RESUMEN
The analysis of protein-linked glycans is of increasing importance, both in basic glycobiological research and during the production process of glycoprotein pharmaceuticals. In many cases, the amount of glycoprotein available for typing the glycans is very low. This, combined with the high branching complexity typical for this class of compounds, makes glycan typing a challenging task. We present here methodology allowing the medium-throughput analysis of N-glycans derived from low picomole amounts of glycoproteins using the standard DNA-sequencing equipment available in any life sciences laboratory. The high sensitivity of the overall analytical process (from glycoprotein to results) is obtained using state-of-the-art deglycosylation procedures combined with a highly efficient and reproducible novel postderivatization cleanup step involving Sephadex G10 packed 96-well filterplates. All sample preparation steps (enzymatic deglycosylation with PNGase F, desalting, derivatization with 8-amino-1,3,6-pyrenetrisulfonic acid, and postderivatization cleanup) are performed using 96-well-based plates. This integrated sample preparation scheme is also compatible with capillary electrophoresis and MALDI-TOF-MS platforms already in use in some glycobiology labs and anticipates the higher throughput that will be offered by the capillary-array-based DNA sequencers currently penetrating the market. The described technology should bring high-performance glycosylation analysis within reach of each life sciences lab and thus help expedite the pace of discovery in the field of glycobiology.
Asunto(s)
Oligosacáridos/química , Análisis de Secuencia de ADN/instrumentación , Análisis de Secuencia/instrumentación , Análisis de Secuencia/métodos , Conformación de Carbohidratos , Secuencia de Carbohidratos , Cromatografía por Intercambio Iónico , Electroforesis en Gel de Poliacrilamida , Colorantes Fluorescentes , Glicoproteínas/química , Glicoproteínas/metabolismo , Glicosilación , Datos de Secuencia Molecular , Oligosacáridos/metabolismo , Polisacáridos/química , Polisacáridos/metabolismo , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Caspases are cysteinyl aspartate-specific proteinases, many of which play a central role in apoptosis. Here, we report the identification of a new murine caspase homologue, viz. caspase-14. It is most related to human/murine caspase-2 and human caspase-9, possesses all the typical amino acid residues of the caspases involved in catalysis, including the QACRG box, and contains no or only a very short prodomain. Murine caspase-14 shows 83% similarity to human caspase-14. Human caspase-14 is assigned to chromosome 19p13.1. Northern blot analysis revealed that mRNA expression of caspase-14 is undetectable in all mouse adult tissues examined except for skin, while it is abundantly expressed in mouse embryos. In contrast to many other caspase family members, murine caspase-14 is not cleaved by granzyme B, caspase-1, caspase-2, caspase-3, caspase-6, caspase-7 or caspase-11, but is weakly processed into p18 and p11 subunits by murine caspase-8. No aspartase activity of murine caspase-14 could be generated by bacterial or yeast expression. Transient overexpression of murine caspase-14 in mammalian cells did not elicit cell death and did not interfere with caspase-8-induced apoptosis. In conclusion, caspase-14 is a member of the caspase family but no proteolytic or biological activities have been identified so far. The high constitutive expression levels in embryos and specific expression in adult skin suggest a role in ontogenesis and skin physiology.
Asunto(s)
Caspasas/genética , Secuencia de Aminoácidos , Animales , Caspasa 1/química , Caspasa 14 , Caspasas/biosíntesis , Caspasas/química , Línea Celular , Clonación Molecular , Células HeLa , Humanos , Masculino , Ratones , Datos de Secuencia Molecular , Especificidad de Órganos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transcripción Genética , TransfecciónRESUMEN
Seven members of the murine caspase (mCASP) family were cloned and functionally characterized by transient overexpression: mCASP-1 (mICE), mCASP-2 (Ich1), mCASP-3 (CPP32), mCASP-6 (Mch2), mCASP-7 (Mch3), mCASP-11 (TX) and mCASP-12. mCASP-11 is presumably the murine homolog of human CASP-4. Although mCASP-12 is related to human CASP-5 (ICErel-III), it is most probably a new CASP-1 family member. On the basis of sequence homology, the caspases can be divided into three subfamilies: first, mCASP-1, mCASP-11 and mCASP-12; second, mCASP-2; third, mCASP-3, mCASP-6 and mCASP-7. The tissue distribution of the CASP-1 subfamily transcripts is more restricted than that of the CASP-3 subfamily transcripts, suggesting that the transcriptional regulation of the CASP members within one subfamily is related, but is quite different between the CASP-1 and the CASP-3 subfamilies. Transient overexpression of each of the seven CASPs induced apoptosis in mammalian cells. Only two, mCASP-1 as well as mCASP-3, were able to process precursor interleukin (IL)-1beta to biologically active IL-1beta. In addition, mCASP-3 is the predominant PARP-cleaving enzyme in vivo.
Asunto(s)
Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Secuencia de Aminoácidos , Animales , Apoptosis/genética , Caspasa 1 , Clonación Molecular , Humanos , Interleucina-1/genética , Interleucina-1/metabolismo , Ratones , Datos de Secuencia Molecular , Poli(ADP-Ribosa) Polimerasas/metabolismo , Biosíntesis de Proteínas , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Distribución Tisular , Transcripción GenéticaRESUMEN
The cytoplasmic beta-catenin protein is implicated in signal transduction and associates with both the cell-cell adhesion protein E-cadherin and the tumor suppressor gene product APC. We determined the primary structure of the human beta-catenin gene (CTNNB1) by analysis of cDNA and genomic clones. The size of the complete gene was determined to be 23.2 kb. Restriction mapping and partial sequence analysis revealed 16 exons. All splice donor and acceptor sites were conformable to the GT/AG rule. The exon size ranged from 61 to 790 bp. Half of the introns were smaller than 550 bp, with the smallest being 84 bp and the longest being 6700 bp. The intron-exon boundaries did not coincide either with conserved sites in the 12 armadillo repeat sequences of beta-catenin or with intron-exon boundaries in the armadillo gene of Drosophila. A major site for transcription initiation was identified as an A residue 214 nucleotides upstream of the ATG initiation codon. The resulting transcript is 3362 nucleotides long. Compared to the previously published mRNA sequence, additional residues were identified, 16 at the 5' end and 766 at the 3' end of the mRNA. An alternative splice acceptor site within exon 16 reduced the 3' UTR sequence by 159 bp. Polymerase chain reaction on cDNA from 14 human cell lines demonstrated the general occurrence of both splice variants. The 5'-flanking region is highly GC-rich and lacks a CCAAT box, but contains a TATA box and potential binding sites for several transcription factors, such as NF kappa B, SP1, AP2, and EGR1. Both a 437-bp fragment and a 6-kb fragment, containing about 4.7 kb of the 5'-flanking region in addition to the noncoding exon 1 and 1 kb of intron 1, showed clear promoter activity when these fragments were linked to a secreted alkaline phosphatase reporter gene and transfected into a mouse epithelial cell line.
Asunto(s)
Proteínas del Citoesqueleto/genética , Genes/genética , Transactivadores , Empalme Alternativo , Secuencia de Aminoácidos , Composición de Base , Secuencia de Bases , Línea Celular , Clonación Molecular , Islas de CpG/genética , Exones/genética , Humanos , Intrones/genética , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , Mapeo Restrictivo , Análisis de Secuencia de ADN , beta CateninaRESUMEN
E-cadherin is a Ca(2+)-dependent epithelial cell-cell adhesion molecule. Downregulation of E-cadherin expression often correlates with strong invasive potential and poor prognosis of human carcinomas. By using recombinant lambda phage, cosmid, and P1 phage clones, we isolated the full-length human E-cadherin gene (CDH1). The gene spans a region of approximately 100 kb, and its location on chromosome 16q22.1 was confirmed by FISH analysis. Detailed restriction mapping and partial sequence analysis of the gene allowed us to identify 16 exons and a 65-kb-long intron 2. The intron-exon boundaries are highly conserved in comparison with other "classical cadherins." In intron 1 we identified a 5' high-density CpG island that may be implicated in transcription regulation during embryogenesis and malignancy.
Asunto(s)
Cadherinas/genética , Cromosomas Humanos Par 16 , Genes , Secuencia de Aminoácidos , Secuencia de Bases , Adhesión Celular , Mapeo Cromosómico , Clonación Molecular , Elementos de Facilitación Genéticos , Exones , Regulación de la Expresión Génica , Humanos , Hibridación Fluorescente in Situ , Intrones , Datos de Secuencia Molecular , Invasividad Neoplásica , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
A pseudogene (CTNNAP1) for the human alpha E-catenin gene was isolated from a human genomic phage library. The pseudogene sequence shows 90% similarity to the alpha E-catenin mRNA at the nucleotide level. Thirty-eight stop codons in all three reading frames and multiple other mutations were found, indicating that the pseudogene does not encode a functional protein. No introns were found in the region corresponding to the open reading frame of the alpha E-catenin cDNA, and two direct repeats flank this same region. Hence, the pseudogene can be classified as a processed pseudogene. Polymerase chain reaction with pseudogene-specific primers on genomic DNA and cDNA from human cell lines and healthy blood donors demonstrated the general occurrence of the pseudogene and the lack of its transcription. By fluorescence in situ hybridization the pseudogene was mapped to human chromosome 5q22 and the alpha E-catenin gene to the formerly disputed locus 5q31. This is the first report of a pseudogene for a member of the cadherin-catenin cell-cell adhesion complex.
Asunto(s)
Cromosomas Humanos Par 5 , Proteínas del Citoesqueleto/genética , Genes , Seudogenes , Secuencia de Bases , Línea Celular , Mapeo Cromosómico , Proteínas del Citoesqueleto/clasificación , ADN Complementario/genética , Biblioteca de Genes , Humanos , Hibridación Fluorescente in Situ , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Reacción en Cadena de la Polimerasa , Secuencias Repetitivas de Ácidos Nucleicos , Homología de Secuencia de Ácido Nucleico , alfa CateninaRESUMEN
A 7.4 kb segment of chromosome II was sequenced and analysed. This segment is part of the 25 kb insert of cosmid clone alpha 1004.10 which is located on the left arm of chromosome II. Sequence analysis revealed four open reading frames (ORFs), of which two had been characterized previously (SSA3, AAR2) and one was not identified. The other ORF was precisely 600 bp long and the deduced protein sequence predicted a very basic protein (pI = 11.1; molecular weight = 22.5 kDa). Evidence was found that the ORF is the S40 ribosomal protein gene (RPG) S8. Consensus splice signals were found in the 5' leader sequence and also potential RPG-specific sequences. Chromoblot analysis revealed a second copy of the S8 RPG on chromosome IV or VIII. This copy is also closely linked to an hsp70 protein gene, SSA4.
Asunto(s)
Genes Fúngicos/genética , Proteínas HSP70 de Choque Térmico/genética , Familia de Multigenes/genética , Proteínas Ribosómicas/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Secuencia de Bases , Mapeo Cromosómico , Cósmidos , Ligamiento Genético , Datos de Secuencia Molecular , Mapeo Restrictivo , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
The nucleotide sequence of a 1558 bp DNA fragment from the right arm of chromosome III of Saccharomyces cerevisiae contains an open reading frame of 954 nucleotides with coding potential for a protein with high similarity to the ubiquitous cyclophilins which are both peptidyl-prolyl cis-trans isomerases and cyclosporin A-binding proteins. It should, therefore, represent the third gene (SCC3) of this kind from S. cerevisiae. SCC3 is present in a single copy in the genome of S. cerevisiae and results in a constitutively expressed 1.2 kb transcript during cell growth. Its putative protein product (Scc3) contains two hydrophobic cores, one at the amino terminal, 20 amino acids long, which could serve as a signal peptide, and the other one at the carboxyl end with a structure similar to a transmembrane helix. These findings suggest that Scc3 could be a secretory or, more likely, a transmembrane protein. The only cyclophilin with similar structure to that of Scc3 is ninaA from Drosophila melanogaster, a transmembrane protein which seems to be implicated in the correct folding and/or intercalation of rhodopsin in the endoplasmic reticulum of the fly photoreceptors (Stamnes, M.A. et al., Cell 65, 219-227, 1991). In addition, the amino and the carboxy regions of Scc3 and ninaA share a significant level of homology, which suggests that they have a similar function, albeit for different target proteins.
Asunto(s)
Isomerasas de Aminoácido/genética , Proteínas Portadoras/genética , Ciclosporinas/genética , ADN de Hongos/química , Saccharomyces cerevisiae/genética , Isomerasas de Aminoácido/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Southern Blotting , Proteínas Portadoras/química , Clonación Molecular , Ciclosporinas/química , ADN de Hongos/análisis , Drosophila melanogaster/genética , Escherichia coli/enzimología , Escherichia coli/genética , Genes Fúngicos , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Isomerasa de Peptidilprolil , Mapeo Restrictivo , Saccharomyces cerevisiae/químicaRESUMEN
From a mouse hybridoma cell line secreting a monoclonal antibody directed against the tumour marker human placental alkaline phosphatase, mRNA coding for the H and L chains of this antibody was isolated and cloned as cDNA. Sequence analysis of the H and L chain cDNAs confirmed the IgG2b,kappa subtype previously established. Recloning the H and L chain cDNA information into SV40-based vectors enabled us to obtain expression of functional immunoglobulin upon cotransfection into COS or CHO dhfr- cells. This illustrates that non-lymphoid cells also have the capacity to assemble active immunoglobulins.
Asunto(s)
Fosfatasa Alcalina/inmunología , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/genética , Isoenzimas/inmunología , Placenta/enzimología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Clonación Molecular , ADN/genética , Proteínas Ligadas a GPI , Regulación de la Expresión Génica , Vectores Genéticos , Humanos , Hibridomas , Datos de Secuencia Molecular , Plásmidos , ARN Mensajero/genética , Proteínas Recombinantes/genética , Homología de Secuencia de Ácido Nucleico , TransfecciónRESUMEN
The type II restriction enzymes CauI and CauII, isolated from Chloroflexus aurantiacus, recognize and cleave (at the position indicated by an arrow) the sequences G decreases G A/T CC and CC decreases G/C GG, respectively. These conclusions are supported by the results from restriction site mapping, sequence analysis by partial chemical degradation, end-group analysis after lambda exonuclease treatment and computer-assisted comparison of DNA sequence data.
Asunto(s)
Enzimas de Restricción del ADN/metabolismo , Desoxirribonucleasas de Localización Especificada Tipo II , Bacterias/enzimología , Secuencia de Bases , Especificidad por SustratoRESUMEN
Bacteriophage MS2 RNA is 3,569 nucleotides long. The nucleotide sequence has been established for the third and last gene, which codes for the replicase protein. A secondary structure model has also been proposed. Biological properties, such as ribosome binding and codon interactions can now be discussed on a molecular basis. As the sequences for the other regions of this RNA have been published already, the complete, primary chemical structure of a viral genome has now been established.