RESUMEN
We have identified, cloned and sequenced three tuf-like genes from Streptomyces ramocissimus (Sr.), the producer of the antibiotic kirromycin which inhibits protein synthesis by binding the polypeptide chain elongation factor EF-Tu. The tuf-1 gene encodes a protein with 71% amino acid residues identical to the well characterized elongation factor Tu of Escherichia coli (Ec.EF-Tu). The genetic location of tuf-1 downstream of a fus homologue and the in vitro activity of Sr.EF-Tu1 show that tuf-1 encodes a genuine EF-Tu. The putative Sr.EF-Tu2 and Sr.EF-Tu3 proteins are 69% and 63% identical to Ec.EF-Tu. Homologues of tuf-1 and tuf-3 were detected in all five Streptomyces strains investigated, but tuf-2 was found in S. ramocissimus only. The three tuf genes were expressed in E. coli and used to produce polyclonal antibodies. Western blot analysis showed that Sr.EF-Tu1 was present at all times under kirromycin production conditions in submerged and surface-grown cultures of S. ramocissimus and in germinating spores. The expression of tuf-2 and tuf-3 was, however, below the detection level. Surprisingly, Sr.EF-Tu1 was kirromycin sensitive, which excludes the possibility that EF-Tu is involved in the kirromycin resistance of S. ramocissimus.
Asunto(s)
Proteínas Bacterianas/genética , Genes Bacterianos , Factor Tu de Elongación Peptídica/genética , Streptomyces/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/biosíntesis , Secuencia de Bases , Clonación Molecular , Farmacorresistencia Microbiana/genética , Escherichia coli/genética , Regulación de la Expresión Génica , Datos de Secuencia Molecular , Factor Tu de Elongación Peptídica/biosíntesis , Piridonas/metabolismo , Piridonas/farmacología , Proteínas Recombinantes de Fusión/biosíntesis , Alineación de Secuencia , Homología de Secuencia , Especificidad de la Especie , Esporas Bacterianas , Streptomyces/efectos de los fármacos , Streptomyces/metabolismo , Streptomyces/fisiologíaRESUMEN
A method is described for obtaining transgenic plants with a high level of expression of the introduced gene. Tobacco protoplasts were transformed with an expression construct containing a translational fusion between mature alpha-amylase from Bacillus licheniformis and the signal peptide of the tobacco PR-S protein. A total number of 5200 transformed protoplasts was cultured to microcalli and screened for alpha-amylase expression by incubation on media containing starch followed by staining with iodine. The calli were divided into four classes, based on the resulting halo sizes on the plates. The halo sizes were found to correlate with the expression levels in transgenic plants regenerated from the calli. The expression levels varied between 0 and 0.5% of soluble leaf protein in the regenerated transgenic plants. Wider implications of this method are discussed.
Asunto(s)
Nicotiana/enzimología , Plantas Modificadas Genéticamente/enzimología , Plantas Tóxicas , alfa-Amilasas/genética , Bacillus/enzimología , Bacillus/genética , Expresión Génica/genética , Plantas Modificadas Genéticamente/genética , Plásmidos/genética , Señales de Clasificación de Proteína/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Nicotiana/genética , Transformación Genética/genética , alfa-Amilasas/metabolismoRESUMEN
As a first example of the feasibility of producing industrial bulk enzymes in plants, we have expressed Bacillus licheniformis alpha-amylase in transgenic tobacco, and applied the seeds directly in starch liquification. The enzyme was properly secreted into the intercellular space, and maximum expression levels of about 0.3% of total soluble protein were obtained. No apparent effect of the presence of the enzyme on plant phenotype was observed. The molecular weight of the enzyme produced in tobacco was around 64 kD. The difference, compared to 55.2 kD for the bacterial enzyme, was found to result from complex-type carbohydrate chains attached to the protein. Application studies on the liquefaction of starch were done with transgenic seeds containing the recombinant alpha-amylase. The resulting hydrolysis products were virtually identical with those obtained from degradation with alpha-amylase from Bacillus licheniformis.
Asunto(s)
Bacillus/enzimología , Nicotiana/enzimología , Plantas Tóxicas , Almidón/metabolismo , alfa-Amilasas/metabolismo , Proteínas Bacterianas/metabolismo , Espacio Extracelular/metabolismo , Vectores Genéticos/genética , Glicosilación , Fenotipo , Plantas Modificadas Genéticamente/enzimología , Transformación Genética/genéticaRESUMEN
We have developed the yeast Kluyveromyces lactis as a host organism for the production of the milk-clotting enzyme chymosin. In contrast to Saccharomyces cerevisiae, we found that this yeast is capable of the synthesis and secretion of fully active prochymosin. Various signal sequences could be used to efficiently direct the secretion of prochymosin in Kluyveromyces, but not in S. cerevisiae. We conclude that the efficient synthetic and secretory capacity of this heterologous protein is a property of the yeast Kluyveromyces. These results have led to the development of a large scale production process for chymosin.
Asunto(s)
Quimosina/biosíntesis , Precursores Enzimáticos/biosíntesis , Kluyveromyces/genética , Proteínas Recombinantes de Fusión/biosíntesis , Saccharomycetales/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , Quimosina/genética , Quimosina/metabolismo , Clonación Molecular , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Kluyveromyces/metabolismo , Datos de Secuencia Molecular , Plásmidos , Procesamiento Proteico-Postraduccional , Señales de Clasificación de Proteína/genética , Señales de Clasificación de Proteína/fisiología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Especificidad de la EspecieRESUMEN
Tertiary interactions involving hairpin or interior loops of RNA can lead to extended quasi-continuous double helical stem regions, consisting of coaxially stacked segments of duplex RNA, bridged by single-stranded connections. This type of compact folding plays a role in various strategic regions of RNA molecules. Their role in ribosome functioning, RNA splicing and recognition of tRNA-like structures is discussed.
Asunto(s)
Modelos Genéticos , Conformación de Ácido Nucleico , ARN Viral/análisis , Secuencia de Bases , Computadores , Empalme del ARN , ARN de Transferencia/análisis , Ribosomas/fisiologíaRESUMEN
The structure of the tRNA-like 3' terminus of tobacco mosaic virus (TMV) RNA has been studied. A 3' -terminal fragment possessing the tRNA-like properties was probed with chemical modification and enzymatic digestions. A model of the secondary structure is proposed for the last 105 nucleotides. The corresponding region of other tobamoviral RNAs can be folded in an identical secondary structure. A three-dimensional model for the tRNA-like structure is given which is compared with those proposed earlier for the tRNA-like 3' termini of turnip yellow mosaic virus (TYMV) RNA and brome mosaic virus (BMV) RNA. A new building principle which we discovered previously by studying the latter RNAs appears to be applied twice in the tRNA-like structure of TMV RNA. The determination of the minimal length requirement for recognition of CTP, ATP:tRNA nucleotidyl-transferase reveals a size of 100 nucleotides in agreement with the models proposed.
RESUMEN
Various plant viral RNAs possess a 3' terminus with tRNA-like properties. These viral RNAs are charged with an amino acid upon incubation with the cognate aminoacyl-tRNA synthetase and ATP. We have studied the structure of end-labelled 3'-terminal fragments of turnip yellow mosaic virus RNA and brome mosaic virus RNA 2 with chemical modifications of the adenosine and cytidine residues and with enzymatic digestions using RNase T1, nuclease S1 and the double-strand-specific ribonuclease from cobra venom. The data indicate that the 3' termini of these plant viral RNAs lack a cloverleaf structure as found in classical tRNA. The three-dimensional folding, however, reveals a striking resemblance with classical tRNA. The models proposed are supported by phylogenetic data. Apparently distinct three-dimensional solutions have evolved to meet the requirements for faithful recognition by tRNA-specific enzymes. The way in which the aminoacyl acceptor arms of these tRNA-like structures are constructed reveal novel features in RNA folding which may have a bearing on the secondary and tertiary structures of RNA in general. The dynamic behaviour of brome mosaic virus RNA 2 in solution presumably is illustrative of conformational transitions, which RNAs generally undergo on changing the ionic conditions.
Asunto(s)
Virus del Mosaico/análisis , ARN de Transferencia , ARN Viral , Modelos Moleculares , Conformación de Ácido NucleicoRESUMEN
The 3' terminus of TYMV RNA, which possesses tRNA-like properties, has been studied. A 3' terminal fragment of 112 nucleotides was obtained by cleavage with RNase H after hybridization of a synthetic oligodeoxynucleotide to the viral RNA. The accessibility of cytidine and adenosine residues was probed with chemical modification. Enzymatic digestion studies were performed with RNase T1, nuclease S1 and the double-strand specific RNase from the venom of the cobra Naja naja oxiana. A model is proposed for the secondary structure of the 3' terminal region of TYMV RNA comprising 86 nucleotides. The main feature of this secondary structure is the absence of a conventional acceptor stem as present in canonical tRNA. However, the terminal 42 nucleotides can be folded in a tertiary structure which bears strong resemblance with the acceptor arm of canonical tRNA. Comparison of this region of TYMV RNA with that of other RNAs from both the tymovirus group and the tobamovirus group gives support to our proposal for such a three-dimensional arrangement. The consequences for the recognition by TYMV RNA of tRNA-specific enzymes is discussed.
Asunto(s)
Virus del Mosaico/genética , ARN de Transferencia/genética , ARN Viral/genética , Secuencia de Bases , Conformación de Ácido Nucleico , Hibridación de Ácido Nucleico , Ribonucleasas/metabolismo , Especificidad por SustratoRESUMEN
The spectrum of mutations induced by ionizing radiation (gamma-rays) was determined in the lacI gene of E. coli. Base substitution was the principal type of mutational event following ionizing radiation. Both transitions and transversions were produced, and no strong specificity for a particular base pair was observed. The spectra of spontaneous and of ionizing-radiation-induced base-pair changes differed significantly at several locations within the lacI gene. The location of 3 of these differences corresponded to sites of spontaneous deamination "hot spots" from which we conclude that gamma-rays do not cause extensive deamination. The specific locus rate was calculated as 4.5 X 10(-10) mutations per rad per gene copy per cell, and the nucleotide substitution rate was 2.2 X 10(-12) per rad. The frameshift mutation, trpE997, was not reverted by gamma-rays.
Asunto(s)
Escherichia coli/genética , Operón Lac/efectos de la radiación , Mutación , Proteínas Represoras/genética , Factores de Transcripción/genética , Composición de Base/efectos de la radiación , ADN Bacteriano/efectos de la radiación , Rayos gamma , Código Genético , Transcripción GenéticaRESUMEN
Late after infection of KB cells with adenovirus 5 an extra protein becomes associated with messenger ribonucleoprotein particles present in the polysomes. This protein has a molecular weight of 100000 and is identical to the virus coded '100K' protein found previously. The extra protein is firmly attached to the messenger ribonucleoprotein complexes. Its binding resists exposure to high salt concentrations as used in puromycin/high-salt dissociation and equilibrium centrifugation in Cs2SO4 gradients. In this respect it resembles the binding of two other proteins of Mr 74000 and 48000 which are commonly found in messenger ribonucleoprotein particles of various eukaryotic cells. The identity between the messenger ribonucleoprotein protein of Mr 100000 and the "100K' protein present in the soluble part of the cytoplasm was established by sodium dodecylsulphate/polyacrylamide gel electrophoresis, isoelectric focusing and peptide mapping after limited proteolysis with Staphylococcus aureus protease.