Expression of Arabidopsis cystathionine γ-synthase (CGS), the key step in methionine biosynthesis, is feedback regulated at the step of mRNA degradation in response to <S>-adenosyl-L-methiomine (SAM). The first exon coding region of CGS is necessary and sufficient for this regulation. A short stretch of amino acid sequence (MTO1 region) is particularly important for this regulation. A nascent peptide emerges after passing through the exit tunnel of ribosome. We suggest a model that the nascent peptide and SAM interact inside the exit tunnel. We introduced amino acid substitutions at Cys-80 and Ser-81, and analyzed their response to SAM and <S>-adenosyl-L-ethionine (SAE) which has ethyl group in place of methyl, and found differences in their response. Here, we suggest that Cys-80 and Ser-81 play a role in recognition of methionine and ethionine.

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A number of eukaryotic mRNAs contain upstream open reading frames (uORFs) within the 5' untranslated region. In several genes, the peptide encoded by the uORF has been shown to have a role in translational regulation of downstream ORF. So far, only one example of such a regulation has been reported in plants.
To further identify Arabidopsis genes whose translation is controlled by the uORF-encoded peptide, we first searched for uORF-containing genes that are predicted to encode transcription factors or to be involved in amino acid biosynthesis, by using Arabidopsis full-length cDNA database. Then, we tested amino acid sequence-dependency of effect of uORFs on translation of downstream ORF. From this screen, we found several uORFs that influence downstream translation in an amino acid sequence-dependent manner. Intriguingly, in one of the identified genes, its expression was suggested to be regulated by a combination of alternative splicing and uORF peptide-mediated translational regulation.

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The 20 amino acids, except for methionine and tryptophan, are codoed for by 2 to 6 codons called synonymous codons. Synonymous codons are not used with equal frequency in protein coding sequences, and are used differently by different organisms. It has been thought that the codon usage is correlated with the translation efficiency. Using an in vitro translation system from tobacco chloroplasts, we devised an in vitro assay for relative translation rates of synonymous codons. We found that translation efficiencies of synonymous codons are not always correlated with codon usage in tobacco chloroplasts. Here, we analyzed the effect of selection of synonymous codons on the translation efficiency of protein coding region from several tobacco chloroplast mRNAs.

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Tobacco chloroplast genome encodes about 80 protein genes that are expressed by chloroplast gene expression system. Many chloroplast mRNAs lack SD-like sequences, which are found in the 5'-untranslated region of most of bacterial mRNAs. A common or specific trans-acting factor is thought to be involved in translation initiation of such chloroplast mRNAs but is still obscured. We have analyzed translation initiation of chloroplast mRNAs using in vitro translation and gel shift assays. Here we show that a trans-acting factor could be shared by some chloroplast mRNAs such as atpB and psbN to facilitate translation initiation. This potential trans-acting factor preferentially bound to a U-rich region upstream of initiation codon of these mRNAs. Mechanism of translation initiation in chloroplasts will be discussed.

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The ndhK gene encodes a subunit of the chloroplast NADH dehydrogenase, and is co-transcribed with the adjacent ndhC and J genes on the tobacco chloroplast genome. The ndhK gene product is translated from polycistronic mRNAs but not from monocistronic ones. According to tobacco chloroplast in vitro translation analyses, we defined that the major initiation site of tobacco ndhK cistron is the AUG codon that located 4 nt upstream from the ndhC stop codon, and ndhK translation depends exclusively on ndhC termination codon. Removal of the ndhC 5' UTR still supported substantial translation of ndhK cistron. This result suggested that ndhK cistron should be translated by an additional unknown mechanism in addition to translational coupling in a termination codon-dependent mannar.

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Chloroplast transcripts are generally processed by complex reactions including RNA cleavage, RNA splicing, RNA editing, trimming to produce mature mRNAs. The 5 untranslated regions of chloroplast mRNAs are some dozens to several hundreds in nucleotide length. Chloroplasts include usually processed and unprocessed mRNA species. As it is difficult to investigate effects of 5 processing on translation in vivo, we used our in vitro translation system. Translation reactions were performed under template limiting and linearly progressing conditions. We here used atpI and psaC mRNAs from tobacco chloroplasts. The atpI mRNA is transcribed from -208 and from -131, and processed at -85. Both the unprocessed mRNAs were translated in similar efficiency while the processed mRNA was translated very efficiently. The psaC mRNA is transcribed from -234 and processed at -173. The processed mRNA was also translated more efficiently than the unprocessed mRNA.

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Chloroplasts of higher plant have own genomes and its gene expression systems, and major gene expression is regulated by translation level. Therefore understanding of translation regulation mechanisms is essential.
It has been regarded that translation is regulated by 5' untranslated region (5' UTR) which is upstream sequence of coding region on mRNA. But, in practice, it is not always true that strong 5' UTR which binds to any coding regions show strong translation activity. It is regarded that coordination with 5' UTR and coding region especially around sequences of the start codon is important.
In this study, we performed chloroplast in vitro translation experiments using one of the strongest translation activity psbA mRNA in tobacco chloroplasts, and assayed the effects of adjacent region of start codon on translation.

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RNA editing is a posttranscriptional process that alters genetic information in RNA level. In plants, a specific C nucleotide is converted to U in chloroplast and mitochondrial mRNA, and adenosine in the wobble position of several nuclear and chloroplast encoded tRNAs is deaminated to inosine. Deaminases have been reported as RNA editing enzymes in mammal and yeast. But catalytic factors for RNA editing in plants, have not been identified. Thus we attempt to characterize a deaminase gene family of Arabidopsis.
Arabidopsis genome contains 17 genes which encode the deaminase domain. Six of them belong to the group of adenosine deaminases acting on tRNA, while none of them belongs to the group of cytidine deaminases for mRNA editing in animal. Structure, expression pattern and subcellular localization of Arabidopsis deaminases are reported.

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Nuclear genes encoding tRNA^Tyr and tRNA^Met-e are interrupted by introns in plants. Pre-tRNA splicing requires the concerted action of three enzymes in yeast cells and plants: an endonuclease (Sen), a tRNA ligase (tRL) and a 2'-phosphotransferase (Pt). We have investigated the subcellular localization of the enzymes. The results show that all enzymes are localized predominantly in the nucleus. tRL and Pt, both containing chloroplast transit signals (CTSs) at the N-termini, are also targeted to the chloroplasts. Moreover, Pt was targeted to the mitochondria. Nucleotide analysis revealed that the context around the first initiation codon in tRL genes from several plants is not optimal for the efficient initiation of translation in plants, suggesting the possibilities that the first AUG is partially skipped to start a translation at the second AUG (or third AUG). Taken together, an alternative initiation in the translation level may result in a multiple targeting to cellular compartments.

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Commonly, treanslation starts from the first initiation codon. It has been reported that there are mRNAs that have short ORFs upstream the major ORF. In this case, these short ORFs, upstream ORF (uORF), are accepted to be regulators of the major ORF because they may affect on its translation. We found that OsMac1 mRNA was composed of three types of splaicing variants with difference in the 5'UTR. There were three uORFs in all splicing variants of OsMac1 mRNA. We analyzed the effect of these mRNAs to translation of major ORF. We constructed a reporter gene encoding β-glucuronitase following 5'UTRs of these splicing variants. Apparent GUS activities were detected in the transformant cells containing each reporter genes. Strength of the GUS activity was different on the transformants. These results suggest that alternative splicing on the mRNA of OsMac1 was involved in regulation of translation of the major ORF.

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Eukaryotic DNA is packed in higher ordered structure of chromatin, whose states are thought to confer basis of epigenetic regulation. Such states are heritable over mitosis or meiosis, however, the molecular mechanism of chromatin reconstitution coupled with DNA replication and repair is still largely unknown. To address this issue, we have focused on function of Arabidopsis BRU1/TSK/MGO3 that was identified as a nuclear factor involved in both DNA repair response and epigenetic regulation of heterochromatic genes. BRU1 has been proposed to have a role for chromatin reconstitution after DNA replication and/or repair. In this study, we show that BRU1 ensures tissue specific expression pattern of genes, required for meristem formation and seed development. Together with global expression profiles in bru1, the result suggests that bru1 possibly affects a wider range of epigenetic regulation. Our recent trial to purify BRU1 complex by TAP system will also be presented.

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One of the Japanese cedars, Cryptomeria japonica, can be induced to form reproductive organs upon growth regulator applications, temperature shifts and photoperiod manipulations. Two- and 3-year-old saplings of C. japonica remained dormant when maintained at 15C with the 8 hour photoperiod. When these saplings were treated with GA₃ (100ppm or 500ppm) and kept in the same condition for 7 weeks and then transferred to the condition, 20/15C (day/night) with a natural photoperiod, in November, reproductive organs appeared after the vegetative growth. In order to establish an experimental system for the molecular analyses of epigenetics of the reproductive process, the location of the reproductive organs on the saplings was examined to locate the sites of induction. The effect of another condition, 30/25C (day/night) with a natural photoperiod, was also studied. The reproductive induction process will be discussed based on the male/female cone distribution in the branches of the saplings.

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Proteasome is a large multisubunit complex that degrades damaged or ubiquitinated proteins. Although it is one of the most important features of plant proteasome that the most subunits are coded by duplicated genes, specific functions of each paralogous subunit are still unclear.
We have demonstrated that loss-of-function mutant of the specific paralogous protein shows aberrant response to nutritional stress. Furthermore, it has been reported that peptidase activities of proteasome were changed in response to nutritional and oxidative stress, suggesting that plant proteasome enables to transform structure and function to response to environmental stress conditions. To evaluate this hypothesis, we tried to identify the subunit composition of proteasome under various stress conditions.
We have succeeded in established high-throughput method to identify and quantify composition of paralogous proteins with affinity purification and combination of 2D-PAGE and MS analysis. Proteasome transformation in response to environmental stress will be discussed.

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Arabidopsis AtbZIP10 is a bZIP transcription factor which participates in various cellular environmental responses as programmed cell death, disease resistance and osmoregulation. Our preliminary experiment supposed that Arabidopsis group-C bZIP transcription factors (bZIP-C: AtbZIP9,10,25,63) is very unstable in plant cells. Here we tried to figure out the mechanism of selective degradation of AtbZIP10 via ubiquitination by focusing on 26S proteasome-mediated degradation. Time-dependent degradation of AtbZIP10-GFP transiently expressed in Arabidopsis mesophyll protoplasts was inhibited by 26S proteasome inhibitor, MG-132. Deletion analysis of AtbZIP10-GFP demonstrated that this degradation can be inhibited by lacking domain II, which is highly conserved among bZIP-C. Since domain II contains some highly conserved lysine residues, known as ubiquitination target, bZIP-C including AtbZIP10 would be regulated by selective degradation via ubiquitination. Since now ubiquitination of AtbZIP10 has been confirmed by in vitro ubiquitination assay, we'll present this result, too.

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FtsH protease is an ATP dependent protease. This protease localized on thylakoid membrane. Main function of FtsH protease is considered to be quality control of membrane proteins in the thylakoid membrane. D1 protein which is important component of photosystem II is one of the substrate proteins of FtsH protease. FtsH deficit mutant shows variegated phenotype, thus this protease plays an important role in the biogenesis of chloroplast. Structure of this enzyme is consisted from three domains, transmembrane domain, ATPase domain, and protease domain. Substrate protein forced to be denatured in the ATPase domain, successively transferred to the protease domain. The denatured substrate was digested in the protease domain. Arabidopsis contained 12 isoforms of FtsH proteases in the nuclear genome. Mutation in FtsH5 showed strong variegated phenotype. In this study, we constructed an expression system of ATPase domain in FtsH5. Biochemical and kinetic properties of expressed protein were analyzed.

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The main function of FtsH protease in plant cell is quality control of membrane protein. D1 protein in photosystem II is one of the main targets of FtsH protease, because D1 protein is easily damage during the photosynthesis. FtsH protease participates in this turnover. FtsH protease is an ATP dependent metalloprotease and is composed from the following three domains; transmembrane, ATPase and protease domain. We constructed over-expression system of protease domain in FtsH from tobacco. Protein was collected as inclusion body. It was activated by urea denatured and dialyzed. We used FITC (fluorescein isothiocyanate)-casein for substrate protein. Biochemical analysis revealed that the activity was stimulated by higher concentration of magnesium ion, and pH optimum showed alkalescent condition. Beta-casein was selectively digested, though alpha-casein, bovine serum albumin, or Rubisco was not degraded by the protease domain. Cleavage site of this digested beta-casein clarified by peptide sequencing.

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FtsH protease is an ATP dependent metalloprotease, which localized on thylakoid membrane. Main function of FtsH protease is to degrade photo-damaged D1 protein in the reaction center of photosystem2. This protease is composed from transmembrane domain, ATPase domain and protease domain. In this study, we focused on the functional analysis of ATPase domain of FtsH protease in var2 subunit. FtsH protease from Arabidopsis was composed from at least four types of subunits, though that from E.coli was homohexamer. We constructed an expression vector of ATPase domain and transformed into BL21-CodonPlus(DE3)-RIL. Purification was performed using Ni-Chelating Sepharose. The quality of this protein was verified by western blotting and peptide sequencing. We examined biochemical analysis to determine the optimum condition of this enzyme. We also investigated effect of inhibitors. Kinetic parameters, Km,Vmax and Kcat, were calculated in the optimum condition.

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Plastids originated from photosynthetic bacteria like cyanobacteria, threfore plastid ribosomes belong to the eubacterial 70S class. Furthermore plastid ribosomes have seven plastid-specific ribosomal proteins (PSRPs). PSRPs are conserved across land plants, although their functions remain unknown.
To elucidate the function of PSRP-1, we made psrp-1 disruptant and psrp-1 overexpressing strain of Synechocystis sp. PCC 6803, and examined their growth. The growth of these psrp-1 mutants are the same as wild-type at 30 degrees C on solid medium. However, at 40 degrees C, growth of the psrp-1 disruptant was significantly suppressed in comparison with wild-type and the psrp-1 overexpressing strain. While, in solid medium containing glucose at 30 degrees C, growth of the psrp-1 disruptant was better than that of wild-type, and growth of the psrp-1 overexpressing strain was suppressed. These results suggest that PSRP-1 negatively regulates translation.

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In Arabidopsis thaliana, the microRNA miR398 post-transcriptionally downregulates the expression of CuZn-SOD and involves in stress-response and Cu homeostasis. Since the target sequence of miR398 for CuZn-SOD gene is located in the cording region, it seems that plant CuZn-SODs are regulated in similar fashion. However, in maize, anther mechanism might be possible because of the preferential localization of chloroplastic CuZn-SOD (SOD-1), and the absence of Fe-SOD activity in the cells. We have cloned maize SOD-1 gene and Fe-SOD cDNA. To elucidate the gene expression of CuZn-SOD in maize, we analyzed gene response against environmental stress and Cu availability. SOD-1 gene contained Cu-responsive element, TnnnGCTG in promoter region and miR398 target sequence with 19/21 match in exon 4. Seedlings were treated with Cu limitation or excess and their responses were analyzed. In addition, effects of cold treatment, drought, MV were investigated. We will discuss those results with relation to microRNA involvement.

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Although known as a harmful food ingredient of plant origin, oxalate has been considered in plants to contribute to cellular protection against heavy metal toxicity by exerting chelating effects. During our research of functional characterization of an NO₂-inducible germin-like protein (RmGLP2) from azalea leaves¹, we found that recombinant RmGLP2 proteins, when overexpressed in tobacco cultured cells, were destined for the apoplast where they exhibited enzymatic activities for both superoxide dismutase and oxalate oxidase (OxO). In parallel with the increased OxO activity in the apoplast, there was a marked decrease in oxalate levels in the transformed cells. In the presence of heavy metals, transformed cells showed the hypersensitivity and significantly impaired biomass production. These results strongly suggest the plant physiological importance of oxalate accumulation in suppressing heavy metal toxicity.
¹Kondo et al. (2008) Biochem. Biophys. Res. Commun. 377: 857-861.

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The first step of plant-specific protein O-glycosylation is the hydroxylation of proline residue catalyzed by prolyl 4-hydroxylases (P4Hs). To investigate this modification, we have been analyzing P4Hs in tobacco. This time we cloned three full-length cDNAs for type2 P4H (NtP4H2.1, NtP4H2.2, NtP4H2.3). All the encoded proteins contain N-terminal signal peptide, catalytic domain and C-terminal Tox1 domain, the function of which is unknown. Membrane fractionation analysis using a specific antibody for NtP4H2.2 revealed NtP4H2.2 distributes in fractions containing Golgi apparatus. NtP4H2.2 fused with GFP co-localized with a cis-Golgi marker protein in tobacco BY-2 cells. Almost all NtP4H2.2 was recovered in sedimentable fraction after the sonication of microsome. These results indicate type2 P4H is a membrane protein localized predominantly in the Golgi apparatus. We are currently investigating the function of the Tox1 domain and results of this analysis will be presented.

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The cysteine protease which has a thiol group in the active center participates in the protein maturation and degradation process in response to the external stimuli, and in various physiological processes such as germination, morphogenesis, and programmed cell death. It is becoming clearer that they are involved in perceiving both biotic and abiotic stresses, signaling and execution
Actinidine of Kiwifruit belongs to a papain family, and the crystal structure is known, and there is also a report that it consists of the isozymes with different pI.
1. We isolated three cysteine proteases with different activity by anion ion exchange chromatography from Actindia (peaks 1, 2 and 3). Until now the crystal structure of peaks 1 and 2 was clarified.
2. Gene amplification was carried out by genomic PCR and RT-PCR by using primers based on the actinidine gene of a kiwi fruit. The clone with the DNA sequence corresponding to the exons has been amplified by RT-PCR.

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In higher plants, sulfate taken up from the environment is converted to adenosine 5'-phosphosulfate (APS) by ATP sulfurylase. APS can be a substrate of sulfur reduction, or can be phosphorylated to yield 3'-phosphoadenosine 5'-phosphosulfate, a donor for sulfation of a range of metabolites. The Arabidopsis genome contains four ATP sulfurylase genes (ATPS1, ATPS2, ATPS3, ATPS4). Physiological function of each ATPS was analyzed in their knockout mutants. atps1, atps2 and atps3 exhibited lower levels of ATP sulfurylase activity than wild-type plants. Expression of ATPS-GFP fusion constructs in Arabidopsis plants suggested that ATPS1, ATPS3 and ATPS4 localized exclusively in chloroplasts whereas ATPS2 expressed in both cytosol and chloroplasts. In addition, cell type-specificities of ATPS were different from each other. It is suggested that each ATPS plays a specific role in the sulfur metabolism.

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Recently, a stable nuclear transformation technique was established with two marine diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana . However each system accommodates only one selection system, which limits the capacity of diatom transformation systems. The purpose of this research is to isolate auxotrophic mutants of marine diatoms and to establish the phenotypic complementation system of the genetic lesion. P. tricornutum and T. pseudonana were mutagenized by 1mg/mL ENU ( N -ehtyl- N - nitrosourea) solution for 30min. Then, they were cultured on ASW (artificial sea water) agar medium containing 0.6mM 5-fluoro orotic acid (5-FOA) and 0.5mM uracil. 5-FOA resistant clones were innoculated to several ASW agar plate containing 1.8mM 5-FOA and 0.5mM uracil. As a result, a few clones of uracil auxotrophic mutants of P. tricornutum was isolated. The results from characterization of these mutant will be discussed.

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Genetic analyses have revealed that leucine-rich repeat receptor-like kinases (LRR-RLKs) play important roles in plant signal transduction. However, biochemical properties of most LRR-RLKs remain unknown because it is difficult to obtain sufficient proteins for biochemical analyses. Therefore, we optimized LRR-RLK expression in Saccharomyces cerevisiae.
First, to determine the most appropriate strain for LRR-RLK expression, LRR-RLKs we expressed in several strains and found that BY2777, which is a mutant strain of vacuolar proteases is appropriate strain for it. In yeast, it is known that proper folding and secretion of some protein can be facilitated by fusing it to yeast-derived polypeptide Hsp150Δ. Therefore, we also examined whether Hsp150Δcan promote the secretion of LRR-RLK in yeast. Secretions of some LRR-RLKs are found to be improved by fusing them to Hsp150Δ. We are now optimizing LRR-RLK solubilization conditions and confirming their kinase activity for the high-throughput assessment of protein quality.

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Monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) are the major lipids in chloroplasts. These galactoglycerolipids are known to play an important role apart from their involvement in chloroplast biogenesis and photoautotrophic growth. In Arabidopsis grown under Pi-limited conditions, an accumulation of DGDG occurs concomitantly with a decrease of phospholipids and expression of typeB MGD gene is increased. It is unknown whether this Pi starvation-induced lipid remodeling is common to plants. Here, we cloned two types of sesame MGDG synthase genes and analyzed the lipid composition and gene expression in Pi-starved sesame seedlings. As a result, we observed the accumulations of not only DGDG but MGDG in sesame roots under Pi starvation, while in Arabidopsis, only DGDG accumulates. MGD gene expressions were similar between sesames and Arabidopsis. Hence, we concluded that sesames also have the lipid remodeling, but it is distinct from Arabidopsis one in respect of MGDG accumulation.

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We examined the role of putative rice (Oryza sativa L.) monogalactosyldiacylglycerol synthase (UDPgalactose: 1,2-diacylglycerol 3-b-D-galactosyl transferase; EC 2.4.1.46, MGDG synthase) gene OsMGD under salt and drought stresses by overexpressing in tobacco. OsMGD transgenic plants showed better growth and vigor under salt stress in vitro and maintained higher photosynthesis and PSII photon yield under salt and drought stresses than wild type. Transgenic plants had significantly higher contents of monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG) and lipid-P than wild type. Upon salt stress, MGDG and DGDG maintained significantly higher levels than wild type. Overall fatty acid composition of MGDG and DGDG in wild type and OsMGD transgenic plants was basically unchanged during 72 h of salt stress, except for slight decreases in levels of α-linolenate of both galactolipids only in wild type plants. This indicated that OsMGD overexpressed plants maintained greater stability of galactolipid composition, membrane structure and functions to cope with salt tolerance.

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Although lipids are rich in reproductive organ in plant, physiological roles of each lipid species have not yet well understood. We have studied the role of sterols on the development of reproductive organs by reverse-genetic strategy using Arabidopsis.
HMG-CoA reductase, the key enzyme of sterol biosynthesis, is encoded by HMG1 and HMG2 in the Arabidopsis genome. hmg1 shows dwarfing, early senescence and male sterility, and hmg1 hmg2 were male gametphyte lethal. Microscopic analyses showed pollen-coat was absent in hmg1 and hmg1 hmg2 microspores were stunted.
Next, we focused the steroidal backbone synthetic step. Oxidosqualene is cyclized to cycloartenol by CAS1 and lanosterol by LAS1. The knockout allele of CAS1, cas1-2, was male gametophyte lethal. However, the microspores of cas1-2 were less damaged than those of hmg1 hmg2. These results suggest that metabolites from mevalonate to oxidosqualene and/or sterol biosynthetic pathway via lanosterol are important for the development of microspores.

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Sugars modulate many vital metabolic and developmental processes in plants, however, the exact mechanisms involved in morphogenesis, developmental signalling and stress tolerance remain largely unknown. Here we report the characterization of a novel Arabidopsis thaliana mutant, sweetie, with drastically altered morphogenesis, and a strongly modified carbohydrate metabolism leading to elevated levels of trehalose, trehalose-6-phosphate and starch. Genes implicated in sugar metabolism, sugar transport, senescence, ethylene biosynthesis and abiotic stress were found to be upregulated in sweetie. However, SWEETIE encodes a protein that is unrelated to any known enzyme involved in sugar metabolism. We suggest that SWEETIE plays an important regulatory function that influences multiple metabolic, hormonal and stress-related pathways, leading to altered gene expression and pronounced changes in the accumulation of sugar, starch and ethylene.

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Sphingolipids are ubiquitous and essential components in the membrane of eukaryotic cells and also known as bioactive molecules. In plant sphingolipids, C4 positions of the LCBs are commonly hydroxylated by dihydrosphingosine C4 hydroxylase (DSH). There were five DSH genes (OsDSH1 to OsDSH5) in the rice genome. The expression patterns of OsDSH1 to OsDSH5 were analyzed by the GUS reporter assay as well as RT-PCR. All five genes showed different expression patterns with each other. When DSH enzyme-activities of these OsDSHs were determined by yeast complementation test, it was shown that OsDSH1 and OsDSH4 efficiently restored the yeast DSH mutation (Δsur2), suggesting they functioned as DSH. The transformants with RNAi to OsDSH1 showed severe reduction of their fertilities. On the other hand, the overexpressed-transformants of OsDSH5 showed the dwarf phenotype. These results suggested that functions of OsDSHs diverge in the tissues in with specific gene expression.

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