Band 382, Heft 2

While only about ten percent of the databank entries are defined as glycoproteins, it has been estimated recently that more than half of all proteins are glycoproteins. Mucintype Oglycosylation is a widespread posttranslational modification of proteins found in the entire animal kingdom, but also in higher plants. The structural complexity of the chains initiated by Olinked GalNAc exceeds that of Nlinked chains by far. The process during which serine and threonine residues of proteins become modified is confined to the cis to trans Golgi compartments. The initiation of this process by peptidyl GalNActransferases is ruled by the sequence context of putative Oglycosylation sites, but also by epigenetic regulatory mechanisms, which can be mediated by enzyme competition. The cellular repertoir of glycosyltransferases with their distinct donor sugar and acceptor sugar specificities, their sequential action at highlyordered surfaces, and their localizations in subcompartments of the Golgi finally determine the cellspecific Oglycosylation profile. Dramatic alterations of the glycosylation machinery are observed in cancer cells, resulting in aberrantly Oglycosylated proteins that expose previously masked peptide motifs and new antigenic targets. The functional aspects of Olinked glycans, which comprise among many others their potential role in sorting and secretion of glycoproteins, their influence on protein conformation, and their multifarious involvement in cell adhesion and immunological processes, appear as complex as their structures.

Our growing comprehension of the biological roles of glycan moieties has created a clear need for expression systems that can produce mammaliantype glycoproteins. In turn, this has intensified interest in understanding the protein glycosylation pathways of the heterologous hosts that are commonly used for recombinant glycoprotein expression. Among these, insect cells are the most widely used and, particularly in their role as hosts for baculovirus expression vectors, provide a powerful tool for biotechnology. Various studies of the glycosylation patterns of endogenous and recombinant glycoproteins produced by insect cells have revealed a large variety of O and Nlinked glycan structures and have established that the major processed O and Nglycan species found on these glycoproteins are (Galß1,3)GalNAcOSer/Thr and Man3(Fuc)GlcNAc2-NAsn, respectively. However, the ability or inability of insect cells to synthesize and compartmentalize sialic acids and to produce sialylated glycans remains controversial. This is an important issue because terminal sialic acid residues play diverse biological roles in many glycoconjugates. While most work indicates that insect cellderived glycoproteins are not sialylated, some wellcontrolled studies suggest that sialylation can occur. In evaluating this work, it is important to recognize that oligosaccharide structural determination is tedious work, due to the infinite diversity of this class of compounds. Furthermore, there is no universal method of glycan analysis; rather, various strategies and techniques can be used, which provide glycobiologists with relatively more or less precise and reliable results. Therefore, it is important to consider the methodology used to assess glycan structures when evaluating these studies. The purpose of this review is to survey the studies that have contributed to our current view of glycoprotein sialylation in insect cell systems, according to the methods used. Possible reasons for the disagreement on this topic in the literature, which include the diverse origins of biological material and experimental artifacts, will be discussed. In the final analysis, it appears that if insect cells have the genetic potential to perform sialylation of glycoproteins, this is a highly specialized function that probably occurs rarely. Thus, the production of sialylated recombinant glycoproteins in the baculovirusinsect cell system will require metabolic engineering efforts to extend the native protein glycosylation pathways of insect cells.

Several inherited disorders affecting the biosynthetic pathways of Nglycans have been discovered during the past years. This review summarizes the current knowledge in this rapidly expanding field and covers the molecular bases of these disorders as well as their phenotypical consequences.

During the evolution of the eukaryotic cell, genes encoding proteins involved in the metabolism of mitochondrial DNA (mtDNA) have been transferred from the endosymbiont into the host genome. Mitochondrial singlestranded DNAbinding (mtSSB) proteins serve as an excellent argument supporting this aspect of the endosymbiotic theory. The crystal structure of the human mtSSB, together with an abundance of biochemical and genetic data, revealed several exciting features of mtSSB proteins and enabled a detailed comparison with their prokaryotic counterparts. Moreover, identification of a novel member of the mtSSB family, mitochondrial telomerebinding protein of the yeast Candida parapsilosis, has raised interesting questions regarding mtDNA metabolism and evolution.

A large number of studies on the structure of Nglycosidically linked oligosaccharides from glycoproteins of different organs and/or different species have been carried out in the past using various combinations of techniques such as monosaccharide analysis, permethylation, peracteylation, exoglycosidase sequencing, normal and reversed phase HPLC, mass spectrometry and nuclear magnetic resonance spectroscopy. Although it is widely accepted that the processing of Nglycans in the ER and Golgi of mammalian cells follows the same principal metabolic rules, analyses have revealed that the glycosylation pattern of a particular protein may differ depending on the cell type in which it is expressed. Nglycans from brain glycoproteins have been shown to include a variety of hybrid and complextype structures with structural features that are not so commonly found on glycoproteins from other organs and which have, therefore, been classified as brainspecific. Comparison of the Nglycans of glycoproteins from homogenates of rat, mouse and human brains confirm that, in general, glycoproteins from human brain show a similar profile of brainspecific Nglycans as glycoproteins from mouse and rat brain.

Glycosphingolipids from the parasitic liver flukes F a sciola gigantica and Fasciola hepatica w e re isolated and their carbohydrate moieties were structurally analysed by methylation analysis, exoglycosidase t reatment, ontarget exoglycosidase cleavage and matrixassisted laser desorption/ionisation timeofflight mass spectrometry. For both liver fluke species, the ceramide monohexosides Gal1-ceramide and Glc1-ceramide were found in relative amounts of 1.0 to 0.1, respectively. From F. gigantica, the ceramide dihexoside was isolated in sufficient amounts to be structurally determined as lactosylceramide, Gal?4- Glc1-ceramide, while for both liver fluke species the ceramide trihexoside was shown to be Gal?4 G a lb4- Glc1-ceramide, which is designated as either globotriaosylceramide, Pblood group antigen or CD77 leucocyte cluster of diff e rentiation antigen. To our knowledge, this is the first report on the expression of globoseries glycosphingolipids in nonmammalian species. Ceramide analysis of ceramide monohexosides yielded as major components octadecanoic and 2 h y d roxyoctadecanoic fatty acids together with C18- and C20-phytosphingosines. By the use of an anti CD77 monoclonal antibody and the Escherichia coli Shiga toxin B1 subunit, globotriaosylceramide could be immunolocalised to the tegument of F. hepatica cryosections. The sharing of CD77 between liver flukes and their mammalian hosts fits in with the concept of molecular mimicry, which is closely parallel to the established imitation of host CD15 (Lewis X) displayed by the blood fluke Schistosoma mansoni.

A TBLASTN search of the Drosophila melanogaster expressed sequence tag (EST) database with the amino acid sequence of human UDPNacetylglucosamine: ?-3-Dmannoside ?-1,2-Nacetylglucosaminyltransferase I (GnT I, EC 2.4.1.101) as probe yielded a clone (GM01211) with 56% identity over 36 carboxyterminal amino acids. A 550 base pair (bp) probe derived from the EST clone was used to screen a Drosophila cDNA library in ?ZAP II and two cDNAs lacking a start ATG codon were obtained. 5Rapid amplification of cDNA ends (5RACE) yielded a 2828 bp cDNA containing a fulllength 1368 bp open reading frame encoding a 456 amino acid protein with putative Nterminal cytoplasmic (5 residues) and hydrophobic transmembrane (20 residues) domains. The protein showed 52% amino acid sequence identity to human GnT I. This cDNA, truncated to remove the Nterminal hydrophobic domain, was expressed in the baculovirus/Sf9 system as a secreted protein containing an Nterminal (His)[6] tag. Protein purified by adsorption to and elution from nickel beads converted Man?1-6(Man?1-3)Man?octyl (M3-octyl) to Man?1-6(GlcNAc?1-2Man?1-3)Man?octyl. The K values (0.7 and 0.03 mM for M3-octyl and UDPGlc NAc respectively), temperature optimum (37 C), pH optimum (pH 5 to 6) and divalent cation requirements (Mn > Fe, Mg, Ni > Ba, Ca, Cd, Cu) were similar to mammalian GnT I. TBLASTN searches of the Berkeley Drosophila Genome Project database with the Drosophila GnT I cDNA sequence as probe allowed localization of the gene to chromosomal region 2R; 57A9. Comparison of the cDNA and genomic DNA sequences allowed the assignment of seven exons and six introns; all introns showed GTAG splice site consensus sequences. This is the first insect GnT I gene to be cloned and expressed.

The SialylTn antigen (Sialyl?Ser/Thr) is expressed as a cancerassociated antigen on the surface of cancer cells. Its presence is associated with a poor prognosis in patients with colorectal and other cancers. We previously reported that SialylTn expression in LSC human colon cancer cells could be explained by a specific lack of the activity of core 1 ?3-Galtransferase (Brockhausen et al., Glycoconjugate J. 15, 595 603, 1998) and an inability to synthesize the common Oglycan core structures. To support this mechanism, or find other mechanisms to explain SialylTn antigen expression, we investigated the Oglycosylation pathways in clonal rat colon cancer cell lines that were selected for positive or negative expression of SialylTn antigen, and compared these pathways to those in normal rat colonic mucosa. Normal rat colonic mucosa had very active glycosyltransferases synthesizing Oglycan core structures 1 to 4. Several sialyl, sulfo and fucosyltransferases were also active. An M type core 2 ?6-GlcNActransferase was found to be present in rat colon mucosa and all of the rat colon cancer cells. Oglycosylation pathways in rat colon cancer cells were significantly different from normal rat colonic mucosa; for example, rat colon cancer cells lost the ability to synthesize Oglycan core 3. All rat colon cancer cell lines, regardless of the SialylTn phenotype, expressed glycosyltransferases assembling complex Oglycans of core 1 and core 2 structures (unlike human LSC colon cancer cells which lack core 1 ?3-Galtransferase activity). It was the activity of CMPsialic acid:GalNAcmucin ?6-sialyltransferase that coincided with SialylTn expression. SialylTn negative cells had a several fold higher activity of core 2 ?6-GlcNActransferase which synthesizes complex Oglycans that may mask adjacent SialylTn epitopes. The results suggest a new mechanism controlling SialylTn expression in cancer cells.

Total synthesis and structural characterization of a novel 6-Osulfo deNacetylsialyl Lewis X, which was originally discovered as a minor byproduct of the parent 6-Osulfo Nacetylsialyl Lewis X, a highaffinity endogenous ligand for human Lselectin, are described. The total synthesis has been achieved by a highly efficient, regio and α stereoselective glycosylation of Ntrifluoroacetylneuraminic acid, selective protections of the 3- and 6-hydroxyl groups of Nacetylglucosamine that undergo fucosylation and sulfation, and construction of the glycolipid structure containing a ceramide. The structure of 6-Osulfo deNacetylsialyl Lewis X ganglioside was characterized by fast atom bombardment mass spectrometry (FABMS).

Lateral assemblies of sphingolipids, glycosphingolipids and cholesterol, termed rafts, are postulated to be present in biological membranes and to function in important cellular phenomena. We probed whether rafts are heterogeneous by determining the relative distribution of two gangliosides, GM1 and GD3, in artificial supported monolayers, in intact rat primary cerebellar granule neurones, and in membrane rafts isolated from rat cerebellum. Fluorescence resonance energy transfer (FRET) using fluorophore labelled cholera toxin B subunit (which binds GM1) and mAb R24 (which binds GD3) revealed that GM1 spontaneously selfassociates but does not cocluster with GD3 in supported monolayers and on intact neurones. Cholera toxin and immunocytochemical labelling of isolated membrane rafts from rat cerebellum further demonstrated that GM1 does not colocalise with GD3. Furthermore, whereas the membrane raft resident proteins Lyn and caveolin both colocalise with GD3 in isolated membrane rafts, GM1 appears in separate and distinct aggregates. These data support prior reports that membrane rafts are heterogeneous, although the mechanisms for establishing and maintaining such heterogeneity remain to be determined.

Eight pyridylamino (PA) derivatives of fucosecontaining oligosaccharides, which occur as free oligosaccharides in human milk and also are derived from glycosphingolipids, have been analyzed by highperformance liquid chromatography (HPLC) on normalphase and reversedphase columns, and by matrixassisted laser desorption/ionization timeofflight MALDITOF) mass spectrometry. Six out of eight PAoligosaccharides were clearly separated by both normal and reversedphase HPLC at a column temperature of 40 C, but two PAoligosaccharides, lactoNfucopentaose II [Gal?1-3(Fuc?1-4)GlcNAc?1- 3Gal?1-4GlcPA] and lactoNfucopentaose III Gal?1-4(Fuc?1-3)GlcNAc?1-3Gal?1-4GlcPA], were not separated. The two unresolved PAoligosaccharides were finally separated by reversedphase HPLC at a column temperature of 11 C. MALDITOF mass spectra of PAoligosaccharides demonstrated pseudomolecular ions as the predominant signals, therefore information about the molecular mass of each PAoligosaccharide was easily obtained. Postsource decay (PSD) MALDITOF mass spectra of PAoligosaccharides gave information about the carbohydrate sequences and carbohydrate species of each PAoligosaccharide by detecting the ions responsible for the cleavage of the glycosidic bonds. The detection limits of the PAoligosaccharides by HPLC, MALDITOF mass spectrometry, and PSD MALDITOF mass spectrometry were 20 fmol, 20 fmol, and 2 pmol, respectively. These results suggest that a system including HPLC and MALDITOF mass spectrometry or HPLC and PSD MALDITOF mass spectrometry is quite useful for the structural characterization of subpmol or pmol levels of fucosecontaining oligosaccharides, and that these methods could be used for the analysis of various types of oligosaccharides derived from glycoproteins and glycosphingolipids.

Gangliosides from histopathologicallydefined human cerebrumresembling remnant and cerebellum from 37 and 30 gestational weekold anencephaluses were identified using mass spectrometry and high performance thin layer chromatography combined with immunochemical analysis in comparison to respective normal newborn/fetal and adult brain regions. A novel strategy of nanoelectrospray ionization quadrupole timeofflight tandem MS has been developed for identification of ganglioside components in complex mixtures. By morphoanatomical and histological investigation the anencephalic cerebral remnant was found to be aberrant, while the anencephalic cerebellum was defined as normal. Total ganglioside concentrations in the anencephalic cerebral remnant and the cerebellum were 34% and 13% lower in relation to the agematched controls. In the cerebral remnant, GD3, GM2 and GT1b were elevated, while GD1a was decreased in the anencephalic cerebral remnant, but enriched in anencephalic cerebellum. GQ1b was reduced in both anencephalic regions. Gg[4]Cer, GM1b and GD1?, members of the ?series biosynthetic pathway, and neolactoseries gangliosides were found to be present in anencephalic, as well as in normal, fetal and adult brain tissues, indicating the occurrence of these biosynthetic pathways in human brain. In both cerebral and cerebellar anencephalic tissues, GM1b, GD1?, nLM1 and nLD1 were expressed at a higher rate in relation to normal tissue. It can be demonstrated that the anencephalic cerebral remnant, as a primitive brain structure, represents a naturallyoccurring model to study the ganglioside involvement in induction of aberrant brain development.

The isolation and characterization of acidic lipids from both Trichomonas vaginalis and Tritrichomonas foetus have been carried out using radiolabeling, a combination of high performance liquid and thin layer chromatographic techniques, and mass spectrometry. Unique among the eukaryotes, these organisms produce phosphatidylglycerols and Oacyl phosphatidylglycerol like compounds. In this study, the molecular weight distributions of the phosphatidylglycerols and acyl phosphatidylglycerols were determined by negativeion liquid secondary ionization mass spectrometry (LSIMS) and the fatty acyl groups within each molecular species were assessed by collision induced decomposition tandem mass spectrometry (CID MS/MS). Both species were found to contain primarily oleic acid in the sn-2 position. The lipids of T. vaginalis had approximately equal amounts of C16 and C18 in the sn-1 position, with varying degrees of unsaturation, especially in the C18 species. The T. foetus lipids had C18 almost exclusively, but also varied in the unsaturation. Other acidic lipids included inositol phosphosphingolipids and inositol diphosphosphingolipids.

Acid sphingomyelinase is a watersoluble, lysosomal glycoprotein that catalyzes the degradation of membrane bound sphingomyelin into phosphorylcholine and ceramide. Sphingomyelin itself is an important component of the extracellular leaflet of various cellular membranes. The aim of the present investigation was to study sphingomyelin hydrolysis as a membrane bound process. We analyzed the degradation of sphingomyelin by recombinant, highly purified acid sphingomyelinase in a detergentfree, liposomal assay system. In order to mimic the in vivo intralysosomal conditions as closely as possible a number of negatively charged, lysosomally occuring lipids including bis(monoacylglycero)phosphate and phosphatidylinositol were incorporated into substratecarrying liposomes. Dolichol and its phosphate ester dolicholphosphate were also included in this study. Bis(monoacylglycero)phosphate and phosphatidylinositol were both effective stimulators of sphingomyelin hydrolysis. Dolichol and dolicholphosphate also significantly increased sphingomyelin hydrolysis. The influence of membrane curvature was investigated by incorporating the substrate into small (SUVs) and large unilamellar vesicles (LUVs) with varying mean diameter. Degradation rates were substantially higher in SUVs than in LUVs. Surface plasmon resonance experiments demonstrated that acid sphingomyelinase binds strongly to lipid bilayers. This interaction is significantly enhanced by anionic lipids such as bis(monoacylglycero)phosphate. Under detergent free conditions only the sphingolipid activator protein SAPC had a pronounced influence on sphingomyelin degradation in both neutral and negatively charged liposomes, catalyzed by highly purified acid sphingomyelinase, while SAPA, B and D had no noticeable effect on sphingomyelin degradation.

The first two steps in mammalian biosynthesis of Nacetylneuraminic acid, an important carbohydrate moiety in biological recognition systems, are performed by the bifunctional enzyme UDPNacetylglucosamine 2-epimerase/Nacetylmannosamine kinase. A subclone of the human B lymphoma cell line BJAB K20, lacking UDPNacetylglucosamine 2- epimerase/Nacetylmannosamine kinase mRNA as well as epimerase activity, displayed hyposialylated, functionally impaired cell surface glycoconjugates. Here we show that this cell line surprisingly still retains Nacetylmannosamine kinase activity. A gel filtration analysis of BJAB K88 control cells, which express UDPNacetylglucosamine 2-epimerase/Nacetylmannosamine kinase, revealed two Nacetylmannosamine kinase activity peaks, one coeluting with UDPNacetylglucosamine 2-epimerase activity and one coeluting with Nacetylglucosamine kinase. For this enzyme previous studies already showed ManNAc kinase activity in vitro. In contrast, the hyposialylated BJAB K20 subclone displayed only the Nacetylmannosamine kinase peak, comigrating with Nacetylglucosamine kinase. The CMPNacetylneuraminic acid content of both K88 and K20 cells and the sialylation of cell surface glycoconjugates of K20 cells could be significantly increased by supple menting the medium with Nacetylmannosamine. This Nacetylmannosamineinduced increase was drastically reduced by cosupplementation with Nacetylglucosamine only in K20 cells. We therefore propose the phosphorylation of Nacetylmannosamine as a hitherto unrecognized role of Nacetylglucosamine kinase in living cells.

The enzymatic access to nucleotideactivated oligosaccharides by a glycosidasecatalyzed transglycosylation reaction was explored. The nucleotide sugars UDPGlcNAc and UDPGlc were tested as acceptor substrates for ?galactosidase from Bacillus circulans using lactose as donor substrate. The UDPdisaccharides Gal(?1-4)GlcNAc(?1-UDP) (UDPLacNAc) and Gal(?1 4) Glc(?1-UDP) (UDPLac) and the UDPtrisaccharides Gal(?1-4)Gal(?1-4)GlcNAc(?1- UDP and Gal(?1 4 ) Gal(?1 4 ) Glc(?1-UDP) were formed stereo and regioselectively. Their chemical structures were characterized by [1]H and [13]C NMR spectroscopy and fast atom bombardment mass spectrometry. The synthesis in frozen solution at -5 C instead of 30 C gave significantly higher product yields with respect to the acceptor substrates. This was due to a remarkably higher product stability in the small liquid phase of the frozen reaction mixture. Under optimized conditions, at 5 C and pH 4.5 with 500 mM lactose and 100 mM UDPGlcNAc, an overall yield of 8.2% (81.8 mol, 62.8 mg with 100% purity) for Gal(b1 4 ) GlcNAc(?1-UDP) and 3.6% (36.1 mol, 35 mg with 96% purity) for Gal(?1-4)Gal(?1-4)GlcNAc(?1- UDP) was obtained. UDPGlc as acceptor gave an overall yield of 5.0% (41.3 mol, 32.3 mg with 93% purity) for Gal(?1-4)Glc(?1-UDP) and 1.6% (13.0 mol, 12.2 mg with 95% purity) for Gal(?1-4)Gal(?1- 4)Glc(?1-UDP). The analysis of other nucleotide sugars revealed UDPGal, UDPGalNAc, UDPXyl and dTDP, CDP, ADP and GDPGlc as further acceptor substrates for ?galactosidase from Bacillus circulans.

Bacterial sialidases represent important colonization or virulence factors. The development of a rational basis for the design of antimicrobials targeted to sialidases requires the knowledge of the exact roles of their conserved amino acids. A recombinant enzyme of the small (43 kDa) sialidase of Clostridium perfringens was used as a model in our study. Several conserved amino acids, identified by alignment of known sialidase sequences, were altered by sitedirected mutagenesis. All recombinant enzymes were affinitypurified and the enzymatic characteristics were determined. Among the mutated enzymes with modifications in the environment of the 4-hydroxyl group of bound sialic acids, D54N and D54E exhibited minor changes in substrate binding. However, a reduced activity and changes in their pH curves indicate the importance of a charged group at this area. R56K, which is supposed to bind directly to sialic acids as in the homologous Salmonella typhimurium sialidase, showed a 2500-fold reduced activity. The amino acids Asp-62 and Asp-100 are probably involved in catalysis, indicated by reduced activities and altered temperature and pH curves of mutant enzymes. Exchanging Glu-230 with threonine or aspartic acid led to dramatic decreases in activity. This residue and Y 347 are supposed to be crucial for providing a suitable environment for catalysis. However, unaltered pH curves of mutant sialidases exclude their direct involvement in protonation or deprotonation events. These results indicate that the interactions with the substrates vary in different sialidases and that they might be more complex than suggested by mere static Xray structures.

The formation of Nglycosidic linkages of glycoproteins involves the ordered assembly of the common Glc[3]Man[9]GlcNAc[2] coreoligosaccharide on the lipid carrier dolichyl pyrophosphate. Whereas early mannosylation steps occur on the cytoplasmic side of the endoplasmic reticulum with GDPMan as donor, the final reactions from Man[5]GlcNAc[2]PPDol to Man[9]Glc Nac[2]PPDol on the lumenal side use DolPMan. We have investigated these later stages in vitro using a detergentsolubilized enzyme extract from yeast membranes. Mannosyltransfer from DolPMan to [[3]H]Man[5]GlcNAc[2]PPDol with formation of all intermediates up to Man[9]GlcNAc[2]PPDol occured in a rapid, time and proteindependent fashion. We find that the initial reaction from Man[5]GlcNAc[2]PPDol to Man[6]GlcNAc[2]PPDol is independent of metal ions, but further elongations need Mn[2+] that can be partly replaced by Mg[2+] or Ca[2+]. Zn[2+] or Cd[2+] ions were found to inhibit formation of Man[7-9]GlcNAc[2]PPDol, but do not affect synthesis of Man[6]GlcNAc[2]PPDol. Extension did not occur when the acceptor was added as a free Man[5]GlcNAc[2] oligosaccharide or when GDPMan was used as mannosyldonor. The alg3 mutant was described to accumulate Man[5]GlcNAc[2]PPDol. We expressed a functional active HAepitope tagged ALG3 fusion and succeeded to selectively immunoprecipitate the DolPMan:Man[5]GlcNAc[2]PPDol mannosyltransferase activity from the other enzymes of the detergent extract involved in the subsequent mannosylation reactions. This demonstrates that Alg3p represents the mannosyltransferase itself and not an accessory protein involved in the reaction.

Mithramycin inhibits transcription by binding to G/Crich sequences, thereby preventing regulatory protein binding. However, it is also possible that mithramycin inhibits gene expression by preventing intramolecular triplex DNA assembly. We tested this hypothesis using the DNA triplex adopted by the murine cmyb protooncogene. The 5regulatory region of cmyb contains two polypurine:polypyrimidine tracts with imperfect mirror symmetry, which are highly conserved in the murine and human cmyb sequences. The DNA binding drugs mithramycin and distamycin bind to one of these regions as determined by DNase I protection assay. Gel mobility shift assays, nuclease and chemical hypersensitivity and 2Dgel topological analyses as well as triplexspecific antibody binding studies confirmed the formation of purine*purine:pyrimidine inter and pyrimidine*purine:pyrimidine intramolecular triplex structures in this sequence. Mithramycin binding within the triplex target site displaces the major groovebound oligonucleotide, and also abrogates the supercoildependent HDNA formation, whereas distamycin binding had no such effects. Molecular modeling studies further support these observations. Triplexspecific antibody staining of cells pretreated with mithramycin demonstrates a reversal of chromosomal triplex structures compared to the nontreated and distamycintreated cells. These observations suggest that DNA minor groovebinding drugs interfere with gene expression by precluding intramolecular triplex formation, as well as by physically preventing regulatory protein binding.

The precise roles of signal transducers and activators of transcription (STATs) in cytokinetriggered control of cell physiology are not sufficiently well understood. We have established cell lines in which the individual functional contributions of STAT6 and STAT5a/b to interleukin(IL) 3 and -4-dependent processes can be readily studied. Mutants of STAT6, STAT5a and 5b lacking the transcriptional transactivation domain were fused to the green fluorescent protein (GFP) and expressed in the murine proB cell line Ba/F3 in a regulatable fashion. The expression of these truncated STAT variants could be tightly controlled over a wide range by doxycycline in the medium. They specifically bound to cognate DNA elements upon cytokine stimulation and acted dominantnegatively on the transcription of respective reporter genes in response to IL-3 and -4. The system was applied to the question of STAT contributions to cytokinedependent cell proliferation. Expression of dominantnegative STAT6 had no significant effect on cell growth in response to both IL-3 and IL-4. In contrast, truncated STAT5 interfered with cell proliferation in response to IL-3, and, interestingly, also to IL-4. The results support our earlier findings on a role of STAT5 in IL-4-induced intracellular signaling and indicate that STAT5b in particular is involved in IL-4 receptortriggered control of cell proliferation.