Proc Natl Acad Sci U S A, 1994 Mar 15, 91(6), 1998 - 2002 Sequence-specific transcriptional activation is essential for growth suppression by p53; Pietenpol JA et al.; Although several biochemical features of p53 have been described, their relationship to tumor suppression remains uncertain . We have compared the ability of p53-derived proteins to act as sequence-specific transcriptional (SST) activators with their ability to suppress tumor cell growth, using an improved growth-suppression assay . Both naturally occurring and in vitro derived mutations that abrogated the SST activity of p53 lost the ability to suppress tumor cell growth . Additionally, the N- and C-terminal ends of p53 were shown to be functionally replaceable with foreign transactivation and dimerization domains, respectively, with concordant preservation of both SST and tumor-suppressive properties . Only the central region of p53, conferring specific DNA binding, was required to suppress growth by such hybrid proteins . The SST activity of p53 thus appeared to be essential for the protein to function as a tumor suppressor. Biochemistry, 1994 Mar 15, 33(10), 3071 - 8 Recognition of DNA by GAL4 in solution: use of a monomeric protein-DNA complex for study by NMR; Baleja JD et al.; The complex of a monomer of GAL4 with DNA has been investigated by two-dimensional 1H nuclear magnetic resonance (NMR) spectroscopy . Previous X-ray analysis has revealed a structure in which a dimer of the N-terminal 65-residue fragment of GAL4 forms a complex, 27 kDa in molecular mass, with a 19 base pair full-binding-site DNA {Marmorstein, R., Carey, M., Ptashne, M., & Harrison, S . C . (1992) Nature 356, 408-414} . We have developed a smaller system, half in molecular mass, which is amenable for detailed analysis using NMR . Titration of a 10 base pair half-binding-site DNA with GAL4-(65) shows 1:1 binding, illustrating that one monomer of the protein binds in a specific manner to half-site DNA . The components of the protein-DNA complex are mainly in fast exchange on the NMR chemical shift time scale, with an equilibrium dissociation constant of 161 +/- 12 microM . With a basis of chemical shift data for free GAL4 protein and for the free half-site DNA, the fast exchange facilitates 1H resonance assignments in the complex since cross-peak positions can be examined at different protein:DNA ratios . Chemical shift changes in the DNA reveal the base pairs that are important for recognition by GAL4 . Intermolecular NOE cross-peaks are also observed in spectra of the protein-DNA complex . Their identification places the C-terminal end of the first alpha-helix (residues 12-17) in a position such that the amino acids are able to read the DNA sequence in a manner entirely consistent with the X-ray structure of the related complex.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1994 Mar 15, 33(10), 2815 - 23 Crystal structure of the K12M/G15A triosephosphate isomerase double mutant and electrostatic analysis of the active site; Joseph-McCarthy D et al.; The crystal structure of the yeast triosephosphate isomerase (TIM) double mutant K12M/G15A has been solved to 2 A by X-ray diffraction, and the effects of changing the positively charged lysine to the neutral methionine have been analyzed . The mutant enzyme was crystallized in the presence of the tight-binding inhibitor phosphoglycolohydroxamate, under standard conditions for obtaining crystals of the enzyme-inhibitor complex . The crystals obtained were of the same crystal form as the unliganded wild-type enzyme . The three-dimensional structure confirms that the Lys-12 to Met mutation prevents the enzyme from binding substrate and reveals that the reason is electrostatic and not steric . The substrate-binding loop is in its open position and the Met side chain points away from the active site . Overall, the mutant structure is very similar to that of the wild-type unliganded enzyme . The electrostatic potential at the active site of the mutant enzyme is, however, very different from that of the wild type . It has been postulated previously that Lys-12 may play a role in stabilizing the negative charge in the transition state . This K12M/G15A structure suggests that the active-site Lys, which is strictly conserved, is required for TIM to be able to bind its dianionic substrate. Biochemistry, 1994 Mar 15, 33(10), 2809 - 14 Triosephosphate isomerase requires a positively charged active site: the role of lysine-12; Lodi PJ et al.; The role of lysine-12 at the active site of yeast triosephosphate isomerase has been elucidated by a combination of site-directed mutagenesis, Fourier transform infrared spectroscopy, enzyme kinetics, and X-ray crystallography . Several lines of evidence suggest that the mutant isomerase in which lysine has been changed to methionine cannot bind substrate . This mutant enzyme has no detectable catalytic activity, and infrared experiments show no evidence of binding dihydroxyacetone phosphate nor dihydroxyacetone sulfate to the active site . Furthermore, crystals of the enzyme grown in the presence of phosphoglycolohydroxamate, a potent reaction intermediate analog, show an open active site with no inhibitor bound . Mutation of lysine-12 to arginine produces a protein with a value Km elevated by a factor of 22, a Vmax reduced by a factor of 180, and a Ki for phosphoglycolohydroxamate elevated by a factor of 290 . Mutation of lysine-12 to histidine produces an enzyme that shows virtually no catalytic activity at neutral pH, but below pH 6.1 this enzyme is active, suggesting that protonation of the histidine in this mutant is required for activity . These studies, together with the structural results reported in an accompanying paper, provide convincing evidence that a positive charge is required for substrate binding at the active site of triosephosphate isomerase and that lysine-12 provides this positive charge. Structure, 1994 Mar 15, 2(3), 201 - 8 Laue diffraction study on the structure of cytochrome c peroxidase compound I; Fulop V et al.; BACKGROUND: Cytochrome c peroxidase from yeast is a soluble haem-containing protein found in the mitochondrial electron transport chain where it probably protects against toxic peroxides . The aim of this study was to obtain a reliable structure for the doubly oxidized transient intermediate (termed compound I) in the reaction of cytochrome c peroxidase with hydrogen peroxide . This intermediate contains a semistable free radical on Trp191, and an oxyferryl haem group . RESULTS: Compound I was produced in crystals of yeast cytochrome c peroxidase by reacting the crystalline enzyme with hydrogen peroxide in a flow cell . The reaction was monitored by microspectrophotometry and Laue crystallography in separate experiments . A nearly complete conversion to compound I was achieved within two minutes of the addition of hydrogen peroxide, and the concentration of the intermediate remained at similar levels for an additional half an hour . The structure of the intermediate was determined by Laue diffraction . The refined Laue structure for compound I shows clear structural changes at the peroxide-binding site but no significant changes at the radical site . The photographs were processed with a new software package (LEAP), overcoming many of the former problems encountered in extracting structural information from Laue exposures . CONCLUSIONS: The geometry of the haem environment in this protein allows structural changes to be extremely small, similar in magnitude to those observed for the Fe2+/Fe3+ transition in cytochrome c . The results suggest that these molecules have evolved to transfer electrons with a minimal need for structural adjustment. EMBO J, 1994 Mar 15, 13(6), 1434 - 42 Recognition of a DNA operator by a dimer composed of two different homeodomain proteins; Goutte C et al.; The yeast homeodomain proteins a1 and alpha 2 interact to form a heterodimer that binds DNA with high specificity . The DNA recognition element consists of two similar half sites, arranged with dyad symmetry and separated by a fixed number of base pairs . We demonstrate that in the a1 alpha 2-DNA complex, one of these half-sites is bound by a1 while the other is bound by alpha 2 . These assignments allow a comparison of the chemical and nuclease protection patterns produced by both proteins when bound together to the hsg operator . Contrary to simple expectations, we propose that the a1 and alpha 2 homeodomains are arranged on the DNA in tandem, despite the fact that the recognition sequence is dyad symmetric. J Biotechnol, 1994 Mar 15, 33(1), 63 - 70 Ionic adsorption of catalase on bioskin: kinetic and ultrastructural studies; Solas MT et al.; Bioskin is a natural polymer produced by Acetobacter xylinum and several yeasts in culture . It contains glucosamine and N-acetyl galactosamine which promote ionic adsorption of catalase at the adequate pH value . High values of ionic strength are required to enzyme desorption . Adsorption of catalase on bioskin fibers has been visualized by scanning electron microscopy associated to a dispersion X-ray analyzer . At low enzyme density, the affinity of the immobilized catalase for hydrogen peroxide was 30% lower than that of the free enzyme . This affinity decreased dramatically at higher density of immobilized enzyme and could not be increased by agitation of the enzyme reaction mixture . Immobilized catalase retains about 70% of its initial activity after 16 d storage, whereas soluble enzyme is completely inactivated after 3 d at room temperature . The haeme group of catalase is not protected after immobilization since it is accessible to both EDTA and phloroglucinol, chelating agents which inactivate catalase by removing the iron atom from the haeme group. Biochemistry, 1994 Mar 8, 33(9), 2422 - 9 Secondary structure of fibronectin type 1 and epidermal growth factor modules from tissue-type plasminogen activator by nuclear magnetic resonance; Smith BO et al.; A segment of human tissue-type plasminogen activator (t-PA) corresponding to the fibronectin type 1 (F1) and epidermal growth factor-like (G) pair of modules, residues 1-91, has been produced as a recombinant protein in Saccharomyces cerevisiae, with a single conservative Cys to Ser substitution . The sequence-specific assignment of the 1H and 15N nuclear magnetic resonances from the pair of modules has been completed using 2D 1H nuclear magnetic resonance (NMR) spectra in conjunction with 3D, 15N-edited, 1H and 2D 15N-1H NMR spectra . Slowly exchanging amide protons have been identified, and estimates of a number of backbone 3JNH-C alpha H coupling constants were obtained by line-shape-fitting . The secondary structure of the F1 module in the pair closely matches that previously determined for the isolated F1 module from t-PA, and that of the G module conforms to the "consensus" G module structure determined previously from several isolated G modules . In the module pair, the residues linking the two modules appear to form an extended beta-strand, the carboxy-terminal end of which makes up a third strand of the major beta-sheet of the G module . The intermodule interface is defined by NOEs between residues in the ranges 22-24 in the F1 module and 65-72 in the G module . The NMR data indicate that there is little or no reorientation of the two modules with respect to one another but rather that they combine with a fixed hydrophobic contact dominated by the side chain of leucine-22. Biochemistry, 1994 Mar 8, 33(9), 2402 - 8 Characterization of the guanidine hydrochloride-denatured state of iso-1-cytochrome c by infrared spectroscopy; Bowler BE et al.; Infrared spectroscopy has been used to monitor residual ordered structure in the denatured state of wild-type and two mutants of iso-1-cytochrome c . The technique used involves a careful digital subtraction procedure that removes spectral contributions from buffer, water vapor, and the denaturant guanidine hydrochloride . Reliable and reproducible spectra can be produced using these methods . The data for iso-1-cytochrome c show upon denaturation a shift of the structure-sensitive amide I infrared band away from the spectral region associated with random structure . Second-derivative resolution enhancement of the amide I absorption band uncovers several bands which can be associated with various residual ordered structures in the denatured state . Gradual changes in the amide I band after denaturation are also observed as the guanidine hydrochloride concentration is increased . Two single-site mutants of iso-1-cytochrome c, which have been shown to have more compact denatured states than the wild-type protein, exhibited denatured-state infrared spectra with significant differences from the wild-type protein spectra . These observations provide new insight into the characteristics of protein denatured states. J Biol Chem, 1994 Mar 4, 269(9), 6550 - 7 The influence of beta subunit structure on the stability of Na+/K(+)-ATPase complexes and interaction with K+; Eakle KA et al.; Heterologous expression of the beta subunit of H+/K(+)-ATPase (HK beta) with alpha subunits of Na+/K(+)-ATPase (NK alpha) in yeast leads to the formation of ouabain binding complexes, indicating assembly of the two subunits into active ion pumps (Eakle, K . A., Kim, K . S., Kabalin, M . A., and Farley, R . A . (1992) Proc . Natl . Acad . Sci . U . S . A . 89, 2834-2838) . Complexes of NK alpha and HK beta are less sensitive to inhibition of ouabain binding by K+, suggesting that HK beta lowers the affinity of K+ binding sites . This effect is particularly pronounced when HK beta is combined with the alpha 3 isoform of NK alpha . In this case, titration with K+ yields a biphasic curve, suggesting that there are two nonequivalent sites for K+ binding . Attempts at purifying complexes formed with either alpha 1 + HK beta or alpha 3 + HK beta using SDS extraction of microsomal membranes resulted in the loss of ouabain binding . Controls show that alpha 1 + beta 1 and alpha 3 + beta 1 complexes still retain ouabain binding after SDS extraction under the same conditions . This suggests that the HK beta subunit forms a less stable complex with NK alpha subunits . We have created chimeric beta subunits comprised of the amino-terminal cytoplasmic and transmembrane regions of HK beta combined with the carboxyl-terminal extracellular region of Na+/K(+)-ATPase beta 1 (HN beta 1) and the complementary chimera with amino-terminal cytoplasmic and transmembrane regions of beta 1 combined with the carboxyl-terminal extracellular region of HK beta (NH beta 1) . When NH beta 1 is combined with either alpha 1 or alpha 3, the complexes show profiles of K+ inhibition of ouabain binding that are very similar to HK beta combined with either alpha 1 or alpha 3 . The data suggest that the extracellular region of HK beta is primarily responsible for the effect on apparent K+ affinity . When the HN beta 1 subunit is expressed with the alpha 3 subunit, less than 5% of the amount of ouabain binding complexes are formed compared with HN beta 1 + alpha 1 . This observation suggests that the HN beta 1 subunit either assembles poorly or forms an unstable complex with alpha 3 . After SDS extraction, complexes of alpha 1 + NH beta 1 and alpha 3 + NH beta 1 retain ouabain binding, while alpha 1 + HN beta 1 complexes are sensitive to SDS extraction.(ABSTRACT TRUNCATED AT 400 WORDS) J Biol Chem, 1994 Mar 4, 269(9), 6437 - 43 The use of chemical cross-linking to identify proteins that interact with a mitochondrial presequence; Gaikwad AS et al.; Previous work has shown that when yeast mitochondria are incubated in the presence of the presequence peptide pL4(1-22), the peptide is imported and accumulates within the mitochondrial membranes, presumably at the import sites . If the extramitochondrial concentration of peptide is sufficiently high, enough peptide accumulates within the import sites to prevent the uptake of authentic precursor proteins . We have used chemical cross-linking to probe the interaction of this peptide with yeast mitochondrial proteins . We found that radiolabeled pL4(1-22) could be reproducibly cross-linked to a number of polypeptides . Interestingly, nearly all were membrane proteins . Several of the cross-linked proteins were located in the outer membrane, while others were located in the inner membrane . The interaction between the peptide and many of the cross-linked products was shown to be specific by two independent criteria . First, an excess of unlabeled peptide acted as a competitor in the cross-linking reaction, and, second, treatment of the peptide with the alkylating agent N-ethylmaleimide dramatically reduced its ability to form cross-links . Two of the cross-linked species corresponded to the outer membrane proteins, Mas70p and ISP42 . Significantly, both of these proteins have previously been shown to play critical roles in mitochondrial protein import . While the role of the other cross-linked proteins in the import process remains to be determined, the results of this study demonstrate that our experimental approach may be useful in identifying components of the import machinery as well as proteins that interact with mitochondrial presequences. Plant Mol Biol, 1994 Mar, 24(6), 889 - 901 The tRNA(Ser)-isoacceptors and their genes in Nicotiana rustica: genome organization, expression in vitro and sequence analyses; Teichmann T et al.; The existence of six serine codons results in a complex pattern of tRNA(Ser) isoacceptors in organisms and organelles . According to the original wobble hypothesis, a minimum of three isoacceptors should be sufficient to read the six serine codons . We have isolated five cytoplasmic tRNAs(Ser) from leaves of Nicotiana rustica . Their nucleotide sequences identify them as four different isoacceptors with the anticodons cm5UGA, CGA, IGA and GCU . For tRNA(Ser) with IGA anticodon, two species have been detected which vary only by one nucleotide in the long extra arm . The first three isoacceptors recognize codons of the type UCN whereas the fourth isoacceptor reads the two serine codons AGC and AGU . The tRNA(Ser) sequences were used to design appropriate primers for the amplification of Nicotiana nuclear tRNA(Ser) genes by the polymerase chain reaction (PCR) . A total number of eight tRNA(Ser) genes differing in the coding region were thus identified . Selected PCR DNA fragments were then employed as probes for the isolation of the corresponding genes from a nuclear DNA library of N . rustica . Sequence analyses revealed that five of the isolated seven clones contained tRNA(Ser) genes which are identical in sequence with the five cytoplasmic tRNAs(Ser) mentioned above . None of them contains an intervening sequence . This is the first time that all putative cellular tRNA(Ser) isoacceptors and their corresponding genes have been characterized in an eukaryotic organism . Most of the tRNA(Ser) genes are functional as deduced from in vitro transcription and processing studies . Two of the genes yield pre-tRNAs(Ser) which are not or poorly converted to mature tRNA in a plant extract . The approximate tRNA(Ser) gene number was estimated by hybridization of specific DNA probes to Eco RI-cleaved Nicotiana nuclear DNA . The overall hybridization pattern indicates that members of each particular tRNA(Ser) gene family do not appear to be clustered but distributed randomly throughout the Nicotiana genome. Microsc Res Tech, 1994 Mar 1, 27(4), 284 - 93 A morphological view on mitochondrial protein targeting; van der Klei IJ et al.; Mitochondrial protein targeting includes both intramitochondrial sorting of proteins encoded by the organellar genome and import and subsequent sorting of nuclear encoded precursor proteins . Only a few proteins are encoded by the mitochondrial genome and synthesized in the organellar matrix . These include predominantly inner membrane proteins that are perhaps co-translationally inserted into this membrane . Biochemical data suggest that insertion into the inner membrane may be confined to the inner boundary membrane . Ultrastructurally, however, a preferential association of ribosomes with either inner boundary or cristae membranes has not been established . The majority of the mitochondrial proteins are nuclear encoded and synthesized as precursors in the cytosol . Electron microscopic studies revealed that import of precursor proteins is generally confined to sites where both mitochondrial envelope membranes are closely apposed . In line with these observations, biochemical studies indicated that precursor proteins destined for the inner membrane or matrix have to interact with the energized inner membrane to allow complete passage of the precursor through the outer membrane . As a consequence, the mitochondrial envelope membranes have to be in close proximity at protein import sites . In isolated mitochondria distinct sites (designated as contact sites) exist where both envelope membranes are closely apposed and presumably stably associated . In situ, however, mitochondrial boundary membranes are in close proximity over large areas that cover almost the entire mitochondrial periphery . Consequently, the relative area of the mitochondrial surface, where both boundary membranes are in sufficient proximity for allowing protein translocation, is generally larger in situ compared to that in isolated organelles . Immunocytochemical localization studies showed a rather random distribution of components of the mitochondrial protein translocation machinery over the entire mitochondrial surface and not confined to contact sites . Based on these ultrastructural data and recent biochemical findings we propose that mitochondrial protein import sites are dynamic in nature and include relatively labile regions of close association of the boundary membranes . In vitro, however, mitochondrial protein import may preferentially take place at or near the presumably stable contact sites. Chem Biol Interact, 1994 Mar, 90(3), 235 - 51 The role of coenzyme A in the biotransformation of 2-arylpropionic acids; Hall SD et al.; A number of 2-arylpropionic acid non-steroidal anti-inflammatory drugs ('profens') undergo highly enantioselective inversion from the (R)- to (S)-enantiomer . The mechanism of this inversion reaction involves the initial enantioselective formation of a coenzyme A thioester followed by epimerization and finally hydrolysis to regenerate free acids . Long-chain fatty acyl-CoA synthetase appears to mediate the initial thioester formation and an epimerase of an unknown physiologic function effects the second step . The hydrolases mediating the final step are poorly defined . Available evidence suggests that the liver is quantitatively the most important tissue site of inversion but local tissue inversion may influence the pharmacological and toxicological response of a given organ . Data from isolated rat hepatocytes indicate that other xenobiotics can modulate the formation and hydrolysis of ibuprofenyl-CoA by influencing inversion pathways, non-inversion pathways or both . Interactions between xenobiotics may therefore accentuate inter-individual variability in response to 2-aryl-propionic acids . The formation of 2-arylpropionyl-CoA thioesters in vivo has the potential to disrupt numerous biochemical pathways in addition to enhancing individual exposure to the potent anti-inflammatory (S)-enantiomers. Chem Biol Interact, 1994 Mar, 90(3), 215 - 23 Xenobiotic acyl-CoA formation: evidence of kinetically distinct hepatic microsomal long-chain fatty acid and nafenopin-CoA ligases; Knights KM et al.; Multiplicity of hepatic microsomal coenzyme A ligases catalyzing acyl-CoA thioester formation is an important factor for consideration in relation to the metabolism of xenobiotic carboxylic acids . In this study the kinetic characteristics of rat hepatic microsomal nafenopin-CoA ligase were studied and compared with those of long-chain fatty acid (palmitoyl) CoA ligase . The high affinity component of palmitoyl-CoA formation was inhibited by nafenopin (Ki 53 microM) and ciprofibrate (Ki 1000 microM) . Analagous to palmitoyl-CoA, nafenopin-CoA formation was catalyzed by an apparent high affinity low capacity isoform (Km 6 +/- 2.5 microM, Vmax 0.33 +/- 0.12 nmol/mg per min) which was inhibited competitively by palmitic acid (mean Ki 1.7 microM, n = 5) and R-ibuprofen (mean Ki 10.8 microM, n = 5) whilst ciprofibrate and clofibric acid were ineffective as inhibitors . The intrinsic metabolic clearance of nafenopin to nafenopin-CoA (Vmax/Km 0.057 +/- 0.011 nmol/mg/min/ +/- M) was similar to that reported recently for the formation of ibuprofenyl-CoA by rat liver microsomes . Evidence of both a substantial difference between the Km and Ki for nafenopin and lack of commonality with regard to xenobiotic inhibitors suggests that the high affinity microsomal nafenopin-CoA and long-chain fatty acid-CoA ligases are kinetically distinct . Thus until the current 'long-chain like' xenobiotic-CoA ligases are fully characterised in terms of substrate specificity, inhibitor profile, etc, it will be impossible to rationalize (and possibly predict) the metabolism and hence toxicity of xenobiotic carboxylic acids forming acyl-CoA thioester intermediates. Development, 1994 Mar, 120(3), 473 - 81 Cytosolic interaction between deltex and Notch ankyrin repeats implicates deltex in the Notch signaling pathway; Diederich RJ et al.; Genetic data from Drosophila have suggested a functional relationship between the novel cytoplasmic protein encoded by the deltex locus and the transmembrane receptor encoded by Notch . We have demonstrated a direct interaction between these proteins from expression studies conducted in cultured cells, in yeast, and in the imaginal wing disc . deltex binds specifically to the Notch ankyrin repeats, a region that is crucial for Notch signaling and that constitutes the most conserved domain among Notch family members . In addition, we present a new Notch allele, Nsu42c, that is associated with a missense mutation within the fifth ankyrin repeat . In addition to representing a new class of viable Notch allele, this mutation behaves similarly to mutations of deltex and further implicates the ankyrin repeats in Notch function. J Cell Biol, 1994 Mar, 124(6), 915 - 25 An internal region of the peroxisomal membrane protein PMP47 is essential for sorting to peroxisomes; McCammon MT et al.; Targeting sequences on peroxisomal membrane proteins have not yet been identified . We have attempted to find such a sequence within PMP47, a protein of the methylotrophic yeast, Candida boidinii . This protein of 423 amino acids shows sequence similarity with proteins in the family of mitochondrial carrier proteins . As such, it is predicted to have six membrane-spanning domains . Protease susceptibility experiments are consistent with a six-membrane-spanning model for PMP47, although the topology for the peroxisomal protein is inverted compared with the mitochondrial carrier proteins . PMP47 contains two potential peroxisomal targeting sequences (PTS1), an internal SKL (residues 320-322) and a carboxy terminal AKE (residues 421-423) . Using a heterologous in vivo sorting system, we show that efficient sorting occurs in the absence of both sequences . Analysis of PMP47-dihydrofolate reductase (DHFR) fusion proteins revealed that amino acids 1-199 of PMP47, which contain the first three putative membrane spans, do not contain the necessary targeting information, whereas a fusion with amino acids 1-267, which contains five spans, is fully competent for sorting to peroxisomes . Similarly, a DHFR fusion construct containing residues 268-423 did not target to peroxisomes while residues 203-420 appeared to sort to that organelle, albeit at lower efficiency than the 1-267 construct . However, DHFR constructs containing only amino acids 185-267 or 203-267 of PMP47 were not found to be associated with peroxisomes . We conclude that amino acids 199-267 are necessary for peroxisomal targeting, although additional sequences may be required for efficient sorting to, or retention by, the organelles. EMBO J, 1994 Mar 1, 13(5), 1123 - 31 Activation of MEK family kinases requires phosphorylation of two conserved Ser/Thr residues; Zheng CF et al.; MEK is a family of dual specific protein kinases which activate the extracellular signal-regulated kinases by phosphorylation of threonine and tyrosine residues . MEK itself is activated via serine phosphorylation by upstream activator kinases, including c-raf, mos and MEK kinase . Here, we report the activation phosphorylation sites of human MEK1 and yeast STE7 kinase as determined by a combination of biochemical and genetic approaches . In human MEK1, substitution of either serine residue 218 or 222 with alanine completely abolished its activation by epidermal growth factor-stimulated Swiss 3T3 cell lysates or immunoprecipitated c-raf, suggesting that both serine residues are required for MEK1 activation . Phosphopeptide analysis demonstrated that serine residues 218 and 222 of human MEK1 are the primary sites for phosphorylation by c-raf . These two serine residues are highly conserved in all members of the MEK family, including the yeast STE7 gene product, a MEK homolog in the yeast mating pheromone response pathway . Mutation of the corresponding residues in STE7 completely abolished the biological functions of this gene . These data demonstrate that MEK is activated by phosphorylation of two adjacent serine/threonine residues and this activation mechanism is conserved in the MEK family kinases. Proc Natl Acad Sci U S A, 1994 Mar 1, 91(5), 1848 - 52 Structure, function, and chromosome mapping of the growth-suppressing human homologue of the murine gas1 gene; Del Sal G et al.; We describe the isolation, growth-suppressing activity, and chromosomal localization of the human homologue of the murine growth-arrest-specific gene gas1 . Overexpression of h-gas1 is able to block cell proliferation in the A549 lung carcinoma and the T24 bladder carcinoma cell lines . No effect was observed when h-gas1 was introduced into the osteosarcoma cell line SAOS-2 and into the adenovirus-type-5 transformed cell line 293 . This finding is related to our previous evidence that simian virus 40-transformed NIH 3T3 cells are also refractory to murine gas1 overexpression, suggesting that the retinoblastoma and/or p53 gene products have an active role in mediating the growth-suppressing effect of gas1 . We also show that h-gas1 is on chromosome 9q21.3-22.1, in a region considered to be a fragile site . Altogether, the results raise the possibility that h-gas1 may be a target for genetic alterations leading to its inactivation in tumor cells. Proc Natl Acad Sci U S A, 1994 Mar 1, 91(5), 1652 - 6 Human transcription factor IIIC box B binding subunit; L'Etoile ND et al.; Transcription factor IIIC (TFIIIC) is a multisubunit basic TF for RNA polymerase III . It initiates transcription complex assembly on tRNA and related genes by binding to the internal box B promoter element and is also required for transcription of 5S rRNA and other stable nuclear and cytoplasmic RNAs transcribed by polymerase III . In mammalian cells, regulation of TFIIIC activity controls overall polymerase III transcription in response to growth factors and viral infection . Here, we report the cloning and sequencing of a full-length cDNA (and genomic DNA from the transcription initiation region) encoding the box B binding subunit of human TFIIIC, the 243-kDa alpha subunit . Specific antisera raised against the encoded protein super shifts a TFIIIC-box B DNA complex during an electrophoretic mobility shift assay and immunodepletes TFIIIC transcriptional activity from a partially purified TFIIIC fraction, proving that the cDNA encodes a component of TFIIIC . The human protein shows surprisingly little similarity to the box B binding subunit of yeast TFIIIC. Proc Natl Acad Sci U S A, 1994 Mar 1, 91(5), 1619 - 23 Delineation of a small region within the major transactivation domain of the human glucocorticoid receptor that mediates transactivation of gene expression; Dahlman-Wright K et al.; Previous deletion analysis localized the major transactivation function of the human glucocorticoid receptor to a 185-amino acid segment close to the N terminus of the receptor protein . This region was named tau 1 {Hollenberg, S . M . & Evans, R . M . (1988) Cell 55, 899-906} . To delineate the smallest active region within tau 1, we have systematically tested the transactivation capacity of deletion derivatives of the tau 1 domain, fused to the glucocorticoid receptor DNA-binding domain, in yeast cells . Internal scanning deletions suggested that residues near the C terminus of tau 1 are most important for activity . Deletions of N-terminal and C-terminal sequences identified a 41-amino acid "core" region near the C terminus of tau 1 that is crucial for tau 1 function . Small peptide fragments containing the tau 1 core region are competent for transactivation, while regions outside the tau 1 core are not active . We have previously demonstrated that the intact tau 1 domain squelches the activity of a minimal promoter in vivo and in vitro, suggesting involvement of interactions with a component/components of the basal transcription machinery in the mechanism of transactivation . This activity was maintained in the tau 1 core-containing segments. Mol Cell Biol, 1994 Mar, 14(3), 1979 - 85 Importance of a flanking AT-rich region in target site recognition by the GC box-binding zinc finger protein MIG1; Lundin M et al.; MIG1 is a zinc finger protein that mediates glucose repression in the yeast Saccharomyces cerevisiae . MIG1 is related to the mammalian Krox/Egr, Wilms' tumor, and Sp1 finger proteins . It has two fingers and binds to a GCGGGG motif that resembles the GC boxes recognized by these mammalian proteins . We have performed a complete saturation mutagenesis of a natural MIG1 site in order to elucidate its binding specificity . We found that only three mutations within the GC box retain the ability to bind MIG1: G1 to C, C2 to T, and G5 to A . This result is consistent with current models for zinc finger-DNA binding, which assume that the sequence specificity is determined by base triplet recognition within the GC box . Surprisingly, we found that an AT-rich region 5' to the GC box also is important for MIG1 binding . This AT box is present in all natural MIG1 sites, and it is protected by MIG1 in DNase I footprints . However, the AT box differs from the GC box in that no single base within it is essential for binding . Instead, the AT-rich nature of this sequence seems to be crucial . The fact that AT-rich sequences are known to increase DNA flexibility prompted us to test whether MIG1 bends DNA . We found that binding of MIG1 is associated with bending within the AT box . We conclude that DNA binding by a simple zinc finger protein such as MIG1 can involve both recognition of the GC box and flanking sequence preferences that may reflect local DNA bendability. Mol Cell Biol, 1994 Mar, 14(3), 1956 - 63 Characterization of a novel 23-kilodalton protein of unactive progesterone receptor complexes; Johnson JL et al.; Immunoprecipitation of unactivated avian progesterone receptor results in the copurification of hsp90, hsp70, and three additional proteins, p54, p50, and p23 . p23 is also present in immunoaffinity-purified hsp90 complexes along with hsp70 and another protein, p60 . Antibody and cDNA probes for p23 were prepared in an effort to elucidate the significance and function of this protein . Antibodies to p23 detect similar levels of p23 in all tissues tested and cross-react with a protein of the same size in mice, rabbits, guinea pigs, humans, and Saccharomyces cerevisiae, indicating that p23 is a conserved protein of broad tissue distribution . These antibodies were used to screen a chicken brain cDNA library, resulting in the isolation of a 468-bp partial cDNA clone encoding a sequence containing four sequences corresponding to peptide fragments isolated from chicken p23 . This partial clone was subsequently used to isolate a full-length human cDNA clone . The human cDNA encodes a protein of 160 amino acids that does not show homology to previously identified proteins . The chicken and human cDNAs are 88% identical at the DNA level and 96.3% identical at the protein level . p23 is a highly acidic phosphoprotein with an aspartic acid-rich carboxy-terminal domain . Bacterially overexpressed human p23 was used to raise several monoclonal antibodies to p23 . These antibodies specifically immunoprecipitate p23 in complex with hsp90 in all tissues tested and can be used to immunoaffinity isolate progesterone receptor complexes from chicken oviduct cytosol. Mol Cell Biol, 1994 Mar, 14(3), 1689 - 97 Mutations in an essential U2 small nuclear RNA structure cause cold-sensitive U2 small nuclear ribonucleoprotein function by favoring competing alternative U2 RNA structures; Zavanelli MI et al.; Mutations in stem-loop IIa of yeast U2 RNA cause cold-sensitive growth and cold-sensitive U2 small nuclear ribonucleoprotein function in vitro . Cold-sensitive U2 small nuclear RNA adopts an alternative conformation that occludes the loop and disrupts the stem but does so at both restrictive and permissive temperatures . To determine whether alternative U2 RNA structure causes the defects, we tested second-site mutations in U2 predicted to disrupt the alternative conformation . We find that such mutations efficiently suppress the cold-sensitive phenotypes and partially restore correct U2 RNA folding . A genetic search for additional suppressors of cold sensitivity revealed two unexpected mutations in the base of an adjacent stem-loop . Direct probing of RNA structure in vivo indicates that the suppressors of cold sensitivity act to improve the stability of the essential stem relative to competing alternative structures by disrupting the alternative structures . We suggest that many of the numerous cold-sensitive mutations in a variety of RNAs and RNA-binding proteins could be a result of changes in the stability of a functional RNA conformation relative to a competing structure . The presence of an evolutionarily conserved U2 sequence positioned to form an alternative structure argues that this region of U2 is dynamic during the assembly or function of the U2 small nuclear ribonucleoprotein. Mol Cell Biol, 1994 Mar, 14(3), 1582 - 93 Species-specific interaction of the glutamine-rich activation domains of Sp1 with the TATA box-binding protein; Emili A et al.; We have used protein-blotting and protein affinity chromatography to demonstrate that each of the two glutamine-rich activation domains of the human transcription factor Sp1 can bind specifically and directly to the C-terminal evolutionarily conserved domain of the human TATA box-binding protein (TBP) . These activation domains of Sp1 also bind directly to Drosophila TBP but bind much less strongly to TBP from the yeast Saccharomyces cerevisiae . The abilities of the Sp1 activation domains to interact directly with the TBPs of various species correlate well with their abilities to activate transcription in extracts derived from the same species . We also show that a glutamine-rich transcriptional activating region of the Drosophila protein Antennapedia binds directly to TBP in a species-specific manner that reflects its ability to activate transcription in vivo . These results support the notion that TBP is a direct and important target of glutamine-rich transcriptional activators. Biochim Biophys Acta, 1994 Mar 1, 1217(2), 207 - 10 Guanine nucleotide exchange factor for eukaryotic initiation factor-2 . Cloning of cDNA for the delta-subunit of rabbit translation initiation factor-2B; Price NT et al.; Peptide sequence data for rabbit eIF-2B delta were obtained and used to design redundant oligonucleotides for PCR . RNA was isolated from rabbit liver and used to direct the synthesis of total cDNA . A rabbit eIF-2B delta transcript was then amplified by PCR and sequenced . The PCR product was used to isolate a clone from a rabbit liver cDNA library . RACE (rapid amplification of cDNA ends) was used to obtain further 5' sequence . Subsequently, a full length cDNA was obtained from a rabbit reticulocyte library . PCR was used to confirm that the sequence is the same for the liver factor . The sequence obtained shows strong homology to that of yeast eIF-2B delta, the GCD2 gene product. Oncogene, 1994 Mar, 9(3), 791 - 9 Mechanism of action of a dominant-negative mutant of c-Jun; Brown PH et al.; The AP-1 transcriptional activating complex, made up of Jun and Fos protein, is involved in controlling many cellular processes such as cell proliferation, differentiation and transformation . We have previously characterized a dominant-negative mutant of c-Jun called TAM-67 which forms dimers with c-Jun and c-Fos, and binds DNA as a homodimer or heterodimer with c-Jun or c-Fos . This dominant-negative mutant is a potent inhibitor of AP-1 mediated transactivation, as well as c-jun/ras and TPA/ras-induced transformation . The present report describes experiments designed to elucidate the exact molecular mechanism of this dominant-negative inhibitor . The DNA binding kinetics of both TAM-67:TAM-67 homodimers as well as TAM-67:Fos heterodimers were studied and compared to those of c-Jun and other transactivation-deficient mutants of c-Jun . These studies demonstrated that the TAM-67 proteins have similar DNA binding kinetics to c-Jun and other Jun mutant proteins . Thus, the deletion of the amino-terminal end of the Jun protein does not significantly alter the protein's affinity for DNA . In addition, to determine whether TAM-67 functions through the formation of homodimers, or through interactions with endogenous c-Jun or c-Fos, we constructed a pair of chimeric proteins made by replacing the leucine zipper of TAM-67 with the leucine zippers of GCN4 and c-Fos . These chimeric proteins, termed TAM/GCN4 and TAM/Fos, were then tested for their ability to bind DNA, inhibit c-Jun-induced transactivation, and inhibit TPA/ras-mediated transformation . The results of these studies show that while both chimeric proteins bind equally well to DNA, only the TAM/Fos protein, and not the TAM/GCN4 protein, inhibits AP-1-induced transactivation and TPA/ras-induced transformation . When compared to the TAM-67 protein, the TAM/Fos protein is an equally potent inhibitor of transactivation and transformation . These results suggest that TAM-67 inhibits AP-1-mediated processes through a 'quenching' mechanism by inhibiting the function of endogenous Jun and/or Fos proteins . The implications of these mechanistic findings on the development of potent inhibitors of signal transduction pathways are discussed. Mol Biochem Parasitol, 1994 Mar, 64(1), 1 - 10 Isolation and characterization of the gene encoding histone H2A from Trypanosoma cruzi; Puerta C et al.; In the present paper we report the isolation and characterization of the sequence of two genomic DNA fragments coding for the histone H2A of Trypanosoma cruzi . An analysis of the predicted amino acid sequence shows the presence of the amino-terminal motif characteristic of the H2A histones proteins and the Lys-Lys motif reported to be the site for the ubiquitin attachment . Southern blots of total parasite DNA probed with the H2A sequence suggested that the T . cruzi histone H2A gene is encoded in two independent gene clusters . The molecular karyotyping of the parasite indicated that these two clusters locate in a single chromosome of about 700 kb in length . The T . cruzi H2A mRNA is polyadenylated as are the basal histone mRNAs of higher eukaryotes and the histone mRNAs of yeast . By polymerase chain reaction amplification and sequencing and by S1 mapping we determined respectively the 5' and 3' end of the gene showing that the miniexon is added to the mRNA 71 nucleotides upstream of the ATG initiation codon and that the polyadenylation site locates in nucleotide position 773-775 close to invert repeats. Electrophoresis, 1994 Mar-Apr, 15(3-4), 482 - 90 The molecular chaperones HSP28, GRP78, endoplasmin, and calnexin exhibit strikingly different levels in quiescent keratinocytes as compared to their proliferating normal and transformed counterparts: cDNA cloning and expression of calnexin; Honore B et al.; We have identified nine molecular chaperones in human keratinocytes by one or a combination of three methods: (i) reaction with antibodies raised against the purified proteins, (ii) microsequencing of two-dimensional (2-D) gel purified proteins, or (iii), by cloning of the cDNA and expression of its encoded protein in transformed human amnion cells using the vaccinia virus expression system . The expression levels of each of the molecular chaperones were analyzed in quiescent, normal proliferating, and simian virus SV40 transformed K14 keratinocytes by cutting the corresponding protein spots from dried 2-D gels and counting the radioactivity by liquid scintillation . The most striking observation was the strong up-regulation (936%) of the small heat shock protein HSP28 in the quiescent keratinocytes, a fact that is in line with recent data indicating that the murine homologue (HSP25) may act as a growth inhibitor . Several chaperones that localize to the endoplasmic reticulum and that are involved in the secretory pathway (GRP78, GRP78v, endoplasmin, and calnexin) were expressed at approximately similar levels in normal proliferating and K14 keratinocytes but were down-regulated by 50% or more in the quiescent cells, implying that these cells may possess an impaired ability to secrete certain proteins . Both GRP78 and endoplasmin genes have similar sequences in the promoter regions, suggesting that they may be partly co-regulated at the transcriptional level (McCauliffe et al., J . Biol . Chem . 1992, 267, 2557-2562).(ABSTRACT TRUNCATED AT 250 WORDS) Mol Microbiol, 1994 Mar, 11(5), 805 - 10 Cdc7 protein kinase for DNA metabolism comes of age; Sclafani RA et al.; The Cdc7 protein kinase is the product of an essential cell cycle gene, and is involved in three aspects of DNA metabolism: mitotic DNA replication, meiotic DNA recombination, and replication-dependent DNA repair . The mechanism by which Cdc7 regulates each of its cellular functions is an issue of considerable interest . Recently, much of the research regarding the regulation of cell cycle progression has focused on the regulatory action of cyclins on their catalytic counterparts . We propose that the function of Cdc7 in cell cycle progression is mediated in a similar manner, in that Dbf4, a protein whose transcript level is known to fluctuate in the cell cycle, is essential for Cdc7 kinase activity . The periodic association of Dbf4 with Cdc7 may account for the regulation of Cdc7 kinase function and progression through the cell cycle. Genes Dev, 1994 Mar 1, 8(5), 525 - 37 NOT1(CDC39), NOT2(CDC36), NOT3, and NOT4 encode a global-negative regulator of transcription that differentially affects TATA-element utilization; Collart MA et al.; The yeast HIS3 TR and TC TATA elements support basal transcription, but only TR can respond to transcriptional activators . Four genes, NOT1(CDC39), NOT2(CDC36), NOT3, NOT4, act as general negative regulators and preferentially affect TC-dependent transcription . Allele-specific suppression, a two-hybrid interaction, and biochemical confractionation suggest that NOT1 and NOT2 are nuclear proteins associated in a discrete, 500-kD complex . NOT4 interacts with NOT1 and NOT3 in the two-hybrid assay, and overexpression of NOT3 or NOT4 suppresses not1 and not2 mutations . Repression by the NOT proteins is not attributable to inhibition of transcriptional activators, does not involve the CYC8/TUP1 negative regulatory complex, and is distinct from repression by nucleosomes or by the SPT4, 5, 6 proteins that affect chromatin structure . We propose that the NOT protein inhibit the basic RNA polymerase II transcription machinery, possibly by affecting TFIID function. Curr Biol, 1994 Mar 1, 4(3), 264 - 7 RNA splicing . U2 fulfils a commitment; Hodges PE et al.; Genetic and biochemical studies of pre-mRNA splicing have recently converged to elucidate an early step in the process: the targeting of the U2 small nuclear ribonucleoprotein particle to the pre-mRNA. Curr Biol, 1994 Mar 1, 4(3), 203 - 14 Characterization of a phosphatidylinositol-specific phosphoinositide 3-kinase from mammalian cells; Stephens L et al.; BACKGROUND: As phosphoinositides can serve as signalling molecules within cells, the enzymes responsible for their synthesis and cleavage are likely to be involved in the transduction of signals from the cell surface through the cytoplasm . The precise role of the phosphoinositide 3-kinase that has been cloned from mammalian cells is not known, but it has been implicated in receptor-stimulated mitogenesis, glucose uptake and membrane ruffling . The enzyme can use phosphatidylinositol (PtdIns), PtdIns 4-phosphate and PtdIns (4,5)-bisphosphate as substrates in vitro, but it seems to phosphorylate PtdIns (4,5)-bisphosphate preferentially in vivo . The VPS34 gene product of yeast, by contrast, is a phosphoinositide 3-kinase homologue implicated in vacuolar protein sorting that apparently utilizes only PtdIns as a substrate . The significance of this difference in lipid-substrate preference and its relationship to the functions of the two phosphoinositide kinases is unknown . RESULTS: We have characterized a distinct PtdIns-specific phosphoinositide 3-kinase activity in mammalian cells . Unlike the previously identified, broad-specificity mammalian phosphoinositide kinase, this enzyme is resistant to the drug wortmannin and uses only PtdIns as a substrate in vitro; it therefore has the capacity to generate PtdIns 3-phosphate specifically . The newly characterized enzyme, which was purified by chromatography from cytosol, has biochemical and pharmacological characteristics distinct from those of the broad-specificity enzyme . CONCLUSIONS: The enzyme we have characterized may serve to generate PtdIns 3-phosphate for fundamentally different roles in the cell from those of PtdIns (3,4)-bisphosphate and/or PtdIns (3,4,5)-trisphosphate . Furthermore, the functions of the VSP34 gene product, which may not be relevant to the broad-specificity mammalian phosphoinositide 3-kinase, may be related to those of the enzyme we describe. Curr Biol, 1994 Mar 1, 4(3), 238 - 41 Replication and transcription . Silence of the ORCs; Kelly TJ et al.; The origin recognition complex, a multi-protein complex known to bind to replication origins, has now been implicated in transcriptional silencing, providing another link between DNA replication and transcription. Nucleic Acids Res, 1994 Feb 25, 22(4), 597 - 603 Analysis of the SIP3 protein identified in a two-hybrid screen for interaction with the SNF1 protein kinase; Lesage P et al.; The Saccharomyces cerevisiae SIP3 gene was identified in a two-hybrid screen for proteins that interact in vivo with the SNF1 protein kinase, which is necessary for release of glucose repression . We showed that the C-terminal part of SIP3, recovered through its ability to interact with SNF1, strongly activates transcription when tethered to DNA . We have cloned and sequenced the entire SIP3 gene . The predicted 142-kD SIP3 protein contains a putative leucine zipper motif located in its C terminus . The native SIP3 protein also interacts with DNA-bound SNF1 and activates transcription of a target gene . A complete deletion of the SIP3 gene did not confer phenotypes characteristic of snf1 mutants . However, in a mutant deficient for the SNF1 kinase activity due to loss of the SNF4 stimulatory function, increased dosage of SIP3 partially restored expression of the glucose-repressible SUC2 gene . Overexpression of the C terminus of SIP3 caused defects in growth and SUC2 expression which were remedied by overexpressing SNF1 . Taken together, these genetic data suggest that SIP3 is functionally related to the SNF1 protein kinase pathway. J Mol Biol, 1994 Feb 25, 236(3), 786 - 99 The role of a conserved internal water molecule and its associated hydrogen bond network in cytochrome c; Berghuis AM et al.; High resolution three-dimensional structures for the N52I and N52I-Y67F yeast iso-1-cytochrome c variants have been completed in both oxidation states . The most prominent structural difference observed in both mutant proteins is the displacement of a conserved, internally bound water molecule (Wat166) from the protein matrix . In wild-type yeast iso-1-cytochrome c the position and orientation of this water molecule is found to be dependent on the oxidation state of the heme iron atom . Overall our results suggest the function of Wat166 and its associated hydrogen bond network is threefold . First, the presence of Wat166 provides a convenient mechanism to modify the hydrogen bond network involving several residues near the Met80 ligand in an oxidation state dependent manner . Second, Wat166 is necessary for the maintenance of the spatial relationships between nearby side-chains and the hydrogen bond interactions formed between these groups in this region of the protein . An essential part of this role is ensuring the proper conformation of the side-chain of Tyr67 so that it forms a hydrogen bond interaction with the heme ligand Met80 . This hydrogen bond influences the electron withdrawing power of the Met80 ligand and is therefore a factor in controlling the midpoint reduction potential of cytochrome c . Elimination of this interaction in the N52I-Y67F mutant protein or elimination of Wat166 in the N52I protein with the subsequent disruption in the position and interactions of the Tyr67 side-chain, leads to a drop of approximately 56 mV in the observed midpoint reduction potential of the heme group . Third, Wat166 also appears to mediate increases in the mobility of three nearby segments of polypeptide chain when cytochrome c is in the oxidized state . Previous studies have proposed these changes may be related to oxidation state dependent interactions between cytochrome c and its redox partners . Coincident with the absence of Wat166, such mobility changes are not observed in the N52I and N52I-Y67F mutant proteins . It is possible that much of the increased protein stability observed for both mutant proteins may be due to this factor . Finally, our results show that neither heme iron charge nor heme plane distortion are responsible for oxidation state dependent conformational changes in the pyrrole A propionate region . Instead, the changes observed appear to be driven by the change in conformation that the side-chain of Asn52 experiences as the result of oxidation state dependent movement of Wat166. Science, 1994 Feb 25, 263(5150), 1153 - 6 Phosphorylation of the transcription factor PHO4 by a cyclin-CDK complex, PHO80-PHO85; Kaffman A et al.; Induction of the yeast gene PHO5 is mediated by the transcription factors PHO2 and PHO4 . PHO5 transcription is not detectable in high phosphate; it is thought that the negative regulators PHO80 and PHO85 inactivate PHO2 and PHO4 . Here it is reported that PHO80 has homology to yeast cyclins and interacts with PHO85, a p34cdc2/CDC28-related protein kinase . The PHO80-PHO85 complex phosphorylates PHO4; this phosphorylation is correlated with negative regulation of PHO5 . These results demonstrate the existence of a cyclin-cdk complex that is used for a regulatory process other than cell-cycle control and identify a physiologically relevant substrate for this complex. Biochemistry, 1994 Feb 22, 33(7), 1743 - 52 Evidence that CYP2C19 is the major (S)-mephenytoin 4'-hydroxylase in humans; Goldstein JA et al.; The present study assesses the role of members of the human CYP2C subfamily in the 4'-hydroxylation of (S)-mephenytoin . When recombinant CYP2C proteins were expressed using a yeast cDNA expression system, 2C19 stereospecifically 4'-hydroxylated (S)-mephenytoin with a turnover number at least 10 times higher than that of human liver microsomes . 2C9 (both Ile359 and Leu359 alleles) and 2C18 (Thr385 and Met385 alleles) metabolized this substrate at a rate 100-fold lower than 2C19, and metabolism by these 2C proteins was not stereospecific for the S-enantiomer . 2C8 exhibited very little mephenytoin 4'-hydroxylase activity . In contrast, the Ile359 allele of 2C9 had a high turnover number for the hydroxylation of tolbutamide, while the Leu359 allele was less active toward this substrate . Immunoblot analysis of 16 human liver donor samples indicated that (S)-mephenytoin 4'-hydroxylase activity correlated with the hepatic CYP2C19 content, but it did not correlate with the hepatic content of CYP2C9 . Moreover, direct sequencing of the polymerase chain reaction (PCR) products of 2C9 mRNA from six of these human livers through areas of known allelic variations indicated that the identity of the allele of 2C9 (Cys144 vs Arg, Tyr358 vs Cys, Ile359 vs Leu, or Gly417 vs Asp) did not appear to influence (S)-mephenytoin 4'-hydroxylase activity in these samples . These data indicate that 2C19 is the principal determinant of (S)-mephenytoin 4'-hydroxylase activity in human liver. J Chromatogr A, 1994 Feb 18, 662(1), 13 - 26 Multiple-site binding interactions in metal-affinity chromatography . I . Equilibrium binding of engineered histidine-containing cytochromes c; Todd RJ et al.; Mechanisms of protein retention in immobilized metal-affinity chromatography (IMAC) have been probed using a set of Saccharomyces cerevisiae iso-1-cytochrome c histidine variants constructed by site-directed mutagenesis . Proteins containing a single accessible histidine exhibit Langmuir-type isotherms with maximum protein binding capacities between 5 and 10% of the maximum copper loading and the capacity of the support to bind imidazole . A simple model that assumes that the copper sites are densely packed and can be blocked by protein adsorption yields binding constants for single-histidine proteins that are similar to the binding constant for free imidazole . Proteins containing multiple accessible histidines do not exhibit simple Langmuir-type behavior; they appear to interact with the support by simultaneous coordination to more than one metal ion, the result of which is to increase the apparent binding affinity by as much as a factor of 1000 . The protein binding constant depends on the availability of copper sites: binding is significantly weaker at low surface concentrations of copper that presumably cannot support multiple-site interactions . The protein binding capacity drops to zero at copper loadings less than one-half the maximum, indicating that immobilized iminodiacetic acid ligands are sufficiently close together that two can coordinate a single copper ion, which precludes its interaction with a protein . Protein adsorption via multiple-site coordination has important consequences for the optimization of IMAC separations and the design of new IMAC supports. J Biol Chem, 1994 Feb 18, 269(7), 4891 - 4 Involvement of the basic repeat domain of TATA-binding protein (TBP) in transcription by RNA polymerases I, II, and III; Kim TK et al.; The TATA-binding protein (TBP) plays a central role in transcription initiation by nuclear RNA polymerases I, II, and III . With knowledge of the three-dimensional structure of TBP, mutational analyses were focused on the highly exposed basic repeat domain in yeast TBP in order to identify amino acid residues which could discriminate transcription functions of different RNA polymerases . One mutation (K156L) was found to specifically abolish transcription by RNA polymerase I and another mutation (K138L) specifically abolished transcription by RNA polymerase III, while each maintained the ability to support in vitro transcription by the other two RNA polymerases . Along with previous studies, these results indicate that the basic repeat domain of TBP is important not only for transcription by RNA polymerase II but also for transcription by RNA polymerases I and III and, further, that the region has distinct sites for interactions which are specific for RNA polymerases I and III. J Biol Chem, 1994 Feb 18, 269(7), 4754 - 60 Trypanosoma brucei RNA polymerase II is phosphorylated in the absence of carboxyl-terminal domain heptapeptide repeats; Chapman AB et al.; Formation of an RNA polymerase II transcription initiation complex requires binding of a polymerase that contains a non-phosphorylated largest subunit carboxyl-terminal domain (CTD) . Polymerase binding is followed by elongation after phosphorylation of the CTD by a CTD kinase . Phosphorylation sites are within the repeating heptapeptide motifs which characterize the CTD of all eukaryotic RNA polymerase IIs . In contrast to all other eukaryotes studied, the trypanosome genome contains two genetic loci which encode the large subunit of RNA polymerase II; both genes lack CTD heptapeptide repeat structures . We have examined whether Trypanosoma brucei RNA polymerase II, despite its unique CTD domain, is phosphorylated when isolated from elongating transcription complexes . Elongating trypanosome RNA polymerases were photoaffinity labeled during nuclear run-on assays . The identity of the labeled proteins was established by immunoblotting and immunoprecipitation using polymerase-specific antisera . Analysis of the largest subunit of RNA polymerase II revealed the expected 195-kDa species and an additional larger 220-kDa species . The apparent molecular weight of this larger form of RNA polymerase II decreased incrementally as a function of incubation with increasing concentrations of calf intestinal phosphatase . These results show that extensive phosphorylation of the largest subunit of RNA polymerase-II is a conserved feature between trypanosomes and higher eukaryotes despite the absence of a typical CTD domain. Eur J Biochem, 1994 Feb 15, 220(1), 149 - 53 Effects of pH on carboxypeptidase-Y-catalyzed hydrolysis and aminolysis reactions; Christensen U; The pH dependencies of serine carboxypeptidase-Y-catalysed hydrolysis and aminolysis reactions using L-amino acids and L-amino acid amides as nucleophiles, have been studied and analyzed . The results reveal two catalytically important ionizing groups of the enzyme with rather similar pK values (5-6), the active site His397 and a possibly Glu residue, which is not only important in interactions with carboxylic groups of substrates and nucleophiles {Liao, D.-I., Breddam, K., Sweet, R . M., Bullock, T . & Remmington, S . J . (1992) Biochemistry 31, 9796-9812}, but also indirectly play a role in catalysis . This explains the pH behaviour of hydrolysis of both peptide and ester substrates and further, that L-amino acid amides are better nucleophiles in aminolysis reactions than L-amino acids. EMBO J, 1994 Feb 15, 13(4), 888 - 97 The splicing factor PRP2, a putative RNA helicase, interacts directly with pre-mRNA; Teigelkamp S et al.; The RNA helicase-like splicing factor PRP2 interacts only transiently with spliceosomes . To facilitate analysis of interactions of PRP2 with spliceosomal components, PRP2 protein was stalled in splicing complexes by two different methods . A dominant negative mutant form of PRP2 protein, which associates stably with spliceosomes, was found to interact directly with pre-mRNAs, as demonstrated by UV-crosslinking experiments . The use of various mutant and truncated pre-mRNAs revealed that this interaction requires a spliceable pre-mRNA and an assembled spliceosome; a 3' splice site is not required . To extend these observations to the wild-type PRP2 protein, spliceosomes were depleted of ATP; PRP2 protein interacts with pre-mRNA in these spliceosomes in an ATP-independent fashion . Comparison of RNA binding by PRP2 protein in the presence of ATP or gamma S-ATP showed that ATP hydrolysis rather than mere ATP binding is required to release PRP2 protein from pre-mRNA . As PRP2 is an RNA-stimulated ATPase, these experiments strongly suggest that the pre-mRNA is the native co-factor stimulating ATP hydrolysis by PRP2 protein in spliceosomes . Since PRP2 is a putative RNA helicase, we propose that the pre-mRNA is the target of RNA displacement activity of PRP2 protein, promoting the first step of splicing. EMBO J, 1994 Feb 15, 13(4), 879 - 87 A dominant negative mutation in the conserved RNA helicase motif 'SAT' causes splicing factor PRP2 to stall in spliceosomes; Plumpton M et al.; To characterize sequences in the RNA helicase-like PRP2 protein of Saccharomyces cerevisiae that are essential for its function in pre-mRNA splicing, a pool of random PRP2 mutants was generated . A dominant negative allele was isolated which, when overexpressed in a wild-type yeast strain, inhibited cell growth by causing a defect in pre-mRNA splicing . This defect was partially alleviated by simultaneous co-overexpression of wild-type PRP2 . The dominant negative PRP2 protein inhibited splicing in vitro and caused the accumulation of stalled splicing complexes . Immunoprecipitation with anti-PRP2 antibodies confirmed that dominant negative PRP2 protein competed with its wild-type counterpart for interaction with spliceosomes, with which the mutant protein remained associated . The PRP2-dn1 mutation led to a single amino acid change within the conserved SAT motif that in the prototype helicase eIF-4A is required for RNA unwinding . Purified dominant negative PRP2 protein had approximately 40% of the wild-type level of RNA-stimulated ATPase activity . As ATPase activity was reduced only slightly, but splicing activity was abolished, we propose that the dominant negative phenotype is due primarily to a defect in the putative RNA helicase activity of PRP2 protein. Proc Natl Acad Sci U S A, 1994 Feb 15, 91(4), 1445 - 9 n-Sec1: a neural-specific syntaxin-binding protein; Pevsner J et al.; We have identified n-Sec1, a rat brain homolog of the yeast Sec1p protein that participates in the constitutive secretory pathway between the Golgi apparatus and the plasma membrane . The rat brain cDNA is predicted to encode a 68-kDa protein with 65% amino acid identity to Drosophila rop, 59% identity to Caenorhabditis elegans unc-18, and 27% identity to Saccharomyces cerevisiae Sec1p . By RNA blot analysis, n-Sec1 mRNA expression is neural-specific . An anti-peptide antiserum directed against the n-Sec1 carboxyl terminus detects a 68-kDa protein in rat brain cytosol and membranes, but not in peripheral tissues . In the presence of syntaxin 1a, a plasma membrane protein implicated in synaptic vesicle docking, n-Sec1 becomes membrane-associated . n-Sec1 binds to syntaxin 1a, 2, and 3 fusion proteins coupled to agarose beads, but not to syntaxin 4 fusion protein or beads coupled to a variety of other proteins . These findings indicate that n-Sec1 is a neural-specific, syntaxin-binding protein that may participate in the regulation of synaptic vesicle docking and fusion. Proc Natl Acad Sci U S A, 1994 Feb 15, 91(4), 1285 - 9 Retroviruses in invertebrates: the gypsy retrotransposon is apparently an infectious retrovirus of Drosophila melanogaster; Kim A et al.; Retroviruses are commonly considered to be restricted to vertebrates . However, the genome of many eukaryotes contains mobile sequences known as retrotransposons with long terminal repeats (LTR retrotransposons) or viral retrotransposons, showing similarities with integrated proviruses of retroviruses, such as Ty elements in Saccharomyces cerevisiae, copia-like elements in Drosophila, and endogenous proviruses in vertebrates . The gypsy element of Drosophila melanogaster has LTRs and contains three open reading frames, one of which encodes potential products similar to gag-specific protease, reverse transcriptase, and endonuclease . It is more similar to typical retroviruses than to LTR retrotransposons . We report here experiments showing that gypsy can be transmitted by microinjecting egg plasma from embryos of a strain containing actively transposing gypsy elements into embryos of a strain originally devoid of transposing elements . Horizontal transfer is also observed when individuals of the "empty" stock are raised on medium containing ground pupae of the stock possessing transposing elements . These results suggest that gypsy is an infectious retrovirus and provide evidence that retroviruses also occur in invertebrates. Cell, 1994 Feb 11, 76(3), 531 - 41 The human U1A snRNP protein regulates polyadenylation via a direct interaction with poly(A) polymerase; Gunderson SI et al.; The human U1 snRNP-specific U1A protein autoregulates its production by binding its own pre-mRNA and inhibiting polyadenylation . The mechanism of this regulation has been elucidated by in vitro studies . U1A protein is shown not to prevent either binding of cleavage and polyadenylation specificity factor (CPSF) to its recognition sequence (AUUAAA) or to prevent cleavage of U1A pre-mRNA . Instead, U1A protein bound to U1A pre-mRNA inhibits both specific and nonspecific polyadenylation by mammalian, but not by yeast, poly(A) polymerase (PAP) . Domains are identified in both proteins whose removal uncouples the polyadenylation activity of mammalian PAP from its inhibition via RNA-bound U1A protein . Finally, U1A protein is shown to specifically interact with mammalian PAP in vitro . The possibility that this interaction may reflect a broader role of the U1A protein in polyadenylation is discussed. J Biol Chem, 1994 Feb 11, 269(6), 3968 - 75 Effect of acidic ribosomal phosphoprotein mRNA 5'-untranslated region on gene expression and protein accumulation; Bermejo B et al.; Constructions were made from genes encoding ribosomal acidic phosphoproteins YP1 beta (L44') and YP2 beta (L45) from Saccharomyces cerevisiae in which different parts of the 5'-untranslated regions were included . The constructs were inserted into centromeric plasmids under the control of the GAL1 promoter and expressed in yeast strains in which the genes coding for each acidic protein family, P1 and P2, had been disrupted . Deletions in the 5' region of the two genes have been found to oppositely affect their expression . Deletion of most of this region strongly stimulates the expression of YP2 beta (L45), increasing the translation efficiency of the mRNA, and generating a 6-fold excess of protein in the cell . A similar deletion in the rpYP1 beta gene represses the expression of the protein, reducing drastically the amount of the mRNA in the cell . The overexpression of rpYP2 beta affects the cell growth by inhibiting protein synthesis at the level of initiation . Reduction of the YP2 beta(L45) overproduction by growing in controlled concentrations of glucose abolishes the inhibitory effect . The excess protein, probably as a high molecular weight complex, apparently interferes with the joining of the 60 S subunit to the initiation complex generating the accumulation of polysome half-mers . In addition, the results indicate the existence of a regulatory mechanism by which each one of the two acidic proteins controls the expression of the other polypeptide . YP1 beta(L44') represses the expression of YP2 beta(L45), while this protein stimulates the expression of YP1 beta(L44'). Nucleic Acids Res, 1994 Feb 11, 22(3), 514 - 21 Dynamics of transfer RNAs analyzed by normal mode calculation; Nakamura S et al.; Normal mode calculation is applied to tRNAPhe and tRNAAsp, and their structural and vibrational aspects are analyzed . Dihedral angles along the phosphate-ribose backbone (alpha, beta, gamma, epsilon, zeta) and dihedral angles of glycosyl bonds (chi) are selected as movable parameters . The calculated displacement of each atom agrees with experimental data . In modes with frequencies higher than 130 cm-1, the motions are localized around each stem and the elbow region of the L-shape . On the other hand, collective motions such as bending or twisting of arms are seen in modes with lower frequencies . Hinge axes and bend angles are calculated without prior knowledge . Movements in modes with very low frequencies are combinations of hinge bending motions with various hinge axes and bend angles . The thermal fluctuations of dihedral angles well reflect the structural characters of transfer RNAs . There are some dihedral angles of nucleotides located around the elbow region of L-shape, which fluctuate about five to six times more than the average value . Nucleotides in the position seem to be influential in the dynamics of the entire structure . The normal mode calculation seems to provide much information for the study of conformational changes of transfer RNAs induced by aminoacyl-tRNA synthetase or codon during molecular recognition. Nucleic Acids Res, 1994 Feb 11, 22(3), 404 - 11 A fast word search algorithm for the representation of sequence similarity in genomic DNA; Lefevre C et al.; Representation of sequence similarity by dot matrix plots is a method widely used for comparing biological sequences . The user is presented with an overall view of similarity between two sequences . Computation of this plot has been reconsidered here . An improvement is proposed through the preprocessing of the data into an automation recognizing the word structure of a sequence . The main advantage of this approach is to systematically eliminate the repetitions during word comparison . Simple heuristics are also considered to greatly speed up pattern matching . As a result, large sequences are handled very efficiently . This is illustrated by a comparison of large genomic DNA . The algorithm has been implemented in an interactive application on a microcomputer. Genes Dev, 1994 Feb 1, 8(3), 313 - 27 The MAP kinase Fus3 associates with and phosphorylates the upstream signaling component Ste5; Kranz JE et al.; Activation of the Saccharomyces cerevisiae MAP kinase Fus3 is thought to occur via a linear pathway involving the sequential action of three proteins: Ste5, a protein of unknown function, Ste11, a MAPKK kinase homolog, and Ste7, a MAPK kinase homolog which phosphorylates and activates Fus3 . In this report, we present evidence for a novel mechanism of Fus3 activation that involves a direct association with Ste5, a protein not predicted to interact with Fus3 . First, overexpression of Ste5 suppresses fus3 point mutations in an allele-specific manner and increases Fus3 kinase activity in vitro . Second, Ste5 associates with Fus3 in vivo as demonstrated by the two-hybrid system and by two methods of copurification . Third, Ste5 and Fus3 associate prior to pheromone stimulation even when Fus3 is inactive, and in strains lacking Ste7 and Ste11 . Fourth Ste5 is phosphorylated by Fus3 in purified complexes and copurifies with an additional protein kinase(s) . These observations suggest the possibility that Ste5 promotes signal transduction by tethering Fus3 to its activating protein kinase(s). Mol Cell Biol, 1994 Feb, 14(2), 970 - 81 Role of the histone amino termini in facilitated binding of a transcription factor, GAL4-AH, to nucleosome cores; Vettese-Dadey M et al.; Facilitated, "cooperative" binding of GAL4-AH to nucleosomal DNA occurred in response to inhibition from the core histone amino termini . The binding of GAL4-AH (which contains the DNA-binding and dimerization domains of GAL4) to nucleosome cores containing multiple binding sites initiated at the end of a nucleosome core and proceeded in a cooperative manner until all sites were occupied . However, following tryptic removal of the core histone amino termini, GAL4-AH binding appeared to be noncooperative, similar to binding naked DNA . Binding of GAL4-AH to nucleosomes bearing a single GAL4 site at different positions indicated that inhibition of GAL4 binding was largely mediated by the histone amino termini and primarily occurred at sites well within the core and not near the end . When the histone amino termini were intact, binding of GAL4-AH to sites near the center of a nucleosome core was greatly enhanced by the presence of additional GAL4 dimers bound to more-accessible positions . These data illustrate that the binding of a factor to more-accessible sites, near the end of a nucleosome, allows facilitated binding of additional factors to the center of the nucleosome, thereby overcoming repression from the core histone amino termini . This mechanism may contribute to the binding of multiple factors to complex promoter and enhancer elements in cellular chromatin. Mol Cell Biol, 1994 Feb, 14(2), 1459 - 64 The carboxy-terminal region of mammalian HSP90 is required for its dimerization and function in vivo; Minami Y et al.; The majority of mouse HSP90 exists as alpha-alpha and beta-beta homodimers . Truncation of the 15-kDa carboxy-terminal region of mouse HSP90 by digestion with the Ca(2+)-dependent protease m-calpain caused dissociation of the dimer . When expressed in a reticulocyte lysate, the full-length human HSP90 alpha formed a dimeric form . A plasmid harboring human HSP90 alpha cDNA was constructed so that the carboxy-terminal 49 amino acid residues were removed when translated in vitro . This carboxy-terminally truncated human HSP90 alpha was found to exist as a monomer . In contrast, loss of the 118 amino acid residues from the amino terminus of human HSP90 alpha did not affect its in vitro dimerization . Introduction of an expression plasmid harboring the full-length human HSP90 alpha complements the lethality caused by the double mutations of two HSP90-related genes, hsp82 and hsc82, in a haploid strain of Saccharomyces cerevisiae . The carboxy-terminally truncated human HSP90 alpha neither formed dimers in yeast cells nor rescued the lethal double mutant. Mol Cell Biol, 1994 Feb, 14(2), 1266 - 77 tRNA genes as transcriptional repressor elements; Hull MW et al.; Eukaryotic genomes frequently contain large numbers of repetitive RNA polymerase III (pol III) promoter elements interspersed between and within RNA pol II transcription units, and in several instances a regulatory relationship between the two types of promoter has been postulated . In the budding yeast Saccharomyces cerevisiae, tRNA genes are the only known interspersed pol III promoter-containing repetitive elements, and we find that they strongly inhibit transcription from adjacent pol II promoters in vivo . This inhibition requires active transcription of the upstream tRNA gene but is independent of its orientation and appears not to involve simple steric blockage of the pol II upstream activator sites . Evidence is presented that different pol II promoters can be repressed by different tRNA genes placed upstream at varied distances in both orientations . To test whether this phenomenon functions in naturally occurring instances in which tRNA genes and pol II promoters are juxtaposed, we examined the sigma and Ty3 elements . This class of retrotransposons is always found integrated immediately upstream of different tRNA genes . Weakening tRNA gene transcription by means of a temperature-sensitive mutation in RNA pol III increases the pheromone-inducible expression of sigma and Ty3 elements up to 60-fold. J Biochem (Tokyo), 1994 Feb, 115(2), 175 - 8 Protozyme: emerging evidence in nature; Anraku Y et al.; The VMA1 locus in Saccharomyces cerevisiae contains a nested genetic element, the VDE gene, and expresses two functional proteins . A single VMA1 translational product seems to catalyze a self protein splicing in which an internal domain is excised out to produce a site-specific DNA endonuclease and the N- and C-domains are ligated by a transpeptidation reaction to yield a catalytic subunit of the vacuolar ATPase . Accumulating evidence in the past few years suggests that the VMA1 locus encodes a protozyme (for protos en zyme) which has dual roles in life as a protean catalyst for self protein splicing and for self gene homing . Four protozymes that share a common mechanism in protein splicing have been found in four organisms covering three major phylogenic trees. Yeast, 1994 Feb, 10(2), 265 - 9 SED6 is identical to ERG6, and encodes a putative methyltransferase required for ergosterol synthesis; Hardwick KG et al.; Luminal endoplasmic reticulum (ER) proteins carry a sorting signal that allows them to be retrieved from the Golgi apparatus by a specific receptor . In yeast, this receptor is encoded by the ERD2 gene . Although retrieval of ER proteins does not appear to be an essential process, cells lacking ERD2 do not grow . Several multicopy suppressors of this growth defect have been isolated . The sequence of one of these, SED6, is presented here . Its product contains motifs characteristic of methyltransferases, and it is identical to ERG6, the presumed structural gene for S-adenosylmethionine:delta 24-sterol-C-methyltransferase . The gene is located adjacent to PDR4, near the centromere of chromosome XIII. Biol Chem Hoppe Seyler, 1994 Feb, 375(2), 105 - 12 Different potential of cellular and viral activators of transcription revealed in oocytes and early embryos of Xenopus laevis; Xu L et al.; Many protein domains for transcriptional activation also function when fused to a heterologous DNA binding domain . In mammalian/HeLa cells, we have previously characterized the activation domains of several transcription factors using GAL4 fusion proteins . Here we have tested their transcriptional activity in oocytes and developing embryos of the clawed toad Xenopus laevis . We find that the "acidic" C-terminal domain of the herpesvirus VP16 (= Vmw65) activator, which is active from yeast to man, is also very active in the two Xenopus systems . The constitutive nature of this viral domain may have evolved to be refractory to cellular defense mechanisms . By contrast, activation domains from cellular eukaryotic transcription factors (TFE3, ITF2, MTF-1) are differentially active in oocytes and early embryos . This indicates that their activity can be regulated by protein modification and/or availability of specific coactivators . We have also compared VP16 induced enhancement of transcription from remote and promoter-proximal positions . In both oocytes and late blastula embryos, activation from a promoter-proximal position was more than 50 fold, while only a moderate stimulation (3-8 fold) was observed from remote positions . This may mean that frog oocyte and early embryos are not yet fully geared for gene control by remote enhancers, i.e . respond predominantly to close-by regulatory sequences . The fact that cellular enhancers are naturally located at various distances from the responsive promoters may thus be exploited by multicellular organisms for differential gene control at early and late stages of development. Curr Opin Immunol, 1994 Feb, 6(1), 113 - 24 Mechanisms of leukocyte motility and chemotaxis; Downey GP; Motility is a complex process that depends on the coordination of many cellular functions, including the conversion of information from the environment into a series of coordinated responses that culminate in directed cell movement . Major advances have been made in the understanding of many functions involved in motility, such as transmembrane signaling events, leading to alterations in the actin cytoskeleton, and interactions between adhesion receptors and components of the cytoskeleton, providing a link between the extracellular and intracellular environments . Studies using yeast (Saccharomyces cerevisiae), slime molds (Dictyostelium discoideum) and nematodes (Caenorhabditis elegans) have advanced our understanding of the molecular biology of cytoskeletal proteins and have important implications for mammalian leukocyte motility. Curr Opin Cell Biol, 1994 Feb, 6(1), 41 - 9 Centrosome and kinetochore movement during mitosis; Ault JG et al.; During the past year important progress has been made in refining our understanding of how chromosomes become equally distributed to daughter cells during mitosis . Unlike the situation in diatoms and yeast, it now appears that spindle pole (centrosome) separation during spindle formation and anaphase B is mediated in vertebrates primarily by an astral pulling, and not a pushing, mechanism . Kinetochore motility is directionally unstable, which has important consequences for how chromosomes move to the equator of the forming spindle . Finally, the observation that sister chromatid disjunction occurs even in the presence of high levels of maturation promoting factor reveals that the series of biochemical events responsible for this phenomenon is not an obligatory part of the pathway by which the cell exits mitosis. Biokhimiia, 1994 Feb, 59(2), 282 - 7 {Heterologous expression of functionally active human cytochrome P-450s . Cytochrome P-450IIIA4 catalyzes the biotransformation of the anabolic steroid hormone methandrostenolone}; Krynetskii EIu et al.; The expression of the cytochrome P450IIIA4 gene in the Saccharomyces cerevisiae yeast using the shuttle vector pYeDP1-8/2 has been carried out . The microsomal fraction isolated from the transformed yeast cells was used for biotransformation of the anabolic steroid hormone-methandrostenolone (MA) . The microsomal oxidation products were analyzed by HPLC and two-dimensional TLC . It was shown that microsomes of the yeasts expressing human cytochrome P450IIIA4 catalyze the MA conversion into its 6 beta-hydroxy derivative . An identical product is formed via a reaction catalyzed by human liver microsomes . The use of the heterological system of cytochrome P450IIIA4 expression has made it possible to establish its role in MA metabolism . The experimental system simulates the first phase of the drug biotransformation in liver cells. Rheum Dis Clin North Am, 1994 Feb, 20(1), 1 - 28 Systemic lupus erythematosus . Antibodies to DNA, DNA-binding proteins, and histones; Reeves WH et al.; Pathogenic autoantibodies to DNA are frequently associated with autoantibodies to chromatin-associated proteins such as histones or the Ku (p70/p80) antigens . In view of the increasing evidence that autoantibody production is antigen-driven, and because DNA is packaged with proteins in the cell, we suggest that anti-DNA antibodies may arise in response to DNA-protein complexes rather than naked DNA . Recent studies of the specificities of autoantibodies directed against the components of nucleosomes and transcriptional complexes are consistent with this hypothesis . The possible clinical significance of immune recognition of various types of chromatin complexes in autoimmune disease is discussed. Bioessays, 1994 Feb, 16(2), 101 - 6 Molecular perspectives of chromosome pairing at meiosis; Moens PB; Ideas about the mechanisms that regulate chromosome pairing, recombination, and segregation during meiosis have gained in molecular detail over the last few years . The purpose of this article is to survey briefly the shifts in paradigms and experiments that have generated new perspectives . It has never been very clear what it is that brings together the homologous chromosomes at meiotic prophase . For a while it appeared that the synaptonemal complex might be the nuclear organelle responsible for synapsis, but the supporting evidence has not been entirely convincing . Whatever the mechanism, it has always been assumed that homologous synapsis creates the opportunity for homologous DNA sequences to initiate recombination . At present, alternative ideas are developing . Attractive is the concept that double strand DNA repair mechanisms, that find and use the undamaged homologue for repair, have evolved into a meiotic mechanism for the recognition and pairing of homologous sequences . Subsequent intimate synapsis of homologous chromosomes in the context of the synaptonemal complex may serve later functions in the regulation of interference and segregation at first anaphase . A number of areas that are being tested at present and some that may be investigated in the future are discussed at the end of the review. Plant Mol Biol, 1994 Feb, 24(3), 407 - 16 MAP kinases: universal multi-purpose signaling tools; Jonak C et al.; MAP (mitogen-activated protein) kinases are serine/threonine protein kinases and mediate intracellular phosphorylation events linking various extracellular signals to different cellular targets . MAP kinase, MAP kinase kinase and MAP kinase kinase kinase are functional protein kinase units that are conserved in several signal transduction pathways in animals and yeasts . Isolation of all three components was also shown in plants and suggests conservation of a protein kinase module in all eukaryotic cells . In plants, MAP kinase modules appear to be involved in ethylene signaling and auxin-induced cell proliferation . Therefore, coupling of different extracellular signals to different physiological responses is mediated by MAP kinase cascades and appears to have evolved from a single prototypical protein kinase module which has been adapted to the specific requirements of different organisms. Mol Pharmacol, 1994 Feb, 45(2), 228 - 36 Sex-related differences in the expression of cytochrome P450 in hamsters: cDNA cloning and examination of the expression of three distinct CYP2C cDNAs; Sakuma T et al.; Sex-related differences in the expression of cytochrome P450 isozymes in hamsters were investigated . Three distinct cDNA clones (assigned as CYP2C25, CYP2C26, and CYP2C27) were isolated from liver cDNA libraries from male and female hamsters, using rat CYP2C11 cDNA as a probe . Sequence analysis revealed that these three forms were very similar to each other (> 90% identity of nucleotide sequences) and belong to the CYP2C gene subfamily . The CYP2C25, CYP2C26, and CYP2C27 cDNA clones consisted of an open reading frame encoding 490 amino acids . CYP2C25, CYP2C26, and CYP2C27 proteins expressed in Saccharomyces cerevisiae catalyzed the hydroxylation of tolbutamide and the N-demethylation of aminopyrine and benzphetamine . Only CYP2C25 showed testosterone hydroxylase (16 beta) activity . Northern blot analyses with specific oligonucleotide probes demonstrated that expression of CYP2C27 was male specific and male predominant in kidneys and livers, respectively . No sex-related difference was observed in the expression of CYP2C25 and CYP2C26 . These results support our previous findings that sex-related differences in the expression of liver microsomal cytochromes P450 occur in hamsters. Insect Biochem Mol Biol, 1994 Feb, 24(2), 191 - 201 The smaller protein formed as a ubiquitin fusion in Drosophila is processed from ubiquitin and found on the 60S ribosomal subunit; Redman KL; The only gene in Drosophila melanogaster for a 52 amino acid ribosomal protein (CEP52) is fused to a ubiquitin coding sequence . This study examines expression and proteolytic processing of the encoded fusion protein . Most antibody preparations made against a portion of human CEP52 readily detect the insect protein . The size of the immunoreactive polypeptide indicates that CEP52 is cleaved from ubiquitin and this apparent proteolytic processing was confirmed by amino-terminal sequence analysis of CEP52 isolated by two-dimensional gel electrophoresis . Ribosomes from embryonic, larval and adult Drosophila melanogaster contain equivalent amounts of CEP52 and the protein is associated with the large ribosomal subunit . Stained two-dimensional gels indicate that the quantity of CEP52 associated with ribosomes is similar to that of other ribosomal proteins of corresponding size . A previous investigation had indicated the possibility of intact ubiquitin-CEP52 fusion protein in Dictyostelium discoideum, Saccharomyces cerevisiae and Drosophila melanogaster . One of three antibody preparations used in this study of insect CEP52 reacts with a 40S subunit protein that is the correct size to be the uncleaved fusion protein . However, the putative fusion protein does not react with ubiquitin antibodies and has negligible positive charge at pH5, demonstrating that it is not unprocessed ubiquitin-CEP52. Transplantation, 1994 Feb, 57(3), 327 - 35 Porcine platelet antigens recognized by human xenoreactive natural antibodies; Platt JL et al.; Hyperacute xenograft rejection of porcine organs by primates is initiated by the binding of recipient natural antibodies to endothelium in the donor organ . We showed previously that human natural antibodies recognize 3 porcine endothelial cell glycoproteins with molecular masses of 115 kDa, 125 kDa, and 135 kDa; we called the glycoproteins gp115/135 . We also showed that porcine platelets contain glycoprotein antigens that are the same or very similar to gp115/135 from endothelial cells . The studies reported here were aimed at identifying these porcine platelet antigens and evaluating their potential relevance for the pathogenesis of xenograft injury . The importance of gp115/135 as targets of human natural antibodies was supported by the demonstration that (a) antibodies against porcine platelet gp115/135 are absorbed from the blood of nonhuman primates during the perfusion of a porcine kidney; (b) purified gp115/135 glycoproteins at concentrations < 500 pM, block the binding of human natural antibodies to cultured porcine endothelial cells; (c) the binding of human antibodies to porcine platelet extracts is significantly decreased by removal of gp115 or gp135 from the extracts; (d) antibody binding to gp115/135 initiates the activation of complement . Amino-terminal sequencing of gp115/135 revealed that gp115 has significant homology to human integrin beta 3 chains, gp125 to human alpha IIb chains, and gp135 to human alpha 2 chains . Ligand binding properties of the porcine glycoproteins were consistent with the identity of the antigens revealed by amino acid sequencing and molecular weight . Human natural antibodies also recognized a 250 kDa porcine platelet glycoprotein which was found to be homologous to human von Willebrand factor (vWF) . Antibodies against vWF in the blood of nonhuman primates are absorbed during ex vivo perfusion of a porcine organ . The identification of gp115/135 as integrins, the functions of which in endothelium might include cell signaling and maintenance of barrier function, provides potential insight into the pathogenesis of the rejection reaction in which these processes are manifestly aberrant . The identification of vWF as a potential target antigen raises several questions, including whether vWF provides one basis for antibody binding to xenogeneic endothelium or whether upon secretion from an endothelial cell vWF might actually block antibody binding. J Bioenerg Biomembr, 1994 Feb, 26(1), 101 - 15 Modeling a conformationally sensitive region of the membrane sector of the fungal plasma membrane proton pump; Monk BC et al.; A molecular model for transmembrane segments 1 and 2 from the fungal proton pumping ATPase has been developed, and this structure is predicted to form a helical hairpin loop structure in the membrane . This region was selected because it is highly conformationally active and is believed to be an important site of action for clinically important therapeutics in related animal cell enzymes . The hairpin loop is predicted to form an asymmetric tightly packed structure that is stabilized by an N-cap between D140 and V142, by hydrogen bonding between residues in the turn region and the helices, and by pi-pi interactions between closely apposed aromatic residues . A short four-residue S-shaped turn is stabilized by hydrogen bonding but is predicted to be conformationally heterogeneous . The principal effect of mutations within the hairpin head region is to destabilize the local close packing of side groups which disrupts the pattern of hydrogen bonding in and around the turn region . Depending on the mutation, this causes either a localized or a more global distortion of the primary structure in the hairpin region . These altered structures may explain the effects of mutations in transmembrane segments 1 and 2 on ATP hydrolysis, sensitivity to vanadate, and electrogenic proton transport . The conformational sensitivity of the hairpin structure around the S-turn may also account for the effects of SCH28080 and possibly ouabain in blocking ATPase function in related animal cell enzymes . Finally, the model of transmembrane segments 1 and 2 serves as a template to position transmembrane segments 3 and 8 . This model provides a new view of the H(+)-ATPase that promotes novel structure/function experimentation and could serve as the basis for a more detailed model of the membrane sector of this enzyme. Protein Sci, 1994 Feb, 3(2), 345 - 55 Rational design of a three-heptad coiled-coil protein and comparison by molecular dynamics simulation with the GCN4 coiled coil: presence of interior three-center hydrogen bonds; Rozzelle JE Jr et al.; alpha-Helical coiled coils have a 7-residue repeating pattern (abcdefg) where a and d are usually hydrophobic . We have designed a 2-stranded 44-residue coiled-coil protein (P44) consisting of 2 22-residue alpha-helices linked by 2 terminal disulfide groups to test whether the disulfide bridges could stabilize a 3-heptad coiled coil . P44 should be stabilized by intrahelical hydrogen bonds, interhelical disulfide and salt bridges, and interior hydrophobic interactions . A computer model of P44 was built and its stability was studied by molecular dynamics simulation with explicit water . This doubly crosslinked 3-heptad coiled coil did not unfold during a 300-ps simulation with explicit water . This doubly crosslinked 3-heptad coiled coil did not unfold during a 300-ps simulation . But reduced P44 with 4 thiol groups did unfold . For comparison, the 62-residue crystal structure of the 4-heptad coiled coil of transcription activator GCN4 did not unfold during a 300-ps simulation . Thus P44 may be a stable folded protein in aqueous solution . These simulations revealed the presence of 2 local hydrogen bond networks involving intra-helical 3-center hydrogen bonds in the hydrophobic interior of the coiled coils of GCN4 and P44 . The NH hydrogen at d makes a 3-center hydrogen bond whose major component is to the i - 4 C = O oxygen at g and minor component is to the solvent-inaccessible i - 3 C = O oxygen at a . Likewise, the NH hydrogen at g makes a 3-center hydrogen bond with the i - 4 C = O oxygen at c and the buried i - 3 C = O oxygen at d. J Med Primatol, 1994 Feb-May, 23(2-3), 146 - 50 Cell type-dependence for Vpu function; Geraghty RJ et al.; Human immunodeficiency virus type 1 Vpu has been shown to facilitate virus release from HeLa cells . We demonstrated that Vpu expression is not required for efficient virus release from Cos 1 and CV-1 cells . A yeast GAL4 transcriptional activation system was used to screen for cellular proteins that may interact with Vpu . One such protein was identified which we provisionally designate "Vpu interactive protein" or VIP. Cell Mol Neurobiol, 1994 Feb, 14(1), 9 - 25 Isolation and in situ localization of a cDNA encoding a Kex2-like prohormone convertase in the nematode Caenorhabditis elegans; Gomez-Saladin E et al.; 1 . A cDNA that encodes a Kex2-like prohormone convertase (PC) containing an active site similar to that of mammalian PC2 has been isolated from C . elegans . Total RNA was isolated from a mixed population of strain BA713 worms . After poly-(A)-selection and reverse transcription, degenerate/nested polymerase chain reactions (PCR) were performed using primers based on conserved regions within the active sites of the known vertebrate and invertebrate endoproteases . 2 . Two distinct 300-bp PCR products that shared homologies with the active sites of known Kex2-like endoproteases were isolated . These two PCR products were used to screen a C . elegans cDNA library . 3 . The complete cDNA for a Kex2-like endoprotease, designated CELPC2, was isolated and determined to be 2527 bp in length . This size was confirmed by northern analysis . The deduced amino acid sequence for the CELPC2 cDNA is very similar to the known Kex2-like endoproteases, especially at conserved regions within the active sites, but not identical to any one of them . The strongest structural homology was to vertebrate and invertebrate PC2 sequences . 4 . In situ hybridization suggests that CELPC2 is synthesized primarily in cells associated with the circumpharyngeal nerve ring and the dorsorectal ganglion. Arch Biochem Biophys, 1994 Feb 1, 308(2), 380 - 6 Kinetics of the reduction of cytochrome b5 with mutations in its membrane-binding domain; Wu FF et al.; In an attempt to understand which amino acids in the membrane anchor of cytochrome b5 might be determinants of its ability to support the cytochrome P450-catalyzed oxidation of selected substrates, the synthetic rat cytochrome b5 gene has been mutated by site-directed mutagenesis . The mutant proteins have been expressed in Saccharomyces cerevisiae, purified and assayed for their ability to support the cytochrome P450-catalyzed metabolism of the cytochrome b5 requiring substrate methoxyflurane (G . Vergeres and L . Waskell, 1992, J . Biol . Chem . 267, 12583-12591) . The rate of reduction of the cytochromes b5 by cytochrome P450 reductase has been examined by stopped-flow spectrophotometry to determine whether an altered rate of reduction of cytochrome b5 could explain the observed activity of cytochrome b5 in the purified reconstituted mixed-function oxidase system . A mutant in which the 22-amino-acid membrane anchor was replaced by a sequence of 22 leucines was unable to support methoxyflurane metabolism in the reconstituted system and was reduced by cytochrome P450 reductase at a rate (k = 4.5 x 10(-3) s-1) slow enough to explain this finding . Comparison of the rate of reduction of this mutant cytochrome b5 in 0.025% Tergitol and 40 microM dilauroylphosphatidylcholine suggests that this slow rate of reduction may be explained partially by aggregation of the polyleucine protein . The Pro115Stop mutant protein, which has been truncated by 19 amino acids in its COOH terminus resulting in a protein with one-half of the putative membrane anchor, supports methoxyflurane oxidation at 12-20% of the rate of the wild type protein . In addition it is reduced by cytochrome P450 reductase at a rate which should be capable of supporting a normal rate of production formation . The fact that the Pro115Stop mutant can be reduced at a rate capable of supporting a normal rate of methoxyflurane oxidation but in fact only supports methoxyflurane oxidation at 30% of the normal rate suggests that the mutant protein is deficient in its interactions with cytochrome P450 . The mutant proteins, Pro115Ala and Ala116Pro, behaved essentially as did the wild type protein demonstrating that the presence (Pro115Ala) or absence (Ala116Pro) of an alpha helix in the middle of the putative membrane-binding domain of cytochrome b5 was not a determinant of the interaction of cytochrome b5 with cytochrome P450 reductase and cytochrome P450.(ABSTRACT TRUNCATED AT 400 WORDS) AIDS, 1994 Feb, 8(2), 169 - 81 Evaluation of monoclonal antibodies to HIV-1 envelope by neutralization and binding assays: an international collaboration; D'Souza MP et al.; OBJECTIVE: To characterize a purified panel of monoclonal antibodies (MAb) to epitopes in HIV-1 envelope V3, CD4-binding region, C4 and gp41 . DESIGN: Neutralization and/or binding activity data were obtained from 21 laboratories on a coded panel consisting of seven human MAb, seven mouse MAb, recombinant human CD4 immunoadhesin {CD4-immunoglobulin G (IgG)}, normal human and normal murine Ig . METHODS: Laboratories performed a variety of neutralization assays and antigen binding assays with HIVIIIB, HIVMN and other laboratory strains of HIV-1 . RESULTS: For a single MAb, there was up to a 10(3) range of neutralizing antibody titers between laboratories . The range in titers appeared to depend on the sensitivity of the neutralization assay . Two methods were used to consolidate the data from all laboratories, the geometric mean titer (GMT) and the median neutralizing titer (MNT) . The panel of MAb were also analyzed by a variety of assays that measure binding activity to native or denatured epitopes . The relative binding activity of the MAb did not appear to correlate with neutralizing activity . CONCLUSION: Neutralization results from any single laboratory did not correlate with the collective data . The relative potency (rank order) of the MAb in the panel were equivalent when determined by GMT or MNT . These values may be useful to individual laboratories for estimating the sensitivity of their neutralization assays . The study also identified potential reference reagents with which neutralizing activity could be compared. Plant Mol Biol, 1994 Feb, 24(3), 545 - 7 Isolation and characterization of a rice cDNA similar to the S-phase-specific cyc07 gene; Kidou S et al.; We isolated a rice cDNA clone (T151) which encodes an open reading frame of 262 amino acids . This clone is similar to the S-phase-specific cyc07 gene of Catharanthus roseus . Expression of this gene is much higher in callus than in seedlings and regulated by external stresses such as high osmotic pressure, salinity, low temperature and submergence. J Biol Chem, 1994 Jan 28, 269(4), 2847 - 51 Cloning and expression of the human gene for transaldolase . A novel highly repetitive element constitutes an integral part of the coding sequence; Banki K et al.; A novel highly repetitive retrotransposable element was cloned based on a limited sequence homology to the human T-cell leukemia virus and a related endogenous retroviral sequence, HRES-1 . This repetitive element was found to constitute an integral part of the coding sequence of the human gene for transaldolase . In comparison with the intronless yeast gene, structural analysis of the human transaldolase genomic locus revealed that the human gene is comprised of five exons, second and third of which uniquely developed by insertion of a retrotransposable element . The 1329-base pair full-length cDNA, clone 4/2-4/1, contains an open reading frame coding for a protein of 336 amino acids with a predicted molecular mass of 38 kDa . This protein shows a 58% overall sequence homology with the 37-kDa yeast transaldolase . Antibodies raised against a 22-kDa recombinant polypeptide expressed from a 474-base pair 5' fragment of clone 4/2-4/1, containing repetitive exons 2 and 3, cross-reacted with yeast transaldolase and recognized the 38-kDa native human protein . Detection of a retrotransposon in the coding sequence of the human transaldolase gene demonstrates the importance of these repetitive elements in evolution of the eukaryotic genome. Gene, 1994 Jan 28, 138(1-2), 207 - 11 Isolation of a mouse cDNA encoding Rab23, a small novel GTPase expressed predominantly in the brain; Olkkonen VM et al.; The full-length cDNA encoding Rab23, a novel Ras-related small GTPase, was isolated using the sequence of a previously described {Chavrier et al., Gene 112 (1992) 261-264} short cDNA fragment and the rapid amplification of cDNA ends (RACE) PCR techniques . The deduced amino acid sequence was not very closely related to any previously described small GTPase, but was within the Rab subfamily . A Northern analysis revealed that the rab23 mRNA is predominantly expressed in the brain, which places the protein, together with Rab3a and Rab15, in the group of small GTPases characteristic of the nervous system. J Chromatogr A, 1994 Jan 28, 659(2), 289 - 98 Application of temperature-induced phase partitioning at ambient temperature for enzyme purification; Alred PA et al.; Aqueous two-phase partition and temperature-induced phase separation using a non-ionic, random copolymer composed of 20% ethylene oxide, 80% propylene oxide (EO20 PO80) has been used for purification of glucose-6-phosphate dehydrogenase, hexokinase and 3-phosphoglycerate kinase from bakers' yeast . This EO20PO80 copolymer has a cloud point of 18 degrees C, at which temperature it phase separates from water . Enzymes were first partitioned at 4 degrees C in an initial EO20PO80-dextran T500 aqueous two-phase system . This system had an upper copolymer-rich phase and a lower dextran-rich phase . After phase separation had occurred the upper EO20PO80-rich phase was removed and placed at 24 degrees C . This resulted in formation of a new two-phase system with an upper water phase and a lower phase containing 98% copolymer and 2% water . Enzymes were recovered exclusively in upper water phase leaving a polymer-rich lower phase free of contamination . The phase diagram for the system EO20PO80 and dextran T500 at 4 degrees C has been determined. Biochemistry, 1994 Jan 25, 33(3), 687 - 92 Characterization of folding intermediates using prolyl isomerase; Veeraraghavan S et al.; Structure-reactivity relationships of human peptidyl prolyl cis-trans isomerase (PPI) toward the two slow folding reactions of yeast iso-2 cytochrome c have been used to characterize the structure of folding intermediates in the vicinity of critical prolines . We propose that the relative catalytic efficiency of PPI for the protein substrate relative to a peptide substrate, (kcat/Km)rel, is a measure of structure in folding intermediates . The structural stability of slow-folding intermediates as detected by changes in (kcat/Km)rel was investigated using two structural perturbants: guanidine hydrochloride and site-directed mutagenesis . Neither of the two slow folding reactions for wild-type cytochrome c is catalyzed at low denaturant concentrations . However, both phases are catalyzed at moderate concentrations of guanidine hydrochloride . A mutation in cytochrome c enhances catalysis of the fluorescence-detected slow folding phase . For protein substrates destabilized by denaturants or mutation, we suggest that increases in (kcat/Km)rel result from a loosening of the substrate structure, providing better access of peptidyl prolyl isomerase to critical proline(s). J Biol Chem, 1994 Jan 21, 269(3), 2086 - 92 Reconstitution in vitro of the valyl-tRNA synthetase-elongation factor (EF) 1 beta gamma delta complex . Essential roles of the NH2-terminal extension of valyl-tRNA synthetase and of the EF-1 delta subunit in complex formation; Bec G et al.; Valyl-tRNA synthetase from mammalian cells is isolated exclusively as a complex with elongation factor (EF) 1H (the "heavy" form of eukaryotic EF-1, composed of subunits alpha, beta, gamma, and delta) . In a previous study, the 140-kDa valyl-tRNA synthetase subunit dissociated from the purified rabbit liver complex was shown to display hydrophobic properties, unlike the corresponding yeast cytoplasmic enzyme of 125 kDa (Bec, G., and Waller, J.-P . (1989) J . Biol . Chem . 264, 21138-21143) . Compared to the sequence of yeast cytoplasmic valyl-tRNA synthetase, that of the human enzyme displays an NH2-terminal extension of approximately 200 amino acid residues that bears strong sequence similarity to the NH2-terminal moiety of EF-1 gamma (Hsieh, S . L., and Campbell, R . D . (1991) Biochem . J . 278, 809-816) . We now show that this NH2-terminal extension can be selectively excised by elastase treatment of the isolated rabbit valyl-tRNA synthetase, without impairing catalytic activity . To examine the role of the NH2-terminal extension of mammalian valyl-tRNA synthetase in complex formation and to identify the subunit(s) of EF-1H responsible for binding the enzyme, reconstitution experiments were undertaken . Native or truncated valyl-tRNA synthetases were incubated with the isolated EF-1 subunits beta gamma and delta, either separately or in combination, and the ensuing products were analyzed by chromatography on DEAE-Sepharose FF and Superose 6 . The results demonstrate that the NH2-terminal extension of valyl-tRNA synthetase is required for complex formation and that the enzyme-binding site(s) resides on the EF-1 delta subunit . Moreover, although the EF-1 beta gamma binary complex does not bind valyl-tRNA synthetase, it is nevertheless required for assembly of a complex of defined quaternary structure by preventing the formation of high molecular weight aggregates generated in the presence of EF-1 delta alone. J Biol Chem, 1994 Jan 21, 269(3), 1940 - 4 The reconstituted ADP/ATP carrier activity has an absolute requirement for cardiolipin as shown in cysteine mutants; Hoffmann B et al.; Although the site-directed C73S mutation in the ADP/ATP carrier (AAC) AAC2 gene from Saccharomyces cerevisiae produced a glycerol-positive strain, indicating that the mutant AAC is active, on isolation and reconstitution in egg yolk phosphatidylcholine, the C73S AAC had no transport activity, whereas the wild-type AAC was fully active . Only on addition of cardiolipin was an exchange activity with the C73S AAC obtained . The AACs isolated from the other cysteine mutants did not (C244S) or only marginally (C271S) require cardiolipin for transport on reconstitution . {3H}Carboxyatractylate binding as a measure of incorporated AAC molecules was unchanged on addition of cardiolipin in all mutants, indicating that cardiolipin does not increase the incorporation of the AAC . It also shows that cardiolipin is required only for translocation and not for binding . The activity of the C73S mutant AAC shows half-saturation with cardiolipin at 2% by weight or at 1.15 mol % in the phosphatidylcholine vesicles . Other acidic phospholipids tested such as phosphatidylserine and phosphatidic acid did not activate . Among various cardiolipin derivatives, the selectivity for cardiolipin is high . Only monolysocardiolipin still retains 12% activity . After removal of the bulk of phospholipid, the content of bound phospholipids was assayed by 31P NMR . By unmasking with SDS, in the wild-type AAC and in the C73S AAC, 6.4 mol and only 1.3 and 2.9 mol of bound cardiolipin/mol of AAC dimer are found, respectively . Presumably, on isolation, cardiolipin is lost from the more labile C73S mutant AAC . Although the absolute requirement for cardiolipin is unique for the C73S AAC, it is concluded that in this mutant, the unmasking of the cardiolipin requirement demonstrates a general cardiolipin requirement of the wild-type AAC and of AACs from other sources. J Biol Chem, 1994 Jan 21, 269(3), 2299 - 306 Hydrolytic cleavage of nascent RNA in RNA polymerase III ternary transcription complexes; Whitehall SK et al.; Highly purified yeast RNA polymerase III ternary complexes were found to possess a hydrolytic chain retracting activity that cleaves nascent RNA from its 3'-OH end . Most of the shortened transcripts were capable of resuming RNA chain elongation, indicating that they remain stably associated with the enzyme-DNA complex . Analysis of the products of cleavage indicated that retraction primarily occurred in dinucleotide increments, but that mononucleotides were also excised at lower frequency . The ribonuclease activity was totally dependent on the presence of a divalent cation and was stimulated by the addition of non-cognate ribonucleotides . The inclusion of ATP in the reaction enhanced both the rate and extent of transcript cleavage . Evidence suggesting that the hydrolytic activity is intrinsic to RNA polymerase III and factor-in