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HIC1(hypermethylated in cancer 1) is a transcriptional repressor containing five Krüppel-like C2H2 zinc fingers and an N-terminal dimerization and autonomous repression domain called BTB/POZ. Here, we demonstrate that full-length HIC1 proteins are modified bothin vivoandin vitrowith O-linkedN-acetylglucosamine (O-GlcNAc). This is a highly dynamic glycosylation found within the cytosolic and the nuclear compartments of eukaryotes. Analysis of[3H]Gal-labeled tryptic peptides indicates that HIC1 has three major sites forO-GlcNAc glycosylation. Using C-terminal deletion mutants, we have shown thatO-GlcNAc modification of HIC1 proteins occurred preferentially in the DNA-binding domain. Nonglycosylated and glycosylated forms of full-length HIC1 proteins separated by wheat germ agglutinin affinity purification, displayed the same specific DNA-binding activity in electrophoretic mobility shift assays proving that theO-GlcNAc modification is not directly implicated in the specific DNA recognition of HIC1. Intriguingly, N-terminal truncated forms corresponding to BTB-POZ-deleted proteins exhibited a strikingly differential activity, as the glycosylated truncated forms are unable to bind DNA whereas the unglycosylated ones do. Electrophoretic mobility shift assays performed with separated pools of glycosylated and unglycosylated forms of a construct exhibiting only the DNA-binding domain and the C-terminal tail of HIC1 (residues 399–714) and supershift experiments with wheat germ agglutinin or RL-2, an antibody raised againstO-GlcNAc residues, fully corroborated these results. Interestingly, these truncated proteins areO-GlcNAc modified in their C-terminal tail (residues 670–711) and not in the DNA-binding domain, as for the full-length proteins. Thus, theO-GlcNAc modification of HIC1 does not affect its specific DNA-binding activity and is highly sensitive to conformational effects, notably its dimerization through the BTB/POZ domain. [ABSTRACT FROM AUTHOR]

The tumor suppressor protein, p53, selectively binds to supercoiled (sc) DNA lacking the specific p53 consensus binding sequence (p53CON). Using p53 deletion mutants, we have previously shown that the p53 C-terminal DNA-binding site (CTDBS) is critical for this binding. Here we studied supercoil-selective binding of bacterially expressed full-length p53 using modulation of activity of the p53 DNA-binding domains by oxidation of cysteine residues (to preclude binding within the p53 core domain) and/or by antibodies mapping to epitopes at the protein C-terminus (to block binding within the CTDBS). In the absence of antibody, reduced p53 preferentially bound scDNA lacking p53CON in the presence of 3 kb linear plasmid DNAs or 20 mer oligonucleotides, both containing and lacking the p53CON. Blocking the CTDBS with antibody caused reduced p53 to bind equally to sc and linear or relaxed circular DNA lacking p53CON, but with a high preference for the p53CON. The same immune complex of oxidized p53 failed to bind DNA, while oxidized p53 in the absence of antibody restored selective scDNA binding. Antibodies mapping outside the CTDBS did not prevent p53 supercoil-selective (SCS) binding. These data indicate that the CTDBS is primarily responsible for p53 SCS binding. In the absence of the SCS binding, p53 binds sc or linear (relaxed) DNA via the p53 core domain and exhibits strong sequence-specific binding. Our results support a hypothesis that alterations to DNA topology may be a component of the complex cellular regulatory mechanisms that control the switch between latent and active p53 following cellular stress. [ABSTRACT FROM AUTHOR]

During a search for cDNAs encoding plant sterol acyltransferases, we isolated four full-length cDNAs from Arabidopsis thaliana that encode proteins with substantial identity with animal lecithin : cholesterol acyltransferases (LCATs). The expression of one of these cDNAs, AtLCAT3 (At3g03310), in various yeast strains resulted in the doubling of the triacylglycerol content. Furthermore, a complete lipid analysis of the transformed wild-type yeast showed that its phospholipid content was lower than that of the control (void plasmid-transformed) yeast whereas lysophospholipids and free fatty acids increased. When microsomes from the AtLCAT3-transformed yeast were incubated with di-[1-14C]oleyl phosphatidylcholine, both the lysophospholipid and free fatty acid fractions were highly and similarly labelled, whereas the sameincubation with microsomes from the control yeast produced a negligible labelling of these fractions. Moreover when microsomes from AtLCAT3- transformed yeast were incubated with either sn-1- or sn-2- [1-14C]acyl phosphatidylcholine, the distribution of the labelling between the free fatty acid and the lysophosphatidylcholine fractions strongly suggested a phospholipaseA1 activity for AtLCAT3. The sn-1 specificity of this phospholipase was confirmed by gas chromatography analysis of the hydrolysis of 1-myristoyl, 2-oleyl phosphatidylcholine. Phosphatidylethanolamine and phosphatidic acid were shown to be also hydrolysed by AtLCAT3, although less efficiently than phosphatidylcholine. Lysophospatidylcholine was a weak substrate whereas tripalmitoylglycerol and cholesteryl oleate were not hydrolysed at all. This novel A. thaliana phospholipase A1 shows optimal activity at pH 6-6.5 and 60-65 deg;C and appears to be unaffected by Ca2+. Its sequence is unrelated to all other known phospholipases. Further studies are in progress to elucidate its physiological role. [ABSTRACT FROM AUTHOR]

We have used footprinting techniques on a wide range of natural and synthetic footprinting substrates to examine the sequence-selective interaction of the bis-daunorubicin antibiotic WP631 with DNA. The ligand produces clear DNase I footprints that are very different from those seen with other anthracycline antibiotics such as daunorubicin and nogalamycin. Footprints are found in a diverse range of sequences, many of which are rich in GT (AC) or GA (TC) residues. As expected, the ligand binds well to the sequences CGTACG and CGATCG, but clear footprints are also found at hexanucleotide sequences such GCATGC and GCTAGC. The various footprints do not contain any particular unique di-, tri- or tetranucleotide sequences, but are frequently contain the sequence (G/C)(A/T)(A/T)(G/C). All sequences with this composition are protected by the ligand, though it can also bind to some sites that differ from this consensus by one base pair. [ABSTRACT FROM AUTHOR]

Cellular functions are ultimately linked to metabolic fluxes brought about by thousands of chemical reactions and transport processes. The synthesis of the underlying enzymes and membrane transporters causes the cell a certain ‘effort’ of energy and external resources. Considering that those cells should have had a selection advantage during natural evolution that enabled them to fulfil vital functions (such as growth, defence against toxic compounds, repair of DNA alterations, etc.) with minimal effort, one may postulate the principle of flux minimization, as follows: given the available external substrates and given a set of functionally important ‘target’ fluxes required to accomplish a specific pattern of cellular functions, the stationary metabolic fluxes have to become a minimum. To convert this principle into a mathematical method enabling the prediction of stationary metabolic fluxes, the total flux in the network is measured by a weighted linear combination of all individual fluxes whereby the thermodynamic equilibrium constants are used as weighting factors, i.e. the more the thermodynamic equilibrium lies on the right-hand side of the reaction, the larger the weighting factor for the backward reaction. A linear programming technique is applied to minimize the total flux at fixed values of the target fluxes and under the constraint of flux balance (= steady-state conditions) with respect to all metabolites. The theoretical concept is applied to two metabolic schemes: the energy and redox metabolism of erythrocytes, and the central metabolism of Methylobacterium extorquens AM1 . The flux rates predicted by the flux-minimization method exhibit significant correlations with flux rates obtained by either kinetic modelling or direct experimental determination. Larger deviations occur for segments of the network composed of redundant branches where the flux-minimization method always attributes the total flux to the thermodynamically most favourable branch. Nevertheless, compared with existing methods of structural modelling, the principle of flux minimization appears to be a promising theoretical approach to assess stationary flux rates in metabolic systems in cases where a detailed kinetic model is not yet available. [ABSTRACT FROM AUTHOR]

Little is known about the functions of DNA polymerase λ (Pol λ) recently identified in mammals. From the genomic sequence information of rice and Arabidopsis, we found that Pol λ may be the only member of the X-family in higher plants. We have succeeded in isolating the cDNA and recombinant protein of Pol λ in a higher plant, rice ( Oryza sativa L. cv. Nipponbare) ( OsPol λ). OsPol λ had activities of DNA polymerase, terminal deoxyribonucleotidyl transferase and deoxyribose phosphate lyase, a marker enzyme for base excision repair. It also interacted with rice proliferating cell nuclear antigen (OsPCNA) in a pull-down assay. OsPCNA increased the processivity of OsPol λ. Northern blot analysis showed that the level of OsPol λ expression correlated with cell proliferation in meristematic and meiotic tissues, and was induced by DNA-damaging treatments. These properties suggest that plant Pol λ is a DNA repair enzyme which functions in plant meristematic and meiotic tissues, and that it can substitute for Pol β and terminal deoxyribonucleotidyl transferase. [ABSTRACT FROM AUTHOR]

This article presents corrigendum of various articles, published in the "European Journal of Biochemistry." The paper by A. G. Kourmanova et al. contains errors on pages 2350, 2352 and 2353. On page 2352, in the section entitled "Assessment of the Quantitative Ratio of the Three Variants of mlp Genes in Mistletoe Genomic DNA and Their Transcripts in mRNA' the first sentence should read "Two Primers, Universal for All Obtained Variants of Mistletoe Lectin Gene Sequences Were Designed."

Information on the regulation and structure–function relation of enzymes involved in DNA precursor synthesis is pivotal, as defects in several of these enzymes have been found to cause depletion or deletion of mitochondrial DNA resulting in severe diseases. Here, the effect of amino acid 106 on the enzymatic properties of the cell-cycle-regulated human cytosolic thymidine kinase 1 (TK1) is investigated. On the basis of the previously observed profound differences between recombinant TK1 with Val106 (V106WT) and Met106 (V106M) in catalytic activity and oligomerization pattern, we designed and characterized nine mutants of amino acid 106 differing in size, conformation and polarity. According to their oligomerization pattern and thymidine kinetics, the TK1 mutants can be divided into two groups. Group I (V106A, V106I and V106T) behaves like V106WT, in that pre-assay exposure to ATP induces reversible transition from a dimer with low catalytic activity to a tetramer with high catalytic activity. Group II (V106G, V106H, V106K, V106L and V106Q) behaves like V106M in that they are permanently high activity tetramers, irrespective of ATP exposure. We conclude that size and conformation of amino acid 106 are more important than polarity for the catalytic activity and oligomerization of TK1. The role of amino acid 106 and the sequence surrounding it for dimer–tetramer transition was confirmed by cloning the putative interface fragment of human TK1 and investigating its oligomerization pattern. [ABSTRACT FROM AUTHOR]

A thermostable enzyme from the hyperthermophilic sulphate-reducing archaeon, Archaeoglobus fulgidus , was expressed and characterized on the assumption that it is homologous to exonuclease III from Escherichia coli . Sequence similarity database searches were performed based on the amino acid sequence of exonuclease III. The 774 bp long gene was isolated from a culture sample and cloned into different vectors. Expression proved successful by transforming pET28_Af_Exo in Origami B(DE3) containing a tRNA plasmid with extra copies of argU , ileY and leuW tRNA genes as a host strain. The lack of thioredoxin reductase (trxB ) and glutathione reductase (gor ) in Origami B(DE3) allowed formation of disulfide bridges in the cytosol. Purification was performed by heat treatment of the soluble fraction at 80 °C for 30 min followed by a two-step ion exchange chromatography. The activity of the enzyme could be maintained. Optimal activity was achieved at 80 °C and at a pH of 7. Within the characterization of the protein we could not find any data verifying exonucleolytic activity in the presence of Mg[sup 2+] as described [Ankenbauer, W., Laue, F., Sobek, H., & Greif, M. (2000), patent number WO2001023583]. Instead strong DNA binding properties of the enzyme and nicking activities of double stranded DNA comparable to unspecific DNases could be observed. In contrast to exonuclease III from Escherichia coli , the xthA gene product of Archaeoglobus fulgidus is able to degrade supercoiled plasmids and shows no preferences for blunt or recessed 3′-termini of linear double stranded DNA. The enzyme is inhibited by EDTA and shows only weak activity when replacing Mg[sup 2+] with Ca[sup 2+] ions. [ABSTRACT FROM AUTHOR]

The transcription patterns of 64 linear double stranded DNA templates obtained with T7 RNA polymerase were investigated. These templates consisted of 17 nucleotide-long sequences under the control of the minimal bacteriophage T7 promoter and represented all possible combinations of nucleotides at positions +8, +10 and +11. Two clearly distinct types of template were identified, which produced the range of transcription patterns observed: (a) those that yielded 17-nucleotide-long RNA as the only detectable run-off product (only 15% of the total), and (b) templates that in addition to the expected full-length RNA, produced other products longer than 17 nucleotides. Self-complementarity analysis of the expected run-off transcripts showed that those obtained from the first type of template were able to form stable intermolecular duplexes with non-base-paired 3′-ends. However, the second type of template yielded RNAs able to generate energetically favorable intermolecular duplexes with 3′-end complementarity, therefore yielding an RNA-primed RNA-template. The gel-purified 17-nucleotide-long RNAs transcribed from the latter yielded longer products when incubated under in vitro transcription conditions in the absence of a DNA template. No extension was observed when assaying the 17-nucleotide RNA products resulting from the first type of template. We observed that just a single nucleotide change within the DNA template could convert the RNA product from an RNA-primed template into a nonextendible dimer thus leading to a drastic switch of the 17-nucleotide product yield from less than 10% to 100%. Further, two type B DNA templates were extended by two nucleotides at the 3′-end, to produce RNA transcripts theoretically unable to form 3′-end base-paired duplexes. The full-length products of these modified DNA templates were found to be nonextendible by T7 RNA polymerase under the standard in vitro transcription conditions. [ABSTRACT FROM AUTHOR]

We have identified a novel mammalian gene, termed nicolin 1 gene (NICN1 ), that is present in human, dog and mouse, whereas it is absent from the available genome sequences of nonmammalian organisms. The NICN1 gene consists of six exons and spans about 6 kb of genomic DNA. It encodes a 213 amino acid protein that does not belong to any known protein family. Experiments using green fluorescent protein (GFP)-tagged nicolin 1 fusion proteins indicate that nicolin 1 is a nuclear protein. Northern analysis and semiquantitative RT-PCR demonstrated that the 2.5 kb NICN1 mRNA is expressed in a tissue-specific manner. The highest NICN1 expression levels are found in brain, testis, liver, and kidney. On the other hand the NICN1 expression is weak in spleen, leukocytes, small intestine and colon. The NICN1 gene is also expressed during development. [ABSTRACT FROM AUTHOR]

Dnmt3a is a de novo DNA methyltransferase that modifies unmethylated DNA. In contrast Dnmt1 shows high preference for hemimethylated DNA. However, Dnmt1 can be activated for the methylation of unmodified DNA. We show here that the Dnmt3a and Dnmt1 DNA methyltransferases functionally cooperate in de novo methylation of DNA, because a fivefold stimulation of methylation activity is observed if both enzymes are present. Stimulation is observed if Dnmt3a is used before Dnmt1, but not if incubation with Dnmt1 precedes Dnmt3a, demonstrating that methylation of the DNA by Dnmt3a stimulates Dnmt1 and that no physical interaction of Dnmt1 and Dnmt3a is required. If Dnmt1 and Dnmt3a were incubated together a slightly increased stimulation is observed that could be due to a direct interaction of these enzymes. In addition, we show that Dnmt1 is stimulated for methylation of unmodified DNA if the DNA already carries some methyl groups. We conclude that after initiation of de novo methylation of DNA by Dnmt3a, Dnmt1 becomes activated by the pre-existing methyl groups and further methylates the DNA. Our data suggest that Dnmt1 also has a role in de novo methylation of DNA. This model agrees with the biochemical properties of these enzymes and provides a mechanistic basis for the functional cooperation of different DNA MTases in de novo methylation of DNA that has also been observed in vivo . [ABSTRACT FROM AUTHOR]

From the antennae of the moth Mamestra brassicae , we have identified a lepidopteran G protein α subunit belonging to the Gq family, through immunological detection in crude antennal extract and antennal primary cell cultures, followed by molecular cloning. The complete cDNA sequence (1540 bp) contains an open reading frame encoding a protein of 353 amino acids. This deduced sequence possesses all of the characteristics of the Gq family and shares a very high degree of amino-acid sequence identity with vertebrate (80% with mouse or human Gqα) and invertebrate subunits (varying between 60 and 87% for Gqα from organisms as diverse as sponge and Drosophila ). The expression pattern of the Gq subunit in adult antennae was associated with the olfactory sensilla suggesting a specific role in olfaction. These data provide molecular evidence for a component of the phosphoinositide signaling pathway in moth antennae: this G protein α subunit may be involved in the olfaction transduction process through interaction with G-protein-coupled receptors, stimulating the phospholipase C mediated second messenger pathway. [ABSTRACT FROM AUTHOR]

Two-dimensional NMR and molecular dynamics simulations have been used to determine the three-dimensional structures of two hairpin DNA structures: d-CTAGAG GATCCUTTTGGATCCT (abbreviated as U1-hairpin) and d-CTAGAGGATCCTTUTGGATCCT (abbreviated as U3-hairpin). The 1 H resonances of both of these hairpin structures have been assigned almost completely. NMR restrained molecular dynamics and energy minimization procedures have been used to describe the three-dimensional structures of these hairpins. This study and concurrent NMR structural studies on two other d-CTAGAGGA TCCTUTTGGATCCT (abbreviated as U2-hairpin) and d-CTAGAGGATCCTTTUGGATCCT (abbreviated as U4-hairpin) have shed light upon various interactions reported between Echerichia coli uracil DNA glycosylase (UDG) and uracil-containing DNA. The backbone torsion angles, which partially influence the local conformation of U12 and U14 in U1 and U3-hairpins, respectively, are probably locked in the trans conformation as in the case of U13 in the U2-hairpin. Such a stretched-out backbone conformation in the vicinity of U12 and U14 is thought to be the reason why the K m value is poor for U1- and U3-hairpins as it is for the U2-hairpin. Furthermore, the bases U12 and U14 in both U1- and U3-hairpins adopt an anti conformation, in contrast with the base conformation of U13 in the U2-hairpin, which adopts a syn conformation. The clear discrepancy observed in the U-base orientation with respect to the sugar moieties could explain why the V max value is 10- to 20-fold higher for the U1- and U3-hairpins compared with the U2-hairpin. Taken together, these observations support our interpretation that the unfavourable backbone results in a poor K m value, whereas the unfavourable nucleotide conformation results in a poor V max value. These two parameters therefore make the U1- and U3-hairpins better... [ABSTRACT FROM AUTHOR]

We report the constitution and molecular characterization of a novel transglutaminase (EC 2.3.2.13) that starts to accumulate specifically in the nucleus in the starfish (Asterina pectinifera ) embryo after progression through the early blastula stage. The cDNA for the nuclear transglutaminase was cloned and the cDNA-deduced sequence defines a single open reading frame encoding a protein with 737 amino acids and a predicted molecular mass of 83 kDa. A comparison of this transglutaminase with other members of the gene family revealed an overall sequence identity of 33–41%. A special sequence feature of this transglutaminase, which is not found in other transglutaminases, is the presence of nuclear localization signal-like sequences in the N-terminal region. Microinjection of hybrid constructs that encode the N-terminal segment fused to reporter proteins into the germinal vesicle of an oocyte produced chimeric proteins by transcription-coupled translation. It was found that the N-terminal segment alone was sufficient to effect nuclear accumulation of an otherwise cytoplasmic protein. These results suggest that the nuclear accumulation of the transglutaminase may play an important role in nuclear remodeling during early starfish embryogenesis. [ABSTRACT FROM AUTHOR]

DNA of prokaryotes is in a nonequilibrium structural state, characterized as ‘active’ DNA supercoiling. Alterations in this state affect many life processes and a homeostatic control of DNA supercoiling has been suggested [Menzel, R. & Gellert, M. (1983) Cell 34 , 105–113]. We here report on a new method for quantifying homeostatic control of the high-energy state of in vivo DNA. The method involves making small perturbation in the expression of topoisomerase I, and measuring the effect on DNA supercoiling of a reporter plasmid and on the expression of DNA gyrase. In a separate set of experiments the expression of DNA gyrase was manipulated and the control on DNA supercoiling and topoisomerase I expression was measured [part of these latter experiments has been published in Jensen, P.R., van der Weijden, C.C., Jensen, L.B., Westerhoff, H.V. & Snoep, J.L. (1999) Eur. J. Biochem. 266 , 865–877]. Of the two regulatory mechanisms via which homeostasis is␣conferred, regulation of enzyme activity or regulation of enzyme expression, we quantified the first to be responsible for 72% and the latter for 28%. The gene expression regulation could be dissected to DNA gyrase (21%) and to topoisomerase I (7%). On a scale from 0 (no homeostatic control) to 1 (full homeostatic control) we quantified the homeostatic control of DNA supercoiling at 0.87. A 10% manipulation of either topoisomerase I or DNA gyrase activity results in a 1.3% change of DNA supercoiling only. We conclude that the homeostatic regulation of the nonequilibrium DNA structure in wild-type Escherichia coli is almost complete and subtle (i.e. involving at least three regulatory mechanisms). [ABSTRACT FROM AUTHOR]

Two DNA hairpin motifs (5′-GCGAAGC-3′ and 5′-ACGA AGT-3′), both stabilized by a 5′-GAA loop, have been used to design novel intramolecular double hairpin structures (5′-GCGAAGCACGAAGT-3′ and 5′-ACGAAGTGCG AAGC-3′) in which coaxial stacking of the two hairpin components generates a double-stranded stem region effectively with a single-strand break in the middle of the sequence at either the TG or CA step between unconnected 3′ and 5′ terminal bases. We have investigated by NMR the conformation and dynamics of the DNA at the strand break site. We show that mutual stacking significantly enhances the stability of each hairpin. Further, the anthracycline antibiotic nogalamycin binds cleanly to the 5′-TG (5′-CA) site formed by the mutually stacked hairpins despite the break in the sugar-phosphate backbone on one strand. The complex resembles the structure of nogalamycin–DNA complexes with the drug bound at 5′-TG sites in intact duplex sequences, with π-stacking interactions probably the single dominant stabilizing interaction. [ABSTRACT FROM AUTHOR]

Fractionation of nuclear extracts from posterior silk glands of mulberry silkworm Bombyx mori, resolved the transcription factor TFIIIC into two components (designated here as TFIIIC and TFIIIC1) as in HeLa cell nuclear extracts. The reconstituted transcription of tRNA genes required the presence of both components. The affinity purified TFIIIC is a heteromeric complex comprising of five subunits ranging from 44 to 240 kDa. Of these, the 51-kDa subunit could be specifically crosslinked to the B box of tRNAGly1. Purified swTFIIIC binds to the B box sequences with an affinity in the same range as of yTFIIIC or hTFIIIC2. Although an histone acetyl transferase (HAT) activity was associated with the TFIIIC fractions during the initial stages of purification, the HAT activity, unlike the human TFIIIC preparations, was separated at the final DNA affinity step. The tRNA transcription from DNA template was independent of HAT activity but the repressed transcription from chromatin template could be partially restored by external supplementation of the dissociated HAT activity. This is the first report on the purification and characterization of TFIIIC from insect systems. [ABSTRACT FROM AUTHOR]

To search for novel regulatory regions, we examined the features of chromatin structure in the rat B29/Ig-β gene and its flanking regions by determining DNase I hypersensitive sites (DHS) in plasmacytoma-derived Y3 cells. Six Y3 cell-specific DHS were detected at -8.6, promoter, +0.7, +4.4, +6.0, and +8.7 kb. The DHS at +4.4, +6.0, and +8.7 kb were present in the intergenic region between B29/Ig-β and growth hormone (GH) genes and were mapped inside conserved sequences in rat and humans. In transient transfection into Y3 cells, 2.9-kb DNA containing the +4.4 and +6.0-kb DHS demonstrated six times more enhancing activity than B29/Ig-β promoter alone. Three intergenic DHS each possessed enhancing activity that was highest in the +4.4-kb region. In the electrophoretic mobility shift assay, a major band shift was demonstrated with Y3 nuclear extract and 0.3-kb DNA containing the +4.4-kb region with a conserved 0.22-kb sequence. By footprint analysis, 20 bases in the middle of the 0.3-kb DNA were protected by Y3 nuclear extract in which the consensus binding site for the OCT family was present. Deletion of the footprinted region reduced enhancing activity to that of the B29/Ig-β promoter alone. The sequence responsible for the major band shift and transcriptional enhancing activity in the conserved +4.4-kb region thus coincided with the 20-bp footprinted region. [ABSTRACT FROM AUTHOR]

We isolated 13 tobacco calmodulin (CaM) genes, NtCaM1–13, and analyzed their expression profile in response to pathogen infection and wounding using specific DNA probes for individual CaM genes and specific antibodies for CaM proteins in groups I (NtCaM1/2), II (NtCaM3/4/5/6/7/8/11/12 and 9/10) and III (NtCaM13), respectively. Synchronous cell death in tobacco mosaic virus (TMV)-infected N-gene-containing tobacco leaves accompanied a predominant accumulation of NtCaM1, 2 and 13 transcripts and NtCaM13-type protein, which is a possible ortholog of soybean defense-involved CaM (SCaM-4), preceding induction of PR-1 and PR-3 defense genes. Accumulation of NtCaM1, 2, 3 and 4 transcripts was induced within 30 min after wounding and NtCaM1-type protein accumulated transiently after wounding. NtCaM13-type protein, which was found at a low level in healthy leaves, decreased instantly after wounding. The treatment with a proteasome inhibitor, lactacystin, enhanced wound-induced accumulation of NtCaM1-type protein and inhibited wound-induced decrease of NtCaM13-type protein, suggesting that proteasome activity is involved in the degradation of these CaMs. Thus, our results indicate that levels of individual CaM proteins are differentially regulated both transcriptionally and post-transcriptionally in tobacco plants that are exposed to stresses such as pathogen-induced hypersensitive cell death and wounding. [ABSTRACT FROM AUTHOR]

The elucidation of digestive processes in the Anopheles gambiae gut leading to the utilization of the blood meal will result in a deeper understanding of the physiology of blood digestion and its impact on parasite–vector interactions. Accordingly, the identification of digestive serine proteases in A. gambiae has implications for the development of alternative strategies for the control of mosquito-borne diseases. We report here on the cDNA and genomic cloning and on the expression analysis of two closely related chymotrypsin genes, Anchym1 and Anchym2. Genomic cloning revealed that Anchym1 and Anchym2, which map on chromosomal division 25D, are clustered in tandem within 6 kb, both genes being interrupted by two short introns. After blood feeding, transcription of Anchym1 and Anchym2 is induced in the midgut epithelium, followed by secretion of the translated products into the midgut lumen where the Anchym1 and Anchym2 zymogens are activated by partial tryptic digestion. The amino-acid residues forming the substrate pocket of Anchym1 and Anchym2 suggested chymotryptic cleavage specificity. This was confirmed by mass spectrometry analysis and Edman degradation sequencing of proteolytic products generated by the recombinant, trypsin-activated Anchym1. [ABSTRACT FROM AUTHOR]

S1 proteins A–D are liberated from thoroughly washed nuclei by mild digestion with DNase I or RNase A, and extracted selectively at pH 4.9 from the reaction supernatants. Here, we characterized the S1 proteins, focusing on protein D2, the most abundant S1 protein in the rat liver, and on protein C2 as well. Using a specific antibody, McAb 351, they were shown to occur in the extranucleolar nucleoplasm, and to be extracted partly in the nuclear soluble fraction. We demonstrate that the S1 proteins in this fraction exist constituting heterogeneous nuclear ribonucleoproteins (hnRNPs), through direct binding to hnRNAs, as revealed by centrifugation on density gradients, immunoprecipitation, and UV cross-linking. In hnRNPs, protein D2 occurred at nuclease-hypersensitive sites and C2 in the structures that gave rise to 40 S RNP particles. By microsequencing, protein D2 was identified with a known protein, CArG box motif-binding factor A (CBF-A), which has been characterized as a transcriptional repressor, and C2 as its isoform protein. In fact, CBF-A expressed from its cDNA was indistinguishable from protein D2 in molecular size and immunoreactivity to McAb 351. Thus, the present results demonstrate that S1 proteins C2 and D2 are novel hnRNP proteins, and suggest that the proteins C2 and D2 act in both transcriptional and post-transcriptional processes in gene expression. [ABSTRACT FROM AUTHOR]

The steroid hormone, 20-hydroxyecdysone (20E), directs Drosophila metamorphosis via a heterodimeric receptor formed by two members of the nuclear hormone receptors superfamily, the product of the EcR (EcR) and of the ultraspiracle (Usp) genes. Our previous study [Niedziela-Majka, A., Kochman, M., Ożyhar, A. (2000) Eur. J. Biochem. 267, 507–519] on EcR and Usp DNA-binding domains (EcRDBD and UspDBD, respectively) suggested that UspDBD may act as a specific anchor that preferentially binds the 5′ half-site of the pseudo-palindromic response element from the hsp27 gene promoter and thus locates the heterocomplex in the defined orientation. Here, we analyzed in detail the determinants of the UspDBD interaction with the hsp27 element. The roles of individual amino acids in the putative DNA recognition α helix and the roles of the base pairs of the UspDBD target sequence have been probed by site-directed mutagenesis. The results show how the hsp27 element specifies UspDBD binding and thus the polar assembly of the UspDBD/EcRDBD heterocomplex. It is suggested how possible nucleotide deviations within the 5′ half-site of the element may be used for the fine-tuning of the 20E-response element specificity and consequently the physiological response. [ABSTRACT FROM AUTHOR]

High-mobility-group proteins HMG-1 and HMG-I/Y bind at overlapping sites within the A/T-rich enhancer element of the pea plastocyanin gene. Competition binding experiments revealed that HMG-1 enhanced the binding of HMG-I/Y to a 31-bp region (P31) of the enhancer. Circularization assays showed that HMG-1, but not HMG-I/Y, was able to bend a linear 100-bp DNA containing P31 so that the ends could be ligated. HMG-1, but not HMG-I/Y, showed preferential binding to the circular 100-bp DNA compared with the equivalent linear DNA, indicating that alteration of the conformation of the DNA by HMG-1 was not responsible for enhanced binding of HMG-I/Y. Direct interaction of HMG-I/Y and HMG-1 in the absence of DNA was demonstrated by binding of 35S-labeled proteins to immobilized histidine-tagged proteins, and this was due to an interaction of the N-terminal HMG-box-containing region of HMG-1 and the C-terminal AT-hook region of HMG-I/Y. Kinetic analysis using the IAsys biosensor revealed that HMG-1 had an affinity for immobilized HMG-I/Y (Kd = 28 nm) similar to that for immobilized P31 DNA. HMG-1-enhanced binding of HMG-I/Y to the enhancer element appears to be mediated by the formation of an HMG-1–HMG-I/Y complex, which binds to DNA with the rapid loss of HMG-1. [ABSTRACT FROM AUTHOR]

Examines the binding of p53 protein core domain to supercoiled (SC) DNA. Distribution of the protein between SC and linearized DNA; Biological activity of the protein; Use of gel electrophoresis and immunoblotting in determining the protein binding.

The Gadd45γ (growth arrest and DNA damage-inducible) gene is activated transcriptionally by at least two kinds of agents: DNA damaging agent such as methyl methanesulfonate (MMS) and UV radiation, or cytokines such as interleukin (IL)-6, IL-2 and granulocyte colony-stimulating factor (G-CSF). To investigate the sequences and transcription factors involved in induction of Gadd45γ after treatment with IL-6, the human gene was cloned and sequenced. We found C/EBP (CCAAT/enhancer-binding protein) family proteins, major transcription factors in the IL-6 signal transduction pathway, could regulate the transcriptional activity of the Gadd45γ promoter. In addition, a noncanonical C/EBP-binding site within the Gadd45γ promoter where C/EBPβ and C/EBPδ could bind, was identified by electrophoretic mobility shift assay (EMSA) and reporter gene analysis. Furthermore, we found a coordinated expression profile between Gadd45γ mRNA and C/EBPs (β and δ) protein during the differentiation of M1 cells: the amount of Gadd45γ transcripts became maximal when both C/EBPβ and C/EBPδ levels were high, on day 1 of differentiation of M1 cells after treatment with IL-6. These findings suggest that mitotic growth arrest coupled to M1 cell differentiation is mediated by C/EBPs stimulation of growth arrest-associated genes such as Gadd45γ. [ABSTRACT FROM AUTHOR]

High mobility group (HMG) 1 protein is a highly abundant and an evolutionarily conserved chromosomal protein with two homologous DNA-binding domains (HMG boxes), A and B, attached by a short basic region to an acidic C-terminal tail. The protein has been implicated in a number of fundamental biological processes including DNA replication, transcription, recombination and repair. We demonstrate that HMG1 is able to enhance cohesive-end and blunt-end DNA ligation by T4 DNA ligase via its B domain. The C-terminal flanking sequence of the B domain (seven basic residues out of ≈ 18) and a number of conserved amino-acid residues within the HMG box (mainly basic or hydrophobic) are required for efficient stimulation of ligation. Pull-down assays, electron and scanning force microscopy revealed that HMG1 can associate two DNA molecules via their ends even in the absence of complementary overhangs. We propose that HMG1 protein may be involved in the rejoining of DNA breaks by different DNA ligases due to its ability to bring DNA duplexes and their termini into a close proximity while leaving the ends accessible for ligation. [ABSTRACT FROM AUTHOR]

Dihydrolipoamide succinyltransferase (E2o) is the structural and catalytic core of the 2-oxoglutarate dehydrogenase (OGDH) complex. The cDNA encoding porcine E2o (PE2o) has been cloned. The PE2o cDNA spans 2547 bases encoding a presequence (68 amino-acid residues) and a mature protein (387 residues, Mr = 41 534). Recombinant porcine E2o (rPE2o) (residues 1–387), C- and N-terminal truncated PE2os, and site-directed mutant PE2os were overexpressed in Escherichia coli via the expression vector pET-11d and purified. The succinyltransferase activity of the rPE2o was about 2.2-fold higher than that of the native PE2o. Electron micrographs of the rPE2o negatively stained showed a cube-like structure very similar to that of the native PE2o. Deletion of five amino-acid residues from the C-terminus resulted in a complete loss of both enzymatic activity and formation of the cube-like structure, but the deletion of only the last two residues had no effect on either function, suggesting the important roles of the C-terminal leucine triplet (Leu383–384–385). Substitution of Ser306 with Ala, and Asp362 with Asn, Glu or Ala in the putative active site, and Leu383–384–385 with Ala or Asp abolished both functions. Substitution of His358 with Cys resulted in an 8.5-fold reduction in kcat, with little change in Km values for dihydrolipoamide and succinyl-CoA. However, self-assembly was not affected. These data indicate that Ser306, Asp362 and the Leu383–384–385 triplet are important residues in both the self-assembly and catalytic mechanism of PE2o. [ABSTRACT FROM AUTHOR]

Determines the conformation of sugar phosphate backbone geometry in an 11-mer A-tract DNA duplex, code named AAA. Specification of the sugar pucker and sequence-specific torsional angle preferences of the C3'-O3', C4'-C5' and C5'-O5' bonds; Coupling constants; Torsion of the C3'-)3' bonds; Effect of cross relaxation on measured coupling constants.

Focuses on the identification of cyclic DNA clones of hydddroxycinnamoyl-CoA tyramine N-hydroxycinnamoyltransferase (THT) from tobacco. Properties of the fusion protein expressed in E. Coli; Comparison of THT to acyltransferase; Generation of antibodies against THT.

Examines the stabilization of deoxyribonucleic acid triple helices by a series of mono- and disubstituted amidoanthraquinones. Indication of less efficiency; Summary of circulated relative binding affinities of ligands; Examination of energies for each complex.

Examines the cloning of a cDNA coding for a putative human mitochondrial thioredoxin reductase (TrxR) and its genomic organization. Discussion of cDNA cloning of hTrxR2 and sequence analysis; Localization of hTrxR2 gene; Analysis of hTrxR2 using Northern-blot.

Examines the crystal structure of d(AGGGGCCCCT) in two crystal forms to determine the conformation and dynamics of the [sub n](dC)[sub n]. Structure of A-DNA duplexes; Factors influencing the formation of A-DNA; Interactions of counter ions with A-DNA.

Examines the role of adenylate kinases in the synthesis of adenine nucleotides. Identification of nucleotide and amino acid sequences of human cDNA; Expression and characterization of cDNA clones; Description of functional domains of nucleoside monophosphate kinases.

Examines the formation of intermolecular triplexes on DNA fragments. Use of DNase I footprinting; Availability of DNA fragments for triple-helix formation; Effect of the triplexes on the length of the oligopurine tract.

Presents a study which characterized thermostable RecA protein from Thermus thermopilus. Analysis of the protein's interaction with single-stranded DNA; Overexpression of Thermus thermophilus recA gene in Escherichia Coli.

The mode of action of ribosome-inactivating proteins (RIPs) has, for many years, been considered to be depurination of a specific adenyl residue of ribosomal RNA, resulting in inhibition of protein synthesis. Recently, this view has been challenged by the observation that many RIP preparations have significant DNase activity in addition to their N-glycosidase activity. In this study, we have investigated the putative DNase activity of two RIPs, ricin and pokeweed antiviral protein (PAP), and show that, in both cases, the DNase activity is due to the presence of contaminating nucleases. The N-glycosidase and DNase activities of PAP were separately and specifically inactivated by chemical modification and heat. Gel filtration of ricin allowed physical separation of the two activities. Furthermore, neither recombinant PAP nor recombinant ricin A-chain purified from Escherichia coli displayed DNase activity. [ABSTRACT FROM AUTHOR]

cDNA encoding NG,NG-dimethylarginine dimethylaminohydrolase from rat kidney had been cloned [Kimoto, M., Sasakawa, T., Tsuji, H., Miyatake, S., Oka, T., Nio, N. & Ogawa, T. (1997) Biochim. Biophys. Acta 1337, 6-10]. The enzyme hydrolyzes NG,NG-dimethyl-L-arginine and NG-monomethyl-L-arginine, which are known as endogenous inhibitors for the nitric oxide-generating system. In the present study, human NG,NG-dimethylarginine dimethylaminohydrolase has been purified to homogeneity from liver and characterized. The cDNA clone encoding human NG,NG-dimethylarginine dimethylaminohydrolase was isolated from a human kidney λgt10 library using a probe prepared from a plasmid containing the entire coding region of rat NG,NG-dimethylarginine dimethylaminohydrolase. Its open reading frame encoded a protein of 285 amino acids with a molecular mass of 31 121 Da. The deduced amino acid sequence exhibits 93 % identity with that of rat. The cDNA was expressed as a fusion protein in Escherichia coli and the recombinant protein exhibited enzyme activity which is the same as that of natural enzyme. [ABSTRACT FROM AUTHOR]

Recent observations that several trans-platinum complexes exhibit antitumor activity including activity in cisplatin-resistant tumor cells, violates the classical structure/activity relationships of platinum(II) complexes. According to these relationships, only bifunctional platinum(II) complexes with cis-oriented leaving ligands should be therapeutically active. In order to contribute to the understanding of mechanisms underlying the antitumor activity of these new trans-platinum analogs, various biochemical and biophysical methods as well as molecular modeling techniques were employed to study the modifications of DNA by antitumor trans-[PtCl2(NH3)(quinoline)]. The results indicated that trans-[PtCl2(NH3)(quinoline)] coordinated monofunctionally to DNA with a similar rate as transplatin. The overall rate of the rearrangement to bifunctional adducts was also similar to that observed in the case of DNA modification by transplatin, i.e. it was relatively slow (after 48 h ≈34 % adducts remained monofunctional). In contrast to transplatin, however, trans-[PtCl2(NH3)(quinoline)] formed considerably more interstrand cross-links after 48 h (≈30 %) with a much shorter half-time (≈5 h) (≈12 % for transplatin, t1/2 > 11 h). The results also suggested that the quinoline ligand in all or in a significant fraction of DNA adducts of trans-[PtCl2(NH3)(quinoline)], in which platinum is coordinated to base residues, was well positioned to interact with the duplex. The adducts of trans-[PtCl2(NH3)(quinoline)] terminated in vitro RNA synthesis preferentially at guanine residues. Surprisingly, the type and extent of conformational alterations induced in DNA indicates that trans-[PtCl2(NH3)(quinoline)] behaves in some respects like cisplatin, as indicated by the fact that... [ABSTRACT FROM AUTHOR]

The cDNA of soybean aggtutinin (SBA), a glycoprotein lectin, obtained from the mRNA of soybean seeds at mid-maturation, was cloned in a λgl 10 phage and subcloned in a pUC-8 plasmid. Probing with a fragment of the lectin gene [Vodkin, L. O., Rhodes, P. R. & Goldberg, R. B. (1983) Cell 34, 10231031} afforded a clone of 1012 nucleotides containing the complete coding region of 858 nucleotides for the precursor to soybean agglutinin. The deduced amino acid sequence contains the 253 residues of the mature lectin and an hydrophobic N-terminal signal peptide of 32 amino acids. Expression in Escherichia coli of the eDNA coding for the precursor to the lectin or for the mature lectin led to the accumulation of large quantities of inclusion bodies, from which mature SBA was :isolated in small yield (up to 1 mg/l). It was identical with the native lectin in the hemagglutinating activity and carbohydrate specificity, Nterminal sequence and oligomeric structure, but, because it was not glycosylated, its subunit mass was lower by 2 kDa. Our findings show that pre-SBA is processed into the mature form in the bacteria, and that, contrary to what has been suggested [Nagai, K. & Yamaguchi, H. (1993) J. Biochem. (Tokyo) 113, 123-125], glycosylation is not essential for the folding of the lectin, nor for its subunit assembly into, a biologically active tetramer. To obtain recombinant SBA in secreted form, the pre-SBA cDNA was subcloned in pTM1 vector and the construct inserted into vaccinia virus. When monkey BS-C-1 cells were infected by the virus, using a double expression protocol, recombinant lectin was secreted into the growth medium, from which it was isolated by immunoaffinity chromatography at a yield similar to that from the bacteria. Except for its lower hemagglutinating activity, the product was indistinguishable from native SBA in all properties tested. It was also susceptible to digestion by endo-β-N-acetylglucosaminidase H or N-glycanase which caused a decrease of 2 kDa in its subunit mass and gave the same results on lectin blot analysis, indicating that it too is a glycoprotein with a single oligomannose unit. [ABSTRACT FROM AUTHOR]

A Trichoderma reesei cDNA encoding a previously unknown protein with a C-terminal cellulosebinding domain was obtained by complementation screening of a T. reesei cDNA library in a sec1 yeast mutant impaired in protein secretion. The T. reesei protein shows amino acid similarity over its entire length to the Agaricus bisporus cellulose-induced protein CELl whose function is not known. These two proteins form a new glycosyl hydrolase family, number 61. Expression of the T. reesei cDNA in yeast showed that it encoded a protein with endoglucanase activity and thus the protein was named EGIV and the corresponding gene egl4. Polyclonal antibodies were prepared against EGIV produced in Escherichia coil and detected a 56-kDa protein in the T. reesei culture supernatant. Northern hybridisation revealed that T. reesei egl4 is regulated in the same manner as other cellulase genes of this fungus. [ABSTRACT FROM AUTHOR]

Bacterium Sphingomonas sp. strain RW1 is, under aerobic conditions, able to degrade dibenzofuran and dibenzo-p-dioxin. The first step of the pathway is performed by a ring-dihydroxylating enzyme. Bünz and Cook have reported the purification and characterization of this dioxin dioxygenase and a ferredoxin able to transfer electrons to the dioxygenase [Bünz, E V. & Cook, A. M. (I993) J. Bacteriol. 175, 6467-6475]. The gene encoding this [2Fe-2S] ferredoxin was identified by screening a genomic library constructed in pLAFR3 with a probe generated by a nested-PCR amplification. Primers for the amplification were designed based on the N-terminus sequence of the purified fierredoxin and on sequence comparisons with related proteins. Several cosmids were obtained and the ferredoxin gene, fdx1, was subcloned from one of them. The nucleotide sequence of a 4.6-kb DNA fragment encompassing the ferredoxin gene was determined. While in the case of all known multi-component dioxygenases, genes encoding the α and β subunits are found to be contiguous with the gene of the specific electron cagier, the fdx1 gene in Sphingomonas sp. RW1 does not appear to be directly linked with the dioxin dioxygenase genes. Rather, it is clustered with genes apparently encoding two atypical decarboxylases/isomerases and a glutathione S-transferase. The ferredoxin gene was hyperexpressed and the recombinant ferredoxin was purified. Spectroscopic characterization of Fdx1 demonstrated the presence of a putidaredoxin-type [2Fe-2S] cluster in this protein. Its redox potential was determined to be -245 (± 5) mV versus the normal hydrogen electrode at 25°C, pH 8.0. Therefore, the protein is closely related to [2Fe-2S] ferredoxins known to be electron donors to monooxygenases involved in hydroxylation of aromatic compounds. Thus, this report provides clear evidence that a putidaredoxin-type [2Fe-2S] ferredoxin, namely Fdx1, is able to transfer electrons to the dioxin dioxygenase of Sphingomonas sp. RW1. [ABSTRACT FROM AUTHOR]

Two slightly differing cDNA clones corresponding to the wound-inducible form of a previously characterized seed myrosinase-associated protein (MyAP) have been isolated from Brassica napus L. The transcripts corresponding to the induced MyAP (iMyAP) were found to be developmentally regulated in various vegetative organs. Both young and old leaves exhibited wound-inducible iMyAP expression. Furthermore, in the young plant, wounding resulted in a systemic increase in leaves located both acropetally and basipetally to the wounded leaf. Also, the iMyAP transcripts were induced by methyl jasmonate, jasmonic acid and abscisic acid. The different inductions could be antagonized by salicylic acid. A general responsiveness in methyl-jasmonate-treated leaves was demonstrated by in situ hybridization. No effect on the amount of iMyAP transcript was detected after feeding the plants with the ethylene precursor 1aminocyclopropane-1-carboxylic acid. The similarity between MyAP and a lipase from Arabidopsis thaliana indicated a possible function in liberating acylated glucosinolates from their acyl group, thereby making them available for hydrolysis by the myrosinase enzymes. We also report on a reduction in the amount of myrosinase transcripts derived from the vegetatively expressed MB-gene family after treatment with exogenously applied salicylic acid or abscisic acid. [ABSTRACT FROM AUTHOR]

The type II DNA-methyltransferase (cytosine N4-specific) M.PvuII was overexpressed in Escherichia coli, starting from the internal translation initiator at Met14. Selenomethionine was efficiently incorporated into this short form of M.PvuII by a strain prototrophic for methionine. Both native and selenomethionyl M.PvuII were purified to apparent homogeneity by a two-column chromatography procedure. The yield of purified protein was approximately 1.8 mg/g bacterial paste. Mass spectrometry analysis of selenomethionyl M.PvuII revealed three major forms that probably differ in the degree of selenomethionine incorporation and the extent of selenomethionine oxidation. Amino acid sequencing and mass spectrometry analysis of selenomethionine-containing peptides suggests that Met30, Met51, and Met261 were only partially replaced by selenomethionine. Furthermore, amino acid 261 may be preferentially oxidized in both native and selenomethionyl form. Selenomethionyl and native M.PvuII were crystallized separately as binary complexes of the methyl donor S-adenosyl-L-methionine in the monoclinic space group P21. Two complexes were present per asymmetric unit. Six out of nine selenium positions (per molecule), including the three that were found to be partially substituted, were identified crystallographically. [ABSTRACT FROM AUTHOR]

Reports on the screening of a cDNA library of timothy grass pollen for homologous isoforms of major group-V allergens by low stringency hybridization with a Phl p 5 (Phleum pratense) probe. Expression of eight isoforms in Escherichia coli K12 and purification to homogeneity; Comparison of immunoglobulin E (IgE) reactivities and allergenic reactivities of three isovariants and an allergen of a different group in a selected group of four grass pollen allergic patients by immunoblot, histamine-release and skin-prick tests.

CYP1 protein is a yeast transcriptional regulator which contains a zinc cluster in its DNA-binding domain. It was recently shown by selecting random CYP1 binding sites that CYP1 protein recognizes with a higher affinity targets containing the CGGNNNTANCGG consensus sequence. Notably, this ideal sequence is however not found in wild-type CYP1 target sites. In order to investigate how CYP1 protein actually binds to its targets, mutations were introduced in three of them (UAS1-A/CYC1, UAS1-A/CYB2, UAS/CYC7) and the consequences towards the binding of purified CYP1-(1-200)-peptide were analyzed. Our data support the following conclusions: (a) When the sequence element contains two CGGs and no TA, both CGGs are essential for binding. (b) If the sequence element contains only the right CGG and the TA, both are sufficient but indispensable for the binding of CYP1. (c) When two CGCs and the TA are present, the right CGC, and not the left one, is essential for the binding of CYP1. (d) CYP1-(1-200)-peptide is usually a monomer in solution but binds DNA as a dimer. Finally, we found evidence for the presence of two half-sites with different measured affinities in the asymmetric sequences of some CYP1 targets. [ABSTRACT FROM AUTHOR]

We previously described the structural organization of P25, a member of the major-intrinsic-protein family found in the digestive tract of homopteran sap-sucking insects [Beuron, E, Le Cahérec, E., Guillam, M. T., Cavalier, A., Garret, A., Tassan, J. P., Delamarche, C., Schultz, P., Mallouh, V., Rolland. J. P., Hubert, J-F., Gouranton, J. & Thomas, D. (1995) J. Biol. Chem. 270, 17414–17422]. We demonstrated, by means of introducing P25 tetramers into the membranes of Xenopus oocytes, that this protein exhibits functional properties similar to those of aquaporin I, the archetypal water channel [Le Cahérec, F., Bron, P., Verbavatz, J. M., Garret, A., Morel, G., Cavalier, A., Bonnec, G., Thomas, D., Gouranton, J. & Hubert. J. F. (1996) .1. Cell Sci. 109, 1285–12951. In the present work, we cloned a full-length cDNA from a Cicadella viridis library with an open reading frame of 765 bp that encoded a 26-kDa protein whose sequence was 43, 40, 36 and 36% identical to aquaporins 1, 2, z and tonoplast intrinsic protein γ, respectively. Translation of the corresponding RNA in Xenopus oocytes generated a polypeptide that was specifically recognized by polyclonal antibodies raised against native P25. Expression of the protein in Xenopus oocyte membranes was assessed by immunocytochemistry and led to a 15-fold increase of osmotic membrane water permeability. This increase was inhibited by HgCl2. The permeability had an Arrberdus activation energy of I 1.7 kJ/mol. We called this protein Cicadella aquaporin (AQPcic). The oocytes expressing Cicadella aquaporin were less sensitive to HgCl2 than oocytes expressing aqua- porin I. In the Xenopus oocyte system. Cicadella aquaporin failed to transport glycerol, urea and ions. It exhibited permeabilities to ethylene glycol and formamide similar to those measured for aquaporin 1 under the same conditions. [ABSTRACT FROM AUTHOR]

Cyclin-dependent kinases (Cdks) play a central role in the regulation of the eukaryotic cell cycle. A novel gene encoding a Cdk-like protein, Pjmrk, has been isolated from the human malaria parasite Plasmodium falciparum. The gene has no introns and comprises an open reading frame encoding a protein of 324 amino acids with a predicted molecular mass of 38 kDa. Database searches revealed a striking simi larity to the Cdk subfamily with the highest similarity to human MO15 (Cdk7). The overall sequence of Pfmrk shares 62% similarity and 46% identity with human MOI5, in comparison to the 49–58% similarity and 34–43% identity with other human Cdks. Pfmrk contains two unique inserts: one consisting of 5 amino acids just before the cyclin-binding motif and the other composed of 13 amino acids within the T-loop equivalent region. Southern blots of genomic DNA digests and chromosomal separations showed that Pfmrk is a single copy gene conserved between several parasite strains and is located on chromosome 10. A 2500-nucleotide transcript of this gene is expressed predominantly in the sexual blood stages (gametocytes). suggesting that Pfmrk may be involved in sexual stage development. [ABSTRACT FROM AUTHOR]

A 30-kDa host factor (polyhedrin-promoter-binding protein; PPBP) specifically binds to sequences critical for transcription from the baculovirus polyhedrin (p29) gene initiator promoter [Burma, S., Mukherjee, B., Jain, A., Habib, S. & Hasnain, S. E. (1994) J. Biol. Chem. 269, 2750–2757; Mukherjee, B., Burma. S. & Hasnain, S. E. (1995) J. Biol. Chem. 270. 4405–4411]. A host factor also binds, in gel shift assays, to the very-late p10 gene promoter through DNA sequence motifs similar to the PPBP · p29 interaction. The p10 · host factor complex was specifically competed out with oligonucleotides containing p29 cognate sequence motifs AATAAA and TAAGTATT, but this did not occur when these motifs were replaced with random sequences. From ultraviolet cross-linking analysis, the molecular mass of this host factor was estimated to be approximately 30 kDa. Experiments were performed to investigate if this host factor displayed any differences in affinity and turnover with respect to the p29 and p10 untranslated leader sequences known to be important for temporal fine tuning and the late burst of transcription. Half-life determination of the p10-binding protein revealed similar binding affinities for the initiator elements of both the promoters, but higher affinity for the p10 5`-untranslated region (≈30 min versus ≈10 min). The involvement of a similar host factor binding to both the p10 and p29 promoters indicates the possibility of a similar mode of transcription initiation from these two very-late promoters. [ABSTRACT FROM AUTHOR]

The promoter of the murine Ki-ras proto-oncogene contains a (C+G)-rich homopurine . homopyrimidine (R . Y) sequence that is essential for transcription activity. We have designed two G-rich oligonucleotides, d(TGGGTGGGTGGTTGGGTGGG) (20GT) and d(AGGGAGGGAGGAAGGGAGGG) (20AG), that have the potential to bind the critical Ki-ras sequence via triplex-helix formation. Band-shift experiments have shown that 20GT binds the Ki-ras R . Y duplex with a delta G value of -40 ±- 5 kJ/mol, while 20AG appeared to have a lower affinity under the experimental conditions adopted: 50 mM Tris/HCl, pH 7.4, 50 mM NaCl, 5 mM MgCl&sub2;, 25 degrees C. In the absence of Mg&sup2+;, 20GT did not bind to the Ki-ras R . Y target, while 20AG exhibited the same affinity observed in the magnesium-containing buffer. To gain insight into the solution properties of 20GT and 20AG, we have performed several experiments including polyacrylamide gel electrophoresis (PAGE), hydroxyapatite chromatography, ultraviolet absorption melting and circular dichroism (CD). We found that 20AG rapidly self-associates into presumably a duplex, even at low concentration (< 1 µM), while 20GT forms aggregates slowly, a process favoured by high oligonucleotide concentrations (> 25 µM). The critical Ki-ras sequence was inserted in Bluescript KS+, downstream from the T7 promoter, to investigate to what extent 20AG and 20GT, which are directed against the R . Y target, are able to inhibit T7 RNA polymerase transcription, under near-physiological conditions. Transcription experiments conducted in vitro at pH 7.4 have shown that oligonucleotide 20GT produced a remarkable repression of T7 RNA polymerase activity in the concentration range (10-25 &micro;M), whereas 20AG had little effect on transcription. [ABSTRACT FROM AUTHOR]