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.

Comparison of capillary zone and immunosubtraction with agarose gel and immunofixation electrophoresis for detecting and identifying monoclonal gammopathies.

Capillary zone electrophoresis (CZE) and immuno-subtraction electrophoresis (ISE) were evaluated for ability to detect and immunotype monoclonal proteins, compared with agarose gel electrophoresis (AGE) and immunofixation electrophoresis (IFE), respectively. Six hundred seventeen serum samples were analyzed with CZE and AGE to determine sensitivity and specificity in detecting IFE-confirmed monoclonal gammopathies. Both techniques detected all monoclonal spikes due to IgM (n = 8), IgG (n = 38), and free light chains (n = 3). Agarose gel electrophoresis, however, detected only 11 of 14 (79%) IgA monoclonal spikes detected with CZE. In a second study, 78 serum samples, 48 of which had a monoclonal gammopathy confirmed with IFE, were evaluated with ISE. Only 60% to 75% of the monoclonal gammopathies were correctly immunotyped with ISE by 4 readers blinded to the IFE immunotype. Thus CZE was more sensitive than AGE in detecting low concentrations of monoclonal proteins, but ISE is less accurate than IFE in determining the immunotype of the monoclonal gammopathy.

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.

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.

CYP86A1 from Arabidopsis thaliana encodes a cytochrome P450-dependent fatty acid omega-hydroxylase.

The A. thaliana EST database was screened using consensus motifs derived from P450 families CYP52 and CYP4 catalyzing the omega-hydroxylation of fatty acids and alkanes in Candida and in mammals. One EST cDNA fragment was detected in this way and the corresponding full-length cDNA was cloned from a cDNA library of A. thaliana. This cDNA coded the first member of a new plant P450 family and was termed CYP86A1. The deduced peptide sequence showed highest homology with P450s from families 4 and 52. To confirm the catalytic function, CYP86A1 was expressed in a yeast overexpressing its own NADPH-P450 reductase. Efficient expression was evidenced by spectrophotometry, SDS-PAGE and catalytic activity. CYP86A1 was found to catalyze the omega-hydroxylation of saturated and unsaturated fatty acids with chain lengths from C12 to C18 but not of hexadecane. Genomic organization analyzed by Southern blot suggested a single gene encoding CYP86A1 in A. thaliana.

Further progress towards a catalogue of all Arabidopsis genes: analysis of a set of 5000 non-redundant ESTs.

Nearly 7000 Arabidopsis thaliana-expressed sequence tags (ESTs) from 10 cDNA libraries have been sequenced, of which almost 5000 non-redundant tags have been submitted to the EMBL data bank. The quality of the cDNA libraries used is analysed. Similarity searches in international protein data banks have allowed the detection of significant similarities to a wide range of proteins from many organisms. Alignment with ESTs from the rice systematic sequencing project has allowed the detection of amino acid motifs which are conserved between the two organisms, thus identifying tags to genes encoding highly conserved proteins. These genes are candidates for a common framework in genome mapping projects in different plants.

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.

Molecular characterization of a beta-type proteasome subunit from Arabidopsis thaliana co-expressed at a high level with an alpha-type proteasome subunit early in the cell cycle.

Proteasomes are the essential components of complexes involved in an extralysosomal energy- and ubiquitin-dependent proteolytic pathway. The first alpha-type proteasome subunit in plants has recently been described. In this work, the sequence of the first beta-type proteasome subunit in plants, isolated from Arabidopsis thaliana cDNA libraries is reported. The mRNA accumulation of both subunits was analysed and compared with those of the ubiquitin and histone mRNAs, in different tissues and during re-initiation of mitotic activity. It is demonstrated that in plants, as in animal cells, the transcripts of both the alpha-type and beta-type proteasome subunits accumulate to high levels during cell proliferation, in parallel with mRNAs coding for a ubiquitin fusion protein and several polyubiquitins, but earlier than those coding for histone H4 whose expression is known to be coupled to DNA synthesis. These results suggest that, as in animal cells, proteasomes may be involved in the progression of the cell cycle.

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.

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.

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 hybridization 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.

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

A cDNA clone isolated from an Arabidopsis thaliana cell suspension culture library showed considerable similarities to the proteasome 28 kDa alpha 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/of division.

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.