RESUMEN
Random peptide libraries displayed on the surface of filamentous bacteriophage are widely used as tools for the discovery of ligands for biologically relevant macromolecules, including antibodies, enzymes, and cell surface receptors. Phage display results in linkage of an affinity-selectable function (the displayed peptide) to the DNA encoding that function, allowing selection of individual binding clones by iterative cycles of in vitro panning and in vivo amplification. Critical to the success of a panning experiment is the complexity of the library: the greater the diversity of clones within the library, the more likely the library contains sequences that will bind a given target with useful affinity. A method for construction of high-complexity (> or = 10(9) independent clones) random peptide libraries is presented. The key steps are highly efficient binary ligation under conditions where the vector is relatively dilute, with only a modest molar excess of insert, followed by efficient electrotransformation into Escherichia coli. Library design strategies and a protocol for rapid sequence characterization are also presented.
Asunto(s)
ADN/química , Técnicas Genéticas , Biblioteca de Péptidos , Secuencia de Aminoácidos , Secuencia de Bases , Mapeo Epitopo/métodos , Escherichia coli , Biblioteca de Genes , Cinética , Datos de Secuencia Molecular , Plásmidos/metabolismo , TransfecciónRESUMEN
BACKGROUND: The accuracy of measurement of curves in idiopathic scoliosis has been extensively studied; however, we know of only one article in the literature concerning the accuracy of measurement of curves in congenital scoliosis. That article stated that intraobserver variability was +/- 9.6 degrees and interobserver variability was +/- 11.8 degrees. METHODS: Sixty-nine curves in fifty patients with congenital scoliosis were measured on two separate occasions by seven different observers with varying experience in curve measurement. RESULTS: Mean intraobserver variance ranged from 1.9 degrees to 5.0 degrees, with an average of 2.8 degrees (95% confidence limit, +/- 3 degrees) for the seven observers. The interobserver variance was 3.35 degrees (95% confidence limit, 7.86 degrees). CONCLUSIONS: It is possible to measure curves in congenital scoliosis with much greater accuracy than previously reported. In the clinical situation in which a skilled observer can measure two radiographs at the same time, an accuracy of +/- 3 degrees can be expected 95% of the time.
Asunto(s)
Escoliosis/congénito , Escoliosis/diagnóstico por imagen , Columna Vertebral/diagnóstico por imagen , Historia Moderna 1601- , Humanos , Variaciones Dependientes del Observador , Radiografía , Reproducibilidad de los ResultadosRESUMEN
Selenocysteine (Sec) incorporation requires the TGA opal codon and a downstream Sec insertion sequence (SECIS), which can be partially randomized and cloned into M13 pIII fusion constructs for phage display. This combinatorial approach provides a convenient non-radioactive assay that couples phage production to opal suppression. Two SECIS libraries were prepared, with the immediate downstream nucleotide either randomized (TGAN) or fixed as thymidine (TGAT). The TGAN library resulted in a majority of clones with a downstream purine and selenium-independent phage production, implicating the endo-genous tryptophan-inserting opal suppression pathway. Although the addition of sodium selenite to the growth medium did not affect phage production, it did increase the level of Sec insertion, as shown by the chemical reactivity of the resulting phage. The TGAT phage library yielded clones with strictly selenium-dependent phage production and reactivity consistent with the presence of Sec. These clones were prone to spontaneous mutation upon further propagation, however, resulting in loss of the selenium-dependent phenotype. We conclude that the immediate downstream nucleotide determines whether the endogenous opal suppression pathway competes with co-translational Sec insertion.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Proteínas de Escherichia coli , Escherichia coli/genética , Proteínas de Transporte de Monosacáridos , Nucleótidos/genética , Proteínas/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/genética , Selenocisteína/genética , Selenocisteína/metabolismo , Secuencia de Aminoácidos , Bacteriófago M13/efectos de los fármacos , Bacteriófago M13/genética , Bacteriófago M13/crecimiento & desarrollo , Bacteriófago M13/metabolismo , Secuencia de Bases , Proteínas Portadoras/química , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Clonación Molecular , Codón/genética , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Escherichia coli/virología , Vectores Genéticos/genética , Proteínas de Unión a Maltosa , Mutación/genética , Biblioteca de Péptidos , Biosíntesis de Proteínas/genética , Proteínas/química , Proteínas/genética , Purinas/metabolismo , Distribución Aleatoria , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Selenio/metabolismo , Selenio/farmacología , Selenoproteínas , Análisis de Secuencia de Proteína , Selenito de Sodio/metabolismo , Selenito de Sodio/farmacología , Supresión Genética/genética , Timidina/genética , Timidina/metabolismo , Triptófano/genética , Triptófano/metabolismoRESUMEN
Random peptide libraries are displayed on filamentous bacteriophage as fusions to either the minor coat protein, pIII, or the major coat protein, pVIII. We have devised a means of isolating the peptide displayed on a phage clone by transferring it to the N-terminus of the maltose-binding protein (MBP) of Escherichia coli encoded by malE. Transfer of a peptide sequence to monomeric MBP eliminates phage-encoded amino acids downstream of the insert peptide as well as avidity effects caused by multivalent display on phage. Peptide:MBP fusions are also easily affinity purified on amylose columns. The pMal-p2 vector was engineered to accept phage DNA encoding pIII- and pVIII-displayed peptides fused to their respective leader sequences. Both types of leader sequence were shown to target the peptide:MBP fusions to the periplasm of E. coli. A streamlined procedure for transferring peptides to MBP was applied to clones that had been isolated from a panel of pVIII-displayed peptide libraries by screening with an HIV-1-specific monoclonal antibody (Ab). By enzyme-linked immunosorbent assay, the Ab bound each of the peptide:MBP fusions and required the presence of a disulfide bridge within each peptide. Some of the peptide:MBP fusions were also analyzed using surface plasmon resonance. Thus, our study shows the value of malE fusion vectors in characterizing phage-displayed peptides.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Proteínas Portadoras/genética , Proteínas de Escherichia coli , Proteínas de Transporte de Monosacáridos , Biblioteca de Péptidos , Proteínas de Unión Periplasmáticas , Secuencia de Aminoácidos , Bacteriófagos/genética , Secuencia de Bases , Proteínas Portadoras/metabolismo , Clonación Molecular , ADN Recombinante , Escherichia coli , Vectores Genéticos , Anticuerpos Anti-VIH/inmunología , VIH-1/inmunología , Humanos , Proteínas de Unión a Maltosa , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Procesamiento Proteico-Postraduccional , Señales de Clasificación de Proteína/genética , Señales de Clasificación de Proteína/metabolismo , Proteínas Recombinantes de Fusión/genéticaRESUMEN
STUDY DESIGN: A review of a cohort of 310 consecutive patients who underwent anterior spinal fusion was performed to evaluate the accuracy of hospital ICD-9-CM complication coding. OBJECTIVES: To better understand the clinical significance of conclusions suggested by studies that rely on electronic administrative databases for their data source. SUMMARY OF BACKGROUND DATA: Despite their availability, there have been no studies to date that have evaluated the accuracy of ICD-9-CM administrative databases as they relate to the actual clinical experience in spinal procedures. METHODS: A physician and a research technician independently reviewed the primary medical records for the occurrence of complications. This data was compared with the hospital-acquired ICD-9-CM coded complications. RESULTS: The physician reviewer identified 152 complications in 119 patients, with 32 different types of complications. The research abstracter identified 175 complications in 130 patients, with 34 different types of complications identified. Hospital ICD-9-CM coding identified 105 complications in 80 patients, including only 11 different ICD-9-CM codes. Overall, 27% of ICD-9-CM complication codes were listed as "unspecified or unclassified complications, reactions, or misadventures," and contained no meaningful clinical information. Cardiac and pulmonary complications were over-estimated and wound infections and genitourinary and gastrointestinal complications were underestimated by ICD-9-CM coding. CONCLUSIONS: Studies of complications of spinal procedures using data derived from hospital ICD-9-CM complication codes may be intrinsically flawed because the data available to researchers from these electronic databases may be inaccurate.
Asunto(s)
Sistemas de Información en Hospital/normas , Fusión Vertebral/efectos adversos , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Complicaciones Posoperatorias , Estudios RetrospectivosRESUMEN
We describe the construction and uses of a set of four multipurpose cloning vectors: LITMUS 28, 29, 38 and 39. The vectors feature the high-copy pUC origin and an M13 origin for single-stranded DNA production as well as polylinker sites for most commercially available restriction enzymes that recognize nondegenerate hexanucleotide sites and yield 4-base sticky ends upon cleavage. Sites are arranged, without overlaps, to permit linker addition to blunt-ended fragments and unidirectional nested deletions and are within the lacZ alpha gene to facilitate blue-white screening. Finally, the polylinkers are flanked by a pair of opposing modified T7 promoters to allow in vitro transcription of either strand of a cloned insert with T7 RNA polymerase. Selective unidirectional transcription from one promoter is achieved by cleaving the other at an internal restriction site (AflII or SpeI). Both modified promoters are fully active under standard RNA probe synthesis conditions. In Southern blots of Dirofilaria immitis genomic DNA, an RNA probe prepared from LITMUS performed equivalently to the same RNA probe made from a wild-type promoter vector and a DNA probe prepared by random priming.
Asunto(s)
Clonación Molecular , Vectores Genéticos , Transcripción Genética , Secuencia de Bases , Southern Blotting , Datos de Secuencia Molecular , Sondas ARNRESUMEN
Protein splicing involves the excision of an internal domain from a precursor protein and the ligation of the external domains so as to generate two new proteins. Study of this process has recently been facilitated by the isolation of a precursor and a branched intermediate from a thermophilic protein splicing element expressed in a foreign protein context. Two aspects of protein splicing are examined in this paper. We demonstrate a succinimide at the C-terminus of the spliced internal protein, implicating cyclization of asparagine in resolution of the branched intermediate, and we identify an alkali-labile bond in the branched intermediate. A revised protein splicing model based on these experimental results is presented.
Asunto(s)
Procesamiento Proteico-Postraduccional , Proteínas/metabolismo , Succinimidas/metabolismo , Álcalis , Secuencia de Aminoácidos , Secuencia de Bases , Secuencia Conservada , Cartilla de ADN , Datos de Secuencia Molecular , Proteínas/química , Homología de Secuencia de AminoácidoRESUMEN
The Vent DNA polymerase gene from Thermococcus litoralis contains two in-frame insertions that must be spliced out to form the mature polymerase. Primer extension and cDNA PCR revealed no evidence of spliced RNA to account for this editing. In contrast, pulse-chase analysis indicated that expression constructs lacking the first insertion produced a protein precursor in Escherichia coli that was processed post-translationally to form polymerase and I-TliI, the endonuclease protein that is the product of the second insertion. At least one intermediate, which migrated more slowly than the precursor and may be branched, was also detected. Amino acid substitutions at the splice junction slowed or blocked the protein splicing reaction. Processing occurs in several heterologous systems, indicating either self-splicing or ubiquitous splicing factors. Processing occurs in a mutant lacking I-TliI endonuclease activity, establishing the independence of splicing and endonuclease activities.
Asunto(s)
Archaea/enzimología , ADN Polimerasa Dirigida por ADN/metabolismo , Procesamiento Proteico-Postraduccional , Secuencia de Aminoácidos , Archaea/metabolismo , Secuencia de Bases , ADN Bacteriano , ADN Polimerasa Dirigida por ADN/genética , Datos de Secuencia Molecular , Empalme del ARN , Homología de Secuencia de AminoácidoAsunto(s)
Aminoácidos/metabolismo , Mutagénesis Sitio-Dirigida , Ingeniería de Proteínas , ARN de Transferencia , Supresión Genética , Aminoácidos/química , Anticodón , Biomarcadores , Técnicas In Vitro , Biosíntesis de Proteínas , ARN Ligasa (ATP) , ARN de Transferencia Aminoácido-Específico/química , Aminoacil-ARN de Transferencia/química , Transcripción GenéticaRESUMEN
An amber suppressor tRNA was prepared in vitro by runoff transcription with T7 RNA polymerase. Both full-length tRNA and truncated tRNA lacking the 3' terminal pCpA from the acceptor stem could be synthesized from the same DNA template. Truncated runoff suppressor tRNA could be enzymatically ligated to phenylalanyl-pCpA to generate aminoacylated full-length suppressor tRNA (Phe-tRNA(CUA)). Phe-tRNA(CUA) is capable of suppressing an amber (UAG) mutation in vitro with equivalent efficiency as suppressor prepared by anticodon-loop replacement of a naturally-isolated tRNA. The ease of suppressor tRNA preparation using this method, compared to anticodon-loop replacement, greatly facilitates the use of chemically acylated suppressor tRNA's for site-specifically incorporating unnatural amino acids into proteins.
Asunto(s)
Aminoacil-ARN de Transferencia/metabolismo , Supresión Genética , Acilación , Secuencia de Bases , Clonación Molecular , Codón , Análisis Mutacional de ADN , Datos de Secuencia Molecular , Biosíntesis de Proteínas , Aminoacil-ARN de Transferencia/síntesis química , ARN de Transferencia de Fenilalanina , Moldes Genéticos , Transcripción GenéticaRESUMEN
Methodology is described for the synthesis and chemical aminoacylation of the hybrid dinucleotide 5'-phospho-2'-deoxyribocytidylylriboadenosine (pdCpA). Ligation of aminoacylated pdCpA to a truncated amber suppressor tRNACUA (-CA) using T4 RNA ligase generates an aminoacylated suppressor tRNA which can be used for site-specific incorporation of unnatural amino acids into proteins. Both the ligation and in vitro suppression efficiencies are the same when either pCpA or pdCpA is used. The use of deoxycytidine simplifies the chemistry involved in the synthesis of the dinucleotide pCpA. In addition, these results demonstrate that ribocytidine is not required for recognition of the aminoacylated tRNA during protein synthesis.
Asunto(s)
Fosfatos de Dinucleósidos , Aminoacil-ARN de Transferencia/síntesis química , ARN de Transferencia , Cromatografía Líquida de Alta Presión , Cromatografía en Capa Delgada , Indicadores y Reactivos , Espectroscopía de Resonancia Magnética/métodos , Estructura Molecular , Espectrofotometría , Supresión GenéticaRESUMEN
The incorporation of unnatural amino acids into proteins by site-specific mutagenesis provides a valuable new methodology for the generation of novel proteins that possess unique structural and functional features.
Asunto(s)
Mutación , Proteínas/genética , Acilación , Aminoácidos , ARN de Transferencia/genética , Supresión Genética , beta-Lactamasas/genéticaRESUMEN
A new method has been developed that makes it possible to site-specifically incorporate unnatural amino acids into proteins. Synthetic amino acids were incorporated into the enzyme beta-lactamase by the use of a chemically acylated suppressor transfer RNA that inserted the amino acid in response to a stop codon substituted for the codon encoding residue of interest. Peptide mapping localized the inserted amino acid to a single peptide, and enough enzyme could be generated for purification to homogeneity. The catalytic properties of several mutants at the conserved Phe66 were characterized. The ability to selectively replace amino acids in a protein with a wide variety of structural and electronic variants should provide a more detailed understanding of protein structure and function.
Asunto(s)
Aminoácidos , Proteínas , Electroforesis en Gel de Poliacrilamida , Escherichia coli/enzimología , Mutación , Biosíntesis de Proteínas , ARN de Transferencia/aislamiento & purificación , beta-LactamasasRESUMEN
Energy metabolism and body composition were investigated in perfluorodecanoic acid (PFDA)-treated rats (single i.p. dose of 20, 40 or 80 mg/kg) and their respective pair-fed counterparts 7 days after dosing. Cumulative feed intake and body weight were decreased in a dose-dependent manner. However, PFDA-treated rats either gained less weight or lost more weight (dependent on the dose administered) than their pair-fed, vehicle-treated counterparts, even though feed intake in these two treatment groups was similar. Energy expenditure, determined indirectly by quantifying oxygen consumption and carbon dioxide production, decreased in a dose-dependent fashion, yet it was similar in PFDA-treated rats and their pair-fed counterparts at a given dose. Body composition analysis indicated a dose-dependent decrease in carcass water and protein content in both PFDA-treated rats and pair-fed partners, while total amount of ash remained unchanged in all treatment groups. These alterations in body composition are compatible with a negative energy balance. Even though PFDA-treated rats had a lower body weight than their pair-fed counterparts at each dose level, they were found to have a greater carcass fat content. Thus, at maintenance (i.e., zero change of body weight) PFDA-treated rats require a higher caloric intake associated with a greater body fat content than vehicle-treated animals.