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2.
Nat Biotechnol ; 16(9): 851-6, 1998 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-9743119

RESUMEN

We have used comparative genomics to identify 26 Escherichia coli open reading frames that are both of unknown function (hypothetical open reading frames or y-genes) and conserved in the compact genome of Mycoplasma genitalium. Not surprisingly, these genes are broadly conserved in the bacterial world. We used a markerless knockout strategy to screen for essential E. coli genes. To verify this phenotype, we constructed conditional mutants in genes for which no null mutants could be obtained. In total we identified six genes that are essential for E. coli (yhbZ, ygjD, ycfB, yfil, yihA, and yjeQ). The respective orthologs of the genes yhbZ, ygjD, ycfB, yjeQ, and yihA are also essential in Bacillus subtilis. This low number of essential genes was unexpected and might be due to a characteristic of the versatile genomes of E. coli and B. subtilis that is comparable to the phenomenon of nonorthologous gene displacement. The gene ygjD, encoding a sialoglycoprotease, was eliminated from a minimal genome computationally derived from a comparison of the Haemophilus influenzae and M. genitalium genomes. We show that ygjD and its ortholog ydiE are essential in E. coli and B. subtilis, respectively. Thus, we include this gene in a minimal genome. This study systematically integrates comparative genomics and targeted gene disruptions to identify broadly conserved bacterial genes of unknown function required for survival on complex media.


Asunto(s)
Escherichia coli/genética , Genoma Bacteriano , Secuencia de Aminoácidos , Bacillus subtilis/genética , Secuencia de Bases , Cartilla de ADN , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido
3.
J Biol Chem ; 272(33): 20611-8, 1997 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-9252376

RESUMEN

Interleukin-5 (IL-5), a disulfide-linked homodimer, can be induced to fold as a biological active monomer by extending the loop between its third and fourth helices (Dickason, R. R., and Huston, D. P. (1996) Nature 379, 652-655). We have designed eight monomeric IL-5 proteins to optimize biological activity and stability of the monomer. This was achieved by (i) inserting the joining loop at three different positions, (ii) by introducing an additional intramolecular disulfide bridge onto these backbones, and (iii) by creating circular permutations to fix the position of the carboxyl-terminal helix relative to the three other helices. The proteins dimerize with Kd values ranging from 20 to 200 microM and are therefore monomeric at the picomolar concentrations where they are biologically active. Introduction of a second disulfide confers increased stability, but this increased rigidity results in lower activity of the protein. Contrary to wild type IL-5, mutation of the betac contact residue on the first helix, Glu12, to Lys, into the circularly permutated constructs, did not abolish TF-1 proliferative and eosinophil activation activities. These results indicate that activation of the IL-5 receptor complex is not mediated solely by Glu12 on the first helix, and alternative mechanisms are discussed.


Asunto(s)
Interleucina-5/química , Receptores de Interleucina/química , Sitios de Unión , Dimerización , Humanos , Estructura Secundaria de Proteína , Receptores de Interleucina-5
4.
Recept Channels ; 5(3-4): 201-7, 1997.
Artículo en Inglés | MEDLINE | ID: mdl-9606724

RESUMEN

A fluorescent unnatural amino acid was introduced biosynthetically at known sites into the G protein-coupled neurokinin (tachykinin) NK2 receptor by suppression of UAG nonsense codons with the aid of a chemically misacylated synthetic tRNA specifically designed for the incorporation of unnatural amino acids during heterologous expression in Xenopus oocytes. A systematic UAG-scanning mutagenesis in NK2 extra- or intracellular loops and proximal transmembrane domains established that readthrough at some UAG sites may represent a limitation to the range of applicability of the nonsense suppression methodology. Fluorescence-labeled NK2 mutants containing an unique fluorescent nitrobenzoxadiazoyl-diaminopropionic acid residue at known sites were shown to be functionnally active. Intermolecular distances were determined by measuring the fluorescence resonance energy transfer (FRET) between the fluorescent unnatural amino acid and a fluorescently labeled NK2 heptapeptide antagonist in a native membrane environment. These distances confirmed the seven transmembrane topology for G protein-coupled receptors and determined a structural model for NK2 ligand-receptor interactions. The peptide is inserted between the fifth and sixth transmembrane domains, thus suggesting that antagonism may be caused by preventing correct packing of the helices required for receptor function.


Asunto(s)
Colorantes Fluorescentes , Receptores de Neuroquinina-2/metabolismo , Rodaminas , Secuencia de Aminoácidos , Animales , Sitios de Unión , Transferencia de Energía , Ligandos , Datos de Secuencia Molecular , Mutagénesis , Oocitos/metabolismo , Receptores de Neuroquinina-2/genética , Xenopus
5.
J Biol Chem ; 271(33): 19991-8, 1996 Aug 16.
Artículo en Inglés | MEDLINE | ID: mdl-8702716

RESUMEN

A general method for understanding the mechanisms of ligand recognition and activation of G protein-coupled receptors has been developed. A study of ligand-receptor interactions in the prototypic seven-transmembrane neurokinin-2 receptor (NK2) using this fluorescence-based approach is presented. A fluorescent unnatural amino acid was introduced at known sites into NK2 by suppression of UAG nonsense codons with the aid of a chemically misacylated synthetic tRNA specifically designed for the incorporation of unnatural amino acids during heterologous expression in Xenopus oocytes. Fluorescence-labeled NK2 mutants containing an unique 3-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-2,3-diaminopropionic acid (NBD-Dap) residue at either site 103, in the first extracellular loop, or 248, in the third cytoplasmic loop, were functionally active. The fluorescent NK2 mutants were investigated by microspectrofluorimetry in a native membrane environment. Intermolecular distances were determined by measuring the fluorescence resonance energy transfer (FRET) between the fluorescent unnatural amino acid and a fluorescently labeled NK2 heptapeptide antagonist. These distances, calculated by the theory of Förster, permit to fix the ligand in space and define the structure of the receptor in a molecular model for NK2 ligand-receptor interactions. Our data are the first report of the incorporation of a fluorescent unnatural amino acid into a membrane protein in intact cells by the method of nonsense codon suppression, as well as the first measurement of experimental distances between a G protein-coupled receptor and its ligand by FRET. The method presented here can be generally applied to the analysis of spatial relationships in integral membrane proteins such as receptors or channels.


Asunto(s)
Receptores de Neuroquinina-2/química , Animales , Secuencia de Bases , Células CHO , Cloruros/metabolismo , Cricetinae , Cartilla de ADN/química , Transferencia de Energía , Colorantes Fluorescentes , Proteínas de Unión al GTP , Genes Supresores , Humanos , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis , Aminoacil-ARN de Transferencia/química , Aminoacil-ARN de Transferencia/metabolismo , Receptores de Neuroquinina-2/antagonistas & inhibidores , Espectrometría de Fluorescencia , Relación Estructura-Actividad , Xenopus laevis
6.
J Biol Chem ; 271(11): 5965-7, 1996 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-8626375

RESUMEN

CD40 ligand (CD40L) is a glycoprotein expressed on the surface of activated helper T cells, basophils, mast cells, and eosinophils. Binding of CD40L to its receptor CD40 on the B cell surface induces B cell proliferation, adhesion, and immunoglobulin class switching. We have identified soluble cleavage products of human CD40L in the supernatant of a stimulated human T cell clone. Subcellular fractionation experiments have shown that the transmembrane CD40L is processed inside the microsomes and that its cleavage is stimulation-dependent. The native human soluble CD40L is trimeric and, when used in conjunction with interleukin-4, induces B cell proliferation.


Asunto(s)
Antígenos CD40/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Linfocitos B/inmunología , Ligando de CD40 , Línea Celular , Humanos , Ligandos , Activación de Linfocitos , Microsomas/inmunología , Microsomas/metabolismo , Conformación Proteica , Procesamiento Proteico-Postraduccional , Solubilidad , Linfocitos T/inmunología
7.
J Biol Chem ; 270(13): 7025-8, 1995 Mar 31.
Artículo en Inglés | MEDLINE | ID: mdl-7706236

RESUMEN

CD40 ligand (CD40L) is expressed on the surface of activated CD4+ T cells, basophils, and mast cells. Binding of C40L to its receptor, CD40, on the surface of B cells stimulates B cell proliferation, adhesion and differentiation. A preparation of soluble, recombinant CD40L (Tyr-45 to Leu-261), containing the full-length 29-kDa protein and two smaller fragments of 18 and 14 kDa, has been shown to induce differentiation of B cells derived either from normal donors or from patients with X-linked hyper-IgM syndrome (Durandy, A., Schiff, C., Bonnefoy, J.-Y., Forveille, M., Rousset, F., Mazzei, G., Milili, M., and Fischer, A. (1993) Eur. J. Immunol. 23, 2294-2299). We have now purified each of these fragments to homogeneity and show that only the 18-kDa fragment (identified as Glu-108 to Leu-261) is biologically active. When expressed in recombinant form, the 18-kDa protein exhibited full activity in B cell proliferation and differentiation assays, was able to rescue of B cells from apoptosis, and bound soluble CD40. Sucrose gradient sedimentation shows that the 18-kDa protein sediments as an apparent homotrimer, a result consistent with the proposed trimeric structure of CD40L. This demonstrates that a soluble CD40L can stimulate CD40 in a manner indistinguishable from the membrane-bound form of the protein.


Asunto(s)
Antígenos CD/metabolismo , Linfocitos B/inmunología , Glicoproteínas de Membrana/biosíntesis , Secuencia de Aminoácidos , Animales , Formación de Anticuerpos , Apoptosis , Ligando de CD40 , Línea Celular , Ácido Glutámico , Haplorrinos , Humanos , Inmunoglobulina M/biosíntesis , Síndromes de Inmunodeficiencia/inmunología , Interleucina-4/farmacología , Riñón , Leucina , Ligandos , Activación de Linfocitos , Sustancias Macromoleculares , Glicoproteínas de Membrana/aislamiento & purificación , Glicoproteínas de Membrana/farmacología , Ratones , Peso Molecular , Tonsila Palatina/inmunología , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Valores de Referencia , Homología de Secuencia de Aminoácido , Transfección
8.
FEBS Lett ; 354(2): 195-9, 1994 Nov 07.
Artículo en Inglés | MEDLINE | ID: mdl-7957923

RESUMEN

Gq alpha and G11 alpha differ from other G protein alpha subunits in that they have unique, conserved 6 residue amino-terminal extensions. Wild-type and amino-terminal mutants of Gq alpha expressed in COS cells were analyzed for their ability to functionally couple with co-expressed neurokinin NK2 receptor. Wild-type, T2A and delta 2-7 Gq alpha were able to stimulate agonist driven phospholipase C (PLC) activity in identical manners. Other activities of these two amino-terminal mutants including aluminum fluoride stimulated PLC activity, palmitoylation, interaction with G beta gamma subunits and GTP gamma S-induced trypsin resistance are also similar to the wild-type alpha subunit. This demonstrates that the NK2 receptor is able to functionally interact with the alpha subunit of Gq and that the first seven amino-acids of Gq alpha are not required for any of the alpha subunit functions tested. In contrast to the T2A and delta 2-7 mutants, a C9,10A Gq alpha mutant was not able to couple to either the NK2 receptor or PLC, as assessed by high-affinity agonist binding and activation of PLC either in intact cells or in vitro. The C9,10A protein was able to assume a GTP gamma S-induced trypsin-resistant conformation and partitioned primarily to the pelletable fraction in a manner similar to the wild-type protein. However, it was not labeled with [3H]palmitic acid. This suggests that blocking palmitoylation at the amino-terminus of Gq alpha results in a loss of functional activity which reflects an inability to interact with both the receptor and downstream signaling targets.


Asunto(s)
Proteínas de Unión al GTP/metabolismo , Ácidos Palmíticos/metabolismo , Receptores de Neuroquinina-2/metabolismo , Compuestos de Aluminio/farmacología , Animales , Línea Celular , Citidina Difosfato Diglicéridos/metabolismo , Detergentes , Activación Enzimática , Fluoruros/farmacología , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/genética , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Mutagénesis , Ácido Palmítico , Plásmidos , Receptores de Neuroquinina-2/genética , Proteínas Recombinantes/metabolismo , Solubilidad , Relación Estructura-Actividad , Transfección , Fosfolipasas de Tipo C/metabolismo
9.
Semin Cell Biol ; 5(5): 295-302, 1994 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-7881069

RESUMEN

Red and far-red light control of plant growth and development is mediated by the photoreceptor phytochrome. The way plants utilize red and far-red light is unique in nature, as are the molecular properties of phytochrome, the molecule that provides the mechanistic basis for this type of light perception. Much of what we know about how plants perceive red light has come from research on the structure and function of this photoreceptor. This review discusses the main structural features of phytochrome and some new ideas concerning the relationship between phytochrome structure and function. We propose that phytochrome functions as a dimer and that receptor recognition of phytochrome depends on its gross conformation. We also describe a conserved amino acid repeat within the phytochrome molecule and propose that this repeat is important for dimerization and/or phototransformation.


Asunto(s)
Fitocromo/química , Fenómenos Fisiológicos de las Plantas , Secuencia de Aminoácidos , Luz , Datos de Secuencia Molecular , Células Fotorreceptoras , Transducción de Señal
10.
Biochemistry ; 32(32): 8239-45, 1993 Aug 17.
Artículo en Inglés | MEDLINE | ID: mdl-8347623

RESUMEN

We have produced defined fragments of the oat PhyA AP3 protein using an in vitro translation system and analyzed the quaternary structure of these fragments by size exclusion chromatography. Sequences between amino acids S599 and L683 are shown to dimerize by this in vitro assay and by a lambda repressor-based in vivo assay. A subset of this dimerization region, V623-S673, which has previously been identified as being involved in interdomain interactions on the basis of the behavior of overlapping constructs in a lambda repressor assay for protein-protein interaction, is shown by both assays to be necessary but insufficient for dimerization. Sequences between L685 and R815, which are unable to dimerize by themselves, are shown to interact with sequences between S599 and L683. Sequences E1069-Q1129, also previously suggested to be involved in dimerization, are shown here not to be required for phytochrome dimerization. These results based on an in vitro assay have confirmed some of the results previously obtained using an in vivo assay and extend these earlier results by revealing new protein-protein interactions. This dissection of sequences involved in phytochrome dimerization taken together with previous work has enabled us to propose a model for the behavior of the dimerization region where the core structure involved in dimerization is located on both sides of a region around residue 750 found at the surface.


Asunto(s)
Fragmentos de Péptidos/química , Fitocromo/química , Bacteriófago lambda/genética , Secuencia de Bases , Cromatografía en Gel , Grano Comestible/química , Sustancias Macromoleculares , Datos de Secuencia Molecular , Peso Molecular , Fragmentos de Péptidos/biosíntesis , Fitocromo/biosíntesis , Fitocromo/genética , Reacción en Cadena de la Polimerasa , Biosíntesis de Proteínas , Conformación Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Represoras/genética , Relación Estructura-Actividad
11.
Plant Mol Biol ; 21(6): 1191-4, 1993 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-8490138

RESUMEN

When expressed in Escherichia coli, a truncated form of phytochrome (oat PHYA AP3 residues 464-1129) self associates to form a series of products ranging in size from monomers to aggregates of greater than 20 subunits. When these same phytochrome sequences are coexpressed with the chaperonins GroEL and GroES, the truncated phytochrome migrates as a native-like dimer in size exclusion chromatography and no higher-order aggregates were detected. GroEL and GroES inhibition of phytochrome aggregation in E. coli presumably occurs via the suppression of folding pathways leading to incorrectly folded phytochrome.


Asunto(s)
Proteínas Bacterianas/genética , Proteínas de Choque Térmico/genética , Fitocromo/genética , Proteínas Bacterianas/metabolismo , Chaperoninas , Clonación Molecular , Escherichia coli , Proteínas de Escherichia coli , Proteínas de Choque Térmico/metabolismo , Immunoblotting , Fitocromo/metabolismo , Pliegue de Proteína
12.
Plant Cell ; 4(12): 1519-30, 1992 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-1467651

RESUMEN

We have identified a mutant of pea cultivar Alaska that has many of the characteristics normally associated with light-grown seedlings even when grown in complete darkness. We have designated this mutant lip1, for light independent photomorphogenesis. Etiolated wild-type pea seedlings are white to slightly yellow in color and have a distinct morphology characterized by elongated epicotyls and buds containing unexpanded leaves with small, undifferentiated cells. In contrast, mutant seedlings grown under the same conditions are yellow in color and have short epicotyls and expanded leaves showing clear cellular differentiation. Transmission electron microscopy revealed partially developed, agranal plastids in the dark-grown mutant, unlike wild-type seedlings that contain etioplasts with prolamellar bodies. The mutant also exhibits a much shorter lag period for chlorophyll accumulation when etiolated seedlings are transferred from darkness to white light. The dark-grown mutant has 10-fold less spectrally detectable phytochrome, which can be attributed to a 10-fold reduction in the level of the PHYA polypeptide. Cab, Fed1, and RbcS transcripts are present in dark-grown mutant seedlings at levels comparable to those produced in light-grown material. The levels of these transcripts show a normal decrease when green plants grown for 15 days in a light/dark cycle are transferred to continuous darkness. However, transcript levels remain high during dark treatment of seedlings grown for 9 days in continuous light, indicating that the dark adaptation response in this mutant is developmentally plastic. The lip1 mutant has several features in common with the deetiolated Arabidopsis mutants det1, det2, and cop1. However, there are also several important differences, including varying effects on phytochrome levels, organ-specific gene expression, plastid development, and response to dark adaptation.


Asunto(s)
Fabaceae/genética , Luz , Mutación , Plantas Medicinales , Citocininas/farmacología , Oscuridad , Fabaceae/crecimiento & desarrollo , Fabaceae/efectos de la radiación , Expresión Génica , Giberelinas/farmacología , Morfogénesis/genética , Morfogénesis/efectos de la radiación , Fitocromo/metabolismo , Pigmentación
13.
Plant Cell ; 4(2): 161-71, 1992 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-1633492

RESUMEN

We have used a novel assay based on protein fusions with lambda repressor to identify two small regions within phytochrome's carboxy-terminal domain that are capable of mediating dimerization. Using an in vivo assay, fusions between the DNA binding, amino-terminal domain of lambda repressor and fragments from oat PhyA phytochrome have been assayed for increased repressor activity, an indicator of dimerization. In this assay system, regions of oat phytochrome between amino acids V623-S673 and N1049-Q1129 have been shown to increase repressor activity. These short spans are highly conserved between proteins belonging to the phytochrome PhyA family. Embedded within these sequences are four segments that could potentially form amphipathic alpha helices. Two of the segments are well conserved between PhyA phytochrome and phytochromes encoded by the phyB and phyC genes, suggesting that heterodimers might form by way of subunit interaction at these sites.


Asunto(s)
Proteínas de Unión al ADN , Fitocromo/química , Secuencia de Aminoácidos , Sitios de Unión , Clonación Molecular , Datos de Secuencia Molecular , Fitocromo/genética , Plantas Modificadas Genéticamente/química , Plantas Modificadas Genéticamente/genética , Unión Proteica , Conformación Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Represoras/genética , Proteínas Virales , Proteínas Reguladoras y Accesorias Virales
14.
Plant Physiol ; 92(2): 474-9, 1990 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-16667300

RESUMEN

A method for the preparation of developmentally staged microspores and young pollen from maize (Zea mays) has been devised. The preparations are of sufficient purity and quantity for biochemical analysis, including the analysis of steady-state protein and RNA populations associated with each stage. A major transition in protein populations occurs during the developmental period that encompasses microspore mitosis, the asymmetric nuclear division producing the vegetative and generative nuclei. Several differences between early and late stage proteins can be detected by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two-dimensional gel electrophoresis of proteins reveals that over half of the steady-state proteins differ between the younger and older stages, either quantitative or qualitative. One protein that increases in relative abundance about fourfold is actin. In vitro translation of RNA isolated from staged microspores demonstrates changes in microspore gene expression during the same developmental period.

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