Spontaneous neural activity of the anterodorsal lobe and entopeduncular nucleus in adult zebrafish: a putative homologue of hippocampal sharp waves.

Spontaneous neural activity is instrumental in the formation and maintenance of neural circuits that govern behavior. In mammals, spontaneous activity is observed in the spinal cord, brainstem, diencephalon, and neocortex, and has been most extensively studied in the hippocampus. Using whole-brain in vitro recordings we establish the presence of spontaneous activity in two regions of the zebrafish telenchephalon: the entopeduncular nucleus (EN) and the anterodorsal lobe (ADL). The ADL is part of the lateral telencephalic pallium, an area hypothesized to be functionally equivalent to the mammalian hippocampus. In contrast, the EN has been hypothesized to be equivalent to the mammalian basal ganglia. The observed spontaneous activity is GABA modulated, sensitive to glutamate and chloride transporter antagonists, and is abolished by sodium pump blockers; moreover, the spontaneous activity in the ADL is a slow multiband event (∼100 ms) characterized by an embedded fast ripple wave (∼150-180 Hz). Thus, the spontaneous activity in the ADL shares physiological features of hippocampal sharp waves in rodents. We suggest that this spontaneous activity is important for the formation and maintenance of neural circuits in zebrafish and argue that applying techniques unique to the fish may open novel routes to understand the function of spontaneous activity in mammals.

The zebrafish brain in research and teaching: a simple in vivo and in vitro model for the study of spontaneous neural activity.

Recently, the zebrafish (Danio rerio) has been established as a key animal model in neuroscience. Behavioral, genetic, and immunohistochemical techniques have been used to describe the connectivity of diverse neural circuits. However, few studies have used zebrafish to understand the function of cerebral structures or to study neural circuits. Information about the techniques used to obtain a workable preparation is not readily available. Here, we describe a complete protocol for obtaining in vitro and in vivo zebrafish brain preparations. In addition, we performed extracellular recordings in the whole brain, brain slices, and immobilized nonanesthetized larval zebrafish to evaluate the viability of the tissue. Each type of preparation can be used to detect spontaneous activity, to determine patterns of activity in specific brain areas with unknown functions, or to assess the functional roles of different neuronal groups during brain development in zebrafish. The technique described offers a guide that will provide innovative and broad opportunities to beginner students and researchers who are interested in the functional analysis of neuronal activity, plasticity, and neural development in the zebrafish brain.

Dynamics of sleep-wake cyclicity across the fetal period in sheep (Ovis aries).

All adult mammals examined thus far exhibit sleep bout durations that follow an exponential distribution and wake bout durations that follow a power-law distribution. In altricial rodents such as rats and mice, exponential distributions of sleep bouts are found soon after birth, but the power-law distribution of wake bouts does not emerge until the third postnatal week. Also, both sleep and bouts consolidate across the early postnatal period. It is not known whether similar developmental processes occur in precocial species during the prenatal period. Here we characterize sleep-wake development in a precocial species, the domestic sheep (Ovis aries), from 114 to 148 days gestational age (DGA). Sleep and wake bout durations exhibited exponential distributions throughout the fetal period with some evidence of an emerging exponential-to-power-law transition for wake bouts toward the end of gestation. Both sleep and wake bouts consolidated in an orderly fashion across development and there was little evidence of circadian variation, even in the oldest subjects. These results indicate that similar patterns of sleep-wake organization are found prenatally in a precocial species as are found postnatally in altricial species. Data from more species are needed to fully realize the benefits of a developmental comparative approach for understanding the forces that have shaped the ontogeny and phylogeny of mammalian sleep.

Modulation of hypothalamus and amygdalar activation levels with stimulus valence.

In spite of long-standing evidence showing that the hypothalamus is instrumental in generating behaviors associated with positive and negative emotions, little is known about the role of the hypothalamus in normal human emotional processing. Recent findings have suggested that the hypothalamus plays a role beyond mere control of HPA-axis function; this is also supported by the existence of rich anatomical connections between the hypothalamus and the amygdala, a region known for its important role in emotional processing. However, evidence of emotion-induced hypothalamic activity from neuroimaging studies has been inconsistent, possibly due to methodological limitations (e.g., low spatial resolution). Taking advantage of recent improvements in fMRI technology we set out to explore a possible valence-dependent modulation of hypothalamic activity. Using second order parametric analysis of high-resolution BOLD fMRI, we assessed hypothalamic activation patterns during passive viewing of visual stimuli of varying valence, and compared the results with the activity pattern in the amygdalae, i.e. nuclei with known valence-dependent activity profiles. We show that both hypothalamic and amygdalar activation is modulated by the second-order stimulus valence term, i.e., there is increased neural activity following the processing of both positive and negative stimuli. Our results suggest that the hypothalamus may serve a role in generating emotions broader than generally assumed.

Active medullary control of atonia in week-old rats.

Muscle atonia is a central feature of adult REM sleep which has recently been demonstrated to be a component of sleep in rats as young as 2 days of age (P2). The neural generation of atonia, which depends on mesopontine and medullary structures, is not fully understood in adults and has never been described in infants. In the present experiments we used electrical stimulation in decerebrated pups to identify an inhibitory area within the medial medulla of P7-10 rats. Muscle tone inhibition was consistently found on or near the midline within the ventromedial medulla, dorsal to the inferior olive, in an area that includes the nucleus gigantocellularis, nucleus paramedianus, and raphe obscurus. Chemical infusions in the same region revealed inhibitory responses to quisqualic acid but not to carbachol or corticotropin-releasing factor. Next, extracellular recordings within the medullary inhibitory area revealed neurons with atonia-on profiles; tone-on neurons were also found, typically at more lateral sites. Finally, in non-decerebrated pups, chemical lesions within the inhibitory area resulted in significant reductions in atonia durations, as well as decoupling of atonia from a second component of infant sleep, myoclonic twitching; specifically, twitches occasionally occurred during periods of high muscle tone, a condition reminiscent of "REM without atonia" as described in adults. In summary, we document the existence of an area within the ventromedial medulla of infant rats that (i) causes atonia when stimulated; (ii) contains units that exhibit atonia-related discharge profiles during sleep-wake cycling; and (iii) when lesioned, results in the partial loss of atonia and decoupling of the components of sleep. All together, these findings demonstrate that muscle atonia is actively regulated very early in ontogeny.

Hypothalamic contribution to sleep-wake cycle development.

Infant mammals cycle rapidly between sleep and wakefulness and only gradually does a more consolidated sleep pattern develop. The neural substrates responsible for this consolidation are unknown. To establish a reliable measure of sleep-wake cyclicity in infant rats, nuchal muscle tone was measured in 2-, 5-, and 8-day-old rats, as were motor behaviors associated with sleep (i.e. myoclonic twitching) and wakefulness (e.g. kicking, stretching). Sleep-wake cycles of 2-day-old rats were characterized by short periods of muscle atonia followed by equally short periods of high tone. In 8-day-olds, sleep periods lengthened significantly and disproportionately in relation to awake periods. Next, locus coeruleus (LC) lesions in 8-day-olds resulted in rapid sleep-wake cycling similar to that exhibited by 2-day-olds; in addition, LC lesions had no effect on the duration of awake periods. Finally, transections caudal, but not rostral, to the anterior hypothalamus also reinstated rapid cycling in 8-day-olds, again without affecting the duration of awake periods. This last finding implicates neural structures within the anterior hypothalamus (e.g. ventrolateral preoptic area) in the modulation of sleep-wake cyclicity. The temporal coherence of atonia and myoclonic twitching was not disrupted by any of the manipulations. These results suggest the presence of a bistable mesopontine circuit governing rapid sleep-wake cycling that does not include the LC and that comes increasingly under hypothalamic control during the first postnatal week. This circuit may represent a basic building block with which other sleep components become integrated during ontogeny.

Different glycosphingolipid composition in human neutrophil subcellular compartments.

The binding of a number of carbohydrate-recognizing ligands to glycosphingolipids and polyglycosylceramides of human neutrophil subcellular fractions (plasma membranes/secretory vesicles of resting and ionomycin-stimulated cells, specific and azurophil granules) was examined using the chromatogram binding assay. Several organelle-related differences in glycosphingolipid content were observed. The most prominent difference was a decreased content of the GM3 ganglioside in plasma membranes of activated neutrophils. Gangliosides recognized by anti-VIM-2 antibodies were detected mainly in the acid fractions of azurophil and specific granules. Slow-migrating gangliosides and polyglycosylceramides with Helicobacter pylori-binding activity were found in all acid fractions. A non-acid triglycosylceramide, recognized by Gal(alpha)4Gal-binding Escherichia coli, was detected in the plasma membrane/secretory vesicles but not in the azurophil and specific granules. Although no defined roles of glycosphingolipids have yet been conclusively established with respect to neutrophil function, the fact that many of the identified glycosphingolipids are stored in granules, is in agreement with their role as receptor structures that are exposed on the neutrophil cell surface upon fusion of granules with the plasma membrane. Accordingly, we show that neutrophil granules store specific carbohydrate epitopes that are upregulated to the plasma membrane upon cell activation.

Identification of proteins from Escherichia coli using two-dimensional semi-preparative electrophoresis and mass spectrometry.

Escherichia coli is a gram-negative bacterium that causes sepsis and infections of the nervous system, and the digestive and urinary tracts. The availability of the complete nucleotide sequence encoding the E. coli K-12 genome has made this organism an excellent model for proteomic studies. Semi-preparative two-dimensional electrophoresis, including liquid phase isoelectric focusing (IEF), one-dimensional sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and gel elution, have for the first time been used in combination with matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS), electrospray tandem mass spectrometry and database searching for rapid separation of proteins from a uropathogenic strain of E. coli. The identity of 30 proteins, including the membrane protein nmpC, was obtained using this approach.

Adhesion of type 1-fimbriated Escherichia coli to abiotic surfaces leads to altered composition of outer membrane proteins.

Phenotypic differences between planktonic bacteria and those attached to abiotic surfaces exist, but the mechanisms involved in the adhesion response of bacteria are not well understood. By the use of two-dimensional (2D) polyacrylamide gel electrophoresis, we have demonstrated that attachment of Escherichia coli to abiotic surfaces leads to alteration in the composition of outer membrane proteins. A major decrease in the abundance of resolved proteins was observed during adhesion of type 1-fimbriated E. coli strains, which was at least partly caused by proteolysis. Moreover, a study of fimbriated and nonfimbriated mutants revealed that these changes were due mainly to type 1 fimbria-mediated surface contact and that only a few changes occurred in the outer membranes of nonfimbriated mutant strains. Protein synthesis and proteolytic degradation were involved to different extents in adhesion of fimbriated and nonfimbriated cells. While protein synthesis appeared to affect adhesion of only the nonfimbriated strain, proteolytic activity mostly seemed to contribute to adhesion of the fimbriated strain. Using matrix-assisted laser desorption ionization-time of flight mass spectrometry, six of the proteins resolved by 2D analysis were identified as BtuB, EF-Tu, OmpA, OmpX, Slp, and TolC. While the first two proteins were unaffected by adhesion, the levels of the last four were moderately to strongly reduced. Based on the present results, it may be suggested that physical interactions between type 1 fimbriae and the surface are part of a surface-sensing mechanism in which protein turnover may contribute to the observed change in composition of outer membrane proteins. This change alters the surface characteristics of the cell envelope and may thus influence adhesion.

Glycosphingolipid binding specificities of rotavirus: identification of a sialic acid-binding epitope.

The glycosphingolipid binding specificities of neuraminidase-sensitive (simian SA11 and bovine NCDV) and neuraminidase-insensitive (bovine UK) rotavirus strains were investigated using the thin-layer chromatogram binding assay. Both triple-layered and double-layered viral particles of SA11, NCDV, and UK bound to nonacid glycosphingolipids, including gangliotetraosylceramide (GA1; also called asialo-GM1) and gangliotriaosylceramide (GA2; also called asialo-GM2). Binding to gangliosides was observed with triple-layered particles but not with double-layered particles. The neuraminidase-sensitive and neuraminidase-insensitive rotavirus strains showed distinct ganglioside binding specificities. All three strains bound to sialylneolactotetraosylceramide and GM2 and GD1a gangliosides. However, NeuAc-GM3 and the GM1 ganglioside were recognized by rotavirus strain UK but not by strains SA11 and NCDV. Conversely, NeuGc-GM3 was bound by rotaviruses SA11 and NCDV but not by rotavirus UK. Thus, neuraminidase-sensitive strains bind to external sialic acid residues in gangliosides, while neuraminidase-insensitive strains recognize gangliosides with internal sialic acids, which are resistant to neuraminidase treatment. By testing a panel of gangliosides with triple-layered particles of SA11 and NCDV, the terminal sequence sialyl-galactose (NeuGc/NeuAcalpha3-Galbeta) was identified as the minimal structural element required for the binding of these strains. The binding of triple-layered particles of SA11 and NCDV to NeuGc-GM3, but not to NeuAc-GM3, suggested that the sequence NeuGcalpha3Galbeta is preferred to NeuAcalpha3Galbeta. Further dissection of this binding epitope showed that the carboxyl group and glycerol side chain of sialic acid played an important role in the binding of such triple-layered particles.

Pathogen-host protein-carbohydrate interactions as the basis of important infections.

Microbe-host protein-carbohydrate interactions are most likely the essential first step to produce an infection, although this has been strictly proven only in a few cases. Improved glycotechnology will help identification of new carbohydrate receptors and this knowledge may be used to identify microbial carbohydrate-binding proteins by affinity proteomics approaches. In some cases such conserved proteins may prove to be successful vaccine components, in other cases, like influenza, saccharide analogues may be the only rational alternative. The prognosis may be, based on these improvements, that infection medicine will make considerable progress in the near future.

Inhibition of nonopsonic Helicobacter pylori-induced activation of human neutrophils by sialylated oligosaccharides.

Certain strains of Helicobacter pylori have nonopsonic neutrophil-activating capacity. Some H. pylori strains and the neutrophil-activating protein of H.pylori (HPNAP) bind selectively to gangliosides of human neutrophils. To determine if there is a relationship between the neutrophil-activating capacity and the ganglioside-binding ability, a number of H. pylori strains, and HPNAP, were incubated with oligosaccharides, and the effects on the oxidative burst of subsequently challenged neutrophils was measured by chemiluminescence and flow cytometry. Both by chemiluminescence and flow cytometry a reduced response was obtained by incubation of H.pylori with sialic acid-terminated oligosaccharides, whereas lactose had no effect. The reductions obtained with different sialylated oligosaccharides varied to some extent between the H. pylori strains, but in general 3'-sialyllactosamine was the most efficient inhibitor. Challenge of neutrophils with HPNAP gave no response in the chemiluminescence assay, and a delayed moderate response with flow cytometry. Preincubation of the protein with 3'-sialyllactosamine gave a slight reduction of the response, while 3'-sialyllactose had no effect. The current results suggest that the nonopsonic H. pylori-induced activation of neutrophils occurs by lectinophagocytosis, the recognition of sialylated glycoconjugates on the neutrophil cell surface by a bacterial adhesin leads to phagocytosis and an oxidative burst with the production of reactive oxygen metabolites.

Lactobacillus johnsonii La1 shares carbohydrate-binding specificities with several enteropathogenic bacteria.

The carbohydrate-binding specificities of the probiotic lactic acid bacterium Lactobacillus johnsonii La1 (a health-beneficial bacterial strain able to be incorporated into the human intestinal microflora) were investigated in vitro. First various soluble complex carbohydrates were tested as potential inhibitors of the strain adhesion onto Caco-2 intestinal epithelial cells, and then bacterial binding to glycolipids immobilized on TLC plates was probed. Two major carbohydrate-binding specificities of Lactobacillus johnsonii La1 were identified. A first one for an Endo-H treated yeast cell wall mannoprotein carrying mainly O:-linked oligomannosides, and a second one for the gangliotri- and gangliotetra-osylceramides (asialo-GM1). Similar carbohydrate-binding specificities are known to be expressed on cell surface adhesins of several enteropathogens, enabling them to adhere to the host gut mucosa. These findings corroborate the hypothesis that selected probiotic bacterial strains could be able to compete with enteropathogens for the same carbohydrate receptors in the gut.

A strain of human influenza A virus binds to extended but not short gangliosides as assayed by thin-layer chromatography overlay.

A human strain of influenza virus (A, H1N1) was shown to bind in an unexpected way to leukocyte and other gangliosides when compared with avian virus (A, H4N6) as assayed on TLC plates. The human strain bound only to species with about 10 or more sugars, while the avian strain bound to a wide range of gangliosides including the 5-sugar gangliosides. By use of specific lectins, antibodies, and FAB and MALDI-TOF mass spectrometry an attempt was done to preliminary identify the sequences of leukocyte gangliosides recognized by the human strain. The virus binding pattern did not follow binding by VIM-2 monoclonal antibody and was not identical with binding by anti-sialyl Lewis x antibody. There was no binding by the virus of linear NeuAcalpha3- or NeuAcalpha6-containing gangliosides with up to seven monosaccharides per mol of ceramide. Active species were minor NeuAcalpha6-containing molecules with probably repeated HexHexNAc units and fucose branches. This investigation demonstrates marked distinctions in the recognition of gangliosides between avian and human influenza viruses. Our data emphasize the importance of structural factors associated with more distant parts of the binding epitope and the complexity of carbohydrate recognition by human influenza viruses.

The human gastric colonizer Helicobacter pylori: a challenge for host-parasite glycobiology.

The Gram-negative bacterium Helicobacter pylori was first described in 1983 and currently represents one of the most active single research topics in biomedicine. It is specific for the human stomach and chronically colonizes a majority of the global population, which results in a symptom-free local inflammation. In 10-20% of carriers, gastroduodenal disease develops, including gastric or duodenal ulcer, and atrophic gastritis, which is a precondition to gastric cancer. A probable long coevolution of microbe and homo sapiens in a restricted niche has apparently generated a complex and sophisticated interplay. Access to complete bacterial genome sequences assists in a comparative functional characterization. A dynamic glycosylation of both microbe and host cells is of growing interest to analyze. Several glycoforms of bacterial surface lipopolysaccharides show advanced molecular mimicry of host epitopes and a distinct phase variation. An unusually large family of 32 outer membrane proteins probably reflects the complex interrelationship with the host. The unique diversity found for carbohydrate-binding specificities may be mediated by these surface proteins, of which the Lewis b-binding adhesin is the only known example so far, and these binding activities are subject to phase variation. The host mucosa glycosylation may also vary with different conditions, allowing a modulated crosstalk between microbe and host. The bacterium actively stimulates the host inflammatory response, apparently for nutritional purposes, and there is no evidence for a spontaneous elimination of the microbe. Colonization appears to be preventive for upper stomach and esophageal diseases. Current antibiotic treatment eradicates the microbe and cures ulcer disease. Alternative approaches must, however, be developed for a potential global prevention of disease.

Identification of protein vaccine candidates from Helicobacter pylori using a preparative two-dimensional electrophoretic procedure and mass spectrometry.

Helicobacter pylori is an important human gastric pathogen for which the entire genome sequence is known. This microorganism displays a uniquely complex pattern of binding to complex carbohydrates presented on host mucosal surfaces and other tissues, through adhesion molecules (adhesins) on the microbial cell surface. Adhesins and other membrane-associated proteins are important targets for vaccine development. The identification and characterization of cell-surface proteins expressed by H. pylori is a prerequisite for the development of vaccines designed to interfere with bacterial colonization of host tissues. However, identification of membrane proteins is difficult using a traditional proteomics approach employing 2D-PAGE. We have used a novel approach in the identification of microbial proteins that employs a rapid preparative two-dimensional electrophoretic separation followed by mass spectrometry and database searches. No pre-enrichment of bacterial membranes was required. The entire process, from sample preparation to protein identification, can be completed in less than 18 hours, and the presence of proteins can be monitored after both the first- and second-dimensional separations using mass spectrometry. We were able to identify 40 proteins from a detergent-solubilized H. pylori preparation; over one-third of these were membrane or membrane-associated proteins. A functionally characterized low-abundance membrane protein, the Leb-binding adhesin, was found in this group. The use of this rapid 2D electrophoretic separation in proteomic studies of H. pylori is expected to speed up the identification of expressed virulence proteins and vaccine targets in this and other microbial pathogens.

The assembly and secretion of apolipoprotein B-48-containing very low density lipoproteins in McA-RH7777 cells.

We have used an extraction procedure, which released membrane-bound apoB-100, to study the assembly of apoB-48 VLDL (very low density lipoproteins). This procedure released apoB-48, but not integral membrane proteins, from microsomes of McA-RH7777 cells. Upon gradient ultracentrifugation, the extracted apoB-48 migrated in the same position as the dense apoB-48-containing lipoprotein (apoB-48 HDL (high density lipoprotein)) secreted into the medium. Labeling studies with [(3)H]glycerol demonstrated that the HDL-like particle extracted from the microsomes contains both triglycerides and phosphatidylcholine. The estimated molar ratio between triglyceride and phosphatidylcholine was 0.70 +/- 0.09, supporting the possibility that the particle has a neutral lipid core. Pulse-chase experiments indicated that microsomal apoB-48 HDL can either be secreted as apoB-48 HDL or converted to apoB-48 VLDL. These results support the two-step model of VLDL assembly. To determine the size of apoB required to assemble HDL and VLDL, we produced apoB polypeptides of various lengths and followed their ability to assemble VLDL. Small amounts of apoB-40 were associated with VLDL, but most of the nascent chains associated with VLDL ranged from apoB-48 to apoB-100. Thus, efficient VLDL assembly requires apoB chains of at least apoB-48 size. Nascent polypeptides as small as apoB-20 were associated with particles in the HDL density range. Thus, the structural requirements of apoB to form HDL-like first-step particles differ from those to form second-step VLDL. Analysis of proteins in the d < 1.006 g/ml fraction after ultracentrifugation of the luminal content of the cells identified five chaperone proteins: binding protein, protein disulfide isomerase, calcium-binding protein 2, calreticulin, and glucose regulatory protein 94. Thus, intracellular VLDL is associated with a network of chaperones involved in protein folding. Pulse-chase and subcellular fractionation studies showed that apoB-48 VLDL did not accumulate in the rough endoplasmic reticulum. This finding indicates either that the two steps of apoB lipoprotein assembly occur in different compartment or that the assembled VLDL is transferred rapidly out of the rough endoplasmic reticulum.

Use of an affinity proteomics approach for the identification of low-abundant bacterial adhesins as applied on the Lewis(b)-binding adhesin of Helicobacter pylori.

Microbial attachment to host cell surfaces is considered to be the first essential step for colonization and infection. In most known cases, attachment is mediated by a specific protein-carbohydrate interaction. We have used a carbohydrate-containing crosslinking probe to select bacterial surface adhesins for trypsin digestion, MALDI-TOF mass spectrometry and identification against genome sequence. The present paper describes this functional proteomics approach for identification of the recently cloned low-abundant Lewis(b)-binding adhesin of Helicobacter pylori. Protein identification was obtained through the enrichment of approximately 300 fmol of adhesin from solubilized cells.

Novel carbohydrate binding site recognizing blood group A and B determinants in a hybrid of cholera toxin and Escherichia coli heat-labile enterotoxin B-subunits.

The B-subunits of cholera toxin (CTB) and Escherichia coli heat-labile enterotoxin (LTB) are structurally and functionally related. However, the carbohydrate binding specificities of the two proteins differ. While both CTB and LTB bind to the GM1 ganglioside, LTB also binds to N-acetyllactosamine-terminated glycoconjugates. The structural basis of the differences in carbohydrate recognition has been investigated by a systematic exchange of amino acids between LTB and CTB. Thereby, a CTB/LTB hybrid with a gain-of-function mutation resulting in recognition of blood group A and B determinants was obtained. Glycosphingolipid binding assays showed a specific binding of this hybrid B-subunit, but not CTB or LTB, to slowly migrating non-acid glycosphingolipids of human and animal small intestinal epithelium. A binding-active glycosphingolipid isolated from cat intestinal epithelium was characterized by mass spectrometry and proton NMR as GalNAcalpha3(Fucalpha2)Galbeta4(Fucalpha3)Glc NAcbeta3Galbeta4Glc NAcbeta3Galbeta4Glcbeta1Cer. Comparison with reference glycosphingolipids showed that the minimum binding epitope recognized by the CTB/LTB hybrid was Galalpha3(Fucalpha2)Galbeta4(Fucalpha3)GlcNAc beta. The blood group A and B determinants bind to a novel carbohydrate binding site located at the top of the B-subunit interfaces, distinct from the GM1 binding site, as found by docking and molecular dynamics simulations.

Purification and characterization of RNA polymerase II holoenzyme from Schizosaccharomyces pombe.

We have purified the RNA polymerase II holoenzyme from Schizosaccharomyces pombe to near homogeneity. The Mediator complex is considerably smaller than its counterpart in Saccharomyces cerevisiae, containing only nine polypeptides larger than 19 kDa. Five of these Mediator subunits have been identified as the S. pombe homologs to Rgr1, Srb4, Med7, and Nut2 found in S. cerevisiae and the gene product of a previously uncharacterized open reading frame, PMC2, with no clear homologies to any described protein. The presence of Mediator in a S. pombe RNA polymerase II holoenzyme stimulated phosphorylation of the C-terminal domain by TFIIH purified from S. pombe. This stimulation was species-specific, because S. pombe Mediator could not stimulate TFIIH purified from S. cerevisiae. We suggest that the overall structure and mechanism of the Mediator is evolutionary conserved. The subunit composition, however, has evolved to respond properly to physiological signals.