Perturbations of the ZED1 pseudokinase activate plant immunity.

The Pseudomonas syringae acetyltransferase HopZ1a is delivered into host cells by the type III secretion system to promote bacterial growth. However, in the model plant host Arabidopsis thaliana, HopZ1a activity results in an effector-triggered immune response (ETI) that limits bacterial proliferation. HopZ1a-triggered immunity requires the nucleotide-binding, leucine-rich repeat domain (NLR) protein, ZAR1, and the pseudokinase, ZED1. Here we demonstrate that HopZ1a can acetylate members of a family of 'receptor-like cytoplasmic kinases' (RLCK family VII; also known as PBS1-like kinases, or PBLs) and promote their interaction with ZED1 and ZAR1 to form a ZAR1-ZED1-PBL ternary complex. Interactions between ZED1 and PBL kinases are determined by the pseudokinase features of ZED1, and mutants designed to restore ZED1 kinase motifs can (1) bind to PBLs, (2) recruit ZAR1, and (3) trigger ZAR1-dependent immunity in planta, all independently of HopZ1a. A ZED1 mutant that mimics acetylation by HopZ1a also triggers immunity in planta, providing evidence that effector-induced perturbations of ZED1 also activate ZAR1. Overall, our results suggest that interactions between these two RLCK families are promoted by perturbations of structural features that distinguish active from inactive kinase domain conformations. We propose that effector-induced interactions between ZED1/ZRK pseudokinases (RLCK family XII) and PBL kinases (RLCK family VII) provide a sensitive mechanism for detecting perturbations of either kinase family to activate ZAR1-mediated ETI.

Arabidopsis glycosylphosphatidylinositol-anchored protein LLG1 associates with and modulates FLS2 to regulate innate immunity.

Plants detect and respond to pathogen invasion with membrane-localized pattern recognition receptors (PRRs), which recognize pathogen-associated molecular patterns (PAMPs) and activate downstream immune responses. Here we report that Arabidopsis thaliana LORELEI-LIKE GPI-ANCHORED PROTEIN 1 (LLG1), a coreceptor of the receptor-like kinase FERONIA, regulates PRR signaling. In a forward genetic screen for suppressors of enhanced disease resistance 1 (edr1), we identified the point mutation llg1-3, which suppresses edr1 disease resistance but does not affect plant growth and development. The llg1 mutants show enhanced susceptibility to various virulent pathogens, indicating that LLG1 has an important role in plant immunity. LLG1 constitutively associates with the PAMP receptor FLAGELLIN SENSING 2 (FLS2) and the elongation factor-Tu receptor, and forms a complex with BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED RECEPTOR KINASE 1 in a ligand-dependent manner, indicating that LLG1 functions as a key component of PAMP-recognition immune complexes. Moreover, LLG1 contributes to accumulation and ligand-induced degradation of FLS2, and is required for downstream innate immunity responses, including ligand-induced phosphorylation of BOTRYTIS-INDUCED KINASE 1 and production of reactive oxygen species. Taken together, our findings reveal that LLG1 associates with PAMP receptors and modulates their function to regulate disease responses. As LLG1 functions as a coreceptor of FERONIA and plays central roles in plant growth and development, our findings indicate that LLG1 participates in separate pathways, and may suggest a potential connection between development and innate immunity in plants.

Patterns of constitutively phosphorylated kinases in B cells are associated with disease severity in common variable immunodeficiency.

Patients with common variable immunodeficiency (CVID) constitute a clinically and immunologically heterogeneous group characterized by B-cell dysfunction with hypogammaglobulinemia and defective immunoglobulin class switch of unknown etiology. Current classification systems are insufficient to achieve precise disease management. Characterization of signaling pathways essential for B-cell differentiation and class switch could provide new means to stratify patients. We evaluated constitutive and induced signaling by phospho-specific flow cytometry in 26 CVID patients and 18 healthy blood donors. Strong responses were induced both in CVID and healthy donor B cells upon activation. In contrast, constitutive phosphorylation levels of STAT3,-5,-6, Erk, PLC-γ and Syk were significantly increased in CVID B cells only. Hierarchical clustering revealed a subgroup of CVID patients with elevated constitutive phosphorylation of Syk and PLC-γ. All these patients had non-infectious complications, indicating that a distinct phosphorylation pattern of kinases in B cells identifies a clinically important subgroup of CVID patients.

Reduced muscle mitochondrial enzyme activity in MuSK-immunized mice.

AIM: Muscle specific kinase (MuSK) antibody-positive myasthenia gravis(MG) patients might present with clinical and electrophysiological signs of muscle atrophy. In this study, we investigated the potential contribution of mitochondrial dysfunction to muscle atrophy induced by MuSK immunity. METHODS: Mitochondrial enzyme expression was investigated in muscle samples of MuSK-immunized, acetylcholine receptor (AChR)-immunized, and complete Freund's adjuvant (CFA)-immunized C57BL/6 (B6) mice using histochemical methods. Mitochondrial enzyme activity was also investigated in MuSK- and CFA-immunized mice. RESULTS: Histochemical analysis showed normal muscle fiber activity on succinate dehydrogenase (SDH) and cytochrome oxidase (COX) stains in all immunization groups. However, MuSK-immunized mice had more ragged-red fibers on modified Gomori-trichrome (MGT) stain and more pronounced type 1 muscle fiber atrophy. MuSK-immunized mice also showed reduced citrate synthase, SDH, and NADH-cytochrome c-reductase activity. DISCUSSION: Our results suggest that MuSK-immunity might induce muscle atrophy through mitochondrial dysfunction.

Involvement of the early growth response protein 1 in vascular pathophysiology: an overview.

Hyperactivation of proliferative and growth promoting pathways underlies the progression of vessel remodeling, leading to vascular dysfunction. An upregulation of early growth response protein 1 (Egr-1), a zinc finger transcription factor has been observed in several models of vascular diseases. In the vasculature, Egr-1 expression can be induced by multiple hormonal, metabolic and external stimuli, such as growth factors, cytokines, reactive oxygen species, hyperglycaemia and stretch-induced stress. The structure of the Egr-1 promoter allows both its auto-regulation and its binding with several regulatory transcription cofactors like the serum response factor and the cAMP response element binding protein. Pharmacological and genetic studies have revealed the involvement of several signaling pathways that contribute to the expression of Egr-1. Among them, the mitogen-activated protein kinase pathway has emerged as a predominant signaling cascade that regulates Egr-1 transcription in response to various stimuli. Moreover, targeted deletion of Egr-1 by DNAzymes, antisense oligonucleotides or RNA interference has also helped in defining the importance of Egr-1 in the pathophysiology of vascular diseases. Neointimal formation and expression of genes directly linked with proinflammatory processes have been demonstrated to be enhanced by Egr-1 expression and activity. This review provides an overview on the signaling components implicated in Egr-1 expression and discusses its potential involvement in vascular pathophysiology.

Mycobacterium tuberculosis requires phosphate-responsive gene regulation to resist host immunity.

Mycobacterium tuberculosis persists in the tissues of mammalian hosts despite inducing a robust immune response dominated by the macrophage-activating cytokine gamma interferon (IFN-γ). We identified the M. tuberculosis phosphate-specific transport (Pst) system component PstA1 as a factor required to resist IFN-γ-dependent immunity. A ΔpstA1 mutant was fully virulent in IFN-γ(-/-) mice but attenuated in wild-type (WT) mice and mice lacking specific IFN-γ-inducible immune mechanisms: nitric oxide synthase (NOS2), phagosome-associated p47 GTPase (Irgm1), or phagocyte oxidase (phox). These phenotypes suggest that ΔpstA1 bacteria are sensitized to an IFN-γ-dependent immune mechanism(s) other than NOS2, Irgm1, or phox. In other species, the Pst system has a secondary role as a negative regulator of phosphate starvation-responsive gene expression through an interaction with a two-component signal transduction system. In M. tuberculosis, we found that ΔpstA1 bacteria exhibited dysregulated gene expression during growth in phosphate-rich medium that was mediated by the two-component sensor kinase/response regulator system SenX3-RegX3. Remarkably, deletion of the regX3 gene suppressed the replication and virulence defects of ΔpstA1 bacteria in NOS2(-/-) mice, suggesting that M. tuberculosis requires the Pst system to negatively regulate activity of RegX3 in response to available phosphate in vivo. We therefore speculate that inorganic phosphate is readily available during replication in the lung and is an important signal controlling M. tuberculosis gene expression via the Pst-SenX3-RegX3 signal transduction system. Inability to sense this environmental signal, due to Pst deficiency, results in dysregulation of gene expression and sensitization of the bacteria to the host immune response.

RNAi screen for kinases and phosphatases that play a role in antigen presentation by dendritic cells.

Effective CD8(+) T-cell responses against tumor or microbial antigens that are not directly expressed in antigen-presenting cells (APCs) depend on the cross-presentation of these antigens on MHC class I in APCs. To identify signaling molecules that regulate cross-presentation, we used lentiviral-based RNA interference to test the roles of hundreds of kinases and phosphatases in this process. Our study uncovered eight previously unknown genes, consisting of one positive and seven negative regulators of antigen cross-presentation. Depletion of Acvr1c, a type I receptor for TGF-ß family of signaling molecules, led to an increase in CD80 and CD86 co-stimulator surface expression and secreted IL-12 in mouse bone marrow-derived DCs, as well as antigen-specific T-cell proliferation.

The human RNA kinase hClp1 is active on 3' transfer RNA exons and short interfering RNAs.

RNA interference allows the analysis of gene function by introducing synthetic, short interfering RNAs (siRNAs) into cells. In contrast to siRNA and microRNA duplexes generated endogenously by the RNaseIII endonuclease Dicer, synthetic siRNAs display a 5' OH group. However, to become incorporated into the RNA-induced silencing complex (RISC) and mediate target RNA cleavage, the guide strand of an siRNA needs to display a phosphate group at the 5' end. The identity of the responsible kinase has so far remained elusive. Monitoring siRNA phosphorylation, we applied a chromatographic approach that resulted in the identification of the protein hClp1 (human Clp1), a known component of both transfer RNA splicing and messenger RNA 3'-end formation machineries. Here we report that the kinase hClp1 phosphorylates and licenses synthetic siRNAs to become assembled into RISC for subsequent target RNA cleavage. More importantly, we reveal the physiological role of hClp1 as the RNA kinase that phosphorylates the 5' end of the 3' exon during human tRNA splicing, allowing the subsequent ligation of both exon halves by an unknown tRNA ligase. The investigation of this novel enzymatic activity of hClp1 in the context of mRNA 3'-end formation, where no RNA phosphorylation event has hitherto been predicted, remains a challenge for the future.

Regulation of secretory pathway kinase or kinase-like proteins in human cancers.

Secretory pathway kinase or kinase-like proteins (SPKKPs) are effective in the lumen of the endoplasmic reticulum (ER), Golgi apparatus (GA), and extracellular space. These proteins are involved in secretory signaling pathways and are distinctive from typical protein kinases. Various reports have shown that SPKKPs regulate the tumorigenesis and progression of human cancer via the phosphorylation of various substrates, which is essential in physiological and pathological processes. Emerging evidence has revealed that the expression of SPKKPs in human cancers is regulated by multiple factors. This review summarizes the current understanding of the contribution of SPKKPs in tumorigenesis and the progression of immunity. With the epidemic trend of immunotherapy, targeting SPKKPs may be a novel approach to anticancer therapy. This study briefly discusses the recent advances regarding SPKKPs.

Opposing forces in asthma: regulation of signaling pathways by kinases and phosphatases.

Allergic asthma is a chronic inflammatory disease that involves a sustained T-helper-2 (Th2) type immune response and the recruitment of inflammatory cells such as lymphocytes, mast cells, and eosinophils to the lung. The complex cellular interactions involved in this disease are dependent on cellular responses generated following the integration of environmental signals through the cell signaling pathways. Therefore, the elucidation of the biochemical cascades involved in cellular responses contributing to asthma development has been of great interest in this field. Much attention has been given to the activities of kinases, enzymes that catalyze the addition of a phosphate group to a protein, thus allowing them to modify the function of the target enzyme. This resulted in the identification of key kinases and various cellular processes involved in disease development. New investigations have also begun to unravel the importance of phosphatases, which catalyze the removal of a phosphate group from their target protein. Together, these studies reveal a signaling picture in allergic asthma that is far more complex than originally thought. Herein, we review critical mechanisms of asthma development and discuss how kinases and phosphatases are likely to regulate the development of disease through their effect on these various mechanisms.

RALF-FERONIA Signaling: Linking Plant Immune Response with Cell Growth.

Plants perceive various external and internal signals to self-modulate biological processes through members of the receptor-like kinase (RLK) family, among which Catharanthus roseus receptor-like kinase 1-like (CrRLK1L) proteins with their ligands, rapid alkalinization factor (RALF) peptides, have attracted considerable interest. FERONIA (FER), a CrRLK1L member, was initially reported to act as a major plant cell growth modulator in distinct tissues. Subsequently, the RALF-FER pathway was confirmed to function as an essential regulator of plant stress responses, including but not limited to immune responses. Furthermore, the RALF-FER pathway modulates immune responses and cell growth in a context-specific manner, and the vital roles of this pathway are beginning to be appreciated in crop species. The recent remarkable advances in understanding the functions and molecular mechanisms of the RALF-FER pathway have also raised many interesting questions that need to be answered in the future. This review mainly focuses on the roles of FER and other CrRLK1L members in modulating immune responses in the context of cell growth in response to their RALF peptide ligands and presents a brief outlook for future research.

ptsI gene in the phosphotransfer system is a potential target for developing a live attenuated Salmonella vaccine.

Salmonella enterica serovar Typhimurium causes invasive non­typhoidal Salmonella diseases in animals and humans, resulting in a high mortality rate and huge economic losses globally. As the prevalence of antibiotic­resistant Salmonella has been increasing, vaccination is thought to be the most effective and economical strategy to manage salmonellosis. The present study aimed to investigate whether dysfunction in the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS), which is critical for carbon uptake and survival in macrophages, may be adequate to generate Salmonella­attenuated vaccine strains. A Salmonella strain (KST0555) was generated by deleting the ptsI gene from the PTS and it was revealed that this auxotrophic mutant was unable to efficiently utilize predominant carbon sources during infection (glucose and glycerol), reduced its invasion and replication capacity in macrophages, and significantly (P=0.0065) lowered its virulence in the setting of a mouse colitis model, along with a substantially decreased intestinal colonization and invasiveness compared with its parent strain. The reverse transcription­quantitative PCR results demonstrated that the virulence genes in Salmonella pathogenicity island-1 (SPI-1) and -2 (SPI-2) and the motility of KST0555 were all downregulated compared with its parent strain. Finally, it was revealed that when mice were immunized orally with live KST0555, Salmonella­specific humoral and cellular immune responses were effectively elicited, providing protection against Salmonella infection. Thus, the present promising data provides a strong rationale for the advancement of KST0555 as a live Salmonella vaccine candidate and ptsI as a potential target for developing a live attenuated bacterial vaccine strain.

Identification and verification immune-related regulatory network in acne.

Acne is a common inflammatory skin disease with the dysregulation of innate and adaptive immunity. However, the underlying mechanism of acne has not been completely elucidated. In this study, we identified gene signatures and the immune-related regulatory network in acne using integrated bioinformatics methods. Here, 303 Differentially expressed genes (DEGs) and 28 Hub genes were identified in acne (GSE53795 and GSE108110), which were associated with the inflammation-related signaling pathway. Subsequently, the CIBERSORT algorithm revealed the increased proinflammatory cells in acne. Moreover, we identified 3 kinases (FGR, HCK and LYN) and 2 transcription factors (TFs) (IRF8 and ZBTB16) from DEGs as the key genes, which regulated immune cell infiltration via targeting immune-related genes in acne. The upregulated 3 kinases (FGR, HCK and LYN) and IRF8, and the downregulated ZBTB16 were also confirmed in GSE6475 and in Acne mice. Based on the expression levels of these key genes, the tissues could be divided into 2 clusters using consensus cluster analysis. GSEA analysis showed that inflammation-related signaling pathways significantly enriched in cluster 2, indicating the important role of kinase and TFs on immune regulation in acne. Finally, we found that isotretinoin and trifarotene (CD5789) treatment repressed the expression of immune genes but not the expression of the kinases and TFs, indicating that kinases and TFs may be novel therapeutic target for acne. In conclusion, 3 kinases and 2 TFs were identified and validated as key regulators in the immune-related regulatory networks in acne, providing a more comprehensive understanding and novel therapeutic targets of acne.

Antibodies to the retina N-acetylgalactosaminylphosphotransferase modulate N-cadherin-mediated adhesion and uncouple the N-cadherin transferase complex from the actin-containing cytoskeleton.

Embryonic chick neural retina cells have at their surface an N-Acetylgalactosaminylphosphotransferase (GalNAcPTase) which is associated with, and glycosylates, the calcium-dependent cell-cell adhesion molecule, N-cadherin (Balsamo, J., and J. Lilien. 1990. J. Biol. Chem. 265:2923-2928). In this manuscript, we demonstrate that antibodies directed against the GalNAcPTase, as well as anti-N-cadherin antibodies, are able to inhibit adhesion of chick neural retina cells to a cell monolayer, to immobilized N-cadherin, or to immobilized anti-N-cadherin antibody. These results indicate that anti-GalNAcPTase antibodies modulate the function of N-cadherin, interfering with the formation of N-cadherin-mediated adhesions. We also demonstrate that actin is associated with the N-cadherin/GalNAcPTase complex and that binding of anti-GalNAcPTase antibodies to intact cells results in dissociation of actin from the complex. We suggest that the GalNAcPTase modulates N-cadherin function by altering its interaction with the cytoskeleton.

Immunolocalization of myosin I heavy chain kinase in Acanthamoeba castellanii and binding of purified kinase to isolated plasma membranes.

The actin-activated Mg(2+)-ATPase activities of Acanthamoeba myosins I are known to be maximally expressed only when a single threonine (myosin IA) or serine (myosins IB and IC) is phosphorylated by myosin I heavy chain kinase. The purified kinase is highly activated by autophosphorylation and the rate of autophosphorylation is greatly enhanced by the presence of acidic phospholipids. In this paper, we show by immunofluorescence and immunoelectron microscopy of permeabilized cells that myosin I heavy chain kinase is highly concentrated, but not exclusively, at the plasma membrane. Judged by their electrophoretic mobilities, kinase associated with purified plasma membranes may differ from the cytoplasmic kinase, possibly in the extent of its phosphorylation. Purified kinase binds to highly purified plasma membranes with an apparent KD of approximately 17 nM and a capacity of approximately 0.8 nmol/mg of plasma membrane protein, values that are similar to the affinity and capacity of plasma membranes for myosins I. Binding of kinase to membranes is inhibited by elevated ionic strength and by extensive autophosphorylation but not by substrate-level concentrations of ATP. Membrane-bound kinase autophosphorylates to a lesser extent than free kinase and does not dissociate from the membranes after autophosphorylation. The co-localization of myosin I heavy chain kinase and myosin I at the plasma membrane is of interest in relation to the possible functions of myosin I especially as phospholipids increase kinase activity.

Two Arabidopsis Receptor-like Cytoplasmic Kinases SZE1 and SZE2 Associate with the ZAR1-ZED1 Complex and Are Required for Effector-Triggered Immunity.

Plants utilize intracellular nucleotide-binding leucine-rich repeat domain-containing receptors (NLRs) to recognize pathogen effectors and induce a robust defense response named effector-triggered immunity (ETI). The Arabidopsis NLR protein HOPZ-ACTIVATED RESISTANCE 1 (ZAR1) forms a precomplex with HOPZ-ETI-DEFICIENT 1 (ZED1), a receptor-like cytoplasmic kinase (RLCK) XII-2 subfamily member, to recognize the Pseudomonas syringae effector HopZ1a. We previously described a dominant mutant of Arabidopsis ZED1, zed1-D, which displays temperature-sensitive autoimmunity in a ZAR1-dependent manner. Here, we report that the RLCKs SUPPRESSOR OF ZED1-D1 (SZE1) and SZE2 associate with the ZAR1-ZED1 complex and are required for the ZED1-D-activated autoimmune response and HopZ1a-triggered immunity. We show that SZE1 but not SZE2 has autophosphorylation activity, and that the N-terminal myristoylation of both SZE1 and SZE2 is critical for their plasma membrane localization and ZED1-D-activated autoimmunity. Furthermore, we demonstrate that SZE1 and SZE2 both interact with ZAR1 to form a functional complex and are required for resistance against P. syringae pv. tomato DC3000 expressing HopZ1a. We also provide evidence that SZE1 and SZE2 interact with HopZ1a and function together with ZED1 to change the intramolecular interactions of ZAR1, leading to its activation. Taken together, our results reveal SZE1 and SZE2 as critical signaling components of HopZ1a-triggered immunity.

Application of Monoclonal Antibodies to Detect and Compare the Levels of Streptococcus mutans in Adolescents Undergoing Orthodontic Treatment with Those Not Undergoing Treatment.

The purpose of this study was to detect Streptococcus mutans by using monoclonal antibodies (mAbs) against S. mutans that cause dental caries and compare the levels of the bacterium between the saliva of adolescents undergoing orthodontic treatment (OT) and those not undergoing treatment (NT). Saliva samples, collected from 25 OT adolescents (with a mean age of 12.84 years) and 25 NT adolescents (mean age of 12.4 years), were analyzed by Dentocult-SM and enzyme-linked immunosorbent assay using mAbs against Ag I/II (ckAg I/II) and GTF B (ckGTF B), GTF C (ckGTF C), and GTF D (ckGTF D) of S. mutans. The DMFT index was slightly higher in the OT group (5.12 in OT and 4.96 in NT) and the level of S. mutans (≥105 CFU/mL) was higher in OT (72%) than in NT (56%). The detected levels of ckAg I/II, ckGTF B, ckGTF C, and ckGTF D were slightly higher in OT than in NT. The results of this study indicate that use of mAbs against S. mutans yields sensitive detection for the bacterium in saliva samples and shows that it has a reliable connection to the number of S. mutans and decayed, missing, filled teeth (DMFT), suggesting that the levels of S. mutans in saliva can be defined and compared by the application of the mAbs.

Immunological properties of rat phosphoglycerate mutase isozymes.

In mammalian tissues three phosphoglycerate mutase (D-phosphoglycerate 2,3-phosphomutase, EC 5.4.2.1) isozymes result from the homo-dimeric and hetero-dimeric combinations of two subunits (types M and B). Whereas rabbit antisera against type M subunit (purified from rat muscle) and against type BB isozyme (purified from rat brain) possessed a high degree of specificity, both antisera reacted with type BB and MM isozymes, as demonstrated by immunoneutralization and ELISA. Both the M subunit and B subunit were more immunoreactive than their respective dimeric isozymes. Subunits type M and B may possess common antigenic determinants, and some of these determinants may be sterically hindered in their dimeric structures.

Antigenic structure of adenylate kinase from porcine skeletal muscle--II. Immunochemical cross-reactivity of fragments from adenylate kinase.

Specific antibody to a fragment [CBb(2-56)] of porcine muscle adenylate kinase was purified from goat antiserum against adenylate kinase on an immunoadsorbent column. This anti-CBb antibody cross-reacted in solid phase radioimmunoassay with two other CNBr-fragments of adenylate kinase, CBfN(81-125) and CBfC(126-194). This cross-reactivity explained their high inhibition activities in quantitative precipitin reaction between adenylate kinase and goat antiserum shown in the previous work. Cysteinyl residues of the enzyme (positions 25 and 187) were S-cyanylated with 2-nitro-5-thiocyanobenzoic acid and the enzyme was cleaved at these residues. Fragment 1-24 thus obtained was purified. The fragment 1-24, composing the N-terminal half of CBb(2-56), accounted for full activity of CBb to anti-CBb in radioimmunoassay. Hence antigenic region(s) of CBb(2-56) exist in its N-terminal half, 2-24, and this determinant(s) may be closely related to the cross-reactivity among CB-fragments. CBfN also bound to the antibody fraction which had not been adsorbed to CBb-Sepharose (non-anti-CBb). CBfN carried additional antigenic regions. In conclusion, we propose that the antigenic reactive region(s) of adenylate kinase responsible for the cross-reactivity of the CB-fragments are as follows: -Glu-Glu-Lys-Leu-Lys-Lys- (2-7), -Glu-Glu-Phe-Lys-Arg-Lys- (103-108), -Glu-Glu-Thr-Ile-Lys-Lys- (143-148).

A bifunctional fusion between beta-glucuronidase and neomycin phosphotransferase: a broad-spectrum marker enzyme for plants.

We have used an in vivo selection approach to isolate a gene encoding a bifunctional fusion peptide between Escherichia coli beta-glucuronidase (GUS) and neomycin phosphotransferase II (NPT-II) from transposon Tn5 in the NH2-GUS::NPT-II-COOH configuration. The fused gene is predicted to encode a fusion peptide 885 amino acids long, and was shown in E. coli to synthesize a 97-kDa GUS+ NPT-II+ gene product. Gel-filtration chromatography suggested that, while the native GUS may be active as a dimer and NPT-II as a monomer, the elution profile of the fusion protein is consistent with that of a trimer. The fusion marker has been produced and defined in transgenic Nicotiana tabacum plants, where both the chimeric gene and the gene product were stable. The bifunctional gene enabled direct KmR selection at the callus stage and enzymatic or histochemical assessment of the steady-state production of GUS activity in regenerated plants. In addition to allowing structure-function determination for the GUS and NPT-II domains of the fusion peptide, the gus::npt-II gene simplifies vector constructs where both marker domains are desired.