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 beta-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.

Multiple A-type cyclins express sequentially during the cell cycle in Nicotiana tabacum BY2 cells.

Four full-length and one partial cDNA clones encoding four different A-type cyclins were isolated from a tobacco S-phase-specific library. The corresponding mRNAs displayed sequential appearance and disappearance during the cell cycle of highly synchronized suspension-cultured tobacco cells. Sequence analysis showed that the plant A-type cyclins can be subdivided into three distinct structural groups that are likely to be represented in every plant species. Two of the isolated tobacco cyclins belonging to the same group were highly expressed throughout S and G2 phases but showed different kinetics of induction at the G1/S transition. Another one belonging to a second group was induced at mid-S phase and expressed until mid-M phase. A similar expression pattern was previously reported for a tobacco cyclin belonging to the third group. This sequential expression of multiple A-type cyclins in one type of plant cells makes a clear distinction from the situation in animal cells in which only one A-type cyclin exists in a given species. Furthermore, the expression of the different A-type cyclin genes responded differently upon a block at mid-S phase by DNA synthesis inhibitors. These results suggest that the multiple A-type cyclins act at different steps of the plant cell cycle and, therefore, exert distinct functions. In contrast, the expression of B-type cyclins was restricted to a narrow window corresponding to the M phase.

Histone H1 overexpressed to high level in tobacco affects certain developmental programs but has limited effect on basal cellular functions.

Histone H1, a major structural component of chromatin fiber, is believed to act as a general repressor of transcription. To investigate in vivo the role of this protein in transcription regulation during development of a multicellular organism, we made transgenic tobacco plants that overexpress the gene for Arabidopsis histone H1. In all plants that overexpressed H1 the total H1-to-DNA ratio in chromatin increased 2.3-2.8 times compared with the physiological level. This was accompanied by 50-100% decrease of native tobacco H1. The phenotypic changes in H1-overexpressing plants ranged from mild to severe perturbations in morphological appearance and flowering. No correlation was observed between the extent of phenotypic change and the variation in the amount of overexpressed H1 or the presence or absence of the native tobacco H1. However, the severe phenotypic changes were correlated with early occurrence during plant growth of cells with abnormally heterochromatinized nuclei. Such cells occurred considerably later in plants with milder changes. Surprisingly, the ability of cells with highly heterochromatinized nuclei to fulfill basic physiological functions, including differentiation, was not markedly hampered. The results support the suggestion that chromatin structural changes dependent on H1 stoichiometry and on the profile of major H1 variants have limited regulatory effect on the activity of genes that control basal cellular functions. However, the H1-mediated chromatin changes can be of much greater importance for the regulation of genes involved in control of specific developmental programs.

Identification of cis-elements regulating the expression of an Arabidopsis histone H4 gene.

Protein-DNA interactions in the proximal region of an Arabidopsis H4 histone gene promoter were analyzed by DMS in vivo footprinting combined with LMPCR amplification. Interactions were identified over six particular sequence motifs, five of which were previously shown to bind proteins in maize histone H3 and H4 promoters and are commonly found in the corresponding regions of other plant histone gene promoters. These motifs are located within a 126 bp fragment which was previously shown to confer preferential expression in meristems of transgenic plants. The contribution of each cis-element to the overall expression level and specificity was investigated by testing individual or combined mutations in transgenic Arabidopsis plants. All five motifs behaved as positive cis-elements of unequal strength. The GCCAAT-like sequence GCCACT behaved as a strong positive cis-element but had no influence on the specificity. In contrast, the nonamer AGATCGACG and to a lesser extent the closely linked hexamer CCGTCG proved to be essential for meristem-specific expression. Involvement of the highly conserved histone-specific octamer CGCGGATC in specific expression was revealed at some stages of meristem development. Importance of these three cis-elements, nonamer, hexamer, and octamer, was further confirmed by the fact that combining mutations of two of them either abolished the promoter activity or completely modified the promoter specificity. Mutation of the fifth cis-element, a degenerate copy of the octamer, little perturbed the promoter function. However disruption of both octamers had a dramatic negative effect, thus suggesting that the two copies cooperate to achieve maximal function in the wild-type promoter, possibly by mobilizing the proliferation-specific factors binding to the nonamer and CCGTCG cis-elements.

Functional expression of Saccharomyces cerevisiae CYP51A1 encoding lanosterol-14-demethylase in tobacco results in bypass of endogenous sterol biosynthetic pathway and resistance to an obtusifoliol-14-demethylase herbicide inhibitor.

Nicotiana tabacum protoplasts have been transformed by Agrobacterium tumefaciens containing a T-DNA in which the gene CYP51A1 encoding lanosterol-14-demethylase (LAN14DM) from Saccharomyces cerevisiae is under the control of a cauliflower mosaic virus (CaMV) 35S promoter. Two transformants strongly expressed the LAN14DM as shown by Northern and Western experiments. These transgenic calli were killed by LAB 170250F (LAB) (a phytotoxic fungicide inhibiting both plant obtusifoliol-14-demethylase (OBT14DM) and LAN14DM) but were resistant to gamma-ketotriazole (gamma-kt), a herbicide which has been shown to inhibit OBT14DM but not LAN14DM at a concentration that was lethal to control calli. However, these transgenic calli were killed by mixtures of gamma-kt plus fungicide inhibitors of LAN14DM such as ketoconazole, itraconazole or flusilazole which alone were not effective. Further analysis of the transgenic calli grown in the presence of gamma-kt showed that their delta 5-sterol content was close to that of untreated control calli obtained from protoplasts transformed with control plasmid; this is in agreement with evidence that the LAN14DM expressed from the transgene could bypass the blocked OBT14DM by using the plant substate obtusifoliol. In contrast, control calli when treated with gamma-kt, displayed a sterol content strongly enriched in 14 alpha-methyl sterols and depressed in physiological delta 5-sterols. When the transgenic calli were cultured in mixtures of gamma-kt and LAN14DM inhibitors sterol compositions enriched in 14 alpha-methyl sterols were obtained, reflecting a strong inhibition of both 'endogenous' OBT14DM and 'exogenous' LAN14DM. Taken together these results show that in tobacco calli transformed with CYP51A1, resistance to a triazole herbicide arises from expression of a functional LAN14DM enzyme; its activity in transgenic tissues creates a bypass of the sterol biosynthetic pathway at the 14-demethylase level when this latter is blocked by an OBT14DM herbicide inhibitor.

Constitutive and cell-division-inducible protein-DNA interactions in two maize histone gene promoters.

Protein-DNA interactions in the promoter regions of two maize histone genes have been analyzed by DNase I and DMS in vivo footprinting combined with LMPCR amplification. Both promoters present a bimodular structure characterized by a proximal cell division-specific set of interactions and a distal region which displays constitutive footprints but enhancement of these footprints upon cell proliferation. The inducible region contains two cis-elements common to all replication-dependent plant histone genes, one of them having previously been shown to be a target for the wheat nuclear protein HBP-2. In the constitutive region, the first demonstration for the existence of a transcription factor binding to the highly conserved plant histone-specific octamer CGCGGATC is provided. Exchange of cell-type-specific factors is postulated to occur at that site. Additional immediate upstream constitutive elements binding regulatory proteins include a degenerate octameric sequence, a CCAAT-box, a CACCC sequence and composite ACGTCA/ACGTGG hexameric sequences binding HBP-1-related trans-acting factors. The close proximity of these elements within the constitutive region and the redundancy of some of them suggest complex cooperation and competition mechanisms contributing to achieve the final expression level and likely also to mediate the interplay between constitutive and inducible factors.

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 as cis elements. This promoter region is able to direct faithful expression of the GUS reporter gene in meristematic tissues of transgenic tobacco plants.

Modular organization and development activity of an Arabidopsis thaliana EF-1 alpha gene promoter.

The activity of the Arabidopsis thalana A1 EF-1 alpha gene promoter was analyzed in transgenic Arabidopsis plants. The 5' upstream sequence of the A1 gene and several promoter deletions were fused to the beta-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-1 alpha 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-1 alpha upstream sequences than when these upstream sequences were replaced by the 35S enhancer. Similarly, a synthetic oligonucleotide corresponding to an A1 EF-1 alpha upstream cis-acting element (the TEF1 box), is able to restore partially the original activity when fused to a TEF1-less EF1-alpha promoter but has no significant effect when fused to an enhancer-less 35S promoter.

Histones and histone genes in higher plants: structure and genomic organization.

The primary structure of the plant histone genes has been deduced from the comparison of the nucleotide sequences of 23 genes and 14 cDNAs from eight different species. These data confirmed the extreme conservation of histones H3 and H4 in plant and animal kingdoms. Histone H2B is more variable than H2A and the histone H1 is the less conserved histone. Some interesting observations concerning the non-conserved regions of H2A and H2B in their extended C- and N-terminal regions are reported. Only three plant histone genes were found to possess intervening sequences: one H1 gene and two H3.3 like genes. The most striking differences found between the two kingdoms are the absence from plant histone genes of the palindromic structure existing downstream of the animal genes and the fact that plant histone mRNAs are polyadenylated. This suggests that the post-transcriptional regulation of expression of histone genes is different in the two kingdoms. In plants the multiple copies of the histone genes are organized into multigenic families. In the complex genome of maize the multiple copies of the genes are highly dispersed on the genome.

A 126 bp fragment of a plant histone gene promoter confers preferential expression in meristems of transgenic Arabidopsis.

The tissue-specific pattern of expression directed by the H4A748 Arabidopsis histone promoter was investigated by analysis of beta-glucuronidase (GUS) activity in transgenic Arabidopsis containing H4A748-GUS gene fusions. As determined by fluorimetric and histochemical tests, the H4A748 promoter directs preferential expression in meristems of young seedlings and adult plants. The low activity found in nonproliferating tissues may relate to basal constitutive expression of the histone promoter and/or to endoreduplication occurring in some tissues. The endogenous histone mRNA levels parallel the GUS activity found in different tissues. Analysis of the regulatory properties of 5' deleted promoters showed that multiple positive elements exist between -900 and -219 and that the proximal region of the promoter to -219 is sufficient to establish the full tissue-specific pattern of expression. Further deletion to -93 nearly abolished the promoter activity thus suggesting that the 126 bp fragment located between -219 and -93 contains the elements responsible for the specific expression pattern. The presence of several remarkable sequences within this fragment is discussed.

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.

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

Chimeric genes containing the beta-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.

Subfamilies of histone H3 and H4 genes are located on most, possibly all of the chromosomes in maize.

It has been previously shown that in the genome of maize the multiple copies of the histone H3 and H4 multigenic families are organized into eight to ten subfamilies each containing a variable number of copies. Each subfamily is characterized by a specific proximal environment and thus can be revealed by blot-hybridization with its specific 5' probe. Restriction fragment length polymorphism (RFLP) combined with monosomic analysis was used to localize several H3 and H4 subfamilies on maize chromosomes. H3 and H4 genes were found to be located on most, possibly all of the chromosomes, revealing a remarkably dispersed organization of these multigenic families.

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.

Nucleotide sequence and expression of a maize H1 histone cDNA.

The first complete amino acid sequence of a H1 histone of a monocotyledonous plant was deduced from a cDNA isolated from a maize library. The encoded H1 protein is 245 amino acid-long and shows the classical tripartite organization of this class of histones. The central globular region of 76 residues shows 60% sequence homology with H1 proteins from dicots but only 20% with the animal H1 proteins. However, several of the amino acids considered as being important in the structure of the nucleosome are conserved between this protein and its animal counterparts. The N-terminal region contains an equal number of acidic and basic residues which appears as a general feature of plant H1 proteins. The 124 residue long and highly basic C-terminal region contains a 7-fold repeated element KA/PKXA/PAKA/PK. Southern-blot hybridization showed that the H1 protein is encoded by a small multigene family. Highly homologous H1 gene families were also detected in the genomes of several more or less closely related plant species. The general expression pattern of these genes was not significantly different from that of these genes encoding the core-histones neither during germination nor in the different tissues of adult maize.

Polyadenylation of histone H3 and H4 mRNAs in dicotyledonous plants.

The histone H3 and H4 genes are shown to be expressed in both Arabidopsis plantlets and transitory multicellular suspension. The 5'- and 3'-ends of the H4 mRNAs have been localized on two H4 genes previously sequenced, H4A748 and H4A777. S1-nuclease mapping and reverse-transcriptase-primer-elongation experiments revealed the existence of two start points for transcription, located 31 and 37 nucleotides downstream from the TATA-box. The 3'-end of the mRNA corresponding to H4A748 was localized at 177 nt after the stop codon. The other gene, H4A777, most probably is not expressed. In addition to a long 3'-untranslated region, the H4 mRNA was shown to be polyadenylated in both plantlets and cell-suspension. This observation was extended to the H3 mRNAs of Arabidopsis and of two other dicots, tobacco and sunflower. Previous results on maize H3 and H4 mRNAs suggest that polyadenylation is a common feature for histone mRNAs in higher plants.

The histone H3 and H4 mRNAs are polyadenylated in maize.

Northern blot analysis revealed that the histone H3 and H4 mRNAs are of unusual large size in germinating maize embryos. S1-mapping experiments show that the 3'-untranslated regions of the mRNAs transcribed from 3 H3 and 2 H4 maize genes previously described are much longer than in the non-polyadenylated histone mRNAs which represent a major class in animals. Moreover, oligo d(T) cellulose fractionation of RNAs isolated at different developmental stages indicates that more than 99% of the maize H3 and H4 mRNAs are polyadenylated. A putative polyadenylation signal is present in all five genes 17 to 27 nucleotides before the 3'-ends of the mRNAs.

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

Two 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.

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.

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

Two 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.