RESUMEN
The complementarity-determining regions (CDRs) of a human kappa light chain were replaced with CDRs from a murine gamma-1 heavy chain and, by use of molecular modeling, key heavy chain framework residues were identified and thus included to preserve the native conformation of the heavy chain CDRs. Co-expression of this hybrid human kappa chain (V[HB]C[L]) with a human kappa chain counterpart (V[L]C[L], engineered to contain murine light chain CDRs) resulted in the secretion of high levels of a heterodimeric protein (V[HB]C[L]::V[L]C[L]) termed 'kappabody'. This protein also had equivalent affinity for antigen as the Fab' of the parent murine IgG1. High-level secretion was also observed for the hybrid chain as homodimers (V[HB]C[L]::V[HB]C[L]), which is not observed for chimeric chains consisting of a heavy chain variable region and light chain constant region, i.e. V[H]C[L] homodimers or single chains are not secreted. This indicates that regions within the variable domain, required for secretion of light chains, reside outside of the hypervariable regions (CDRs) and that the heavy chain CDRs and supporting residues do not prevent secretion. These results demonstrate the possibility of designing small, single-domain molecules possessing a given binding activity which may be secreted at high levels from mammalian cells.
Asunto(s)
Cadenas Pesadas de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/inmunología , Cadenas kappa de Inmunoglobulina/inmunología , Ingeniería de Proteínas , Proteínas Recombinantes de Fusión/genética , Secuencia de Aminoácidos , Animales , Afinidad de Anticuerpos , Unión Competitiva , Dimerización , Electroforesis en Gel de Poliacrilamida , Expresión Génica , Humanos , Hibridomas , Cadenas Pesadas de Inmunoglobulina/química , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/química , Región Variable de Inmunoglobulina/genética , Cadenas kappa de Inmunoglobulina/química , Cadenas kappa de Inmunoglobulina/genética , Cinética , Ratones , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/química , Análisis de Secuencia , TransfecciónRESUMEN
This paper reports the discovery and initial characterization of two small plasmids, pCf1 and pCf2, in the marine diatom Cylindrotheca fusiformis. Extracted diatom DNA separates into two bands in CsCl-Hoechst 33258 dye gradients. Upon agarose gel electrophoresis of a sample of the upper band of the gradient we observed, in addition to high molecular weight (genomic) chloroplast and mitochondrial DNA, pairs of lower molecular weight bands. These bands contained two species of circular plasmid DNA molecules, as shown by electron microscopy. The nucleotide composition of the plasmids, and chloroplast and mitochondrial DNAs is similar, as indicated by their co-banding in the gradients. They were cloned, and their restriction maps determined, showing that pCf1 is 4.27 and pCf2 4.08 kb in size. By hybridization analysis, we showed that pCf1 and pCf2 share regions of similarity, but not identity. Neither plasmid hybridizes with mitochondrial DNA. Both plasmids hybridize with chloroplast DNA, and pCf2 also hybridizes with nuclear DNA.
Asunto(s)
Núcleo Celular/metabolismo , Cloroplastos/metabolismo , ADN/genética , Eucariontes/genética , Plásmidos , Clonación Molecular , ADN/ultraestructura , ADN Mitocondrial/genética , Electroforesis en Gel de Agar , Escherichia coli/genética , Microscopía Electrónica , Hibridación de Ácido Nucleico , Mapeo RestrictivoRESUMEN
We have discovered plasmids in 5 of 18 diatom species surveyed. In several species, more than one type of plasmid is present. Several of the plasmids show similarity by hybridization previously characterized plasmids in Cylindrotheca fusiformis (J. D. Jacobs et al., unpublished data). Additionally, there is similarity between the plasmids found in C. fusiformis and chloroplast DNA in three diatom species. These results add to the evidence that the plasmids have features of mobile genetic elements.
Asunto(s)
Eucariontes/genética , Plásmidos , Clonación Molecular , ADN/análisis , Especificidad de la EspecieRESUMEN
A mouse/human chimeric antibody has been constructed by using variable light and variable heavy regions from a murine hybridoma specific for human carcinoembryonic antigen (CEA) (CEM231.6.7). These V regions were combined with kappa and gamma-1 constant region genes cloned from human lymphocytes. The chimeric constructs were sequentially electroporated into murine non-Ig-producing myeloma (P3.653) and hybridoma (SP2/0) cell. Significant differences were seen in expression levels between the two cell types. High levels of expression (24 to 32 micrograms/ml/10(6) cells) were seen with several of the anti-CEA SP2/0 transfectomas but not with the P3.653 cells. The SP2/0 transfectoma lines were adapted to serum-free, chemically defined media and grown in large scale fermentation cultures where they continued to secrete high levels of antibody. The chimeric antibodies remain reactive against human CEA with affinity constants comparable to that of the parental hybridoma antibody. High level expression will make practical the production of chimeric antibodies for in vivo therapeutic and diagnostic purposes.
Asunto(s)
Anticuerpos Monoclonales/genética , Especificidad de Anticuerpos , Antígeno Carcinoembrionario/inmunología , Quimera , Clonación Molecular , Genes de Inmunoglobulinas , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Afinidad de Anticuerpos , Secuencia de Bases , ADN/aislamiento & purificación , Ensayo de Inmunoadsorción Enzimática , Humanos , Hibridomas/análisis , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/genética , Ratones , Datos de Secuencia Molecular , TransfecciónRESUMEN
We showed that the heat killing curve for exponentially growing Saccharomyces cerevisiae was biphasic. This suggests two populations of cells with different thermal killing characteristics. When exponentially growing cells separated into cell cycle-specific fractions via centrifugal elutriation were heat shocked, the fractions enriched in small unbudded cells showed greater resistance to heat killing than did other cell cycle fractions. Cells arrested as unbudded cells fell into two groups on the basis of thermotolerance. Sulfur-starved cells and the temperature-sensitive mutants cdc25, cdc33, and cdc35 arrested as unbudded cells were in a thermotolerant state. Alpha-factor-treated cells arrested in a thermosensitive state, as did the temperature-sensitive mutant cdc36 when grown at the restrictive temperature. cdc7, which arrested at the G1-S boundary, arrested in a thermosensitive state. Our results suggest that there is a subpopulation of unbudded cells in exponentially growing cultures that is in G0 and not in G1 and that some but not all methods which cause arrest as unbudded cells lead to arrest in G0 as opposed to G1. It has been shown previously that yeast cells acquire thermotolerance to a subsequent challenge at an otherwise lethal temperature during a preincubation at 36 degrees C. We showed that this acquisition of thermotolerance was corrected temporally with a transient increase in the percentage of unbudded cells during the preincubation at 36 degrees C. The results suggest a relationship between the heat shock phenomenon and the cell cycle in S. cerevisiae and relate thermotolerance to transient as well as to more prolonged residence in the G0 state.
Asunto(s)
Saccharomyces cerevisiae/crecimiento & desarrollo , Ciclo Celular , División Celular , Calor , Cinética , Mutación , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genéticaRESUMEN
A serologic survey was conducted on 628 white-tailed deer (Odocoileus virginianus) in 1976 and 1979-1980. Tests for antibodies to the etiologic agents of infectious bovine rhinotrancheitis (IBR), parainfluenza 3 (PI3), leptospirosis, and brucellosis produced positive results of 15%, 20%, 3% and 0%, respectively. Adult deer had significantly higher prevalence of antibodies to IBR virus and PI3 virus than fawns. These data provide a basis for monitoring these disease agents in Minnesota's white-tailed deer.
Asunto(s)
Anticuerpos Antibacterianos/análisis , Anticuerpos Antivirales/análisis , Ciervos/inmunología , Animales , Animales Salvajes/inmunología , Animales Salvajes/microbiología , Brucelosis/inmunología , Ciervos/microbiología , Herpesvirus Bovino 1/inmunología , Leptospira/inmunología , Leptospirosis/inmunología , Virus de la Parainfluenza 3 Humana/inmunologíaRESUMEN
We describe the construction of a cosmid cloning vector, pMT555, which allows positive selection for the presence of an inserted DNA fragment. The vector contains sequences which enable its replication and selection in either E. coli or Saccharomyces cerevisiae. We demonstrate that pMT555 may be used for the efficient construction of total genomic banks from small quantities of donor DNA. The positive selection permits the stable maintenance of the cosmid in E. coli and the faithful replication of inserted sequences.
Asunto(s)
Cósmidos , Elementos Transponibles de ADN , Escherichia coli/genética , Saccharomyces cerevisiae/genética , Plásmidos , Selección Genética , Transformación GenéticaRESUMEN
The methods of centrifugal elutriation, two-dimensional gel electrophoresis, and dual isotopic labeling were applied to the study and identification of a number of purified yeast proteins. The location of polypeptide spots corresponding to specific proteins was determined on two-dimensional gels. A dual-label method was used to determine the rates of synthesis through the cell cycle of the identified proteins as well as to confirm the results of previous studies from our laboratory on unidentified proteins. The identified proteins, and the more generally defined phosphorylated, heat shock, and heat stroke proteins were found to follow the general pattern of exponential increase in rate of synthesis through the cell cycle. In addition, colorimetric enzyme activity assays were used to examine the catabolic enzyme alpha-glucosidase (EC 3.2.1.20). Both the activity and synthesis of alpha-glucosidase were found to be nonperiodic with respect to the cell cycle. These data contrast with earlier reports of periodicity, which employed induction and selection synchrony to study enzyme expression through the yeast cell cycle.
Asunto(s)
Ciclo Celular , Proteínas Fúngicas/biosíntesis , Biosíntesis de Proteínas , Proteínas , Saccharomyces cerevisiae/metabolismo , Separación Celular , Electroforesis en Gel de Poliacrilamida , Proteínas de Choque Térmico , Fosfoproteínas/biosíntesis , alfa-Glucosidasas/metabolismoRESUMEN
A study has been made of the effects of a casamino acids shift-up on a prototrophic strain of yeast growing under conditions of ammonium repression. The shift-up produced an increase in growth rate some 120 min after the addition of amino acids to the medium. This growth rate increase was slightly preceded by an increase in the rate of accumulation of DNA. In contrast, the rate of accumulation of protein increased immediately and that of RNA 15-20 min after the shift. RNA was initially accumulated at a rate greater than that required to sustain the new steady state. This was shown to be due to an increase in the rate of synthesis of the rRNA species derived from the 35S precursor. The rate of synthesis of 5S rRNA and of tRNA increased much later and to a lesser extent than that of the 35S derived species. The implications of these results for general theories of regulation of RNA synthesis are discussed.