RESUMEN

A range of oligosaccharide ester derivatives (OEDs) have been designed as drug delivery matrices for controlled release. The synthetic hormone analogue, leuprolide, was encapsulated within these matrices using hydrophobic ion pairing and solvent spray drying. The particles produced modified the release of leuprolide in vitro (dissolution in phosphate buffered saline) and in vivo (subcutaneous and pulmonary delivery in the rat). Release rate was dependent on drug loading and could be manipulated by choice of OED and by combining different OEDs in different ratios. Leuprolide encapsulated in the OEDs retained biological activity as evidenced by elevation in plasma luteinising hormone levels following subcutaneous injection of leuprolide recovered from OED particles in vitro prior to in vivo administration.

Asunto(s)

Leuprolida/química , Oligosacáridos/química , Poliésteres/química , Administración por Inhalación , Animales , Cromatografía Líquida de Alta Presión , Preparaciones de Acción Retardada/química , Composición de Medicamentos , Estabilidad de Medicamentos , Inyecciones Subcutáneas , Lactosa/análogos & derivados , Lactosa/química , Leuprolida/sangre , Ratas , Temperatura , Trehalosa/análogos & derivados , Trehalosa/química

RESUMEN

To establish whether the thromboxane A2 (TXA2) receptor (TP) functionally couples to the Gq family of heterotrimeric G proteins in vivo, we have coexpressed the cDNAs coding for the human platelet/placental TP alpha isoform (TP alpha) and the alpha subunits of Gq or G11 in human embryonic kidney (HEK) 293 cells. TP activation in response to ligand stimulation was monitored by analyzing mobilization of intracellular calcium (Ca++i) in FURA2/AM-loaded transfected HEK 293 and in platelets. Second, we wished to examine the possible interaction of the isoprostane 8-epi prostaglandin F2 alpha with the TP alpha, in transfected HEK 293 cells and with the TPs expressed in platelets. Thus both the prostaglandin endoperoxide/TXA2 analog (U46619) and the 8-epi PGF2 alpha were utilized as ligand probes of TP alpha activation. The results demonstrate that each ligand induced elevations of Ca++i levels in HEK 293 cells, cotransfected with either the TP alpha and G alpha q or the TP alpha and G alpha 11, and also in platelets. Initial stimulation of these cells with U46619 or 8-epi PGF 2 alpha desensitized a subsequent rise in [Ca++]i in response to U46619 or 8-epi PGF 2 alpha, respectively. Moreover, prestimulation with U46619 desensitized a subsequent rise in Ca++i concentration in response to 8-epi PGF 2 alpha, and vice versa. These responses were blocked by the TP antagonist SQ29,548 in both cell types. In contrast, prestimulation of the transfected HEK 293 cells or platelets with thrombin did not desensitize a subsequent rise in [Ca++]i in response to U46619 or 8-epi PGF 2 alpha. After stimulation with either U46619 or 8-epi PGF 2 alpha, no significant rise in Ca++i levels was observed in HEK 293 cells transfected with the TP alpha receptor only or in control cells transfected with the vector pCMV5. These results demonstrate that the TP alpha isoform functionally couples with either Gq or G11 in vivo, whether activated by a PG/TXA2 analog or by the F2 isoprostane 8-epi PGF2 alpha.

Asunto(s)

Dinoprost/análogos & derivados , Proteínas de Unión al GTP/metabolismo , Receptores de Tromboxanos/efectos de los fármacos , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico , Calcio/metabolismo , Línea Celular , ADN Complementario , Dinoprost/farmacología , Humanos , Endoperóxidos de Prostaglandinas Sintéticos/farmacología , Receptores de Tromboxanos/genética , Receptores de Tromboxanos/metabolismo , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tromboxano A2/análogos & derivados , Tromboxano A2/farmacología

RESUMEN

The amino acid sequence of rat N-syndecan core protein was deduced from the cloned cDNA sequence. The sequence predicts a core protein of 442 amino acids with six structural domains: an NH2-terminal signal peptide, a membrane distal glycosaminoglycan attachment domain, a mucin homology domain, a membrane proximal glycosaminoglycan attachment domain, a single transmembrane domain, and a noncatalytic COOH-terminal cytoplasmic domain. Transfection of human 293 cells resulted in the expression of N-syndecan that was modified by heparan sulfate chain addition. Heparitinase digestion of the expressed proteoglycan produced a core protein that migrated on SDS-polyacrylamide gels at an apparent molecular weight of 120, 000, identical to N-syndecan synthesized by neonatal rat brain or Schwann cells. Rat genomic DNA coding for N-syndecan was isolated by hybridization screening. The rat N-syndecan gene is comprised of five exons. Each exon corresponds to a specific core protein structural domain, with the exception of the fifth exon, which contains the coding information for both the transmembrane and cytoplasmic domains as well as the 3'-untranslated region of the mRNA. The first intron is large, with a length of 22 kilobases. The expression of N-syndecan was investigated in late embryonic, neonatal, and adult rats by immunoblotting and Northern blotting analysis. Among the tissues and developmental stages studied, high levels of N-syndecan expression were restricted to the early postnatal nervous system. N-syndecan was expressed in all regions of the nervous system, including cortex, midbrain, spinal cord, and peripheral nerve. Immunohistochemical staining revealed high levels of N-syndecan expression in all brain regions and fiber tract areas.

Asunto(s)

Encéfalo/metabolismo , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/genética , Proteoglicanos/biosíntesis , Proteoglicanos/genética , Médula Espinal/metabolismo , Secuencia de Aminoácidos , Animales , Animales Recién Nacidos , Secuencia de Bases , Línea Celular , Mapeo Cromosómico , Clonación Molecular , ADN Complementario , Exones , Expresión Génica , Biblioteca Genómica , Humanos , Inmunohistoquímica , Intrones , Riñón , Glicoproteínas de Membrana/química , Datos de Secuencia Molecular , Señales de Clasificación de Proteína/química , Señales de Clasificación de Proteína/metabolismo , Proteoglicanos/química , Ratas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Sindecano-3 , Transfección

Asunto(s)

Regulación de la Expresión Génica , Regiones Promotoras Genéticas , Receptores de Tromboxanos/biosíntesis , Northern Blotting , Exones , Humanos , Leucemia Eritroblástica Aguda , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Receptores de Tromboxanos/genética , Mapeo Restrictivo , Células Tumorales Cultivadas

RESUMEN

Recombinant forms of the human thromboxane A2 (TXA2) receptor composed of the carboxyl-terminal amino acid residues 220-343 were phosphorylated in vitro by both cAMP-dependent protein kinase A (PKA) and protein kinase C (PKC), and these phosphorylations were competed for by synthetic peptides corresponding to the proposed carboxyl-terminal cytoplasmic tail and/or the third extracellular loop of the receptor, respectively. Exogenous addition of PKA or PKC to membrane preparations of human embryonic kidney 293 cells, transfected with the TXA2 receptor, typically reduced TXA2 receptor binding by 10 and 30%, respectively. In vivo inhibition of PKC or PKA in the transfected human embryonic kidney 293 cells increased TXA2 receptor binding to 121.4% (+/- 5.3%) and 110.4% (+/- 4.6%), respectively, relative to control cells. In vivo activation of PKC in the platelet-like human erythroleukemia (HEL) cells by the phorbol ester phorbol 12-myristate 13-acetate (PMA) resulted in an initial reduction followed by a time-dependent increase in TXA2 receptor ligand binding. Stimulation of HEL cells with the TXA2 receptor agonist [15-(alpha,2 beta(5Z)-3 alpha(E,3S)-4 alpha)]-7-[3- (3-hydroxy-4-(p-iodophenoxy)-1-butenyl)-7-oxabicyclo-[-2.2,1-]hept -2-yl]- 5-heptenoic acid or basic fibroblast growth factor, alone or together, resulted in a marked decrease in TXA2 receptor binding. Northern blot studies in HEL cells demonstrated that PMA stimulation induced the expression of the TXA2 receptor gene with mRNA levels peaking following PMA stimulation for 4-8 h. This induction is consistent with the presence of a phorbol ester response element in promoter I of the TXA2 receptor gene. Dexamethasone did not induce the expression of the receptor gene, despite the presence of a glucocorticoid response element in promoter II of the TXA2 receptor gene. In summary, our results indicate that the cellular responses to TXA2 are mediated both by phosphorylation of the TXA2 receptor by different protein kinases and by regulated expression of the TXA2 receptor gene.

Asunto(s)

Compuestos Bicíclicos Heterocíclicos con Puentes , Receptores de Tromboxanos/metabolismo , Compuestos Bicíclicos con Puentes/farmacología , Células Cultivadas , Clonación Molecular , Activación Enzimática , Ácidos Grasos Insaturados/farmacología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Regulación de la Expresión Génica , Humanos , Fosforilación , Inhibidores de Proteínas Quinasas , Proteínas Quinasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Tromboxanos/efectos de los fármacos , Receptores de Tromboxanos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Tumorales Cultivadas

RESUMEN

The heterotrimeric guanine nucleotide-binding G proteins, composed of α, ß, and γ subunits, act as signal transducers between cell surface receptors and downstream effector molecules, leading to changes in intracellular second messengers. The superfamily of ras-related low-molecular-mass GTP-binding G proteins is involved in a number of cellular functions, including cell differentiation and growth control, actin polymerization and cytoskeleton arrangement, and intracellular vesicular transport. The heterotrimeric G proteins and the ras-related low-molecular-mass G proteins are modified in vivo by a number of lipid groups, including palmitate, myristate, heterogeneous fatty-acyl groups (C12:0, C14:1, or C14:2 fatty-acyl groups), and C15 farnesyl or C20 geranylgeranyl isoprenoids. Lipid modification of G proteins increases the hydrophobicity of the proteins. In this review, we describe the various types of lipid modification of G proteins and discuss the significance of lipid modification with respect to G-protein function.

RESUMEN

We report on the influence of choice of promoter and RNA polymerase, 5'-untranslated regions and ribosome binding sites, codon usage, leader peptide coding sequences and poly A tail in the 3'-untranslated region on the synthesis of porcine somatotropin (PST) in Escherichia coli. A total of 12 different constructs were tested in this study for the production of porcine somatotropin (PST) in E. coli. Several factors have significant effects on PST synthesis. In the presence of a strong promoter and a strong ribosome binding site, the next most important factor seems to be the combination of sequences at the 5'-end of the mRNA including both the 5'-untranslated region and the start of the coding sequence. Codon usage in the 5'-coding sequence per se is not important in determining the level of PST synthesis where high level expression is achieved from a strong ribosome binding site. However, where low level synthesis of recombinant PST (rPST) is achieved, codon usage in the 5'-coding sequence is important in determining the level of PST synthesis. Leader sequences dramatically reduce the level of PST synthesis. The presence of a poly A tail in the 3'-untranslated region has no significant effect on PST synthesis.

Asunto(s)

Escherichia coli/metabolismo , Hormona del Crecimiento/biosíntesis , Animales , Secuencia de Bases , Biotecnología , Clonación Molecular , Codón/genética , Escherichia coli/genética , Expresión Génica , Vectores Genéticos , Hormona del Crecimiento/genética , Datos de Secuencia Molecular , Plásmidos/genética , Biosíntesis de Proteínas , Señales de Clasificación de Proteína/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Porcinos

RESUMEN

For efficient transcription from the rat ribosomal DNA (rDNA) promoter by RNA polymerase I in vitro, at least two transcription factors, rat UBF and rat SL-1, are required. Transcription cannot take place in vitro in the absence of SL-1. On the other hand, there is considerable difference of opinion concerning the necessity for UBF in in vitro transcription mediated by RNA polymerase 1, and the requirement for UBF is not clear. Mammalian cells code for UBF1 and UBF2, two forms of UBF that differ in HMG box-2, one of four HMG boxes or DNA-binding domains. We have used a monospecific antibody raised to recombinant rat UBF to determine whether UBF1 and UBF2 are required for RNA polymerase I-mediated transcription. This antibody can detect as little as 1.35 x 10(-15) moles of UBF1 or UBF2 in an immunoblot. Fractionated extracts that were competent for transcription had no detectable UBF1 or UBF2 when assayed in immunoblots with this antiserum. This evidence supports the hypothesis that UBF is not required for transcription of the rat rDNA promoter in vitro and most likely functions as an auxillary transcription factor. In addition, we have fractionated rat UBF1 from UBF2 and tested each of them in in vitro transcription assays in which the 45S or spacer rDNA promoter template is limiting. UBF1 can activate transcription from either the 45S or spacer promoter under these conditions, whereas UBF2 cannot. This implies that there is a functional difference in the transactivation of RNA polymerase I by UBF1 and UBF2 in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

ADN Ribosómico/genética , Proteínas de Unión al ADN/fisiología , Proteínas del Complejo de Iniciación de Transcripción Pol1 , Regiones Promotoras Genéticas , ARN Ribosómico/genética , Factores de Transcripción/fisiología , Transcripción Genética , Animales , Línea Celular , Chlorocebus aethiops , ADN de Neoplasias/genética , ADN Recombinante/genética , Fibroblastos/metabolismo , Neoplasias Hepáticas Experimentales , ARN Polimerasa I/metabolismo , Ratas , Células Tumorales Cultivadas

RESUMEN

To investigate the mechanism of transcription of the rat ribosomal DNA (rDNA) promoter, a series of 23 linker-scanning mutants were constructed and assayed in transfected CHO cells and with cell-free extracts. With minor variation, the results of the in vitro and in vivo assays paralleled one another. For example, these assays demonstrated that the mutagenesis of bases from -133 to -124, and those from -106 to -101 of the rDNA promoter significantly inhibited transcription both in vivo and in vitro. Both of these sites lie within the upstream promoter element (UPE) of the rDNA promoter. Several constructs, in particular one that mutated the bases between -49 and -45, were better promoters in vivo than the wild-type promoter. DNAse footprinting experiments with purified UBF, an RNA polymerase I transcription factor, demonstrated the importance of the bases between -106 and -101 for the binding of that factor, providing a positive correlation between the transcription experiments and the binding of UBF to the rDNA promoter.

Asunto(s)

ADN Ribosómico/genética , Proteínas de Unión al ADN/metabolismo , Proteínas del Complejo de Iniciación de Transcripción Pol1 , Regiones Promotoras Genéticas/genética , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Células CHO , Extractos Celulares , Cricetinae , ADN/metabolismo , Análisis Mutacional de ADN , Datos de Secuencia Molecular , Mutación/genética , ARN Polimerasa I/metabolismo , Ratas , Transfección/genética

RESUMEN

The phosphorylation, DNA-binding and dimerization properties of both forms of the RNA polymerase I transcription factor UBF were studied and compared. Tryptic peptide maps of in vivo 32P-labeled UBF contained four phospho-peptides. Two of these peptides are predicted to derive from the serine-rich, carboxyl-terminal of UBF. This region contains nine consensus phosphorylation sites for casein kinase II, and is one of the regions phosphorylated in vitro by casein kinase II. Analysis of the DNA-binding properties of recombinant forms of UBF1 and UBF2 by Southwestern blots revealed: (1) a role for the NH2-terminal 102 amino acid domain of UBF1/UBF2 in DNA-binding; (2) the importance of the bases from -106 to -101 of the rat ribosomal DNA promoter for the binding of UBF; and (3) functional differences between UBF1 and UBF2. Glutaraldehyde cross-linking and overlay assays using recombinant forms of UBF1 and UBF2 demonstrated that the molecules can form both homodimers and heterodimers. These assays also demonstrated that the NH2-terminal 102 amino acids of UBF plays a significant role in dimerization and that other domains contribute to dimerization. The dimerization properties of recombinant forms of UBF1 and UBF2 were different, suggesting that the HMG box 2 of UBF1, which is partially deleted in UBF2, also contributes to UBF dimerization.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Proteínas del Complejo de Iniciación de Transcripción Pol1 , ARN Polimerasa I/metabolismo , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Caseína Quinasas , Electroforesis en Gel de Poliacrilamida , Glutaral/farmacología , Datos de Secuencia Molecular , Mapeo Peptídico , Fosforilación , Regiones Promotoras Genéticas , Proteínas Quinasas/metabolismo , Ratas

RESUMEN

We have analyzed the expression, phosphorylation, and localization of the ribosomal DNA transcription factors UBF1 and UBF2 in Chinese hamster ovary cells in response to serum deprivation. In vivo labeling experiments demonstrate that UBF1 and UBF2 are phosphoproteins. Phosphoamino acid analysis of the in vivo labeled proteins demonstrate that UBF is phosphorylated on serine residues. Following serum deprivation there is no alteration in the cellular levels of UBF1 and UBF2 as determined by Western blotting, but there is an 80% reduction in the level of phosphorylation of UBF compared with logarithmically growing cells. Following serum deprivation there is a redistribution of UBF between the nucleolus, the nucleus, and the cytoplasm. Phosphatase-treated UBF demonstrated a reduced ability to rescue transcription by RNA polymerase I from the rDNA spacer promoter in vitro. These findings suggest that phosphorylation of UBF is a prerequisite for transactivation of RNA polymerase I.

Asunto(s)

Sangre , Proteínas de Unión al ADN/metabolismo , Proteínas del Complejo de Iniciación de Transcripción Pol1 , Factores de Transcripción/metabolismo , Fosfatasa Alcalina/metabolismo , Animales , Western Blotting , Células CHO , Cricetinae , Cricetulus , Proteínas de Unión al ADN/genética , Escherichia coli/enzimología , Escherichia coli/metabolismo , Técnica del Anticuerpo Fluorescente , Expresión Génica , Fosforilación , ARN Mensajero/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Factores de Transcripción/genética , Transcripción Genética

RESUMEN

Two transcription factors, rat UBF (rUBF) and rat SL-1 are required for the efficient transcription of the rat promoter in vitro. In vitro studies have established that two broadly defined cis-acting domains, the core promoter element and the upstream promoter element, cooperate to direct correct transcription by RNA polymerase I. The ability of UBF to bind to two linker-scanning mutants of the upstream promoter element, which did not respond to the addition of UBF in in vitro transcription assays, was assessed by DNase footprinting. UBF protected the same region of the promoter in the linker-scanning mutant in BSM 129/124 as it did in the wild-type, but did not yield a typical footprint over the promoter in the linker-scanning mutant BSM 106/101. Previously we reported that promoters with mutant core promoters elements, either the guanine at -16 or -7 substituted by an adenine, were inactive in vitro unless the assays were supplemented with UBF. Those results suggested that the binding of UBF upstream of the core was required for the promotion of transcription. The interactions between the core and upstream promoter elements were assessed by constructing double mutants of the promoter. In two constructs the conserved guanines at either -16 or -7 were altered in a deletion mutant (-86) that did not respond to UBF. In a third construct the guanine at -16 in BSM 129/124 was changed to an adenine. These bidomain mutant constructs did not respond to the addition of UBF in an in vitro transcription assay, confirming that the rescue of the core promoter mutants requires an intact and functional upstream promoter element.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

ADN Ribosómico/genética , Proteínas de Unión al ADN/metabolismo , Proteínas del Complejo de Iniciación de Transcripción Pol1 , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismo , Transcripción Genética , Animales , Secuencia de Bases , Línea Celular , Cricetinae , Regulación de la Expresión Génica , Prueba de Complementación Genética , Datos de Secuencia Molecular , Mutación , ARN Ribosómico/metabolismo , Ratas , Transfección

RESUMEN

The structure of the rat homologue of the RNA polymerase I transcription factor UBF was investigated. The sequence of the protein was deduced from the sequence of overlapping cDNAs isolated from a cDNA library and from clones of the products generated by the polymerase chain reaction from random-primed, first-strand cDNA. The sequences of these clones indicated that there were two mRNAs for UBF and that the encoded proteins were similar but not identical. One form of rat UBF was essentially identical to human UBF. The second class of UBF mRNA contained an in-frame "deletion" in the coding region that results in the deletion of 37 amino acids from the predicted protein sequence. This deletion reduces the predicted molecular size of the encoded form of UBF by approximately 4400 from 89.4 kDa to 85 kDa and significantly alters the structure of one of the four HMG-1 homology regions (HMG box-2) in that form of UBF. Evidence for the existence of two mRNAs in rat cells was confirmed by a probe protection assay, and we provide evidence that other vertebrate cells contain these same two forms of UBF mRNA. These results are consistent with the observation that UBF purified from four different vertebrates migrates as two bands upon SDS/PAGE. It has been hypothesized that the HMG motifs are the DNA-binding domains of UBF. Altering one of these "boxes," as in the second form of UBF, may alter the functional characteristics of the transcription factor. Thus, the existence of different forms of UBF may have important ramifications for transcription by RNA polymerase I.

Asunto(s)

Proteínas de Unión al ADN/genética , Proteínas del Complejo de Iniciación de Transcripción Pol1 , ARN Polimerasa I/metabolismo , Factores de Transcripción/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN/genética , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , Secuencias Reguladoras de Ácidos Nucleicos

RESUMEN

We have analyzed the inducible synthesis of recombinant porcine somatotropin (rPST) from the phage T7 gene 10 promotor on the vector pET3a [Rosenberg et al., Gene 56 (1987) 125-135]. Low-level synthesis of phage T7 lysozyme is crucial for high-level synthesis (40%) of rPST, which is greatly reduced if T7 lysozyme synthesis is absent or too high. The synthesis of rPST mRNA is optimized in those constructs coding for low levels of T7 lysozyme, with a reduction in mRNA levels in constructs coding for higher levels of T7 lysozyme or no lysozyme. The rPST can be readily purified following a single chromatographic step and is biologically active as determined by the tibia test following administration to hypophysectomized rats.

Asunto(s)

ARN Polimerasas Dirigidas por ADN/metabolismo , Hormona del Crecimiento/genética , Muramidasa/metabolismo , Regiones Promotoras Genéticas , Fagos T/enzimología , Animales , Escherichia coli/genética , Vectores Genéticos , Hormona del Crecimiento/biosíntesis , Hormona del Crecimiento/aislamiento & purificación , Plásmidos , Ratas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Porcinos , Fagos T/genética , Transcripción Genética

Asunto(s)

Escherichia coli/genética , Genes Bacterianos , Genes Virales , Vectores Genéticos , Biosíntesis de Proteínas , Fagos T/genética , Secuencia de Bases , Datos de Secuencia Molecular , Plásmidos , Mapeo Restrictivo , Temperatura

RESUMEN

Mutations in the acceptor stem, the 5-methyluridine-pseudouridine-cytidine (TFC) arm, and the anticodon of Salmonella tRNA2Gly can cause -1 frameshifting. The potential for standard base pairing between acceptor stem positions 1 and 72 is disrupted in the mutant sufS627. This disruption may interfere with the interaction of the tRNA with elongation factor-Tu.GTP or an as-yet-unspecified domain of the ribosome. The potential for standard base pairing in part of the TFC stem is disrupted in mutant sufS625. The nearly universal C-61 base of the TFC stem is altered in mutant sufS617, and the TFC loop is extended in mutant sufS605. These changes are expected to interfere with the stability of the TFC loop and its interaction with the D arm. The mutation in mutant sufS605, and possibly other mutants, alters nucleoside modification in the D arm. Three mutants, sufS601, sufS607, and sufS609, have a cytidine substituted for the modified uridine at position 34, the first anticodon position. None of the alterations grossly disrupts in-frame triplet decoding by the mutant tRNAs. The results show that -1 frameshifting in vivo can be caused by tRNAs with normal anticodon loop size and suggest that alternative conformational states of the mutant tRNAs may allow them to read a codon in frame or to shift reading frame.

Asunto(s)

Anticodón/genética , ADN Bacteriano/genética , Mutación , ARN de Transferencia Aminoácido-Específico/genética , ARN de Transferencia de Glicerina/genética , ARN de Transferencia/genética , Salmonella/genética , Secuencia de Bases , Clonación Molecular , Datos de Secuencia Molecular , Conformación de Ácido Nucleico

RESUMEN

sufS was found to suppress the only known suppressible-1 frameshift mutation, trpE91, at a site identified as GGA and mapped within the single gene of the only tRNA that can decode GGA in Escherichia coli. It mapped to the same gene in Salmonella typhimurium. sufS alleles were recessive, and dominant alleles could not be isolated. This is in contrast to all other tRNA structural gene mutations identified thus far that cause frameshift suppression. The recessiveness implies that all sufS alleles are poor competitors against their wild-type tRNA(Gly2) counterparts. The base G immediately 5' of the GGA suppression site influenced the level but was not critical for suppression by sufS601. From this result, it is inferred that sufS601 causes frameshifting by doublet decoding.

Asunto(s)

Codón/genética , Genes Bacterianos , Genes Recesivos , Mutación , ARN Mensajero/genética , ARN de Transferencia/genética , Supresión Genética , Alelos , Secuencia de Bases , Escherichia coli/genética , Datos de Secuencia Molecular , Mapeo Nucleótido , ARN Bacteriano/genética , Salmonella/genética

RESUMEN

Three new full-length cDNAs coding for porcine somatotropin (PST) have been cloned. The sequence data indicate a high degree of polymorphism in the PST sequence. All six known PST sequences are different.

Asunto(s)

ADN/ultraestructura , Hormona del Crecimiento/genética , Polimorfismo Genético , Animales , Clonación Molecular , Codón , Técnicas In Vitro , Porcinos