RESUMEN
Intron-bearing replacement histone H3 genes in Arabidopsis and other plants are highly and constitutively expressed. We demonstrate that the introns located within the 5'-untranslated regions (5'-UTR) of the two Arabidopsis replacement H3 genes will abolish the cell cycle dependence of an endogenous histone H4 promoter. We demonstrate that these introns, functionally combined with their endogenous promoters, could produce the high and constitutive expression of the replacement H3 genes observed in planta. They strongly increase gene expression whatever the promoter, from the strong 35S CaMV promoter to complete and resected promoters of cell cycle-dependent and replacement histone genes. Quantitative analysis of the extent of reporter gene enhancement in different parts of developing transgenic plantlets, ranging from 2-fold to 70-fold, supports the notion that trans-acting factors are responsible for this effect. Such factors appear most abundant in roots.
Asunto(s)
Arabidopsis/genética , Histonas/genética , Intrones/genética , Transgenes/genética , Caulimovirus/genética , ADN de Plantas/genética , Regulación de la Expresión Génica de las Plantas , Glucuronidasa/genética , Glucuronidasa/metabolismo , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Plantas Modificadas Genéticamente , Regiones Promotoras Genéticas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Distribución Tisular , Transformación GenéticaRESUMEN
The control of cell cycle expression of histone genes in plants is incompletely understood. A new histone H3 gene was cloned from alfalfa (Medicago sativa) that codes for the replication-dependent histone H3.1 variant protein. Despite lacking all promoter sequence motifs that have been associated with cell cycle-dependent histone gene expression in plants, northern analysis of synchronized cells clearly linked gene expression to DNA replication. TTAATNA was recognized as a new sequence element in the 3' untranslated regions of this and all other cell cycle-dependent histone H3 genes of dicotyledonous plants. It is not found in the replication-independent histone H3 genes.
Asunto(s)
Ciclo Celular/genética , Histonas/genética , Medicago sativa/genética , Regiones Promotoras Genéticas , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Cricetinae , Femenino , Regulación de la Expresión Génica de las Plantas , Datos de Secuencia Molecular , Biosíntesis de Proteínas , Secuencias Reguladoras de Ácidos Nucleicos , Homología de Secuencia de Ácido NucleicoRESUMEN
One genomic and six cDNA clones for the replacement histone H3.2 protein of alfalfa (Medicago sativa) were isolated and sequenced. By gene organization they represent 3 distinct genes. PCR methods were used to confirm that only three intron-bearing histone H3.2 genes of this type exist per haploid genome. They co-exist with approximately 56 copies of the previously characterized replication-dependent, intronless histone H3.1 variant gene. Comparison of the relative expression of few constitutive H3.2 genes with the high S phase expression of the abundant cell cycle-dependent H3.1 genes by mRNA levels and protein synthesis measurements revealed that the replacement histone H3.2 genes are very highly expressed. Structural analysis of the genomic replacement H3.2 gene revealed a unique feature. A repeated polypyrimidine sequence motif in the 5' untranslated region of this gene replaces the ubiquitous intron present in all known replacement H3 genes. A hypothesis is presented that this motif and other, non-randomly distributed polypyrimidine sequences in the introns of replacement histone H3 genes of alfalfa and Arabidopsis, may affect nucleosome assembly. Chromatin repression of these replacement genes would be avoided, consistent with the high, constitutive expression of replacement H3 histone genes in plants.
Asunto(s)
Genes de Plantas , Histonas/genética , Medicago sativa/genética , Secuencia de Aminoácidos , Arabidopsis/química , Arabidopsis/genética , Secuencia de Bases , Ciclo Celular/fisiología , Células Cultivadas , Cromatografía Líquida de Alta Presión , Clonación Molecular , Electroforesis en Gel de Agar , Dosificación de Gen , Regulación de la Expresión Génica de las Plantas , Histonas/biosíntesis , Histonas/química , Intrones/genética , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
The stability of histone H3 transcripts in alfalfa for replication-dependent and -independent gene variants was measured by northern analysis under conditions of inhibition of transcription and/or translation. Replication-dependent histone H3.1 transcripts were about three-fold less stable than the equally polyadenylated mRNA for replacement variant H3.2 histone. In actively growing suspension cultures treated with dactinomycin half-lives of 2 and 7 h were observed for H3.1 and H3.2 mRNAs, respectively. mRNA stabilities were also measured indirectly by histone protein synthesis. The translation inhibitor cycloheximide strongly increased mRNA levels for both histone H3 variants. The dependence of histone mRNA turnover on translation in animals also appears to exist in plants. The combination of inhibition of transcription and translation by dactinomycin and cycloheximide was used in an indirect assessment of H3 mRNA stability throughout the cell cycle in partially synchronized and cycle-arrested cultures. Destabilization of replication-dependent histone H3.1 mRNA was detected in non-S phase cells.
Asunto(s)
Histonas/genética , Medicago sativa/genética , ARN Mensajero/metabolismo , ARN de Planta/metabolismo , Ciclo Celular , Células Cultivadas , Cicloheximida/farmacología , Dactinomicina/farmacología , Desoxiadenosinas/farmacología , Medicago sativa/crecimiento & desarrollo , Medicago sativa/metabolismo , Biosíntesis de Proteínas/efectos de los fármacosRESUMEN
A detailed method is presented for the creation of head-to-tail multimers of short blunt restriction fragments, ligated into a plasmid vector in a single-tube reaction. Random priming of the concatemer insert readily yields hybridization probes of high specificity, unattainable from the short monomer fragments.
Asunto(s)
Sondas de ADN/síntesis química , ADN Ligasas , ADN de Plantas/genética , Digoxigenina , Vectores Genéticos , Histonas/genética , Medicago sativa/genética , Hibridación de Ácido Nucleico , Proteínas de Plantas/genéticaRESUMEN
Northern analysis has revealed substantial differences in mRNA accumulation of the two histone H3 gene variants represented by pH3c-1 and pH3c-11 cDNA clones. Both in partially synchronized cell suspension cultures and in protoplast-derived cells from alfalfa, Medicago varia, the maximal level of the histone H3-1 gene transcript coincided with the peak in [(3)H]thymidine incorporation. Histone H3-11 mRNA was detectable in cells throughout the period of the cell cycle studied. Various stress factors such as medium replacement, enzyme digestion of the cell wall, osmotic shock, and auxin treatment considerably increased the level of the histone H3-11 transcript. In alfalfa (Medicago sativa), the presence of H3-11 mRNA in unorganized tissues of microcallus suspension and in somatic embryos induced by auxin treatment supports the idea that this H3 variant exists in a continously active state of transcription. During embryo development, the early globular stage embryos showed increased accumulation of histone H3-11 mRNA in comparison with the later stages. The highest level of the histone H3-1 transcript was detectable 1 day after treatment of callus tissues with 2,4-dichlorophenoxyacetic acid. Somatic embryos contained appreciable levels of histone H3-1 transcripts at all stages of somatic embryo development. These observations suggest that the histone H3-1 gene belòngs to the class of replication-dependent histone genes. The histone H3-11 gene showed characteristics of a constitutively expressed replacement-type histone gene, with a specific characteristic that external factors can influence the level of gene transcription.