RESUMO
The mAbs HyHEL-8, HyHEL-26 (HH8, and HH26, respectively) recognize epitopes on hen egg-white lysozyme (HEL) highly overlapping with the structurally defined HH10 epitope, while the structurally related XRPC-25 is specific for DNP and does not bind HEL. All four Abs appear to use the same Vk23 germ line gene, and all but HH8 use the same VH36-60 germ line gene. Of the three anti-HEL Abs, the sequences of HH26 variable regions are closest to those encoded by the respective germ line sequences. HH8 utilizes a different member of the VH36-60 gene family. Thus, the same Vk and VH genes, combined with somatically derived sequence differences, are used to recognize the unrelated Ags HEL and DNP. In contrast, different VH36-60 germ line genes are used to bind the same antigen (e.g. HH8 vs HH10 and HH26). While the affinities of HH10, HH8, and HH26 for HEL vary by less than 10-fold, their affinities for mutated Ag vary over several orders of magnitude. Analyses of Fab binding kinetics with natural species variants and site-directed mutants of lysozyme indicate that these cross-reactivity differences reflect the relative sensitivities of both the association and dissociation rates to antigenic mutation: HH8 has relatively mutation-insensitive association and dissociation rates, HH10 has a relatively mutation-sensitive association rate but more variable dissociation rates, and HH26 has variable association and dissociation rates. Only a few amino acid differences among the antibodies produce the observed differences in the robustness of the association and dissociation rates. Our results suggest that association and dissociation rates and mutation sensitivity of these rates may be independently modulated during antibody repertoire development.
Assuntos
Anticorpos Monoclonais/química , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Afinidade de Anticorpos , Especificidade de Anticorpos , Reações Antígeno-Anticorpo , Epitopos/química , Epitopos/genética , Genes de Imunoglobulinas , Variação Genética , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , Região Variável de Imunoglobulina/química , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/metabolismo , Cinética , Muramidase/química , Muramidase/genética , Muramidase/imunologia , Mutagênese Sítio-DirigidaAssuntos
Códon de Terminação/análise , Análise Mutacional de DNA/métodos , Proteínas de Ligação a DNA , Mutação , Reação em Cadeia da Polimerase/métodos , Proteína da Polipose Adenomatosa do Colo , Neoplasias Colorretais/genética , Proteínas do Citoesqueleto/genética , Humanos , Linfócitos/fisiologia , Proteína 2 Homóloga a MutS , Síndromes Neoplásicas Hereditárias/genética , Biossíntese de Proteínas , Proteínas Proto-Oncogênicas/genética , Transcrição GênicaRESUMO
The technique of in vitro transcription/translation (IVTT) has become an important method of detecting mutations that result in a prematurely terminated protein. Subsequent characterization of the mutations by cloning and sequencing the RT-PCR products, however, is often difficult and time consuming. This is due in large part to the altered metabolism to which transcripts containing translation terminating mutations are subject. Recent data has shown that mRNAs with nonsense or frame shift mutations are often selectively degraded, so that mutation bearing transcripts are significantly less abundant that wild-type transcripts and, after cloning, mutant clones are correspondingly scarce. We have developed a reliable method of identifying the cDNA clones containing translation terminating mutations by a 'second round' of IVTT. Clones are subjected to PCR and IVTT using similar conditions as in the initial IVTT reaction and are identified unequivocally as either wild-type or mutant prior to sequencing. Wasteful 'blind' sequencing is thus avoided as well as possible misidentification of taq polymerase errors as the mutation of interest.
Assuntos
Proteínas de Ligação a DNA/genética , Genes da Neurofibromatose 1 , Testes de Mutagenicidade , Reação em Cadeia da Polimerase/métodos , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Bases , Clonagem Molecular , Primers do DNA , Reparo do DNA , DNA Complementar , Éxons , Mutação da Fase de Leitura , Proteínas Fúngicas/genética , Triagem de Portadores Genéticos , Humanos , Dados de Sequência Molecular , Proteína 1 Homóloga a MutL , Proteína 2 Homóloga a MutS , Proteínas de Saccharomyces cerevisiae , Transcrição GênicaRESUMO
Hereditary nonpolyposis colorectal cancer (HNPCC) is a common autosomal dominant disease caused by germline mutations in DNA mismatch repair genes. The mutational spectrum in these genes appears to be diverse, in both the distribution and the nature of the mutations. However, most described mutations generate a premature stop codon and ultimately result in the synthesis of a truncated protein. We have employed an in vitro transcription/translation assay to identify germline mutations in DNA mismatch repair genes from patients suspected of belonging to HNPCC kindreds. Our results suggest that this approach will be highly effective in identifying mutations in these patients and may lead to a reliable diagnostic test for the pre-symptomatic identification of HNPCC.
Assuntos
Neoplasias Colorretais Hereditárias sem Polipose/genética , Reparo do DNA/genética , Mutação , Sequência de Bases , Análise Mutacional de DNA , Humanos , Dados de Sequência MolecularRESUMO
BACKGROUND & AIMS: Hereditary nonpolyposis colorectal cancer (HNPCC) has been linked recently to a defect in repairing mismatched nucleotides in DNA. The aim of this study was to screen for germline mutations that result in prematurely truncated proteins in two of the mismatch repair genes identified at this time, hMLH1 and hMSH2, in a consecutive series of patients belonging to familial aggregations of colorectal cancer. METHODS: Nineteen individuals with colorectal cancer from 19 families were consecutively referred because of a strong positive family history of colorectal cancer. Premature truncation mutations in hMLH1 and hMSH2 were sought from lymphocyte RNA by using an in vitro transcription/translation (IVTT) assay. RESULTS: Protein truncating mutations in the hMLH1 or hMSH2 genes were found in 50% of families with HNPCC (6 of 12) but were not observed in any of the remaining familial aggregations that did not fulfill the standard criteria for HNPCC. In some of the IVTT-positive samples, the mutations were characterized by genomic sequencing. CONCLUSIONS: IVTT may be a practical method to accomplish primary screening of germline mutations in DNA mismatch pair genes in HNPCC; however, a broader approach is necessary to obtain a more complete picture of the mutational spectrum in HNPCC and other familial aggregations of colorectal cancer.
Assuntos
Neoplasias Colorretais Hereditárias sem Polipose/genética , Testes Genéticos/métodos , Biossíntese de Proteínas , Transcrição Gênica , Sequência de Bases , Humanos , Dados de Sequência Molecular , Mutação , Reação em Cadeia da PolimeraseRESUMO
Neurofibromatosis 1 (NF1) is a common genetic disorder characterized by abnormalities of tissues derived from the neural crest. To define germ-line mutations in the NF1 gene, we studied 20 patients with familial or sporadic cases of NF1 diagnosed clinically and one patient with only café-au-lait spots and no other diagnostic criteria. A protein truncation assay identified abnormal polypeptides synthesized in vitro from five RT-PCR products that represented the entire NF1 coding region. Truncated polypeptides were observed in 14 individuals. The mutations responsible for the generation of abnormal polypeptides were characterized by DNA sequencing. Thirteen previously unpublished mutations were characterized in the 14 individuals. The mutation 2027insC was observed in two unrelated individuals; the other 12 mutations were unique. The sequence changes included seven nonsense and four frameshift mutations that created premature translation termination signals, and two large in-frame deletions that led to the synthesis of truncated polypeptides. One of the mutations was found in the child with a single clinical diagnostic criterion, providing her with a presumptive diagnosis of NF1. Our results confirm that truncating mutations are frequent in both familial and sporadic NF1 cases. The identification of mutations in 14 of 21 individuals studied (67%) suggests that the use of protein truncation assays will rapidly accelerate the rate of identification of NF1 mutations. Because we scanned the entire NF1 coding region in each individual, the distribution of NF1 truncating mutations was discerned for the first time. The mutations were relatively evenly distributed throughout the coding region with no evidence for clustering.
Assuntos
Genes da Neurofibromatose 1 , Mutação , Sequência de Bases , Linhagem Celular , Criança , Análise Mutacional de DNA , Primers do DNA , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Dados de Sequência Molecular , Neurofibromina 1 , Polimorfismo Genético , Proteínas/genéticaAssuntos
Análise Mutacional de DNA , Genes da Neurofibromatose 1 , Testes Genéticos/métodos , Neurofibromatose 2/genética , Reação em Cadeia da Polimerase/métodos , Proteínas/química , Sistema Livre de Células , Primers do DNA , DNA Complementar/genética , Humanos , Neurofibromina 1 , Biossíntese de Proteínas , Proteínas/genética , Deleção de Sequência , Transcrição GênicaRESUMO
Baculoviruses establish systemic infections within susceptible insect hosts, even though host tissues are surrounded by basal laminae, extracellular matrices that exclude particles smaller than these viruses. Using a recombinant Autographa californica M nuclear polyhedrosis virus containing a lacZ reporter gene under the control of a constitutive promoter, we followed the progression of infection in Trichoplusia ni larvae. We discovered that infection of the larval insect tracheal system (and not hemocytes, as thought previously) provides the major conduit for this virus to pass through basal laminae and to spread throughout the host. Tracheal epidermal cells, the only known cellular components of the tracheal system, share a common lymph system. Locally these cells contact one another by interdigitating cytoplasmic extensions called epidermal feet. These two features of the tracheal system are likely to facilitate the rapid systemic spread of the virus. The findings reported here have major implications for the fields of insect pathology and biological control and usher in an important consideration regarding host-range factors.
Assuntos
Lepidópteros/microbiologia , Nucleopoliedrovírus/patogenicidade , Animais , Vetores Genéticos , Larva , Lepidópteros/anatomia & histologia , Traqueia/citologia , Traqueia/microbiologiaRESUMO
The complex formed between hen egg white lysozyme (HEL) and the monoclonal antibody HyHEL-10 Fab fragment has an interface composed of van der Waals interactions, hydrogen bonds, and a single ion pair. The antibody overlaps part of the active site cleft. Putative critical residues within the epitope region of HEL, identified from the x-ray crystallographic structure of the complex, were replaced by site-directed mutagenesis to probe their relative importance in determining affinity of the antibody for HEL. Twenty single mutations of HEL at three contact residues (Arg-21HEL, Asp-101HEL, and Gly-102HEL) and at a partially buried residue (Asn-19HEL) in the epitope were made, and the effects on the free energies of dissociation were measured. A correlation between increased amino acid side-chain volume and reduced affinity for HELs with mutations at position 101 was observed. The D101GHEL mutant is bound to HyHEL-10 as tightly as wild-type enzyme, but the delta delta Gdissoc is increased by about 2.2 kcal (9.2 kJ)/mol for the larger residues in this position. HEL variants with lysine or histidine replacements for arginine at position 21 are bound 1.4-2.7 times more tightly than those with neutral or negatively charged amino acids in this position. These exhibit 1/40 the affinity for HyHEL-10 Fab compared with wild type. There is no side-chain volume correlation with delta delta Gdissoc at position 21. Although Gly-102HEL and Asn-19HEL are in the epitope, replacements at these positions have no effect on the affinity of HEL for the antibody.
Assuntos
Anticorpos Monoclonais/metabolismo , Epitopos/metabolismo , Muramidase/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação de Anticorpos , Ligação Competitiva , Galinhas , Epitopos/química , Epitopos/genética , Cinética , Matemática , Muramidase/química , Muramidase/genética , Mutagênese Sítio-Dirigida , TermodinâmicaAssuntos
Muramidase/metabolismo , Mutagênese Sítio-Dirigida , Proteínas/metabolismo , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos/metabolismo , Antígenos/metabolismo , Evolução Biológica , Galinhas , Clonagem Molecular , Coturnix , Escherichia coli/genética , Variação Genética , Cinética , Dados de Sequência Molecular , Muramidase/biossíntese , Muramidase/genética , Biossíntese de Proteínas , Proteínas/isolamento & purificação , Codorniz , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Saccharomyces cerevisiae , Homologia de Sequência de Aminoácidos , Transfecção , PerusRESUMO
The purpose of this study was to compare the role of frequency selectivity in measures of auditory and vibrotactile temporal resolution. In the first experiment, temporal modulation transfer functions for a sinusoidally amplitude modulated (SAM) 250-Hz carrier revealed auditory modulation thresholds significantly lower than corresponding vibrotactile modulation thresholds at SAM frequencies greater than or equal to 100 Hz. In the second experiment, auditory and vibrotactile gap detection thresholds were measured by presenting silent gaps bounded by markers of the same or different frequency. The marker frequency F1 = 250 Hz preceded the silent gap and marker frequencies after the silent gap included F2 = 250, 255, 263, 310, and 325 Hz. Auditory gap detection thresholds were lower than corresponding vibrotactile thresholds for F2 markers less than or equal to 263 Hz, but were greater than the corresponding vibrotactile gap detection thresholds for F2 markers greater than or equal to 310 Hz. When the auditory gap detection thresholds were transformed into filter attenuation values, the results were modeled well by a constant-percentage (10%) bandwidth filter centered on F1. The vibrotactile gap detection thresholds, however, were independent of marker frequency separation. In a third experiment, auditory and vibrotactile rate difference limens (RDLs) were measured for a 250-Hz carrier at SAM rates less than or equal to 100 Hz. Auditory RDLs were lower than corresponding vibrotactile RDLs for standard rates greater than 10 Hz. Combination tones may have confounded auditory performance for standard rates of 80 and 100 Hz. The results from these experiments revealed that frequency selectivity influences auditory measures of temporal resolution, but there was no evidence of frequency selectivity affecting vibrotactile temporal resolution.