Showing papers in "The EMBO Journal in 1987"

GUS fusions: beta‐glucuronidase as a sensitive and versatile gene fusion marker in higher plants.

Abstract: We have used the Escherichia coli beta-glucuronidase gene (GUS) as a gene fusion marker for analysis of gene expression in transformed plants. Higher plants tested lack intrinsic beta-glucuronidase activity, thus enhancing the sensitivity with which measurements can be made. We have constructed gene fusions using the cauliflower mosaic virus (CaMV) 35S promoter or the promoter from a gene encoding the small subunit of ribulose bisphosphate carboxylase (rbcS) to direct the expression of beta-glucuronidase in transformed plants. Expression of GUS can be measured accurately using fluorometric assays of very small amounts of transformed plant tissue. Plants expressing GUS are normal, healthy and fertile. GUS is very stable, and tissue extracts continue to show high levels of GUS activity after prolonged storage. Histochemical analysis has been used to demonstrate the localization of gene activity in cells and tissues of transformed plants.

Human proto-oncogene c-kit: a new cell surface receptor tyrosine kinase for an unidentified ligand.

Abstract: Structural features of v-kit, the oncogene of HZ4 feline sarcoma virus, suggested that this gene arose by transduction and truncation of cellular sequences. Complementary DNA cloning of the human proto-oncogene coding for a receptor tyrosine kinase confirmed this possibility: c-kit encodes a transmembrane glycoprotein that is structurally related to the receptor for macrophage growth factor (CSF-1) and the receptor for platelet-derived growth factor. The c-kit gene is widely expressed as a single, 5-kb transcript, and it is localized to human chromosome 4 and to mouse chromosome 5. A c-kit peptide antibody permitted the identification of a 145,000 dalton c-kit gene product that is inserted in the cellular plasma membrane and is capable of self-phosphorylation on tyrosine residues in both human glioblastoma cells and transfected mouse fibroblasts. Our results suggest that p145c-kit functions as a cell surface receptor for an as yet unidentified ligand. Furthermore, carboxy- and amino-terminal truncations that occurred during the viral transduction process are likely to have generated the transformation potential of v-kit.

Transforming growth factor beta modulates the expression of collagenase and metalloproteinase inhibitor.

Abstract: Exposure of quiescent MRC-5 human fibroblasts to growth factors such as epidermal growth factor, basic fibroblast growth factor or embryonal carcinoma-derived growth factor resulted in the induction of mRNA transcripts encoding the metalloproteinases collagenase and stromelysin and the specific metalloproteinase inhibitor TIMP, whilst expression of collagen and fibronectin was relatively unaffected Exposure of quiescent cells to growth factors in the presence of transforming growth factor beta (TGF-beta) resulted in inhibition of collagenase induction and a synergistic increase in TIMP expression TGF-beta alone did not significantly induce metalloproteinase or TIMP expression These effects on mRNA transcripts were reflected in increased secretion of TIMP protein and collagenase activity Nuclear run-off analysis of growth factor-induced transcription revealed that the TGF-beta modulation of TIMP and collagenase expression was due to transcriptional mechanisms The observations suggest that TGF-beta exerts a selective effect on extracellular matrix deposition by modulating the action of other growth factors on metalloproteinase and TIMP expression

Selection of AUG initiation codons differs in plants and animals.

Abstract: The influence of the nucleotide at position -3 relative to the AUG initiation codon on the initiation of protein synthesis was studied in two different in vitro translation systems using synthetic mRNAs. The four mRNAs, transcribed from cDNAs directed by an SP6 promoter, were identical except for mutations at nucleotide -3. In each case, translation of mRNAs produced a single protein of Mr = 12,600. Relative translational efficiencies showed a hierarchy in the reticulocyte lysate system (100, 85, 61 and 38% for A, G, U and C in position -3, respectively) but no differences in the wheat germ system. Differential mRNA degradation or polypeptide chain elongation were excluded as causes of the differences observed in translation in the reticulocyte lysate. mRNA competition increased the differences observed in translational efficiencies in reticulocyte lysate but showed no effect in wheat germ. Analysis of 61 plant and 209 animal mRNA sequences revealed qualitative and quantitative differences between the consensus sequences surrounding AUG initiation codons. Whereas the consensus sequence for animals was CACCAUG that for plants was AACAAUGGC. Both the structural and functional findings suggest that the factors which select AUG initiation codons in plants and animals differ significantly.

Two signals mediate hormone-dependent nuclear localization of the glucocorticoid receptor.

Abstract: We have detected nuclear localization signals within the 795 amino acid rat glucocorticoid receptor Using a transient expression assay, we monitored by immunofluorescence the subcellular distribution of receptor derivatives and beta-galactosidase-receptor fusion proteins Two distinct nuclear localization signals, NL1 and NL2, were defined NL1 maps to a 28 amino acid segment closely associated, but not coincident with the DNA binding domain; NL2 resides within a 256 amino acid region that also includes the hormone binding domain Most importantly, nuclear localization of fusion proteins containing either the full-length receptor or the NL2 region alone is fully hormone-dependent; similar results were obtained with the wild-type receptor, provided the analysis was performed in medium lacking serum and phenol red The rate of hormone-induced nuclear localization of an NL2-containing fusion protein is consistent with the rapid kinetics of hormone-regulated transcription mediated by the receptor Thus, hormonal control of nuclear localization contributes to the modulation of glucocorticoid receptor transcriptional regulatory activity

Engineering herbicide resistance in plants by expression of a detoxifying enzyme.

Abstract: Phosphinothricin (PPT) is a potent inhibitor of glutamine synthetase in plants and is used as a non-selective herbicide. The bar gene which confers resistance in Streptomyces hygroscopicus to bialaphos, a tripeptide containing PPT, encodes a phosphinothricin acetyltransferase (PAT) (see accompanying paper). The bar gene was placed under control of the 35S promoter of the cauliflower mosaic virus and transferred to plant cells using Agrobacterium-mediated transformation. PAT was used as a selectable marker in protoplast co-cultivation. The chimeric bar gene was expressed in tobacco, potato and tomato plants. Transgenic plants showed complete resistance towards high doses of the commercial formulations of phosphinothricin and bialaphos. These data present a successful approach to obtain herbicide-resistant plants by detoxification of the herbicide.

The regulatory c1 locus of Zea mays encodes a protein with homology to myb proto-oncogene products and with structural similarities to transcriptional activators.

Abstract: The structure of the wild-type c1 locus of Zea mays was determined by sequence analysis of one genomic and two cDNA clones. The coding region is composed of three exons (150 bp, 129 bp and one, at least 720 bp) and two small introns (88 bp and 145 bp). Transcription of the mRNAs corresponding to the two cDNA clones cLC6 (1.1 kb) and cLC28 (2.1 kb) starts from the same promoter. Both cDNAs are identical except that cLC28 extends further at its 3' end. A putative protein, 273 amino acids in length was deduced from the sequence of both transcripts. It contains two domains, one basic and the other acidic and might function as a transcriptional activator. The basic domain of this c1-encoded protein shows 40% sequence homology to the protein products of animal myb proto-oncogenes.

Distinct primary structures, ligand-binding properties and tissue-specific expression of four human muscarinic acetylcholine receptors.

Abstract: To investigate the molecular basis for the diversity in muscarinic cholinergic function, we have isolated the genes encoding the human M1 and M2 muscarinic receptors (mAChR) as well as two previously undiscovered mAChR subtypes, designated HM3 and HM4 The amino acid sequence of each subtype reflects a structure consisting of seven, highly conserved transmembrane segments and a large intracellular region unique to each subtype, which may constitute the ligand-binding and effector-coupling domains respectively Significant differences in affinity for muscarinic ligands were detected in individual mAChR subtypes produced by transfection of mammalian cells Each subtype exhibited multiple affinity states for agonists; differences among subtypes in the affinities and proportions of such sites suggest the capacity of mAChR subtypes to interact differentially with the cellular effector-coupling apparatus Subtype-specific mRNA expression was observed in the heart, pancreas and a neuronal cell line, indicating that the regulation of mAChR gene expression contributes to the differentiation of cholinergic activity

Differences in B cell growth phenotype reflect novel patterns of Epstein-Barr virus latent gene expression in Burkitt's lymphoma cells

Abstract: Recently established Epstein-Barr virus (EBV)-positive Burkitt's lymphoma (BL) cell lines, carrying chromosomal translocations indicative of their malignant origin, have been monitored for their degree of in vitro progression towards a more 'lymphoblastoid' cell surface phenotype and growth pattern, and for their expression of three EBV latent gene products which are constitutively present in all virus-transformed normal lymphoblastoid cell lines (LCLs). BL cell lines which stably retained the original tumour biopsy phenotype on serial passage were all positive for the nuclear antigen EBNA 1 but did not express detectable amounts of two other 'transforming' proteins, EBNA 2 and the latent membrane protein (LMP). This novel pattern of EBV gene expression was also observed on direct analysis of BL biopsy tissue. All three viral proteins became detectable, however, in BL cell lines which had progressed towards a more LCL-like phenotype in vitro. This work establishes a link between B cell phenotype and the accompanying pattern of EBV latent gene expression, and identifies a novel type of EBV:cell interaction which may be unique to BL cells.

Characterization of the herbicide-resistance gene bar from Streptomyces hygroscopicus.

Abstract: A gene which confers resistance to the herbicide bialaphos (bar) has been characterized. The bar gene was originally cloned from Streptomyces hygroscopicus, an organism which produces the tripeptide bialaphos as a secondary metabolite. Bialaphos contains phosphinothricin, an analogue of glutamate which is an inhibitor of glutamine synthetase. The bar gene product was purified and shown to be a modifying enzyme which acetylates phosphinothricin or demethylphosphinothricin but not bialaphos or glutamate. The bar gene was subcloned and its nucleotide sequence was determined. Interspecific transfer of this Streptomyces gene into Escherichia coli showed that it could be used as a selectable marker in other bacteria. In the accompanying paper, bar has been used to engineer herbicide-resistant plants.

The nerve growth factor: thirty-five years later.

Abstract: "Embryogenesis is in some way a model system. It has always been distinguished by the exactitude even puctilio, of its anatomical descriptions. An experiment by one of the great masters of embryology could be made the text of a discourse on scientific method. But something is wrong, or has been wrong. There is no theory of development in the sense in which Mendelism is a theory that accounts for the results of breeding experiments. There has therefore been little sense of progression or timeliness about embryological research. Of many papers delivered at embryological meetings, however good they may be in themselves.. .one too often feels that they might have been delivered five years beforehand without making anyone much the wiser, or deferred for five years without making anyone conscious of a great loss". (1) This feeling of frustration so incisively conveyed by these considerations by P. Medawar, prevaded, in the forties, the field of experimental embryology which had been enthusiastically acclaimed in the mid-thirties, when the upper lip of the amphibian blastopore brought this area of research to the forefront of the biological stage. The side branch of experimental neuroembryology, which had stemmed out from the common tree and was entirely devoted to the study of the trophic interrelations between neuronal cell populations and between these and the innervated organs and tissues was then in its initial vigorous growth phase. It in turn suffered from a sharp decrease in the enthusiasm that had inflamed the pioneers in this field, ever since R. G. Harrison delivered his celebrated lecture on this topic at the Royal Society in London in 1935 (2). Although the alternate 'wax and wane' cycles are the rule rather than the exception in all fields of human endeavor, in that of biological sciences the 'wane' is all too often indicative of a justified loss of faith in the rational and methodical approach that had at first raised so much hope. A brief account of the state-of-the-art of experimental neuroembryology in the forties, when interest in this approach to the study of the developing nervous system was waning, is a prerequisite for understanding the sudden unforseeable turn of events which resulted in the discovery of the Nerve Growth Factor. EXPERIMENTAL NEUROEMBRYOLOGY IN THE EARLY FORTIES

Overexpression of the EGF receptor-related proto-oncogene erbB-2 in human mammary tumor cell lines by different molecular mechanisms

Abstract: Amplification of the erbB/EGF receptor and a structurally related gene, designated erbB-2, have previously been detected in a variety of human tumors In a series of human mammary tumor cell lines, analysis of transcripts of these genes revealed elevated levels of one or the other in more than 60% of tumors analyzed Eight cell lines demonstrated erbB-2 mRNA levels ranging from 4- to 128-fold above those of normal controls erbB-2 expression was evaluated in comparison to the expression level of actin observed in these cell lines There was no evidence of an aberrantly sized erbB-2 transcript in any of these lines Immunoblot analysis indicated elevation in levels of the 185-kd product of the erbB-2 gene expressed by these cells In four lines erbB-2 gene amplification in the absence of an apparent gene rearrangement was demonstrated In a representative cell line of this type, SK-BR-3, the amplified erbB-2 gene copies were located in an aberrant chromosomal location Four additional cell lines, which demonstrated 4- to 8-fold overexpression of erbB-2 mRNA, did not exhibit gene amplification In a representative cell line of this type ZR-75-1, an apparently normal chromosomal location was found for the erbB-2 gene Our findings indicate that overexpression of the erbB-2 gene in mammary tumor cell lines is frequent and associated with different genetic abnormalities

Molecular cloning and expression of cDNA encoding a murine myeloid leukaemia inhibitory factor (LIF).

Abstract: Leukaemia inhibitory factor (LIF) can induce macrophage differentiation in M1 murine myeloid leukaemic cells and suppress their proliferation in vitro. It does not stimulate the proliferation of normal progenitor cells and is apparently distinct from known colony-stimulating factors. We have used oligo-nucleotides complementary to partial amino acid sequence of LIF to isolate a LIF clone from a T lymphocyte cDNA library. When this cDNA was coupled to a yeast expression vector (YEpsec1) and introduced into yeast cells, a molecule with the biological properties characteristic of native LIF was secreted into the growth medium. The amino acid sequence of LIF established it to be a unique molecular entity, distinct from the other known haemopoietic growth factors. Since LIF is encoded by a unique gene, two biochemically separable forms of LIF probably represent post-transcriptional or posttranslational variants of the same gene product. In contrast to several other haemopoietic regulators, the 0.8- to 1-kb LIF mRNA was expressed constitutively in two murine T lymphocyte cell lines examined, and its abundance was not enhanced by stimulation with concanavalin A. Cloning, sequencing and expressing LIF has resolved several discrepancies in the literature concerning the identity of factors capable of inducing differentiation of murine myeloid leukaemic cells in vitro.

Sequence-specific positioning of nucleosomes over the steroid-inducible MMTV promoter.

Abstract: We have investigated chromatin organization over the MMTV LTR, a promoter regulated by steroid hormones. The studies were performed on cell lines containing BPV-based episomal constructs. Nucleosome positioning was determined by localization of sites sensitive to the enzyme micrococcal nuclease, or to the chemical MPE-Fe(II). Experiments with both reagents indicate that nucleosomes are specifically positioned in MMTV LTR chromatin. In the absence of hormone a regular cutting pattern is obtained, with cleavage sites at +136, -60, -250, -444, -651, -826 and -1019 relative to the Cap site. In the presence of hormone the cutting pattern is unchanged, except for a region between -60 and -250 that becomes hypersensitive to MPE-Fe(II). This region contains the DNA sequences to which steroid receptor complexes bind during transcriptional activation. Our results indicate that this region is associated in chromatin from uninduced cells with a macromolecular complex (probably a nucleosome core), and this complex is displaced (or modified) upon binding of activated receptor.

Biological function of `pathogenesis-related' proteins: four PR proteins of tobacco have 1,3-β-glucanase activity

Abstract: Three of the ten acidic `pathogenesis-related' (PR) proteins known to accumulate in Nicotiana tabacum cv Samsun NN reacting hypersensitively to tobacco mosaic virus, namely −O, −N and −2, have been shown to have 1,3-β-glucanase (EC 3.2.1.39) activity. By using sera raised against each protein purified to homogeneity close serological relationships have been demonstrated between the three proteins. The same specific sera cross-reacted with a basic protein which is also a 1,3-β-glucanase induced by virus infection and which can be considered as a new basic pathogenesis-related protein of tobacco. Protein PR-O and the basic 1,3-β-glucanase display about the same specific enzymatic activity, i.e. 50-fold and 250-fold higher than specific activities of proteins PR-N and -2 respectively.

Structure and expression of human B cell stimulatory factor-2 (BSF-2/IL-6) gene.

Abstract: The chromosomal DNA segment of human B cell stimulatory factor-2 (BSF-2/IL-6) was isolated and characterized by nucleotide sequence analysis. The human BSF-2/IL-6 gene consists of five exons and four introns and its organization shows a distinctive similarity to granulocyte colony-stimulating factor gene. The two genes have the same number of exons and introns and the size of each exon is strikingly similar. The BSF-2/IL-6 mRNA was found to be constitutively expressed in a human T cell leukemia virus-1 transformed T cell line, TCL-Na1, a bladder cell carcinoma line, T24, and an amnion derived cell line, FL. The BSF-2/IL-6 mRNA was also found to be inducible with interleukin-1 beta in an astrocytoma line, U373 and a glioblastoma line, SK-MG-4. S1 mapping and primer extension analyses showed the presence of multiple initiation sites and the preferential utilization of a different initiation site for each individual tissue tested.

The yeast ubiquitin genes: a family of natural gene fusions.

Abstract: Ubiquitin is a 76-residue protein highly conserved among eukaryotes. Conjugation of ubiquitin to intracellular proteins mediates their selective degradation in vivo. We describe a family of four ubiquitin-coding loci in the yeast Saccharomyces cerevisiae. UB11, UB12 and UB13 encode hybrid proteins in which ubiquitin is fused to unrelated ('tail') amino acid sequences. The ubiquitin coding elements of UB11 and UB12 are interrupted at identical positions by non-homologous introns. UB11 and UB12 encode identical 52-residue tails, whereas UB13 encodes a different 76-residue tail. The tail amino acid sequences are highly conserved between yeast and mammals. Each tail contains a putative metal-binding, nucleic acid-binding domain of the form Cys-X2-4-Cys-X2-15-Cys-X2-4-Cys, suggesting that these proteins may function by binding to DNA. The fourth gene, UB14, encodes a polyubiquitin precursor protein containing five ubiquitin repeats in a head-to-tail, spacerless arrangement. All four ubiquitin genes are expressed in exponentially growing cells, while in stationary-phase cells the expression of UB11 and UB12 is repressed. The UB14 gene, which is strongly inducible by starvation, high temperatures and other stresses, contains in its upstream region strong homologies to the consensus 'heat shock box' nucleotide sequence. Elsewhere we show that the essential function of the UB14 gene is to provide ubiquitin to cells under stress.

Characterization and localization of the even-skipped protein of Drosophila.

Abstract: On the basis of homeo box cross-homology we have isolated the pair-rule gene even-skipped (eve) of Drosophila. The eve transcription unit appears to be less than 1.5 kb in length, and encodes a single mRNA of approximately 1.4 kb. The nucleotide sequence of genomic and cDNA clones indicates that the eve protein is composed of 376 amino acid residues, and that its homeo domain shares only approximately 50% amino acid identity with the homeo domains of previously characterized genes. Using antibodies raised against a beta-galactosidase fusion protein we show that the eve protein is distributed in a series of seven transverse stripes at the cellular blastoderm stage, and is localized primarily within the nuclear regions of those embryonic cells that express the gene. After gastrulation, seven weakly stained stripes of eve expression appear, resulting in a transient pattern that consists of a total of 14 evenly spaced stripes. Both the original and new stripes gradually disappear during germ band elongation. A second expression pattern emerges during neurogenesis, whereby eve protein is detected in discrete subsets of neurons in each of the ventral ganglia.

Molecular cloning of sequences from wingless, a segment polarity gene in Drosophila: the spatial distribution of a transcript in embryos.

Abstract: In Drosophila the process of segmentation depends on the function of coordinate, gap, pair-rule and segment-polarity genes. Mutations in segment-polarity genes cause defects in the pattern of every segment. Here the cloning of sequences from a segment-polarity gene, wingless, and the in situ localization of a transcript in embryos are described. The transcript is first detected in the anterior and posterior regions of the blastoderm embryo at cellularization, and accumulates in a series of stripes in the extended germ band, one stripe per metameric unit. Each stripe is localized to the most posterior cells of each parasegment. The signal is predominantly epidermal, and transcript accumulates only transiently in the mesoderm and nervous system. This pattern of expression is discussed with respect to models of pattern formation in segmental units.

Microtubules containing detyrosinated tubulin are less dynamic.

Abstract: Peptide antibodies specific for tyrosinated (tyr-tubulin) or detyrosinated alpha-tubulin (glu-tubulin) have been generated for studying the relative stability of microtubules enriched in either form of alpha-tubulin Treatment of Vero cells with nocodazole has revealed that interphase microtubules rich in glu-tubulin (glu-microtubules) are resistant to higher concentrations of the microtubule-disrupting drug than the microtubules containing only tyr-tubulin (tyr-microtubules) Glu-tubulin is enriched in centrioles and mid-bodies, but absent from the first interphase microtubules that have repolymerized in late telophase Tubulin (including both forms) has been labeled with rhodamine (rh-tubulin) and microinjected into Vero cells to study in vivo the dynamic properties and incorporation rates of tubulin into microtubules rich in either glu- or tyr-tubulin Tyr-microtubules are significantly more rapidly labeled by the microinjected rh-tubulin than glu-microtubules Ten minutes after injection, rh-tubulin is present in virtually all tyr-microtubules The half-time of turnover of glu-microtubules is approximately 1 h Even several hours after microinjection, some of the glu-microtubules have consistently not incorporated visible amounts of rh-tubulin These results suggest that tyr- and glu-microtubules respectively represent relatively dynamic and stable subclasses of interphase microtubules

Independent and synergistic activity of rol A, B and C loci in stimulating abnormal growth in plants.

Abstract: The Ri plasmid A4 of Agrobacterium rhizogenes contains within its T-DNA genetic information able to trigger root formation in infected plants. Tobacco plants regenerated from transformed roots display the hairy root (hr) syndrome. We show that DNA fragments containing the rol B locus alone are able to induce root formation both in tobacco and kalanchoe tissues. The rol A and the rol C loci by themselves are also able to induce root formation in tobacco but not in kalanchoe. This capacity to induce root formation in either host is greatly increased when the rol A and/or C loci are combined with the rol B locus. Root induction is shown to be correlated with the expression of the rol loci. Transgenic plants exhibit all the characteristics of the hairy root syndrome only when all three loci are present and expressed. Although the activity of the rol encoded functions is synergistic, each of them appears to independently influence host functions involved in the determination of root differentiation.

Molecular cloning of the beta-subunit of human prolyl 4-hydroxylase. This subunit and protein disulphide isomerase are products of the same gene.

Abstract: Prolyl 4-hydroxylase (EC 1.14.11.2), an alpha 2 beta 2 tetramer, catalyses the formation of 4-hydroxyproline in collagens by the hydroxylation of proline residues in peptide linkages. We report here the isolation of cDNA clones coding for the beta-subunit of prolyl 4-hydroxylase from a human hepatoma lambda gt11 library and a corresponding human placenta library. Five overlapping clones covering all the coding sequences and almost all the non-coding sequences were characterized. The size of the mRNA hybridizing with these clones in Northern blotting is approximately 2.5 kb. The clones encode a polypeptide of 508 amino acid residues, including a signal peptide of 17 amino acids. These human sequences were found to be very similar to those recently reported for rat protein disulphide isomerase (EC 5.3.4.1). The degree of homology between these two proteins was 84% at the level of nucleotide sequences or 94% at the level of amino acid sequences. Southern blot analyses of human genomic DNA with a cDNA probe for the beta-subunit indicated the presence of only one gene containing these sequences. The product of a single gene thus appears to possess two different enzymatic functions depending on whether it is present in cells in monomer form or in the prolyl 4-hydroxylase tetramer.

Complementary DNA for human glioblastoma-derived T cell suppressor factor, a novel member of the transforming growth factor-beta gene family.

Abstract: Human glioblastoma cells secrete a peptide, termed glioblastoma-derived T cell suppressor factor (G-TsF), which has suppressive effects on interleukin-2-dependent T cell growth. As shown here, complementary DNA for G-TsF reveals that G-TsF shares 71% amino acid homology with transforming growth factor-beta (TGF-beta). In analogy to TGF-beta it is apparently synthesized as the carboxy-terminal end of a precursor polypeptide which undergoes proteolytic cleavage to yield the 112 amino-acid-long mature form of G-TsF. Comparison of the amino-terminal sequence of G-TsF with that of porcine TGF-beta 2 and bovine cartilage-inducing factor B shows complete homology, which indicates that we have cloned the human analogue of these factors. It is tempting to consider a role for G-TsF in tumor growth where it may enhance tumor cell proliferation in an autocrine way and/or reduce immunosurveillance of tumor development.

Sequence-specific interactions of a pea nuclear factor with light-responsive elements upstream of the rbcS-3A gene.

Abstract: Pea nuclear extracts were used in gel retardation assays and DNase I footprinting experiments to identify a protein factor that specifically interacts with regulatory DNA sequences upstream of the pea rbcS-3A-gene This factor, designated GT-1, binds to two short sequences (boxes II and III) in the -150 region that are known to function as light-responsive elements (LREs) in transgenic tobacco Binding of GT-1 to homologous sequences further upstream (boxes II and III in the -220 region) indicates that these boxes comprise the redundant LRE that functions in vivo when boxes II and III are deleted In both box II and box II, methylation interference experiments demonstrate that two adjacent G residues are critical for GT-binding Single Gs present in boxes III and III are also important Since GT-1 is present in nuclear extracts from leaves of light-grown and dark-adapted pea plants, its regulatory role does not depend on de novo synthesis Thus if GT-1 binds differentially in vivo it must be postranslationally modified or sterically blocked from binding by another factor in response to light

Structure and expression of human thrombomodulin, a thrombin receptor on endothelium acting as a cofactor for protein C activation.

Abstract: We have deduced the entire 575-amino acid sequence of the human thrombomodulin precursor from cDNA clones. The precursor starts with an 18-residue signal peptide domain, followed by the NH2-terminal domain, a domain with six epidermal growth factor-like structures, an O-glycosylation site-rich domain, a 24-residue transmembrane domain and a cytoplasmic domain. Simian COS cells transfected with the expression vector pSV2 containing thrombomodulin cDNA synthesized immunoreactive and functionally active thrombomodulin.

Three dimensional structure of porcine pancreatic alpha-amylase at 2.9 A resolution. Role of calcium in structure and activity.

Abstract: The crystal structure of porcine pancreatic alpha-amylase (PPA) has been solved at 2.9 A resolution by X-ray crystallographic methods. The enzyme contains three domains. The larger, in the N-terminal part, consists of 330 amino acid residues. This central domain has the typical parallel-stranded alpha-beta barrel structure (alpha beta)8, already found in a number of other enzymes like triose phosphate isomerase and pyruvate kinase. The C-terminal domain forms a distinct globular unit where the chain folds into an eight-stranded antiparallel beta-barrel. The third domain lies between a beta-strand and a alpha-helix of the central domain, in a position similar to those found for domain B in triose phosphate isomerase and pyruvate kinase. It is essentially composed of antiparallel beta-sheets. The active site is located in a cleft within the N-terminal central domain, at the carboxy-end of the beta-strands of the (alpha beta)8 barrel. Binding of various substrate analogues to the enzyme suggests that the amino acid residues involved in the catalytic reaction are a pair of aspartic acids. A number of other residues surround the substrate and seem to participate in its binding via hydrogen bonds and hydrophobic interactions. The 'essential' calcium ion has been located near the active site region and between two domains, each of them providing two calcium ligands. On the basis of sequence comparisons this calcium binding site is suggested to be a common structural feature of all alpha-amylases. It represents a new type of calcium-protein interaction pattern.(ABSTRACT TRUNCATED AT 250 WORDS)

Fluoride complexes of aluminium or beryllium act on G-proteins as reversibly bound analogues of the gamma phosphate of GTP.

Abstract: Fluoride activation of G proteins requires the presence of aluminium or beryllium and it has been suggested that AIF4- acts as an analogue of the gamma-phosphate of GTP in the nucleotide site. We have investigated the action of AIF4- or of BeF3- on transducin (T), the G protein of the retinal rods, either indirectly through the activation of cGMP phosphodiesterase, or more directly through their effects on the conformation of transducin itself. In the presence of AIF4- or BeF3-, purified T alpha subunit of transducin activates purified cyclic GMP phosphodiesterase (PDE) in the absence of photoactivated rhodopsin. Activation is totally reversed by elution of fluoride or partially reversed by addition of excess T beta gamma. Activation requires that GDP or a suitable analogue be bound to T alpha: T alpha-GDP and T alpha-GDP alpha S are activable by fluorides, but not T alpha-GDP beta S, nor T alpha that has released its nucleotide upon binding to photoexcited rhodopsin. Analysis of previous works on other G proteins and with other nucleotide analogues confirm that in all cases fluoride activation requires that a GDP unsubstituted at its beta phosphate be bound in T alpha. By contrast with alumino-fluoride complexes, which can adopt various coordination geometries, all beryllium fluoride complexes are tetracoordinated, with a Be-F bond length of 1.55 A, and strictly isomorphous to a phosphate group. Our study confirms that fluoride activation of transducin results from a reversible binding of the metal-fluoride complex in the nucleotide site of T alpha, next to the beta phosphate of GDP, as an analogue of the gamma phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)

Transformed human cells produce a new fibronectin isoform by preferential alternative splicing of a previously unobserved exon.

Abstract: Purification and amino acid sequence analysis of a proteolytic fragment of fibronectin (FN) from transformed human cells demonstrated that a high percentage of these FN molecules contains an extra amino acid sequence which is present only in a very low percentage of FN molecules from normal fibroblasts and is undetectable in plasma FN. This new amino acid sequence introduces into the FN molecule a site very sensitive to a number of proteolytic enzymes. By analyzing the cellular mRNA and genomic clones, we have demonstrated that this sequence derives from a differential splicing pattern of the FN mRNA precursors, which leads in transformed cells to a high-level expression of an extra type III homology repeat (ED-B) coded for by a previously unobserved exon. Here we also report the complete sequence of this new exon. These results demonstrate that in malignant cells the mechanisms regulating the splicing of FN mRNA precursors are altered.

Structural features required for ligand binding to the beta-adrenergic receptor.

Abstract: On the basis of the homology between the amino acid sequences of the beta-adrenergic receptor (beta AR) and the opsin proteins we have proposed that the ligand binding domain lies within the seven transmembrane hydrophobic regions of the protein, which are connected by hydrophilic regions alternatively exposed extracellularly and intracellularly. We have systematically examined the importance of each of these regions by making a sequential series of deletions in the gene for the hamster beta AR which encompass most of the protein coding region. The ability of the corresponding mutant receptors to be expressed, localized to the cell membrane, and bind beta-adrenergic ligands has been analyzed, using transient expression in COS-7 cells. The hydrophobic regions and the hydrophilic segments immediately adjacent to the membrane cannot be removed without affecting the processing and membrane localization of the beta AR. However, most of the hydrophilic regions appear to be dispensable for ligand binding. In addition, we observed that substitution of the conserved cysteine residues at positions 106 and 184 dramatically altered the ligand binding characteristics of the beta AR, suggesting the occurrence of a disulfide bond between these two residues in the native protein. These data are discussed in terms of the tertiary structure of the beta AR.

T cell suppressor factor from human glioblastoma cells is a 12.5-kd protein closely related to transforming growth factor-beta.

Abstract: T cell suppressor factor produced by human glioblastoma cells inhibits T cell proliferation in vitro and more specifically interferes with interleukin-2 (IL-2)-dependent T cell growth. Here we report the purification of this factor from conditioned medium of the human glioblastoma cell line 308. Amino-terminal sequence analysis of the 12.5-kd protein demonstrates that eight out of the first 20 amino acids are identical to human transforming growth factor-beta. Purified glioblastoma-derived T cell suppressor factor and transforming growth factor-beta from porcine platelets inhibit both IL-2-induced proliferation of ovalbumin-specific T helper cells and lectin-induced thymocyte proliferation with similar specific activities. If released by glioblastoma cells in vivo, the factor may contribute to impaired immunosurveillance and to the cellular immunodeficiency state detected in the patients.