Gabriel Philipps

Cell Cycle Regulation of the Tobacco Ribonucleotide Reductase Small Subunit Gene Is Mediated by E2F-like Elements

Ribonucleotide reductase (RNR) is a key enzyme involved in the DNA synthesis pathway. The RNR-encoded genes are cell cycle regulated and specifically expressed in S phase. The promoter of the RNR2 gene encoding for the small subunit was isolated from tobacco. Both in vivo and in vitro studies of the DNA–protein interactions in synchronized BY2 tobacco cells showed that two E2F-like motifs were involved in multiple specific complexes, some of which displayed cell cycle–regulated binding activities. Moreover, these two elements could specifically interact with a purified tobacco E2F protein. Involvement of the E2F elements in regulating the RNR2 promoter was checked by functional analyses in synchronized transgenic BY2 cells transformed with various RNR2 promoter constructs fused to the luciferase reporter gene. The two E2F elements were involved in upregulation of the promoter at the G1/S transition and mutation of both elements prevented any significant induction of the RNR promoter. In addition, one of the E2F elements sharing homology with the animal E2F/cell cycle–dependent element motif behaved like a repressor when outside of the S phase. These data provide evidence that E2F elements play a crucial role in cell cycle regulation of gene transcription in plants.

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

Molecular characterization of tobacco ribonucleotide reductase RNR1 and RNR2 cDNAs and cell cycle-regulated expression in synchronized plant cells

Eukaryotic ribonucleotide reductase (RNR), the enzyme involved in the synthesis of the deoxyribonucleotides, consists of two R1 and R2 subunits whose activities and gene expression are differentially regulated during the cell cycle and are preferentially induced at the G1/S transition. We have isolated three cDNA clones from a tobacco S phase library, two encoding the large R1 subunit, the first cloned in plants, and one encoding the small R2 subunit. From Southern blot hybridization we deduce that RNR2 is encoded by a single-copy gene whereas RNR1 is encoded by a small multigene family. The level of RNR mRNA is cell-cycle regulated showing a maximum in S phase. In mid-S phase, RNR2 transcripts show a higher maximum level than RNR1 transcripts. Analysis of the effects of various cell cycle inhibitors added to freshly subcultured stationary phase cells leads to the conclusion that RNR gene induction at the entry of the cells into the cell cycle takes place in late G1-early S phase. Addition of DNA synthesis-blocking agents to cycling cells synchronized in mid-S phase resulted in an enhancement of RNR transcript level, thus suggesting that RNR gene expression may be linked to the DNA synthesis rate by a feedback-like regulatory mechanism.

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.

cDNA nucleotide sequence and expression of a maize cytoplasmic ribosomal protein S13 gene

The complete amino acid sequence of a cytoplasmic ribosomal protein S13 of maize was deduced from the cDNA isolated from a maize cDNA library. The encoded protein is 151 amino acids long and shows a homology of 73% with the corresponding protein S13 of rat. Southern blots analysis shows that the maize protein S13 is encoded by a small multigene family conserved in plant species closely related to maize. The S13 RNAs accumulate preferentially in proliferating tissues and cells and their transcription occurs in parallel to the DNA synthesis.

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.

Study of phase-specific gene expression in synchronized tobacco cells

Although the basic mechanisms which control the progression through the cell cycle appear to be conserved in all higher eukaryotes, the unique features of the plant developmental programme must be somehow reflected in a plant-specific regulation of the factors which control cell division. In the last few years, considerable progress has been achieved in identifying the major components of the cell cycle in plants. The question of how these components direct expression of specific genes at specific stages of the cell cycle, and how they are themselves regulated, constitutes a challenge for the present and the next years. This review summarizes our current knowledge at molecular and biochemical levels of cell cycle-regulated expression in the model system, the synchronized tobacco BY2 cell suspension, and discusses the results in comparison to those obtained by different methods and in other plant systems.

Nucleotide sequence and expression of two cDNA coding for two histone H2B variants of maize

The complete amino acid sequences of two variants of histone H2B of maize were deduced from the cDNAs isolated from a maize cDNA library. The two encoded proteins are 150 (H2B(1)) and 149 (H2B(2)) amino acids long and shows the classical organization of H2B histones. The hydrophobic C-terminal region is highly conserved as compared to that of the animal counterparts with only 21 changes (13 conservative) among the 90 residues. Between the N-terminal part and the C-terminal region we note the presence of a basic cluster (9 residues) characteristic of histones H2B. The N-terminal third is extended as compared to the animal consensus H2B and has the same size as the H2B histone of wheat. Up to 9 acidic residues and a five time repeated pentapeptide PA/KXE/KK are present in this region. Southern-blot hybrization showed that the H2B histones are encoded by a multigenic family like the other core histones (H3 and H4) of plants. The general expression pattern of these genes was not significantly different from that of the H3 and H4 genes neither in germinating seeds nor in different tissues of adult maize.

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.