RESUMEN
A database containing mapped partial cDNA sequences from Caenorhabditis elegans will provide a ready starting point for identifying nematode homologues of important human genes and determining their functions in C. elegans. A total of 720 expressed sequence tags (ESTs) have been generated from 585 clones randomly selected from a mixed-stage C. elegans cDNA library. Comparison of these ESTs with sequence databases identified 422 new C. elegans genes, of which 317 are not similar to any sequences in the database. Twenty-six new genes have been mapped by YAC clone hybridization. Members of several gene families, including cuticle collagens, GTP-binding proteins, and RNA helicases were discovered. Many of the new genes are similar to known or potential human disease genes, including CFTR and the LDL receptor.
Asunto(s)
Caenorhabditis elegans/genética , Genes de Helminto , Secuencia de Aminoácidos , Animales , Clonación Molecular , Colágeno/genética , ADN/genética , Expresión Génica , Biblioteca de Genes , Humanos , Datos de Secuencia Molecular , Familia de Multigenes , Homología de Secuencia de Aminoácido , Especificidad de la EspecieRESUMEN
The sequences of three cosmids (90 kilobases) from the Huntington's disease region in chromosome 4p16.3 have been determined. A 30,837 base overlap of DNA sequenced from two individuals was found to contain 72 DNA sequence polymorphisms, an average of 2.3 polymorphisms per kilobase (kb). The assembled 58 kb contig contains 62 Alu repeats, and eleven predicted exons representing at least three expressed genes that encode previously unidentified proteins. Each of these genes is associated with a CpG island. The structure of one of the new genes, hda1-1, has been determined by characterizing cDNAs from a placental library. This gene is expressed in a variety of tissues and may encode a novel housekeeping gene.
Asunto(s)
Cromosomas Humanos Par 4 , Cósmidos , ADN/genética , Enfermedad de Huntington/genética , Polimorfismo Genético , Secuencias Repetitivas de Ácidos Nucleicos , Secuencia de Aminoácidos , Secuencia de Bases , Bandeo Cromosómico , Mapeo Cromosómico , Clonación Molecular , Codón/genética , Elementos Transponibles de ADN , Femenino , Expresión Génica , Biblioteca de Genes , Humanos , Células Híbridas , Datos de Secuencia Molecular , Placenta/fisiología , Mutación Puntual , Embarazo , Proteínas/genéticaRESUMEN
Automated partial DNA sequencing was conducted on more than 600 randomly selected human brain complementary DNA (cDNA) clones to generate expressed sequence tags (ESTs). ESTs have applications in the discovery of new human genes, mapping of the human genome, and identification of coding regions in genomic sequences. Of the sequences generated, 337 represent new genes, including 48 with significant similarity to genes from other organisms, such as a yeast RNA polymerase II subunit; Drosophila kinesin, Notch, and Enhancer of split; and a murine tyrosine kinase receptor. Forty-six ESTs were mapped to chromosomes after amplification by the polymerase chain reaction. This fast approach to cDNA characterization will facilitate the tagging of most human genes in a few years at a fraction of the cost of complete genomic sequencing, provide new genetic markers, and serve as a resource in diverse biological research fields.
Asunto(s)
Secuencia de Bases , Encéfalo/fisiología , ADN/genética , Biblioteca de Genes , Proyecto Genoma Humano , Secuencia de Aminoácidos , Automatización , Mapeo Cromosómico , Expresión Génica , Humanos , Datos de Secuencia Molecular , Familia de Multigenes , Reacción en Cadena de la Polimerasa , Homología de Secuencia de Ácido NucleicoRESUMEN
A computer program, MOD (mixed oligo designer), which aids in planning site-directed mutagenesis experiments using highly substituted oligodeoxyribonucleotides (oligos), is described. The program calculates the relationship between the degree of oligo substitution and the mutation frequency, in order to achieve an optimal level of mutagenesis. The program can be used on a wide variety of computers and runs under a number of different operating systems.
Asunto(s)
Mutagénesis Sitio-Dirigida , Oligodesoxirribonucleótidos/síntesis química , Programas Informáticos , Algoritmos , Secuencia de Bases , Distribución Binomial , Datos de Secuencia Molecular , Mutagénesis InsercionalRESUMEN
SPLICE, a software tool for the extraction of sequences from files in GenBank tape format, has been developed. The program can analyze the features table in this format and use any of the information provided to write the corresponding sequences into a standard sequence file format suitable for use with sequence analysis programs. Sequences that are present as several subsequent fragments in a single GenBank file, such as those encoding a peptide, can be spliced together by the program. Further, sequences that are present in more than one Genbank file, such as an exon which spans several different files, can also be spliced into one sequence. SPLICE runs under the MS/DOS and Unix operating systems, can be called as a sub-process by other programs and can process batches of files.
Asunto(s)
ADN/química , Biblioteca de Genes , Programas Informáticos , Animales , Secuencia de Bases , Cobayas , Datos de Secuencia MolecularRESUMEN
We have written a computer program, BIGPROBE, which facilitates the design of long nucleic acid probes from the partial or complete amino acid sequence of a protein. BIGPROBE relies upon information on codon usage, intercodon dinucleotide frequency, and potential probe self-complementarity. We have examined the accuracy with which the program predicts coding sequences using sample human and rat genes and probe lengths of 30-60 nucleotides. Rat probe sequences selected by BIGPROBE using either codon usage or dinucleotide frequency data alone averaged 86-92% homology with the known exons of the corresponding gene sequences. Predictive accuracy with rat gene probes could be improved to 89-94%, depending upon probe length, by applying codon usage and dinucleotide frequency data in combination. Similar accuracy was achieved for human genes.
Asunto(s)
Secuencia de Bases , Computadores , Microcomputadores , Mapeo Nucleótido/métodos , Oligonucleótidos/genética , Programas Informáticos/métodos , Secuencia de Aminoácidos , Animales , Codón , Humanos , Ratas , Homología de Secuencia de Ácido NucleicoRESUMEN
The genome of the mouse (Mus musculus) contains a family of repeated DNA sequences defined by a 1.3 kb EcoRI fragment. Restriction maps of ten cloned fragments from this family have been determined. The fragments were of seven different types, based on the patterns of digestion obtained with AvaII, HindIII, and TaqI restriction enzymes. These seven unique sets of sequences fell into two classes, as defined by the position of a single HindIII site. Portions of fragments from each of the two classes were sequenced. Although certain regions of the repeat were highly conserved between classes, there was more intraspecific sequence divergence among the sequenced regions than has been observed for the short interspersed Alu family of repeated sequences in mammals. Sequences of both HindIII classes were found to be present within the mouse X chromosome; we can conclude that both classes must also be present on other mouse chromosomes.
Asunto(s)
ADN , Ratones/genética , Animales , Secuencia de Bases , Clonación Molecular , Enzimas de Restricción del ADN , Desoxirribonucleasa EcoRI , Procesamiento Automatizado de Datos , Mapeo Nucleótido , Plásmidos , Homología de Secuencia de Ácido NucleicoRESUMEN
Based on the analysis of genomic DNA from single healthy animals of each of five primate species, nucleotide and predicted amino acid sequences of the infectious amyloid precursor gene of higher apes (Gorilla and Pan) and Old World (Macaca) and New World (Ateles, Saimiri) monkeys showed 95-99% homology to the human sequences, corresponding to their phylogenetic distance from humans. Two of 18 amino acids that differed from humans resulted from nucleotide changes at sites of mutations in humans with familial forms of spongiform encephalopathy (a deleted codon within the codon 51-91 region of 24 bp repeats and a substitution at codon 198). In each of the five animals, codon 129 specified methionine, the more common of the two polymorphic genotypes in humans. Because genotypic homology did not correlate with experimental transmission rates of human spongiform encephalopathy, primary structural similarity of the infectious amyloid precursor protein in humans and experimental primates may not be an important factor in disease transmissibility.
Asunto(s)
Amiloide/genética , Primates/genética , Enfermedades por Prión/transmisión , Precursores de Proteínas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cebidae/genética , ADN/genética , ADN/aislamiento & purificación , Cartilla de ADN , Gorilla gorilla/genética , Hominidae/genética , Humanos , Macaca mulatta/genética , Datos de Secuencia Molecular , Pan troglodytes/genética , Filogenia , Mutación Puntual , Proteínas Priónicas , Priones , Saimiri/genética , Homología de Secuencia de AminoácidoRESUMEN
We recently described a new approach for the rapid characterization of expressed genes by partial DNA sequencing to generate 'expressed sequence tags'. From a set of 600 human brain complementary DNA clones, 348 were informative nuclear-encoded messenger RNAs. We have now partially sequenced 2,672 new, independent cDNA clones isolated from four human brain cDNA libraries to generate 2,375 expressed sequence tags to nuclear-encoded genes. These sequences, together with 348 brain expressed sequence tags from our previous study, comprise more than 2,500 new human genes and 870,769 base pairs of DNA sequence. These data represent an approximate doubling of the number of human genes identified by DNA sequencing and may represent as many as 5% of the genes in the human genome.
Asunto(s)
Química Encefálica/genética , ADN/análisis , Biblioteca de Genes , Lugares Marcados de Secuencia , Bases de Datos Bibliográficas , HumanosRESUMEN
Mutations in the human phosphofructokinase muscle subunit gene (PFKM) are known to cause myopathy classified as glycogenosis type VII (Tarui disease). Previously described molecular defects include base substitutions altering encoded amino acids or resulting in abnormal splicing. We report a mutation resulting in phosphofructokinase deficiency in three patients from an Ashkenazi Jewish family. Using a reverse transcription PCR assay, PFKM subunit transcripts differing by length were detected in skeletal muscle tissue of all three affected subjects. In the longer transcript, an insertion of 252 nucleotides totally homologous to the structure of the 10th intron of the PFKM gene was found separating exon 10 from exon 11. In addition, two single base transitions were identified by direct sequencing: [exon 6; codon 95; CGA (Arg) to TGA (stop)] and [exon 7; codon 172; ACC (Thr) to ACT (Thr)] in either transcript. Single-stranded conformational polymorphism and restriction enzyme analyses confirmed the presence of these point substitutions in genomic DNA and strongly suggested homozygosity for the pathogenic allele. The nonsense mutation at codon 95 appeared solely responsible for the phenotype in these patients, further expanding genetic heterogeneity of Tarui disease. Transcripts with and without intron 10 arising from identical mutant alleles probably resulted from differential pre-mRNA processing and may represent a novel message from the PFKM gene.