Abstract: It is shown that in immunological, structural and functional terms the uncoating protein, which catalyses ATP-dependent dissociation of clathrin triskelia from clathrin-coated vesicles is intimately related to two major stress proteins of mammalian cells. These proteins of hitherto unknown functions have polypeptide mol. wts. of 73 kd and 72 kd, respectively. They are normal cell constituents which are synthesized in increased abundance under adverse environmental circumstances, such as non-physiological temperatures or treatment with amino acid analogues.

Metabolism of c-myc gene products: c-myc mRNA and protein expression in the cell cycle.

Abstract: The presence and synthesis of c-myc protein and mRNA in the cell cycle has been studied. We find that c-myc mRNA is present, at equivalent levels, at all times in the cell cycle with the possible exception of mitosis. Furthermore, we demonstrate that this mRNA is transcribed in both G1 and G2 phases. An analysis of the c-myc protein in vivo shows that de novo synthesis occurs in G1 and G2 and the protein turns over with a half-life of approximately 20-30 min in both phases. Furthermore, the level of c-myc protein rapidly increases in cell populations when they re-initiate the cell cycle, thereafter decreasing as the culture reaches quiescence. The results therefore suggest that expression of c-myc can be rapidly modulated and that it is activated during the G0 to G1 transition, but is expressed thereafter in the cell cycle.