Nicole Chaubet

Modular organization and developmental activity of an Arabidopsis thaliana EF-1α gene promoter

The activity of the Arabidopsis thalana A1 EF-1α gene promoter was analyzed in transgenic Arabidopsis plants. The 5′ upstream sequence of the A1 gene and several promoter deletions were fused to the β-glucuronidase (GUS) coding region. Promoter activity was monitored by quantitative and histochemical assays of GUS activity. The results show that the A1 promoter exhibits a modular organization. Sequences both upstream and downstream relative to the transcription initiation site are involved in quantitative and tissue-specific expression during vegetative growth. One upstream element may be involved in the activation of expression in meristematic tissues; the downstream region, corresponding to an intron within the 5′ non-coding region (5′IVS), is important for expression in roots; both upstream and downstream sequences are required for expression in leaves, suggesting combinatorial properties of EF-1α cis-regulatory elements. This notion of specific combinatorial regulation is reinforced by the results of transient expression experiments in transfected Arabidopsis protoplasts. The deletion of the 5′IVS has much more effect on expression when the promoter activity is under the control of A1 EF-1α upstream sequences than when these upstream sequences were replaced by the 35S enhancer. Similarly, a synthetic oligonucleotide corresponding to an A1 EF-1α upstream cis-acting element (the TEF1 box), is able to restore partially the original activity when fused to a TEF1-less EF1-α promoter but has no significant effect when fused to an enhancer-less 35S promoter.

A plant histone gene promoter can direct both replication-dependent and -independent gene expression in transgenic plants

SummaryChimeric genes containing the β-glucuronidase (GUS) gene under the control of different Arabidopsis histone H3 and H4 promoters were found to be highly expressed in transient expression experiments using tobacco protoplasts. The activity of one of these promoters, H4A748, was further analyzed. The kinetics of H4A748-GUS activity are very similar to these of a CaMV 35S-GUS constitutive gene during protoplast culture. No increase in H4A748-GUS activity was found after 24 h of protoplast culture when DNA synthesis starts, nor was the GUS activity affected when an inhibitor of DNA synthesis was included in the culture medium. This failure to detect any replication-dependent activity is most likely to be due to the fact that transient transcription of the introduced construct is restricted to the first 24 h following transfection. Stable integration of the H4A748-GUS gene into tobacco plants showed that the histone promoter could confer increased expression in meristematic tissues but it is also expressed to significant levels in non-proliferating tissues. Protoplasts prepared from these transgenic tobacco plants were cultivated under different conditions that affect DNA synthesis. Analysis of H4A748-GUS activity revealed (i) the existence of a basal replication-independent activity and (ii) a replication-dependent activity induced in parallel with DNA synthesis. These results show that the histone H4 promoter is able to direct both replication-dependent and -independent gene expression.

Genomic organization and nucleotide sequences of two histone H3 and two histone H4 genes of Arabidopsis thaliana

SummaryTwo histone H3 and two histone H4 genes have been cloned from a λgtWESλ·B Arabidopsis thaliana gene library. From their nucleotide sequences and from studies on their genomic organization, the following conclusions can be drawn:1)The nucleotide sequences of the two H3 coding regions show only 85% homology, but encode the same proteins. The Arabidopsis H3 has the same amino acid sequence as its counterpart in corn, but differs from that of pea and wheat by replacement in position 90 of a serine by an alanine. The two H4 coding regions have 97% sequence homology and encode the same protein, identical to the sequence of their counterpart in pea, corn and one H4 variant in wheat.2)The 5′-flanking regions of the 4 genes contain the classical histone-gene-specific consensus sequences, except H3A725 which lacks the GATCC-like pentamer. The conserved octanucleotide 5′-CGCGGATC-3′ which was previously found in the 5′-flanking sequences of corn and wheat H3 and H4 genes is also present in all four genes described here approximately 200 to 250 nucleotides upstream from the initiation ATG.The 5′-flanking regions of the H4 genes display extensive sequence homology, whereas those of the H3 genes do not.3)The 3′-flanking regions do not possess the classical histone-gene-specific T hyphenated dyad symmetry motif.4)Each H3 and H4 gene exists as 5 to 7 copies per haploid genome.

Nucleotide sequences of two corn histone H3 genes. Genomic organization of the corn histone H3 and H4 genes.

SummaryTwo histone H3 genes have been cloned from a λgtWESλ.B corn genomic library. The nucleotide sequences show 96% homology and both encode the same protein, which differs from its counterpart in wheat and pea by one amino acid substitution. The 5′-flanking regions of the two corn H3 genes contain the classical histone-gene-specific consensus sequences and possess several regions of extensive nucleotide homology. A conserved octanucleotide 5′-CGCGGATC-3′ occurs at approximately 200 nucleotides upstream from the initiation ATG codon. This octanucleotide was found to exist in all of the 7 plant histone genes sequenced so far. Codon usage is characterized by a very high frequency of C (67%) and G (28%) at the third position of the codons, those ending by A (1%) and T (4%) being practically excluded.Comparison of Southern blots of EcoRI, EcoRV and BamHI digested genomic DNA suggests that the corn H3 and H4 genes are not closely associated. The H3 genes exist as 60 to 80 copies and the H4 genes as 100 to 120 copies per diploid genome. re]19851002 rv]19851212 ac]19851216

Organ-specific expression of different histone H3 and H4 gene subfamilies in developing and adult maize

The steady-state levels of H3 and H4 mRNAs transcribed from three H3 and two H4 multigene subfamilies were studied during germination and in different organs of maize. During germination the five subfamilies are expressed in parallel to DNA synthesis, but a 5-fold difference in the quantity of mRNAs transcribed per gene copy was found from our subfamily to another. In adult plants H3 and H4 mRNA levels are highest in organs containing meristematic tissues but also high in non-proliferating tissues. No strict tissue specificity expression could be detected but some subfamilies show preferential expression in some tissues.

Organization of the histone H3 and H4 multigenic families in maize and in related genomes

SummaryFive cloned histone H3 and H4 genes from maize have specific 5′ non-transcribed regions. Blot hybridization of each 5′ region to DNA from different maize inbred lines showed that the H3 and H4 multigenic families are organized into subfamilies. Each subfamily has a specific environment and contains a different (from 4–16) number of gene copies. H3 and H4 subfamilies with similar environments as those found in maize were shown to exist in the genomes of more or less related plants, including perennial teosinte, sorgho, sugar cane and Coïx. Such observations may contribute to establishing phylogenetic relationships at a molecular level between different plants and thus highlight some of the evolutionary mechanisms of the genomes of higher plants.

Nuclease sensitivity and functional analysis of a maize histone H3 gene promoter

A 1 kb region of a maize H3 histone gene promoter has been analysed at a structural and functional level. Micrococcal nuclease digestion of isolated nuclei showed that the promoter region is organized into nucleosomes but a zone extending over approximately one nucleosome (20 to 230 bp upstream of the TATA box) displays remarkable accessibility to digestion. Three DNase I-hypersensitive sites were found within this zone at the vicinity of consensus sequences, some of which are already known to act ascis elements. This promoter region is able to direct faithful expression of the GUS reporter gene in meristematic tissues of transgenic tobacco plants.

Functional analysis of the promoter region of a maize (Zea mays L.) H3 histone gene in transgenic Arabidopsis thaliana

A 1023 bp fragment and truncated derivatives of the maize (Zea mays L.) histone H3C4 gene promoter were fused to the ß-glucuronidase (GUS) gene and introduced via Agrobacterium tumefaciens into the genome of Arabidopsis thaliana. GUS activity was found in various meristems of transgenic plants as for other plant histone promoters, but unexplained activity also occurred at branching points of both stems and roots. Deletion of the upstream 558 bp of the promoter reduced its activity to an almost basal expression. Internal deletion of a downstream fragment containing plant histone-specific sequence motifs reduced the promoter activity in all tissues and abolished the expression in meristems. Thus, both the proximal and distal regions of the promoter appear necessary to achieve the final expression pattern in dicotyledonous plant tissues. In mesophyll protoplasts isolated from the transformed Arabidopsis plants, the full-length promoter showed both S phase-dependent and -independent activity, like other plant histone gene promoters. Neither of the 5′-truncated nor the internal-deleted promoters were able to direct S phase-dependent activity, thus revealing necessary cooperation between the proximal and distal parts of the promoter to achieve cell cycle-regulated expression. The involvement of the different regions of the promoter in the different types of expression is discussed.

Replication-independent cis-acting element of a maize histone gene promoter

Abstract A chimeric gene containing the β-glucoronidase (GUS) coding sequence fused to the maize histone H4C7 gene promoter was expressed in transient assays in tobacco mesophyll protoplasts revealing the replication-independent activity of the plant histone gene promoter. Functional analysis of 5′ deletions of the H4C7 promoter suggested the presence of antagonistic activities in a region encompassing the plant histone-specific octamer and a hybrid hexameric motif, and revealed an essential cis-acting positive element between nucleotides -82 and -36 relative to the TATA box. Furthermore, point mutations introduced in the CCATCGAAC motif present in this region drastically reduced the activity of the H4C7 promoter. We conclude this nonameric sequence, present in the 5′ flanking DNA sequence of most plant histone genes, to be a positive cis-acting element controlling the basal replication-independent activity of the H4C7 promoter.

In vivo footprinting by LMPCR procedure

A number of approaches have been developed to elucidate molecular mechanisms of gene regulation at the transcriptional level. In vivo footprinting, however, is the only method providing information of when and how proteins actually occupy a given regulatory region of the DNA in the living cell.