RESUMEN

Histidine triad nucleotide-binding (HinT) proteins are dimeric proteins that bind to purines and are found in all three kingdoms: the eukarya, bacteria and archaea. In eukaryotes, HinT proteins have been detected intracellularly, but their function is unknown. Until now, knowledge about HinT proteins in prokaryotes was restricted to sequence similarities and nucleotide-binding studies. In this study, we provide evidence that, in the cell wall-less prokaryote, Mycoplasma hominis, the gene encoding the HinT protein forms an operon with two other genes. These genes encode the species-specific membrane proteins, P60 and P80, which are associated within the mycoplasma membrane. The finding that HinT interacts with this complex by binding to P80 provides novel insight into the organization of bacterial HinT proteins.

Asunto(s)

Proteínas Bacterianas/metabolismo , Proteínas Portadoras/genética , Citosol/metabolismo , Lipoproteínas/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mycoplasma hominis/metabolismo , Proteínas Bacterianas/genética , Proteínas Portadoras/metabolismo , Membrana Celular/metabolismo , Clonación Molecular , Lipoproteínas/genética , Datos de Secuencia Molecular , Mycoplasma hominis/genética , Mycoplasma hominis/crecimiento & desarrollo , Pruebas de Precipitina , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Resonancia por Plasmón de Superficie

RESUMEN

AMP-activated protein kinase (AMPK) and cAMP-dependent protein kinase (cAMPK) have been reported to phosphorylate sites on phosphorylase kinase (PhK). Their target residues Ser 1018 and Ser 1020, respectively, are located in the so-called multi-phosphorylation domain in the PhK alpha subunit. In PhK preparations, only one of these serines is phosphorylated, but never both of them. The aim of this study was to determine whether phosphorylation by cAMPK or AMPK would influence subsequent phosphorylation by the other kinase. Surprisingly, employing four different PhK substrates, it could be demonstrated that, in contradiction to previous reports, PhK is not phosphorylated by AMPK.

Asunto(s)

Complejos Multienzimáticos/metabolismo , Músculo Esquelético/enzimología , Fosforilasa Quinasa/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Quinasas Activadas por AMP , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cartilla de ADN , Datos de Secuencia Molecular , Complejos Multienzimáticos/aislamiento & purificación , Fosforilasa Quinasa/aislamiento & purificación , Fosforilación , Proteínas Serina-Treonina Quinasas/aislamiento & purificación , Conejos , Especificidad por Sustrato

RESUMEN

Mycoplasma hominis, a cell-wall-less prokaryote, was shown to be cytoadherent by the participation of a 100-kDa membrane protein (P100). To identify the gene encoding P100, peptides of P100 were partially sequenced to enable the synthesis of P100-specific oligonucleotides suitable as probes for the detection of the P100 gene. With this strategy, we identified a genomic region of about 10. 4 kb in M. hominis FBG carrying the P100 gene. Analysis of the complete deduced protein sequence suggests that P100 is expressed as a pre-lipoprotein with a structure in the N-terminal region common to peptide-binding proteins and an ATP- or GTP-binding P-loop structure in the C-terminal region. Downstream of the P100 gene, an additional four open reading frames putatively encoding the four core domains of an active transport system, OppBCDF, were localized. The organization of the P100 gene and oppBCDF in a transcriptionally active operon structure was demonstrated in Northern blot and reverse transcription-PCR analyses, as all gene-specific probes detected a common RNA of 9.5 kb. Primer extension analysis revealed that the transcriptional initiation site was localized 323 nucleotides upstream of the methionine-encoding ATG of the P100 gene. The peptide-binding character of the P100 protein was confirmed by fluorescence spectroscopy and strongly suggests that the cytoadherence-mediating lipoprotein P100 represents OppA, the substrate-binding domain of a peptide transport system in M. hominis.

Asunto(s)

Proteínas Bacterianas , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Lipoproteínas/genética , Lipoproteínas/metabolismo , Proteínas de Transporte de Membrana , Mycoplasma hominis/genética , Secuencia de Aminoácidos , Adhesión Bacteriana/genética , Secuencia de Bases , Transporte Biológico/fisiología , Northern Blotting , Proteínas Portadoras/análisis , Mapeo Cromosómico , Lipoproteínas/análisis , Datos de Secuencia Molecular , Mycoplasma hominis/metabolismo , Sondas de Oligonucleótidos , Oligopéptidos/metabolismo , Operón/genética , Plásmidos , ARN Bacteriano/análisis , ARN Mensajero/análisis , Espectrometría de Fluorescencia

RESUMEN

Based on the partial peptide sequence of inositol 1,4, 5-trisphosphate 3-kinase purified with 135 000-fold enrichment from chicken erythrocytes, cDNA-fragments were cloned by RT-PCR using degenerate oligonucleotides. Subsequent hybridization screening of an embryonic chicken cDNA library and 5'-RACE yielded a cDNA-contig of 2418 bp, encoding a 452 amino acid protein. The amino acid sequence shows the highest degree of homology with A-isoforms of inositol 1,4,5-trisphosphate 3-kinase (65% identities), whereas homology towards B and C isoforms was lower (57% and 52% amino acid identities respectively). These findings reveal a new tissue-specific pattern of A-isoform expression, a form which so far has only been found in brain and testes. Two overlapping lambda-genomic clones for chicken inositol 1,4,5-trisphosphate 3-kinase, isolated by hybridization screening, covered 18 499 bp of genomic sequence. This contig included four exons: three of them were present in all cDNA clones, whereas one was only represented in a single cDNA clone. In addition, the sequence of the latter differed from the other cDNAs by an in-frame deletion of 72 bp within the coding region for the catalytic domain of the enzyme. This divergent cDNA suggests the existence of alternative splice products, at least in embryonic tissue.A comparison of the position of introns, with the respective introns known from the corresponding gene from Caenorhabditis elegans, revealed a high degree of conservation of intron positions between vertebrates and invertebrates. Functional data for the enzyme suggests that the conserved exons represent defined functional protein modules.

Asunto(s)

Eritrocitos/enzimología , Intrones/genética , Invertebrados/genética , Isoenzimas/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Vertebrados/genética , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Pollos , Clonación Molecular , Secuencia Conservada , ADN Complementario/química , ADN Complementario/genética , ADN Recombinante , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Genes/genética , Biblioteca Genómica , Invertebrados/enzimología , Isoenzimas/aislamiento & purificación , Datos de Secuencia Molecular , Fosfotransferasas (Aceptor de Grupo Alcohol)/aislamiento & purificación , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido

RESUMEN

The clp60 gene encoding P60, a conserved lipoprotein of Mycoplasma hominis, was cloned and sequenced from both the type strain PG21 and the isolate FBG. Both open reading frames were identical in length, comprising 1746 nucleotides. The deduced amino acid sequences differed in 16 out of 582 amino acids. As expected, none of these divergences mapped within the epitope that was recognized by mAb CG4 in all of the 198 isolates of M. hominis analyzed so far. This conserved epitope was narrowed down to amino acids 454 through 464 within the C terminus of P60. For the expression of the recombinant homolog P60, P60rec, in E. coli the TGA codons of clp60 were substituted for TGG codons prior to cloning of clp60 into the expression plasmid pQE41. The expression of P60rec as a fusion protein with dihydrofolate reductase carrying an N-terminal His-tag enabled the purification of large amounts of P60rec in a soluble form.

Asunto(s)

Proteínas Bacterianas/metabolismo , Escherichia coli/genética , Lipoproteínas/genética , Proteínas de la Membrana/metabolismo , Mycoplasma hominis/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Secuencia de Bases , Clonación Molecular , Mapeo Epitopo , Lipoproteínas/química , Lipoproteínas/aislamiento & purificación , Proteínas de la Membrana/química , Proteínas de la Membrana/aislamiento & purificación , Datos de Secuencia Molecular , Plásmidos , Transcripción Genética

RESUMEN

A Toxoplasma gondii tachyzoite expression library was screened with immune sera from T. gondii infected patients. Among others, one gene product reacted strongly with human sera and was further investigated. The gene called B10 was shown to encode a 41 kDa antigen. The complete genomic nucleotide sequence of the B10 protein has been analysed and was shown to contain one intron with conserved splice junctions. Southern blot analysis indicated that B10 is a single-copy gene. The corresponding 1.5 kb cDNA encodes a 318 amino acid sequence of mainly hydrophilic character with a putative signal sequence of 19 amino acids and no further trans-membrane domain. Immunofluorescence assays and immunoblots with a preparation of excreted-secreted antigens (ESA) suggested that the native protein is secreted into the parasitophorous vacuole and its delimiting membrane, indicating that B10 is a member of the ESA family of T. gondii. Recombinant B10 protein exhibited a strong reactivity with human serum samples both in ELISA and in immunoblots.

Asunto(s)

Antígenos de Protozoos/genética , Genes Protozoarios , Proteínas Protozoarias/genética , Toxoplasma/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antiprotozoarios/sangre , Especificidad de Anticuerpos , Antígenos de Protozoos/química , Secuencia de Bases , Northern Blotting , Southern Blotting/métodos , Línea Celular , ADN Complementario/análisis , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente Directa , Humanos , Immunoblotting , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Proteínas Protozoarias/química , Conejos , Proteínas Recombinantes/biosíntesis , Toxoplasmosis/inmunología , Toxoplasmosis/parasitología

RESUMEN

The gene encoding p50, an adhesin of Mycoplasma hominis, was identified, cloned, and sequenced. Comparison of the derived amino acid sequence with the N-terminal amino acids sequenced by the Edman reaction of the native protein revealed that p50 is expressed as a 467-amino-acid precursor. Posttranslational modification leads to a 441-amino-acid lipoprotein with an extended, predominantly helical structure and a leucine zipper. Computer analysis of the amino acid sequence identified a threefold-repetitive sequence motif comprising approximately three-quarters of the total protein. Different regions of the p50 polypeptide chain were expressed in Escherichia coli. Western blot (immunoblot) analysis of the E. coli lysates revealed that the epitopes of four p50-specific monoclonal antibodies were localized in the middle and C-terminal part of the protein. Epitope mapping by exonuclease III digestion showed that all of the four monoclonal antibodies bound within the same region of the threefold-repetitive amino acid sequence motif. The repeats, which were highly homologous but not identical in structure, could be differentiated by the monoclonal antibodies.

Asunto(s)

Adhesinas Bacterianas/química , Anticuerpos Monoclonales/inmunología , Mycoplasma hominis/química , Adhesinas Bacterianas/genética , Adhesinas Bacterianas/inmunología , Secuencia de Aminoácidos , Secuencia de Bases , Mapeo Epitopo , Datos de Secuencia Molecular , Transcripción Genética