RESUMEN
Biological and serological assays have been used to define four subregions for the I region of the major histocompatibility complex (MHC) in the order I-A, I-B, I-J, and I-E. The I-J subregion presumably encodes the I-J polypeptide of the elusive T-cell suppressor factors. Restriction enzyme site polymorphisms and DNA sequence analyses of the I region from four recombinant mouse strains were used to localize the putative I-B and I-J subregions to a 1.0-kilobase (kb) region within the E beta gene. Sequencing this region from E beta clones derived from the two mouse strains: B10.A(3R), I-Jb and B10.A(5R), I-Jk initially used to define the I-J subregion revealed that these regions are identical, hence the distinct I-Jb and I-Jk molecules cannot be encoded by this DNA. In addition, the DNA sequence data also refute the earlier mapping of the I-B subregion. Analysis of the DNA sequences of three parental and four I region recombinants reveals that the recombinant events in three of the recombinant strains occurred within a 1-kb region of DNA, supporting the proposition that a hotspot for recombination exists in the I region. The only striking feature of this hotspot is a tetramer repeat (AGGC)n that shows 80 percent homology to the minisatellite sequence which may facilitate recombination in human chromosomes.
Asunto(s)
Complejo Mayor de Histocompatibilidad , Recombinación Genética , Animales , Secuencia de Bases , Enzimas de Restricción del ADN , Genes MHC Clase II , Ratones , Secuencias Repetitivas de Ácidos Nucleicos , Factores Supresores Inmunológicos/genéticaRESUMEN
A gene encoding a novel CACCC box-binding protein that binds to the promoter region of the human T-cell receptor (TCR) V beta 8.1 gene and the mouse TCR alpha gene silencer has been cloned. This gene, termed ht beta, contains four zinc fingers of the class Cys2-X12-His2 that may be responsible for DNA binding and a highly negatively charged region that defines a putative transcriptional activation domain. Analysis of the expression of ht beta mRNA revealed similar expression levels and patterns in various cell lines. The bacterially expressed ht beta protein can bind to the CACCC box in both the human TCR V beta 8.1 gene promoter and the mouse TCR alpha gene silencer. The CACCC box is essential for efficient transcription of the V beta 8.1 promoter. Cotransfection with an ht beta expression plasmid and a reporter vector indicated that ht beta can activate human TCR V beta 8.1 gene transcription. ht beta also is able to counteract the silencing effect of the mouse TCR alpha gene silencer. The CACCC box has been found in almost all V beta 8.1 gene subfamily members and in both TCR alpha and beta gene enhancers in humans and mice. These results suggest that the CACCC box-binding protein may have an important regulatory function for TCR gene expression in alpha beta T cells versus gamma delta T cells.
Asunto(s)
Proteínas de Unión al ADN/genética , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Linfocitos T/metabolismo , Factores de Transcripción/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Regulación de la Expresión Génica , Genes , Humanos , Ratones , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/química , Regiones Promotoras Genéticas , ARN Mensajero/genética , Mapeo Restrictivo , Transcripción Genética , Dedos de ZincRESUMEN
A peptide sequence was identified by phage display technology that could be used as an alternative to chymopapain for the release of hematopoietic progenitor cells captured by anti-CD34 monoclonal antibodies. This was achieved by affinity selection screening (biopanning) of a random hexapeptide sequence phage display library. Four rounds of biopanning were performed to enrich for phage clones with specific affinity for anti-CD34 monoclonal antibody, 9C5. DNA sequence analyses of these phage clones revealed an enrichment of two predominant sequences, QQGWFP and TQGSFW. These two clones also shared a consensus sequence motif, QGxF, that exhibited 50% and 67% homology with a region spanning amino acids 14-19 of the mature CD34 antigen. Based on these data, synthetic peptides were generated and assessed for their ability to release 9C5 from CD34+ cells. Using a flow cytometric assay, it was found that the synthetic peptide, 9069N, effectively released 9C5 from the CD34-expressing cell line, KG1a, in a concentration-dependent manner (77% and 99% release of 9C5 at 0.14 and 0.70 mM peptide concentrations, respectively). In the Isolex 300i immunomagnetic selection system, this peptide was shown to be effective at releasing 9C5 sensitized CD34+ hematopoietic progenitors from sheep anti-mouse IgG Dynabeads. Thus, a synthetic peptide, which specifically and efficiently released immunomagnetically selected hematopoietic progenitor cells from paramagnetic beads, was identified. This reagent is a significant advance in the selection of hematopoietic progenitors in that it does not alter cell surface antigens. As such, further phenotypic characterization or immunoselection can be performed.
Asunto(s)
Antígenos CD34/inmunología , Bacteriófagos/genética , Células Madre Hematopoyéticas/inmunología , Péptidos/inmunología , Secuencia de Aminoácidos , Secuencia de Bases , Cartilla de ADN , Células Madre Hematopoyéticas/citología , Humanos , Separación Inmunomagnética , Péptidos/química , Péptidos/genéticaRESUMEN
Simpson-Golabi-Behmel syndrome (SGBS) is an X-linked overgrowth disorder recently shown to be caused by mutations in the heparan sulfate proteoglycan GPC3 [Pilia et al., Nat Genet; 12:241-247 1996]. We have used Southern blot analysis and polymerase chain reaction amplification of intra-exonic sequences to identify four new GPC3 mutations and further characterize three previously reported SGBS mutations. De novo GPC3 mutations were identified in 2 families. In general, the mutations were unique deletions ranging from less than 0.1 kb to more than 300 kb in length with no evidence of a mutational hot spot discerned. The lack of correlation between the phenotype of 18 affected males from these 7 families and the location and size of the GPC3 gene mutations suggest that SGBS is caused by a nonfunctional GPC3 protein.
Asunto(s)
Deleción Cromosómica , Heparitina Sulfato/genética , Mutación , Proteoglicanos/genética , Anomalías Múltiples/genética , Autorradiografía , Southern Blotting , Sondas de ADN , Genotipo , Proteoglicanos de Heparán Sulfato , Humanos , Masculino , Linaje , Fenotipo , Reacción en Cadena de la Polimerasa , Cromosoma X/genéticaAsunto(s)
Bacteriófago phi X 174/enzimología , Replicación del ADN , ADN Viral/biosíntesis , Proteínas Bacterianas/metabolismo , ADN/metabolismo , ADN Primasa , ADN de Cadena Simple/biosíntesis , Monoéster Fosfórico Hidrolasas/metabolismo , ARN Nucleotidiltransferasas/metabolismo , Proteínas Virales/metabolismo , Replicación ViralRESUMEN
A case is presented of a live-born infant with nonimmune hydrops fetalis who survived for 9 h. Neuropathological examination revealed extensive neuronal loss and gliosis in the subcortical gray nuclei suggestive of anoxic brain damage some weeks before birth. In addition the cerebellum was found to be hypoplastic and immature. Possible pathogenetic mechanisms in relation to the hydrops are discussed. In view of the scanty documentation of cerebral lesions in the literature, more detailed examinations of the central nervous system in all cases of hydrops are suggested.
Asunto(s)
Encéfalo/patología , Edema/patología , Enfermedades Fetales/patología , Hipoxia Encefálica/patología , Adulto , Femenino , Humanos , Recién Nacido , Masculino , Placenta/patología , EmbarazoRESUMEN
The Escherichia coli dnaC protein, purified to homogeneity from overproducing plasmid strains, is a polypeptide of 31,000 daltons (determined on a denaturing gel). The native molecular weight as calculated from the sedimentation coefficient of 2.75 S and Stokes radius of 24.5 A is 29,000. dnaC protein is N-ethylmaleimide sensitive (Wickner, S., Berkower, L., Wright, M., and Hurwitz J. (1973) Proc. Natl. Acad. Sci. U. S. A. 70, 2369-2373), and has 3 sulfhydryl groups as determined with [14C]p-chloromercuribenzoate. The activity was assayed by complementation of a mutant dnaC extract or by reconstitution of a purified protein system which converts phi X174 single-stranded DNA to the duplex replicative form. In this conversion the dnaC protein is required during the initial prepriming stage of phi X174 DNA replication. Antiserum against dnaC protein specifically inhibits this stage but not the subsequent priming and elongation steps carried out by primase and the PolIII holoenzyme. Requirement for dnaC protein was also manifested in the in vitro replication of a plasmid DNA containing the E. coli origin of replication (oriC) by complementation of a mutant extract and specific inhibition by dnaC antiserum.
Asunto(s)
Proteínas Bacterianas/genética , Escherichia coli/genética , Genes Bacterianos , Genes , Adenosina Trifosfatasas/metabolismo , Aminoácidos/análisis , Proteínas Bacterianas/aislamiento & purificación , Replicación del ADN , Etilmaleimida/farmacología , Cinética , PlásmidosRESUMEN
The Escherichia coli dnaB and dnaC proteins form a tight complex in the presence of ATP (Wickner, S., and Hurwitz, J., (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 921-925). The complexed dnaC protein is resistant to inhibition by the sulfhydryl reagent, N-ethylmaleimide. This protection is not observed when ATP is substituted by AMP, ADP, adenyl 5'-yl imidodiphosphate, or adenosine-5'-O-(3-thiotriphosphate); dATP provides partial protection. A sedimentation coefficient of 15.2 S determined by glycerol gradient sedimentation and a Stokes radius of 64 A determined by gel filtration suggests a molecular weight in the range of 400,000. The complex isolated by DEAE-cellulose chromatography contains six dnaC protein monomers of 29,000 daltons contains six dnaC protein monomers of 29,000 daltons contains six dnaC protein monomers of 29,000 daltons per dnaB protein hexamer (300,000 daltons) consistent with a calculated weight of 474,000. The isolated dnaB-dnaC protein complex functions in vitro in the replication of phage phi X174 single-stranded DNA to the duplex replicative form. Tritium-labeled dnaC protein, absent from an isolated prepriming com-dnaC protein, absent from an isolated prepriming complex intermediate, was nevertheless bound to the phiX replicative form DNA synthesized in vitro. These results suggest that stable inclusion od dnaC protein in the priming complex bound to DNA requires a completely assembled primosome.
Asunto(s)
Proteínas Bacterianas/genética , Escherichia coli/genética , Genes Bacterianos , Genes , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Replicación del ADN , Etilmaleimida/farmacología , Cinética , Conformación ProteicaRESUMEN
Eight cases are presented of arhinencephaly and its associated malformations, which included 2 examples of holoprosencephaly and 3 of agenesis of the corpus callosum. Additional features included cortical malformations, anomalies of the long tracts and of the optic pathway, cerebellar hypoplasia and dentato-olivary dysplasia. Each of these components covered a wide spectrum ranging in severity from extreme to minimal. Craniofacial dysmorphism, and cardiac, renal and endocrine disorders were present in some cases. Only 2 cases were associated with chromosomal abnormalities, 1 with trisomy 13, the other with partial trisomy 7(7q+). Of possible environmental factors, maternal diabetes was recorded in 1 case. While all cases can be classified into broad categories, the individual variations render each case apparently unique.
Asunto(s)
Anomalías Múltiples/patología , Diencéfalo/anomalías , Telencéfalo/anomalías , Adulto , Agenesia del Cuerpo Calloso , Niño , Diencéfalo/patología , Femenino , Humanos , Lactante , Recién Nacido , Discapacidad Intelectual , Masculino , Tabique Pelúcido/anomalías , Telencéfalo/patologíaRESUMEN
The alpha beta T-cell receptor (TCR) recognizes antigenic peptides bound to major histocompatibility complex (MHC) molecules. In contrast to the antibody combining site, for which the antigen contact or complementarity-determining residues (CDRs) have been precisely defined, the location and function of the corresponding CDR regions of the alpha and beta TCR chains are not known. To develop a model system for systematic analysis of the CDRs of the alpha beta TCR, we isolated a panel of murine T-cell clones that recognize a lysozyme peptide containing residues 74-88 bound to either Ab or Abm-12 MHC class II molecules. Although these two MHC molecules differ by only three amino acid residues within the A beta chain, each of the T-cell clones was specific for peptide bound to the self-MHC molecule and did not recognize the same peptide bound to the other MHC molecule. The structural basis for this exquisite ligand specificity of the TCRs was analyzed by isolation and characterization of alpha and beta chain genes from five closely related T-cell clones. Comparison of predicted amino acid sequences mapped the ligand specificity differences to residues present within the alpha chain variable region segment and the alpha and beta chain variable-joining region junction regions. Thus with current models of TCR-ligand interactions, the results suggest that residues 26-30 of the alpha chain variable region may constitute one of the CDR regions of the TCR.
Asunto(s)
Reordenamiento Génico de Linfocito T , Antígenos de Histocompatibilidad Clase II/inmunología , Muramidasa/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/química , Linfocitos T/inmunología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células Clonales , Ratones , Datos de Secuencia Molecular , Muramidasa/química , Péptidos/química , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Alineación de Secuencia , Relación Estructura-ActividadRESUMEN
Recombinant lambda phages containing the genes for dnaZ protein (the gamma subunit of DNA polymerse III holoenzyme), primase (dnaG protein) and dnaC protein from Escherichia coli and Salmonella typhimurium were isolated. Each gene cloned from S. typhimurium has extensive DNA sequence homology to the corresponding E. coli gene. Clones selected by complementation of a dnaA temperature-sensitive mutant appear similar to other isolated suppressors of dnaA (Projan and Wechsler 1981). Derivatives of each cloned fragment suitable for overproduction of the protein were constructed. Of those tested, only the phage containing the E. coli dnaZ gene resulted in significant overproduction.
Asunto(s)
Bacteriófago lambda/genética , Clonación Molecular , Replicación del ADN , ADN Bacteriano/genética , Escherichia coli/genética , Genes Bacterianos , Genes , Proteínas Bacterianas/genética , ADN Ligasas/metabolismo , ADN Polimerasa I/metabolismo , Enzimas de Restricción del ADN , Plásmidos , Polinucleótido 5'-Hidroxil-Quinasa/metabolismoRESUMEN
Using Southern DNA hybridization techniques, restriction enzyme site polymorphisms have been used to correlate the molecular maps of the murine major histocompatibility complex (MHC) I region with the genetic map derived from analyses of recombinant mouse strains. The data indicated that the DNA that maps between the I-A and I-E subregions is limited to 3.4 kilobases (kb) and includes the 3' end of the E beta gene. According to classical genetic mapping by recombinational analysis of serological markers, this region should encode the I-B and I-J subregions. These observations are surprising in two respects. First, 3.4 kb is a small amount of DNA to encode even one complete murine gene. Second, this region, which putatively encodes the I-J gene, appears to reside at least partially within the E beta gene. To analyze these apparent paradoxes, further, we cloned the 3.4-kb region in question from six I-region combinant strains [B10.A(3R), B10.a(5R), B10.A(4R), B10.GD, B10.HTT, and B10.S(9R)] and four strains used in the derivation of the recombinants (B10.D2, B10.A, C57BL/10, and ASW) into a lambda phage vector. By direct restriction enzyme mapping of polymorphic sites, we have confirmed the previously identified boundaries of the I-A and I-E subregions and have narrowed the estimate of the distance between these subregions to approximately 2.0 kb of DNA. This 2.0-kb region encompasses part of the intron between the first- (beta 1) and second-domain (beta 2) exons and the second-domain exon (beta 2) of the E beta gene.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Complejo Mayor de Histocompatibilidad , Animales , Clonación Molecular , Haplotipos , Ratones , Polimorfismo de Longitud del Fragmento de Restricción , Recombinación GenéticaRESUMEN
A simple and rapid strategy for DNA sequence analysis based on the Sanger chain-termination method is described. This procedure utilizes full-sized inserts of 1 to 4 kb of DNA cloned into M13 bacteriophage vectors. After the sequence of the first 600-650 bp of the insert DNA has been determined with the commercially available universal vector primer, a specific oligonucleotide is synthesized utilizing the sequence data obtained from the 3' end of the sequence and used as a primer to extend the sequence analysis for another 600-650 nucleotides. Additional primers are synthesized in a similar manner until the nucleotide sequence of the entire insert DNA has been determined. General guidelines for the selection of oligonucleotide length and composition and the use of unpurified primers are discussed. The use of the specific-primer-directed approach to dideoxynucleotide sequence analysis, in association with highly purified single-stranded template DNA, reduces considerably the time required for the analysis of large segments of DNA.
Asunto(s)
Secuencia de Bases , ADN Bacteriano/genética , Bacteriófagos/genética , Composición de Base , Clonación Molecular , Elementos Transponibles de ADN , Escherichia coli/genética , Vectores Genéticos , OligodesoxirribonucleótidosRESUMEN
The authors describe 13 cocaine-exposed infants with optic nerve abnormalities, delayed visual maturation, and prolonged eyelid edema. Prolonged and potentially vision-threatening eyelid edema is a new clinical entity. The pharmacology of cocaine, its easy access to fetal circulation, and its neurotropic characteristics can be used to explain optic nerve abnormalities and delayed visual maturation. In infants with any of these eye abnormalities, a careful investigation for cocaine abuse is advisable.
Asunto(s)
Anomalías Inducidas por Medicamentos/etiología , Cocaína/efectos adversos , Anomalías del Ojo/inducido químicamente , Efectos Tardíos de la Exposición Prenatal , Edema/inducido químicamente , Enfermedades de los Párpados/inducido químicamente , Femenino , Humanos , Lactante , Intercambio Materno-Fetal , Enfermedades del Nervio Óptico/inducido químicamente , Embarazo , Trastornos de la Visión/inducido químicamenteRESUMEN
The nucleotide sequence of the Escherichia coli dnaC gene and the primary structure of the dnaC protein were determined. The NH2-terminal amino acid sequence of the dnaC protein matched that predicted from the nucleotide sequence of the 735-base pair coding region. The dnaC gene lacks characteristic promoter structures; neither the "Pribnow box" nor the "-35 sequence" was detected within 222 base pairs upstream from the initiator ATG codon. There is, however, a typical Shine-Dalgarno sequence 7-10 base pairs before the ATG codon. An upstream open reading frame, separated by just 2 base pairs from the coding region of dnaC, encodes the COOH-terminal half of the dnaT product (protein i; Masai, H., Bond, M. W., and Arai, K. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 1256-1260). The dnaC protein contains 245 amino acids with a calculated molecular weight of 27,894 consistent with the observed value (29,000). Similar to dnaG and dnaT, dnaC uses several minor codons; the significance of these minor codons to the low level expression of the protein product in E. coli cells remains to be determined. The in vitro site-directed mutagenesis method was employed to determine the functional region involved in interaction with dnaB protein. The first cysteine residue located in the NH2-terminal region of the dnaC protein (Cys69) was shown to be important for this activity. Overall sequence homology between dnaC protein and lambda P protein, functionally analogous to the dnaC protein in the lambda phage DNA replication, is not extensive. There are, however, several short stretches of homologous regions including the NH2-terminal eight amino acids and the Cys78 region of dnaC protein.
Asunto(s)
Proteínas Bacterianas , Cisteína , ADN Helicasas , Escherichia coli , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Secuencia de Bases , Codón , ADN Bacteriano/genética , AdnB Helicasas , Escherichia coli/genética , Datos de Secuencia Molecular , Mutación , Fragmentos de Péptidos , Homología de Secuencia de Ácido NucleicoRESUMEN
The molecular analysis of crossing-over within the mouse major histocompatibility complex provides a useful approach for the study of the structural characteristics of meiotic recombination. In this study five intra-I-region recombinants, each derived from Ik/Ib heterozygotes, were characterized for restriction-fragment length polymorphisms (RFLPs) characteristic of the I region of the two parental strains. Southern blot analysis of intra-I recombinant strains A.TBR2, A.TBR3, A.TBR5, A.TBR13, and A.TBR17 using six I-region DNA probes revealed that the point of crossing-over in all five recombinants occurred within a 6.2-kb KpnI-EcoRI segment located within the E beta gene. The segments of DNA containing the crossover point from each of the recombinant chromosomes were cloned by screening partial genomic libraries constructed in lambda gt7 bacteriophage. Construction of partial restriction maps of the cloned segments from the parental and recombinant chromosomes permitted the boundaries of the area containing the crossover site to be narrowed to a 4.0-kb segment located almost entirely within an intron of the E beta gene. The recognition that the points of crossing-over in all five recombinants studied are clustered in a relatively small area of the I region provides further evidence for a hot spot of recombination associated with the E beta gene.
Asunto(s)
Intercambio Genético , Genes MHC Clase I , Meiosis , Animales , Mapeo Cromosómico , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Hibridación de Ácido Nucleico , Polimorfismo de Longitud del Fragmento de Restricción , Recombinación GenéticaRESUMEN
We describe two children with deficiency of short-chain L-3-hydroxyacyl-CoA dehydrogenase, a new disorder of the mitochondrial beta-oxidation of straight-chain fatty acids. The patients presented with fasting-induced vomiting, and ketosis and low blood glucose, features typical of ketotic hypoglycemia were documented in one. Enzyme assays were performed in cultured skin fibroblasts. In whole fibroblast preparations there was reduced enzyme activity but high residual activity due to the presence of a nonmitochondrial enzyme. In isolated fibroblast mitochondria the residual enzyme activities were 5 and 6% of the normal controls. Activity in an obligate heterozygote was intermediate, suggesting that this is an autosomal recessive disorder.
Asunto(s)
3-Hidroxiacil-CoA Deshidrogenasas/deficiencia , Mitocondrias/enzimología , Ácidos Mirísticos/metabolismo , Palmitatos/metabolismo , Células Cultivadas , Ácidos Grasos/metabolismo , Femenino , Humanos , Lactante , Masculino , Ácido Mirístico , Oxidación-Reducción , Piel/citologíaRESUMEN
We described previously a simple test on petri plates for detecting chemical carcinogens as mutagens, using an especially sensitive set of bacterial strains to detect mutagenic acitivty and a mammalian liver extract for carcinogen activity. We now extend the utility of the method by introducing two new bacterial strains which can detect with great sensitivity many carcinogens which we did not detect before or detected with less sensitivity. Among these carcinogens are aflatoxin B-1, sterigmatocystin, benzyl chloride, benzo[a]-pyrene, 7,12-dimethylbenzanthracene, 1'-acetoxysafrole, and the nitrofuran food additive furylfuramide (AF-2). The new strains TA100 and TA98 contain an R factor plasmid, pKM101, in our standard tester strains TA1535 and TA1538. The R factor increases mutagenesis with certain mutagens, but not others. We present evidence that the mutagens that become more effective work through an error-prone recombinational repair.
Asunto(s)
Carcinógenos , Farmacorresistencia Microbiana , Herencia Extracromosómica , Mutágenos , Salmonella typhimurium/efectos de los fármacos , Bioensayo , Mesilatos/farmacología , Mutación/efectos de los fármacos , Nitrofuranos/farmacologíaRESUMEN
A Prader-Willi syndrome patient is described who has a de novo balanced translocation, (4;15)(q27;q11.2)pat, with breakpoints lying between SNRPN exons 2 and 3. Parental-origin studies indicate that there is no uniparental disomy and no apparent deletion. This patient expresses ZNF127, SNRPN exons 1 and 2, IPW, and D15S227E (PAR1) but does not express either SNRPN exons 3 and 4 or D15S226E (PAR5), as assayed by reverse transcription-PCR, of peripheral blood cells. Methylation studies showed normal biparental patterns of inheritance of loci DN34/ZNF127, D15S63, and SNRPN exon 1. Results for this patient and that reported by Sun et al. support the contention that an intact genomic region and/or transcription of SNRPN exons 2 and 3 play a pivotal role in the manifestations of the major clinical phenotype in Prader-Willi syndrome.