RESUMEN
A series of group 13 metal complexes featuring the beta-diketiminate ligand [[(C(6)H(3)-2,6-i-Pr(2))NC(Me)](2)CH](-) (i.e., [Dipp(2)nacnac](-), Dipp = C(6)H(3)-2,6-i-Pr(2)) have been prepared and spectroscopically and structurally characterized. The chloride derivatives Dipp(2)nacnacMCl(2) (M = Al (3), Ga (5), In (8)) were isolated in good yield by the reaction of 1 equiv of Dipp(2)nacnacLi.Et(2)O (2) and the respective metal halides. The iodide derivatives Dipp(2)nacnacMI(2) (M = Al (4), Ga (6), In (9)), which are useful for reduction to afford M(I) species, were made by a variety of routes. Thus, 4 was obtained by treatment of the previously reported Dipp(2)nacnacAlMe(2) with I(2), whereas the gallium analogue 6 was obtained as a product of the reaction of "GaI" with Dipp(2)nacnacLi.Et(2)O, and 9 was obtained by direct reaction of InI(3) and the lithium salt. The methyl derivatives Dipp(2)nacnacMMe(2) (M = Ga (7), In (10)), which are analogous to the previously reported Dipp(2)nacnacAlMe(2), were synthesized by the reaction of GaMe(3) with Dipp(2)nacnacH (1) or by reaction of the indium chloride derivative 8 with 2 equiv of MeMgBr in diethyl ether. The compounds 3-10 exist as colorless, air- and moisture-sensitive crystalline solids. Their X-ray crystal structures feature nearly planar C(3)N(2) arrays in the Dipp(2)nacnac ligand backbone with short C-C and C-N distances that are consistent with a delocalized structure. However, there are large dihedral angles between the C(3)N(2) plane and the N(2)M metal coordination plane which have been attributed mainly to steric effects. The relatively short M-N distances are consistent with the coordination numbers of the metals and the normal/dative character of the nitrogen ligands. The compounds were also characterized by (1)H and (13)C NMR spectroscopy. (1)H NMR data for 7 revealed equivalent methyl groups whereas the spectrum of 10 displayed two In-Me signals which indicated that ring wagging was slow on the (1)H NMR time scale.
RESUMEN
Examples of compounds with gallium-boron donor-acceptor bonds, HC[MeC(2,6-pri2C6H3)N]2Ga-->B(C6F5)3 3 and (eta 5-C5Me5)Ga-->B(C6F5)3 4 have been prepared by treatment of the free gallanediyls with B(C6F5)3; the structures of both compounds were determined by X-ray crystallography.
RESUMEN
Gene targeting in mouse embryonic stem (ES) cells was used to replace (i) the mouse immunoglobulin heavy chain (IgH) Cgamma2a gene segment (mCgamma2a) with the human Cgamma1 gene segment (hCgamma1), and (ii) the mouse immunoglobulin light chain (IgL) Ckappa gene segment (mC kappa) with its human counterpart (hC kappa). ES cells carrying these gene conversions were used to generate chimeric mice that transmitted the human alleles through the germ line. Mice homozygous for both gene alterations were generated by breeding. Serum from homozygous mutant mice contained comparable amounts of antibodies with chimeric kappa or mouse lambda light chains but only small fractions of basal serum IgG or antibodies elicited against immunizing agents contained chimeric heavy chains. A relative increase in immunogen-specific hCgamma1 antibodies was seen following immunization in combination with the saponin adjuvant QS-21. The effect of this was to shift the IgG1-dominated response to an IgG subclass profile that included significant amounts of IgG2a, IgG2b and IgG3 and chimeric IgG. The amounts of antibody secreted by hybridomas derived from mutant and wild-type mice were similar. Sequencing confirmed correct splicing of hCgamma1 and hCkappa gene segments to mouse J gene segments in hybridoma Ig gene transcripts. In conclusion, IgHhCgamma1/IgLhCkappa double mutant mice provide a useful animal model for deriving humanized antibodies with potential applications in immunotherapy and diagnostics in vivo as well as for investigating hCgamma1 associated functions.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/genética , Cadenas gamma de Inmunoglobulina/biosíntesis , Cadenas gamma de Inmunoglobulina/genética , Cadenas kappa de Inmunoglobulina/biosíntesis , Cadenas kappa de Inmunoglobulina/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Animales , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Secuencia de Bases , Clonación Molecular , Embrión de Mamíferos , Femenino , Citometría de Flujo , Marcación de Gen , Humanos , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/sangre , Inmunoglobulina G/genética , Cadenas gamma de Inmunoglobulina/inmunología , Cadenas kappa de Inmunoglobulina/inmunología , Masculino , Ratones , Ratones Endogámicos , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/inmunología , Bazo/citología , Bazo/metabolismo , Células Madre/metabolismoRESUMEN
Carcinoembryonic antigen (CEA) has been shown to be one of the best markers for in vivo tumor targeting of radiolabeled antibodies, despite the fact that it is localized predominantly at the apical side of human colon carcinoma cells within the fairly closed pseudolumen structures formed by these tumors. Due to this particular histological localization, a large proportion of the CEA molecules may remain inaccessible to the intravenously injected radiolabeled anti-CEA antibodies of IgG isotype, which are widely used in the clinic. In order to improve targeting, we made a recombinant dimeric IgA, which should have the capacity to translocate from the basolateral to the apical side of the pseudolumen formed by colon carcinoma cells after binding to the polyIg receptor (pIgR). A genomic chimeric mouse-human IgA2 construct was made using one of our most specific anti-CEA hybridomas, CE-25. The chimeric IgA (chIgA) was expressed in the Sp2/0 myeloma cell line. The secreted recombinant antibody was found to consist mostly of a dimeric form of IgA with a molecular weight of about 350 kDa. The dimeric chIgA was shown to translocate efficiently in vitro across a monolayer of epithelial cells expressing the pIgR and to retain full CEA binding activity.
Asunto(s)
Anticuerpos Antineoplásicos/biosíntesis , Antígeno Carcinoembrionario/inmunología , Inmunoglobulina A/biosíntesis , Proteínas Recombinantes de Fusión/biosíntesis , Animales , Anticuerpos Antineoplásicos/química , Anticuerpos Antineoplásicos/inmunología , Afinidad de Anticuerpos , Unión Competitiva , Transporte Biológico/inmunología , Línea Celular , Epitelio/inmunología , Humanos , Inmunoglobulina A/química , Inmunoglobulina A/inmunología , Ratones , Ratones Desnudos , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/inmunología , TransfecciónRESUMEN
We report the isolation, sequence, and identification of a cDNA encoding the human kinesin light-chain (KLC) protein. The cDNA molecule consisted of 276 nucleotides of 5' untranslated region, the complete coding sequence of 1,710 nucleotides, and 322 nucleotides of 3' untranslated region. It encoded a polypeptide of 569 amino acids and a deduced molecular mass of 64,789 daltons. The predicted secondary internal structure of the KLC molecule consisted of about 27 contiguous repeats, each of approximately 21 amino acid residues, and could be divided into three domains. The amino-terminal domain consisted of heptad repeats typical of the rod domain of several cytoskeletal proteins. The central and carboxy-terminal domains consist of 21-mer repeats. KLC mRNA was expressed in most tissues analyzed. The gene, which was expressed in bacteria and Chinese hamster ovary cells, was provisionally assigned to the long arm of human chromosome 14.
Asunto(s)
Cinesinas/genética , Proteínas Asociadas a Microtúbulos/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Southern Blotting , Células Cultivadas , Cromosomas Humanos Par 14 , Clonación Molecular , ADN Complementario , Células HeLa , Humanos , Proteínas Asociadas a Microtúbulos/química , Datos de Secuencia Molecular , Pruebas de Precipitina , Estructura Secundaria de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Homología de Secuencia de Aminoácido , Linfocitos T/metabolismoRESUMEN
Mouse mAb TES-C21(C21) recognizes an epitope on human IgE and, therefore, has potential as a therapeutic agent in patients with IgE-mediated allergies such as hay fever, food and drug allergies and extrinsic asthma. The clinical usefulness of mouse antibodies is limited, however, due to their immunogenicity in humans. Mouse C21 antibody was humanized by complementarity determining region (CDR) grafting with the aim of developing an effective and safe therapeutic for the treatment of IgE-mediated allergies. The CDR-grafted, or reshaped human, C21 variable regions were carefully designed using a specially constructed molecular model of the mouse C21 variable regions. A key step in the design of reshaped human variable regions is the selection of the human framework regions (FRs) to serve as the backbones of the reshaped human variable regions. Two approaches to the selection of human FRs were tested: (i) selection from human consensus sequences and (ii) selection from individual human antibodies. The reshaped human and mouse C21 antibodies were tested and compared using a biosensor to measure the kinetics of binding to human IgE. Surprisingly, a few of the reshaped human C21 antibodies exhibited patterns of binding and affinities that were essentially identical to those of mouse C21 antibody.
Asunto(s)
Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Inmunoglobulina E/inmunología , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/biosíntesis , Especificidad de Anticuerpos , Secuencia de Bases , Células Cultivadas , Humanos , Hipersensibilidad/tratamiento farmacológico , Regiones Constantes de Inmunoglobulina/biosíntesis , Regiones Constantes de Inmunoglobulina/genética , Regiones Constantes de Inmunoglobulina/inmunología , Cadenas Pesadas de Inmunoglobulina/biosíntesis , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas Ligeras de Inmunoglobulina/biosíntesis , Cadenas Ligeras de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/biosíntesis , Región Variable de Inmunoglobulina/inmunología , Inmunoterapia Adoptiva/métodos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Ingeniería de Proteínas , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/inmunología , Homología de Secuencia de Aminoácido , Especificidad de la EspecieRESUMEN
Tumour localisation and tumour to normal tissue ratios of a chimeric anti-carcinoembryonic antigen (CEA) monoclonal antibody (MAb), in intact form and as an F(ab')2 fragment labelled with 125I and 131I, were compared in groups of nude mice bearing four different colon cancer xenografts, T380, Co112 or LoVo, of human origin, or a rat colon cancer transfected with human CEA cDNA, called '3G7'. For each tumour, three to four mice per time point were analysed 6, 12, 24, 48 and 96 h after MAb injection. In the different tumours, maximal localisation of intact MAb was obtained at 24 to 48 h, and of F(ab')2 fragment 12 to 24 h after injection. Among the different tumours, localisation was highest with colon cancer T380, with 64% of the injected dose per gram (% ID/g) for the intact MAb and 57% for its F(ab')2 fragment, while in the three other tumours, maximal localisation ranged from 14 to 22% ID g-1 for the intact MAb and was about 11% for the F(ab')2. Tumour to normal tissue ratios of intact MAb increased rapidly until 24 h after injection and remained stable or showed only a minor increase thereafter. In contrast, for the F(ab')2 fragment, the tumour to normal tissue ratios increased steadily up to 4 days after injection reaching markedly higher values than those obtained with intact MAb. For the four different xenografts, tumour to blood ratios of F(ab')2 were about 2, 3 and 5 to 16 times higher than those of intact antibodies at 12, 24 and 96 h after injection, respectively.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Antígeno Carcinoembrionario/inmunología , Neoplasias del Colon/metabolismo , Fragmentos Fab de Inmunoglobulinas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Animales , Anticuerpos Monoclonales/sangre , Neoplasias del Colon/sangre , Humanos , Fragmentos Fab de Inmunoglobulinas/sangre , Radioisótopos de Yodo/metabolismo , Ratones , Ratones Desnudos , Proteínas Recombinantes de Fusión/sangre , Distribución Tisular , Trasplante HeterólogoRESUMEN
The structural organization of anti-idiotypic (id) antibodies has been investigated mostly in haptenic systems. No information is available about the structural characteristics of anti-id antibodies in major histocompatibility complex (MHC) antigenic systems, although these data may contribute to our understanding of the molecular basis of their functional role in the immune response. Therefore, we have determined the nucleotide and derived amino acid sequence of the VH and VL regions of the anti-id monoclonal antibodies (mAb) F5-444, F5-830, F5-963, F5-1126, F5-1336 and F5-1419, which had been elicited with the syngeneic anti-HLA-DR1, 4, w14, w8, 9 mAb AC1.59. The six anti-id mAb are heterogenous in their VH and VL region gene usage. This structural heterogeneity is not correlated with their target specificity and with their ability to elicit anti-HLA-DR antibodies. The latter characteristic is markedly influenced by a limited number of amino acid substitutions, since mAb F5-444, which induces anti-HLA-DR antibodies, differs only in two residues in complementarity-determining regions and in five residues in framework regions from mAb F5-1126, which does not induce anti-HLA-DR antibodies. The heterogeneity in VH and VL region gene usage by the six anti-id mAb in the HLA-DR system is at variance with the restricted VH and VL region gene usage by syngeneic anti-id mAb in several haptenic systems. Furthermore, at variance with haptenic systems, the primary structure of the D segments of the anti-id mAb is not correlated with their ability to induce anti-HLA-DR antibodies. On the other hand, the frequency of D-D fusion events underlying the derivation of the D segments of the six anti-id mAb in the HLA-DR system and their average length are similar to those found in anti-id mAb in haptenic systems. In addition, like in the latter systems, somatic mutations appear to contribute to the generation of diversity of anti-id mAb in the HLA-DR system.
Asunto(s)
Anticuerpos Antiidiotipos/química , Anticuerpos Monoclonales/química , Antígenos HLA-DR/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antiidiotipos/genética , Anticuerpos Monoclonales/genética , Secuencia de Bases , Genes de Inmunoglobulinas , Cadenas Pesadas de Inmunoglobulina/química , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas J de Inmunoglobulina/química , Cadenas Ligeras de Inmunoglobulina/química , Cadenas Ligeras de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/química , Región Variable de Inmunoglobulina/genética , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , MutaciónRESUMEN
We have constructed genes expressing single-chain antigen binding proteins (scFv) which recognize the human erbB-2 receptor. These genes encode the heavy and light chain variable domains of an erbB-2 receptor specific monoclonal antibody, MAb FRP5, connected by a peptide linker. In order to express a bifunctional molecule, a bacterial alkaline phosphatase gene was fused 3' to the scFv gene. The scFv(FRP5) and scFv(FRP5)-alkaline phosphatase fusion protein (scFv(FRP5)-PhoA) expressed in E. coli specifically recognize the human erbB-2 protein and compete with MAb FRP5 for binding to the receptor. The bound scFv(FRP5)-PhoA protein can be detected directly on tumor cells using a substrate for alkaline phosphatase, showing that the chimeric protein retains both binding and enzymatic activity.
Asunto(s)
Fosfatasa Alcalina/metabolismo , Anticuerpos Monoclonales/metabolismo , Clonación Molecular , Plásmidos/química , Proteínas Proto-Oncogénicas/metabolismo , Fosfatasa Alcalina/genética , Secuencia de Aminoácidos , Unión Competitiva , Escherichia coli/genética , Femenino , Genes Bacterianos , Humanos , Inmunohistoquímica , Datos de Secuencia Molecular , Plásmidos/genética , Receptor ErbB-2RESUMEN
Based on previous experiments in nude mice, showing that fluoresceinated monoclonal antibodies against carcinoembryonic antigen localized specifically in human carcinoma xenografts and could be detected by laser-induced fluorescence, we performed a feasibility study to determine whether this immunophotodiagnosis method could be applied in the clinic. Six patients, with known primary colorectal carcinoma, received an i.v. injection of 4.5 or 9 mg of mouse-human chimeric anti-carcinoembryonic antigen monoclonal antibody coupled with 0.10-0.28 mg of fluorescein (molar ratio 1/10 to 1/14). The monoclonal antibody was also labeled with 0.2-0.4 mCi of 125I (1 Ci = 37 GBq). Photodetection of the tumor was done ex vivo on surgically resected tissues for the six patients and in vivo by fluorescence rectosigmoidoscopy for the sixth patient. Upon laser irradiation, clearly detectable heterogeneous green fluorescence from the dye-antibody conjugate was visually observed on all six tumors; almost no such fluorescence was detectable on normal mucosa. The yellowish tissue autofluorescence, which was emitted from both tumor and normal mucosa, could be subtracted by real-time image processing. Radioactivity measurements confirmed the specificity of tumor localization by the conjugate; tissue concentrations of up to 0.059% injected dose per g of tumor and 10 times less (0.006%) per g of normal mucosa were found. The overall results demonstrate the feasibility of tumor immunophotodiagnosis at the clinical level.
Asunto(s)
Antígeno Carcinoembrionario/análisis , Carcinoma/diagnóstico , Neoplasias Colorrectales/diagnóstico , Afinidad de Anticuerpos , Antígeno Carcinoembrionario/inmunología , Técnica del Anticuerpo Fluorescente , Humanos , Proteínas Recombinantes de FusiónRESUMEN
Hitherto anti-CEA monoclonal antibodies (MAbs), normally of mouse origin, have been used primarily for clinical diagnosis of colorectal cancer, either as a tumor marker in serum to monitor tumor recurrence, or latterly as a means to localize in vivo CEA-bearing tumors, and metastases in patients. In vivo diagnosis using mouse anti-CEA MAbs has so far had limited clinical utility because the antibodies elicit a strong anti-mouse immunoglobulin immune response on repeated administration in man. This problem has been addressed by the development of various strategies for "humanization" of mouse anti-CEA MAbs by genetic manipulation of immunoglobulin genes. Such humanized, engineered antibodies markedly attenuate the antigenic response directed against the MAb, such that safe, repeated administration to patients has become feasible. Such humanized anti-CEA antibodies can thus be radioactively-labelled and applied for in vivo monitoring and detection of recurrent malignant disease, or used for therapeutic strategies which similarly take advantage of the ability of the antibodies to target cytotoxic agents selectively to tumor cells. The application of these novel procedures for manipulating MAb structure presents entirely new opportunities for diagnosis and treatment of human colorectal cancer.
Asunto(s)
Antígeno Carcinoembrionario/inmunología , Neoplasias Colorrectales/terapia , Ingeniería Genética , Inmunoterapia/métodos , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/uso terapéutico , Neoplasias Colorrectales/diagnóstico , HumanosRESUMEN
Mouse-human chimeric monoclonal antibodies (MAbs) of 3 different human IgG sub-classes directed against carcinoembryonic antigen (CEA) have been produced in SP-0 cells transfected with genomic chimeric DNA. F(ab')2 fragments were obtained by pepsin digestion of the purified chimeric MAbs of human IgG1, IgG2 and IgG4 sub-class and of parental mouse MAb IgG1. The 4 F(ab')2 fragments exhibit similar molecular weight by SDS-PAGE. They were labelled with 125I or 131I and high binding (80 to 87%) to purified unsolubilized CEA was observed. In vivo, double labelling experiments indicate that the longest biological half-life and the highest tumour-localization capacity is obtained with F(ab')2 from chimeric MAb of human IgG2 sub-class, whereas F(ab')2 from chimeric MAb IgG4 give very low values for these 2 parameters. F(ab')2 from chimeric MAb IgG1 and from parental mouse MAb yield intermediate results in vivo. Our findings should help to select the appropriate human IgG sub-class to produce chimeric or reshaped MAb F(ab')2 to be used for tumour detection by immunoscintigraphy and for radioimmunotherapy.
Asunto(s)
Fragmentos Fab de Inmunoglobulinas/inmunología , Inmunoglobulina G/inmunología , Proteínas Recombinantes de Fusión/inmunología , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Afinidad de Anticuerpos , Antígeno Carcinoembrionario/inmunología , Neoplasias del Colon/inmunología , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Inmunoglobulina G/genética , Inmunoglobulina G/metabolismo , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Relación Estructura-ActividadRESUMEN
Normal rats were injected intravenously with 131I- and 125I-labeled intact murine and chimeric mouse-human monoclonal antibodies directed against carcinoembryonic antigen or with the corresponding F(ab')2 fragments. At different times after injection, individual animals were killed and radioactivity of blood and major organs, including bones and bone marrow, was determined. Ratios comparing radioactivity concentration in different tissues with that of bone marrow were calculated and found to remain stable during several effective half-lives of the antibodies. Mean bone marrow radioactivity was 35% (range, 29%-40%) of that of blood and 126% (range, 108%-147%) of that of liver after injection of intact Mabs or F(ab')2 fragments. In nude rats bearing human colon carcinoma xenografts producing carcinoembryonic antigen, relative bone marrow radioactivity was slightly lower than that in normal rats.
Asunto(s)
Anticuerpos Monoclonales/farmacocinética , Médula Ósea/metabolismo , Fragmentos Fab de Inmunoglobulinas/farmacocinética , Animales , Anticuerpos Monoclonales/administración & dosificación , Antígeno Carcinoembrionario/inmunología , Neoplasias del Colon/metabolismo , Femenino , Humanos , Fragmentos Fab de Inmunoglobulinas/administración & dosificación , Inyecciones Intravenosas , Radioisótopos de Yodo , Ratones , Dosis de Radiación , Ratas , Ratas Desnudas , Distribución Tisular , Trasplante HeterólogoRESUMEN
Radioimmunodetection of tumours with monoclonal antibodies is becoming an established procedure. Positron emission tomography (PET) shows better resolution than normal gamma camera single photon emission tomography and can provide more precise quantitative data. Thus, in the present study, these powerful methods have been combined to perform radioimmuno PET (RI-PET). Monoclonal antibodies directed against carcinoembryonic antigen (CEA) an IgG, its F(ab')2 and a mouse-human chimeric IgG derived from it were labelled with 124I, a positron-emitting radionuclide with a convenient physical half-life of four days. Mice, xenografted with a CEA-producing human colon carcinoma, were injected with the 124I-MAb and the tumours were visualized using PET. The concentrations of 124I in tumour and normal tissue were determined by both PET and direct radioactivity counting of the dissected animals, with very good agreement. To allow PET quantification, a procedure was established to account for the presence of radioactivity during the absorption correction measurement (transmission scan). Comparison of PET and tissue counting indicates that this novel combination of radioimmunolocalization and PET (RI-PET) will provide, in addition to more precise diagnosis, more accurate radiation dosimetry for radioimmunotherapy.
Asunto(s)
Anticuerpos Monoclonales , Antígeno Carcinoembrionario/inmunología , Neoplasias del Colon/diagnóstico por imagen , Tomografía Computarizada de Emisión , Animales , Anticuerpos Monoclonales/farmacocinética , Neoplasias del Colon/inmunología , Neoplasias del Colon/metabolismo , Humanos , Radioisótopos de Yodo , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Trasplante HeterólogoRESUMEN
The repetitive fraction of the genome of the eukaryotic slime mould Physarum polycephalum is dominated by the Tp1 family of highly repetitive retrotransposon-like sequences. Tp1 elements consist of two terminal direct repeats of 277bp which flank an internal domain of 8.3kb. They are the major sequence component in the hypermethylated (M+) fraction of the genome where they have been found exclusively in scrambled clusters of up to 50kb long. Scrambling is thought to have arisen by insertion of Tp1 into further copies of the same sequence. In the present study, sequence analysis of cloned Tp1 elements has revealed striking homologies of the predicted amino acid sequence to several highly conserved domains characteristic of retrotransposons. The relative order of the predicted coding regions indicates that Tp1 elements are more closely related to copia and Ty than to retroviruses. Self-integration and methylation of Tp1 elements may function to limit transposition frequency. Such mechanisms provide a possible explanation for the origin and organisation of M + DNA in the Physarum genome.
Asunto(s)
Elementos Transponibles de ADN/genética , Physarum/genética , Secuencia de Aminoácidos , Codón , Endonucleasas/genética , Endopeptidasas/genética , Biblioteca Genómica , Metilación , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , ADN Polimerasa Dirigida por ARN/genética , Secuencias Repetitivas de Ácidos Nucleicos , Alineación de Secuencia , Homología de Secuencia de Ácido NucleicoRESUMEN
The use of conventional DNA cloning procedures to obtain productively rearranged Ig genes from B cell hybridomas for structure/function analysis of immunoglobulins is tedious and time-consuming. Here we describe a procedure based on PCR which permits rapid, selective isolation of DNA segments containing individual hybridoma-specific Ig gene rearrangements. The method, an adaptation of the so-called 'inverted PCR' technique (IPCR), can be applied most efficiently to specific genes where a preliminary restriction map is available from Southern blot analysis of the hybridoma genomic DNA. To achieve amplification of a given rearranged Ig locus, small amounts of total hybridoma DNA are digested to completion with a chosen restriction endonuclease and the fragments circularised by DNA ligase. Cleavage of the DNA circles using a second restriction enzyme, chosen specifically to cut 3' to a rearranged V-(D)-J exon, leads to linear DNA segments where the rearranged gene is now flanked by segments of known nucleotide sequence derived originally from the 3' region of the Ig H or L chain gene locus. This permits the selection of oligonucleotides that provide convergent primers for specific amplification of DNA segments containing the required gene rearrangement. Amplified DNA fragments can be cloned and rapidly characterised by sequence analysis.
Asunto(s)
Linfocitos B/fisiología , Reordenamiento Génico de Linfocito B , Genes de Inmunoglobulinas , Hibridomas , Región Variable de Inmunoglobulina/genética , Animales , Secuencia de Bases , Southern Blotting , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/genética , Ratones , Datos de Secuencia Molecular , Oligonucleótidos , Reacción en Cadena de la Polimerasa , Mapeo RestrictivoRESUMEN
The repetitive fraction of the genome of the eukaryotic slime mould Physarum polycephalum is dominated by Tp1, a family of retrotransposon-like sequences. Tp1 elements are arranged in scrambled clusters probably arising from integration of the element into copies of its own sequence. The present report describes a second sequence family, Tp2, which has been identified within cloned DNA segments of scrambled Tp1 sequences. Like Tp1, the Tp2 element is structurally related to retrotransposons, having long terminal direct repeats and being flanked by an apparent target site duplication, but its relatively short length (1.68 kb) indicates that it is probably incapable of encoding all the functions necessary for its own mobilisation. Analysis of the coding potential of the Tp2 element supports this view, although a striking homology to a nucleic acid binding domain common to many retrotransposons was identified. As with Tp1, putative regulatory signals can be identified in the LTRs of Tp2. Identical arrangements of Tp2 with respect to Tp1 in more than one independently derived clone indicate that non-functional copies of Tp2 may be mobilised as part of a Tp1 transcriptional unit.
Asunto(s)
Elementos Transponibles de ADN , Physarum/genética , Secuencia de Aminoácidos , Secuencia de Bases , Codón , Genes Fúngicos , Datos de Secuencia Molecular , Secuencias Repetitivas de Ácidos Nucleicos , Mapeo Restrictivo , Transcripción GenéticaRESUMEN
A procedure was devised for the identification and specific cloning of functionally rearranged variable region immunoglobulin (Ig) gene segments from genomic DNA of a murine hybridoma cell line which produces a high-affinity monoclonal antibody (MAb) directed against human carcinoembryonic antigen (CEA). The cloned, functionally-rearranged murine Ig H-chain and L-chain variable region gene segments were incorporated into plasmid vectors capable of directing the expression of a chimaeric mouse-human antibody molecule with human (gamma 4, kappa) constant region sequences. Expression plasmids were transfected into a mouse myeloma cell line by electroporation and transfectomas secreting functional chimaeric antibody selected. Chimaeric antibody generated by transfectomas was analysed and shown to compete effectively with its murine counterpart for binding to the CEA epitope, and to have an equivalent antigen-binding affinity. This anti-CEA recombinant antibody should find application in in vivo diagnosis by immunoscintigraphy of human colonic carcinoma, and possibly also in therapy of the disease, overcoming some of the difficulties associated with the repeated use of non-human immunoglobulins in human patients.
Asunto(s)
Anticuerpos Monoclonales/genética , Antígeno Carcinoembrionario/inmunología , Clonación Molecular , Animales , Anticuerpos Monoclonales/biosíntesis , Secuencia de Bases , Quimera , Genes de Inmunoglobulinas , Humanos , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Transcripción GenéticaRESUMEN
Microorganisms have been used traditionally by industry as sources of natural products, or as sources of enzymes capable of mediating specific chemical transformations. This situation has changed radically in recent years, a time during which we have seen a dramatic increase in the number and range of potential biotechnological applications of enzymes and their genetically-engineered variants. An increasing number of enzymes, receptors and other proteins have now been structurally characterized, and their genes isolated as a basis for producing recombinant proteins for genetic analysis of their structure and function. These innovations have necessitated development of associated technologies for large-scale production of proteins in bioreactors, appropriate strategies for quality control, and new analytical tools for structural characterization of recombinant gene products. Some recombinant proteins are already in an advanced stage of development for use either as new-generation therapeutics, as target molecules for "intelligent" drug screening, or as biological components of biosensors. As the predictive power of protein model building improves, the diversity of applications of such technology will increase further as it becomes feasible to generate totally synthetic proteins with specifically-tailored properties.