Masami Sekine

Phosphorylation of Retinoblastoma-Related Protein by the Cyclin D/Cyclin-Dependent Kinase Complex Is Activated at the G1/S-Phase Transition in Tobacco

In mammals, D-type cyclin-associated kinases mainly regulate the G1/S transition by phosphorylating the retinoblastoma (Rb) protein. We previously demonstrated that in tobacco, cyclin D (Nicta; CycD3;3) is complexed with the PSTAIRE-containing cyclin-dependent kinase (CDKA) from tobacco. Here, we show that Nicta; CycD3;3–associated kinases phosphorylate both the tobacco Rb-related protein (NtRb1) and histone H1. Although NtRb1 kinase activity was detected only during the middle G1- to early S-phase, histone H1 kinase activity was observed as two peaks in G1- to S-phase and G2/M- to M-phase. Importantly, we show that the proportion of cells in the G1-phase was reduced in transgenic Bright Yellow-2 cells overexpressing Nicta; CycD3;3-GFP. Mutational analyses revealed that phosphorylation of Thr-191 in Nicta; CycD3;3 possibly is required for both full kinase activity and localization predominantly to the nucleus. These data suggest that Nicta; CycD3;3 acts as a rate-limiting regulator in the G1/S transition by forming active complexes with CDKA or its related kinases to phosphorylate Rb-related protein and potentially plays a novel role during G2/M and mitosis.

Isolation and characterization of the E2F‐like gene in plants

The transcription factor E2F regulates the expression of genes involved in the progression of G1/S transition and DNA replication in mammalian cells. We cloned and characterized a cDNA (NtE2F) corresponding to a E2F homolog of tobacco (Nicotiana tabacum). The transcription of NtE2F was induced as cells progressed from G1 to the S phase and expressed much earlier than that of the proliferating cell nuclear antigen (PCNA) gene. We demonstrated that NtE2F can interact with the tobacco retinoblastoma (Rb)‐related protein in a yeast two‐hybrid assay. To further characterize NtE2F, the trans‐activation activity of NtE2F was examined by using a transient assay in the tobacco Bright Yellow‐2 (BY‐2) cells with NtE2F fused to the DNA‐binding domain of the yeast transcriptional activator GAL4. NtE2F activated the transcription of the β‐glucuronidase (GUS) reporter gene driven by a cauliflower mosaic virus (CaMV) 35S core promoter containing the GAL4‐binding sequence. This is the first report of the identification of a functionally equivalent E2F‐like gene in plants.

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.

Distinct classes of mitotic cyclins are differentially expressed in the soybean shoot apex during the cell cycle.

Protein phosphorylation by the complexes of cyclin and cyclin-dependent kinase plays a key role in cell cycle progression in all eukaryotes. The amplification by polymerase chain reaction of a cyclin box from developing root nodules and root apices of soybean showed the expression of a number of different molecular species of mitotic cyclins in plant meristems, and they were classified into five distinct groups based on their sequence similarities. The complete soybean cyclin cDNAs, cyc1Gm to cyc5Gm, corresponding to each group were isolated, and their predicted amino acid sequences showed clear similarities to mitotic cyclins identified from various organisms. These genes are expressed predominantly in such meristematic tissues as root and shoot apices and young developing nodules. Double-target in situ hybridization involving histone H4 as an S-phase marker allowed us to estimate the phases during which these cyclin genes are abundantly expressed. The results indicated that cyc5Gm is expressed in G2-to-M phases and cyc3Gm is expressed from late S-to-G2 phases. These expression patterns, together with the sequence criteria, strongly suggest that cyc3Gm and cyc5Gm encode the plant cognates for A- and B-type cyclins, respectively. In addition, the expression of cyc1Gm was restricted during a short period in S phase, suggesting that it belongs to a novel class of plant cyclins. Sequence comparison of 18 plant mitotic cyclins cloned thus far showed that they can be divided into four distinct structural groups with different functions in cell cycle progression.

Expression of a foreign gene in Chlamydomonas reinhardtii chloroplast.

Chimeric genes for expression of a foreign gene in the Chlamydomonas reinhardtii chloroplast were constructed. These chimeric genes are composed of the promoter from chloroplast genes, rbcL, psbA, and atpA, 5- and 3-untranslated regions, and the Escherichia coli beta-glucuronidase (GUS) structural gene (uidA) as a foreign gene. Three types of chloroplast transformants (RG, PG, and AG), which contained the rbcL-uidA, psbA-uidA, and atpA-uidA chimeric genes integrated in the chloroplast genome, were generated by particle bombardment. The AG transformant grown under photoautotrophic conditions showed the highest GUS activity (130 nmol/min/mg protein) so far reported in C. reinhardtii, and the accumulated GUS protein accounted for 0.08% of the total soluble proteins. GUS activity in RG was 12% of that in AG, and no activity was detected in PG. We also measured the GUS activity from transformants grown under heterotrophic conditions, but the culture conditions made little difference in activity levels. The difference in the amount of accumulated GUS protein in the transformants was paralleled by the difference in the level of transcripts, and the pattern of gene expression was not the same as that of the endogenous genes in the chloroplast.

Transcriptional activation of tobacco E2F is repressed by co-transfection with the retinoblastoma-related protein: Cyclin D expression overcomes this repressor activity.

Evidence is emerging that the E2F family of transcription factors plays an important role in the regulation of gene expression at the G1/S transition in plants. Here, we show that in the tobacco proliferating cell nuclear antigen (PCNA), whose transcript is specifically expressed at G1/S phase, the two E2F binding sites are synergistically responsible for transcriptional activation at G1/S phase in synchronized tobacco BY-2 cells transformed with promoter constructs fused to a reporter gene. In addition, we have isolated the tobacco DP cDNA (NtDP) and showed that significant activation of the reporter gene was observed in transient expression assays by concomitantly transfecting with plasmids expressing NtE2F and NtDP. This transcriptional activation was repressed by co-transfection with a plasmid expressing NtRBR1; in vitro pull-down assays also revealed that NtRBR1 binds directly to NtE2F, thereby potentially blocking the transcriptional activation of NtE2F. Importantly, this repressor activity was cancelled when NtRBR1 was further co-transfected with a plasmid expressing cyclin D but not with cyclin A or cyclin B. These results are discussed with respect to the repression activity of NtRBR1 on the NtE2F/NtDP complex.

5' Untranslated region of the HSP18.2 gene contributes to efficient translation in plant cells

Maximizing the amount of protein translated per unit mRNA is an important goal in establishing expression systems. The 5 untranslated region (5-UTR) of mRNA is known to play an important role in determining the rate of translation. The full length 5-UTR from the Arabidopsis thaliana heat shock protein (HSP) gene,HSPI8.2 gene, was inserted into the cloning site between the cauliflower mosaic virus 35S RNA promoter and beta-glucuronidase (GUS) gene in the pBI221 plasmid. When this construct was transfected into Nicotiana tabacum (tobacco) BY-2 protoplasts, the level of protein was about 10-fold higher than that of unmodified pB1221. The accumulation of each transcript was the same level. We also demonstrated that the 5-UTR of the HSP18.2 gene enhances the rate of translation in stable transgenic BY-2 clones and Arabidopsis T87 protoplasts. The 5-UTRs of the other Arabidopsis HSP genes -HSP17.4, HSP81-1,HSP81-2, andHSP81-3 - also conferred efficient translation. These 5-UTRs ofHSP genes may be of use in increasing the expression of foreign proteins. In combination with a strong promoter, it can be used in the development of efficient protein production systems.

Metabolic engineering of cultured Tobacco cells

Construction of a gene expression system in tobacco cultured cells (BY2) was studied. A 925 bp promoter fragment of a heat-shock protein gene (HSP18.2) of Arabidopsis thaliana showed clear heat-shock response of expression of the beta-glucuronidase (GUS) reporter gene in BY2 cells. Similar results were observed in a 500 mL flask and 3-L jar fermentor. Isolation of strong promoters in BY2 cells was tried. cDNA clones, in which the mRNA level is high in log-phase cells and the copy number in the genome is low, were isolated. These clones showed high homology with F1-ATPase (mitochondria type), elongation factor 1-alpha, and a gene with an unknown function of A. thaliana (clone 27), respectively. A 5-flanking region of clone 27 showed 6.2 times the promoter activity of the CaMV35S promoter in BY2 cells. Three cDNA clones, which are expressed in the stationary growth phase of BY2 cells, were isolated by a differential screening. These clones showed high sequence homologies to alcohol dehydrogenase, pectin esterase, and extensin. Promoters of these genes will be useful in gene expression in high cell-density culture.

Isolation of growth-phase-specific promoters from cultured tobacco cells.

Strong promoters are required under several culture conditions for effective transgene expression in tobacco BY2 cells. We have isolated the promoter fragments of 4 genes exhibiting high homology to those of Arabidopsis thaliana 108C1T7 (unknown function) and F1-ATPase-delta, alcohol dehydrogenase and pectin esterase genes from a genomic DNA library of BY2 cells. Two of the four genes were strongly expressed during every phase of growth of BY2 cells, and the other two were expressed only during the stationary phase. Each of the promoter fragments was ligated to the GUS reporter gene and introduced into the chromosome of BY2 cells by Agrobacterium-mediated transformation. Growth-phase-dependent expression of the GUS gene was reproduced under the control of all 4 promoters observed with the original genes. Significantly higher expression was observed under the control of Nt108p during every phase of cell growth and under the control of NtADHp and NtPESp during the stationary phase than that under the control of the CaMV35S promoter.

Expression pattern of tobacco cyclin genes

We have previously isolated three cDNA clones (Ntcyc25, Ntcyc27, Ntcyc29) encoding mitotic cyclins from tobacco (Nicotiana tabacum), and in this report we describe the expression patterns of these genes. RNA gelblot analysis showed that the mitotic cyclin genes were expressed predominantly in actively dividing meristematic tissues such as the shoot apex and young developing leaves. In situ hybridization analysis indicated that while theNtcyc29 gene was expressed consistently at low levels in shoot apex, but its transcripts were found at relatively high levels in the flower bud, indicating the possibility that theNtcyc29 product plays a role in flower development. TheNtcyc25 andNtcyc29 genes were expressed differentially in the root apex during the cell cycle, confirming the result obtained using synchronized-cultured tobacco cells. These results suggest that the transcriptionally regulated cyclin genes may be important in controlling cell division in tobacco.