RESUMEN
The study of native genomes has been greatly facilitated by the use of direct genomic sequencing and footprinting strategies. This review provides an overview of the techniques involved and presents some highlights of the biological results obtained with these methods.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Animales , Secuencia de Bases , Técnicas Genéticas , Biblioteca Genómica , Humanos , Reacción en Cadena de la PolimerasaRESUMEN
The report that human growth hormone pre-mRNA is not processed in transgenic plant tissues (A. Barta, K. Sommergruber, D. Thompson, K. Hartmuth, M.A. Matzke, and A.J.M. Matzke, Plant Mol. Biol. 6:347-357, 1986) has suggested that differences in mRNA splicing processes exist between plants and animals. To gain more information about the specificity of plant pre-mRNA processing, we have compared the splicing of the soybean leghemoglobin pre-mRNA with that of the human beta-globin pre-mRNA in transfected plant (Orychophragmus violaceus and Nicotiana tabacum) protoplasts and mammalian (HeLa) cells. Of the three introns of leghemoglobin pre-mRNA, only intron 2 was correctly and efficiently processed in HeLa cells. The 5' splice sites of the remaining two introns were faithfully recognized, but correct processing of the 3' sites took place only rarely (intron 1) or not at all (intron 3); cryptic 3' splice sites were used instead. While the first intron in human beta-globin pre-mRNA was not spliced in transfected plant protoplasts, intron 2 processing occurred at a low level, indicating that some mammalian introns can be recognized by the plant intron-splicing machinery. However, excision of intron 2 proved to be incorrect, involving the authentic 5' splice site and a cryptic 3' splice site. Our results indicate that the mechanism of 3'-splice-site selection during intron excision differs between plants and animals. This conclusion is supported by analysis of the 3'-splice-site consensus sequences in animal and plant introns which revealed that polypyrimidine tracts, characteristic of animal introns, are not present in plant pre-mRNAs. It is proposed that an elevated AU content of plant introns is important for their processing.
Asunto(s)
Plantas/metabolismo , Precursores del ARN/metabolismo , Empalme del ARN , Secuencia de Bases , Células HeLa/metabolismo , Humanos , Leghemoglobina/genética , Datos de Secuencia Molecular , Plantas/genética , Protoplastos/metabolismo , Especificidad de la EspecieRESUMEN
We have analyzed the junction regions of inserted elements within the human amylase gene complex. This complex contains five genes which are expressed at high levels either in the pancreas or in the parotid gland. The proximal 5'-flanking regions of these genes contain two inserted elements. A gamma-actin pseudogene is located at a position 200 base pairs upstream of the first coding exon. All of the amylase genes contain this insert. The subsequent insertion of an endogenous retrovirus interrupted the gamma-actin pseudogene within its 3'-untranslated region. Nucleotide sequence analysis of the inserted elements associated with each of the five human amylase genes has revealed a series of molecular events during the recent history of this gene family. The data indicate that the entire gene family was generated during primate evolution from one ancestral gene copy and that the retroviral insertion activated a cryptic promoter.
Asunto(s)
Amilasas/genética , Evolución Biológica , Elementos Transponibles de ADN , Genes Virales , Genes , Isoenzimas/genética , Páncreas/enzimología , Seudogenes , Retroviridae/genética , Glándulas Salivales/enzimología , Actinas/genética , Animales , Secuencia de Bases , Southern Blotting , Humanos , Ratones , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , Primates/genética , Homología de Secuencia de Ácido NucleicoRESUMEN
Cosmid clones containing 250 kilobases of genomic DNA from the human amylase gene cluster have been isolated. These clones contain seven distinct amylase genes which appear to comprise the complete multigene family. By sequence comparison with the cDNAs, we have identified two pancreatic amylase genes and three salivary amylase genes. Two truncated pseudogenes were also recovered. Intergenic distances of 17 to 22 kilobases separate the amylase gene copies. Within the past 10 million years, duplications, gene conversions, and unequal crossover events have resulted in a very high level of sequence similarity among human amylase gene copies. To identify sequence elements involved in tissue-specific expression and hormonal regulation, the promoter regions of the human amylase genes were sequenced and compared with those of the corresponding mouse genes. The promoters of the human and mouse pancreatic amylase genes are highly homologous between nucleotide -160 and the cap site. Two sequence elements thought to influence pancreas-specific expression of the rodent genes are present in the human genes. In contrast, similarity in the 5' flanking sequences of the salivary amylase genes is limited to several short sequence elements whose positions and orientations differ in the two species. Some of these sequence elements are also associated with other parotid-specific genes and may be involved in their tissue-specific expression. A glucocorticoid response element and a general enhancer element are closely associated in several of the amylase promoters.
Asunto(s)
Amilasas/genética , Evolución Biológica , Genes , Secuencia de Bases , Clonación Molecular , Cósmidos , ADN/genética , Enzimas de Restricción del ADN , Exones , Humanos , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Páncreas/enzimología , Regiones Promotoras Genéticas , Seudogenes , Saliva/enzimología , Homología de Secuencia de Ácido NucleicoRESUMEN
The mode of action of a synthetic cytostatic quinone was studied in Escherichia coli. 1. At concentrations of 1.5-6 mug/ml, 5,8-dioxo-6-amino-7-chloroquinoline rapidly inhibits growth and protein synthesis in E. coli. The synthesis of RNA is immediately affected in E. coli rel+ whereas in E. coli rel- the accumulation of RNA can proceed on addition of the quinone. This indicates that the inhibition of RNA synthesis in the stringent strain is a consequence of the regulatory phenomenon governed by the rel gene. 2. Chloramphenicol, known to abolish the stringent control mechanism, added simultaneously with the quinone allows the accumulation of RNA to proceed in the stringent strain. 3. Guanosine tetraphosphate accumulates in quinone-treated E. coli rel+ but not in the relaxed mutant strain. 4. Addition of amino acids reverses all inhibitory effects observed in quinone treated stringent and relaxed cells. 5. It is concluded that the bacteriostatic effect of 5,8-dioxo-6-amino-7-chloroquinoline on E. coli is caused by an apparent intracellular amino acid starvation.
Asunto(s)
Aminoquinolinas/farmacología , Proteínas Bacterianas/biosíntesis , Escherichia coli/metabolismo , Nucleótidos de Guanina/biosíntesis , ARN Bacteriano/biosíntesis , Alanina/metabolismo , Aminoácidos/farmacología , Radioisótopos de Carbono , Cloranfenicol/farmacología , Escherichia coli/efectos de los fármacos , Cetonas/farmacología , Fenilalanina/metabolismo , Fosfatos/metabolismo , Radioisótopos de Fósforo , Tritio , Uridina/metabolismoRESUMEN
At concentrations of 1-1.6 mug/ml, 5,8-dioxo-6-amino-7-chloroquinoline causes auxotrophy for leucine in Escherichia coli MRE 600. With increasing concentrations of this quinone additional amino acids are required for growth. The amount of leucine in the pool of free amino acids is not decreased after treatment of E. coli with the quinone. Transfer RNALeu, however, is charged with leucine less than 10% in quinone-treated cells of E. coli, whereas in control cells the degree of aminoacylation is about 85%. From these data we conclude that the quinone causes auxotrophy for leucine by interacting with the charging process of tRNALeu. Quinone was found to inhibit leucyl-tRNA synthetase activity in purified extracts of E. coli with E. coli tRNA as substrate.
Asunto(s)
Aminoacil-ARNt Sintetasas/antagonistas & inhibidores , Aminoquinolinas/farmacología , Escherichia coli/enzimología , Leucina-ARNt Ligasa/antagonistas & inhibidores , Aminoácidos/metabolismo , Escherichia coli/efectos de los fármacos , Cetonas/farmacología , Leucina , Biosíntesis de Proteínas/efectos de los fármacos , Quinonas/farmacología , ARN Bacteriano/metabolismo , ARN de Transferencia/metabolismoAsunto(s)
ADN/genética , Plantas/genética , Precursores del ARN/genética , Procesamiento Postranscripcional del ARN , Transcripción Genética , Transfección , Células Cultivadas , Medios de Cultivo , Técnicas Genéticas , Indicadores y Reactivos , Plásmidos , Protoplastos/metabolismo , ARN/genética , ARN/aislamiento & purificación , Ultracentrifugación/métodosAsunto(s)
Citosina/análogos & derivados , Daño del ADN , Reparación del ADN , ADN/genética , Escherichia coli/genética , Virus 40 de los Simios/genética , 5-Metilcitosina , Animales , Composición de Base , Secuencia de Bases , Línea Celular , Citosina/metabolismo , ADN/química , ADN/metabolismo , ADN Glicosilasas , Desaminación , Células HeLa , Humanos , Datos de Secuencia Molecular , N-Glicosil Hidrolasas/metabolismoAsunto(s)
Anhidrasas Carbónicas/genética , Genes , Isoenzimas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN/análisis , Enzimas de Restricción del ADN , Ligamiento Genético , Humanos , Ratones , Hibridación de Ácido Nucleico , Plásmidos , Especificidad de la EspecieRESUMEN
To avoid the mutagenic effect of spontaneous hydrolytic deamination of 5-methylcytosine, G.T mispairs, arising in DNA as a result of this process, should always be corrected to G.C pairs. We describe here the identification of a DNA glycosylase activity present in nuclear extracts from HeLa cells, which removes the mispaired thymine to generate an apyrimidinic (AP) site opposite the guanine. We further show, using a specific antibody and inhibitors, that the single nucleotide gap, created upon processing of the AP site, is filled in by DNA polymerase beta. This finding substantiates the proposed role of this enzyme in short-patch DNA repair.
Asunto(s)
Composición de Base , Núcleo Celular/enzimología , ADN Polimerasa I/metabolismo , Reparación del ADN , Endodesoxirribonucleasas/metabolismo , Afidicolina , Secuencia de Bases , Cromatografía en Capa Delgada , Desoxirribonucleasa (Dímero de Pirimidina) , Diterpenos/farmacología , Guanina , Células HeLa/enzimología , Humanos , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/síntesis química , Oligodesoxirribonucleótidos/metabolismo , Mapeo Restrictivo , Especificidad por Sustrato , TiminaRESUMEN
In differentiated cells, only a specific subset of genes is expressed. Recently, several genes have been shown to be transcriptionally inactivated by methylation of cytosine residues, mainly within their promoter sequences. Spontaneous hydrolytic deamination of 5-methylcytosine to thymine, which has been estimated to generate up to 12 G.T mismatched base pairs in the human genome per day, could have a deleterious effect on the expression of such genes. We recently reported that mammalian cells possess a specific repair pathway, which counteracts the mutagenic effects of this deamination by correcting G.T mismatches almost exclusively to G.C pairs. We show here that, in nuclear extracts from HeLa cells, this repair is mediated by excision of the aberrant thymidine monophosphate residue, followed by gap-filling to generate a G.C pair. We also provide preliminary evidence that the initial step of this process involves a DNA glycosylase.
Asunto(s)
Reparación del ADN , Secuencia de Bases , ADN Glicosilasas , Células HeLa , Humanos , Cinética , Datos de Secuencia Molecular , Mutación , N-Glicosil Hidrolasas/metabolismoRESUMEN
We have recently reported the first example of inverse splicing of a eukaryotic pre-mRNA intron using a whole cell extract from the yeast Saccharomyces cerevisiae. The concomitant circularization of the exon in the course of this splicing reaction gave rise to the hypothesis that the circular RNA species, which had been recently discovered in vivo in mammalian cells, were generated by inverse splicing. Here we report the formation of a circular exon in HeLa cell nuclear extracts by an inverse splicing reaction of the second intron of the human beta-globin gene from a pre-mRNA transcript in which the two intron halves flanked an artificially fused, single exon. Our data demonstrate that the mammalian pre-mRNA splicing system has indeed an intrinsic capability of aligning splice sites in reverse order and that this alignment can be followed by a complete splicing reaction, whereby the discontinuous intron sequences are removed. Thus we propose that circular exons in vivo arise as a result of an inverse splicing reaction following the pairing of a 5' splice site with an upstream 3' splice site and that the frequency of this event is influenced by the presence and strength of other, competing splice sites.
Asunto(s)
Exones , Intrones , Precursores del ARN/metabolismo , Empalme del ARN , Extractos Celulares , Núcleo Celular/metabolismo , Globinas/genética , Células HeLa , Humanos , Conformación de Ácido Nucleico , ARN/metabolismo , Precursores del ARN/química , ARN CircularRESUMEN
The bacteriostatic effect of low concentrations of the antibiotic granaticin on Bacillus subtilis is relieved by the addition leucine to the growth medium. In cells treated with granaticin, aminoacylation of leucine tRNA is specifically decreased, but the content of free leucine is not. It is concluded that granaticin interferes with the charging process of leucine tRNA in B. subtilis leading to leucine auxotrophy.
Asunto(s)
Aminoacil-ARNt Sintetasas/antagonistas & inhibidores , Leucina-ARNt Ligasa/antagonistas & inhibidores , Aminoácidos/análisis , Antibacterianos/farmacología , Bacillus subtilis/efectos de los fármacos , Proteínas Bacterianas/biosíntesis , ADN/biosíntesis , Leucina/farmacología , Naftoquinonas , Quinonas/farmacología , ARN/biosíntesis , Factores de TiempoRESUMEN
The antibiotic granaticin interferes in Bacillus subtilis with the charging process of tRNALeu causing both the arrest of protein synthesis and bacteriostasis [A. Ogilvie, K. Wiebauer & W. Kersten (1975) Biochem. J. 152, 511-515]. A concomitant inhibition of RNA synthesis is observed. This inhibition was studied with mutant strains of B. subtilis. 2. Granaticin inhibits protein and RNA synthesis in stringently controlled B. subtilis (rel+) to about the same extent. In a relaxed mutant strain (rel-) of B. subtilis, protein synthesis is also inhibited, but the accumulation of RNA continues after the addition of the drug. 3. Chloramphenicol, which is known to abolish the stringent control mechanism, added simultaneously with granaticin, allows the synthesis of RNA to proceed in the stringent strain. 4. Guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) accumulate in granaticin-treated stringently controlled B. subtilis but not in the rel- mutant. 5. It is concluded that the inhibition of RNA synthesis granaticin can adequately be explained as a stringent response caused by the interference by the drug with leucyl-tRNA synthetase.
Asunto(s)
Antibacterianos/farmacología , Bacillus subtilis/metabolismo , ARN/biosíntesis , Bacillus subtilis/efectos de los fármacos , Proteínas Bacterianas/biosíntesis , Cloranfenicol/farmacología , Nucleótidos de Guanina/metabolismo , Guanosina Trifosfato/metabolismo , Fenilalanina/metabolismo , Quinonas/farmacología , Uridina/metabolismoRESUMEN
Leucyl-tRNA synthetase from Escherichia coli is rapidly inactivated by 6-amino-7-chloro-5,8-dioxoquinoline (quinone), a model substance for cytostatic quinones. Loss of activity follows pseudo-first order kinetics. The quinone masks essential--SH groups that are reactive with N-ethylmaleimide. Specific protection of the enzyme by leucine provides evidence for active site-directed modification. Half-maximal protection is found at a concentration of 150 micron which is identical with the dissociation constant of the enzyme.substrate complex. The competitive inhibitor leucinol also protects the enzyme from inactivation by the quinone. MgATP enhances the protective effect of leucinol about 250-fold, thus substantiating recently published findings on synergistic coupling of ligands to aminoacyl-tRNA synthetases. The results support the assumption that the bacteriostatic quinone directly interferes with leucyl-tRNA synthetase in growing cells. Active-site-directed inhibition of the enzyme could adequately explain the phenotypically observed auxotrophy for leucine of quinone-treated E. coli.
Asunto(s)
Aminoacil-ARNt Sintetasas/metabolismo , Aminoquinolinas/farmacología , Escherichia coli/enzimología , Leucina-ARNt Ligasa/metabolismo , Leucina/metabolismo , Adenosina Trifosfato/farmacología , Sitios de Unión , Etilmaleimida/farmacología , Cinética , Magnesio/farmacología , ARN de TransferenciaRESUMEN
The herpes simplex virus type 1 immediate-early protein ICP4 plays an essential role in the regulation of the expression of all viral genes. It is the major trans activator of early and late genes and also has a negative regulatory effect on immediate-early gene transcription. ICP4 is a sequence-specific DNA-binding protein and has always been purified in a dimeric form. The part of the protein that consists of the entire highly conserved region 2 and of the distal portion of region 1 retains the ability to specifically associate with DNA and to form homodimers in solution. In an attempt to map the dimerization domain of ICP4, we used a dimerization trap assay, in which we screened deletion fragments of this 217-amino-acid stretch for sequences that could confer dimerization properties on a heterologous cellular transcription factor (LFB1), which binds to its cognate DNA sequence only as a dimer. The analysis of these chimeric proteins expressed in vitro ultimately identified a stretch of 34 amino acids (343 to 376) that could still confer DNA-binding activity on the LFB1 reporter protein and thus apparently contained the ICP4 dimerization motif. Consistent with this result, a truncated ICP4 protein containing amino acids 343 to 490, in spite of the complete loss of DNA-binding activity, appeared to retain the capacity to form a heterodimer with a longer ICP4 peptide after coexpression in an in vitro translation system.
Asunto(s)
Herpesvirus Humano 1/genética , Proteínas Inmediatas-Precoces/genética , Secuencia de Aminoácidos , Secuencia de Bases , ADN Viral/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Escherichia coli/genética , Herpesvirus Humano 1/química , Proteínas Inmediatas-Precoces/química , Datos de Secuencia Molecular , Conformación Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Eliminación de Secuencia , Transactivadores/química , Transactivadores/genéticaRESUMEN
Using a cloned cDNA (pGAP30) the nucleotide sequence for chicken glyceraldehyde-3-phosphate dehydrogenase mRNA has been determined. The cDNA insert contains 1051 nucleotides representing the amino acid coding sequence, with the exception of 49 NH2-terminal amino acids, and includes the entire 3'-noncoding region. Sequence information on the missing 5' terminus of the mRNA, not represented in the clone pGAP30, was obtained by extension of the cDNA using an 85-nucleotide-long internal fragment as a primer. Thus the sequence of 310 amino acids of chicken glyceraldehyde-3-phosphate dehydrogenase representing 93% of the complete primary structure could be derived. The coding portion exhibits non-random utilization of synonymous codons with a strong bias for codons with G or C at the third position. The non-coding region contains several octanucleotides which are repeated and shows a potentially stable stem-and-loop structure located towards the end of the mRNA. Hypothetical functional implications of the putative secondary structure are discussed.
Asunto(s)
Clonación Molecular , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Miocardio/enzimología , ARN Mensajero/análisis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Pollos , Conformación de Ácido NucleicoRESUMEN
A gene library was constructed with DNA from strain A mice by using the phage lambda vector lambda 1059. By screening with cloned cDNA for the third component of mouse complement, C3, four different C3 genomic clones were isolated from this library. Two of the recombinant phages carry insertions of 14 and 18 kilobase pairs, respectively, which together cover one complete copy of the C3 gene and several hundred nucleotides of its 5' and 3' flanking sequences. The distance from the 5' end of the gene, which includes the hexanucleotide T-A-T-A-A-A and a translation initiation codon, to its 3' end as defined by the poly(A) attachment site is 24 kilobase pairs. From the genomic DNA sequence, a signal peptide of 24 amino acid residues is predicted at the NH2 terminus of the initial translation product. The signal peptide and the next two amino acids are encoded by the first exon of this gene.
Asunto(s)
Complemento C3/genética , Animales , Secuencia de Bases , Clonación Molecular , ADN/genética , Genes , Ratones , Operón , Péptidos/genética , Poli A/genética , Señales de Clasificación de ProteínaRESUMEN
Mouse liver mRNA species which direct the cell-free synthesis of pro-C3 and pro-C4 polypeptides with an apparent molecular weight of 175,000 and 190,000, respectively, were shown to sediment faster than 28 S. By electrophoresis in a denaturing gel the mRNA for C3 was determined to contain approximately 7,500 ribonucleotides. cDNA was synthesized from size-fractionated mouse liver mRNA and cloned in the plasmid pBR 322. Among the cDNA clones recovered three were identified as being complementary to portions of the mRNA for C3.