Transient Expression Studies Research Articles

Characterization of an Oxidative Stress Inducible Nonspecific Lipid Transfer Protein Coding cDNA and its Promoter from Drought Tolerant Plant Prosopis juliflora

Abiotic stress is the principal cause of crop failure worldwide. Prosopis juliflora is a hardy plant reported to be tolerant to drought, salinity, extremes of soil pH, and heavy metal stress. Here, we report the characterization of a cDNA clone for a putative nonspecific lipid transfer protein (Pj LTP1) found abundantly in a drought stressed leaf cDNA library of P. juliflora and its promoter. A multiple sequence analysis of Pj LTP1 with well-established and officially designated nonspecific lipid transfer proteins allergens revealed high similarity at the amino acid level. Northern analysis of Pj LTP1 in P. juliflora leaves under oxidative stress revealed steady upregulation at the time points analyzed. A 929-bp fragment was isolated from the 5′ end of Pj LTP1, and transient reporter gene expression studies revealed it to be a functional promoter. Several cis-acting elements previously reported to function in stress response were found in this promoter. The possible reasons for changes in gene expression during stress in relation to the host plant’s stress tolerance mechanisms are discussed.

Further Evaluation of the Localization and Functionality of Hemagglutinin Epitope- and Fluorescent Protein-Tagged AtMinD1 inArabidopsis thaliana

Symmetric chloroplast division requires a prokaryote-derived division regulator protein MinD, whose subchloroplastic localization remains to be completely established. We investigated the localization and functionality of AtMinD1 (Arabidopsis thaliana MinD) fused with a dual hemagglutinin epitope (dHA) or a yellow fluorescent protein (YFP). AtMinD1-dHA, which successfully complemented the arc11/atminD1 mutant phenotype, was predominantly located at the envelope membrane and the mid-chloroplast constriction site. Meanwhile, AtMinD1-YFP was non-functional and showed suborganellar localization partly similar to that of AtMinD1-dHA. This prompts us to reevaluate earlier transgenic and transient expression studies using fluorescent protein-tagged AtMinD1.

Specific Interaction between Tomato HsfA1 and HsfA2 Creates Hetero-oligomeric Superactivator Complexes for Synergistic Activation of Heat Stress Gene Expression

In plants, a family of more than 20 heat stress transcription factors (Hsf) controls the expression of heat stress (hs) genes. There is increasing evidence for the functional diversification between individual members of the Hsf family fulfilling distinct roles in response to various environmental stress conditions and developmental signals. In response to hs, accumulation of both heat stress proteins (Hsp) and Hsfs is induced. In tomato, the physical interaction between the constitutively expressed HsfA1 and the hs-inducible HsfA2 results in synergistic transcriptional activation (superactivation) of hs gene expression. Here, we show that the interaction is strikingly specific and not observed with other class A Hsfs. Hetero-oligomerization of the two-component Hsfs is preferred to homo-oligomerization, and each Hsf in the HsfA1/HsfA2 hetero-oligomeric complex has its characteristic contribution to its function as superactivator. Distinct regions of the oligomerization domain are responsible for specific homo- and hetero-oligomeric interactions leading to the formation of hexameric complexes. The results are summarized in a model of assembly and function of HsfA1/A2 superactivator complexes in hs gene regulation.

Mucopolysaccharidosis type I in 21 Czech and Slovak patients: Mutation analysis suggests a functional importance of C‐terminus of the IDUA protein

Mucopolysaccharidosis type I (MPS I) is an autosomal recessive lysosomal storage disorder that is caused by a deficiency of the enzyme α-l-iduronidase (IDUA). Of the 21 Czech and Slovak patients who have been diagnosed with MPS I in the last 30 years, 16 have a severe clinical presentation (Hurler syndrome), 2 less severe manifestations (Scheie syndrome), and 3 an intermediate severity (Hurler/Scheie phenotype). Mutation analysis was performed in 20 MPS I patients and 39 mutant alleles were identified. There was a high prevalence of the null mutations p.W402X (12 alleles) and p.Q70X (7 alleles) in this cohort. Four of the 13 different mutations were novel: p.V620F (3 alleles), p.W626X (1 allele), c.1727 + 2T > G (1 allele) and c.1918_1927del (2 alleles). The pathogenicity of the novel mutations was verified by transient expression studies in Chinese hamster ovary cells. Seven haplotypes were observed in the patient alleles using 13 intragenic polymorphisms. One of the two haplotypes associated with the mutation p.Q70X was not found in any of the controls. Haplotype analysis showed, that mutations p.Q70X, p.V620F, and p.D315Y probably have more than one ancestor. Missense mutations localized predominantly in the hydrophobic core of the enzyme are associated with the severe phenotype, whereas missense mutations localized to the surface of the enzyme are usually associated with the attenuated phenotypes. Mutations in the 130 C-terminal amino acids lead to clinical manifestations, which indicates a functional importance of the C-terminus of the IDUA protein. © 2009 Wiley-Liss, Inc.

Genetic variation of FUT3 in Ghanaians, Caucasians, and Mongolians

The FUT3 gene regulates the expression of Lewis blood group antigens. Several lines of evidence suggest association between expression of these antigens and Helicobacter pylori infection or susceptibility to cardiovascular diseases. Single-nucleotide polymorphisms (SNPs) responsible for the Lewis-negative phenotype have been reported, but systematic sequence analyses have not yet been performed. The polymerase chain reaction product containing the whole FUT3 coding region (1086 bp) was sequenced in three human populations, Ghanaians (n = 106), Caucasians (n = 100), and Mongolians (n = 50). Some haplotypes were determined by sequencing cloned inserts, and the other haplotypes were inferred by the free software program PHASE. Also examined was the functional significance of several newly identified SNPs by a transient expression study. Thirty-two SNPs were found, including 15 novel SNPs, in the three populations. The functional impact of three nonsynonymous SNPs that occurred on the Lewis-positive alleles or weakly enzyme-inactivating alleles was examined. A transient expression study suggested that 478C>T and 968G>C are enzyme-inactivating mutations. Sequence analysis identified many SNPs, but most of them are in complete linkage disequilibrium with previously reported SNPs responsible for the Lewis-negative phenotype. 13G>A, 59T>G, and 202T>C seem to be useful as tag SNPs for detection of Lewis-negative alleles in genotyping of Lewis blood groups and large-scale association studies with diseases in many populations.

The mammalian UPR boosts glycoprotein ERAD by suppressing the proteolytic downregulation of ER mannosidase I

The secretory pathway provides a physical route through which only correctly folded gene products are delivered to the eukaryotic cell surface. The efficiency of endoplasmic reticulum (ER)-associated degradation (ERAD), which orchestrates the clearance of structurally aberrant proteins under basal conditions, is boosted by the unfolded protein response (UPR) as one of several means to relieve ER stress. However, the underlying mechanism that links the two systems in higher eukaryotes has remained elusive. Herein, the results of transient expression, RNAi-mediated knockdown and functional studies demonstrate that the transcriptional elevation of EDEM1 boosts the efficiency of glycoprotein ERAD through the formation of a complex that suppresses the proteolytic downregulation of ER mannosidase I (ERManI). The results of site-directed mutagenesis indicate that this capacity does not require that EDEM1 possess inherent mannosidase activity. A model is proposed in which ERManI, by functioning as a downstream effector target of EDEM1, represents a checkpoint activation paradigm by which the mammalian UPR coordinates the boosting of ERAD.

Transformation of Moss Physcomitrella patens Gametophytes Using a Biolistic Projectile Delivery System

INTRODUCTIONThis method makes use of biolistic bombardment to propel gold particles coated with DNA into protonemal tissue of the moss Physcomitrella patens. It is especially suitable for transient gene expression studies, but it can be used to obtain stable transformants.

Gene expression studies in kiwifruit and gene over-expression in Arabidopsis indicates that GDP-L-galactose guanyltransferase is a major control point of vitamin C biosynthesis.

Vitamin C (L-ascorbic acid, AsA) is an essential metabolite for plants and animals. Kiwifruit (Actinidia spp.) are a rich dietary source of AsA for humans. To understand AsA biosynthesis in kiwifruit, AsA levels and the relative expression of genes putatively involved in AsA biosynthesis, regeneration, and transport were correlated by quantitative polymerase chain reaction in leaves and during fruit development in four kiwifruit genotypes (three species; A. eriantha, A. chinensis, and A. deliciosa). During fruit development, fruit AsA concentration peaked between 4 and 6 weeks after anthesis with A. eriantha having 3–16-fold higher AsA than other genotypes. The rise in AsA concentration typically occurred close to the peak in expression of the L-galactose pathway biosynthetic genes, particularly the GDP-L-galactose guanyltransferase gene. The high concentration of AsA found in the fruit of A. eriantha is probably due to higher expression of the GDP-mannose-3′,5′-epimerase and GDP-L-galactose guanyltransferase genes. Over-expression of the kiwifruit GDP-L-galactose guanyltransferase gene in Arabidopsis resulted in up to a 4-fold increase in AsA, while up to a 7-fold increase in AsA was observed in transient expression studies where both GDP-L-galactose guanyltransferase and GDP-mannose-3′,5′-epimerase genes were co-expressed. These studies show the importance of GDP-L-galactose guanyltransferase as a rate-limiting step to AsA, and demonstrate how AsA can be significantly increased in plants.

Identification of sequence elements regulating promoter activity and replication of a monopartite begomovirus-associated DNA satellite

DNA beta is a circular single-stranded satellite DNA associated with certain monopartite begomoviruses (family Geminiviridae) which causes economically important diseases such as cotton leaf curl disease. DNA beta contains a single gene, betaC1, which encodes a pathogenicity protein responsible for symptom production. Transient expression studies in Nicotiana tabacum using the beta-glucuronidase reporter gene driven by a betaC1 promoter-deletion series of the DNA beta associated with cotton leaf curl Multan virus identified a 68 nt region (between -139 and -207) which is important for betaC1 transcription. This 68 nt region contains a G-box (CACGTG) located 143 nt upstream of the betaC1 start codon. Mutation of the G-box resulted in a significant reduction in betaC1 promoter activity and DNA beta replication efficiency. In addition, the G-box motif was found to bind specifically to a protein(s) in nuclear extracts prepared from tobacco leaf tissues. Our results indicate that interaction of the G-box motif with host nuclear factors is important for efficient gene expression and replication of DNA beta.

Differential regulation of zebrafish metallothionein-II (zMT-II) gene transcription in ZFL and SJD cell lines by metal ions

Two alleles of a zebrafish metallothionein II gene (zMT-II) promoter (zMT-IIA and zMT-IIB) containing 10 MREs in the 5′-flanking region (1514 bp) were identified in zebrafish. These putative MREs were confirmed via electrophoretic mobility shift assay (EMSA) to have binding activities from the cellular and nuclear extracts of a zebrafish cell line, ZFL. Transient gene expression studies using zebrafish liver (ZFL) and caudal fin (SJD) cell lines also confirmed that the most distal cluster of MREs contributed to the maximal induction of zMT-IIA activity by Zn 2+ and that this Zn 2+ induction was dose-dependent. Further transient gene expression assay of the zMT-IIA gene promoter was carried out to study the effects of various metal ions (Zn 2+, Cd 2+, Cu 2+, Hg +, As 3+, As 5+, Cr 3+ and Cr 6+), and Zn 2+ and Cd 2+ were found to be the most efficient MT gene inducers of zMT-II. As 3+ was a weak inducer of zMT-II in the two cell lines, and Hg + caused significant induction only in the SJD cells. No significant induction was found in the other metal ion exposures. EMSA also identified transcription factor(s) of two different sizes from the cytoplasmic and nuclear extracts of the ZFL cells that were able to bind with the MREs, but no increase in MRE binding was detected in the extracts of these cells after Zn 2+ or Cd 2+ treatment, compared with untreated control cells. The mechanisms of MT gene transcription induction via metal ions are discussed herein.

Transient gene expression in guard cell chloroplasts of tobacco using ArF excimer laser microablation

In this paper, we report a novel method for delivering genes into chloroplasts of tobacco cells using laser microablation. The plasmid pLD200-GFP was introduced into chloroplasts of Nicotiana tabacum cv. Xanthi guard cells and transient GFP expression was detected in the chloroplasts after 2-3 d of incubation. The technique uses an argon fluoride (ArF) excimer laser to perforate the cell surface in a 4 mum(2) area in the presence of plasmid coated gold microparticles. Pretreatment of guard cells to promote stomatal closure prior to laser ablation resulted in a significant increase in the survival rate of cells and a transient expression rate of 2-3% in trial number basis was archived. Our method has unique advantages such as avoiding laborious pretreatments that adversely affect cell viability and specific delivery of transgenes into a desired cell in complex leaf tissue. This technique is a potential tool for cell specific transient gene expression studies for elucidation of gene regulation and expression.

A Proteomics Approach for Identification of Single Strand DNA-binding Proteins Involved in Transcriptional Regulation of Mouse μ Opioid Receptor Gene

The pharmacological actions of morphine and morphine-like drugs such as heroin are mediated primarily through the μ opioid receptor. Previously a single strand DNA element of the mouse μ opioid receptor gene (Oprm1) proximal promoter was found to be important for regulating Oprm1 in neuronal cells. To identify proteins binding to the single strand DNA element as potential regulators for Oprm1, affinity column chromatography with the single strand DNA element was performed using neuroblastoma NS20Y cells followed by two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry. We identified five poly(C)-binding proteins: heterogeneous nuclear ribonucleoprotein (hnRNP) K, α-complex proteins (αCP) αCP1, αCP2, αCP2-KL, and αCP3. Binding of these proteins to the single strand DNA element of Oprm1 was sequence-specific as confirmed by supershift assays. In cotransfection studies, hnRNP K, αCP1, αCP2, and αCP2-KL activated the Oprm1 promoter activity, whereas αCP3 acted as a repressor. Ectopic expression of hnRNP K, αCP1, αCP2, and αCP2-KL also led to activation of the endogenous Oprm1 transcripts, and αCP3 repressed endogenous Oprm1 transcripts. We demonstrate novel roles as transcriptional regulators in Oprm1 regulation for hnRNP K and αCP binding to the single strand DNA element.

Genetic variation of FUT2 in Ovambos, Turks, and Mongolians

Many single-nucleotide polymorphisms (SNPs) have been identified in the coding region of the FUT2 locus, which encodes secretor type alpha(1,2)fucosyltransferase. These SNPs are highly population-specific. The 1121-bp polymerase chain reaction (PCR) product containing the whole FUT2 coding region in three human populations, Ovambos (n = 74), Turks (n = 70), and Mongolians (n = 118), was sequenced. The haplotypes consisting of novel SNPs were determined by sequencing cloned inserts, and the haplotypes consisting of already reported SNPs were inferred by free computer software (PHASE). The functional significance of novel SNPs by transient expression study was also examined. Twenty-four SNPs were found including seven novel SNPs (i.e., 4G > A, 244G > A, 442C > A, 489G > A, 569G > A, 665G > A, and 950C > T). A transient expression study suggested that the 244G > A, 569G > A, and 950C > T SNPs are enzyme-inactivating mutations. This study identified 24 SNPs in the FUT2 gene, of which 7 were novel. The frequencies of alleles and genotypes were determined in Ovambos, Turks, and Mongolians. The allelic composition of each population was similar to those of geographically closer populations.

Contribution of the conservative cleavage motif to posttranslational processing of the carboxyl terminal domain of rodent Muc3

In previous works, we showed by transient expression studies in COS-1 cells that the C-terminal domain of rat intestinal membrane mucin (rMuc3) that was cloned in the pSecTag2 plasmid (named as p20) is posttranslationally cleaved twice. One location is between the glycine and the serine within a LS1KGS2IV1V2 motif, and the other is in the 49 kDa membrane-tethered fragment at an undefined site. The sea-urchin sperm protein, enterokinase and agrin module of rMuc3 is responsible for the cleavage and association of the cleaved fragments. The present study demonstrates how the conservative cleavage motif LS1KGS2IV1V2 contributes to posttranslational processing through mutagenesis of each residue in the LS(1)KGS2IV1V2 motif. Mutation of S2 to alanine (p20s2/a) completely prevented cleavage. While p20k/a (in this construct the K is replaced by A) and p20s1/a (in this construct the S1 is replaced by A) (6 and 3%) showed almost the same result as the wild-type p20 transfectant (4%), 79, 39, 22, 17, and 14% of the products from p20g/a (in this construct the G is replaced by A), p20i/a (in this construct the I is replaced by A), p20l/a (in this construct the L is replaced by A), p20v2/a (in this construct the V2 is replaced by A), and p20v1/a (in this construct the V1 is replaced by A) remained uncleaved. The cleaved N-terminal fragment of the p20s1/a transfectant was 26 kDa, but the N-terminal fragments from p20, p20g/a, p20l/a, p20k/a, p20i/a, p20v1/a, and p20v2/a were 30 kDa. The S1 residue was possibly O-glycosylated, which was supported by deglycosylation with O-cocktail (a mixture of glycosidases). The N-terminal fragment of p20s1/a transfected cells was present at high levels in the spent media. Thus, the S2, G, I, L, V2, and V1 residues within the conserved cleavage motif, LS1KGS2IV1V2, are important for cleavage and contribute to the structural formation and conformational stress of the small loop between the beta2 and the beta3 strands. The S1 residue is possibly O-glycosylated, and mutation of S1 residue to alanine does not affect the cleavage of the LS1KGS2IV1V2 motif, but it is important for the dissociation and further release of the cleaved N-terminal fragment from the cell surface.

Molecular investigations of a novel iduronate-2-sulfatase mutant in a Chinese patient

Background Molecular investigations of iduronate-2-sulfatase (IDS) mutants for the X-linked lysosomal storage disease mucopolysaccharidosis type II (MPS II, Hunter disease), commonly depends on transient expression studies to verify a single nucleotide change to be pathogenic. In 2 severely affected patients, IDS missense mutations, c.1016T>C (novel) and c.1016T>G (known) were identified predicting the substitution of an ambivalent cyclic proline and a hydrophilic arginine respectively for the hydrophobic leucine at residue 339. We hypothesized that residue Leu339 may be functionally critical. Methods We performed a study for the 2 mutations by in-situ mutagenesis, in vitro expression, and functional analysis. Results Transient expression revealed that both the missense variants had stable mRNA but their residual enzyme activities remained < 2.5% of normal level. The effect of the missense mutations on protein expression was detected by Western blot analysis. Both the missense mutations synthesized the precursor form but had reduced mature form of IDS. Conclusion The novel mutation p.L339P is a disease-causing mutation affecting maturation of the protein.

Cis-Acting Gene Regulatory Activities in the Terminal Regions ofSleeping BeautyDNA Transposon-Based Vectors

Sleeping Beauty (SB) DNA transposon-based vectors belong to a growing family of nonviral integrating vectors that represent attractive alternatives to conventional virus-based integrating gene vehicles. Because of concerns related to mutagenesis and/or activation of cellular genes by integrating vectors, much attention has been paid to integration site preferences and the ability of vectors to influence expression of neighboring genes. Here, we test the hypothesis that terminal repeats of transposons carry cis-acting regulatory sequences. In transient gene expression studies, we demonstrate that the inverted repeats of SB direct gene expression in HeLa cells to levels that are 3-fold higher than in promoter-deficient controls. Inverted repeats pointing toward the transposon center consistently facilitate the highest levels of activity in a number of cell lines. We show that transposon sequences flanking the inverted repeats of SB are required for positive effects on gene expression and, moreover, that these regions contain both stimulatory and inhibitory cis-acting elements. In the context of an integrated SB vector the regulatory activities of the transposon termini are sufficient to drive expression of selectable marker genes carried by the transposon, indicating that opposing transcriptional activities originating from the transposon termini may influence expression of its genetic cargo. Finally, detection of regulatory properties of the terminal repeats of the active Tc3 element from Caenorhabditis elegans leads to the suggestion that transcriptional activities of the inverted repeats are conserved among Tc1/mariner transposons in nature. Our data suggest that SB-based gene vectors may carry ancient properties of self-regulation with potential relevance for SB-directed therapeutic gene transfer.

Structural Insight into the Interaction between Platelet Integrin αIIbβ3 and Cytoskeletal Protein Skelemin

Skelemin is a large cytoskeletal protein critical for cell morphology. Previous studies have suggested that its two-tandem immunoglobulin C2-like repeats (SkIgC4 and SkIgC5) are involved in binding to integrin beta3 cytoplasmic tail (CT), providing a mechanism for skelemin to regulate integrin-mediated signaling and cell spreading. Using NMR spectroscopy, we have studied the molecular details of the skelemin IgC45 interaction with the cytoplasmic face of integrin alphaIIbbeta3. Here, we show that skelemin IgC45 domains form a complex not only with integrin beta3 CT but also, surprisingly, with the integrin alphaIIb CT. Chemical shift mapping experiments demonstrate that both membrane-proximal regions of alphaIIb and beta3 CTs are involved in binding to skelemin. NMR structural determinations, combined with homology modeling, revealed that SkIgC4 and SkIgC5 both exhibited a conserved Ig-fold and both repeats were required for effective binding to and attenuation of alphaIIbbeta3 cytoplasmic complex. These data provide the first molecular insight into how skelemin may interact with integrins and regulate integrin-mediated signaling and cell spreading.

Cloning and characterization of a tilapia ( Oreochromis aureus) metallothionein gene promoter in Hepa-T1 cells following the administration of various heavy metal ions

Metallothioneins (MTs) are highly conserved intracellular metal-binding proteins that contribute to the homeostasis of essential metals and the detoxification of non-essential heavy metals. MT gene expression is induced by various heavy metal ions, and Zn 2+ is able to bind and activate a transcription factor associated with the MT gene that is known as the metal responsive element (MRE) binding transcription factor-1 (MTF-1). Heavy metals other than Zn 2+, such as Cd 2+ and Cu 2+, fail to activate the binding of MTF-1 to MREs despite their ability to induce the transcription of the MT gene. To study how different metal ions regulate MT gene expression, a tilapia (ti)-MT gene promoter was cloned and its responses to activation by various metal ions measured using a Hepa T1 cell culture model. The tiMT gene promoter contains six functional MREs within 2118 bp 5′ of the translational start site. A transient gene expression study showed the tiMT gene promoter fragment to be responsive to Cd 2+, Cu 2+, Hg 2+, Pb 2+, and Zn 2+. Deletions from the 5′ end and the site-directed mutagenesis of individual MREs in the tiMT gene promoter confirmed that both proximal and distal clusters of MREs were required for the maximal metal induction of the tiMT gene. The distal cluster of MREs greatly enhanced the induction of tiMT gene expression by several of the heavy metal ions, and especially the non-Zn 2+ ions. Individual MREs showed a different responsiveness to metal ions, with MREe being the most potent, MREb being responsive to Zn 2+ but not to other metal ions, and MREa being mainly for the basal expression of the tiMT gene. Electrophoretic mobility shift assay (EMSA) identified a transcription factor that was able to bind most of the MREs, with the exception of MREd, but the binding was only activated by the in vivo administration of Zn 2+, not the administration of Cd 2+ or Cu 2+. In conclusion, the results of this study on a Hepa T1 cell model suggest that the mechanism of MT gene activation by non-Zn 2+ metal ions is different from that of activation by Zn 2+, and that different MREs may be involved in the activation of the tiMT gene by different metal ions without enhancing the binding of MTF-1 to MREs.

PSITE Vectors for Stable Integration or Transient Expression of Autofluorescent Protein Fusions in Plants: Probing Nicotiana benthamiana-Virus Interactions

Plant functional proteomics research is increasingly dependent upon vectors that facilitate high-throughput gene cloning and expression of fusions to autofluorescent proteins. Here, we describe the pSITE family of plasmids, a new set of Agrobacterium binary vectors, suitable for the stable integration or transient expression of various autofluorescent protein fusions in plant cells. The pSITE vectors permit single-step Gateway-mediated recombination cloning for construction of binary vectors that can be used directly in transient expression studies or for the selection of transgenic plants on media containing kanamycin. These vectors can be used to express native proteins or fusions to monmeric red fluorescent protein or the enhanced green fluorescent protein and its cyan and yellow-shifted spectral variants. We have validated the vectors for use in transient expression assays and for the generation of transgenic plants. Additionally, we have generated markers for fluorescent highlighting of actin filaments, chromatin, endoplasmic reticulum, and nucleoli. Finally, we show that pSITE vectors can be used for targeted gene expression in virus-infected cells, which should facilitate high-throughput characterization of protein dynamics in host-virus interactions.

Rubisco gene expression in C4 plants

In leaves of most C(4) plants, ribulose 1,5 bisphosphate carboxylase (Rubisco) accumulates only in bundle sheath (bs) cells that surround the vascular centres, and not in mesophyll (mp) cells. It has been shown previously that in the C(4) dicots amaranth and Flaveria bidentis, post-transcriptional control of mRNA translation and stability mediate the C(4) expression patterns of genes encoding the large and small Rubisco subunits (chloroplast rbcL and nuclear RbcS, respectively). Translational control appears to regulate bs cell-specific Rubisco gene expression during early dicot leaf development, while control of mRNA stability appears to mediate bs-specific accumulation of RbcS and rbcL transcripts in mature leaves. Post-transcriptional control is also involved in the regulation of Rubisco gene expression by light, and in response to photosynthetic activity. Transgenic and transient expression studies in F. bidentis provide direct evidence for post-transcriptional control of bs cell-specific RbcS expression, which is mediated by the 5' and 3' untranslated regions (UTRs) of the mRNA. Comparisons of Rubisco gene expression in these dicots and in the monocot maize indicates possible commonalities in the regulation of RbcS and rbcL genes in these divergent C(4) species. Now that the role of post-transcriptional regulation in C(4) gene expression has been established, it is likely that future studies of mRNA-protein interactions will address long-standing questions about the establishment and maintenance of cell type-specificity in these plants. Some of these regulatory mechanisms may have ancestral origins in C(3) species, through modification of pre-existing factors, or by the acquisition of novel C(4) processes.