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Properties of and Proteins Associated with the Extracellular ATPase of Chicken Gizzard Smooth Muscle
Abstract
The chicken gizzard smooth muscle extracellular ATPase (ecto-ATPase) is a low abundance, high specific activity, divalent cation-dependent, nonspecific nucleotide triphosphatase (NTPase). The ATPase is a 66-kDa glycoprotein with a protein core of 53 kDa (Stout, J.G. and Kirley, T.L. (1994) J. Biochem. Biophys. Methods 29, 61-75). In this study we evaluated the characteristics of a bank of monoclonal antibodies raised against a partially purified chicken gizzard ecto-ATPase. 18 monoclonal antibodies identified by an ATPase capture assay were tested for effects on ATPase activity as well as for their Western blot and immunoprecipitation potential. The five most promising monoclonal antibodies were used to immunopurify the ecto-ATPase. The one-step immunoaffinity purification of solubilized chicken gizzard membranes with all five of these monoclonal antibodies isolated a 66-kDa protein whose identity was confirmed by N-terminal sequence analysis to be the ecto-ATPase. Several of these monoclonal antibodies stimulated ecto-ATPase activity similar to that observed previously with lectins. Western blot analysis revealed that three of the five monoclonal antibodies recognized a major immunoreactive band at 66 kDa (53-kDa core protein), consistent with previous purification results. The other two antibodies recognized proteins of approximately 90 and 160 kDa on Western blots. The 90-kDa co-immunopurifying (and presumably associated or related) protein was identified by N-terminal analysis as LEP100, a glycoprotein that shuttles between the plasma and lysosomal membranes. The approximately 160-kDa co-immunopurifying protein was identified by N-terminal analysis as integrin, a protein involved in extracellular contacts with adhesion molecules. Extended N-terminal sequence analysis of the immunopurified 66-kDa ecto-ATPase revealed some sequence homology with mouse lysosomal associated membrane protein. Tissue distribution of the ecto-ATPase showed that the highest levels of protein were expressed in muscle tissues (cardiac, skeletal, and smooth) and brain.
... Their physiological role is so far unknown. However, several hypotheses have been proposed, such as (1) protection from cytolytic effects of extracellular ATP (Filippini et al. 1990;Redegeld et al. 1991;Steinberg and Di Virgilio 1991), (2) regulation of ectokinase substrate concentration (Plesner 1995), (3) involvement in signal transduction (Margolis et al. 1990;Dubyak and El-Moatassim 1993;Najjar et al. 1993;Clifford et al. 1997) and (4) involvement in cellular adhesion (Knowles 1995;Stout et al. 1995;Kirley 1997). ...
In this work we describe the ability of living Crithidia deanei to hydrolyze extracellular ATP. In intact cells at pH 7.2, a low level of ATP hydrolysis was observed in the absence of any divalent metal (0.41±0.13 nmol Pi h–1 107 cells–1). The ATP hydrolysis was stimulated by MgCl2 and the Mg2+-dependent ecto-ATPase activity was 4.05±0.17 nmol Pi h–1 107 cells–1. Mg2+-dependent ecto-ATPase activity increased linearly with cell density and with time for at least 60 min. The addition of MgCl2 to extracellular medium increased the ecto-ATPase activity in a dose-dependent manner. At 5 mM ATP, half-maximal stimulation of ATP hydrolysis was obtained with 0.93±0.26 mM MgCl2. This stimulatory activity was also observed when MgCl2 was replaced by MnCl2, but not CaCl2 or SrCl2. The apparent K
m for Mg-ATP2– was 0.26±0.03 mM. ATP was the best substrate for this enzyme; other nucleotides, such as ITP, GTP, UTP and CTP, produced lower reaction rates. In the pH range from 6.6 to 8.4, in which the cells were viable, the acid phosphatase activity also present in this cell decreased, while the Mg2+-dependent ATPase activity did not change. This ecto-ATPase activity was insensitive to inhibitors of other ATPase and phosphatase activities, such as oligomycin, sodium azide, bafilomycin A1, ouabain, vanadate, molybdate, sodium fluoride and tartrate. To confirm that this Mg2+-dependent ATPase was an ecto-ATPase, we used the impermeant inhibitor 4, 4′-diisothiocyanostylbene 2′-2′-disulfonic acid as well as suramin, an antagonist of P2 purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg2+-dependent ATPase activity in a dose-dependent manner. The cell surface location of the ATP-hydrolyzing site was also confirmed by cytochemical analysis.
... Recently, high ecto-nucleotidase activity of several protozoan parasites -including Toxoplasma gondii, Entamoeba histolytica, Leishmania tropica, Leishmania amazonesis, Trypanosoma cruzi, Trypanosoma brucei, and Tritrichomonas foetus -has been shown to interfere with the extracellular signaling of the host and affect the virulence and pathogenesis of these organisms [8,9,10,11,12,13,14,15, 16,17]. Thus, it has been suggested that these enzymes play a role in the pathogenicity of these parasites by controlling the host cell response to infection, specifically by: (i) protecting the parasite from the cytolytic effects of extracellular ATP, (ii) regulating ectokinase substrate concentrations, (iii) preventing activation of signal transduction cascades associated with cellular injury, and (iv) facilitating cellular adhesion [18,19,20,21,22,23,24,25,26,27,28], reviewed in [28]. Among ecto-nucleosidases, Ecto-ATPases, or E-ATPases, are cell-surface enzymes that hydrolyze a range of extracellular nucleoside triphosphates (NTPs) and nucleoside diphosphates (NDPs). ...
Herein, we report the biochemical and functional characterization of a novel Ca(2+)-activated nucleoside diphosphatase (apyrase), CApy, of the intracellular gut pathogen Cryptosporidium. The purified recombinant CApy protein displayed activity, substrate specificity and calcium dependency strikingly similar to the previously described human apyrase, SCAN-1 (soluble calcium-activated nucleotidase 1). CApy was found to be expressed in both Cryptosporidium parvum oocysts and sporozoites, and displayed a polar localization in the latter, suggesting a possible co-localization with the apical complex of the parasite. In vitro binding experiments revealed that CApy interacts with the host cell in a dose-dependent fashion, implying the presence of an interacting partner on the surface of the host cell. Antibodies directed against CApy block Cryptosporidium parvum sporozoite invasion of HCT-8 cells, suggesting that CApy may play an active role during the early stages of parasite invasion. Sequence analyses revealed that the capy gene shares a high degree of homology with apyrases identified in other organisms, including parasites, insects and humans. Phylogenetic analysis argues that the capy gene is most likely an ancestral feature that has been lost from most apicomplexan genomes except Cryptosporidium, Neospora and Toxoplasma.
The publication of a report on the molecular cloning of the rat liver ecto-ATPase1 in 1989 was considered a major break-through in ecto-ATPase research, especially since no other ecto-ATPase had been purified at that time. The extensive homology of the cDNA sequence of the rat liver ecto-ATPase with human biliary glycoprotein I (BGPI) also gave hope that the function of the ecto-ATPases might soon be revealed since functional studies of BGPI and related proteins in the carcinoembryonic antigen (CEA) gene family had begun and a cell adhesion function had been suggested2, 3. Subsequent reports on amino acid sequence similarity of the rat liver ecto-ATPase with a rat liver cell adhesion molecule (cell-CAM 105)4, cross-reactivity of the ecto-ATPase and eell-CAM105 with antibodies generated against the other protein5, and functional assays4–6 unambiguously established that the BGP-like cDNA codes for a cell adhesion molecule. However, the important question of the relationship of ATPase activity and cell adhesion function was not addressed. In later reports where consequences of manipulating the cDNA on cell aggregation were described6–8, there was no concomitant evaluation of the ATPase activity of the mutants.
A variety of ecto-enzyme activities exist on the surfaces of all cells. The ecto-nucleotidases, ecto-ATPase, ecto-ADPase, and apyrase or ATP diphosphohydrolase (ecto-ATPDase), are of particular interest at this workshop. These enzymes are not as well understood as most of the other ecto-enzymes. There have been few attempts to purify these enzymes relative to a large number of descriptive studies. This is due in large part to the fact that these integral membrane glycoproteins (purification of which is particularly difficult) are rarely present in high concentration, and affinity chromatography of these enzymes has not always been successful. This paper will present a general strategy for production of monoclonal antibodies to these enzymes exploiting a new hybridoma screening assay. It will also discuss the use of the resulting monoclonal antibodies for immunopurification of the enzymes. The strategy and methods described have been applied to the purification of the chicken oviduct1 and chicken liver2 ecto-ATP diphosphohydrolase and to the chicken gizzard ecto-ATPase3. This paper will emphasize the methods used for purification of the chicken oviduct enzyme. Specific details for each enzyme have been published in the above references. This review will emphasize general methods which may be of use to other workers.
The plasma membrane Ca2+/Mg2+ ecto-ATPase is an acidic glycoprotein, which hydrolyzes different nucleoside triphosphates and is activated by millimolar concentrations of various divalent cations. Unlike transport ATPases, it does not require Mg-ATP as a substrate and is different from the mitochondrial, myofibrillar, and sarcoplasmic reticulum ATPases. This enzyme is present in all tissues of the body including liver, brain, heart, kidney, blood, platelets, endothelium, and smooth muscles. The Ca2+/Mg2+ ecto-ATPase is considered to play diverse physiological roles such as termination of purinergic transmission, regulation of extracellular ATP concentration, gating mechanism for Ca2+ and Mg2+ fluxes, ATP-driven proton pump, cell-to-cell communication as well as cellular differentiation and transformation in a tissue specific manner. The activity of Ca2+/Mg2+ ecto-ATPase is altered by a wide variety of physiological, pharmacological, and pathological interventions which change membrane fluidity and its composition with respect to cholesterol and phospholipid contents. The molecular weight of this enzyme varies from tissue to tissue in the range of 180–240 kDa with subunits of 90, 80, 67, 20, and 10 kDa. The cDNA sequence for the plasma membrane Ca2+/Mg2+ ecto-ATPase from different tissues show homology with different adhesion molecules including CD36, CD39, and CD70. The evidence in the existing literature suggests that the Ca2+/Mg2+ ecto-ATPase is a multifunctional adhesion molecule which exists in different isoforms in various tissues.
The ecto-ATPase from chicken smooth muscle was solubilized, purified, and characterized. Mono- and polyclonal antibodies were raised and the tissue distribution based on Western analysis was determined. N-terminal and internal protein sequences were determined and used to design degenerate oligonucleotide probes to screen a chicken muscle cDNA library. Two overlapping partial clones encoding most of the ecto-ATPase were isolated and sequenced. A unified theory as to the mechanism by which many varied types of molecules modulate ecto-ATPase activity was developed, and the theory was supported by cross-linking data.
Extracellular nucleotides are now recognized as mediators of immune and non-immune cell function. The effects of extracellular nucleotides also vary with tissue. For example, extracellular ATP in micromolar concentrations can form pores in cell membranes, resulting in osmotic changes that are detrimental to the cell1. In bone marrow and thymocytes, extracellular ATP stimulates DNA synthesis, but it inhibits DNA synthesis in spleen, lymph nodes and peripheral blood lymphocytes2. The nucleotide also has cytostatic and cytotoxic effects on some tumor cells3, but the mechanism is unknown. In addition, ATP triggers histamine secretion from mast cells4–6 and the secretion of granules from neutrophils and monocytes7, 8, but inhibits macrophage-9, 10, NK cell-11–13 mediated cytotoxicities. Furthermore, extracellular ATP may serve as a substrate for ectoprotein kinases14 which have been identified on neutrophils13, erythrocytes16, neuronal cells17 and fibroblasts18.
A variety of nucleotides and the nucleoside adenosine can act as extracellular signaling substances. Their function is terminated by extracellular degradation via surface-located enzymes. The breakdown products may be recycled. This review discusses recent developments in the cellular and molecular biology of enzymes involved in extracellular purine metabolism, including diadenosine polyphosphate hydrolase, ATP-diphosphohydrolase (apyrase), nucleotide pyrophosphatase, 5′-nucleotidase, alkaline phosphatase, NAD-glycohydrolase, and adenosine deaminase. The potential of the surface-located enzymes for ADP-ribosylation and phosphorylation of extracellular proteins is also briefly discussed. Drug Dev. Res. 39:337–352, 1996. © 1997 Wiley-Liss, Inc.
Purines have long been known for their roles in extracellular signaling. One of the most interesting functions to come to light recently has been the involvement, particularly of adenosine 5'-triphosphate (ATP), as a neurotransmitter in the central and the sympathetic nervous system. ATP is stored in and released from synaptic nerve terminals, like other neurotransmitters, and is known to act post-synaptically via specific rapidly-conducting, ligand-gated ion channels, the P2x receptors. Another interesting feature is the discovery that ATP is widely found to be a "co-transmitter" at the same synapses in combination with other neurotransmitters such as noradrenaline, acetylcholine, and GABA, altering our picture of the biophysics and biochemistry of neurotransmission at these synapses. We describe here these and other aspects of neurotransmission by ATP being investigated vigorously today, including recent findings on P2x receptors and those on the synaptic inactivation of ATP by ecto-ATPase. We conclude by pointing out possible pharmacological and clinical implications of neurotransmission by ATP.
Ecto-ATPase, a transmembrane enzyme that catalyzes the hydrolysis of extracellular ATP (ATPJ to ADP and inorganic phosphate, is expressed upon cell activation, Ecto-ATPase is inhibited by non-hydrolyzable ATP analogues, which are competitive inhibitors of the catalytic reaction, and the ATP analogue affinity label, 5′-p-(fluorosulfonyl)benzoyl adenosine (5′-FSBA), which irreversibly inhibits the catalytic activity. These nucleotide antagonists do not cross the cell membrane and are specific for ecto-ATPase in T cells, B cells and NK cells. Inhibition of ecto-ATPase by both reversible and irreversible nucleotide ant agonists results in the inhibition of antigen induced cytokine secretion and cytolytic activity of T cells. Likewise, granule release and cytolytic activity of NK cells as well as antibody secretion and spontaneous proliferation by B-cell hybridomas are inhibited. Inhibition of ecto-ATPase does not influence effector cell-target cell conjugate formation, but acts, in part, by regulating the influx of extracellular calcium that is necessary to maintain cellular activation. Thus, further elucidation of ecto-ATPase regulation and expression and its interaction with intracellular signal transduction events will provide a basis for understanding the role of the hydrolysis of ATPe by ecto-ATPase in lymphocyte effector function.
The amino acid sequence of the ecto-ATPase from rat liver was deduced from analysis of cDNA clones and a genomic clone. Immunoblots with antibodies raised against a peptide sequence deduced from the cDNA sequence indicated that the determined amino acid sequence is that of the ecto-ATPase. The deduced sequence predicts a 519-amino acid protein with a calculated molecular mass of 57,388 daltons. There are 16 potential asparagine-linked glycosylation sites in the protein. Hydropathy analysis of the deduced amino acid sequence indicates that the protein has two hydrophobic stretches. One is located at the N-terminal and the other is near the C-terminal end.
A full-length clone encoding the ecto-ATPase was expressed transiently in mouse L cells and human HeLa cells. The cell lysate from the transfected cells contained immunoreactive ecto-ATPase and Ca²⁺-stimulated ATPase activities. The expressed protein is glycosylated and has an apparent molecular weight (100,000) similar to that of the rat liver plasma membrane ecto-ATPase.
Integrin heterodimers mediate a variety of adhesive interactions, including neuronal attachment to and process outgrowth on laminin. We report here the cloning and primary sequence of an M-200 kD integrin alpha subunit that associates with the integrin beta 1 subunit to form a receptor for both laminin and collagen. Similarities in ligand-binding specificity, relative molecular mass and NH2-terminal sequence make this a strong candidate for the rat homologue of the alpha subunit of the human integrin VLA-1. The full-length rat alpha 1 cDNAs encode a protein containing a purative signal sequence and a mature polypeptide of 1,152 amino acids, with extracellular, transmembrane and cytoplasmic domains. Several structural features are conserved with other integrin alpha chains, including (a) a sequence motif repeated seven times in the NH2-terminal half; (b) potential Ca2+/Mg2+ binding sites in repeats 5, 6, and 7, and (c) alignment of at least 14 of 23 cysteine residues. This rat alpha 1 sequence also contains a 206-amino acid I domain, inserted between repeats 2 and 3, that is homologous to I domains found in the same position in the alpha subunits of several integrins (VLA-2, Mac-1, LFA-1, p150). The rat alpha 1 and human VLA-2 apha subunits share greater than 50% sequence identity in the seven repeats and I domain, suggesting that these sequence identities may underlie some of their similar ligand-binding specificities. However, the rat integrin alpha 1 subunit has several unique features, including a 38-residue insert between two Ca2+/Mg2+ binding domains, and a divergent 15-residue cytoplasmic sequence, that may potentially account for unique functions of this integrin.
A complete set of chimeras was made be~ tween the lysosomal membrane glycoprotein LEP100 and the plasma membrane-directed vesicular stomatitis virus G protein, combining a glycosylated lumenal or ectodomain, a single transmembrane domain, and a cytosolic carboxyl-terrninal domain. These chimeras, the parent molecules, and a truncated form of LEPI00 lacking the transmembrane and cytosolic domains were expressed in mouse L cells. Only LEP100 and chimeras that included the cytosolic 11 amino acid carboxyl terminus of LEP100 were targeted to lyso-somes. The other chimeras accumulated in the plasma membrane, and truncated LEP100 was secreted. Chimeras that included the extracellular domain of vesicular stomatitis G protein and the carboxyl terminus of LEP100 were targeted to lysosomes and very rapidly degraded. Therefore, in chimera-expressing cells, virtually all the chimeric molecules were newly synthesized and still in the biosynthesis and lysosomal targeting pathways. The behavior of one of these chimeras was studied in detail. After its processing in the Golgi apparatus, the chimera entered the plasma membrane/endosome compartment and rapidly cycled between the plasma membrane and endosomes before going to lysosomes. In pulse-expression experiments, a large population of chimeric molecules was observed to appear transiently in the plasma membrane by im-munofluorescence microscopy. Soon after protein synthesis was inhibited, this surface population disappeared. When lysosomal proteolysis was inhibited, chimeric molecules accumulated in lysosomes. These data suggest that the plasma membrane/early endo-some compartment is on the pathway to the lysosomal membrane. This explains why mutations that block endocytosis result in the accumulation of lysosomal membrane proteins in the plasma membrane.
A simple method is described for promoting the fusion of mouse myeloma cells in suspension with polyethylene glycol (PEG 1000). By carefully controlling the concentration of PEG and the time of exposure of the cells, it was possible to obtain hybridization frequencies several-hundred-fold higher than those obtained with Sendai virus.
A method for the co-purification of calponin and caldesmon from bovine aorta smooth muscle was established involving heat treatment, ammonium sulfate fractionation (0–30 and 30–50%), CM52 column chromatography, and Ultrogel AcA44 gel filtration. The yields of calponin and caldesmon from 200 g of smooth muscle were 17 and 32 mg, respectively. This simplified, fast, and efficient method for the purification of calponin and caldesmon constitutes a useful tool for studying thin filament-linked regulation of smooth muscle contraction.
Transverse tubule (TT) membranes isolated from chicken skeletal muscle possess a very active magnesium-stimulated ATPase (Mg-ATPase) activity. The Mg-ATPase has been tentatively identified as a 102-kD concanavalin A (Con A)-binding glycoprotein comprising 80% of the integral membrane protein (Okamoto, V.R., 1985, Arch. Biochem. Biophys., 237:43-54). To firmly identify the Mg-ATPase as the 102-kD TT component and to characterize the structural relationship between this protein and the closely related sarcoplasmic reticulum (SR) Ca-ATPase, polyclonal antibodies were raised against the purified SR Ca-ATPase and the TT 102-kD glycoprotein, and the immunological relationship between the two ATPases was studied by means of Western immunoblots and enzyme-linked immunosorbent assays (ELISA). Anti-chicken and anti-rabbit SR Ca-ATPase antibodies were not able to distinguish between the TT 102-kD glycoprotein and the SR Ca-ATPase. The SR Ca-ATPase and the putative 102-kD TT Mg-ATPase also possess common structural elements, as indicated by amino acid compositional and peptide mapping analyses. The two 102-kD proteins exhibit similar amino acid compositions, especially with regard to the population of charged amino acid residues. Furthermore, one-dimensional peptide maps of the two proteins, and immunoblots thereof, show striking similarities indicating that the two proteins share many common epitopes and peptide domains. Polyclonal antibodies raised against the purified TT 102-kD glycoprotein were localized by indirect immunofluorescence exclusively in the TT-rich I bands of the muscle cell. The antibodies substantially inhibit the Mg-ATPase activity of isolated TT vesicles, and Con A pretreatment could prevent antibody inhibition of TT Mg-ATPase activity. Further, the binding of antibodies to intact TT vesicles could be reduced by prior treatment with Con A. We conclude that the TT 102-kD glycoprotein is the TT Mg-ATPase and that a high degree of structural homology exists between this protein and the SR Ca-ATPase.
Using surface immunoprecipitation at 37 degrees C to "catch" the transient apical or basolateral appearance of an endogenous MDCK lysosomal membrane glycoprotein, the AC17 antigen, we demonstrate that the bulk of newly synthesized AC17 antigen is polarly targeted from the Golgi apparatus to the basolateral plasma membrane or early endosomes and is then transported to lysosomes via the endocytic pathway. The AC17 antigen exhibits very similar properties to members of the family of lysosomal-associated membrane glycoproteins (LAMPs). Parallel studies of an avian LAMP, LEP100, transfected into MDCK cells revealed colocalization of the two proteins to lysosomes, identical biosynthetic and degradation rates, and similar low levels of steady-state expression on both the apical (0.8%) and basolateral (2.1%) membranes. After treatment of the cells with chloroquine, newly synthesized AC17 antigen, while still initially targeted basolaterally, appears stably in both the apical and basolateral domains, consistent with the depletion of the AC17 antigen from lysosomes and its recycling in a nonpolar fashion to the cell surface.
Extracellular ATP, at micromolar concentrations, induces significant functional changes in a wide variety of cells and tissues. ATP can be released from the cytosol of damaged cells or from exocytotic vesicles and/or granules contained in many types of secretory cells. There are also efficient extracellular mechanisms for the rapid metabolism of released nucleotides by ecto-ATPases and 5'-nucleotidases. The diverse biological responses to ATP are mediated by a variety of cell surface receptors that are activated when ATP or other nucleotides are bound. The functionally identified nucleotide or P2-purinergic receptors include 1) ATP receptors that stimulate G protein-coupled effector enzymes and signaling cascades, including inositol phospholipid hydrolysis and the mobilization of intracellular Ca2+ stores; 2) ATP receptors that directly activate ligand-gated cation channels in the plasma membranes of many excitable cell types; 3) ATP receptors that, via the rapid induction of surface membrane channels and/or pores permeable to ions and endogenous metabolites, produce cytotoxic or activation responses in macrophages and other immune effector cells; and 4) ADP receptors that trigger rapid ion fluxes and aggregation responses in platelets. Current research in this area is directed toward the identification and structural characterization of these receptors by biochemical and molecular biological approaches.
The α-subunit of an abundant chick gizzard integrin was isolated (T. Kelly, L. Molony, and K. Burridge, 1987, J. Biol. Chem. 262, 17,189–17,199) and fragmented by proteolytic digestion. The N-terminal sequences of the intact polypeptide and of several internal peptides were determined and were found to be highly homologous to the mammalian integrin α1-subunit. Monoclonal antibodies to the chick integrin β1-chain react on immunoblots with the gizzard integrin β-subunit (U. Hofer, J. Syfrig, and R. Chiquet-Ehrismann, 1990, J. Biol. Chem. 265, 14,561–14,565). The chain composition of the abundant chick gizzard integrin is therefore α1β1. Polyclonal antibodies to the avian integrin α1-subunit block attachment of embryonic gizzard cells to human and chick collagen IV completely and inhibit attachment to mouse Engelbreth-Holm-Swarm (EHS) tumor laminin partially. In ELISA-style receptor assays, the isolated α1β1 integrin bound to human and chick collagen IV and to mouse EHS tumor and chick heart laminin. While the binding to collagen IV was abolished by removal of divalent cations, the binding to laminin was not sensitive to EDTA under the conditions used. Collagen I bound the isolated avian α1β1 integrin only weakly. As collagen IV was the only extracellular matrix protein for which a consistent, divalent cation-dependent, binding to the avian α1β1 integrin could be demonstrated in both cellular and molecular assays we suggest that it is a preferred ligand for this integrin.
The potential to exploit self-incompatibility (SI) in plant breeding and its attraction as a system in which to investigate the molecular basis of cell-cell recognition and signaling has resulted in it becoming one of the most intensively studied and exciting topics in plant biology. Much research has encompassed the molecular cloning of genes involved in SI; consequently many reviews of this subject have focused on this area. In this article we have attempted to strike a somewhat different balance, with the objective of giving as broad a picture of the topic of SI as is possible within the confines of a single article. We provide a comprehensive summary of the genetics and population genetics of SI and, in describing progress toward the elucidation of the molecular basis of SI, have surveyed a much wider range of species than has previously been reviewed.