Tandem Construct Research Articles

GYGD pore motifs in neighbouring potassium channel subunits interact to determine ion selectivity.

Cells maintain a negative resting membrane potential through the constitutive activity of background K+ channels. A novel multigene family of such K+ channels has recently been identified. A unique characteristic of these K+ channels is the presence of two homologous, subunit-like domains, each containing a pore-forming region. Sequence co-variations in the GYGD signature motifs of the two pore regions suggested an interaction between neighbouring pore domains. Mutations of the GYGD motif in the rat drk1 (Kv2.1) K+ channel showed that the tyrosine (Y) position was important for K+ selectivity and single channel conductance, whereas the aspartate (D) position was a critical determinant of open state stability. Tandem constructs engineered to mimic the GYGx-GxGD pattern seen in two-domain K+ channels delineated a co-operative intersubunit interaction between the Y and D positions, which determined ion selectivity, conductance and gating. In the bacterial KcsA K+ channel crystal structure, the equivalent aspartate residue (D80) does not directly interact with permeating K+ ions. However, the data presented here show that the D position is able to fine-tune ion selectivity through a functional interaction with the Y position in the neighbouring subunit. These data indicate a physiological basis for the extensive sequence variation seen in the GYGD motifs of two-domain K+ channels. It is suggested that a cell can precisely regulate its resting membrane potential by selectively expressing a complement of two-domain K+ channels.

Two critical cysteine residues implicated in disulfide bond formation and proper folding of Kir2.1.

Inwardly rectifying potassium channels are important in cellular repolarization of many excitable tissues. Amino acid sequence alignment of different mammalian inward rectifier K(+) channels revealed two absolutely conserved cysteine residues in the putative extracellular face, suggesting a possible disulfide bond. Replacement of these cysteine residues in the Kir2.1 channel (i.e., C122 and C154) with either alanine or serine abolished current in Xenopus laevis oocytes although Western blotting established that the channels were fully expressed. The digestion pattern of channels treated with V8 protease combined with Western blotting under reducing and nonreducing conditions confirmed intrasubunit cross-linking of C122 and C154. Whole-cell and single channel current recordings of oocytes expressing tandem tetrameric constructs with one or two of the mutant subunits suggested that insertion of one mutant subunit is sufficient to eliminate channel function. Coexpression studies confirmed that the cysteine mutant channels eliminate wild-type Kir2.1 currents in a dominant-negative manner. Despite these results, sulfhydryl reduction did not alter the functional properties of Kir2.1 currents. Molecular modeling of Kir2.1 with the two cysteines cross-linked predicted that the extracellular loop between the first transmembrane domain and the pore helix contains a beta-hairpin structure. Distinct from the KcsA structure, the disulfide bond together with the beta-hairpin structure is expected to constrain and stabilize the P-loop and selectivity filter. Taken together, these results suggest that intramolecular disulfide bond exists between C122 and C154 of Kir2.1 channel and this cross-link might be required for proper channel folding.

Four color compensation

Four-color immunophenotyping can now be routinely performed using either a single laser or dual laser flow cytometer. When a single laser instrument is used, the fluorochromes evaluated are usually FITC, PE, PE-TR and PE-CY5 (or PerCP). For two-laser excitation APC is generally used in place of PE-TR. Since each tandem dye construct contains PE, three of the four detectors are affected and compensation can be problematic. In this report we show that each tandem conjugated antibody, whether different batches from the same supplier or conjugates from different suppliers all require unique compensation. This inconsistency results in erroneous data, negates the use of single labeled particles as a method for providing adequate compensation and requires dual and triple labeled cells of known pattern to verify compensation. It is also shown that improper compensation can reduce or eliminate completely the detection of fluorescence emission from PECY5 conjugated antibodies. These problems are caused by a variation in energy transfer between PE and either TR or CY5 because the chemistry involved in preparation and conjugation to antibodies is not sufficiently controlled to produce reagents with uniform compensation requirements. The variation in tandem dye compensation can be addressed by either using the same tandem conjugated antibody, by using the same second step tandem reagent to an appropriate first step antibody or by using software compensation. The latter provides an easy solution because a unique compensation matrix can be produced for each antibody tandem conjugate. Cytometry (Comm. Clin. Cytometry) 38:161–175, 1999. © 1999 Wiley-Liss, Inc.

Four color compensation.

Four-color immunophenotyping can now be routinely performed using either a single laser or dual laser flow cytometer. When a single laser instrument is used, the fluorochromes evaluated are usually FITC, PE, PE-TR and PE-CY5 (or PerCP). For two-laser excitation APC is generally used in place of PE-TR. Since each tandem dye construct contains PE, three of the four detectors are affected and compensation can be problematic. In this report we show that each tandem conjugated antibody, whether different batches from the same supplier or conjugates from different suppliers all require unique compensation. This inconsistency results in erroneous data, negates the use of single labeled particles as a method for providing adequate compensation and requires dual and triple labeled cells of known pattern to verify compensation. It is also shown that improper compensation can reduce or eliminate completely the detection of fluorescence emission from PECY5 conjugated antibodies. These problems are caused by a variation in energy transfer between PE and either TR or CY5 because the chemistry involved in preparation and conjugation to antibodies is not sufficiently controlled to produce reagents with uniform compensation requirements. The variation in tandem dye compensation can be addressed by either using the same tandem conjugated antibody, by using the same second step tandem reagent to an appropriate first step antibody or by using software compensation. The latter provides an easy solution because a unique compensation matrix can be produced for each antibody tandem conjugate.

The immunogenicity of various peptide antigens inducing cross-reacting antibodies to a cell surface protein antigen of Streptococcus mutans.

A cell surface protein antigen (PAc) of Streptococcus mutans may be involved in the binding of bacteria to the tooth surface, and has long been focused upon as a candidate for a preventive vaccine of dental caries. Previously the peptide PAc (365-377) was shown to raise an antibody in B10.D2 mice which inhibited the binding of salivary components to the PAc molecule. Using this peptide as a unit peptide, two constructs based on multiple antigenic peptides, and several types of tandem repeats of two or three copies were synthesized to estimate the immunogenicity of these peptides. Increase in the immunogenicity was observed for all constructs with the use of an adjuvant compared to the unit peptide alone. However, the tandem repeat constructs generally induced antibody production in the absence of adjuvant, while the multiple antigenic peptide constructs did not induce antibody production under the same condition. Although such a phenomenon may be restricted to this particular peptide sequence, these results may influence the strategy for the design of peptide vaccines.

Nucleotide sequences surrounding the nonanucleotide promoter motif influence the activity of yeast mitochondrial promoter.

The highly conserved nonanucleotide (5'-TATAAGTAA[+2]) promoter sequence dictates initiation of gene-specific transcription by the mitochondrial (mt) RNA polymerase in yeast mitochondria. However, transcriptional efficiency of the nonanucleotide promoter in different mt genes varies severalfold. To explore the regulatory role of the promoter-proximal template sequence in mt transcription, different deletion, nucleotide (nt) substitution, and tandem promoter constructs were analyzed under in vitro transcription reaction conditions. It has been found that the conserved nonanucleotide promoter plus more than 9 nt of nonconserved sequence 3' to the promoter were absolutely essential for mt gene-specific transcription. In addition, approximately 300 nt of nonspecific DNA sequence 5' to the promoter was also important for efficient transcription. Interestingly, introduction of consecutive T residues in the early transcribed sequence of the template strongly inhibited mt transcription at low nt concentrations (i.e., 5 microM UTP). In contrast, neither other nt clusters nor a bacterial terminator-like sequences at that location inhibited mt transcription. Under the nonproductive reaction conditions, the full-length transcript from the mt polyT template was drastically reduced with the formation of several short abortive oligoribonucleotides. These results suggest that the transcriptional efficacy of the yeast mt promoter is influenced by sequence 3' to the promoter.

Role of phospholipase Cgamma at fertilization and during mitosis in sea urchin eggs and embryos.

It is well known that stimulation of egg metabolism after fertilization is due to a rise in intracellular free calcium concentration. In sea urchin eggs, this first calcium signal is followed by other calcium transients that allow progression through mitotic control points of the cell cycle of the early embryo. How sperm induces these calcium transients is still far from being understood. In sea urchin eggs, both InsP3 and ryanodine receptors contribute to generate the fertilization calcium transient, while the InsP3 receptor generates the subsequent mitotic calcium transients. The identity of the mechanisms that generate InsP3 after fertilization remains an enigma. In order to determine whether PLCgamma might be the origin of the peaks of InsP3 production that punctuate the first mitotic cell cycles of the fertilized sea urchin egg, we have amplified by RT-PCR several fragments of sea urchin PLCgamma containing the two SH2 domains. The sequence shares similarities with SH2 domains of PLCgamma from mammals. One fragment was subcloned into a bacterial expression plasmid and a GST-fusion protein was produced and purified. Antibodies raised to the GST fusion protein demonstrate the presence of PLCgamma protein in eggs. Microinjection of the fragment into embryos interferes with mitosis. A related construct made from bovine PLCgamma also delayed or prevented entry into mitosis and blocked or prolonged metaphase. The bovine construct also blocked the calcium transient at fertilization, in contrast to a tandem SH2 control construct which did not inhibit either fertilization or mitosis. Our data indicate that PLCgamma plays a key role during fertilization and early development.

A V3 loop haptenic peptide sequence, when tandemly repeated, enhances immunogenicity by facilitating helper T-cell responses to a covalently linked carrier protein

Subunit immunogens containing tandemly repeated copies of T- and B-cell epitopes have been shown to be more immunogenic than the respective immunogen containing only a single copy of the sequence. It has been unclear, however, whether the increased immunogenicity of a tandemly repeated B-cell epitope necessarily results from increased helper T-cell responses to intrinsic T-cell epitopes in the tandemly repeated sequences, or to neodeterminant T-cell epitopes created at the junction of adjacent repeat sequences. We examined this question by comparing the immunogenicity in mice of two immunogens containing one or eight tandemly repeated copies of an HIV-1 V3 loop haptenic sequence. Our results show that the tandemly repeated haptenic sequence potentiates the immunogenicity of the protein construct, likely through the facilitation of enhanced B-cell interaction with the tandem repeat construct and the consequent elicitation of increased carrier protein-specific helper T-cell responses.

Enhancer function and novel DNA binding protein activity in the near upstream betaAPP gene promoter.

The role of betaAPP gene transcription and promoter regulation in modifying amyloid beta-peptide (Abeta) levels is not well understood. Increased production of Abeta or changes in Abeta42/Abeta40 ratio by fibroblasts occurs in the presence of mutant presenilin or betaAPP alleles in familial Alzheimer's disease subjects. Both betaAPP mRNA and Abeta levels are increased in trisomy 21. The APP gene promoter is in a class of housekeeping genes and contains two putative consensus sites for the binding of transcription factor AP1. Electrophoretic mobility shift (EMSA) and DNase protection assays using human fibroblast and HeLa nuclear extract identified specific protein binding with novel Sp1-like properties to both a near-upstream and a downstream domain of the betaAPP promoter. The upstream binding activity was localized to a putative AP1 consensus site and its immediate 5'-adjacent GC-rich element. However, c-Jun antibody and competition experiments had no effect on binding to this domain. A series of 5'-deleted betaAPP promoter-reporter gene transfections in HeLa and fibroblast cells showed that the domain-containing region, n.t. -383 to -348, exerts a 2.9-fold activating influence on basal pbetaAPP-reporter transcription. When subcloned to test enhancer function, the 5'-GC element/'AP1 site' tandem construct conferred four-fold greater activity than either element alone and two-fold greater than the more 3'-situated HSE consensus sequence. Phorbol ester treatment had no effect in these reporter assays. This element shares homology and binding properties with a domain immediately 5' to the downstream E-box/USF element. An interaction model involving both domains and looping of interjacent DNA is proposed. We conclude that this newly described binding protein-enhancer complex is required for full betaAPP promoter activation.

Evidence for Dimerization of Dimers in K + Channel Assembly

Voltage-gated K + channels are tetrameric, but how the four subunits assemble is not known. We analyzed inactivation kinetics and peak current levels elicited for a variety of wild-type and mutant Kv1.3 subunits, expressed singly, in combination, and as tandem constructs, to show that 1) the dominant pathway involves a dimerization of dimers, and 2) dimer-dimer interaction may involve interaction sites that differ from those involved in monomer-monomer association. Moreover, using nondenaturing gel electrophoresis, we detected dimers and tetramers, but not trimers, in the translation reaction of Kv1.3 monomers.

Stoichiometry of a Ligand-gated Ion Channel Determined by Fluorescence Energy Transfer

We have developed a method to determine the stoichiometry of subunits within an oligomeric cell surface receptor using fluorescently tagged antibodies to the individual subunits and measuring energy transfer between them. Anti-c-Myc monoclonal antibody (mAb 9-E10) derivatized with a fluorophore (europium cryptate, EuK) was used to individually label c-Myc-tagged alpha1-, beta2-, or gamma2-subunits of the hetero-oligomeric gamma-aminobutyric acid (GABAA) receptor in intact cells. The maximal fluorescent signal derived from the alpha1(c-Myc)beta2gamma2 and the alpha1beta2(c-Myc)gamma2 receptors was twice that obtained with alpha1beta2gamma2(c-Myc), suggesting that there are 2x alpha-, 2x beta-, and 1x gamma-subunits in a receptor monomer. This observation was extended using fluorescence energy transfer. Receptors were half-maximally saturated with EuK-anti-c-Myc mAb, and the remaining alpha1(c-Myc) subunits were labeled with excess anti-c-Myc mAb derivatized with the fluorescence energy acceptor, XL665. On exposure to laser light, energy transfer from EuK to XL665 occurred with alpha1(c-Myc)beta2gamma2 and alpha1beta2(c-Myc)gamma2, but no significant energy transfer was observed with alpha1beta2gamma2(c-Myc) receptors, indicating the absence of a second gamma-subunit in a receptor monomer. We confirm that the GABAA receptor subtype, alpha1beta2gamma2, is composed of two copies each of the alpha- and beta-subunits and one copy of the gamma-subunit (i.e. (alpha1)2(beta2)2(gamma2)1) and conclude that this method would have general applicability to other multisubunit cell surface proteins.

Role of the S4 in cooperativity of voltage-dependent potassium channel activation.

Charged residues in the S4 transmembrane segment of voltage-gated cation channels play a key role in opening channels in response to changes in voltage across the cell membrane. However, the molecular mechanism of channel activation is not well understood. To learn more about the role of the S4 in channel gating, we constructed chimeras in which S4 segments from several divergent potassium channels, Shab, Shal, Shaw, and Kv3.2, were inserted into a Shaker potassium channel background. These S4 donor channels have distinctly different voltage-dependent gating properties and S4 amino acid sequences. None of the S4 chimeras have the gating behavior of their respective S4 donor channels. The conductance-voltage relations of all S4 chimeras are shifted to more positive voltages and the slopes are decreased. There is no consistent correlation between the nominal charge content of the S4 and the slope of the conductance-voltage relation, suggesting that the mutations introduced by the S4 chimeras may alter cooperative interactions in the gating process. We compared the gating behavior of the Shaw S4 chimera with its parent channels, Shaker and Shaw, in detail. The Shaw S4 substitution alters activation gating profoundly without introducing obvious changes in other channel functions. Analysis of the voltage-dependent gating kinetics suggests that the dominant effect of the Shaw S4 substitution is to alter a single cooperative transition late in the activation pathway, making it rate limiting. This interpretation is supported further by studies of channels assembled from tandem heterodimer constructs with both Shaker and Shaw S4 subunits. Activation gating in the heterodimer channels can be predicted from the properties of the homotetrameric channels only if it is assumed that the mutations alter a cooperative transition in the activation pathway rather than independent transitions.

Genetic transformation of the apple rootstock M26 with the RolB gene and its influence on rooting

The apple rootstock M26 ( Malus × domestica Borkh.) was transformed using the binary vector pCMBB: GUS in the Agrobacterium tumefaciens strain C58C1. The vector contained the npt II gene under the nos promotor and a tandem gene construct of the gus and the rol B genes both under the rolB promotor. Two transformed clones were verified with PCR and Southern blot analyses. One clone contained all three genes, whereas in the other clone only rol B and npt II genes were integrated in the plant genome. Rooting experiments using both intact shoots and stem segments showed that integration of the rol B gene increased auxin sensitivity and rooting ability compared with untransformed control shoots. GUS activity driven by the rol B promotor was higly expressed in the root meristem but not in the extended root. In the shoot, GUS activity was confined to the procambial strands. In the bud, the leaf primordia showed rather intense uniform staining. During growth of the leaf the provascular cells of the midrib showed weak GUS expression and developed leaves were almost unstained.

Systemic Infection of Vigna mungo Using the Cloned DNAs of the Blackgram Isolate of Mungbean Yellow Mosaic Geminivirus through Agroinoculation and Transmission of the Progeny Virus by Whiteflies

AbstractAgroinoculation of blackgram (Vigna mungo) by the cloned genomic components (DNA‐A and DNA‐B) of a non‐sap transmissible isolate of mungbean yellow mosaic geminivirus (MYMV) has been demonstrated. It has been shown that MYMV has a bipartite genome. Tandem dimeric constructs of DNA‐A and DNA‐B were infectious when inoculated together and induced systemic yellow mosaic symptoms. Agroinoculation of germinated seeds of blackgram was superior to inoculation of stems of seedlings. Infection of blackgram plants was confirmed by ELISA, dot blot and Southern hybridization. Agroinfected plants contained different forms of DNA characteristic of whitefly transmitted geminiviruses. The virus from agroinfected plants was transmissible by the whitefly Bemisia tabaci.

How does the W434F mutation block current in Shaker potassium channels?

The mutation W434F produces an apparently complete block of potassium current in Shaker channels expressed in Xenopus oocytes. Tandem tetrameric constructs containing one or two subunits with this mutation showed rapid inactivation, although the NH2-terminal inactivation domain was absent from these constructs. The inactivation showed a selective dependence on external cations and was slowed by external TEA; these properties are characteristic of C-type inactivation. Inactivation was, however, incompletely relieved by hyperpolarization, suggesting the presence of a voltage-independent component. The hybrid channels had near-normal conductance and ion selectivity. Single-channel recordings from patches containing many W434F channels showed occasional channel openings, consistent with open probabilities of 10(-5) or less. We conclude that the W434F mutation produces a channel that is predominantly found in an inactivated state.

Random circular permutation of genes and expressed polypeptide chains: application of the method to the catalytic chains of aspartate transcarbamoylase.

Recent studies on proteins whose N and C termini are in close proximity have demonstrated that folding of polypeptide chains and assembly of oligomers can be accomplished with circularly permuted chains. As yet no methodical study has been conducted to determine how extensively new termini can be introduced and where such termini cannot be tolerated. We have devised a procedure to generate random circular permutations of the catalytic chains of Escherichia coli aspartate transcarbamoylase (ATCase; EC 2.1.3.2) and to select clones that produce active or stable holoenzyme containing permuted chains. A tandem gene construct was made, based on the desired linkage between amino acid residues in the C- and N-terminal regions of the polypeptide chain, and this DNA was treated with a suitable restriction enzyme to yield a fragment containing the rearranged coding sequence for the chain. Circularization achieved with DNA ligase, followed by linearization at random with DNase I, and incorporation of the linearized, repaired, blunt-ended, rearranged genes into a suitable plasmid permitted the expression of randomly permuted polypeptide chains. The plasmid with appropriate stop codons also contained pyrI, the gene encoding the regulatory chain of ATCase. Colonies expressing detectable amounts of ATCase-like molecules containing permuted catalytic chains were identified by an immunoblot technique or by their ability to grow in the absence of pyrimidines in the growth medium. Sequencing of positive clones revealed a variety of novel circular permutations. Some had N and C termini within helices of the wild-type enzyme as well as deletions and insertions. Permutations were concentrated in the C-terminal domain and only few were detected in the N-terminal domain. The technique, which is adaptable generally to proteins whose N and C termini are near each other, can be of value in relating in vivo folding of nascent, growing polypeptide chains to in vitro renaturation of complete chains and determining the role of protein sequence in folding kinetics.

Do voltage-gated Kv1.1 and inward rectifier Kir2.1 potassium channels form heteromultimers?

Possible heteromultimer formation between Kv- and Kir-type K + channels was investigated, in connection with the known functional diversity of K + channels in vivo. Voltage-clamp experiments were performed on Xenopus oocytes, either injected with concatenated Kir2.1-Kv1.1 mRNA, or co-injected with Kv1.1 and Kir2.1 mRNA. K + currents could be approximated by the algebraic sum of the 2 K + current types alone. The tandem construct did not show functional expression, although it could be detected by Western blotting. We conclude that Kv1.1 and Kir2.1 α-subunit proteins fail to assemble and do not contribute functional diversity to K + channels.

Subunit interactions in the activation of cyclic nucleotide-gated ion channels

Cyclic nucleotide-gated (CNG) ion channels of retinal photoreceptors and olfactory neurons are multimeric proteins of unknown stoichiometry. To investigate the subunit interactions that occur during CNG channel activation, we have used tandem cDNA constructs of the rod CNG channel to generate heteromultimeric channels composed of wild-type and mutant subunits. We introduced point mutations that affect channel activation: 1) D604M, which alters the relative ability of agonists to promote the allosteric conformational change(s) associated with channel opening, and 2) T560A, which primarily affects the initial binding affinity for cGMP, and to a lesser extent, the allosteric transition. At saturating concentrations of agonist, heteromultimeric channels were intermediate between wild-type and mutant homomultimers in agonist efficacy and apparent affinity for cGMP, cIMP, and cAMP, consistent with a model for the allosteric transition involving a concerted conformational change in all of the channel subunits. Results were also consistent with a model involving independent transitions in two or three, but not one or four, of the channel subunits. The behavior of the heterodimers implies that the channel stoichiometry is some multiple of 2 and is consistent with a tetrameric quaternary structure for the functional channel complex. Steady-state dose-response relations for homomultimeric and heteromultimeric channels were well fit by a Monod, Wyman, and Changeux model with a concerted allosteric opening transition stabilized by binding of agonist.

N-type Inactivation in the Mammalian Shaker K + Channel Kv1.4

Mammalian voltage-gated K+ channels are oligomeric proteins, some of which may be composed in vivo of subunits derived from several similar genes. We have studied N-type inactivation in the rapidly inactivating Kv1.4 channel and, in specific, heteromultimers of this gene product with Kv1.5 noninactivating subunits. Heteromultimeric channels were analyzed for the stoichiometry of Kv1.4:Kv1.5 subunits by observing shifts in the midpoints of steady-state availability from that of homomultimeric channels. This analysis was employed to examine inactivation of heteromultimeric channels expressed in Xenopus oocytes using two model systems: by expression of a Kv1. 4-Kv1.5 tandem fusion construct and by coexpression of native Kv1.4 and Kv1.5 channels across a wide relative concentration range of microinjected mRNA. Additionally, inactivation was examined in coexpression experiments of N-terminal deletion mutants of Kv1.4. We found that (i) a single inactivating subunit conferred inactivation in all hetero-multimers studied; (ii) the rate of inactivation could not be distinguished in channels containing two inactivating subunits from those containing one inactivating subunit; and (iii) large deletions in the linker region between the N-terminal inactivation region and the first membrane-spanning domain had no effect on the rate of inactivation. These data confirm the importance of the proximal N-terminal region in the inactivation of mammalian Kv1.4 channels, and suggest that the inactivation particle remains in close proximity to the permeation pathway even when the channel is in the open state.

Tandem Placement of a Coronavirus Promoter Results in Enhanced mRNA Synthesis from the Downstream-Most Initiation Site

Insertion of the 17-nucleotide promoter region for the bovine coronavirus N gene as part of a 27-nucleotide cassette into the open reading frame of a cloned synthetic defective-interfering (DI) RNA resulted in synthesis of subDI RNA transcripts from the replicating DI RNA genome. Duplicating and triplicating the promoter sequence in tandem caused a progressive increase in the efficiency of subgenomic mRNA synthesis despite a concurrent decrease in the rate of DI RNA accumulation that was not specific to the promoter sequences being added. Although initiation of transcription (leader fusion) occurred at each of the three promoter sites in the tandem construct, almost all of the transcripts were found as a product of the most downstream (3′-most on the genome) promoter. These results show that enhancement of subgenomic mRNA synthesis is a property that can reside within sequence situated near the promoter. A possible role for the plus strand in the downstream promoter choice is suggested.