Claude Gigot

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.

Genes encoding a histone H3.3-like variant in Arabidopsis contain intervening sequences

Two genes encoding a particular H3 histone variant were isolated from Arabidopsis thaliana. These genes differ from the H3 genes previously cloned from Arabidopsis and other plants by several interesting properties: (1) the two genes are located close to each other; (2) their coding regions are interrupted by two or three small introns, the two closest to the initiation codon being located at the same place in the two genes; (3) another, long intron is located in the 5-untranslated region just before the initiation codon of gene I as deduced from the sequence of several corresponding cDNAs, and very likely also of gene II; (4) these genes do not show preferential expression in organs containing meristematic tissues contrary to the classical intronless replication-dependent histone genes, thus suggesting that their expression is not replication-dependent; (5) the protein encoded by both genes is the same and corresponds to a minor H3 variant highly conserved among all the plant species studied up to now. All these characteristics are common with the animal replication-independent H3.3 histone genes and it is assumed that the genes described here are the first example of the equivalent H3.3 gene family in plants. Interestingly, the promoter regions of the two genes have the same general structure as the Arabidopsis intronless genes. Possible implications on the regulation of H3 genes expression are discussed.

Tissue-dependent enhancement of transgene expression by introns of replacement histone H3 genes of Arabidopsis

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.

Multilevel regulation of histone gene expression during the cell cycle in tobacco cells

The respective involvement of transcriptional and post-transcriptional mechanisms in coupling H3 and H4 histone gene expression to the S phase of the cell cycle has been studied in synchronized tobacco cells. Induction of histone gene expression at the G1/S transition is shown to be essentially directed by an increase in the transcription rate in response to cellular signals occurring at the initiation step of DNA replication. Histone gene induction thus precedes the burst of DNA synthesis. However, when the elongation step of DNA replication is ineffective or artificially arrested, feedback mechanisms apparently act at the translation level to avoid overproduction of histone proteins from their mRNAs. At the end of S phase, post-transcriptional mechanisms ensure a rapid degradation of histone mRNAs. Transcription factors are bound to the cis -elements of histone promoters throughout the cell cycle, thus suggesting a post-translational modification of some of them to trigger promoter activation at the G1/S transition. Based on these results, a model is proposed for histone gene transcriptional induction in connection with the components of the cell cycle machinery.

Genomic organization and nucleotide sequences of two corn histone H4 genes

The sea urchin histone H4 gene has been used as a probe to clone two corn histone H4 genes from a lambda gtWES X lambda B corn genomic library. The nucleotide (nt) sequences of both genes showed that the encoded amino acid sequences were identical to that of the H4 of pea and one variant of wheat. The nt sequences of the coding regions showed 92% homology. 5- and 3-flanking regions do not show extensive nt sequence analogies. Southern blotting of the EcoRI digested genomic DNA suggests the existence of multiple H4 genes dispersed throughout the genome.

Cloning and sequence analysis of a cDNA clone from Arabidopsis thaliana homologous to a proteasome α subunit from Drosophila

A cDNA clone isolated from an Arabidopsis thaliana cell suspension culture library showed considerable similarities to the proteasome 28 kDa α subunit of Drosophila [(1990) Gene 90, 235–241]. The 250 amino acid‐long protein encoded by Arabidopsis TAS‐g64 clone has important homologies in its primary structure and in the predicted secondary structure with the PROS‐28.1 clone from Drosophila. The only divergence observed between the two sequences is for the 20 C‐terminal amino acids. This subunit might share important functions in both kingdoms, as revealed by the important conservation between plants and animals. In plant cells it is encoded by a single‐copy gene and probably regulated by stress and/or division.

An internal standard improves the reliability of transient expression studies in plant protoplasts.

Transient expression of foreign genes introduced on a plasmid into isolated plant protoplasts is widely used to study the control of gene expression. Unfortunately, many experimental variables implicated in this technique are difficult or impossible to control, resulting in a disturbing degree of variability between otherwise identical experiments. We have studied the co-expression of two constitutively expressed genes located on the same plasmid. This has allowed us to identify the lot of plasmid DNA as an important source of variation, along with the protoplast lot. Plasmid DNA concentration was found to be of minor importance. Since the variation of expression level of the two genes was identical for the two genes in all experiments, we propose the use of an internal standard in all comparative transient expression studies, which allows the reduction of the variation between experiments by one order of magnitude.

Molecular characterization and cell cycle-regulated expression of a cDNA clone from Arabidopsis thaliana homologous to the small subunit of ribonucleotide reductase

A cDNA clone isolated from an Arabidopsis thaliana cell suspension library showed highly significant homology to the small subunit of ribonucleotide reductase (R2) from different species. The 340 amino acid‐long deduced putative protein contains all the residues that are important for the enzyme activity and structure. In A. thaliana this enzyme is encoded by a single‐copy gene. In synchronized tobacco BY2 cells the corresponding mRNAs specifically accumulate during the S phase of the cell cycle.

Fractionation and characterization of histones from barley (Hordeum vulgare) leaves : Existence of multiple H2A and H2B variants.

AbstractHistones were extracted from purified nuclei isolated from four cereals,viz. barley (Hordeum vulgare), wheat(Triticum aestivum), Aegilops squarrosa and corn (Zea mais), and from tobacco (Nicotiana tabacum). Analysis of the histones on SDS gels showed complex electrophoretic patterns with one species of both H3 and H4, one to three species of H1 and two to five species of H2. Judged from the electrophoretic patterns, the histones from barley, wheat and Aegilops are identical but different from those of corn with respect to H2. Like tobacco, corn showed two H2 components, whereas barley, wheat and Aegilops showed five H2 components.SDS gel electrophoresis of histones extracted from buds and roots of germinating seeds at different steps of germination and from different parts of ten-day-old leaves revealed that the existence in barley of multiple histone 2 variants is not restricted to any particular stage of differentiation of barley.Histones from barley leaves were resolved into four fractions by Biogel P-100 gel filtration and histones 2 were further fractionated by their differential solubility in HCl-ethanol. Each of these five fractions (H1, H3, H4, H2A and H2B, respectively) were characterized by electrophoresis on SDS or Triton-acid-urea gels and by their amino acid compositions as compared with the homologous histones of calf thymus and chicken erythrocytes. This revealed the following:i)H3 and H4 are strictly analogous to their animal counterparts. However, H4 has an unexplained lower electrophoretic mobility in Triton-containing acid-urea gels.ii)H1 contains three components with lower electrophoretic mobilities than H1 from erythrocytes, contains more alanine than lysine and has a lower ratio of basic to acidic residues.iii)Both H2A and H2B contain at least four variants each, with higher molecular weights than in animals and higher lysine to arginine ratios. H2A variants comigrate in acid-urea-Triton gels with chicken erythrocytes H2A, whereas H2B migrate much slower.nIt was concluded that the presence of multiple major variants of H2A and H2B is a frequent but not universal feature in cereals. The existence of these variants is not restricted to the embryonic stage as previously suggested for wheat (31).

Rapid labelling of a nonencapsulated RNA of bromegrass mosaic virus.

Recently Lane and Kaesberg [l] have observed that bromegrass mosaic virus (BMV) contains 5 types of RNA. The mixture of the three largest RNA’s is infectious while the smallest RNA and a minor component seem to have no effect on infectivity. We report in this paper on a study of the incorporation of j2P into the different RNA’s of BMV. We have found that the different RNA’s are labelled and encapsulated at different rates.