Cloning of dexamethasone-induced transcript: a novel glucocorticoid-induced gene that is upregulated in emphysema.

To identify changes in gene expression associated with emphysema, we used differential display to compare RNA extracted from emphysematous lungs with that of unused donor tissues taken at the time of transplant. A differentially expressed sequence was identified corresponding to the 3' end of a novel human complementary DNA (cDNA) of unknown function. The human and mouse cDNA sequences were completed by 5' rapid amplification of cDNA ends. We have named it DEXI for dexamethasone-induced transcript. DEXI messenger RNA (mRNA) was upregulated 147% in emphysematous tissue compared with donor tissue. DEXI mRNA was also upregulated 230% by dexamethasone treatment of A549. The increase in expression of DEXI found in emphysema patients' tissues may be owing to their known treatment with corticosteroids. The human DEXI gene is intronless and the predicted open reading frame encodes a 95-residue acidic protein. Database searches revealed the presence of homologues only in mammals, and a human pseudogene. The protein has a predicted central transmembrane domain and a carboxy-terminal leucine zipper. The human mRNA has a single 1.3-kb transcript. We suggest that the increased expression of DEXI in emphysema may either be relevant to disease progression or be indicative of glucocorticoid responsiveness in treated patients.

Analysis of DEXI/Dexi refines the organization of the mouse 7C and human 15q11-->q13 imprinting clusters.

Identification of imprinted genes in the Prader-Willi/Angelman syndrome deletion region is complicated by the presence of large flanking repeats. While inactive copies of DEXI are located within the repeats, we have now localized the active DEXI gene to 15q11-->q13 outside the PWS/AS deletion and Dexi to mouse chromosome 16, suggesting complex evolution of this genomic region in both species.

Gene-expression profiling of human osteoblasts following treatment with the ionic products of Bioglass 45S5 dissolution.

The effect of the ionic products of Bioglass 45S5 dissolution on the gene-expression profile of human osteoblasts was investigated by cDNA microarray analysis of 1,176 genes. Treatment with the ionic products of Bioglass 45S5 dissolution increased the levels of 60 transcripts twofold or more and reduced the levels of five transcripts to one-half or less than in control. Markedly up-regulated genes included RCL, a c-myc responsive growth related gene, cell cycle regulators such as G1/S specific cyclin D1, and apoptosis regulators including calpain and defender against cell death (DAD1). Other significantly up-regulated genes included the cell surface receptors CD44 and integrin beta1, and various extracellular matrix regulators including metalloproteinases-2 and -4 and their inhibitors TIMP-1 and TIMP-2. The identification of differentially expressed genes by cDNA microarray analysis has offered new insights into the mode of action of bioactive glasses and has proven to be an effective tool in evaluating their osteoproductive properties.

Flotillin-1: gene structure: cDNA cloning from human lung and the identification of alternative polyadenylation signals.

To identify changes in gene expression associated with emphysema, differential display was used to compare RNA extracted from emphysematous lung with that of unused donor tissue taken at the time of transplant. Two expressed clones with sequence homology to the 3' UTR of the murine flotillin-1 cDNA were identified. Flotillin-1 is a plasma membrane protein, which has been associated with detergent-insoluble glycolipid-rich domains and the formation of caveolae. One clone was 95 bp longer than the other. It arose from the use of a second polyadenylation signal and its existence was not due to differential expression nor to polymorphisms in the human flotillin-1 sequence. The 1839 bp human flotillin-1 sequence was completed by 5' RACE from a lung cDNA library. The human mRNA has a 1.9 kbase transcript being highly expressed in brain, heart and lung. The single copy flotillin-1 gene is located at 6p21.3 in the MHC class I region and consists of 13 exons over 15 kb. The ORF encodes a 427 residue protein with a molecular mass 47355 Da, and an isoelectric point 7.08. Human flotillin-1 has a 98% identity with the murine protein and a 47% identity with human flotillin-2. Flotillin-1 belongs to the Band 7.2/stomatin protein family, possessing a hydrophobic N-terminal region, predicted to form a single, outside to inside, transmembrane domain. The long central alpha-helical domain may form a coiled-coil. We have isolated and characterised a cDNA encoding the human flotillin-1 gene, which may play an important role in raft formation.

Human homologues of yeast vacuolar protein sorting 29 and 35.

In the yeast Saccharomyces cerevisiae, a membrane coat complex is required for endosome to Golgi retrograde transport. The vacuolar protein sorting proteins Vps29p, Vps35p, and Vps26p are required for pre-vacuolar/late endosome to Golgi retrieval of the vacuolar hydrolase receptor Vps10p. They form a cargo recognition and concentration subcomplex, termed the inner shell of the retromer coat, prior to vesicle formation by the addition of the membrane-deforming outer shell. We have cloned the human and murine homologues of yeast Vps29p and the human homologue of Vps35p. They encode 182 and 796 residue proteins, with 43 and 29% identity to their respective yeast. The 10.5 kb, 5 exon, VPS29 gene is located on chromosome 12q24 and the 29.6 kb, 17 exon, VPS35 gene is on chromosome 16. In humans, Vps29p, Vps35p, and Hbeta58, the homologue of Vps26p, may form an inner shell of the retromer coat similar to that found in yeast.

Ionic products of bioactive glass dissolution increase proliferation of human osteoblasts and induce insulin-like growth factor II mRNA expression and protein synthesis.

Bioglass 45S5 is an osteoproductive material, which resorbs by releasing its constitutive ions into solution. Treatment with the ionic products of Bioglass 45S5 dissolution in DMEM for 4 days increased human osteoblast proliferation to 155% of control. Two days after treatment, differential gene expression was analyzed by cDNA microarrays. Expression of a potent osteoblast mitogenic growth factor, insulin-like growth factor II (IGF-II), was increased to 290%. Additionally, there was a 168% increase in the concentration of unbound IGF-II protein in the conditioned media of treated osteoblasts. Expression levels of IGFBP-3, an IGF-II carrier protein, metalloproteinase-2 and cathepsin-D were also increased to 200, 340, and 310% of control levels, respectively. Metalloproteinase-2 and cathepsin-D are proteases that cleave IGF-II from its carrier proteins, resulting in the release of the unbound biologically active IGF-II. We suggest that the stimulatory effect of the ionic products of Bioglass 45S5 dissolution on osteoblast proliferation may be mediated by IGF-II.

Molecular cloning of the human and murine 2-amino-3-ketobutyrate coenzyme A ligase cDNAs.

The conversion of L-threonine to glycine in both prokaryotes and eukaryotes takes place through a two-step biochemical pathway involving the enzymes L-threonine dehydrogenase (EC 1.1.1103) and 2-amino-3-ketobutyrate coenzyme A ligase (KBL; EC 2.3.1.29). The genes encoding these enzymes have been described in prokaryotes but not in eukaryotes. We report the cloning of transcripts for KBL, the second enzyme in the pathway, from human and murine lung and a partial transcript from bovine liver. Two peptide sequences from the purified bovine KBL protein, one from the N-terminus and the other from the peptide containing the pyridoxal 5'-phosphate-binding lysine residue [Tong, H. & Davis, L. (1994) J. Biol. Chem. 269, 4057-4064], are identical with regions of the conceptual translation of the transcript obtained from bovine liver. The partial transcript from bovine liver was very similar to the human transcript, being 91% and 92% identical at the nucleotide and amino-acid levels, respectively. The human and murine KBL transcripts are 1.5 kb long, with ORFs encoding proteins of 419 and 416 residues, respectively. The mouse protein has 90% identity with the human protein. The human transcript is strongly expressed in heart, brain, liver and pancreas compared with the lung. The N-termini of both human and mouse proteins have characteristics of mitochondrial import sequences. Both human and murine proteins have 54% identity with the well-characterised prokaryote KLB protein from Escherichia coli. Database searches with the human cDNA sequence enabled us to identify the human KBL gene on chromosome 22q12-13, consisting of nine exons over 9 kb, and a hypothetical Caenorhabditis elegans KLB gene on chromosome IV, consisting of five exons over 2 kb.

Neutrophils enhance expression of inducible nitric oxide synthase in human normal but not cystic fibrosis bronchial epithelial cells.

The bronchial epithelium in cystic fibrosis (CF) expresses very low levels of the inducible form of nitric oxide synthase (iNOS). The product of iNOS, nitric oxide (NO), mediates anti-microbial effects and can reduce neutrophil sequestration in the lung. Heavy neutrophilic infiltration of the pulmonary epithelium is a major feature of the end-stage CF lung. This study hypothesized that the system whereby the pulmonary epithelium protects itself against exaggerated neutrophilic infiltration by producing NO is compromised in CF. Human neutrophils were activated by incubation with cytokines, added to monolayers of normal (16HBE14o-) and CF (CFBE41o-) bronchial epithelial cells and co-cultured for up to 72 h. Marked up-regulation of iNOS protein expression was seen in normal bronchial epithelial cells following neutrophil co-culture but the CF cells showed a significantly smaller increase (p<0.001). To determine whether the relative lack of protein was due to a defect in translation, RT-PCR of iNOS mRNA was carried out and a pattern of mRNA expression was seen paralleling that of the protein. The reduced production of NO by CF compared with normal epithelium was shown by the presence of significantly (p<0.001) less accumulated nitrites in medium after co-culture with neutrophils. In summary, this study shows that the normal production of NO by bronchial epithelium in response to contact with neutrophils is lacking in CF. As NO has been shown to oppose neutrophil sequestration, its relative lack in CF may underlie the heavy neutrophilic infiltration that characterizes the disease.

Inhibition of dendrite formation in mouse melanocytes transiently transfected with antisense DNA to myosin Va.

In mice a molecular motor of the myosin V class (designated myosin Va) is known to be the product of the dilute locus, where a mutation prevents melanosome transport in melanocytes. There is conflicting evidence about whether it has a role in dendrite outgrowth. We investigated its role by transiently transfecting antisense oligonucleotides to inhibit its expression in a melanocyte cell line. We demonstrated mRNA and protein expression of myosin Va in 3 mouse melanocyte lines and 1 human melanoma cell line, using RT-PCR and immunoblotting. Two splice variants were found in human cells whilst only the longer transcript, containing an additional exon, was present in mouse melanocyte lines. The shorter variant was detected in other mouse tissues. Myosin Va protein levels were similar in 3 melanocyte lines with differing amounts of pigmentation, indicating that expression of myosin Va is not tightly coupled to expression of melanin. Immunocytochemistry showed 2 types of myosin Va localisation. A punctate pattern of staining concentrated in the perinuclear region was indicative of organelle association, and the observation of occasional linear punctate staining aligned with F-actin bundles supported the idea that myosin Va has a role in transporting melanosomes along actin filaments. Staining was also intense at tips of dendrites and at sites of dendrite-cell contact, consistent with a possible role in dendrite growth. Transient transfection of antisense phosphorothioate oligodeoxynucleotides targeted against myosin Va mRNA reduced expression of myosin Va protein in cultured mouse melan-a melanocytes by over 70 % 20 h after transfection whereas a control (shuffled sequence) oligonucleotide did not. Upon trypsinisation and replating these cells the capacity of the transfected cells to extend new dendrites was reduced in the cells containing the specific antisense oligonucleotides but unaffected by the control oligonucleotide. Image analysis confirmed that the effect of transfection on morphology was statistically significant (P < 0.01). In contrast when cells were not trypsinised and replated following transfection so that previously existing dendrites could persist, the normal dendritic morphology continued to be observed. We conclude that in addition to its involvement in melanosome transport, myosin Va has a role in the extension of new dendrites by melanocytes but not in maintenance of pre-existing dendrites.

X-linked sideroblastic anaemia due to a mutation in the erythroid 5-aminolaevulinate synthase gene leading to an arginine170 to leucine substitution.

DNA sequencing of the coding region of the erythroid 5-aminolaevulinate synthase (ALAS2) cDNA from a male with pyridoxine-responsive sideroblastic anaemia revealed a missense mutation, a G561T transversion in exon 5 of the gene. Previously, the mutation G561A has been shown to be responsible for sideroblastic anaemia in females and thought to be lethal in males (1). The mutation G561T results in the loss of an MspA1-I cutting site. Analysis of MspA1-I restriction enzyme digests of amplified exon 5 genomic DNA from other family members revealed that the proband's mother, aunt and youngest sister, who were not anaemic, were heterozygous carriers of the mutation. The G561T mutation results in an arginine to leucine substitution at amino acid residue 170. This arginine residue is conserved in both the erythroid and housekeeping ALAS in vertebrates as well as in all other known ALAS proteins and is located in a predicted alpha-helix region close to the amino-terminus of the enzymatic region of the protein.

Hereditary sideroblastic anaemia due to a mutation in exon 10 of the erythroid 5-aminolaevulinate synthase gene.

DNA sequencing of the coding region of the erythroid 5-aminolaevulinate synthase (ALAS2) cDNA from a male with pyridoxine-responsive sideroblastic anaemia revealed a missense mutation C1622G and a closely linked polymorphism C1612A in exon 10 of the gene. Sequence analysis of the genomic DNA from other family members revealed that the proband's mother and daughter were heterozygous carriers of the mutation, consistent with the X-linked inheritance. The C1622G mutation results in a histidine to aspartic acid substitution at amino acid residue 524. The histidine residue is conserved in both the erythroid and housekeeping ALAS proteins in vertebrates, all other known ALAS proteins and other oxamine synthases that have pyridoxal 5'-phosphate as a co-factor. This histidine is located in a predicted loop, preceding a long alpha-helix region near the carboxy-terminus.

Loss of cell surface microvilli on rat basophilic leukaemia cells precedes secretion and can be mimicked using the calmodulin antagonist trifluoperazine.

OBJECTIVE: We studied changes in cell surface morphology following treatment with secretagogue or trifluoperazine in a mast cell model. MATERIALS AND METHODS: Rat basophilic leukaemia (RBL) cells were treated with antigen and or the calmodulin antagonist, 0-50 microM trifluoperazine (TFP). The release of a secretory granule enzyme, beta-hexosaminidase, into the external medium was used as a measure of secretion. Quantitation of cell surface microvilli was determined by using a computer with input from a digitising tablet from scanning electron micrographs. Cytoskeletal proteins present in microvilli were analysed by confocal immunofluorescence. RESULTS: When RBL cells are stimulated to secrete with an antigen, the cell surface is transformed from a microvillous morphology to a ruffled one. The cell surface rearrangement preceded beta-hexosaminidase secretion: the majority of microvilli disappeared rapidly after stimulation (t1/2 of 39 s) whereas secretion can only be measured after a lag of 47 s. The calmodulin antagonist, TFP did not inhibit antigen-induced secretion or loss of microvilli, however TFP alone caused a similar loss of microvilli but was unable to stimulate or potentiate secretion. The microvilli mostly disappeared within 30 s, and a half-maximal effect occurred at approximately 8 microM TFP. Using immunofluorescence, calmodulin was localized to punctate structures on the dorsal cell surface which presumably correspond to the microvilli, and which also stained for F-actin and myosin I. CONCLUSIONS: Loss of cell surface microvilli on RBL cells precedes secretion and could reflect a cytoskeletal rearrangement which facilitates fusion of secretory granules with the membrane. It can be mimicked using trifluoperazine and we suggest it may involve calmodulin-binding components of the microvillus cytoskeleton such as myosin I.

Circular ruffle formation in rat basophilic leukemia cells in response to antigen stimulation.

Rat basophilic leukemia cells have previously been described to undergo striking cell surface changes after IgE-mediated stimulation of histamine secretion, whereby the dorsal surface loses its microvilli and acquires characteristic wavy ruffles. We have found using scanning electron microscopy, phase contrast and immunofluorescence, that a proportion of these cells also exhibit the formation of circular membrane ruffles on their dorsal surface after exposure to an IgE-directed secretagogue; some cells also develop circular membrane ruffles following stimulation by phorbol myristate acetate or by calcium ionophore A23187. A flattened morphology appears to be linked to circular membrane ruffle formation in that these ruffles were found in areas of presumed cell spreading which are largely devoid of intermediate filaments and displaced to one side of the cell's nucleus, and they were not observed on rounded cells. This is in contrast to the wavy ruffles which are found on the entire cell surface including the region overlying the nucleus, and which are observed in rounded cells as well as spread cells. Circular ruffle formation and secretion are triggered by similar concentrations of antigen, but the circular ruffles are formed more slowly and only become abundant at times after most of the histamine has been released. The circular membrane ruffles showed no obvious association with endocytosis, as detected using fluorescein isothiocyanate-dextran as a fluid phase marker. The position of accumulation of endocytotic vesicles occurring subsequent to secretion was not found to be related to the circular membrane ruffles, but was observed around the nucleus. Circular membrane ruffles contain F-actin, and their formation is prevented by cytochalasin D. At least three types of myosin, types I, II and V are present and presumably play a role in circular ruffle formation.

Identification of an arginine452 to histidine substitution in the erythroid 5-aminolaevulinate synthetase gene in a large pedigree with X-linked hereditary sideroblastic anaemia.

The coding region of the erythroid 5-aminolaevulinate synthetase gene (ALAS2) from a large pedigree with pyridoxine-responsive X-linked hereditary sideroblastic anaemia was examined for mutations. In three affected males from this pedigree, single strand conformational polymorphism (SSCP) analysis showed anomalous migration of a PCR product spanning exon 9. Sequencing of amplified genomic DNA from one of these affected males revealed a guanine to adenine transition at nucleotide 1407 of the cDNA sequence in exon 9 of the gene. This mutation results in the loss of an HhaI restriction enzyme digest site. An HhaI digest assay demonstrated the presence of this mutation in other affected males but not in unaffected males and unrelated individuals. The point mutation results in an arginine to histidine substitution at amino acid residue 452. The arginine residue is conserved in both the erythroid and housekeeping ALAS genes in all known vertebrate sequences. This arginine is located in the middle of a predicted alpha-helix.

Analysis of three deletion breakpoints in Xp21.1 and the further localization of RP3.

The gene responsible for X-linked retinitis pigmentosa (xlRP) in Xp21.1 (RP3) was initially localized by deletion analysis to within a 150- to 170-kb region between the CYBB locus and the proximal deletion junction (BBJPROX) from a patient, BB, who suffered from Duchenne muscular dystrophy (DMD), McLeod syndrome, chronic granulomatous disease (CGD), and xlRP. This gene has recently been isolated and was found to be located outside and 400 kb proximal to the BB deletion. Further analysis of BBJPROX has identified the breakpoint junction sequence, showing that it occurs within an Alu repetitive element on the proximal side but with no significant homology to the distal sequence in dystrophin intron 30. Analysis of an overlapping deletion in patient NF, who suffered from DMD, CGD, and McLeod syndrome, shows that this deletion is within 4 kb but extends centromeric to BBJPROX, consistent with the location of RP3 outside the BB deletion region. A sequence with strong homology to a THE-1 transposon-like element was identified 7-13 kb from the proximal BB and NF breakpoints. These elements have been implicated in several highly unstable genomic regions. A third overlapping deletion, in a patient, SB, who suffered from CGD, McLeod syndrome, and xlRP, has here been shown to extend 380 kb proximal to the NF breakpoint, consistent with the finding that RP3 lies outside the BB deletion. This deletion has now been shown to disrupt the RP3 (RPGR) gene. The reason for the retinitis pigmentosa phenotype in patient BB remains unclear, but the most likely explanations include a long-range chromosomal position effect, a small secondary rearrangement, and the presence of a coincident autosomal form of retinitis pigmentosa.

Chicken myosin IB mRNA is highly expressed in lymphoid tissues.

Little is known about the functions of members of the myosin I family in vertebrates. Chicken myosin IB is a member of the amoeba-type subclass of myosin I molecules and tissue localisation studies may provide possible clues to the functions of these myosin I molecules. The expression of the mRNA of this unconventional myosin IB was analysed by in situ hybridization and compared with that of the well characterised brush border myosin I on frozen sections of tissues from the adult domestic chicken. High levels of myosin IB mRNA were found in the intestine and spleen, but were not found in other tissues examined such as brain, heart, lung, liver and kidney. In the intestine, myosin IB mRNA was much more abundant in the lamina propria than in the enterocytes, whereas brush border myosin I mRNA was restricted to the enterocytes. In the spleen, myosin IB mRNA expression was abundant in regions of white pulp, namely germinal centres, periellipsoid lymphocyte sheaths and periarteriolar lymphocyte sheaths. Lymphocytes are the major cell type in both the lamina propria and the white pulp of the spleen, which suggests that chicken myosin IB is highly expressed in lymphocytes. Lymphocyte recirculation depends on their migration through the endothelial layer and it is possible that myosin IB may have a role to play in this type of cell motility.

Identification of a novel gene, ETX1 from Xp21.1, a candidate gene for X-linked retintis pigmentosa (RP3).

A novel gene encoding a 2.2 kilobase transcript has been isolated from the Xp21.1 region of the human X chromosome by exon amplification. The gene, called EXT1, spans 80 kilobases and contains 12 exons, at least two of which are alternatively spliced and have predicted products of 464 and 471 amino acids respectively. Conceptual translation of the open reading frames shows one product with a 30 amino acid signal peptide, which is absent from the alternative transcript, followed by three complement control protein domains, a hydrophobic region with a possible role in membrane anchorage and short 17 amino acid putative cytoplasmic carboxyl terminus. An alternative first exon contains a 39 amino acid open reading frame which is rich in serine and threonine residues and contains a potential chondroitin/dermatan sulphate attachment site. Northern analysis showed ETX1 expression within the retina and heart with lower levels in several other tissues. Since ETX1 lies within the region thought to contain the x-linked retinitis pigmentosa (xIRP) gene, RP3, it was screened for mutation within a set of 45 xIRP patients using single strand conformation analysis and/or chemical cleavage of mismatch using reverse transcription/polymerase chain reaction amplification of polyA+RNA from blood cells. Three low frequently variants (17-23Ldel, P225S, S413F) were found in both patients and controls; one of which (P225S) was found in four of 45 unrelated patient chromosomes and one of 178 control chromosomes (p <0.001). The allelic association between P225S and xIRP alleles suggests a common ancestral chromosome bearing the P225S variant and an RP3 mutation at a neighbouring locus.

Gel electrophoresis of native gelsolin and gelsolin-actin complexes.

BHK gelsolin migrated on non-denaturing 8-25% polyacrylamide gels with an apparent molecular mass of 80 kDa. In the absence of Ca2+ no complex formation occurred between BHK gelsolin and actin. In the presence of Ca2+ two complex species were found: a ternary complex, GA2, of apparent molecular mass of 210 kDa at gelsolin:actin ratio of 1:2, and a novel quaternary complex, GA3, of apparent molecular mass of 247 kDa when actin was in excess. Both cytoplasmic and plasma gelsolin species form GA3 with skeletal muscle actin. No complexes larger than GA3 were observed. The formation of GA3 involves the binding of a third actin to the gelsolin molecule at the site previously assumed to be masked, rather than to the actin molecules already present in GA2. In preference to GA3, GA2 was incorporated into actin filaments stabilized with phalloidin. On chelation of free Ca2+, both GA2 and GA3 dissociated to form the EGTA stable binary complex (GA) with an apparent molecular mass of 140 kDa and free actin.

Isolation and characterization of gelsolin from cultured BHK cells.

The Ca2+-dependent actin-polymerization nucleating protein of the cytoplasmic fraction of Baby hamster kidney (BHK) C13 cells has been isolated by anion-exchange, hydroxyapatite and gel-filtration chromatography. This protein has been identified as a cytoplasmic gelsolin by the following criteria: molecular mass of 90 kDa on SDS-PAGE, immunocrossreactivity with pig plasma gelsolin and similar actin-binding properties to gelsolins purified from other sources. BHK gelsolin forms a 1:2 ternary complex with rabbit muscle actin that is dependent on the presence of Ca2+. The ternary complex is dissociated on chelation of Ca2+ with EGTA to a binary complex and free actin. BHK gelsolin nucleates the polymerization of pyrene-labelled G-actin in a Ca2+-dependent manner. The proportion of unpolymerized monomer is increased in the presence of BHK gelsolin by an amount consistent with capping of the positive filament ends. The rate of actin depolymerization induced by diluting F-actin to below its critical concentration (Cc) is unaffected by the presence of BHK gelsolin in EGTA. However, in the presence of Ca2+ the rate of depolymerization is increased indicating that BHK gelsolin severs actin filaments in a Ca2+-dependent manner.

Gel electrophoresis of native actin and the actin-deoxyribonuclease I complex.

Electrophoresis of monomeric actin (G-actin) on 8-25% acrylamide Pharmacia PhastGels was carried out using gels and agarose buffer strips preequilibrated in buffer containing adenosine triphosphate (ATP), calcium ions (Ca2+) and dithiothreitol. On these gels G-actin ran as a sharp band at an apparent molecular mass of 45 kDa relative to standard proteins which is slightly greater than its actual molecular mass of 42 kDa. Electrophoresis in the absence of these solutes led to denaturation and aggregation of the protein, as reflected by a long streak. Filamentous actin (F-actin) did not enter the gel. The actin monomer-binding protein, deoxyribonuclease I, (DNase I) forms a binary complex with G-actin. The purity and apparent molecular mass 74 kDa of this complex were determined by native gel electrophoresis. By the simple procedure of preequilibrating both gel and buffer strips with appropriate ligands, this technique could be extended to investigate interactions between actin and other G-actin-binding proteins and other proteins whose stability is ligand dependent.