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| Biochim Biophys Acta, 1993 Oct 13, 1170(2), 118 - 24 Substrate and hormone regulation of palmitoyl-CoA synthetase in 7800 C1 Morris hepatoma cells and cultured rat hepatocytes; Wu P et al.; The effects of tetradecylthioacetic acid (TTA), insulin and dexamethasone on palmitoyl-CoA synthetase activity and its mRNA both in 7800 C1 hepatoma cells and cultured rat hepatocytes were studied . (1) When the hepatoma cells were cultivated in the presence of fatty acids or alkyl thioacetic acids (3-thia fatty acids) palmitoyl-CoA synthetase activity was increased several fold . The stronger effect was obtained with TTA, which also increased long-chain acyl-CoA synthetase mRNA significantly . TTA has no inducing effect on butyryl-CoA synthetase and little effect on octanoyl-CoA synthetase in the same cells . Dexamethasone also had inducing effect on palmitoyl-CoA synthetase in the hepatoma cells . Insulin counteracted the induction given by TTA . All of these regulation actions take place at the pretranslational level . (2) In isolated hepatocytes the activity of palmitoyl-CoA synthetase was much higher than in hepatoma cells, but it was lost rapidly in culture . The loss of the enzyme activity was slowed down in the presence of TTA and insulin, either alone or combined . Dexamethasone combined with TTA reversed the loss of enzyme activity, while dexamethasone alone even increased the loss . Analysis of palmitoyl-CoA synthetase mRNA shows that TTA prevents the loss of the enzyme activity by inducing mRNA of the enzyme, dexamethasone enhances the effect of TTA, while insulin stabilizes the enzyme activity in the cultured cells without increasing the mRNA level. Biochemistry, 1993 Oct 12, 32(40), 10912 - 22 Resonance Raman study of the interactions between cytochrome c variants and cytochrome c oxidase; Hildebrandt P et al.; The structural changes in oxidized yeast iso-1-cytochrome c and fully oxidized bovine cytochrome c oxidase that are induced upon complex formation have been analyzed by resonance Raman spectroscopy . The main spectral changes could be ascribed to cytochrome c, which in the case of the wild-type protein are essentially the same as previously observed in the complex of horse heart cytochrome c and bovine cytochrome c oxidase {Hildebrandt et al . (1990) Biochemistry 29, 1661-1668} . These spectral changes are attributed to the formation of the conformational state II (approximately 45%) which exhibits an open heme pocket structure . The structural changes are assumed to be induced by the electrostatic interactions between the negatively charged binding domain on cytochrome c oxidase and the positively charged lysine residues on the front surface of cytochrome c . Substituting one of these lysine residues (i.e., Lys-72) by an alanine significantly lowers the state II content (< 15%), implying that this lysine is essential for controlling the conformational equilibrium of the bound protein . On the other hand, the replacement of lysine-79 by alanine only slightly lowers the state II content (approximately 35%) . However, the analysis of the spectra suggests that lysine-79 may be involved in controlling conformational details within the heme pocket of the bound cytochrome c . Due to the underlying structural changes and the lowered redox potential, formation of state II may be of functional importance for the physiological electron-transfer process by lowering the reorganization energy and increasing the driving force . The spectral changes caused by complex formation that are attributable to cytochrome c oxidase indicate structural changes of the vinyl and formyl substituents while the ground-state conformations of the porphyrin macrocycles are preserved . This finding implies that the conformational changes in the heme pockets of cytochrome c oxidase are much smaller than those in cytochrome c . These changes refer not only to heme a but also to heme a3, located remote from the cytochrome c binding site, pointing to a long-range structural communication between the binding domain and the oxygen reduction site . The possible functional implications of these structural changes are discussed. Biochemistry, 1993 Oct 12, 32(40), 10727 - 35 Limited proteolysis of creatine kinase . Implications for three-dimensional structure and for conformational substrates; Wyss M et al.; Proteinase K, subtilisin, pronase E, elastase, bactotrypsin, and thermolysin are all shown here to cleave native mitochondrial creatine kinase from chicken heart (Mib-CK) very specifically at a single site, either before or after Ala-323 . In analogy with hen egg ovalbumin, where the same proteases all cleaved the polypeptide chain very specifically around Ala-352, Ala-323 of Mib-CK may be located in an exposed surface loop that is sensitive to protease attack . Gel permeation chromatography demonstrated that the two proteolytic fragments of Mib-CK with M(r)'s of approximately 37,000 and approximately 6000 remain associated with each other . Proteinase K cleavage did not influence the octamer to dimer ratio of Mib-CK, indicating that selective cleavage after Ala-323 has no direct effect on dimer-dimer interfaces within the octamer . However, upon addition of MgADP plus creatine and nitrate to induce a transition-state analogue complex of the enzyme, native Mib-CK dissociated much more readily into dimers than proteinase K-digested Mib-CK . Furthermore, proteinase K cleavage of Mib-CK resulted in 2-11-fold decreases in the Vmax values, as well as in 6-23-fold increases in the Km values for phosphocreatine, creatine, and MgATP, whereas the Kd values for both MgATP and creatine were unaffected . Consequently, proteinase K cleavage of Mib-CK does not affect substrate binding per se, but interferes with substrate-induced conformational changes which are essential for catalysis and which mediate the synergism in substrate binding as it is observed with the unmodified enzyme.(ABSTRACT TRUNCATED AT 250 WORDS) Science, 1993 Oct 8, 262(5131), 244 - 8 Regional codon randomization: defining a TATA-binding protein surface required for RNA polymerase III transcription; Cormack BP et al.; The TATA-binding protein (TBP) is required for transcription by all three nuclear RNA polymerases . TBP was subjected to regional codon randomization, a codon-based mutagenesis method that generates complex yet compact protein libraries . Analysis of 186 temperature-sensitive TBP mutants yielded 65 specifically defective in transcription by RNA polymerase III (Pol III) . These mutants map to a limited TBP surface that may interact with Tds4, a component of the Pol III transcription factor TFIIIB . Strains that contain the Pol III-defective derivatives have increased amounts of messenger RNA, which suggests that competition among TBP-interacting factors for limiting quantities of TBP determines the ratio of Pol II and Pol III transcription in vivo. Nature, 1993 Oct 7, 365(6446), 562 - 6 Residues in the TATA-binding protein required to mediate a transcriptional response to retinoic acid in EC cells; Keaveney M et al.; The eukaryotic TATA-binding protein TBP, which is required for transcription by RNA polymerase II, is tightly associated with a particular set of factors in the TFIID complex, and as such provides a target for transcriptional regulation exerted by upstream factors . An embryonic carcinoma (EC) cell-specific activity like that of the viral factor E1A has been implicated in the mediation of transactivation from the retinoic acid receptor to human TBP, but yeast TBP cannot perform this function . Using TBP mutants with an altered TATA-box-binding specificity, we show here that yeast TBP can mediate transcriptional activation in mammalian cells and that its inability to convey retinoic acid-dependent transactivation in EC cells is due to specific residues in its core region . These residues preclude a functional association with the cellular E1A-like activity . TBP is thus a target for retinoic acid-dependent transactivation in EC cells by providing a surface for interaction with the EC cell-specific E1A-like activity. Nature, 1993 Oct 7, 365(6446), 520 - 7 Co-crystal structure of TBP recognizing the minor groove of a TATA element; Kim JL et al.; The three-dimensional structure of a TATA-box binding polypeptide complexed with the TATA element of the adenovirus major late promoter has been determined by X-ray crystallography at 2.25 A resolution . Binding of the saddle-shaped protein induces a conformational change in the DNA, inducing sharp kinks at either end of the sequence TATAAAAG . Between the kinks, the right-handed double helix is smoothly curved and partially unwound, presenting a widened minor groove to TBP's concave, antiparallel beta-sheet . Side-chain/base interactions are restricted to the minor groove, and include hydrogen bonds, van der Waals contacts and phenylalanine-base stacking interactions. J Biol Chem, 1993 Oct 5, 268(28), 21335 - 43 Function of the hydrophilic carboxyl terminus of type II DNA topoisomerase from Drosophila melanogaster . II . In vivo studies; Crenshaw DG et al.; Genetic complementation, protein distribution, and in vivo enzymatic activity by carboxyl-terminal truncation mutations of the Drosophila enzyme were examined . Removal of more than 273 of the 1447 amino acids composing the full-length topoisomerase inactivates the enzyme in vivo and in vitro; removal of 227 amino acids or less has no apparent effect on the ability of the enzyme to substitute for a conditional lethal, or null mutation, of the Saccharomyces cerevisiae top2 gene . Four catalytically active mutants, from which 227 or 240 amino acids are deleted, define an intervening, critical region . Each mutant in this critical region displays different in vivo complementation activity ranging from complete complementation to noncomplementation . Deletion analysis revealed a potent nuclear localization signal within the most distal 60 amino acids, although this is apparently not the only functional signal sequence encoded in the enzyme . Subcellular fractionation and indirect immunofluorescence demonstrate that the truncated enzymes localize to the nucleus, albeit with reduced efficiency compared to wild type . The ability of these mutants, including a mutant in the critical region which does not complement, to catalyze the decatenation of replicated plasmids and the segregation of replicated chromosomes was also examined. J Biol Chem, 1993 Oct 5, 268(28), 21328 - 34 Function of the hydrophilic carboxyl terminus of type II DNA topoisomerase from Drosophila melanogaster . I . In vitro studies; Crenshaw DG et al.; The function of the hydrophilic carboxyl-terminal region of Drosophila DNA topoisomerase II was examined by constructing a series of deletion mutants at the 3'-end of the Drosophila Top2 cDNA . The truncated enzymes were then expressed in Saccharomyces cerevisiae . Deletion of up to 240 out of 1447 total amino acids had no apparent effect on the enzyme's ability to catalyze topisomerization reactions . When 273, or more, amino acids were deleted, the enzyme was no longer active . Examples were found where deletion of less than 240 amino acids inactivated the enzyme . Based on the hydrodynamic properties determined for one of these mutants, the lack of activity was most likely due to misfolding of the polypeptides . The active mutants have similar hydrodynamic properties and heat inactivation profiles as the intact enzyme, suggesting that they are dimeric and stably folded . The carboxyl-terminal 240 amino acids also were not required for interaction with the drug VM26 . The only difference noted between the shortest, active mutant and the full-length enzyme was a decrease in the stability of the interaction of the truncated enzyme with DNA as evidenced by a decrease in the ionic strength at which catalysis was optimal and at which the transition between a processive and distributive mode of supercoil relaxation occurred. J Biol Chem, 1993 Oct 5, 268(28), 21147 - 54 The carboxyl-terminal transactivation domain of human serum response factor contains DNA-activated protein kinase phosphorylation sites; Liu SH et al.; The serum response factor (SRF) is a 67-kDa phosphoprotein that, together with auxiliary factors, modulates transcription of immediate early genes containing serum response elements in their promoters . Here we show that the carboxyl-terminal domain of human SRF is phosphorylated in vivo and is recognized in vitro by the double-stranded DNA-activated serine/threonine-specific protein kinase, DNA-PK . SRF phosphorylation by DNA-PK was stimulated by its cognate binding site . Protein microsequence analysis of a 22-amino acid synthetic SRF peptide and phosphopeptide analysis of genetically altered glutathione S-transferase-SRF fusion proteins identified Ser-435 and Ser-446 of human SRF as sites phosphorylated by DNA-PK . Both serines are followed by glutamine . Changing Gln-436 and Gln-447 to other residues reduced or eliminated phosphorylation by DNA-PK, confirming that these glutamines are important determinants for kinase recognition . The carboxyl-terminal transcription activation domain was mapped within a 71-amino acid region that contains both DNA-PK phosphorylation sites . Amino acid substitutions that interfered with phosphorylation by DNA-PK at Ser-435/446 in GAL4-SRF fusion proteins were reduced in transactivation potency . From these data we suggest that DNA-PK phosphorylation may modulate SRF activity in vivo. J Biol Chem, 1993 Oct 5, 268(28), 21113 - 9 Complete coding sequence, intron/exon organization, and chromosomal location of the gene for the core I protein of human ubiquinol-cytochrome c reductase; Hoffman GG et al.; Core I protein is a nuclear-encoded component of the ubiquinol-cytochrome c reductase complex of the mitochondrial respiratory chain . We have located the gene for the human core I protein in the p21 region of chromosome 3, just upstream of the COL7A1 gene which encodes type VII collagen . The core I gene, which has been sequenced in its entirety, is comprised of 10,417 base pairs, from the major transcription start site to the polyadenylation signal, and contains 13 exons . The predicted polypeptide contains 480 amino acids, of which the first 34 are predicted to constitute a typical mitochondrial leader peptide containing 6 positively charged arginine residues . The predicted human protein shows significant homology with core I protein from Saccharomyces cerevisiae, rather high homology (64% similarity, 46% identity) with the processing enhancing protein, which functions as core I protein in Neurospora crassa, and, surprisingly, highest homology with the small subunit of the mitochondrial processing peptidase of rat (74% similarity, 55% identity) . The predicted human sequence is 87% identical to the reported bovine core I sequence predicted from cDNA cloning, up to residue 298, but the two predicted sequences are widely divergent after that point. J Biol Chem, 1993 Oct 5, 268(28), 20904 - 10 Dominant negative activity of an endogenous thyroid hormone receptor variant (alpha 2) is due to competition for binding sites on target genes; Katz D et al.; Regulation of development and metabolism by thyroid hormone (T3) may be influenced by a non-T3 binding T3 receptor (TR) isoform, TR alpha 2, which can inhibit transcriptional activation by legitimate TRs . Numerous mechanisms have been postulated to explain the dominant negative actions of TR alpha 2, including competition for target genes, formation of inactive heterodimers, and squelching . We have found that excess TR alpha 2 was required to inhibit TR alpha 1-mediated transactivation from multiple T3 response elements (TREs) . Inhibition of T3 action by TR alpha 2 was specific for TRE-containing genes, because a GAL4/TR alpha 1 chimera, which heterodimerized with the 9-cis-retinoic acid receptor (RXR) and activated transcription from the GAL4 binding site in the presence of T3, was not inhibited by TR alpha 2 . In contrast, TR alpha 2 inhibited transactivation by TR alpha/VP16, a chimeric protein containing the N-terminal DNA binding domain (DBD) of TR alpha 1 fused to the transcriptional activation domain of VP16 . Indeed, TR alpha 2 inhibited the binding of TR alpha 1 monomers, homodimers, and RXR-heterodimers to DNA in vitro, whereas the TR alpha 2 C terminus alone did not . Although TR alpha 2 bound to TREs with less affinity than TR alpha 1, it bound directly to target genes in the cell nucleus . Furthermore, a TR alpha 2 mutant which binds more avidly to TREs was a more effective inhibitor of T3 action than wild type TR alpha 2 . Together these data indicate that TR alpha 2 inhibits T3 action by competing for binding to TREs. J Biol Chem, 1993 Oct 5, 268(28), 20870 - 6 Modulation by vitamin B6 of glucocorticoid receptor-mediated gene expression requires transcription factors in addition to the glucocorticoid receptor; Allgood VE et al.; We have investigated the mechanism by which vitamin B6 acts to modulate steroid hormone-mediated gene expression . We show that the level of glucocorticoid-induced gene expression from simple promoters, containing only hormone response elements and a TATA sequence, was not affected by alterations in intracellular vitamin B6 concentration . However, modulation of hormone-induced gene expression was restored with the inclusion of a binding site for the transcription factor nuclear factor 1 (NF1) within the hormone-responsive promoter; glucocorticoid-induced gene expression was reduced by 44% under conditions of elevated intracellular vitamin B6 concentration and enhanced by 98% in mild vitamin deficiency . Under these conditions, neither glucocorticoid receptor sedimentation characteristics, receptor activation, nor DNA binding capacity was affected . Quantitatively analogous effects were detected with estrogen-induced gene expression when an NF1 binding site was removed from or introduced into an estrogen-responsive promoter . NF1-mediated constitutive transcription was not affected by alterations in vitamin concentration . The modulatory effect of vitamin did not require strict positioning of or spacing between the glucocorticoid response element and NF1 binding site . Moreover, a heterologous transcriptional activator, composed of the viral E1a transactivation domain and the GAL4 DNA binding domain, does not substitute for NF1 in restoring vitamin B6 modulation of hormone-induced gene expression . These results suggest that vitamin B6 modulates steroid hormone-mediated gene expression through its influence on a functional or cooperative interaction between steroid hormone receptors and the transcription factor NF1. J Biol Chem, 1993 Oct 5, 268(28), 20721 - 4 Interaction between a complex of RNA polymerase III subunits and the 70-kDa component of transcription factor IIIB; Werner M et al.; A system that detects the formation of complexes between different proteins by linking them to separate domains of the GAL4 transcription activator protein has been used to study protein-protein interactions between four essential and unique subunits of yeast RNA polymerase III (C82, C53, C34 and C31), the 70-kDa component of the initiation transcription factor IIIB (TFIIIB70) and the TATA-binding protein . We found that C82, C34, and C31 are able to combine with each other in vivo and that C34 interacts with TFIIIB70 . These results suggest that C34 and TFIIIB70 are specificity determinants of the RNA polymerase III-TFIIIB interaction. Mol Cell Biol, 1993 Oct, 13(10), 6558 - 71 A family of human phosphodiesterases homologous to the dunce learning and memory gene product of Drosophila melanogaster are potential targets for antidepressant drugs; Bolger G et al.; We have isolated cDNAs for four human genes (DPDE1 through DPDE4) closely related to the dnc learning and memory locus of Drosophila melanogaster . The deduced amino acid sequences of the Drosophila and human proteins have considerable homology, extending beyond the putative catalytic region to include two novel, highly conserved, upstream conserved regions (UCR1 and UCR2) . The upstream conserved regions are located in the amino-terminal regions of the proteins and appear to be unique to these genes . Polymerase chain reaction analysis suggested that these genes encoded the only homologs of dnc in the human genome . Three of the four genes were expressed in Saccharomyces cerevisiae and shown to encode cyclic AMP-specific phosphodiesterases . The products of the expressed genes displayed the pattern of sensitivity to inhibitors expected for members of the type IV, cyclic AMP-specific class of phosphodiesterases . Each of the four genes demonstrated a distinctive pattern of expression in RNA from human cell lines. Mol Cell Biol, 1993 Oct, 13(10), 6501 - 8 Inhibition of E2F-1 transactivation by direct binding of the retinoblastoma protein; Helin K et al.; Loss of a functional retinoblastoma tumor suppressor gene product, pRB, is a key step in the development of many human tumors . pRB is a negative regulator of cell proliferation and appears to participate in control of entry into the S phase of the cell cycle . The recent demonstration that pRB binds to transcription factor E2F has provided a model for the mechanism of pRB-mediated growth regulation . Since adenovirus E1A proteins dissociate the pRB-E2F complexes and stimulate E2F-dependent transcription, it has been suggested that pRB inhibits E2F transactivation . Although some evidence for this hypothesis has been provided, it has not been possible to determine the mechanism of pRB-mediated inhibition of E2F transactivation . In this study, we constructed mutants of E2F-1 that do not bind to pRB yet retain the ability to transactivate the adenovirus E2 promoter through E2F DNA-binding sites . We demonstrated that transactivation mediated by the wild-type E2F-1 protein was inhibited by overexpression of wild-type pRB but not by a naturally occurring mutant of pRB . Transactivation mediated by mutants of E2F-1 which do not bind to pRB was not affected by overexpression of wild-type pRB . Furthermore, when the E2F-1 transactivation domain was fused to the GAL4 DNA-binding domain, pRB inhibited GAL4-E2F-1 transactivation through GAL4 sites . Expression of pRB did not inhibit transactivation mediated by GAL4-E2F-1 mutant constructs which were devoid of pRB binding . In conclusion, these data demonstrate that pRB inhibits E2F-dependent transactivation by direct protein-protein interaction. Mol Cell Biol, 1993 Oct, 13(10), 6170 - 9 Nuclear dot antigens may specify transcriptional domains in the nucleus; Xie K et al.; A bank of 892 human autoimmune serum samples was screened by indirect immunofluorescence on human tissue culture HT-29 cells . Seven serum samples that stain 4 to 10 bright dots in cell lines of several different mammals, including humans, monkeys, rats, and pigs, were identified . Immunofluorescence experiments indicate that these antigens, called nuclear dot (ND) antigens, are distinct from splicing complexes, kinetochores, and other known nuclear structures . An ND antigen recognized by these sera was cloned by immunoscreening a human lambda gt11 expression library . Analysis of seven cDNA clones for the ND antigen indicates that several mRNAs exist, perhaps derived through alternative splicing mechanisms . One major form of the message has an open reading frame of 1,440 bp capable of encoding a 53,000-M(r) protein . Treatment of cells with detergent, salt, or RNase A fails to remove the ND antigen from the nucleus . However, incubation with DNase I obliterates ND staining, indicating that the ND protein directly or indirectly associates with nuclear DNA . Fusion of the ND protein to a LexA DNA binding domain activates transcription in Saccharomyces cerevisiae . A 75-amino-acid domain that activates transcription in both yeast and primate cells has been identified . We suggest that ND antigens may participate in the activation of transcription of specific regions of the genome. Mol Cell Biol, 1993 Oct, 13(10), 5981 - 9 ADA3: a gene, identified by resistance to GAL4-VP16, with properties similar to and different from those of ADA2; Pina B et al.; We describe the isolation of a yeast gene, ADA3, mutations in which prevent the toxicity of GAL4-VP16 in vivo . Toxicity was previously proposed to be due to the trapping of general transcription factors required at RNA polymerase II promoters (S . L . Berger, B . Pina, N . Silverman, G . A . Marcus, J . Agapite, J . L . Regier, S . J . Triezenberg, and L . Guarente, Cell 70:251-265, 1992) . trans activation by VP16 as well as the acidic activation domain of GCN4 is reduced in the mutant . Other activation domains, such as those of GAL4 and HAP4, are only slightly affected in the mutant . This spectrum is similar to that observed for mutants with lesions in ADA2, a gene proposed to encode a transcriptional adaptor . The ADA3 gene is not absolutely essential for cell growth, but gene disruption mutants grow slowly and are temperature sensitive . Strains doubly disrupted for ada2 and ada3 grow no more slowly than single mutants, providing further evidence that these genes function in the same pathway . Selection of initiation sites by the general transcriptional machinery in vitro is altered in the ada3 mutant, providing a clue that ADA3 could be a novel general transcription factor involved in the response to acidic activators. J Cell Biol, 1993 Oct, 123(1), 119 - 26 Presequence and mature part of preproteins strongly influence the dependence of mitochondrial protein import on heat shock protein 70 in the matrix; Voos W et al.; To test the hypothesis that 70-kD mitochondrial heat shock protein (mt-hsp70) has a dual role in membrane translocation of preproteins we screened preproteins in an attempt to find examples which required either only the unfoldase or only the translocase function of mt-hsp70 . We found that a series of fusion proteins containing amino-terminal portions of the intermembrane space protein cytochrome b2 (cyt . b2) fused to dihydrofolate reductase (DHFR) were differentially imported into mitochondria containing mutant hsp70s . A fusion protein between the amino-terminal 167 residues of the precursor of cyt . b2 and DHFR was efficiently transported into mitochondria independently of both hsp70 functions . When the length of the cyt . b2 portion was increased and included the heme binding domain, the fusion protein became dependent on the unfoldase function of mt-hsp70, presumably caused by a conformational restriction of the heme-bound preprotein . In the absence of heme the noncovalent heme binding domain in the longer fusion proteins no longer conferred a dependence on the unfoldase function . When the cyt . b2 portion of the fusion protein was less than 167 residues, its import was still independent of mt-hsp70 function; however, deletion of the intermembrane space sorting signal resulted in preproteins that ended up in the matrix of wild-type mitochondria and whose translocation was strictly dependent on the translocase function of mt-hsp70 . These findings provide strong evidence for a dual role of mt-hsp70 in membrane translocation and indicate that preproteins with an intermembrane space sorting signal can be correctly imported even in mutants with severely impaired hsp70 function. J Cell Biol, 1993 Oct, 123(1), 109 - 17 A dual role for mitochondrial heat shock protein 70 in membrane translocation of preproteins; Gambill BD et al.; The role of mitochondrial 70-kD heat shock protein (mt-hsp70) in protein translocation across both the outer and inner mitochondrial membranes was studied using two temperature-sensitive yeast mutants . The degree of polypeptide translocation into the matrix of mutant mitochondria was analyzed using a matrix-targeted preprotein that was cleaved twice by the processing peptidase . A short amino-terminal segment of the preprotein (40-60 amino acids) was driven into the matrix by the membrane potential, independent of hsp70 function, allowing a single cleavage of the presequence . Artificial unfolding of the preprotein allowed complete translocation into the matrix in the case where mutant mt-hsp70 had detectable binding activity . However, in the mutant mitochondria in which binding to mt-hsp70 could not be detected the mature part of the preprotein was only translocated to the intermembrane space . We propose that mt-hsp70 fulfills a dual role in membrane translocation of preproteins . (a) Mt-hsp70 facilitates unfolding of the polypeptide chain for translocation across the mitochondrial membranes . (b) Binding of mt-hsp70 to the polypeptide chain is essential for driving the completion of transport of a matrix-targeted preprotein across the inner membrane . This second role is independent of the folding state of the preprotein, thus identifying mt-hsp70 as a genuine component of the inner membrane translocation machinery . Furthermore we determined the sites of the mutations and show that both a functional ATPase domain and ATP are needed for mt-hsp70 to bind to the polypeptide chain and drive its translocation into the matrix. Oncogene, 1993 Oct, 8(10), 2833 - 8 Activation of gene transcription by the amino terminus of the N-Myc protein does not require association with the protein encoded by the retinoblastoma suppressor gene RB1; Cziepluch C et al.; N-Myc encodes a nuclear phosphoprotein that contains a basic region (BR), a helix-loop-helix (HLH) and a leucine zipper (Zip) . These motifs are hallmarks of certain transcription factors . In pursuit of the question if N-Myc can activate transcription, we have employed an experimental model involving the yeast transcription factor Gal4 . We have first generated fusion proteins containing the Gal4 DNA-binding domain joined to portions of N-Myc . Subsequently we have analysed if chimeric proteins can transactivate the transcription of a reporter under the control of Gal4 binding sites . Here we show that the amino terminal portion of N-Myc activates transcription . Activation maps to a domain highly conserved among Myc-proteins and to other non-conserved sequences, suggesting functional redundancy . Previous studies had documented in vitro association of the RB1 protein with N-Myc (Rustig et al., 1991) . We here confirm this observation and identify the region encompassing the transactivation domain as responsible for RB1 binding . Analyses of N-Myc transactivation in retinoblastoma cell line WERI lacking a function RB1 protein gave results similar to those with cell lines having an intact RB1 protein, showing that RB1 protein is not required for transactivation by N-Myc . The present findings leave open the question if deregulated expression of N-Myc contributes to tumorigenesis by transcriptional activation of as yet unidentified target genes or by functionally inactivating the protein encoded by the tumor suppressor gene RB1, or by a combination of both. Oncogene, 1993 Oct, 8(10), 2805 - 12 SV40 T antigen abrogates p53-mediated transcriptional activity; Jiang D et al.; Recent evidence suggests that the tumor-suppressor protein p53 functions as a transcriptional regulator to control cell proliferation . An interaction with p53 is required for SV40 T antigen to transform primary cells; however, the effect of T antigen binding on p53 function is not known . In order to determine if an interaction with T antigen results in loss of p53-mediated transcriptional activity, we have used vectors expressing either a p53-GAL4 fusion protein or a wild-type p53 protein in transient co-transfection assays with T-antigen expression vectors . We have demonstrated that coexpression of T antigen significantly reduces both p53-GAL4-mediated transcription from a GAL4-dependent CAT reporter and p53-mediated transcription from a consensus p53 binding site in vivo . Moreover, T antigen was able to reduce binding of p53-GAL4 to its GAL4 binding sequence in gel shift experiments in vitro . These observed activities of T antigen were all dependent upon a functional p53-binding domain . In addition, coexpression of human papillomavirus type 18 E6 protein, able to bind to p53, was able to significantly reduce p53-mediated transcription . These results suggest that an interaction of certain viral oncoproteins with p53 results in loss of transcriptional activity of p53, a function that is important for maintaining normal cell growth. Biol Pharm Bull, 1993 Oct, 16(10), 978 - 81 New lectins from bulbs of Croccus sativum; Oda Y et al.; Four isolectins were isolated from bulbs of Croccus sativum (saffron), using affinity chromatography on mannan-Sepharose 4B, gel filtration and ion-exchange column chromatography in the presence of 8 M urea . The relative molecular masses of these lectins were determined by gel filtration to be approximately 48 kDa . On polyacrylamide gel electrophoresis, relative molecular masses of 8 kDa were obtained, suggesting that the lectins are hexamers . No carbohydrates were detected in the lectins . The lectins agglutinated the yeasts of Saccharomyces cerevisiae at a minimum concentration of 30 micrograms/ml, however, they did not agglutinate animal erythrocytes (human, sheep, rabbit and mouse) even at a concentration of 1000 micrograms/ml . In inhibitory experiments, mannan from Saccharomyces cerevisiae was the most potent inhibitor of C . sativum lectins . While D-mannose showed no inhibition, manno-oligosaccharides with either (alpha 1-2, 1-3) or (alpha 1-6) linkages were potent inhibitors, suggesting that C . sativum lectins recognized manno-oligosaccharide units larger than mannobiose . Ovomucoid also exhibited potent inhibition, however, the other carbohydrates examined did not. Biol Pharm Bull, 1993 Oct, 16(10), 956 - 9 Characterization of lignoceroyl-CoA ligase activity in chicken liver microsomes; Shiraishi T et al.; Lignoceroyl-coenzyme A (CoA) ligase activity was detected in microsomal fractions from chicken liver in the presence of alpha-cyclodextrin as a solubilizing agent of lignoceric acid . Heptakis(2,6-di-O-methyl)-beta-cyclodextrin (dimethyl-beta-cyclodextrin) and hexakis(2,6-di-O-methyl)-alpha-cyclodextrin (dimethyl-alpha-cyclodextrin), among the cyclodextrins tested, were more effective than alpha-cyclodextrin as solubilizing agents . We have characterized lignoceroyl-CoA ligase activity in comparison with palmitoyl-CoA ligase activity . Lignoceroyl-CoA and palmitoyl-CoA ligase activities showed a similar dependency on CoA concentration . However, lignoceroyl-CoA ligase activity exhibited responses to the Mg2+ ion, adenosine triphosphate (ATP), ATP analogues (adenosine monophosphate (AMP) and adenosine diphosphate (ADP)), and heat treatment, which were distinctly different from the responses of palmitoyl-CoA ligase activity . These results are consistent with the idea that lignoceroyl-CoA and palmitoyl-CoA are synthesized by two different enzymes. J Nat Prod, 1993 Oct, 56(10), 1831 - 4 Two bioactive pterocarpans from Erythrina burana; Dagne E et al.; Bioactivity-directed fractionation of the CHCl3 extract of the bark of Erythrina burana afforded phaseollidin {1} and cristacarpin {2} . Both 1 and 2 exhibited moderate but selective activity towards DNA repair-deficient yeast mutants, whereas only 1 was found to be cytotoxic . 13C-nmr spectra of both compounds were assigned. J Nat Prod, 1993 Oct, 56(10), 1772 - 8 New norcucurbitacin and heptanorcucurbitacin glucosides from Fevillea trilobata; Valente LM et al.; From the MeOH extract of the seeds of Fevillea trilobata (Cucurbitaceae) were isolated fevicordin A glucoside {1}, cayaponoside B {2}, cayaponoside D {3}, a new norcucurbitacin glucoside, and a new heptanorcucurbitacin glucoside . The structure of the new norcucurbitacin glucoside, andirobicin A glucoside, was established as 29-nor-1,2,3,4,5,10-dehydro-25-methoxy-2-O-beta-D-glucopyranosyl- 3,16 alpha,20R,22 xi-tetrahydroxy-11-oxocucurbit-23-ene {4}, and that of the novel heptanorcucurbitacin glucoside, andirobicin B glucoside, as 22,23,24,25,26,27,29-heptanor-1,2,3,4,5,10-dehydro-2-O-beta-D-g luc opyranosyl-3,16 alpha-dihydroxycucurbita-11,20-dione {5}. Bioessays, 1993 Oct, 15(10), 667 - 74 Protein splicing: excision of intervening sequences at the protein level; Cooper AA et al.; Protein splicing is an extraordinary post-translational reaction that removes an intact central "spacer" domain (Sp) from precursor proteins (N-Sp-C) while splicing together the N- and C-domains of the precursor, via a peptide bond, to produce a new protein (N-C) . All of the available data on protein splicing fit a model in which these intervening sequences excise at the protein level via a self-splicing mechanism . Several proteins have recently been discovered that undergo protein splicing, and in two such cases, the excised spacer protein is an endonuclease . Such endonucleases are capable of conferring genetic mobility upon the intervening sequences that encodes them . These intervening sequences define a new family of mobile genetic elements that are translated yet remain phenotypically silent by excising at the protein rather than the RNA level. Heredity, 1993 Oct, 71 ( Pt 4), 342 - 51 Evaluation of models of homologue search with respect to their efficiency on meiotic pairing; Loidl J et al.; We have constructed models of how homologous chromosomes come together at meiotic prophase for pairing and recombination . For these models we have calculated how many homology testing events are necessary on average to ensure that each chromosome finds its respective partner . We have identified several conditions that would greatly reduce the effort spent on homology search when compare with a search strategy which is based on fortuitous homologous contracts . The models are discussed in the light of experimental evidence. Eur J Cell Biol, 1993 Oct, 62(1), 13 - 21 The amino terminus of mammalian nucleolin specifically recognizes SV40 T-antigen type nuclear localization sequences; Xue Z et al.; Nucleolin is a major nucleolar protein in mammalian cells that is thought to be involved in ribosome biogenesis . The discovery that nucleolin shuttles between the cytoplasm and the nucleus raises the possibility that it is also involved in transporting ribosomal or nuclear proteins to the nucleus . The three structural domains of nucleolin bear a striking resemblance to the domains of a previously identified yeast protein NSR1, although the two proteins do not share a high degree of sequence similarity . NSR1 specifically recognizes the nuclear localization sequence (NLS) of both the simian virus large T antigen (SV40 T-antigen) and the yeast histone H2B by ligand blot analysis, and is a candidate for a receptor involved in the initial stages of nuclear transport . We report here that nucleolin, either purified from Chinese hamster ovary (CHO) cells or expressed in yeast, also specifically recognizes the wild-type, but not a mutant, histone H2B nuclear localization sequence by ligand blot analysis . The NLS recognition site is located within the N-terminal domain of both proteins . In showing that nucleolin, a protein that moves between the cytoplasm and the nucleus, also has the ability to interact with nuclear localization signals, our data support the idea that shuttling nucleolar proteins play a role in nuclear transport. Eur J Cell Biol, 1993 Oct, 62(1), 1 - 12 Analysis of nucleo-cytoplasmic transport in a thermosensitive mutant of nuclear pore protein NSP1; Nehrbass U et al.; NSP1 is related to a group of nuclear pore proteins termed 'nucleoporins' . We observe that in thermosensitive nsp1 mutants lacZ fusion proteins which contain the nuclear targeting sequence of Mat alpha 2 or Pho2 are located inside the nucleus at the permissive temperature (23 degrees C), but are mislocalized in the cytoplasm at the restrictive temperature (37 degrees C) . No evidence was obtained that the large lacZ reporter protein leaks out from the nucleus . Another nuclear passenger protein consisting of the NLS of ribosomal protein L25 and cytosolic dihydrofolate reductase (DHFR) is also accumulating in the cytoplasm after shifting ts nsp1 cells to 37 degrees C . In the latter case, this could be attributed to an increased leakage of the reporter protein from the nucleus into the cytoplasm . These data suggest that NSP1 mutation affects nuclear transport and nuclear retention, but the effects depend on the used NLS and the reporter protein. Yeast, 1993 Oct, 9(10), 1103 - 5 PFK2, ISP42, ERG2 and RAD14 are located on the right arm of chromosome XIII; Heinisch JJ; An 11 kb yeast DNA insertion isolated from a genomic library by complementation of a phosphofructokinase-deficient strain was used as a source for a partial sequence analysis . Four genes were shown to reside on this fragment, namely PFK2, ISP42, ERG2 and RAD14 . PFK2 was mapped previously to the right arm of chromosome XIII, locating the latter three genes to the same chromosome. Yeast, 1993 Oct, 9(10), 1057 - 63 KTR2: a new member of the KRE2 mannosyltransferase gene family; Lussier M et al.; The KTR2 gene from Saccharomyces cerevisiae was identified by polymerase chain reaction amplification of genomic DNA using primers derived from regions of high homology between the products of three yeast genes, KRE2, YUR1 and KTR1 . The product encoded by the KTR2 gene is a predicted type II membrane protein of 425 amino acid residues with a short cytoplasmic N-terminus, a membrane-spanning region and a large lumenal domain containing residues with a short cytoplasmic N-terminus, a membrane-spanning region and a large lumenal domain containing four potential N-glycosylation sites . Ktr2p has 58% identity with Yur1p, 39% with Ktr1p and 34% with Kre2p . One member of this gene family, KRE2 (also known as MNT1; Hausler and Robbins, 1992), encodes an alpha-1,2 mannosyltransferase which adds the third mannose onto O-linked glycoprotein side-chains (Hausler et al., 1992) . In contrast to KRE2 null mutants, which produce shortened (two-mannose) chains, mutants harboring a KTR2 gene disruption synthesize O-linked chains with the wild-type patterns of five mannose residues . A null mutation in KTR2 leads to partial resistance to killer toxin and hints that KTR2, which encodes a putative mannosyltransferase, is involved in extracellular matrix assembly. Trends Biochem Sci, 1993 Oct, 18(10), 381 - 4 Beat the clock: paradigms for NTPases in the maintenance of biological fidelity; Burgess SM et al.; Kinetic proofreading is a strategy used by cells to reduce the number of errors made when replicating and expressing genetic information . Recent advances in mRNA splicing suggest a variation on the theme of previously described kinetic proofreading mechanisms, which may apply to other multicomponent assembly processes in the cell. Trends Biochem Sci, 1993 Oct, 18(10), 366 - 71 Co-translational protein import into mitochondria: an alternative view; Verner K; Recent in vitro and in vivo experiments suggest that the synthesis and import of mitochondrial proteins are very tightly coupled and that a co-translational import reaction may be mandatory for some proteins . These results are entirely consistent with early experiments which suggested that import occurs co-translationally and that cytosolic polysomes synthesizing mitochondrial proteins are bound to protein import sites on isolated mitochondria . This article discusses and seemingly contradictory reports concerning the involvement of co-translational and post-translational mechanisms in the import process and examines the impact of recent developments in the field. Curr Genet, 1993 Oct, 24(4), 337 - 43 Mitochondrial genome of the dimorphic zygomycete Mucor racemosus; Schramke ML et al.; Mitochondria were isolated from the dimorphic zygomycete Mucor racemosus by differential centrifugation . DNA from the organelles was purified by cesium chloride-ethidium bromide isopycnic centrifugation . Examination of the mitochondrial DNA by electron microscopy revealed a circular chromosome approximately 63.8 kbp in circumference . The chromosome was digested with restriction endonucleases and the resulting DNA fragments were separated by agarose-gel electrophoresis . Electophoretic mobilities and stoichiometry of the fragments indicated a mixed population of mtDNA molecules each with a size of about 63.4 kbp . Physical maps were constructed from analyses of fragments generated in single and double restriction digests and from the hybridization of fragments to probes for the large and small mitochondrial rRNA genes from Saccharomyces cerevisiae . The Mucor mitochondrial chromosome was found to exist in the form of two flip-flop isomers with inverted repeat sequences encoding both rRNA genes. Biotechniques, 1993 Oct, 15(4), 714 - 21 Automated low-redundancy large-scale DNA sequencing by primer walking; Voss H et al.; Low-redundancy automated DNA sequencing by primer walking is described . T7 DNA polymerase is used together with computer-selected walking primers and fluorescein-dATP as internal label to sequence large plasmids or cosmids directly on a standard DNA sequencer with an error rate below 1% up to 500 bases (in the unedited raw data) . The low error rate allows efficient sequencing with low (2-3 times) redundancy . Plasmid subclones covering 20 kb of a cosmid insert were sequenced with an overall redundancy of 2.7 in the course of the European community Saccharomyces cerevisiae genome sequencing project . Neighboring plasmid subclones were linked by direct cosmid sequencing . Sets of ten walking primers are synthesized on the EMBL multiple segmental DNA synthesizer at low costs and used for sequencing with greater than 95% efficiency . The accuracy of the directed approach is improved by simultaneous walking on both strands by designing two primers in opposite directions in the same starting region . One primer is used to confirm sequence data on the opposite strand, and the other primer to obtain new sequence data. Plant Mol Biol, 1993 Oct, 23(2), 409 - 13 A rice (Oryza sativa L.) cDNA encodes a protein sequence homologous to the eukaryotic ribosomal 5S RNA-binding protein; Kim JK et al.; A rice (Oryza sativa L.) cDNA clone coding for the cytoplasmic ribosomal protein L5, which associates with 5 S rRNA for ribosome assembly, was cloned and its nucleotide sequence was determined . The primary structure of rice L5, deduced from the nucleotide sequence, contains 294 amino acids and has intriguing features some of which are also conserved in other eucaryotic homologues . These include: four clusters of basic amino acids, one of which may serve as a nucleolar localization signal; three repeated amino acid sequences; the conservation of glycine residues . This protein was identified as the nuclear-encoded cytoplasmic ribosomal protein L5 of rice by sequence similarity to other eucaryotic ribosomal 5 S RNA-binding proteins of rat, chicken, Xenopus laevis, and Saccharomyces cerevisiae . Rice L5 shares 51 to 62% amino acid sequence identity with the homologues . A group of ribosomal proteins from archaebacteria including Methanococcus vanniellii L18 and Halobacterium cutirubrum L13, which are known to be associated with 5 S rRNA, also related to rice L5 and the other eucaryotic counterparts, suggesting an evolutionary relationship in these ribosomal 5 S RNA-binding proteins. Plant Mol Biol, 1993 Oct, 23(1), 35 - 43 A soybean coproporphyrinogen oxidase gene is highly expressed in root nodules; Madsen O et al.; In plants the enzyme coproporphyrinogen oxidase catalyzes the oxidative decarboxylation of coproporphyrinogen III to protoporphyrinogen IX in the heme and chlorophyll biosynthesis pathway(s) . We have isolated a soybean coproporphyrinogen oxidase cDNA from a cDNA library and determined the primary structure of the corresponding gene . The coproporphyrinogen oxidase gene encodes a polypeptide with a predicted molecular mass of 43 kDa . The derived amino acid sequence shows 50% similarity to the corresponding yeast amino acid sequence . The main difference is an extension of 67 amino acids at the N-terminus of the soybean polypeptide which may function as a transit peptide . A full-length coproporphyrinogen oxidase cDNA clone complements a yeast mutant deleted of the coproporphyrinogen oxidase gene, thus demonstrating the function of the soybean protein . The soybean coproporphyrinogen oxidase gene is highly expressed in nodules at the stage where several late nodulins including leghemoglobin appear . The coproporphyrinogen oxidase mRNA is also detectable in leaves but at a lower level than in nodules while no mRNA is detectable in roots . The high level of coproporphyrinogen oxidase mRNA in soybean nodules implies that the plant increases heme production in the nodules to meet the demand for additional heme required for hemoprotein formation. Indian J Biochem Biophys, 1993 Oct, 30(5), 264 - 9 Inactivation of glyceraldehyde-3-phosphate dehydrogenase with SH-reagents and its relationship to the protein quaternary structure; Malhotra OP et al.; Inactivation of mung bean glyceraldehyde-3-phosphate dehydrogenase (GPDH) with excess iodoacetate or N-ethylmaleimide exhibits pseudo-first order kinetics at pH 7.3 and 8.6 in the absence and presence of NAD+, suggesting that all the reactive SH groups (four per tetrameric GPDH molecule) have equivalent reactivity towards these reagents . This is similar to the D2-symmetry conformation proposed on the basis of thermal inactivation data {Malhotra and Srinivasan, Arch . Biochem . Biophys . 236, 775-781 (1985)} . With p-chloromercury benzoate (p-CMB), the inactivation of GPDH is very fast and its kinetics can be monitored at low reagent concentration only . Keeping a high molar p-CMB: enzyme ratio (= 47), the kinetics were found to be biphasic, with half of the activity being lost in a fast and the remaining in a slow phase, characteristic of C2-symmetry conformation and half site reactivity . The p-CMB inactivation could be largely reversed on the addition of excess cysteine . A comparison of these data with literature reports on this and other GPDHs reveals that all reagents having large non-polar moieties exhibit half site reactivity with this enzyme. J Bioenerg Biomembr, 1993 Oct, 25(5), 459 - 72 Chemical, immunological, enzymatic, and genetic approaches to studying the arrangement of the peptide chain of the ADP/ATP carrier in the mitochondrial membrane; Brandolin G et al.; In the process of oxidative phosphorylation, the exchange of cytosolic ADP3- against mitochondrial ATP4- across the inner mitochondrial membrane is mediated by a specific carrier protein . Two different conformations for this carrier have been demonstrated on the basis of interactions with specific inhibitors, namely carboxyatractyloside (CATR) and bongkrekic acid (BA) . The two conformations, referred to as CATR and BA conformations, are interconvertible, provided that ADP or ATP are present . The functional ADP/ATP carrier is probably organized as a tetramer . In the presence of CATR or BA the tetramer is split into two dimers combined with either of the two inhibitors . The amino acid sequence of the beef heart carrier monomer (297 residues) contains three repeats of about 100 residues each . Experimental results obtained through different approaches, including photolabeling, immunochemistry, and limited proteolysis, can be interpreted on the basis of a model with five or six transmembrane alpha helices per carrier monomer . Two mobile regions involved in the binding of nucleotides and accessible to proteolytic enzymes have been identified . Each of them may be visualized as consisting of two pairs of short amphipathic alpha helices, which can be juxtaposed to form hydrophilic channels facilitating the nucleotide transport . Mutagenesis in yeast is currently being used to detect strategic amino acids in ADP/ATP transport. Genes Dev, 1993 Oct, 7(10), 1862 - 70 The carboxy-terminal tail of the homeo domain protein alpha 2 is required for function with a second homeo domain protein; Mak A et al.; The homeo domain protein alpha 2 from Saccharomyces cerevisiae has two roles in the a/alpha cell: With MCM1, alpha 2 turns off transcription of a-specific genes; with a1 (a second homeo domain protein), alpha 2 represses transcription of haploid-specific genes . From the carboxy-terminal side of the alpha 2 homeo domain extends an unstructured 22-amino-acid residue tail . In this paper we show that the carboxy-terminal tail of alpha 2 is required for formation of a stable a1/alpha 2-operator complex and is thus required for a1/alpha 2-mediated repression of transcription . In contrast, the tail is dispensable for alpha 2/MCM1-mediated repression . These results indicate that a short, unstructured tail mediates the interaction between two homeo domain proteins. Plant Physiol, 1993 Oct, 103(2), 525 - 33 Molecular characterization of PAB2, a member of the multigene family coding for poly(A)-binding proteins in Arabidopsis thaliana; Hilson P et al.; The poly(A) tail of eukaryotic mRNAs associates with poly(A)-binding (PAB) proteins whose role in mRNA translation and stability is being intensively investigated . Very little is known about the structure and function of the PAB genes in plants . We have cloned multiple PAB-related sequences from Arabidopsis thaliana . Results suggest that PAB proteins are encoded by a multigene family . One member of this family (PAB2) is expressed in root and shoot tissues . The complete nucleotide sequence of PAB2 was determined . Study of the predicted PAB2 protein reveals a similarity in structure among vertebrate, insect, yeast, and plant PAB proteins . All contain two highly conserved domains: an amino-terminal sequence formed by four RNA recognition motifs and an uncharacterized carboxyl-terminal region of 69 to 71 amino acids . Possible roles for the carboxyl-terminal conserved domain are discussed in view of recently published data concerning the structure and function of PAB proteins. Mutat Res, 1993 Oct, 319(2), 89 - 101 The mutagenicity of Gramoxone (paraquat) on different eukaryotic systems; el-Abidin Salam AZ et al.; The possible mutagenicity of the herbicide Gramoxone was evaluated using five different living systems: Allium cepa, Vicia faba, yeast, Drosophila melanogaster and human lymphocytes . The results indicate that Gramoxone has mutagenic activity at the cytological level in Allium cepa, Vicia faba and human lymphocytes . All doses were effective in inducing chromosomal abnormalities and a clear dose-response relationship was observed in the various cytological tests . Analysis of chromosomal abnormalities revealed that this herbicide displays clastogenic and turbagenic activities . At the gene mutation level Gramoxone induced gene conversion at the trp-5 locus and reversion at the ilv locus in Saccharomyces cerevisiae . In Drosophila melanogaster, Gramoxone proved to be mutagenic to germ cells and induced a high frequency of sex-linked recessive lethals (SLRL) . At the protein level, Gramoxone had detectable mutagenic effects on the genetic background of two enzymes, Adh and Est-6 . Gramoxone should be considered a mutagenic herbicide. Anal Biochem, 1993 Oct, 214(1), 301 - 12 Anti-hirudin monoclonal antibodies directed toward discontinuous epitopes of the hirudin amino-terminal and epitopes involving the carboxy-terminal hirudin amino acids; Koch C et al.; A panel of eight monoclonal antibodies (MAbs) was obtained against recombinant hirudin variant 2 (rHV2) . Specificities of the eight MAbs indicate that four of them recognize C-terminal amino acid residues (Group A) and four are directed against discontinuous epitopes and recognize a determinant (or determinants) within the 43 N-terminal residues (Group B) . Using these antibodies recombinant hirudins missing one or more C-terminal amino acids can be distinguished from molecules with an intact C-terminus either in enzyme immunoassays (EIAs) or by immunoaffinity chromatography . A sandwich EIA using the combination of two antibodies, one from each group, can quantitate both recombinant hirudin variant 1 (rHV1) and rHV2 with a detection range from 1 to 10 ng/ml in either buffer or plasma . Using only one MAb a competitive antibody capture EIA can quantitate recombinant or natural hirudin variants 1, 2, and 3 with a detection range from 5 to 100 ng/ml for rHV2 with a lysine in position 47 (rHV2K47) . None of the antibodies recognizes hirudin after it is complexed to alpha-thrombin . The ability of any one of these anti-rHV2 antibodies to interfere with hirudin binding to alpha-thrombin as measured by inhibition of thrombin's amidolytic activity correlates with the range of MAb affinity constants (KD = 3.5 x 10(-9) to 1 x 10(-6) M) . Incubating hirudin with one antibody from Group A (KD = 3.5 x 10(-8) M) and one from Group B (KD = 6.0 x 10(-9) M) completely blocks the ability of hirudin to bind alpha-thrombin . This MAb panel is thus useful for probing the recombinant C-terminal integrity of hirudin, for sensitive free hirudin quantitations, and the combined use of two MAbs has potential applications as an antidote for hirudin in vivo. Biochim Biophys Acta, 1993 Sep 29, 1170(1), 44 - 52 Purification of peroxisomes and subcellular distribution of enzyme activities for activation and oxidation of very-long-chain fatty acids in rat brain; Singh I et al.; Brain contains high amounts of very-long-chain (VLC) fatty acids (> C22) . Since mitochondria from liver and skin fibroblasts lack lignoceroyl-CoA ligase, in liver and skin fibroblasts fatty acids are exclusively oxidized in peroxisomes . Findings by Poulos and associates {9} suggested that contrary to liver and cultured skin fibroblasts brain mitochondria contain lignoceroyl-CoA ligase and can oxidize lignoceric acid . The present study was undertaken to develop a procedure for the isolation of subcellular organelles of higher purity from brain and to get a better understanding of the subcellular localization of the oxidation of VLC fatty acids in brain . The enzyme activities for activation and oxidation of palmitic and lignoceric acids were determined in peroxisomes, mitochondria, microsomes and a myelin fraction from rat brain and peroxisomes, mitochondria and microsomes purified from rat liver . Like in liver, brain lignoceroyl-CoA ligase activity in microsomes and peroxisomes was approx . 9 times higher than in mitochondria . In addition to palmitoyl-CoA ligase the antibodies against palmitoyl-CoA ligase inhibited the residual mitochondrial lignoceroyl-CoA ligase activity, meaning that lignoceroyl-CoA ligase activity in mitochondria was derived from palmitoyl-CoA ligase . Accordingly, in peroxisomes lignoceric acid was oxidized at 7 times higher rate than in mitochondria . Mitochondria were able to oxidize lignoceric acid efficiently when supplemented with lignoceroyl-CoA ligase activity from microsomes or myelin . These results show that in brain lignoceric acid is oxidized in peroxisomes and that lignoceroyl-CoA ligase activity is localized in peroxisomes and microsomes, but not in mitochondria . Peroxisomes and microsomes contain both lignoceroyl-CoA and palmitoyl-CoA ligases . Similar to peroxisomes and microsomes, the antibodies against palmitoyl-CoA ligase inhibited only the palmitoyl-CoA ligase activity in myelin but not the lignoceroyl-CoA ligase activity . These results suggest that in addition to palmitoyl-CoA ligase, myelin also contains lignoceroyl-CoA ligase. J Biol Chem, 1993 Sep 25, 268(27), 19923 - 6 ATP-independent loading of the proliferating cell nuclear antigen requires DNA ends; Burgers PM et al.; The proliferating cell nuclear antigen (PCNA) is a processivity subunit for eukaryotic DNA polymerase delta . We present biochemical evidence that yeast PCNA likely adopts a toroidal structure containing an inside cavity through which double-stranded DNA slides . A comparative study of DNA replication reactions on circular versus linear model substrates shows that PCNA can only interact productively with DNA polymerase delta if the substrate is linear . This combined with the observation that a large molar excess of PCNA is required for maximal stimulatory activity is consistent with a model in which PCNA slips onto the end of the DNA in an ATP-independent manner. Nature, 1993 Sep 23, 365(6444), 347 - 9 SEC12 encodes a guanine-nucleotide-exchange factor essential for transport vesicle budding from the ER; Barlowe C et al.; In yeast a type II integral membrane glycoprotein that is essential for transport vesicle budding from the endoplasmic reticulum (ER) is encoded by SEC12 (refs 1-3) . SAR1 was discovered as a multicopy suppressor of the sec12-1ts strain and encodes a GTPase of M(r) 21,000 (21K) also essential for vesicle budding from the ER . Sar1 is a peripherally associated membrane protein which shows enhanced membrane binding in cells containing elevated levels of Sec12 protein (refs 6, 7) . We show here that a purified fragment of Sec12 promotes guanine-nucleotide dissociation from Sar1 whereas the purified mutant Sec12-1 has only 15% of the wild-type activity . GTP hydrolysis by Sar1 is not enhanced by Sec12, but is stimulated more than 50-fold by a mixture of Sec12 and Sec23, a GTPase-activating protein specific for Sar1 (ref . 8) . We propose that Sec12 catalyses Sar1 guanine-nucleotide exchange in a process that recruits Sar1 to a vesicle formation site on the ER membrane. Biochemistry, 1993 Sep 21, 32(37), 9807 - 18 Effect of arginine-48 replacement on the reaction between cytochrome c peroxidase and hydrogen peroxide; Vitello LB et al.; The crystallographic structures of two cytochrome c peroxidase (CcP) mutants, CcP(R48L) and CcP(R48K), have been determined . In addition, the electronic absorption spectrum and the hydrogen peroxide reactivity of these two mutants have been determined between pH 4 and 8 . Both the crystallographic structure and the electronic absorption spectrum of CcP(R48L) are consistent with exclusive pentacoordination of the heme iron between pH 4 and 6.5 . At higher pH, CcP(R48L) forms an alkaline bis-imidazole form of CcP with the distal histidine coordinated to the heme iron . The apparent pKA for this transition is 7.5 in CcP(R48L) . The observed pseudo-first-order rate constant for the reaction between CcP(R48L) and hydrogen peroxide saturates at high peroxide concentrations . The data are consistent with a rate-limiting oxygen-oxygen bond scission at high peroxide concentrations . The observed rate of the bond scission step ranges between 1000 and 1950 s-1, an estimated 2 orders of magnitude slower than for wild-type enzyme . The data suggest that the protonated form of His-52 increases the bond scission step by a factor of 2 . The properties of the CcP(R48K) mutant are significantly different from those of CcP(R48L) . The crystal structure of CcP(R48K) shows Lys-48 occupying the putative peroxide binding site . The electronic absorption spectrum indicates that CcP(R48K) is predominantly pentacoordinate at neutral pH but with detectable amounts of hexacoordinate forms . Two ionizable groups affect the electronic absorption spectrum of CcP(R48K) . An apparent ionization near pH 4 produces an enzyme with increased hexacoordination, while an apparent pKA of 6.9 generates the alkaline bis-imidazole form . The peroxide reaction saturates at high peroxide concentrations for CcP(R48K) and is attributed to a conformational-gating mechanism . The maximum rate for the reaction between CcP(R48K) and hydrogen peroxide is probably limited by the movement of either Lys-48 or His-52 . This rate is 200 and 290 s-1 in nitrate-containing buffers and phosphate buffers, respectively . Evidence is provided that Arg-48 in wild-type enzyme is responsible for nitrate binding in the heme pocket and for stabilizing CcP Compound I. Biochemistry, 1993 Sep 21, 32(37), 9798 - 806 Histidine 52 is a critical residue for rapid formation of cytochrome c peroxidase compound I; Erman JE et al.; The crystal structure and reactivity with hydrogen peroxide are reported for a mutant of a cloned cytochrome c peroxidase {CcP(MI)}, in which the conserved distal His (His-52) is replaced with Leu . The reaction of the H52L enzyme with peroxide was examined as a function of pH in 0.1 M phosphate buffers and buffers in which nitrate was used to adjust the ionic strength . The pH-independent bimolecular rate constant for the reaction of H52L with peroxide was 731 +/- 44 and 236 +/- 14 M-1 s-1 in phosphate and nitrate-containing buffers, respectively . This represents a 10(5)-fold decrease in rate relative to the CcP(MI) parent under comparable conditions . Single-crystal diffraction studies showed that no dramatic changes in the structure or in the accessibility of the heme binding site were caused by the mutation . Rather, the mutation caused significant structural changes only at residue 52 and the nearby active-site water molecules . The residual reactivity of the H52L enzyme with peroxide was pH- and buffer-dependent . In nitrate-containing buffer, the apparent bimolecular rate constant for the reaction with peroxide decreased with decreasing pH; the loss of reactivity correlated with protonation of a group with an apparent pKA = 4.5 . Protonation of the group caused a loss of reactivity with peroxide . This is in contrast to the CcP(MI) parent enzyme, as well as all other mutants that have been examined, where the loss of reactivity correlates with protonation of an enzyme group with an apparent pKA = 5.4.(ABSTRACT TRUNCATED AT 250 WORDS) Science, 1993 Sep 17, 261(5128), 1551 - 7 A role for the transcription factors Mbp1 and Swi4 in progression from G1 to S phase; Koch C et al.; In budding yeast genes that encode G1 cyclins and proteins involved in DNA synthesis are transcriptionally activated in late G1 . A transcription factor, called SBF, is composed of Swi4 and Swi6 proteins and activates transcription of G1 cyclin genes . A different, but related, complex called MBF binds to MCB elements (Mlu I cell cycle box) found in the promoter of most DNA synthesis genes . MBF contains Swi6 and a 120-kilodalton protein (p120) . MBF was purified and the gene encoding p120 (termed MBP1) was cloned . A deletion of MBP1 was not lethal but led to deregulated expression of DNA synthesis genes, indicating a direct regulatory role for MBF in MCB-driven transcription . Mbp1 is related to Swi4 . Strains deleted for both MBP1 and SWI4 were inviable, demonstrating that transcriptional activation by MBF and SBF has an important role in the transition from G1 to S phase. Gene, 1993 Sep 15, 131(2), 299 - 300 Sequence of a cDNA encoding wheat eukaryotic protein synthesis initiation factor 4A; Metz AM et al.; A cDNA encoding wheat translation initiation factor 4A (eIF-4A) was isolated from a wheat cDNA library and sequenced . The deduced wheat eIF-4A amino acid sequence from the cDNA is compared to eIF-4A from Arabidopsis thaliana, tobacco, mouse and Saccharomyces cerevisiae . Putative RNA helicase motifs, and putative ATP-binding and hydrolysis sites are identified. Eur J Biochem, 1993 Sep 15, 216(3), 689 - 707 Peptidylproline cis-trans-isomerases: immunophilins; Galat A; Two sequence-unrelated families of proteins possess peptidylproline cis-trans-isomerase activities (PPIase) . PPIases are highly sequence conserved and multifunctional proteins which are present in many types of cells with a considerably divergent phylogenetic distribution . On the cellular level, PPIases occur in every compartment, both as free species and anchored to membranes . Diverse posttranslational modifications such as glycosylation, N-terminal modifications and phosphorylation constitute the additional functional features of PPIases . Folding, assembly and trafficking of proteins in the cellular milieu are regulated by PPIases . These enzymes accelerate the rate of in-vitro protein folding and they have the ability to bind proteins and act as chaperones . Some PPIases are coregulatory subunits of molecular complexes including heat-shock proteins, glucocorticoid receptors and ion channels . Secreted forms of PPIases are inflammatory and chemotactic agents for monocytes, eosinophils and basophils . The potent and clinically useful immunosuppressants CsA, FK506 or rapamycin bind with high affinities to PPIases (immunophilins) . The binding criterion allows us to sort the PPIases for the following two superfamilies of proteins: the cyclophilins (CsA-binding proteins) and the FKBP (FK506/rapamycin-binding proteins) . Although none of PPIases appeared to be essential for the viability of haploid yeast cells some of the immunophilin/immunosuppressant complexes are toxic both for yeast and mammalian cells . At least seven unlinked genes of cyclophilins and four unlinked genes of FKBP exist in human genomic DNA . Selected immunophilins regulate two different signalling pathways in lymphoid cells, namely the secretion of growth factors by stimulated T-cells and interleukin-2-induced T-cell proliferation . Moreover, selected FKBP mediate the cytotoxic effects of rapamycin in non-lymphoid cells . Accounts of the discovery of PPIases (immunophilins) and their functions are given in this review . A larger spectrum of proteins is analysed in relation to various signal-transduction pathways in lymphoid cells which involve immunophilins or their complexes with the immunosuppressants CsA, FK506 or rapamycin. Cancer Res, 1993 Sep 15, 53(18), 4343 - 8 Camptothecin resistance from a single mutation changing glycine 363 of human DNA topoisomerase I to cysteine; Benedetti P et al.; A full-length human DNA topoisomerase I complementary DNA clone was mutagenized in vitro and the mutagenized DNA was used to replace wild-type human TOP1 complementary DNA in YCpGAL1-hTOP1, a plasmid constructed for the expression of the human enzyme in yeast . A yeast strain devoid of yeast DNA topoisomerase I and permeable to the anticancer drug camptothecin was transformed with the plasmid pool . Assays of DNA topoisomerase I in lysates of camptothecin-resistant transformants identified one with nearly the same level of the enzyme as transformants of unmutagenized YCpGAL1-hTOP1, and a single mutation changing Gly363 to a cysteine was found in this mutant . The G363C mutant enzyme was overexpressed in yeast and partially purified . It differed significantly from wild-type human DNA topoisomerase I similarly expressed and purified: camptothecin-stimulated cleavage of DNA was observed with the wild-type but not the G363C enzyme, and the DNA relaxation activity of the mutant enzyme, unlike that of the wild-type enzyme, was not significantly stimulated by Mg(II) . The positions of the G363C and other previously reported camptothecin resistance mutations in eukaryotic DNA topoisomerase I were discussed in terms of a model in which the active site is an interdomainal cleft. Biochem Biophys Res Commun, 1993 Sep 15, 195(2), 785 - 91 The beta subunit of the Drosophila melanogaster ATP synthase: cDNA cloning, amino acid analysis and identification of the protein in adult flies; Pena P et al.; The cDNA encoding the Drosophila melanogaster beta subunit of H+ ATP synthase has been cloned and sequenced . The predicted mature protein is highly homologous to the equivalent beta subunits of other organisms and is preceded by a signal peptide of 31 amino acids, that although not conserved at primary sequence level has the characteristics of leader peptides present in other mitochondrial proteins . We have raised polyclonal antibodies that specifically recognize the beta H+ ATP synthase subunit present in Drosophila melanogaster protein extracts . This is the first time that a gene of the ATP synthase complex has been characterized in the invertebrate phyla. J Biol Chem, 1993 Sep 15, 268(26), 19177 - 80 Biogenesis of the mitochondrial receptor complex . Two receptors are required for binding of MOM38 to the outer membrane surface; Keil P et al.; Targeting of preproteins to mitochondria is mediated by the receptor complex in the outer membrane that contains two import receptors and the general insertion pore with MOM38 (38-kDa mitochondrial outer membrane protein) as major constituent . As all components of the receptor complex have to be imported from the cytosol themselves, the specificity of their targeting is fundamental for the correct assembly of mitochondria . None of the receptors is involved in its own import; the precursor of the main receptor MOM19 is even targeted without any surface receptor but directly assembles with MOM38 . We report that import of the precursor of MOM38 strictly depended on surface receptors . The import followed a new highly selective mechanism in that both receptors together were needed for the specific binding of the preprotein to the outer membrane surface, which was followed by its assembly into the receptor complex . These findings suggest that targeting of the mitochondrial targeting components involves a complex system of mutual specificity control, ensuring a selective assembly of the components into preexisting import sites. Biochemistry, 1993 Sep 14, 32(36), 9323 - 8 Kinetic analysis of NAD(+)-isocitrate dehydrogenase with altered isocitrate binding sites: contribution of IDH1 and IDH2 subunits to regulation and catalysis; Cupp JR et al.; NAD(+)-dependent isocitrate dehydrogenase from Saccharomyces cerevisiae is an allosterically regulated enzyme that exists as an octamer composed of two nonidentical subunits, designated IDH1 and IDH2 . To determine the contribution of each subunit to regulation and catalysis, a conserved serine residue at the proposed active site of each subunit was mutated to alanine . This mutation in IDH1 resulted in a 6-fold decrease in Vmax and a decrease in cooperativity, but little change in S0.5 for isocitrate . The mutant IDH2, in contrast, exhibited a 60-fold decrease in maximal velocity and a 2-fold reduction in S0.5 for isocitrate, but the cooperativity was unaffected . Responses to the allosteric modifier AMP also differed for the two mutant enzymes . The IDH1 mutant enzyme was not activated by AMP, whereas the IDH2 mutant enzyme exhibited an increase in isocitrate affinity in the presence of AMP similar to that observed with the wild-type enzyme . On the basis of these kinetic results, a model is presented which proposes that IDH1 functions as a regulatory subunit while IDH2 functions in catalysis . To determine if IDH1 or IDH2 alone is catalytically active, we also expressed the individual subunits in yeast strains in which the gene encoding the other subunit had been disrupted . Mitochondrial extracts from strains overexpressing solely IDH1 or IDH2 contained no detectable activity in the presence or absence of AMP . Gel filtration of these extracts showed that both IDH1 and IDH2 behaved as monomers, suggesting that the major subunit interactions within the octamer are between IDH1 and IDH2. Nucleic Acids Res, 1993 Sep 11, 21(18), 4348 - 55 Dimerization of leucine zippers analyzed by random selection; Pu WT et al.; The leucine zipper is a coiled coil that mediates specific dimerization of bZIP DNA-binding domains . A hydrophobic spine involving the conserved leucines runs down the coiled-coil and is thought to stabilize the dimer . We used the method of random selection to further define the primary sequence requirements for homodimer formation and heterodimer formation with Fos . When positions on either side of the hydrophobic spine of GCN4 are diversified to include the corresponding residues of Jun, a large percentage of the resulting sequences form homodimers, and a large percentage form heterodimers with Fos . Basic residues were preferred, but not essential, at position e of zippers which heterodimerize with Fos . When random sequences containing 5 heptad repeat of leucines are subject to a selection for homodimer formation, a diverse set of sequences is isolated . Certain residues are preferred at each position in the heptad repeat, although no essential primary sequence determinants could be identified . No pair of residues not involving the conserved leucines could be identified which strongly promotes homodimerization . These results suggest that factors determining leucine zipper dimerization are complex, with numerous interactions contributing to the association. Cell, 1993 Sep 10, 74(5), 919 - 28 An essential role for phosphatidylinositol transfer protein in phospholipase C-mediated inositol lipid signaling; Thomas GM et al.; Transmembrane signaling by the phospholipase C-beta (PLC-beta) pathway is known to require at least three components: the receptor, the G protein, and the PLC . Recent studies have indicated that if the cytosol is allowed to leak out of HL60 cells, then G protein-stimulated PLC activity is greatly diminished, indicating an essential role for a cytosolic component(s) . We now report the complete purification of one component based on its ability to reconstitute GTP gamma S-mediated PLC activity and identify it as the phosphatidylinositol transfer protein (PI-TP) . Based on the in vitro effects of PI-TP, we surmise that it is involved in transporting PI from intracellular compartments for conversion to PI bisphosphate (PIP2) prior to hydrolysis by PLC-beta 2/PLC-beta 3, the endogenous PLC isoforms present in these cells. Nature, 1993 Sep 9, 365(6442), 176 - 9 A protein translocation defect linked to ubiquitin conjugation at the endoplasmic reticulum; Sommer T et al.; Ubiquitin-conjugating enzymes function in selective proteolysis pathways and catalyse the covalent attachment of ubiquitin to proteolytic substrates . Here we report the identification of an integral membrane ubiquitin-conjugating enzyme . This enzyme, UBC6, localizes to the endoplasmic reticulum (ER), with the catalytic domain facing the cytosol . ubc6 loss-of-function mutants suppress the protein translocation defect caused by a mutation in SEC61, which encodes a key component of a multisubunit protein translocation apparatus of the ER . The expression of the sec61 mutant phenotype requires both the activity of UBC6 and its localization at the ER membrane . This suggests that UBC6 may mediate selective degradation of ER membrane proteins and that the protein translocation defect of sec61 may be caused by proteolysis of components of a structurally distorted mutant translocation apparatus. Biochim Biophys Acta, 1993 Sep 8, 1169(3), 243 - 9 Occurrence and characterization of a dehydratase enzyme in the leek icosanoyl-CoA synthase complex; Lessire R et al.; The presence of a beta-hydroxyacyl-CoA dehydratase involved in the icosanoyl-CoA synthase (EC 2.3.1.119) complex of leek epidermis has been demonstrated using antibodies raised against the purified beta-hydroxyacyl-CoA dehydratase from rat liver . In a first step the leek icosanoyl-CoA synthase activity was measured in the presence of different amounts of this antibody, the results obtained showed a 75% inhibition of the activity using a 8:1 IgG/microsomal protein ratio, whereas only a weak diminution of the activity occurred using pre-immune IgG . The analysis of the reaction products after incubation in the presence of increasing IgG amounts showed a decrease of the fatty acids (the final product) and an accumulation of beta-hydroxy fatty acids using immune IgG, whereas no change occurred in the presence of pre-immune IgG . Moreover, the beta-hydroxyacyl-CoA dehydratase activity was strongly inhibited, whereas in the same conditions, the beta-ketoacyl-CoA reductase and the (trans-2-3) enoyl-CoA reductase activities were not affected . The protein fractions that eluted from the DEAE and Ultrogel columns containing the leek icosanoyl-CoA synthase activity were able to specifically bind the anti beta-hydroxyacyl-CoA dehydratase from rat liver . The cross-reactivity was demonstrated . In immunoblotting experiments using the same antiserum after SDS-PAGE of the purified leek icosanoyl-CoA synthase, only one of the four protein bands constituting the leek icosanoyl-CoA synthase was detected . This protein, having an apparent molecular mass of 65 kDa, could be the dehydratase component of the elongation complex. Gene, 1993 Sep 6, 131(1), 155 - 6 Sequence of a Leishmania major gene encoding an ubiquitin fusion protein; Graeff GR et al.; A gene encoding an ubiquitin-tail protein fusion was isolated from the parasitic protozoan, Leishmania major, and sequenced . The L . major tail protein shares 97, 96, 67, 62, 62 and 61% sequence identity with the tail proteins of Trypanosoma brucei, Trypanosoma cruzi, yeast, Dictyostelium discoideum, human, and Arabidopsis thaliana, respectively . The putative 'zinc finger' nucleic acid-binding domain found in all ubiquitin 'tail' or 'extension' proteins described is also conserved in the L . major sequence . The upstream sequence indicated that this gene is not located at the end of a polyubiquitin sequence. FEBS Lett, 1993 Sep 6, 330(1), 66 - 70 Identification of MIM23, a putative component of the protein import machinery of the mitochondrial inner membrane; Dekker PJ et al.; A screening for yeast mutants impaired in mitochondrial protein import led to the identification of two genes (MPII and MPI2) encoding the essential components MIM44 and MIM17 of the inner membrane import machinery . We analyzed twelve additional mutants obtained in the screening and found two further complementation groups . One group represents mutants of SSC1, the gene encoding mitochondrial hsp70, an essential matrix protein required for protein import across the inner membrane . The second complementation group represents mutants of a new gene (MP13) encoding a 23 kDa integral inner membrane protein (MIM23) . MIM23 is synthesized without a presequence, and its import to the inner membrane requires a membrane potential . MIM23 contains a domain homologous to half of MIM17 . We speculate that MIM23 is a new member of the protein import machinery of the mitochondrial inner membrane. J Mol Biol, 1993 Sep 5, 233(1), 139 - 54 The X-ray structure of the GCN4-bZIP bound to ATF/CREB site DNA shows the complex depends on DNA flexibility; Konig P et al.; The X-ray structure of the DNA binding domain of the yeast transcriptional activator protein GCN4 bound to a DNA fragment containing the sequence of the perfectly symmetrical ATF/CREB site has been solved to 3.0 A resolution . The architecture of this specific recognition complex supports the current model for bZIP proteins: a homodimer of parallel alpha-helices form an interhelix coiled-coil region via the leucine zipper, and the two N-terminal basic regions fit into the major groove of half sites on opposite sides of the DNA double helix . The structure shows that DNA flexibility plays the predominant role in the preservation of protein contacts with the symmetric ATF/CREB site (ATGACGTCAT) as compared to the pseudo-symmetric AP-1 target site (ATGACTCAT), overcoming the positional displacement of functional groups introduced by the additional G.C base-pair at the center of the ATF/CREB sequence. Nippon Rinsho, 1993 Sep, 51(9), 2246 - 51 {Structural analysis of human genome by YAC technologies}; Soeda E; A method for construction of YAC (Yeast Artificial Chromosome) libraries with large inserts has been developed and promoted the ongoing project of human genome . Isolation by PCR screening charges the YAC clone with a unique tag of a pair of PCR primers at the defined chromosome site (Sequence Tagged Sites; STS) . Current evaluation of YAC has revealed that larger YAC has more problems where rearrangements including deletion and chimera occur extensively in DNA molecules, presenting a limited use of this technology in mapping; the contig map with mega YACs will be substituted by some other system such as cosmids with which the human healthy and disease genes will be characterized. J Eukaryot Microbiol, 1993 Sep-Oct, 40(5), 589 - 93 Hydrolase compartmentalization limits rate of digestion in Acanthamoeba; Hohman TC et al.; The kinetics of lysosomal enzyme acquisition by newly formed phagosomes was studied by following the rate of digestion of radiolabeled yeast fed to Acanthamoeba . The distribution of hydrolases among phagosomes was assessed by electron microscopic acid phosphatase cytochemistry and by measurement of three glycosidases in isolated early and late phagosomes . The results show that compartmentalization of hydrolases limit the digestion of large phagocytic loads . The hydrolases appear to be sequestered into the early phagosomes and not to be distributed either by small vesicle transport or phagosome-phagosome fusion to those formed later . We infer from these results that newly internalized surface membrane in phagosomes is not rapidly randomized with internal pools, but is recycled to the surface as a function of the digestive process. Protein Sci, 1993 Sep, 2(9), 1429 - 40 The structure and function of omega loop A replacements in cytochrome c; Murphy ME et al.; The structural and functional consequences of replacing omega-loop A (residues 18-32) in yeast iso-1-cytochrome c with the corresponding loop of Rhodospirillum rubrum cytochrome c2 have been examined . The three-dimensional structure of this loop replacement mutant RepA2 cytochrome c, and a second mutant RepA2(Val 20) cytochrome c in which residue 20 was back substituted to valine, were determined using X-ray diffraction techniques . A change in the molecular packing is evident in the RepA2 mutant protein, which has a phenylalanine at position 20, a residue considerably larger than the valine found in wild-type yeast iso-1-cytochrome c . The side chain of Phe 20 is redirected toward the molecular surface, altering the packing of this region of omega-loop A with the hydrophobic core of the protein . In the RepA2(Val 20) structure, omega-loop A contains a valine at position 20, which restores the original wild-type packing arrangement of the hydrophobic core . Also, as a result of omega-loop A replacement, residue 26 is changed from a histidine to asparagine, which results in displacements of the main-chain atoms near residue 44 to which residue 26 is hydrogen bonded . In vivo studies of the growth rate of the mutant strains on nonfermentable media indicate that the RepA2(Val 20) cytochrome c behaves much like the wild-type yeast iso-1 protein, whereas the stability and function of the RepA2 cytochrome c showed a temperature dependence . The midpoint reduction potential measured by cyclic voltammetry of the RepA2 mutant is 271 mV at 25 degrees C . This is 19 mV less than the wild-type and RepA2(Val 20) proteins (290 mV) and may result from disruption of the hydrophobic packing in the heme pocket and increased mobility of omega-loop A in RepA2 cytochrome c . The temperature dependence of the reduction potential is also greatly enhanced in the RepA2 protein. Mol Cell Biol, 1993 Sep, 13(9), 5738 - 48 Novel members of the mitogen-activated protein kinase activator family in Xenopus laevis; Yashar BM et al.; Mitogen-activated protein (MAP) kinases comprise an evolutionarily conserved family of proteins that includes at least three vertebrate protein kinases (p42, p44, and p55 MAPK) and five yeast protein kinases (SPK1, MPK1, HOG1, FUS3, and KSS1) . Members of this family are activated by a variety of extracellular agents that influence cellular proliferation and differentiation . In Saccharomyces cerevisiae, there are multiple physiologically distinct MAP kinase activation pathways composed of structurally related kinases . The recently cloned vertebrate MAP kinase activators are structurally related to MAP kinase activators in these yeast pathways . These similarities suggest that homologous kinase cascades are utilized for signal transduction in many, if not all, eukaryotes . We have identified additional members of the MAP kinase activator family in Xenopus laevis by a polymerase chain reaction-based analysis of embryonic cDNAs . One of the clones identified (XMEK2) encodes a unique predicted protein kinase that is similar to the previously reported activator (MAPKK) in X . laevis . XMEK2, a highly expressed maternal mRNA, is developmentally regulated during embryogenesis and expressed in brain and muscle . Expression of XMEK2 in yeast cells suppressed the growth defect associated with loss of the yeast MAP kinase activator homologs, MKK1 and MKK2 . Partial sequence of a second cDNA clone (XMEK3) identified yet another potential MAP kinase activator . The pattern of expression of XMEK3 is distinct from that of p42 MAPK and XMEK2 . The high degree of amino acid sequence similarity of XMEK2, XMEK3, and MAPKK suggests that these three are related members of an amphibian family of protein kinases involved in the activation of MAP kinase . Discovery of this family suggests that multiple MAP kinase activation pathways similar to those in yeast cells exist in vertebrates. Mol Cell Biol, 1993 Sep, 13(9), 5659 - 69 Far1 and Fus3 link the mating pheromone signal transduction pathway to three G1-phase Cdc28 kinase complexes; Tyers M et al.; In the yeast Saccharomyces cerevisiae, the Cdc28 protein kinase controls commitment to cell division at Start, but no biologically relevant G1-phase substrates have been identified . We have studied the kinase complexes formed between Cdc28 and each of the G1 cyclins Cln1, Cln2, and Cln3 . Each complex has a specific array of coprecipitated in vitro substrates . We identify one of these as Far1, a protein required for pheromone-induced arrest at Start . Treatment with alpha-factor induces a preferential association and/or phosphorylation of Far1 by the Cln1, Cln2, and Cln3 kinase complexes . This induced interaction depends upon the Fus3 protein kinase, a mitogen-activated protein kinase homolog that functions near the bottom of the alpha-factor signal transduction pathway . Thus, we trace a path through which a mitogen-activated protein kinase regulates a Cdc2 kinase. Radiat Res, 1993 Sep, 135(3), 365 - 71 DNA repair in Chlamydomonas reinhardtii induced by heat shock and gamma radiation; Boreham DR et al.; Saccharomyces cerevisiae and Chlamydomonas reinhardtii respond to a sublethal exposure of ionizing radiation by increasing their resistance to killing by a second exposure . We demonstrate here that the two lower eukaryotes apparently achieve this by different mechanisms . We have shown that induced radioresistance in yeast results from increased capacity for recombinational repair, which we believe to occur in G2-phase haploid cells by recombination between homologous chromosomes . This is not possible in G1-phase haploid cells, which lack a second copy of DNA . Haploid C . reinhardtii cells, however, show induced resistance when irradiated asynchronously or in the G1 phase of the cell cycle . We have shown previously that the development of radiation resistance in yeast is proportional to the magnitude of the inducing dose and clearly demonstrates an oxygen effect . There was no oxygen effect for induced radiation resistance in C . reinhardtii cells, but induction remained proportional to dose . In yeast we have reported that both increased radioresistance and thermotolerance are inducible by a heat shock . Here, C . reinhardtii showed induced thermotolerance but no induced radioresistance in response to a heat stress . We have also determined previously that the induced recombinational DNA repair system in yeast recognizes alkylation lesions and therefore confers increased resistance to mutation by MNNG . In these experiments, C . reinhardtii induced for radioresistance were not more resistant to MNNG mutagenesis . These data indicate that haploid C . reinhardtii has a unique DSB repair mechanism . We propose that one possible mechanism may involve chloroplast DNA in a cooperative chloroplast/nuclear recombinational repair process. Eur J Biochem, 1993 Sep 1, 216(2), 615 - 22 Acyl-CoA synthetase mRNA expression is controlled by fibric-acid derivatives, feeding and liver proliferation; Schoonjans K et al.; Several enzymes of the beta-oxidation pathway have been shown to be induced after stimulation with peroxisomal proliferators, including several hypolipidemic drugs . We investigated the regulation of the long-chain-acyl-CoA synthetase (ACS) gene in the liver . Fenofibrate, a hypolipidemic drug and potent peroxisomal proliferator, induced ACS gene expression in several tissues . In liver, large increases in ACS mRNA levels and ACS activity were observed after fenofibrate administration . Adipose tissue ACS mRNA levels and ACS activity were also stimulated upon fibrate treatment but to a lesser extent in comparison with liver ACS mRNA . Kidney ACS mRNA was only weakly induced, except for the highest dose and the longest treatment period, where a strong induction was observed . In contrast to these tissues, heart ACS mRNA and ACS activity remained almost unchanged after fenofibrate treatment . These effects of fenofibrate could be reproduced by other fibrates such as clofibrate . In addition, it is demonstrated that both nutritional composition and liver proliferation trigger ACS gene expression in liver . Consequently, these data suggest that ACS is a highly regulated enzyme with a potentially important control function in lipid metabolism. Genes Dev, 1993 Sep, 7(9), 1737 - 54 mRNA destabilization triggered by premature translational termination depends on at least three cis-acting sequence elements and one trans-acting factor; Peltz SW et al.; Nonsense mutations in a gene can accelerate the decay rate of the mRNA transcribed from that gene, a phenomenon we describe as nonsense-mediated mRNA decay . Using amber (UAG) mutants of the yeast PGK1 gene as a model system, we find that nonsense-mediated mRNA decay is position dependent, that is, nonsense mutations within the initial two-thirds of the PGK1-coding region accelerate the decay rate of the PGK1 transcript < or = 12-fold, whereas nonsense mutations within the carboxy-terminal third of the coding region have no effect on mRNA decay . Moreover, we find that this position effect reflects (1) a requirement for sequences 3' to the nonsense mutation that may be necessary for translational reinitiation or pausing, and (2) the presence of an additional sequence that, when translated, inactivates the nonsense-mediated mRNA decay pathway . This stabilizing element is positioned within the coding region such that it constitutes the boundary between nonsense mutations that do or do not affect mRNA decay . Rapid decay of PGK1 nonsense-containing transcripts is also dependent on the status of the UPF1 gene . Regardless of the position of an amber codon in the PGK1 gene, deletion of the UPF1 gene restores wild-type decay rates to nonsense-containing PGK1 transcripts. Proc Natl Acad Sci U S A, 1993 Sep 1, 90(17), 8179 - 83 Identification of a gene required for membrane protein retention in the early secretory pathway; Nishikawa S et al.; The yeast SEC12 gene product (Sec12p) is an integral membrane protein required for the protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus . Although this protein is almost exclusively localized in the ER, a significant fraction of Sec12p is modified by an enzyme that resides in the early compartment of the Golgi apparatus, suggesting that Sec12p is cycling between the ER and the early Golgi . We have taken a genetic approach to investigate the retention mechanism of Sec12p . Analysis of mutants that are defective in the retention of the Sec12-Mf alpha 1 fusion protein in the early secretory compartments has identified a gene, RER1 . A recessive mutation in RER1 causes mislocalization of the authentic Sec12p as well as two different Sec12 fusion proteins to the late Golgi apparatus and even to the cell surface . However, the rer1 mutant is not defective in the retention of an ER-resident soluble protein, BiP, suggesting that soluble and membrane proteins are retained in the ER by distinct mechanisms. J Cell Biol, 1993 Sep, 122(5), 1013 - 22 Localization of Drosophila retinal degeneration B, a membrane-associated phosphatidylinositol transfer protein; Vihtelic TS et al.; The Drosophila retinal degeneration B (rdgB) mutation causes abnormal photoreceptor response and light-enhanced retinal degeneration . Immunoblots using polyclonal anti-rdgB serum showed that rdgB is a 160-kD membrane protein . The antiserum localized the rdgB protein in photoreceptors, antennae, and regions of the Drosophila brain, indicating that the rdgB protein functions in many sensory and neuronal cells . In photoreceptors, the protein localized adjacent to the rhabdomeres, in the vicinity of the subrhabdomeric cisternae . The rdgB protein's amino-terminal 281 residues are > 40% identical to the rat brain phosphatidylinositol transfer protein (PI-TP) . A truncated rdgB protein, which contains only this amino-terminal domain, possesses a phosphatidylinositol transfer activity in vitro . The remaining 773 carboxyl terminal amino acids have additional functional domains . Nitrocellulose overlay experiments reveal that an acidic amino acid domain, adjacent to the PI transfer domain, binds 45Ca+2 . Six hydrophobic segments are found in the middle of the putative translation product and likely function as membrane spanning domains . These results suggest that the rdgB protein, unlike the small soluble PI-TPs, is a membrane-associated PI-TP, which may be directly regulated by light-induced changes in intracellular calcium. J Biochem (Tokyo), 1993 Sep, 114(3), 363 - 9 Importance of successive prolines in the carboxy-terminal region of P450 2C2 and P450 2C14 for the hydroxylase activities; Uno T et al.; Proline-480 and proline-481 are highly conserved in the P450 2 family . When these successive prolines of P450 2C2 were replaced by Ala-Thr, its activities of laurate (omega-1)-hydroxylation and benzphetamine N-demethylation were lost . On the other hand, the mutated P450 which retained one of the proline residues was 60-100% active in the (omega-1)-hydroxylation and 100-120% active in the N-demethylation . Pro-Pro (480, 481) to Ala-Thr mutated P450 2C14 was also inactive in testosterone 16 alpha-hydroxylation and benzphetamine N-demethylation, the activities of the wild-type P450 2C14 . In the carboxyterminal region of the P450 2C subfamily, the sequence of P450 2C2 from residues 469-473 is noticeably different from those of the other members of this subfamily . The Ser-473 to Val mutation of P450 2C2 caused a decrease in the (omega-1)-hydroxylase activity to one-fifth but the N-demethylase activity was not much affected . When the sequence of P450 2C2 from residues 469-473 was replaced by that of P450 2C14 (mutation at four residues), the 16 alpha-hydroxylase and N-demethylase activities were increased by seven- and three-fold, respectively, and the (omega-1)-hydroxylase activity was decreased to one-third . The mutated P450 2C2, in which the carboxy-terminal four residue sequence or the proline cluster adjacent to the amino-terminal membrane-anchor signal sequences was deleted, was not accumulated in yeast cells transformed with plasmids directing the synthesis of such P450s.(ABSTRACT TRUNCATED AT 250 WORDS) Mol Biochem Parasitol, 1993 Sep, 61(1), 37 - 48 Molecular characterization of the largest subunit of Plasmodium falciparum RNA polymerase I; Fox BA et al.; Plasmodium species possess developmentally regulated ribosomal RNA (rRNA) genes . This report describes the expression and gene structure of the largest subunit of P . falciparum RNA polymerase I (RNAPI), which is responsible for the synthesis of rRNA . The RNAPI largest subunit gene was present as a single copy gene on chromosome 9 . Three exons encode the 2910-amino acid RNAPI polypeptide (340 140 Da) . A comparison of Plasmodium, Trypanosoma, and Saccharomyces cerevisiae nuclear RNAP largest subunits identified conserved amino acid positions and class-specific amino acid positions . Novel amino acid insertions were found between RNAPI conserved regions A and B (region A'), D and DE1 (region D'), DE2 and E (region DE2'), and F and G (region F') . Leucine zipper domains were found within regions D', DE2, and DE2' . A novel serine-rich repeat domain, a domain with homology to the C-terminal domain of eukaryotic upstream binding factor (UBF), and 4 highly conserved casein kinase II (CKII) Ser/Thr phosphorylation motifs were found within a 127-amino acid sub-region of enlarged region F' . The novel RNAPI serine-rich repeat contained a conserved motif, Ser-X3-Ser, which was also identified in the serine-rich repeat domains of the P . falciparum RNAPII and RNAPIII largest subunits, as well as within a highly homologous serine-rich repeat from trophozoite antigen R45 . The results of this molecular analysis indicate that phosphorylation and dephosphorylation mechanisms regulate the activity of P . falciparum RNAPI. J Nat Prod, 1993 Sep, 56(9), 1451 - 8 A screen for inhibitors of DNA recombination: identification of two new spirostanol glycosides from Chamaedorea linearis; Patil AD et al.; Two new glycosides have been isolated from the MeOH extract of the stem wood and stem bark of an Ecuadorian plant Chamaedorea linearis, and their structures have been determined by spectroscopic means and X-ray analysis of the aglycone to be 1-O-{beta-L-fucopyranosyl-(4'-sulfate)}-25R,5 alpha-spirostane-1 beta, 3 beta-diol {1}) and 1-O-{beta-L-fucopyranosyl-(4'-sulfate)}-25R,5 alpha-spirostane-1 alpha, 3 beta-diol {2} . These compounds were identified in a screen for inhibitors of recombinational DNA repair . Cytotoxic activity was also demonstrated. EMBO J, 1993 Sep, 12(9), 3693 - 701 Evidence for a repair enzyme complex involving ERCC1 and complementing activities of ERCC4, ERCC11 and xeroderma pigmentosum group F; van Vuuren AJ et al.; Nucleotide excision repair (NER), one of the major cellular DNA repair systems, removes a wide range of lesions in a multi-enzyme reaction . In man, a NER defect due to a mutation in one of at least 11 distinct genes, can give rise to the inherited repair disorders xeroderma pigmentosum (XP), Cockayne's syndrome or PIBIDS, a photosensitive form of the brittle hair disease trichothiodystrophy . Laboratory-induced NER-deficient mutants of cultured rodent cells have been classified into 11 complementation groups (CGs) . Some of these have been shown to correspond with human disorders . In cell-free extracts prepared from rodent CGs 1-5 and 11, but not in a mutant from CG6, we find an impaired repair of damage induced in plasmids by UV light and N-acetoxy-acetylaminofluorene . Complementation analysis in vitro of rodent CGs is accomplished by pairwise mixing of mutant extracts . The results show that mutants from groups 2, 3, 5 and XP-A can complement all other CGs tested . However, selective non-complementation in vitro was observed in mutual mixtures of groups 1, 4, 11 and XP-F, suggesting that the complementing activities involved somehow affect each other . Depletion of wild-type human extracts from ERCC1 protein using specific anti-ERCC1 antibodies concomitantly removed the correcting activities for groups 4, 11 and XP-F, but not those for the other CGs . Furthermore, we find that 33 kDa ERCC1 protein sediments as a high mol . wt species of approximately 120 kDa in a native glycerol gradient.(ABSTRACT TRUNCATED AT 250 WORDS) EMBO J, 1993 Sep, 12(9), 3519 - 28 Both Arabidopsis TATA binding protein (TBP) isoforms are functionally identical in RNA polymerase II and III transcription in plant cells: evidence for gene-specific changes in DNA binding specificity of TBP; Heard DJ et al.; Promoters of pol II and pol III transcribed U-snRNA genes in plants have identical sequence elements comprised of a -30 TATA box and an upstream sequence element (USE), located four or three helical turns upstream of the TATA box in pol II and pol III genes, respectively; it is this difference in element spacing that determines the RNA polymerase specificity of the gene . We are interested in identifying factors binding to U-snRNA gene promoters and their role in selection of RNA polymerase . In this work we have investigated possible differences in the activity of the two TATA binding proteins (TBPs) encoded by two different TBP genes of Arabidopsis . Using mutant TBPs with altered DNA binding specificity, similar to those described previously in yeast, we show that two Arabidopsis TBP isoforms are equally active with both pol II and pol III U-snRNA genes and with an mRNA gene transfected into plant protoplasts . In contrast to yeast, where modified TBP permits transcription only from promoters containing the TGTAAA mutant of the consensus (TATAAA) TATA element, altered Arabidopsis TBPs also suppress other TATA box mutants . Similar results were obtained with human and yeast TBP mutants expressed in plant cells . Interestingly, in several cases suppression of different TATA box mutants by altered TBPs was gene or RNA polymerase specific suggesting that assembly of TBP into specific complexes containing different TBP-associated factors may alter DNA binding specificity of the protein. Cell Growth Differ, 1993 Sep, 4(9), 707 - 13 Negative growth selection against rodent fibroblasts targeted for genetic inhibition of farnesyl transferase; Prendergast GC et al.; The Ras oncoprotein must be modified by farnesyl transferase (FTase) for biological activity . Therefore, inhibition of FTase may offer a means to block ras induced cell transformation . To address this hypothesis, we have introduced antisense and dominant inhibitory FTase expression plasmids into a panel of normal, mutant ras-, and mos- transformed rodent fibroblasts in an effort to genetically suppress FTase activity . Antisense FTase constructs reduced colony formation efficiency approximately 29% in normal and approximately 41% in ras-transformed cells relative to control plasmids . In contrast, antisense FTase plasmids did not exhibit a statistically significant effect on colony formation efficiency in mos-transformed transfectants . FTase alpha N199K is a mutant form of the alpha subunit of FTase that exhibits dominant inhibitory activity versus native FTase . Only mos-transformed transfectants exhibited expression of alpha N199K RNA in 15 of 16 fibroblast lines that were randomly selected and characterized . Our data suggest that genetic inhibition of FTase may result in a selection against animal cell growth. Anal Biochem, 1993 Sep, 213(2), 378 - 85 Characterization of absorption and scattering properties of small-volume biological samples using time-resolved spectroscopy; Liu H et al.; With time-resolved spectroscopy, we develop an experimental approach by sample substitution to measure the absorption (mu a) and reduced scattering (mu's) coefficients of small-volume biological samples . To investigate the method, small-volume control samples are substituted into a large-size host medium during increases in the absorber (or scatterer) concentration of the host . By characterizing the deviation of the spectra taken with and without the sample, we determine the matching points where the sample and surrounding medium are optically identical . We show that this method can result in correct values of the mu a and mu's for the sample within 6% error if the matching conditions for both the mu a and mu's are fully realized . The results also indicate that this method can give approximate values of the mu a and mu's in a reasonable range if either the mu a or the mu's matching between the two media is realized . This method has been applied to the studies of absorption properties of a human finger and of scattering properties of yeast. Trends Biochem Sci, 1993 Sep, 18(9), 334 - 8 Cyclosporin A, FK506 and rapamycin: more than just immunosuppression; Kunz J et al.; The mechanisms of action of the immunosuppressive drugs cyclosporin A (CsA), FK506 and rapamycin are strikingly conserved from yeast to human T cells . Recent results obtained with yeast corroborate calcineurin as the target of CsA-cyclophilin and FK506-FKBP complexes, and reveal a phosphatidylinositol 3-kinase homologue as the target of the rapamycin-FKBP complex. Mutat Res, 1993 Sep, 289(1), 39 - 46 Bridge-building between mathematical theory and molecular biology: the REV2 gene as paradigm; Eckardt-Schupp F et al.; The DNA damage-repair theory of R.H . Haynes anticipated the possibility of dose-dependent repair processes . The mathematical formalism developed by Haynes and coworkers on the basis of this theory provided tools to probe for the existence of inducible components of mutation or recombination by analysis of dose-response curves . Subsequently, we found that biological and molecular analysis of the Saccharomyces cerevisiae REV2 gene supported the validity of the postulates derived from the mathematical analysis . In this article, we briefly review the foregoing and summarize evidence that the REV2 gene product might function in DNA damage-inducible repair and mutation processes. Biochemistry, 1993 Aug 31, 32(34), 8842 - 7 P59 (FK506 binding protein 59) interaction with heat shock proteins is highly conserved and may involve proteins other than steroid receptors; Tai PK et al.; P59 {also known as FK506 binding protein 59 (FKBP59) or heat shock protein 56 (hsp56)} and heat shock proteins 90 and 70 (hsp90 and hsp70) associate with steroid receptors and are believed to maintain the receptors in an inactive state . Recently, we showed that p59 purified from human lymphocytes is an immunophilin (FKBP59) which binds both FK506 and rapamycin . It was also demonstrated that immunosuppressant-FKBP59 complexes associate with hsp90, hsp70, and the glucocorticoid receptor {Tai, P.-K . K., Albers, M . W., Chang, H., Faber, L . E., & Schreiber, S . L . (1992) Science 256, 1315-1318} . Here we provide evidence that rabbit uterine p59 also binds FK506 and rapamycin and that p59 or its homologue is associated with nontransformed progesterone receptors of rabbit uterus and chicken oviduct . This suggests that the immunophilin-heat shock protein-steroid receptor interaction is ubiquitous and not limited to immune systems . A FKBP59 homologue complexed with hsp90-hsp70 was also detected in yeast, which suggests that the immunophilin-heat shock protein association has been evolutionarily conserved . In addition, we found that the FKBP59-hsp complexes are more complicated than previously thought, involving other proteins such as actin and a 63-kDa protein, p63 . The association of p63 to the p59 complex was inhibited by FK506 and rapamycin, suggesting that p63 could be a potential target for the immunosuppressive actions of these two drugs.(ABSTRACT TRUNCATED AT 250 WORDS) FEBS Lett, 1993 Aug 30, 329(3), 268 - 72 DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid) inhibits an early step of protein translocation across the mammalian ER membrane; Jungnickel B et al.; Protein translocation across the endoplasmic reticulum (ER) membrane of yeast can be inhibited by agents believed to specifically affect the transport of ATP through the membrane (Mayinger, P . and Meyer, D.I . (1993) EMBO J . 12, 659-666), suggesting the involvement of a translocation component in the lumen of the ER that binds ATP . We demonstrate that one of the inhibitors, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), also affects the translocation of proteins into mammalian microsomes . Translocation is blocked at the point of transfer of the nascent chain from the signal recognition particle (SRP) into the ER-membrane . We also confirm that photoaffinity-labelling of microsomes with 8-azido-ATP inhibits the same early step of protein translocation . Since this step is reported to not require ATP, these results raise the possibility that, in both cases, factor(s) other than ATP-binding components of the translocation machinery are perturbed. Cell, 1993 Aug 27, 74(4), 743 - 56 A transmembrane protein with a cdc2+/CDC28-related kinase activity is required for signaling from the ER to the nucleus; Mori K et al.; In eukaryotic cells, the accumulation of unfolded proteins in the endoplasmic reticulum (ER) triggers a signaling pathway from the ER to the nucleus . Several yeast mutants defective in this pathway map to the ERN1 gene, which protects cells from lethal consequences of stress by signaling for increased expression of BiP and other ER proteins . ERN1 encodes a 1115 amino acid transmembrane protein (Ern1p) whose glycosylated N-terminal portion is located inside microsomes and whose cytoplasmic C-terminal portion carries an essential protein kinase activity . We postulate that Ern1p is the proximal sensor of events in the ER and that binding of ligand causes transduction of information across the ER membrane, leading to activation of a specific set of transcription factors. Nature, 1993 Aug 26, 364(6440), 821 - 4 Protein-kinase-A-dependent activator in transcription factor CREB reveals new role for CREM repressors; Brindle P et al.; Hormonally induced increases in cyclic AMP levels induce phosphorylation of the transcription factor CREB at a serine residue at position 133 by protein kinase A (ref . 1), enhancing its ability to activate transcription without affecting its intracellular location or DNA-binding activity . This effect is dependent on a 60-amino-acid region of CREB that contains Ser133 and is termed the kinase-inducible domain (KID)2, which also occurs in the CREB-related CREM-alpha and -beta proteins, although these are transcriptional repressors . Here we show that the KID domain confers a cAMP-inducible increase on the activity of the Q2 activation domain from CREB and the acidic activation domains from the yeast proteins GAL4 and GCN4 . Remarkably, it retains this ability even when attached to a separate polypeptide bound to an adjacent site in the promoter . KID may therefore be the first of a new class of conditional activators that work through other promoter-bound factors to stimulate gene expression in response to hormonal stimuli. Gene, 1993 Aug 25, 130(2), 271 - 5 Characterization of the pyruvate kinase-encoding gene (pki1) of Trichoderma reesei; Schindler M et al.; The pyruvate kinase-encoding gene (pki1) from Trichoderma reesei was isolated by hybridization to the corresponding Aspergillus nidulans pkiA gene . The 1614-bp nucleotide (nt) sequence of the cloned gene codes for a 538-amino-acid protein . The coding sequence contains a single intron of 246 nt at a position identical to that of intron E in the A . nidulans gene . The PKI protein shows extensive homology to the PKIs of A . nidulans and A . niger (67%) and Saccharomyces cerevisiae (59%) . The 5' non-coding sequence contains a number of motifs typical for yeast glycolytic genes, but so far only rarely found in filamentous fungi. Gene, 1993 Aug 25, 130(2), 259 - 64 Functional domains of the transcriptional activator NUC-1 in Neurospora crassa; Kang S; The NUC-1 regulatory protein directly controls the transcription of these genes and how the activity enzymes in Neurospora crassa . To understand how NUC-1 regulates the transcription of these genes and how the activity of NUC-1 is modulated by other regulatory proteins, two putative functional domains of NUC-1 were analysed: the DNA-binding domain and the regulatory domain . The DNA-binding activity of NUC-1 has not been directly demonstrated; however, results of deletion analysis, sequence analysis of the nuc-1 mutant alleles, and strong sequence similarity with the Saccharomyces cerevisiae PHO4 protein strongly suggest that the basic helix-loop-helix motif of NUC-1 forms a DNA-binding domain . Deletion and mutant analyses revealed that 39 amino acid (aa) residues (aa 463 to 501), or fewer, of NUC-1 are interacting with the negative regulatory factor(s), the PREG and/or PGOV proteins. J Biol Chem, 1993 Aug 25, 268(24), 18259 - 66 Structure/function relationships in hexokinase . Site-directed mutational analyses and characterization of overexpressed fragments implicate different functions for the N- and C-terminal halves of the enzyme; Arora KK et al.; Hexokinases are comprised of two highly homologous approximately 50-kDa halves and are product-inhibited by glucose-6-P . Four amino acid residues, Ser603, Asp657, Glu708, and Glu742, located in the C-terminal half of the tumor mitochondrial enzyme have been shown to be essential for enzyme function (Arora, K . K., Filburn, C . R., and Pedersen, P . L . (1991) J . Biol . Chem . 266, 5359-5362) . Here we have assessed also the role of the N-terminal half of the same enzyme . Site-directed mutagenesis of residues predicted to interact with glucose in the N-terminal half, i.e . Ser155, Asp209, and Glu260, to Ala, have no effect on hexokinase activity . In addition, inhibition by hexose mono- and bisphosphates is unchanged for each of the mutant enzymes . Significantly, the overexpressed N-terminal polypeptide is devoid of catalytic activity but does have the capacity to bind ATP-agarose and be released with ATP and glucose-6-P . In contrast, the overexpressed C-terminal polypeptide is catalytically active and shows the same product inhibition pattern as the complete 100-kDa parent enzyme . These results emphasize that the N-terminal half of tumor hexokinase is essential neither for catalysis nor product modulation . Rather, the N-terminal half may play another role, perhaps in modulation of the ATP/glucose-6-P-dependent binding of the enzyme to tumor mitochondria or by acting as a spacer between the outer mitochondrial membrane and the C-terminal catalytic unit. J Biol Chem, 1993 Aug 25, 268(24), 17878 - 82 Nitric oxide-independent, thiol-associated ADP-ribosylation inactivates aldehyde dehydrogenase; McDonald LJ et al.; Nitric oxide inhibits the activity of glyceraldehyde-3-phosphate dehydrogenase and stimulates NAD-dependent automodification of a cysteine (Dimmeler, S., Lottspeich, F., and Brune, B . (1992) J . Biol . Chem . 267, 16771-16774) . Another NAD-utilizing dehydrogenase that has a catalytic cysteine, aldehyde dehydrogenase (ALDH), was also inhibited by nitric oxide . Unlike glyceraldehyde-3-phosphate dehydrogenase, ALDH was modified in a nitric oxide-independent process by ADP-ribose, but not by NAD . Modification, which proceeded to > 2 mol ADP-ribose.mol ALDH-1, was associated with an exponential decrease in enzyme activity to less than 10% of control . Two types of evidence suggested modification of the ALDH-active site: 1) ADP-ribose inhibited ALDH competitively (Ki = 0.46 mM) with respect to NAD (Km = 0.11 mM) in brief incubations and 2) the presence of substrates protected ALDH from both modification and inhibition by ADP-ribose . The ALDH-ADP-ribose bond was sensitive to base and mercuric ion and stable to acid and neutral hydroxylamine, properties shared with the ADP-ribosylcysteine linkage synthesized enzymatically by pertussis toxin . These data demonstrate a novel means of inactivation of an NAD-dependent enzyme, namely the affinity-based modification of the enzyme NAD site by ADP-ribose, and suggest that nonenzymatic ADP-ribosylation may be responsible for modification of cysteine residues. J Biol Chem, 1993 Aug 25, 268(24), 17662 - 4 Targeting of histone tails by poly(ADP-ribose); Panzeter PL et al.; After Zn2+ finger-mediated binding to a DNA break, poly(ADP-ribose) polymerase becomes automodified with long polymers of ADP-ribose . These nucleic acid-like polymers may facilitate DNA repair by noncovalently interacting with neighboring proteins . Using a novel screening technique, we have identified histones as the predominant poly(ADP-ribose)-binding species in human keratinocytes, rat hepatocytes, frog eggs, and yeast . Polymer binding is confined specifically to the histone domains responsible for DNA condensation, i.e . histone tails . Our results indicate that polymers of ADP-ribose are targeted to sites of DNA strand breaks by poly(ADP-ribose) polymerase and subsequently function to alter chromatin conformation through noncovalent interactions with histone tails. J Biol Chem, 1993 Aug 25, 268(24), 18070 - 5 Deletion of the SH3 domain of Src interferes with regulation by the phosphorylated carboxyl-terminal tyrosine; Okada M et al.; A current model for the regulation of the Src protein-tyrosine kinase proposes that the COOH-terminal phosphotyrosine, Tyr-527, binds to the Src homology 2 (SH2) region in an intramolecular interaction that represses the kinase domain . This model is consistent with the activation of Src by mutations in the SH2 domain or COOH terminus . Mutations in the SH3 domain also activate Src, although this region is not thought to bind phosphotyrosine . Seidel-Dugan et al . (Seidel-Dugan, C., Meyer, B . E., Thomas, S . M., and Brugge, J . S . (1992) Mol . Cell . Biol . 12, 1835-1845) have shown that Src mutants with deletions in the SH2 or SH3 domain transform chicken embryo fibroblasts and have increased kinase activity . These mutant proteins are underphosphorylated at Tyr-527, a change that could in itself activate the mutants . Therefore, it is not possible to distinguish whether the SH2 and SH3 domains are needed for phosphorylation of Tyr-527 or for Src to adopt or maintain the repressed state . We have artificially increased the level of Tyr-527 phosphorylation of SH2 and SH3 deletion mutants by coexpressing them with the Tyr-527 kinase, Csk, in yeast cells . We find that both the SH2 and SH3 domains are needed for inhibition of Src by Csk . The SH2 domain is needed for efficient phosphorylation by Csk, both in yeast cells and in vitro . The SH3 domain is needed for Src to be inhibited when Tyr-527 is phosphorylated by Csk . This suggests that the SH3 domain cooperates with the SH2 domain and phosphorylated Tyr-527 to inhibit the kinase domain . Dephosphorylation of SH3 domain mutants at Tyr-527 in fibroblasts could be a consequence of a failure of the proposed SH2/phosphotyrosine interaction. Science, 1993 Aug 20, 261(5124), 1035 - 8 Group II intron RNA catalysis of progressive nucleotide insertion: a model for RNA editing; Mueller MW et al.; The self-splicing bl1 intron lariat from mitochondria of Saccharomyces cerevisiae catalyzed the insertion of nucleotidyl monomers derived from the 3' end of a donor RNA into an acceptor RNA in a 3' to 5' direction in vitro . In this catalyzed reaction, the site specificity provided by intermolecular base pair interactions, the formation of chimeric intermediates, the polarity of the nucleotidyl insertion, and its reversibility all resemble such properties in previously proposed models of RNA editing in kinetoplastid mitochondria . These results suggest that RNA editing occurs by way of a concerted, two-step transesterification mechanism and that RNA splicing and RNA editing might be prebiotically related mechanisms; possibly, both evolved from a primordial demand for self-replication. J Inorg Biochem, 1993 Aug 15, 51(3), 663 - 76 Changes in global stability and local structure of cytochrome c upon substituting phenylalanine-82 with tyrosine; Greene RM et al.; We have examined the F82Y;C102T variant of Saccharomyces cerevisiae iso-1-cytochrome c using high-resolution proton nuclear magnetic resonance spectroscopy, chemical denaturation, and differential scanning calorimetry . Comparison of proton chemical shifts, paramagnetic shifts, and nuclear Overhauser effects indicates structural changes are localized to the vicinity of position 82 . One alteration involves the rearrangement of the side chain of leucine-85 . Using many more proton assignments than were available in the initial report {G . J . Pielak, R . A . Atkinson, J . Boyd, and R . J . P . Williams, Eur . J . Biochem . 177, 179-185 (1988)}, a second alteration involving an interaction between arginine-13 and tyrosine-82 is observed . The interaction appears to involve a hydrogen bond with the eta-protons of arginine's guanido group acting as donor and tyrosine's phenolic eta-oxygen as acceptor . In spite of this potentially-stabilizing interaction, the free energy of denaturation decreases by approximately 2.4 kcal mol-1 . Results are discussed with respect to alterations in the native and denatured states. J Inorg Biochem, 1993 Aug 15, 51(3), 649 - 53 Dramatic stabilization of ferricytochrome c upon reduction; Hilgen-Willis S et al.; By combining measurements of the free energy of denaturation of the C102T variant of Saccharomyces cerevisiae iso-1-ferricytochrome c with determination of the formal potentials for the native and chemically-denatured states we have determined the free energy of denaturation of the ferro form of the protein . We report that the simplest of all chemical modifications, addition of an electron, increases the stability of ferricytochrome c by approximately 10 kcal mol-1 at 300 K, pH 4.6 . This makes reduced cytochrome c one of the most stable proteins yet investigated. J Biol Chem, 1993 Aug 15, 268(23), 16999 - 7009 Distinct activation domains within cAMP response element-binding protein (CREB) mediate basal and cAMP-stimulated transcription; Quinn PG; Activation of protein kinase A (PKA) by cAMP results in phosphorylation of cAMP response element-binding protein (CREB) and induction of specific gene expression . However, whether CREB participates directly in basal (PKA-independent) transcription is still an open question, and existing studies conflict over the identification of putative basal activation domains . In the present study, the activation domain of CREB, whether fused to the GAL4 DNA binding domain (CRG) or in native CREB, stimulated basal activity of the minimal tk promoter, but not the minimal SV40 early promoter . Cotransfection with PKI, a specific inhibitor of PKA, blocked PKA-induced expression of both promoters, but did not block CRG-mediated basal expression of the tk promoter . In addition, both CRG and a PKA phosphorylation site mutant provided comparable stimulation of basal tk promoter activity . Examination of a series of CREB deletion mutants mapped basal activity to interacting domains, located on either side of the previously identified PKA activation domain (amino acids 98-142) . This PKA-independent activity mapped primarily to a bipartite COOH-terminal basal activation domain (amino acids 165-252) . Its major component bears no obvious homology to previously identified activation domains, whereas a minor component is glutamine-rich . This COOH-terminal domain acts independently and provides the majority of basal activation but requires an NH2-terminal domain (amino acids 41-86) to provide full basal activity . A repressor domain (amino acids 142-165), deletion of which enhanced both basal and PKA-activated transcription, was also identified . This work establishes that CREB contains distinct basal and PKA-activated domains, that they operate independently for both loss of function and gain of function, and that they work on different promoters in different cell types. Proc Natl Acad Sci U S A, 1993 Aug 15, 90(16), 7706 - 10 The N-terminal coiled-coil domain of beta is essential for gamma association: a model for G-protein beta gamma subunit interaction; Garritsen A et al.; We have identified the N terminus of the beta subunit as an essential domain for G-protein beta gamma assembly . A C-terminal fragment, beta 1-(130-340), fails to bind gamma unless coexpressed with the complementary N-terminal fragment, beta 1-(1-129) . Deletion of the N-terminal 33 residues of beta 1, a region identified by computer algorithm to favor coiled-coil formation, abolishes gamma 2 association . On the basis of these findings, we propose a coiled-coil model of beta gamma interaction and refine this by computer-assisted molecular modeling . The model is tested by further mutagenesis: reversing the charge of residues in beta 1 that are hypothesized to be involved in interhelical salt bridges precludes gamma association . Insertions in the coiled-coil region, which disrupt the proposed hydrophobic interface, prevent gamma association . This structural basis for beta gamma dimerization provides a starting point for the design of beta and gamma mutants that can be used to map regions in beta gamma critical for interactions with the alpha subunit, receptors, and effectors. J Biol Chem, 1993 Aug 15, 268(23), 17564 - 70 Mechanism of site-specific recombination . Logic of assembling recombinase catalytic site from fractional active sites; Lee J et al.; The active nucleophilic species in the strand cleavage and strand exchange steps of site-specific recombination by the Flp protein are the active site tyrosine (Tyr-343) of Flp and the 5'-hydroxyl of Flp-nicked DNA, respectively . The target phosphodiester, activated by Flp, can be cleaved by an exogenous nucleophile derived, for example, from H2O2 . Flp variants that are defective in the phosphate activation step and cannot sustain Tyr-343-mediated cleavage also fail to elicit H2O2-mediated cleavage . An Flp mutant lacking Tyr-343, (Flp(Y343F)), can carry out both the strand cleavage and strand exchange reactions in the presence of a age and strand exchange reactions in the presence of a tyrosine analog . These results are consistent with a cis-activation/trans-nucleophilic attack paradigm for strand breakage and strand union . The proposed model conceptually unifies the chemistry and enzymology of the two partial reactions of recombination . The mechanism of Flp action has strong implications for phosphoryl transfer reactions in other site-specific DNA recombination systems and in RNA splicing. J Immunol, 1993 Aug 15, 151(4), 1979 - 88 Characterization of A1, a novel hemopoietic-specific early-response gene with sequence similarity to bcl-2; Lin EY et al.; Granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates hemopoietic cell proliferation, differentiation, and functional activation by inducing the expression of specific genes . As part of an investigation of the regulation of gene expression by GM-CSF, we have previously identified a novel murine GM-CSF-inducible gene, A1 . In this report, we present the complete nucleotide sequence of the A1 mRNA as well as a portion of the 5' flanking region, and describe the expression pattern of the gene . The results demonstrate that A1 is a hemopoietic tissue-specific gene that is expressed in several hemopoietic cell lineages, including T-helper lymphocytes, macrophages, and neutrophils . In murine bone marrow-derived macrophages, A1 gene expression is rapidly and transiently induced by GM-CSF, and the induction was independent of de novo protein synthesis . In addition to GM-CSF, a transient induction of A1 mRNA accumulation was observed in response to LPS in macrophages . This induction is not mediated by IL-1 alpha or IL-6, neither of which stimulate A1 . In the myeloid precursor cell line, 32D cl3, A1 gene expression is stably induced during granulocyte colony-stimulating factor-stimulated myeloid cell differentiation . The A1 message encodes a predicted polypeptide with an M(r) of 20,024 and no signal peptide . The peptide sequence contains a region of 80 amino acids that shows similarity to bcl-2 and to the recently described bcl-2-related gene, MCL1 . These data demonstrate that A1 is a novel early-response gene whose expression is associated with a variety of stimuli and occurs in several hemopoietic cell types. Proc Natl Acad Sci U S A, 1993 Aug 15, 90(16), 7603 - 7 Long-term increases in neurotransmitter release from neuronal cells expressing a constitutively active adenylate cyclase from a herpes simplex virus type 1 vector; Geller AI et al.; Signal-transduction pathways mediate a wide range of short-term changes in the physiology of neuronal systems from invertebrates to mammals . However, examples of long-term changes in neuronal physiology mediated by these pathways have been limited to invertebrate systems . In this report, long-term changes in the physiology of mammalian neurons were studied by using genetic intervention to cause a long-lasting activation of the cAMP pathway . The catalytic domain of yeast adenylate cyclase (cyr), encoding a constitutive enzyme activity, was expressed in neuronal cells infected with a defective herpes simplex virus vector (pHSVcyr) . In PC-12 cells infected with pHSVcyr, increases were seen in cAMP levels, protein kinase A activity, protein phosphorylation, phosphorylation of the tyrosine hydroxylase protein kinase A site (Ser40), and catecholamine release . Infection of sympathetic neurons with pHSVcyr increased cAMP levels, protein phosphorylation, and catecholamine release . Yeast adenylate cyclase immunoreactivity and elevated cAMP levels were localized to the cell bodies of sympathetic neurons . The increase in neurotransmitter release was both Ca(2+)- and activity-dependent and persisted for at least 1 week after infection of the sympathetic neurons, suggesting that sustained physiological activation of the cAMP pathway may mediate long-term changes in the neuronal physiology of mammalian systems. Proc Natl Acad Sci U S A, 1993 Aug 15, 90(16), 7884 - 8 Acute mixed-lineage leukemia t(4;11)(q21;q23) generates an MLL-AF4 fusion product; Domer PH et al.; A chromosomal translocation, t(4;11)-(q21;q23), is associated with an aggressive mixed-lineage leukemia . A yeast artificial chromosome was used to clone the chromosomal breakpoint of this translocation in the RS4;11 cell line . The breakpoint sequences revealed an inverted repeat bordered by a consensus site for topoisomerase II binding and cleavage as well as chi-like elements . The der(11) chromosome encodes a fusion RNA and predicted chimeric protein between the 11q23 gene MLL and a 4q21 gene designated AF4 . The sequence of the complete open reading frame for this fusion transcript reveals the MLL protein to have homology with DNA methyltransferase, the Drosophila trithorax gene product, and the "AT-hook" motif of high-mobility-group proteins . An alternative splice that deletes the AT-hook region of MLL was identified . AF4 is a serine- and proline-rich putative transcription factor with a glutamine-rich carboxyl terminus . The composition of the complete MLL-AF4 fusion product argues that it may act through either a gain-of-function or a dominant negative mechanism in leukemogenesis. Nature, 1993 Aug 5, 364(6437), 544 - 7 Transactivation by NF-IL6/LAP is enhanced by phosphorylation of its activation domain; Trautwein C et al.; One of the members of the bZIP family of transcriptional activators is NF-IL6/LAP (IL-6 DBP, C/EBP beta, CRP2) . NF-IL6/LAP protein is highly expressed in liver nuclei, where it has been implicated as a master regulator of the acute-phase response, induced by interleukin-6 (IL-6) and other inflammatory mediators . Also, NF-IL6/LAP is involved in the activation of the IL-6 promoter in response to IL-1 and bacterial lipopolysaccharide . The control of NF-IL6/LAP expression and activity is complex and poorly understood . Under some conditions the NF-IL6/LAP gene is transcriptionally activated by IL-1 and lipopolysaccharide, whereas in other instances, its binding to cognate DNA sequences is enhanced by cytokines . Additionally, the ability of constitutively expressed NF-IL6/LAP to activate transcription is strongly augmented by IL-6, through an unknown signalling pathway . We now show that stimulation of the protein kinase C pathway increases the phosphorylation of Ser 105 within the activation domain of NF-IL6/LAP, and enhances its transcriptional efficacy. FEMS Immunol Med Microbiol, 1993 Aug, 7(2), 153 - 9 Dextran sulphate enhancement of lipopolysaccharide-induced tumour necrosis factor-alpha production by murine peritoneal macrophages: correlation with macrophage blockade; Kamochi M et al.; Proteose peptone-induced murine peritoneal macrophages (M phi) were preincubated with 100-800 micrograms/ml of dextran sulphate (DS) 500 (M(r) 500,000) or DS1000 (M(r) 1,000,000) . After 2-24 h of the preincubation, the M phi were stimulated with 1 microgram/ml of lipopolysaccharide (LPS) in vitro for 18 h in DS-free culture medium . The culture supernatants were then collected for TNF assay . The LPS-induced TNF activity of M phi supernatant preincubated with DS500 or DS1000 for 6 h was enhanced by up to about ten-fold compared with those preincubated without DS . This enhancing effect was not observed when M phi were preincubated with 100-800 micrograms/ml of low molecular weight DS5 (M(r) 5,000) or neutral dextran (Dex) 500 (M(r) 500,000) . The enhancement of LPS-induced TNF-alpha production from M phi was observed after 2 or 4 h of incubation with DS1000 or DS500, respectively . The phagocytic activity of M phi was determined in vitro by the ingestion index and phagocytic capacity using Saccharomyces cerevisiae . Treatment with DS500 or DS1000 significantly suppressed the phagocytic activity from 2 h after the incubation, but this suppression was not observed in M phi incubated with DS5 or Dex500 . Our experiments indicate that DS500 and DS1000 act directly on M phi and enhance LPS-induced TNF-alpha production from M phi, and that the enhancement is closely related to the suppression of M phi phagocytic function. Proteins, 1993 Aug, 16(4), 408 - 22 A hypothetical complex between crystalline flavocytochrome b2 and cytochrome c; Tegoni M et al.; Flavocytochrome b2 and cytochrome c are physiological electron transfer partners in yeast mitochondria . The formation of a stable complex between them has been demonstrated both in solution and in the crystalline state . On the basis of the three-dimensional structures, using molecular modeling and energy minimization, we have generated a hypothetical model for the interaction of these redox partners in the crystal lattice . General criteria such as good charge and surface complementarity, plausible orientation, and separation distance of the prosthetic groups, as well as more specific criteria such as the stoichiometry determined in the crystal, and the involvement of both domains and of more than one subunit of flavocytochrome b2 led us to discriminate between several possible interaction sites . In the hypothetical model we present, four cytochrome c molecules interact with a tetramer of flavocytochrome b2 . The b2 and c hemes are coplanar, with an edge-to-edge distance of 14 A . The contact surface area is ca . 800 A2 . Several electrostatic interactions involving the flavin and the heme domains of flavocytochrome b2 stabilize the binding of cytochrome c. Mol Cell Biol, 1993 Aug, 13(8), 4806 - 13 Suppression of a defect in the 5' untranslated leader of mitochondrial COX3 mRNA by a mutation affecting an mRNA-specific translational activator protein; Costanzo MC et al.; Translation of the Saccharomyces cerevisiae mitochondrial COX3 mRNA, encoding subunit III of cytochrome c oxidase, specifically requires the action of the nuclear gene products PET54, PET122, and PET494 at a site encoded in the 612-base 5' untranslated leader . To identify more precisely the site of action of the translational activators, we constructed two large deletions of the COX3 mRNA 5' untranslated leader . Both deletions blocked translation without affecting mRNA stability . However, one of the large deletions was able to revert to partial function by a small secondary deletion within the remaining 5' leader sequences . Translation of the resulting mutant (cox3-15) mRNA was still dependent on the nuclear-encoded specific activators but was cold sensitive . We selected revertants of this mitochondrial mutant at low temperature to identify genes encoding proteins that might interact with the COX3 mRNA 5' leader . One such revertant carried a missense mutation in the PET122 gene that was a strong and dominant suppressor of the cold-sensitive defect in the mRNA, indicating that the PET122 protein interacts functionally (possibly directly) with the COX3 mRNA 5' leader . The cox3-15 mutation was not suppressed by overproduction of the wild-type PET122 protein but was very weakly suppressed by overproduction of PET494 and slightly better suppressed by co-overproduction of PET494 and PET122. J Virol, 1993 Aug, 67(8), 4598 - 604 Functional domains of the simian foamy virus type 1 transcriptional transactivator (Taf); Mergia A et al.; The genome of simian foamy virus type 1 encodes a transcriptional transactivator (Taf) that dramatically elevates gene expression directed by the viral long terminal repeat . In this report, we describe the functional domains of simian foamy virus type 1 Taf . Several taf mutants and fusion proteins of Taf and the DNA-binding domain of the Saccharomyces cerevisiae transcriptional transactivator GAL4 were used in this study . Taf contains two potent activation domains . One of the activation domains is located at the amino terminus (positions 1 to 48, with position 1 representing the initiator amino acid methionine) and contains several acidic amino acids . The second activation domain was mapped to a region at the carboxy terminus (positions 277 to 300) . These two domains activate gene expression directed by the viral long terminal repeat independently of each other . No significant amino acid sequence homology between the activation domains is noted . Thus, Taf belongs in part to the family of acidic transcriptional transactivators . The activation domain at the carboxy terminus is conserved among foamy virus transactivators but is not related to other known transcriptional activators . Therefore, the mechanism of gene activation by the carboxy terminus of Taf may be novel . In addition, a potential binding domain rich in basic amino acids (positions 179 to 222) and a highly conserved sequence among foamy virus transactivators (positions 93 to 109) were found to be critical for Taf activity. J Virol, 1993 Aug, 67(8), 4474 - 83 A major transactivator of varicella-zoster virus, the immediate-early protein IE62, contains a potent N-terminal activation domain; Perera LP et al.; Accumulating evidence indicates that the product of the putative immediate-early gene ORF62 (IE62) activates varicella-zoster virus (VZV) genes thought to represent all three kinetic classes, namely, immediate-early (alpha), early (beta), and late (gamma) classes, of VZV genes as well as a variety heterologous gene promoters . However, the mechanism(s) by which IE62 protein mediates transactivation of these diverse VZV and heterologous gene promoters remains to be elucidated . In this study, by using yeast GAL4 protein chimeras, the coding regions of VZV ORF62 possessing activation domains have been assessed . We demonstrate that the VZV IE62 protein contains a potent activation domain in the N-terminal portion of the molecule, encoded within the first 86 codons of ORF62 . The predicted secondary structure profile and the acid-base composition of this IE62 domain resemble those of other transregulatory proteins whose activation is mediated through acidic, hydrophobic elements . In addition, we show that deletion of this activation domain from the 1,310-residue native IE62 protein results in ablation of the transactivator function of IE62 . We also present evidence that the mutant IE62 protein lacking the activation domain, though devoid of transactivation ability, was still capable of interfering with the activation of target promoters by the native, full-length IE62. Proteins, 1993 Aug, 16(4), 384 - 92 Molecular dynamics study of structure and stability of a model coiled coil; Zhang L et al.; This paper employs methods used earlier to study helix propensity in a model alpha-helix . The methods are extended to simulations of a motif structure of the alpha-helical coiled coil, i.e., a structure with a simple amino acid sequence, containing only alanine, leucine, and valine, with leucine and valine forming hydrophobic contacts in the helix interface (positions "d" and "a") . Dynamic simulations of the model coiled-coil structure reproduce characteristic features of the coiled-coil motif seen in experimental studies . Free energy simulations were used to assess the change in stability of the model when a leucine pair or a valine pair in the helix interface was replaced with an alanine pair . A leucine pair at position d was found to contribute 3.4 kcal/mol to the stability of the coiled coil relative to an alanine pair, and a valine pair at position a was found to contribute 0.8 kcal/mol relative to an alanine pair . The value for the leucine pair agrees with reports in two experimental studies with molecules having different amino sequence . The value for the valine pair is reasonable given the smaller size of the valine side chain and the intrinsic low helix propensity of valine . No experimental value was available for comparison. Eur J Biochem, 1993 Aug 1, 215(3), 587 - 94 In vitro formation of a photoreversible adduct of phycocyanobilin and tobacco apophytochrome B; Kunkel T et al.; The light-stable tobacco phytochrome apoprotein (PHYB) expressed in yeast can be assembled with phycocyanobilin to give a photoreversible adduct . The spectral properties of the reconstituted PHYB-phycocyanobilin species were determined by absorbacen and difference absorbance spectroscopies . The holoprotein exhibits absorbance maxima at 408 nm and 712 nm for the far-red-light-absorbing (Pfr) form and 356 nm and 658 nm for the red-light-absorbing (Pr) form . The ligation of the chromophores to the dimeric PHYB apoprotein resulted in a PHYB-phycocyanobilin adduct with the spectral properties of the Pr form . Kinetic analyses of the in vitro reconstitution for PHYB apoprotein under saturating concentrations of phycocyanobilin revealed a pseudo first-order rate constant of 2.8 x 10(-2)s.-1 . The similarity with the reported rate constant for the reconstitution of light-labile phytochrome (PHYA) from oat {Li, L . & Lagarias, J.C . (1992) Phytochrome assembly, J . Biol . Chem . 267, 19,204-19,210} suggests that the mechanisms of chromophore attachment are probably very similar for PHYA and PHYB. Biochem J, 1993 Aug 1, 293 ( Pt 3), 769 - 74 Reduced sulphydryl groups are required for DNA binding of Ku protein; Zhang WW et al.; The Ku protein, a DNA-binding complex that is composed of two subunits of 70 kDa and of 86 kDa, has been suggested to play a role in gene transcription . The dependence of the in vitro DNA-binding activity of affinity-purified Ku protein on reduced cysteine residues has been studied using sulphydryl-modifying agents . Inhibition of the DNA-binding activity was caused by alkylation with N-ethylmaleimide and by crosslinking with azadicarboxylic acid bis(dimethylamide) . Treatment of the protein with a large excess of N-ethylmaleimide after it had bound to DNA did not completely dissociate the complex from the DNA, suggesting that some cysteines may be in direct contact with DNA . Pre-incubation of the protein at 37 degrees C or above caused rapid inactivation of DNA binding . The elevated temperature azadicarboxylic acid bis(dimethylamide) treatments resulted in the formation of a crosslinked product, which was detected by Western blotting . The effects of azadicarboxylic acid bis(dimethylmaleimide) and heat were completely reversible by treatment with a reducing agent, such as dithiothreitol . These results demonstrate that in vitro DNA-binding activity of the Ku protein requires reduced sulphydryl groups . Interestingly, the DNA-binding activity of Ku protein was protected from heat inactivation by the presence of a HeLa cell nuclear extract, suggesting that a nuclear factor or factors may be responsible for the maintenance of the reduced cysteines of the Ku protein in vivo . Thus, the biochemical function of the Ku protein may be regulated through oxidation-reduction of its cysteine residues. EMBO J, 1993 Aug, 12(8), 3227 - 36 Genetic analysis of the leucine heptad repeats of Lac repressor: evidence for a 4-helical bundle; Alberti S et al.; Gel-filtration experiments indicate that a peptide (P2) composed of the basic region of GCN4 fused to the leucine heptad repeats of Lac repressor forms tetrameric aggregates . Gel-shift experiments were performed to determine the orientation of the helices in the tetrameric P2 aggregate . Sandwich-complex formation of peptide P2 with two DNA fragments containing two symmetrical CRE binding sites (5'-ATGACGTCAT-3') at a distance of 21 bp suggests antiparallel aggregation of the Lac leucine heptad repeats . Thus, we conclude that the leucine heptad repeats of Lac repressor have the ability to form homomeric 4-helical bundles with an antiparallel arrangement of the helices . This topology enables the two DNA fragments in the sandwich complexes to be held together by two tetramers of peptide P2 . Replacement of the uncharged amino acids of the helical g and e positions of peptide P2 by the corresponding charged residues of GCN4 (peptide P4) results in a dimeric and parallel aggregation of the leucine heptad repeats, and consequently abolishes the potential to form sandwich structures . Similarly, a hybrid Lac repressor in which the GCN4 leucine zipper replaces the natural Lac leucine heptad repeats forms dimers only . It regains the ability to form tetramers when the charged amino acids in helical positions g and e are replaced by uncharged alanines. EMBO J, 1993 Aug, 12(8), 3023 - 34 Genetic and biochemical characterization of ISP6, a small mitochondrial outer membrane protein associated with the protein translocation complex; Kassenbrock CK et al.; To search genetically for additional components of the protein translocation apparatus of mitochondria, we have used low fidelity PCR mutagenesis to generate temperature-sensitive mutants in the outer membrane translocation pore component ISP42 . A high copy number suppressor of temperature-sensitive isp42 has been isolated and sequenced . This novel gene, denoted ISP6, encodes a 61 amino acid integral membrane protein of the mitochondrial outer membrane, which is oriented with its amino-terminus facing the cytosol . Disruption of the ISP6 gene is without apparent effect in wild type yeast cells, but is lethal in temperature-sensitive isp42 mutants . Immunoprecipitation of the gene product, ISP42p, from mitochondria solubilized under mild conditions reveals a multi-protein complex containing ISP6p and ISP42p. Virology, 1993 Aug, 195(2), 569 - 77 Twenty-one base pair repeat elements influence the ability of a Gal4-Tax fusion protein to transactivate the HTLV-I long terminal repeat; Connor LM et al.; The Tax1 protein of the human T-cell leukemia virus (HTLV-I) is a 40-kDa positive transactivator of viral gene expression . Tax1 does not bind directly to DNA, but associates indirectly with DNA via cellular transcription factors . To further investigate the activation of HTLV-I transcription by Tax1, a chimeric protein containing Tax1 fused to the DNA binding domain of Gal4 was created (Gal4-Tax) . HTLV-I long terminal repeat (LTR) reporter plasmids were constructed in which specific Tax1 responsive elements were replaced with Gal4 binding sites . Cotransfection of Gal4-Tax or Tax1 with HTLV-I LTR reporter constructs containing Gal4 binding sites demonstrated that Gal4 sequences were necessary but not sufficient for maximal activation of the promoter by Gal4-Tax . Sequences surrounding the Gal4 binding sites were important in determining the level of Gal4-Tax activation . Association of Gal4-Tax with promoters which contained six Gal4 binding sites, but which lacked flanking LTR sequences, were weakly transactivated by Gal4-Tax (sevenfold) . In contrast, LTR-CAT reporter constructs containing three Gal4 binding sites flanked by two 21 base pair repeat elements demonstrated a ninefold greater response to Gal4-Tax . These results suggest that cellular transcription factors, which bind the 21 base pair repeat elements, influence the ability of Tax1 to function as a transactivator . Furthermore, this effect is not fully explained by the ability of these factors to physically direct Tax1 to the LTR. Mol Cell Biol, 1993 Aug, 13(8), 4640 - 7 Identification of transcriptional activation and inhibitory domains in serum response factor (SRF) by using GAL4-SRF constructs; Johansen FE et al.; The binding of serum response factor (SRF) to the c-fos serum response element has been shown to be essential for serum and growth factor activation of c-Fos . Since SRF is ubiquitously expressed, it has been difficult to measure the activity of SRF introduced into cells . To assay for functions of SRF in cells, we have changed its DNA binding specificity by fusing it to the DNA binding domain of GAL4 . Transfection of GAL4-SRF constructs into cells has allowed us to identify SRF's transcriptional activation domain as well as domains which inhibit this activity . First, we found that the transcriptional activation domain maps to between amino acids 339 and 508 in HeLa cells and to between amino acids 414 and 508 in NIH 3T3 cells . Second, we show that in the context of GAL4-SRF constructs, there are two separate domains of SRF that can inhibit its activation domain . Although these domains overlap the DNA binding and dimerization domains of SRF, these functions were not required for inhibition . Finally, we show that one of the inhibitory domains is modular in that it can also inhibit activation when it is moved amino terminal to GAL4's DNA binding domain in an SRF-GAL4-SRF construct . The implications of these inhibitory domains for SRF regulation are discussed. Mol Cell Biol, 1993 Aug, 13(8), 4572 - 7 Uncoupling of initiation and reinitiation rates during HeLa RNA polymerase II transcription in vitro; Jiang Y et al.; RNA polymerase II transcription is influenced both by how rapidly a gene is induced and by the rate at which continuous reinitiation occurs after induction . We show here that in vitro the rates of these two critical steps need not be the same . For activator GAL-AH-dependent HeLa transcription, the rate of assembling a preinitiation complex is significantly slower than the rate of reinitiation . Although reinitiation is rapid, it still requires ATP hydrolysis . This unexpected uncoupling of the rates of initiation and reinitiation implies that in regulating mammalian promoter activity, one must consider separately the controls on initiation during induction and the controls on the subsequent reinitiation events. J Virol, 1993 Aug, 67(8), 5030 - 4 Genetic evidence that the Tat proteins of human immunodeficiency virus types 1 and 2 can multimerize in the eukaryotic cell nucleus; Bogerd HP et al.; The formation of dimers or higher-order multimers is critical to the biological activity of many eukaryotic regulatory proteins . However, biochemical analyses of the multimerization capacity of the Tat trans activator of human immunodeficiency virus types 1 (HIV-1) and 2 (HIV-2) have yielded contradictory results . We used the two-hybrid genetic assay for protein-protein interactions in the eukaryote Saccharomyces cerevisiae (S . Fields and O.-K . Song, Nature {London} 340:245-246, 1989) to examine the multimerization of Tat in vivo . Both HIV-1 and HIV-2 Tat are shown to form specific homo- but not heteromultimers in the yeast cell nucleus . Mutational analysis indicates a critical role for the essential core motif of Tat in mediating this interaction but demonstrates that efficient Tat multimerization does not require an intact cysteine motif . These data raise the possibility that the multimerization of Tat may be important for Tat function in higher eukaryotes. Aging (Milano), 1993 Aug, 5(4), 299 - 307 FRAR course on laboratory approaches to aging . Genetic influences on aging in mammals and invertebrates; Johnson TE; A central theme underlies this review: "Genetics offers an important tool for identifying key molecular events that are involved in specifying biological functions." This approach has been used repeatedly to understand such diverse biological phenomena as oncogenesis, development, and the cell cycle, but has only recently been applied to the analysis of organismic aging and senescence . The power of the genetic approach lies in the ability to integrate phenomena that are displayed at multiple observational levels (i.e., from the molecular to the whole organism), and the power to reveal causal factors that are not dependent upon the prejudice of the investigator . I discuss several areas where genetics has been fruitfully applied to the study of the aging processes: human genes identified by "segmental progeroid" mutations; neurological diseases of the elderly; the limited proliferative life span of human somatic cells in tissue culture; studies on the life span of the mouse; and genetic analysis of life span in shorter-lived metazoans (Drosophila melanogaster and Caenorhabditis elegans), and the yeast Saccharomyces cerevisiae. Cell Struct Funct, 1993 Aug, 18(4), 205 - 10 Assembly of envelope structure with vesicles associated with Ku-homologous protein in Xenopus egg extract in the absence of chromatin; Takasuga Y et al.; To study the process of nuclear envelope assembly at the end of mitosis, we developed a chromatin-free in vitro system for assembly of envelope structures in Xenopus interphase egg extract, and examined the participation of Ku-homologous protein in the assembly . The envelope structure assembled spontaneously in the absence of chromatin or DNA between glass plates under a condition that minimized generation of flow of the extract . Morphological study using an electron microscopy has revealed that the membrane surrounding the envelope structure is a double membrane that contains gaps resembling nuclear pore complex . Their assembly was dependent on ATP and was inhibited by the addition of GTP-gamma-S or N-ethylmaleimide . Depletion of a pre-nuclear vesicle by preincubating the interphase egg extracts with large excess of sperm head chromatins impaired the assembly . The membrane vesicle, which was associated with Ku-homologous protein of Xenopus, participated in the assembly as proven by reaction with monoclonal antibody made specific for Ku p70 protein . However, the assembly process of the envelope structure was inhibited only slightly by the antibody, suggesting that the Ku-homologous protein does not participate in the fusion process of vesicles to form the envelope structure. Curr Opin Cell Biol, 1993 Aug, 5(4), 636 - 40 Protein sorting in the endomembrane system of plant cells; Gal S et al.; Although many properties of the targeting of plant endomembrane proteins are similar to mammalian and yeast systems, several clear differences are found that will be stressed in this review . In the past year, significant advances in our understanding of storage protein segregation in the endoplasmic reticulum, compartmentation of Golgi, and the signals for vacuolar protein targeting have been made . This work will form the basis for determining the mechanism of these sorting phenomena. Protein Eng, 1993 Aug, 6(6), 557 - 64 Determination of the structure of symmetric coiled-coil proteins from NMR data: application of the leucine zipper proteins Jun and GCN4; O'Donoghue SI et al.; Previous attempts to determine the solution structures of homodimeric 'leucine zippers' using nuclear magnetic resonance (NMR) spectroscopy have been impeded by the complete symmetry of these coiled-coil molecules, which makes it impossible a priori to distinguish between intra- and intermonomer dipolar connectivities . Consequently, a number of ad hoc approaches have been used in an attempt to derive tertiary solution structures of these molecules from the NMR data . In this paper we present a more rigorous approach for analysing the NMR spectra of symmetric coiled-coil proteins . This analysis is based on calculations of intra- and intermonomer interproton distances in the recently determined crystal structure of the GCN4 leucine zipper {O'Shea, E.K., Klemm, J.D., Kim, P.S . and Alber, T . (1991) Science, 254, 539-543} and in symmetric coiled-coil models of the leucine zippers of GCN4 and the human oncoprotein Jun which we constructed using a dynamic simulated annealing approach . This analysis has enabled the formulation of a set of rules for interpreting the NMR spectra of symmetric coiled-coil proteins and has also led to the prediction of novel dipolar connectivities which we demonstrate in a 2-D NMR spectrum of the homodimeric Jun leucine zipper. J Biomol Struct Dyn, 1993 Aug, 11(1), 1 - 10 Sizing of large DNA molecules by hook formation in a loose matrix; Guo XH et al.; We present details on how to implement a newly developed methodology, Optical Contour Maximization (OCM), for accurately sizing large DNA molecules . Agarose gel containing stained DNA is cast between a slide and a coverslip, and molecules moved by an applied electrical field are observed using fluorescence microscopy . The molecules of interest lie in the interface formed between the coverslip and gel . DNA movement in this region is largely confined to lateral motions . When a DNA molecule snags an obstacle, it elongates, forming a metastable hook that can persist for several seconds . We found that the longest observed hook contour length can be determined from rapidly collected images . This maximized length shows a linear correlation with reported size {X.H . Guo, E.J . Huff & D.C . Schwartz, Nature 359, 783 (1992)} Successful measurements require a critical balance between the voltage needed for full elongation, and the unwanted effects of too large a voltage: fewer metastable hooks form, they dissipate faster, molecule breakage becomes a problem, and faster image collection becomes necessary . We measured apparent contour length as a function of applied voltage and determined an optimal voltage for our apparatus, using a singly anchored 114kb molecule . The measurement precision is estimated from the distribution of results . We expect that OCM will find utility in physical mapping and molecular karyotyping of lower eucaryotes of medical importance. Analyst, 1993 Aug, 118(8), 973 - 8 Electrocatalytic reduction of hydrogen peroxide at a stationary pyrolytic graphite electrode surface in the presence of cytochrome c peroxidase: a description based on a microelectrode array model for adsorbed enzyme molecules; Armstrong FA et al.; Electrochemical reduction of H2O2 at pyrolytic graphite disc electrodes of radius 2.5 mm occurs at readily accessible potentials (600 mV versus the standard hydrogen electrode) in the presence of yeast cytochrome c peroxidase . Introduction of the enzyme into the electrolyte solution initiates large changes in the ellipsometric angles measured for the electrode-solution interface, consistent with time-dependent enzyme adsorption . This process may be correlated with changes in electrochemical activity . Over the same time course, linear-sweep voltammograms are characterized by a transition from a sigmoidal to a peak-type waveform . It is proposed that the time-dependent behaviour may be rationalized by use of a microscopic model for substrate mass transport, in which the two-electron reduction of peroxide occurs at electrocatalytic sites consisting of adsorbed enzyme molecules . A voltammetric theory based on treating the adsorbed redox enzymes as an expanding array of microelectrodes is in excellent agreement with experiment. Yeast, 1993 Aug, 9(8), 907 - 13 Isolation and DNA sequence of the STE14 gene encoding farnesyl cysteine: carboxyl methyltransferase; Ashby MN et al.; We isolated a mutant defective in C-terminal farnesyl cysteine:carboxyl methyltransferase activity from a screen for mutations causing a-specific sterility . A genomic fragment was cloned from a yeast multi-copy library that restored mating . Both the cloned gene and the sterile mutation were allelic to the STE14 gene . A ste14-complementing 2.17 kb BamHI fragment subclone was sequenced and found to encode a 239 amino acid protein with a molecular weight of 27,887 Daltons . The hydrophobicity profile of the methyltransferase reveals the presence of at least five potential transmembrane domains . In comparisons of the C-terminal methyltransferase amino acid sequence with those in the PIR and Swiss protein databases, no significantly similar sequences were found nor were conserved regions from other methyltransferases present. Yeast, 1993 Aug, 9(8), 847 - 57 The multifunctional regulatory proteins ABF1 and CPF1 are involved in the formation of a nuclease-hypersensitive region in the promoter of the QCR8 gene; De Winde JH et al.; The abundant DNA-binding proteins ABF1 and CPF1 are members of a family of global regulators with diverse chromosomal functions in the yeast Saccharomyces cerevisiae . Recent evidence suggests that these protein factors may be involved in establishing and maintaining well-defined chromatin in promoter regions and other genetic elements . We have investigated the involvement of ABF1 and CPF1 in chromatin organization at the QCR8 gene, encoding subunit VIII of the mitochondrial ubiquinol-cytochrome c oxidoreductase . The promoter region of the QCR8 gene contains overlapping binding sites for ABF1 and CPF1 . Nucleosome positioning studies indicate that the QCR8 gene is associated with a phased array of nucleosomes under both catabolite-repressed and derepressed growth conditions . Analysis of binding site mutants reveals that both ABF1 and CPF1 are involved in maintaining a nuclease-hypersensitive region in the QCR8 promoter . The chromatin structure at QCR8 during steady-state growth is, however, mainly dependent on binding of ABF1 to the promoter region . Implications of these findings for the role played by ABF1 and CPF1 in the regulation of mitochondrial biogenesis and other processes important for cell growth and division will be discussed. Yeast, 1993 Aug, 9(8), 835 - 45 Analysis of the inducer-responsive CAR1 upstream activation sequence (UASI) and the factors required for its operation; Kovari LZ et al.; Induced production of arginase (CAR1) enzyme activity and steady-state CAR1 mRNA in Saccharomyces cerevisiae requires wild-type ARG80/ARGRI and ARG81/ARGRII gene products . We demonstrate here that these gene products, along with that of the MCM1 gene, are required for the inducer-dependent USAI-A, UASI-B and UASI-C elements to function but they are not required for operation of inducer-independent CAR1 UASC1 or UASC2 . Through the use of single and multiple point mutations, the CAR1 UASI-B and UASI-C elements were demonstrated to be at least 23 bp in length . Moreover, simultaneous mutation of both ends of an elements gave stronger phenotypes than mutations at either end . The center of the element was more sensitive to mutation than were the ends. Proc Natl Acad Sci U S A, 1993 Aug 1, 90(15), 7215 - 9 Mutations in the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2 alpha) that overcome the inhibitory effect of eIF-2 alpha phosphorylation on translation initiation; Vazquez de Aldana CR et al.; Phosphorylation of eIF-2 alpha in Saccharomyces cerevisiae by the protein kinase GCN2 leads to inhibition of general translation initiation and a specific increase in translation of GCN4 mRNA . We isolated mutations in the eIF-2 alpha structural gene that do not affect the growth rate of wild-type yeast but which suppress the toxic effects of eIF-2 alpha hyperphosphorylation catalyzed by mutationally activated forms of GCN2 . These eIF-2 alpha mutations also impair translational derepression of GCN4 in strains expressing wild-type GCN2 protein . All four mutations alter single amino acids within 40 residues of the phosphorylation site in eIF-2 alpha; however, three alleles do not decrease the level of eIF-2 alpha phosphorylation . We propose that these mutations alter the interaction between eIF-2 and its recycling factor eukaryotic translation initiation factor 2B (eIF-2B) in a way that diminishes the inhibitory effect of phosphorylated eIF-2 on the essential function of eIF-2B in translation initiation . These mutations may identify a region in eIF-2 alpha that participates directly in a physical interaction with the GCN3 subunit of eIF-2B. EMBO J, 1993 Aug, 12(8), 3061 - 71 Purification of NSP1 reveals complex formation with 'GLFG' nucleoporins and a novel nuclear pore protein NIC96; Grandi P et al.; The essential C-terminal domain of NSP1 mediates assembly into the nuclear pore complex (NPC) . To identify components which interact physically with this yeast nucleoporin, the tagged C-terminal domain of NSP1 (ProtA-NSP1) was isolated by affinity chromatography under non-denaturing conditions . The purified complex contains ProtA-NSP1, two previously identified 'GLFG' nucleoporins, NUP49 (NSP49) and p54 and a novel protein designated NIC96 (for Nucleoporin-Interacting Component of 96 kDa) . Conversely, affinity purification of tagged NSP49 enriches for NSP1, the p54 and the NIC96 component . The NIC96 gene was cloned; it encodes a novel 839 amino acid protein essential for cell growth . By immunofluorescence, protein A-tagged NIC96 exhibits a punctate nuclear membrane staining indicative of nuclear pore location . Therefore, affinity purification of tagged nucleoporins has allowed the definition of a subcomplex of the NPC and analysis of physical interactions between nuclear pore proteins. Nature, 1993 Jul 29, 364(6436), 454 - 7 Dimerization and the control of transcription by Krüppel; Sauer F et al.; Kruppel (KR), a Drosophila zinc finger-type transcription factor, can both activate and repress gene expression through interaction with a single DNA-binding site . The opposite regulatory effects of KR are concentration-dependent, and they require distinct portions of KR such as the N-terminal region for activation and the C-terminal region for repression . Here we show that KR is able to form homodimers through sequences located within the C terminus . When these sequences were fused to separated functional parts of the yeast transcription factor GAL4, they reconstituted a functional transcriptional activator on dimerization in vivo . Our results suggest that the KR monomer is a transcriptional activator . At higher concentration KR forms a homodimer and becomes a repressor that functions through the same target sequences as the activator. Biochemistry, 1993 Jul 27, 32(29), 7496 - 502 Peptide analogs of the beef heart mitochondrial F1-ATPase inhibitor protein; Stout JS et al.; Peptide analogs which correspond to the conserved region of the natural ATPase inhibitor protein from beef heart, Candida utilis, and Saccharomyces cerevisiae mitochondria were synthesized by solid-phase methodologies and tested for ATPase inhibitory activity . These peptides were found to be potent inhibitors of F1-ATPase-catalyzed ATP hydrolysis in acidic reaction media, having I50 values of 1.1 +/- 0.4 microM, 10 +/- 5 microM, and 48 +/- 19 microM, respectively . These results closely match those obtained for the naturally occurring inhibitor proteins . Additional peptides that correspond to the beef heart beta-subunit near the binding site of the beef heart inhibitor protein and that possess a substantial homology with the conserved region of the inhibitor protein were synthesized . Several of these peptides were found to be inhibitors of the ATPase activity . The best inhibitor, with an I50 value of 20 +/- 3 microM, was the peptide resembling the beef heart beta-subunit comprising amino acids 394-413 . This peptide most closely resembles the peptides derived from the conserved region of the inhibitor protein . The insertion of five glycine residues between the charge clusters in the beta-394-413 peptide resulted in a peptide which was able to stimulate the hydrolysis of ATP. FEBS Lett, 1993 Jul 26, 327(2), 165 - 71 Identification of an isoform with an extremely large Cys-rich region of PC6, a Kex2-like processing endoprotease; Nakagawa T et al.; In the previous study {1993, J . Biochem . (Tokyo) 113, 132-135} we identified PC6, a member of the Kex2 family of processing endoproteases . In this study, we identified another cDNA encoding an isoform of PC6, and designated it as PC6B and redesignated the originally identified PC6 as PC6A . PC6B had a very large Cys-rich region consisting of 22-times repeats of a Cys-rich motif, and a putative transmembrane domain which is not present in PC6A . A PC6B transcript was found mainly in the intestine, while PC6A transcripts were in various tissues . These results suggest distinct roles of PC6A and PC6B in endoproteolytic processing of precursor proteins. Biochim Biophys Acta, 1993 Jul 25, 1150(1), 89 - 97 Puromycin inhibits protein import into mitochondria by interfering with an intramitochondrial ATP-dependent reaction; Price J et al.; We have performed experiments which demonstrate that puromycin inhibits the import of proteins into mitochondria in in vitro reactions containing mitochondria isolated from the yeast Saccharomyces cerevisiae and precursor proteins synthesized in a nuclease-treated rabbit reticulocyte lysate . Puromycin inhibited the import of several precursor proteins including; a fusion protein consisting of the first 22 N-terminal residues of yeast cytochrome oxidase subunit IV fused to mouse dihydrofolate reductase, both a destabilized and truncated form of this same fusion protein, the beta-subunit of the yeast mitochondrial F1-ATPase and yeast alcohol dehydrogenase III . The insertion of the yeast outer mitochondrial protein porin was not inhibited by puromycin . Puromycin-induced import inhibition could be overcome by adding additional ATP to the import reactions . However, if access of ATP to the mitochondrial matrix was prevented by blocking the adenine nucleotide translocase with carboxyatractyloside, ATP addition was unable to overcome the inhibitory effect of puromycin on protein import . Collectively, these results demonstrate that puromycin inhibits protein import into mitochondria by interfering with an ATP-dependent step in the import process and that the ATP-dependent component in the reaction is located inside the inner mitochondrial membrane . In addition to supporting the view that ATP is required in the matrix for efficient protein import, these results may provide a useful tool for identifying the ATP-binding components of the import apparatus. Nature, 1993 Jul 22, 364(6435), 308 - 13 Normal and oncogenic p21ras proteins bind to the amino-terminal regulatory domain of c-Raf-1; Zhang XF et al.; In higher eukaryotes, the Ras and Raf-1 proto-oncoproteins transduce growth and differentiation signals initiated by tyrosine kinases . The Ras polypeptide and the amino-terminal regulatory domain of Raf-1 (residues 1-257) are shown to interact, directly in vitro and in a yeast expression system . Raf-1 (1-257) binds GTP-Ras in preference to GDP-Ras, and inhibits Ras-GAP activity . Mutations in and around the Ras effector domain impair Ras binding to Raf-1 (1-257) and Ras transforming activity in parallel. Cell, 1993 Jul 16, 74(1), 205 - 14 Mammalian Ras interacts directly with the serine/threonine kinase Raf; Vojtek AB et al.; We have identified proteins that interact with H-Ras using a two hybrid system screen of a mouse cDNA library . Approximately 50% of the clones identified encoded portions of the c-Raf and A-Raf serine/threonine kinases . Overlaps among these clones define a conserved 81 residue region of the N-terminus of Raf as the Ras interaction region . We show that Raf interacts with wild-type and activated Ras, but not with an effector domain mutant of Ras or with a dominant-interfering Ras mutant . Using purified bacterially expressed fusion proteins, we show, furthermore, that Ras and the N-terminal region of Raf associate directly in vitro and that this interaction is dependent on GTP bound to Ras. Biochem J, 1993 Jul 15, 293 ( Pt 2), 437 - 42 Activation of procathepsin B in human hepatoma cells: the conversion into the mature enzyme relies on the action of cathepsin B itself; Mach L et al.; In order to elucidate the processing mechanism of the lysosomal cysteine proteinase, cathepsin B, in mammalian cells, recombinant rat and human cathepsin B precursors were expressed in Saccharomyces cerevisiae . The active-site cysteine residue was changed to serine to prevent autoprocessing . When the purified proenzymes were incubated with the soluble fraction of postnuclear organelles obtained from human hepatoma HepG2 cells, processing to a 33 kDa form corresponding to the mature endogenous single-chain enzyme was observed . Inhibitors of metallo-, serine and aspartic proteinases exerted no significant effect on procathepsin B processing in vitro . However, the processing activity was effectively blocked by cysteine proteinase inhibitors, in particular E-64 and its cathepsin-B-selective derivative CA-074 . Processing positions were identified by using anti-peptide antibodies specific for epitopes in the N- and C-terminal cleavage regions . The single-chain form produced in vitro was thus shown to contain an N-terminal extension of at least four residues relative to the mature lysosomal enzyme, as well as a C-terminal extension present in the proenzyme but usually absent in fully processed cathepsin B . On expression of the wild-type proenzyme in yeast, procathepsin B undergoes autoprocessing, yielding a single-chain form of the active enzyme, which contains similar N- and C-terminal extensions . These results indicate that maturation of procathepsin B in vivo in mammalian tissues relies on the proteolytic activity of cathepsin B itself. Biochem J, 1993 Jul 15, 293 ( Pt 2), 475 - 9 Cytosolic and nuclear spermidine acetyltransferases in growing NIH 3T3 fibroblasts stimulated with serum or polyamines: relationship to polyamine-biosynthetic decarboxylases and histone acetyltransferase; Desiderio MA et al.; The expression (mRNA level of enzymic activity) of cytosolic and nuclear spermidine acetyltransferases was studied in NIH 3T3 fibroblasts, either (1) serum-starved and stimulated to grow by serum refeeding, or (2) treated with inhibitors of ornithine decarboxylase (ODC) (MDL 72.175) and S-adenosylmethionine decarboxylase (AdoMetDC) (MDL 73.811) and stimulated to grow by spermidine . Expression of the known growth-regulated genes for ODC, AdoMetDC and histone acetyltransferase was also examined . The mRNA for spermidine/spermine N1-acetyltransferase (SAT) accumulated after serum refeeding (between 6 and 16 h) and even more after spermidine addition (16 h) . Histone acetyltransferase activity increased after both growth stimuli, whereas spermidine N8-acetyltransferase activity remained unchanged . After serum stimulation, the ODC mRNA level and activity rose between 6 and 16 h, whereas AdoMetDC mRNA accumulation occurred later (16 h) than the increase in enzyme activity (6 h) . Stimulation of ODC and AdoMetDC activities was suppressed by the inhibitors added alone or in combination with spermidine, whereas mRNA accumulation was down-regulated by spermidine . These results indicate that the expression of SAT was growth-controlled and that SAT mRNA level was regulated by polyamines. Proc Natl Acad Sci U S A, 1993 Jul 15, 90(14), 6706 - 10 Suppression of oncogenic Ras by mutant neurofibromatosis type 1 genes with single amino acid substitutions; Nakafuku M et al.; NF1 was first identified as the gene responsible for the pathogenesis of the human genetic disorder neurofibromatosis type 1 . cDNA cloning revealed that its putative protein product has a domain showing significant sequence homology with the mammalian Ras GTPase activating protein and two yeast Saccharomyces cerevisiae proteins, Ira1 and Ira2 . The Ras GTPase activating protein-related domain of the NF1 gene product (NF1-GRD) stimulates GTPase activity of normal Ras proteins but not of oncogenic mutant Ras from both mammalian and yeast cells . Thus, in yeast, NF1-GRD can suppress the heat-shock-sensitive phenotype of ira- cells but not the same phenotype of activated RAS such as RAS2Val19 and RAS2Leu68 . We have screened a pool of mutagenized NF1 expression plasmids and obtained two mutant NF1 cDNA clones that can suppress the heat-shock-sensitive phenotype of RAS2Val19 cells . One clone (NF201) suppressed RAS2Leu68, RAS2Ser41, and RAS2Val19, whereas another clone (NF204) preferentially suppressed RAS2Val19 . When expressed in mammalian cells, these mutant NF1-GRDs were able to induce the morphological reversion of v-ras-transformed NIH 3T3 cells . Both wild-type and mutant NF1-GRDs can stimulate the GTPase activity of normal but not transforming Ras . We suggest that mutant NF1-GRDs may bind tightly to transforming Ras, which stays in GTP-bound conformation, thus preventing the interaction with the putative effector molecule . On the other hand, normal Ras cannot be sequestered since the bound GTP is rapidly hydrolyzed upon interaction with mutant NF1-GRD to yield Ras-GDP, which is readily released from the NF1-GRD and recycled. Proc Natl Acad Sci U S A, 1993 Jul 15, 90(14), 6691 - 5 cDNA structure of the mouse and rat subtilisin/kexin-like PC5: a candidate proprotein convertase expressed in endocrine and nonendocrine cells; Lusson J et al.; By using reverse transcriptase/PCR and oligonucleotide sequences derived from conserved segments (including the conserved RRGDL sequence) of the known proprotein convertases (PCs) PC1, PC2, furin, and PC4, we identified a subtilisin/kexin-like PC called PC5 in both mouse and rat tissues . The composite structure (2.85 kb) was deduced from the analysis of the reverse transcription/PCR products combined with the sequence from a clone isolated from a cDNA library made from corticotropin-activated mouse adrenocortical Y1 cells . The deduced cDNA structures of mouse PC5 and rat PC5 showed that the closest homologue is PACE4 . Furthermore, like furin, Drosophila melanogaster (d) dfurin2, and PACE4, PC5 shows the presence of a C-terminal Cys-rich domain containing either 5 (PC5 and PACE4) or 10 (dfurin2) repeats of the consensus motif Cys-Xaa2-Cys-Xaa3-Cys-Xaa(5-7)-Cys-Xaa2-Cys-Xaa (8-15)-Cys-Xaa3-Cys-Xaa(9-16) . The richest sources of rat PC5 mRNA (3.8 kb) are the adrenal and gut, but it can also be detected in many endocrine and nonendocrine tissues . Corticotropin-stimulated adrenocortical Y1 cells showed an increased expression of PC5 mRNA, suggesting an upregulation by cAMP . In situ hybridization of rat brain sections demonstrated a unique distribution of PC5 compared to PC1, PC2, and furin. J Biol Chem, 1993 Jul 15, 268(20), 14836 - 41 Properties of the SDC25 C-domain, a GDP to GTP exchange factor of RAS proteins and in vitro modulation of adenylyl cyclase; Crechet JB et al.; The SDC25 C-domain, the product encoded by the 3'-terminal part of the Saccharomyces cerevisiae SDC25 gene, acts as a GDP dissociation stimulator on RAS proteins (Crechet, J.B., Poullet, P., Mistou, M . Y., Parmeggiani, A., Camonis, J., Boy-Marcotte, E., Damak, F., and Jacquet, M . (1990b) Science 248, 866-868) . To define further its role in the RAS-adenylyl cyclase pathway, an in vitro system was used, which utilized cell membranes from yeast strains with appropriate genotypes carrying alterations in the positive regulators of adenylyl cyclase activity . The SDC25 C-domain was able to stimulate the adenylyl cyclase activity of membranes from RAS2 cdc25 strains . Our results indicate that the SDC25 C-domain activates adenylyl cyclase by rapidly recycling the active RAS2 . or RAS1.GTP complex from the respective GDP complex . This is also supported by the observation that the stimulation of adenylyl cyclase activity by RAS2T152I, a mutant characterized by a constitutively fast GDP to GTP exchange, was insensitive to the action of the SDC25 C-domain . No direct influence of this GDP dissociation stimulator on adenylyl cyclase was detected . Biochemical evidence was obtained, showing that in the presence of the functional target of RAS, the adenylyl cyclase, the effects of SDC25 C-domain and the catalytic domain of GTPase-activating protein are antagonistic . This in vitro system allowed a quantitative evaluation of the effects of positive and negative effectors of RAS on adenylyl cyclase and the biochemical analysis of conditions inducing a phenotype of permanently activated adenylyl cyclase. Proc Natl Acad Sci U S A, 1993 Jul 15, 90(14), 6508 - 12 Expression and localization of two low molecular weight GTP-binding proteins, Rab8 and Rab10, by epitope tag; Chen YT et al.; Small GTP-binding proteins of the YPT/SEC4/Rab family have been shown to play an essential role in intracellular membrane trafficking . In mammals, Rab8 and Rab10 are the two small GTP-binding proteins identified so far that are closest to SEC4, an essential gene product involved in post-Golgi constitutive secretion in the yeast Saccharomyces cerevisiae . To study the localization of Rab proteins, we have expressed the cDNAs with an influenza virus hemagglutinin (HA) epitope tag at the N terminus . The feasibility of this method was tested by using yeast SEC4 . HA-tagged SEC4 functionally complemented a temperature-sensitive sec4 mutant similarly to wild-type SEC4, indicating that the modified protein retained functional integrity . Monoclonal antibody 12CA5, raised against the HA tag, was used to determine the expression and localization of HA-tagged proteins after transfection . In stably transfected CHO and Swiss 3T3 cells, HA-tagged Rab8 was localized to the cell periphery, with the highest concentration in the ruffling areas . In contrast, epitope-tagged Rab10 expressed in CHO and BHK cells was concentrated on membranes in the perinuclear region . By light microscopy, the staining partially overlapped with that of a Golgi marker, beta-COP . Thus, despite the high degree homology of Rab8 and Rab10 (66% identity), the two proteins are localized to distinct cellular compartments . This approach should provide a general tool for the analyses of other members of the YPT/SEC4/rab gene family. FEBS Lett, 1993 Jul 12, 326(1-3), 145 - 8 Crystal structure of transketolase in complex with thiamine thiazolone diphosphate, an analogue of the reaction intermediate, at 2.3 A resolution; Nilsson U et al.; The crystal structure of the complex of transketolase and thiamine thiazolone diphosphate has been determined at 2.3 A resolution . The complex has a structure which closely resembles that of this enzyme with the cofactor ThDP . This is consistent with the observation that the binding of the analogue to transketolase involves ground state rather than transition state interactions . Since thiamine thiazolone diphosphate resembles an expected intermediate in the catalytic pathway, the structure of the intermediate was modelled from the crystal structure . Based on this model, enzymic groups responsible for binding of the intermediate and proton transfer during catalysis are suggested. Nucleic Acids Res, 1993 Jul 11, 21(14), 3257 - 63 Interactions of USF and Ku antigen with a human DNA region containing a replication origin; Toth EC et al.; By means of a combination of ion-exchange and sequence-specific affinity chromatography techniques, we have purified to homogeneity two protein complexes binding in a human DNA region (B48) previously recognized to contain a DNA replication origin . The DNA sequence used for the protein purification (B48 binding site) contains a binding site for basic-helix-loop-helix DNA binding proteins . The first complex is composed of two polypeptides of 42- and 44-kDa; its size, heat stability, and target DNA sequence suggest that it corresponds to transcription factor USF; furthermore, the 42-kDa polypeptide is recognized by antibodies raised against 43-kDa-USF . The second complex is represented by equimolar amounts of two proteins of 72 and 87 kDa; microsequencing of the two species indicated that they correspond to the human Ku antigen . In analogy with Ku, they produce a regular pattern of footprints without an apparent sequence-specificity, and their binding can be competed by unspecific DNA provided that it contains free ends . The potential role of B48 binding site and of these cognate proteins in origin activation is discussed. Science, 1993 Jul 9, 261(5118), 206 - 8 An NAD derivative produced during transfer RNA splicing: ADP-ribose 1"-2" cyclic phosphate; Culver GM et al.; Transfer RNA (tRNA) splicing is essential in Saccharomyces cerevisiae as well as in humans, and many of its features are the same in both . In yeast, the final step of this process is removal of the 2' phosphate generated at the splice junction during ligation . A nicotinamide adenine dinucleotide (NAD)-dependent phosphotransferase catalyzes removal of the 2' phosphate and produces a small molecule . It is shown here that this small molecule is an NAD derivative: adenosine diphosphate (ADP)-ribose 1"-2" cyclic phosphate . Evidence is also presented that this molecule is produced in Xenopus laevis oocytes as a result of dephosphorylation of ligated tRNA. Biochemistry, 1993 Jul 6, 32(26), 6794 - 801 Oligosaccharyltransferase: synthesis and use of deuterium-labeled peptide substrates as mechanistic probes; Lee J et al.; Chemically synthesized peptide and lipid disaccharide substrates have been used to investigate two possible mechanisms for enzyme-catalyzed N-glycosylation . Using microsomal oligosaccharyltransferase isolated from yeast, the fate of the deuterium in three stereospecifically deuterated peptides has been investigated . In all three cases, the deuterium present in the peptide substrate was retained in the glycopeptide product, as shown clearly by 1H NMR spectral comparisons . The lack of deuterium wash-out during catalysis provides strong evidence against either enol lactone or ketene formation as an intermediate in this reaction. J Mol Biol, 1993 Jul 5, 232(1), 79 - 88 MSS1, a nuclear-encoded mitochondrial GTPase involved in the expression of COX1 subunit of cytochrome c oxidase; Decoster E et al.; Mutations in the MSS1 gene render the yeast cells respiratory deficient only in the presence of the PR454 mutation (paromomycin resistance) in the mitochondrial 15 S ribosomal RNA gene . The MSS1 gene product works in association with the small subunit of mitoribosomes and seems to play some part in mitochondrial translation . The block in the splicing of introns of cytochrome c oxidase subunit 1 could result from a specific impeding of the translation of maturases . Comparison of the MSS1 putative protein with data libraries revealed that it contains, in its second half, the consensus sequences characteristic of GTP binding proteins and is very homologous to the bacterial "genes 50K". J Biol Chem, 1993 Jul 5, 268(19), 14250 - 5 On the simultaneous binding of eukaryotic DNA topoisomerase II to a pair of double-stranded DNA helices; Roca J et al.; Stabilization of crossings of pairs of DNA helices by binding of eukaryotic DNA topoisomerase II was studied by two types of experiments . In one, mixtures of yeast DNA topoisomerase II and supercoiled DNA were incubated with vaccinia virus topoisomerase, and the linking numbers of the DNA products were measured to quantitate supercoils that were constrained by the stoichiometrically bound yeast enzyme molecules; in parallel, the same yeast enzyme-supercoiled DNA mixtures were incubated with a nonhydrolyzable ATP analog AMPPNP (adenosine 5'-(beta, gamma-imido)triphosphate) instead of the vaccinia enzyme, and DNA linking number changes following the addition of AMPPNP were measured to monitor DNA transport mediated by the yeast enzyme and AMPPNP . In the second type of experiments, formation of knotted DNA rings by the addition of AMPPNP to mixtures of yeast DNA topoisomerase II and different topological forms of DNA rings was studied . These experiments indicate that binding of yeast DNA topoisomerase II to DNA crossings is significant, especially in low salt media containing Mg(II), and that this mode of binding strongly affects DNA knotting . It appears, however, that stabilization of DNA crossovers by the eukaryotic type II enzyme is not directly related to its DNA transport activity. J Biol Chem, 1993 Jul 5, 268(19), 14417 - 25 Tests for the fractional active-site model in Flp site-specific recombination . Assembly of a functional recombination complex in half-site and full-site strand transfer; Chen JW et al.; The Arg191-His305-Arg308 (the RHR triad) and Tyr343 of Flp site-specific recombinase correspond to the invariant tetrad residues of the integrase family of proteins . Flp mutants altered at these positions are blocked at the strand cleavage or the strand exchange step of recombination . Hybrid half-site-recombinase complexes formed by step-arrest mutants of Flp have revealed that an Flp monomer occupying a half-site does not cleave that half-site but rather cleaves a half-site occupied by a second Flp monomer . This trans-DNA cleavage is neatly accommodated by a model in which an Flp active site is assembled by contribution of amino acid residues from at least two protein monomers . Using a combination of wild type Flp, single, double, and triple step-arrest Flp mutants, critical predictions of the fractional active-site model have been verified . First, a wild type monomer paired with an RHR triad-Tyr343 double mutant is a catalytically inactive combination . Second, each pairwise combination of a single, double, or triple RHR mutant with Flp (Y343F) yields approximately equivalent levels of catalytic complementation . Half-site to half-site and half-site to full-site crosses suggest that execution of a strand transfer event within a half-site and between a half-site and a full site requires dimeric and tetrameric Flp configurations, respectively. J Biol Chem, 1993 Jul 5, 268(19), 14026 - 32 Powerful dominant negative mutants of the human estrogen receptor; Ince BA et al.; We have identified and characterized three human estrogen receptor (ER) mutants, which, at low concentrations, are capable of blocking the intracellular activity of wild type ER . The mutants, a truncated ER (ER1-530), a point mutant (L540Q), and a frameshift (S554fs), were generated by random chemical mutagenesis of the ER hormone binding domain and screened first for low activity in a yeast selection system . In transient co-transfection assays using ER-deficient Chinese hamster ovary cells, all three mutants exhibited less than 10% of the transcription activation activity of wild type ER, and when co-expressed with wild type ER, each of the mutants effectively suppressed the ability of wild type ER to activate transcription of an estrogen-regulated reporter plasmid . When equal amounts of plasmid encoding the ER mutants and wild type ER were used, S554fs, ER1-530, and L540Q suppressed the activity of wild type ER by 80, 55, and 75%, respectively . At a ratio of 1 part S554fs to 10 parts wild type ER, transcription was still inhibited by 40% . Western blot analysis showed that all three mutants were expressed at approximately the same level as wild type ER . Suppression of transcription was specific for ER, since the mutants did not inhibit progesterone receptor-mediated transcription . Not all mutations leading to inactive ER confer the dominant negative phenotype, as five ER mutants rendered transcriptionally inactive by point mutations between residues 516 and 524 of the ER hormone binding domain were poor inhibitors of wild type ER activity . Binding studies showed that the L540Q and S554fs dominant negative mutants bound 17 beta-estradiol with wild type affinity (Kd = 0.3-0.5 nM), whereas ER1-530 exhibited a 15-fold reduction in affinity for estradiol . The three dominant negative ERs showed significant ability to interact with the estrogen response element (ERE) in promoter interference assays, but ER1-530 and S554fs displayed little or no binding to the ERE in gel mobility shift assays where higher affinity for the DNA may be required for the receptor-ERE complex to remain associated during the electrophoresis . These data support the idea that, in all three mutants, it is loss of function of the COOH-terminal transactivation domain which leads to the dominant negative phenotype . S554fs, a powerful dominant negative mutant, is a good candidate for further studies aimed at suppressing the estrogen-dependent growth of human breast cancer cells. J Biol Chem, 1993 Jul 5, 268(19), 13885 - 92 The acquisition of lysophosphatidylcholine by African trypanosomes; Bowes AE et al.; Bloodstream forms of the African trypanosome, Trypanosoma brucei, can acquire substantial amounts of exogenous lysophospholipid . Lysophosphatidylcholine uptake is through a pathway consisting of three enzymes, phospholipase A1, acyl-CoA ligase, and lysophosphatidylcholine:acyl-CoA acyltransferase . The pathway enables the organism to acquire fatty acids and phospholipid head groups such as choline . Radiolabeling and 13C NMR studies show that two molecules of lysophosphatidylcholine are used to generate one molecule of cellular phosphatidylcholine . The three enzymes are associated with the trypanosomal plasma membrane and are accessible to exogenous substrates . The first enzyme, phospholipase A1, generates free fatty acid from exogenous lysophospholipid, which the second enzyme, a ligase, uses to form acyl-CoA . The fatty acyl-CoA formed by this route is in a separate pool from that derived from exogenous free fatty acid and is used by the third enzyme, acyltransferase, to acylate a second molecule of exogenous lysophospholipid . Acyltransferase is accessible to exogenous and endogenous acyl-CoA . The high activity of this pathway in bloodstream forms, compared with procyclic culture form trypanosomes, suggests that it may play a role in the acquisition of fatty acids for synthesis of the membrane form of the variant surface glycoprotein . Extracellular myristoyllysophosphatidylcholine can be used by trypanosomes as a source of myristate in remodeling the lipid anchor of the variant surface glycoprotein. Mol Endocrinol, 1993 Jul, 7(7), 833 - 9 Positive regulation of the vitamin D receptor by its cognate ligand in heterologous expression systems; Santiso-Mere D et al.; Hormonal vitamin D3 is a major regulator of calcium metabolism and is involved in basic cellular processes, such as those of proliferation and differentiation . These actions are mediated via an intracellular vitamin D3 receptor (VDR), which is a member of the evergrowing steroid hormone receptor superfamily . The interaction between the vitamin D3 ligand and its receptor is thought to be through a classic steroid hormone mechanism . It is notable, however, that 1,25-dihydroxyvitamin D3 {1,25-(OH)2D3} has also been documented as an agent that directly up-regulates endogenous VDR in both intact animals and cultured cells . In this report, we confirm that the levels of recombinantly expressed VDR produced in transiently transfected COS-1 cells also increase several-fold when the cells are treated with 1,25-(OH)2D3 . Additionally, we show that a similar pattern is exhibited in a Saccharomyces cerevisiae expression system . This indicates that the mechanism for VDR up-regulation is conserved in both yeast and mammalian cells . Our results show that up-regulation by 1,25-(OH)2D3 is specific to the VDR, and that 1,25-(OH)2D3 does not affect other expressed receptor proteins, such as those for estrogen and progesterone . Finally, we demonstrate that the mechanism of up-regulation apparently occurs at the level of the protein and is most likely due to altered stability of the occupied receptor . Our observations lead us to propose that in addition to the classically viewed role of hormone in receptor activation, 1,25-(OH)2D3 may serve to amplify signal response via homologous up-regulation. Anal Biochem, 1993 Jul, 212(1), 194 - 205 Determination of endopolyphosphatase using polyphosphate glucokinase; Kowalczyk TH et al.; A method has been developed for determining endopolyphosphatase (polyphosphate depolymerase, EC 3.6.1.10) activity . The enzyme catalyzes the hydrolysis of inorganic polyphosphates {poly(Ps)} by cleaving internal phosphoanhydride bonds without removal of terminal phosphate residues . During the reaction, shorter poly(P) chains are formed and the molar concentration of poly(P) increases . This enzymatic activity is difficult to quantitate, because the substrates and products of the reaction are chemically identical . The commonly used viscometric method lacks sensitivity and cannot be used with shorter poly(P) substrates . The method described here overcomes these problems, and in addition is rapid, simple, and can be used for distinguishing between the endopolyphosphatase and exopolyphosphatase (EC 3.6.1.11) activities . It is based on monitoring the increase in the number of poly(P) chains generated by endopolyphosphatase . For this purpose, the method takes advantage of the specific property of poly(P) glucokinase (EC 2.7.1.63) which utilizes poly(Ps) of different sizes present in the endopolyphosphatase reaction mixture and reduces them to fairly uniform very short-chain product, poly(P)m . The concentration of poly(P)m is expressed in terms of acid-labile phosphorus and is proportional to the duration of the endopolyphosphatase reaction (i.e., the number of original poly(P) chains) and to protein concentration . The increase in the poly(P)m concentration is a relative measure of the endopolyphosphatase activity . Under certain conditions, m equals 3.5 and the activity can be expressed in standard units, since the exact number of poly(P) chains formed by endopolyphosphatase can be calculated from the increase in molar concentration of poly(P)3.5 . Accuracy and advantages of the assay are discussed. Arch Biochem Biophys, 1993 Jul, 304(1), 242 - 7 The structure of the MnIIADP-nitrate-lyxose complex at the active site of hexokinase; Olsen LR et al.; The coordination scheme of Mn2+ in the hexokinase-MnIIADP-nitrate-lyxose complex has been determined by electron paramagnetic resonance (EPR) spectroscopy with 17O-enriched ligands . Nitrate binds to the active site of hexokinase when MnIIADP and a sugar substrate or analogue are present . The binding of nitrate enhances inhibition by glucose when ADP is present and narrows the EPR signals of the enzyme-bound MnIIADP complex in the presence of sugar substrates or analogues . Experiments using regiospecifically 17O-enriched ADP, 17O-enriched nitrate, and 17O-enriched water establish the coordination scheme of Mn2+ . The EPR experiments show that ADP is a beta-monodentate ligand and that nitrate binds directly to Mn2+ . Four water molecules complete the coordination sphere of the enzyme-bound Mn2+ . The dissociation constant (Kd approximately 8 mM) of nitrate for the complex with enzyme, MnIIADP, and lyxose was obtained from titration experiments . These results suggest that nitrate-stabilized, dead-end complexes of hexokinase may be useful in stabilizing the closed conformation of this "hinge-bending" enzyme for crystallographic experiments. Anal Biochem, 1993 Jul, 212(1), 150 - 3 A dependable method for the synthesis of {14C}trehalose; Stambuk BU et al.; A new method for the preparation of {14C}trehalose was developed, based on the ability of yeast cells to accumulate trehalose under stress . The method is simple and reliable . It utilizes a yeast strain in which the gene that encodes for phosphoglucoisomerase has been deleted . Thus, exogenously supplied glucose is not metabolized, but is instead converted to trehalose . The {14C}-trehalose obtained is pure, it is hydrolyzed by trehalase, and it is not susceptible to the action of alpha-glucosidase . The yield of this method is in the order of 35% of the {14C}glucose supplied. Am J Physiol, 1993 Jul, 265(1 Pt 1), E44 - 50 Marked reduction of acyl-CoA synthetase activity and mRNA in intra-abdominal visceral fat by physical exercise; Shimomura I et al.; Several reports have suggested that the reduction of intra-abdominal visceral fat after physical exercise is more prominent than that of subcutaneous fat . We compared some parameters in mesenteric and subcutaneous fats between sedentary and exercised rats (treadmill running; 10-20 m/min, 60 min/day, 7 days) . Tissue weight and cell volume were decreased in mesenteric fat by the exercise . The exercise reduced activity and mRNA levels of acyl-CoA synthetase (ACS; 67 and 26% of those of the sedentary group, respectively), mRNA levels of lipoprotein lipase (LPL; 49% of those of the sedentary group), and GLUT-4 (38% of those of the sedentary group) in the mesenteric fat . In contrast, all of these parameters did not change significantly in the subcutaneous fat . Gastrocnemius muscle was heavier in exercised rats . ACS activity was elevated in the gastrocnemius muscle of the exercised rats (137% of those of sedentary group), although mRNA levels of ACS, LPL, and GLUT-4 did not change in the muscle by the exercise . These observations suggest that mesenteric fat may contribute to switching of distribution of plasma energy flux, including lipid and glucose, from fat tissue to muscle in physical exercise. EMBO J, 1993 Jul, 12(7), 2949 - 57 Additive, cooperative and anti-cooperative effects between identity nucleotides of a tRNA; Putz J et al.; We have investigated the functional relationship between nucleotides in yeast tRNAAsp that are important for aspartylation by yeast aspartyl-tRNA synthetase . Transcripts of tRNAAsp with two or more mutations at identity positions G73, G34, U35, C36 and base pair G10-U25 have been prepared and the steady-state kinetics of their aspartylation were measured . Multiple mutations affect the catalytic activities of the synthetase mainly at the level of the catalytic constant, kcat . Kinetic data were expressed as free energy variation at transition state of these multiple mutants and comparison of experimental values with those calculated from results on single mutants defined three types of relationships between the identity nucleotides of this tRNA . Nucleotides located far apart in the three-dimensional structure of the tRNA act cooperatively whereas nucleotides of the anticodon triplet act either additively or anti-cooperatively . These results are related to the specific interactions of functional groups on identity nucleotides with amino acids in the protein as revealed by the crystal structure of the tRNAAsp/aspartyl-tRNA synthetase complex . These relationships between identity nucleotides may play an important role in the biological function of tRNAs. Genes Dev, 1993 Jul, 7(7B), 1390 - 9 Interactions between PRP9 and SPP91 splicing factors identify a protein complex required in prespliceosome assembly; Legrain P et al.; The PRP9 protein is a yeast splicing factor implicated in the early steps of spliceosome assembly whose sequence contains an amino-terminal putative leucine zipper structure and two carboxy-terminal motifs reminiscent of zinc fingers . Here, we show that the deletion of the second carboxy-terminal motif results in a dominant lethal phenotype . This observation, combined with an in vivo-binding assay for protein-protein interactions, reveals the presence of two distinct binding sites on the PRP9 protein . The carboxy-terminal region contributes to the PRP9 homodimerization, whereas the amino-terminal region binds the SPP91 splicing factor . Further experiments suggest that other factors bind to PRP9 and SPP91 proteins . Finally, we demonstrate that the PRP9 protein acts after the formation of the U1 snRNP-pre-mRNA complex . The existence of a protein complex including the PRP9 factor is discussed. Genes Dev, 1993 Jul, 7(7B), 1341 - 53 Drosophila tissue-specific transcription factor NTF-1 contains a novel isoleucine-rich activation motif; Attardi LD et al.; The Drosophila tissue-specific transcription factor NTF-1 provides a useful model system for studying the mechanisms by which promoter-selective factors control the development of a multicellular organism . A number of promoters that may be targets of NTF-1 regulation have been identified . For example, NTF-1 plays a critical role in the tissue-specific expression of the Drosophila Dopa decarboxylase gene . Additionally, by using in vitro assays, it has been possible to characterize the mechanism of NTF-1 activation, revealing its dependence on specific coactivators, or TAFs . Here, we report the use of both in vivo and in vitro assays to identify the functional domains of NTF-1 . These consist of an unusually large, unique DNA-binding and dimerization domain, as well as a novel, isoleucine-rich activation domain . This 56-amino-acid activation region fails to interact with the putative Sp1 coactivator, dTAFII110, and thus appears to use a mechanism distinct from the glutamine-rich activation domain of Sp1 . Additionally, NTF-1 appears to activate transcription in a species-specific manner, utilizing distinct domains in Drosophila and yeast. Proc Natl Acad Sci U S A, 1993 Jul 1, 90(13), 6018 - 22 Phylogenetic footprinting reveals unexpected complexity in trans factor binding upstream from the epsilon-globin gene; Gumucio DL et al.; The human epsilon-globin gene undergoes dramatic changes in transcriptional activity during development, but the molecular factors that control its high expression in the embryo and its complete repression at 6-8 weeks of gestation are unknown . Although a putative silencer has been identified, the action of this silencer appears to be necessary but not sufficient for complete repression of epsilon gene expression, suggesting that multiple control elements may be required . Phylogenetic footprinting is a strategy that uses evolution to aid in the elucidation of these multiple control points . The strategy is based on the observation that the characteristic developmental expression pattern of the epsilon gene is conserved in all placental mammals . By aligning epsilon genomic sequences (from -2.0 kb upstream to the epsilon polyadenylylation signal), conserved sequence elements that are likely binding sites for trans factors can be identified against the background of neutral DNA . Twenty-one such conserved elements (phylogenetic footprints) were found upstream of the epsilon gene . Oligonucleotides spanning these conserved elements were used in a gel-shift assay to reveal 47 nuclear binding sites . Among these were 8 binding sites for YY1 (yin and yang 1), a protein with dual (activator or repressor) activity; 5 binding sites for the putative stage selector protein, SSP; and 7 binding sites for an as yet unidentified protein . The large number of high-affinity interactions detected in this analysis further supports the notion that the epsilon gene is regulated by multiple redundant elements. Mol Cell Biol, 1993 Jul, 13(7), 4039 - 48 SKN1 and KRE6 define a pair of functional homologs encoding putative membrane proteins involved in beta-glucan synthesis; Roemer T et al.; KRE6 encodes a predicted type II membrane protein which, when disrupted, results in a slowly growing, killer toxin-resistant mutant possessing half the normal level of a structurally wild-type cell wall (1-->6)-beta-glucan (T . Roemer and H . Bussey, Proc . Natl . Acad . Sci . USA 88:11295-11299, 1991) . The mutant phenotype and structure of the KRE6 gene product, Kre6p, suggest that it may be a beta-glucan synthase component, implying that (1-->6)-beta-glucan synthesis in Saccharomyces cerevisiae is functionally redundant . To examine this possibility, we screened a multicopy genomic library for suppression of both the slow-growth and killer resistance phenotypes of a kre6 mutant and identified SKN1, which encodes a protein sharing 66% overall identity to Kre6p . SKN1 suppresses kre6 null alleles in a dose-dependent manner, though disruption of the SKN1 locus has no effect on killer sensitivity, growth, or (1-->6)-beta-glucan levels . skn1 kre6 double disruptants, however, showed a dramatic reduction in both (1-->6)-beta-glucan levels and growth rate compared with either single disruptant . Moreover, the residual (1-->6)-beta-glucan polymer in skn1 kre6 double mutants is smaller in size and altered in structure . Since single disruptions of these genes lead to structurally wild-type (1-->6)-beta-glucan polymers, Kre6p and Skn1p appear to function independently, possibly in parallel, in (1-->6)-beta-glucan biosynthesis. J Cell Biol, 1993 Jul, 122(2), 461 - 71 A cell cycle and mutational analysis of anchorage-independent growth: cell adhesion and TGF-beta 1 control G1/S transit specifically; Han EK et al.; We have examined cell cycle control of anchorage-independent growth in nontransformed fibroblasts . In previous studies using G0-synchronized NRK and NIH-3T3 cells, we showed that anchorage-independent growth is regulated by an attachment-dependent transition at G1/S that resembles the START control point in the cell cycle of Saccharomyces cerevisiae . In the studies reported here, we have synchronized NRK and NIH-3T3 fibroblasts immediately after this attachment-dependent transition to determine if other portions of the fibroblast cell cycle are similarly regulated by adhesion . Our results show that S-, G2-, and M-phase progression proceed in the absence of attachment . Thus, we conclude that the adhesion requirement for proliferation of these cells can be explained in terms of the single START-like transition . In related studies, we show that TGF-beta 1 overrides the attachment-dependent transition in NRK and AKR-2B fibroblasts (lines in which TGF-beta 1 induces anchorage-independent growth), but not in NIH-3T3 or Balb/c 3T3 fibroblasts (lines in which TGF-beta 1 fails to induce anchorage-independent growth) . These results show that (a) adhesion and TGF-beta 1 can have similar effects in stimulating cell cycle progression from G1 to S and (b) the differential effects of TGF-beta 1 on anchorage-independent growth of various fibroblast lines are directly reflected in the differential effects of the growth factor at G1/S . Finally, we have randomly mutagenized NRK fibroblasts to generate mutant lines that have lost their attachment/TGF-beta 1 requirement for G1/S transit while retaining their normal mitogen requirements for proliferation . These clones, which readily proliferate in mitogen-supplemented soft agar, appear non-transformed in monolayer: they are well spread, nonrefractile, and contact inhibited . The existence of this new fibroblast phenotype demonstrates (a) that the growth factor and adhesion/TGF-beta 1 requirements for cell cycle progression are genetically separable, (b) that the two major control points in the fibroblast cell cycle (G0/G1 and G1/S) are regulated by distinct extracellular signals, and (c) that the genes regulating anchorage-independent growth need not be involved in regulating contact inhibition, focus formation, or growth factor dependence. Genes Dev, 1993 Jul, 7(7A), 1133 - 45 Silent domains are assembled continuously from the telomere and are defined by promoter distance and strength, and by SIR3 dosage; Renauld H et al.; In Saccharomyces cerevisiae, telomeres repress transcription of genes located nearby . This region-specific gene inactivation is thought to involve the packaging of telomeric domains into silent chromatin . To gain insight into the mechanism of telomeric silencing, a genetic assay to examine the spread of silencing along the distal right arm of chromosome V was developed . The frequency of silencing a telomere-adjacent URA3 gene decreased with increasing distance of the gene's promoter from the telomere, irrespective of transcriptional orientation . The distance over which telomeric silencing of URA3 was observed was extended by weakening the gene's promoter--specifically, by deleting PPR1, the trans-activator of URA3 . The silent telomeric domain was extended even farther by increasing the gene dosage of SIR3 . These results suggest that a gene's promoter is a key determinant in controlling silencing on that gene and that SIR3 is a crucial component of the silent chromatin domain that initiates at the telomere and is assembled inwardly along the yeast chromosome . Finally, silencing is not observed on the centromeric side of an actively transcribed telomeric gene, demonstrating that the repressed telomeric domain is propagated continuously along the DNA . Taken together, these data reflect the complex and dynamic organization of eukaryotic genomes into functionally distinct regions. Dev Dyn, 1993 Jul, 197(3), 227 - 37 The Etl-1 gene encodes a nuclear protein differentially expressed during early mouse development; Schoor M et al.; Recently, we isolated a novel mouse gene, Etl-1 (Enhancer-trap-locus-1), whose deduced amino acid sequence shows in its C-terminal portion striking homology to the brahma protein (BRM), a transcriptional regulator of homeotic genes in Drosophila, and to SNF2/SWI2, a transcriptional regulator of various genes in Saccharomyces cerevisiae . Here we report the generation of antibodies against the Etl-1 gene product (ETL-1) and describe the subcellular localization as well as the expression and distribution of the ETL-1 protein during mouse pre- and early post-implantation development . ETL-1 is a nuclear protein and is expressed in a biphasic manner during early embryogenesis . Moderate levels of ETL-1 were detected in unfertilized and fertilized eggs but in the latter the protein was not concentrated in the pronuclei and seemed evenly distributed throughout the cytoplasm . In two-cell embryos nuclear ETL-1 protein accumulated transiently and levels decreased during subsequent cleavage development . After the morula stage, ETL-1 levels increased again; in blastocysts high levels of ETL-1 were present in inner cell mass cells whereas trophectoderm cells contained little or no ETL-1 . During subsequent development essentially all cell types except parietal endoderm and trophoblast cells contained high levels of ETL-1 . Our results imply that nuclear ETL-1 is dispensable for the progression to the two cell stage, and suggest that during cleavage ETL-1 might be needed at the onset of embryonic transcription . In blastocysts ETL-1 function might be specifically required in cells of the inner cell mass and later in most cells of the embryo proper and extraembryonic ectoderm lineage. Am J Vet Res, 1993 Jul, 54(7), 1055 - 9 Comparison of the chemiluminescence responses of bovine neutrophils to differently opsonized zymosan particles; Leino L et al.; Stimulatory effects of 6 zymosan preparations on luminol-dependent chemiluminescence (CL) responses of isolated bovine neutrophils were compared . Unopsonized zymosan particles and zymosan particles opsonized with bovine IgG1, IgG2, fresh serum, or serum from which zymosan-specific antibodies, but not complement, had been removed (C3-serum) induced strong CL responses, with nearly equal maximal peaks in the presence of extracellular Ca2+ and Mg2+, whereas the response to fetal bovine serum-opsonized zymosan particles was markedly low . Removal of extracellular divalent cations almost completely blocked the CL reaction triggered by unopsonized, IgG1-opsonized, C3-opsonized, and fetal bovine serum-opsonized zymosan particles . By contrast, no change in the respiratory burst activity induced by serum-opsonized zymosan and only partial reduction in the response to IgG2-opsonized zymosan were seen under these conditions . Further experiments were performed with 4 zymosan preparations on neutrophils isolated from 2 calves with a genetic deficiency of CD11/CD18 membrane antigens . The unopsonized zymosan-induced CL reaction was absent in these cells . A reduced, but clear, response was observed with C3-opsonized zymosan . Unexpectedly, in the absence of extracellular Ca2+ and Mg2+, serum-opsonized zymosan failed to generate the respiratory burst, whereas response to IgG2-opsonized zymosan was normal in the CD11/CD18-deficient neutrophils . These findings indicate that unopsonized zymosan may act in a divalent cation-dependent manner at the receptor for C3bi in bovine neutrophils, as it has been shown to do in the human system . In addition, it seems that IgG2-Fc receptors capable of signaling the respiratory burst in the absence of extracellular Ca2+ and Mg2+ exist on bovine neutrophils. Curr Genet, 1993 Jul-Aug, 24(1-2), 7 - 11 Relationship between chromosomal alpha-specific suppressors of sir4-11 and polymorphism of the HMRa-bearing fragment; Ono B et al.; In the course of studying sir4-11 which is responsible for the non-mating phenotype of asd-homothallism of Saccharomyces cerevisiae, we detected chromosomal alpha-specific suppressors of it . By examining the mating-type cassette constitution of two strains and the spore-clones derived from a diploid culture formed by a mass mating between these strains, we obtained the following results; (1) the HMRa-bearing HindIII-Bg/II restriction fragment was dimorphic, as judged by size, within each tetrad, (2) while one form was common to all tetrads, the other varied among tetrads, (3) spore-clones with the alpha-specific suppressor of sir4-11 had the variant forms while those without had the common form, and (4) while the two parents had the common form, each independent diploid clone had the common form plus a variant form . From these results, we conclude that the mating of cells in certain combinations induces a change of DNA structure at or near HMRa in a mating-pair specific manner and that the change makes HMRa non-derepressible or non-functional when derepressed. J Virol Methods, 1993 Jul, 43(2), 221 - 32 Use of recombinant gp135 to study epitope-specific antibody responses to maedi visna virus; Carey N et al.; The envelope glycoprotein gp135 of the ovine lentivirus maedi visna virus (MVV) is the main target for neutralising antibody in vivo, however little is known about the specific regions of gp135 which elicit this neutralising response . We have used the polymerase chain reaction (PCR) to generate overlapping fragments of the gp135 gene which have been expressed as fusion proteins in the yeast Ty-VLP system . These fusion proteins have been used to analyse the antibody response to gp135 in MVV infected sheep and we are able to identify at least three distinct regions of gp135 to which antibodies are directed . The approach described in this paper provides a rapid and simple method of generating overlapping fusion proteins with which to carry out epitope mapping studies. Biochem Biophys Res Commun, 1993 Jun 30, 193(3), 864 - 71 Type 2 vasopressin receptor gene, the gene responsible nephrogenic diabetes insipidus, maps to Xq28 close to the LICAM gene; Frattini A et al.; Although long contigs have been assembled in Xq28, the interval between the anonymous probe St14 and the color vision locus is still incompletely defined . We report here that the recently cloned gene for type 2 vasopressin receptor (V2R) is physically linked to L1CAM using YACs and cosmids across about 180 kb of the region . Since it is known that L1CAM maps near the color pigment genes, this finding locates V2R in Xq28 in the area where nephrogenic diabetes insipidus (NDI) has been mapped by linkage analysis . The PFGE analysis of the clones positions V2R about 40 kb from the L1CAM gene in a region that appears to contain other unknown genes, since at least four putative CpG islands were identified by restriction analysis with rare cutter enzymes. Philos Trans R Soc Lond B Biol Sci, 1993 Jun 29, 340(1293), 325 - 32 Isolation and characterization of SRF accessory proteins; Dalton S et al.; Many genes which are regulated by growth factors contain a common regulatory element, the serum response element (SRE) . Activation of transcription by the SRE involves a ternary complex formed between a ubiquitous factor, serum response factor (SRF), and a second protein, p62/TCF . We used a yeast genetic screen to isolate cDNAs encoding a protein, SAP-1, with the DNA binding properties of p62/TCF . The SAP-1 sequence contains three regions of homology to the previously uncharacterized Elk-1 protein, which also acts as an SRF accessory protein . Only two of these regions are required for cooperative interactions with SRF in the ternary complex . The third contains several conserved sites for the MAP kinases, whose activity is regulated in response to growth factor stimulation . We discuss the potential role of these proteins in regulation of the c-fos SRE. FEBS Lett, 1993 Jun 28, 325(1-2), 76 - 80 The nuclear pore complex; Hurt EC; Over the past years, significant progress has been made both in the analysis of the structural and molecular organization of the nuclear pore complex (NPC) and the mechanism of nuclear transport . In this minireview, I will focus on some of the recent developments in this field . Structural studies employing high resolution EM have revealed a detailed view of the three-dimensional organization of the NPC . In addition, an isolation procedure which yields highly enriched NPCs from yeast has given insight into the molecular complexity of the NPC organization . By biochemical, immunological and genetic approaches, a series of novel pore proteins were identified . Exploiting yeast as a genetic system, several mutants defective in nuclear import of proteins and export of RNA were selected . By in vitro nuclear transport assays, soluble cytoplasmic factors including NLS (nuclear localization sequence) binding proteins and heat shock proteins required for nuclear accumulation were found . The aim of the future research must be to put these various components of the NPC and nuclear transport machinery in a topological and functional context. FEBS Lett, 1993 Jun 28, 325(1-2), 108 - 11 Structure, function and regulation of plasma membrane H(+)-ATPase; Serrano R; Most antigenic determinants of yeast ATPase are located within its N-terminal part . Amino acids 24-56, required for insertion at the plasma membrane, are highly accessible . The C-terminus behaves as a modulable auto-inhibitory domain in both yeast and plant ATPases . The expression of functional plant enzyme in yeast allows its mutational analysis . Plant tissues involved in active transport, such as the stomata guard cells, phloem, root epidermis and endodermis, are enriched in ATPase . One isoform is phloem-specific . The fact that auxin induces the synthesis of ATPase in corn coleoptiles provides molecular support to the 'Acid growth' theory. J Biol Chem, 1993 Jun 25, 268(18), 13248 - 52 Site specificity of pea histone acetyltransferase B in vitro; Mingarro I et al.; Histone acetyltransferase B from pea embryonic axes has been purified approximately 300-fold by a combination of chromatographic procedures, including affinity chromatography on histone-agarose . The enzyme preparation has been used for the in vitro transfer of acetyl groups from {1-14C}acetyl-CoA to non-acetylated pea histone H4 . Up to three acetyl groups can be introduced into the histone . The resulting mono-, di-, and triacetylated H4 isoforms were separated and sequenced to determine the acetylated sites . Only sites 5, 12, and 16 were used by histone acetyltransferase B, but no clear preference among them was observed . The absence of modification of other potentially acetylatable sites is another indication that acetylation of the different lysine residues in the N-terminal H4 tail serves as a specific signal in different nuclear processes. J Biol Chem, 1993 Jun 25, 268(18), 13062 - 7 p53 binds to the TATA-binding protein-TATA complex; Martin DW et al.; Earlier reports show that p53, both wild type and mutants, may affect transcription . Wild-type p53 activates promoters with p53-binding sites while inhibiting promoters without binding sites . Mutant p53, on the other hand, has been shown to activate transcription from specific promoters . These observations suggest that both wild-type and mutant p53 may interact with a general transcription factor(s) . In this report, we have shown that the cloned TATA-binding protein (TBP) from human and yeast interacts with human p53 . TBP co-immunoprecipitates with wild-type or mutant human p53 when incubated with the p53-specific monoclonal antibody and Protein A-agarose . Wild-type murine p53 has also been found to interact with human TBP . Protein blot assays have demonstrated that the interaction between p53 and human TBP is direct . By gel retention analysis, we have shown that the complex of TBP and p53 (both wild type and mutant) can bind to the TATA box . The similar qualitative binding capability of wild-type and mutant p53 with human TBP and the similarity of the two complexes in binding to the TATA box suggest that the functional discrimination between wild-type and mutant p53 may not lie in their ability to bind TBP . The nature of the p53.TBP or p53.TBP.TATA complex may determine the success of transcription. Nucleic Acids Res, 1993 Jun 25, 21(12), 2907 - 11 The cAMP response element binding protein, CREB, is a potent inhibitor of diverse transcriptional activators; Lemaigre FP et al.; Cyclic AMP response element binding protein (CREB) activates transcription of cAMP response element (CRE)-containing promoters following an elevation of intracellular cAMP . Here we show that CREB and the highly related protein ATF-1 are also potent transcription inhibitors . Strikingly, CREB inhibits transcription of multiple activators, whose DNA-binding domains and activation regions are unrelated to one another . Inhibition requires that the CREB dimerization and DNA-binding domains are intact . However, inhibition is not dependent upon the presence of a CRE in the promoter, and does not involve heterodimer formation between CREB and the activator . The ability of an activator protein to inhibit transcription in such a promiscuous fashion has not been previously reported. FEBS Lett, 1993 Jun 21, 324(3), 309 - 13 Sunlight-induced mutagenicity of a common sunscreen ingredient; Knowland J et al.; We have tested the mutagenicity of a UV-B sunscreen ingredient called Padimate-O or octyl dimethyl PABA, which, chemically speaking, is identical to an industrial chemical that generates free radicals when illuminated . It is harmless in the dark but mutagenic in sunlight, attacking DNA directly . A commercial sunscreen containing Padimate-O behaves in the same way . UV-A in sunlight also excites Padimate-O, although less than UV-B . Some related compounds, including a known carcinogen, behave similarly . As mutagens may be carcinogenic, our results suggest that some sunscreens could, while preventing sunburn, contribute to sunlight-related cancers. Cell, 1993 Jun 18, 73(6), 1223 - 32 The transactivator proteins VP16 and GAL4 bind replication factor A; He Z et al.; Many transcription factors can activate the initiation of DNA replication . We have used affinity chromatography to show that the acidic activation domains of the transcription factors VP16, GAL4, and p53 each bind selectively to human and yeast replication factor A (RPA) . The binding is direct and to the largest subunit of the trimeric RPA complex, RPA-1 . Mutations in VP16 that reduce the ability of GAL4-VP16 to activate polyomavirus DNA replication also compromise the binding of VP16 to RPA . We suggest that transcription factors may interact with RPA either to stabilize single-stranded DNA at a replication origin or to recruit DNA polymerase alpha to the replication initiation complex. Proc Natl Acad Sci U S A, 1993 Jun 15, 90(12), 5633 - 7 Functional domains of transcription factor TFIIB; Buratowski S et al.; Transcription factor TFIIB is an essential component of the RNA polymerase II initiation complex . TFIIB carries out at least two functions: it interacts directly with the TATA-binding protein (TBP) and helps to recruit RNA polymerase II into the initiation complex . The sequence of TFIIB reveals a potential zinc-binding domain and an imperfect duplication of approximately 70 amino acids . Mutagenesis of cysteine codons within the putative zinc finger results in mutant proteins that bind normally to TBP but are unable to recruit RNA polymerase II-TFIIF into the initiation complex . Changing the two most highly conserved amino acids in the TFIIB repeats reduces the ability of TFIIB to interact with TBP . Therefore, the two functions of TFIIB can be assigned to two separable functional domains of the protein. Proc Natl Acad Sci U S A, 1993 Jun 15, 90(12), 5628 - 32 Delineation of two functional regions of transcription factor TFIIB; Barberis A et al.; Human transcription factor TFIIB, a protein of 316 amino acids, was subjected to limited proteolysis in order to define stable structural domains . We find that the C-terminal region of TFIIB, residues 106-316, is relatively stable, while the N-terminal region is very sensitive to proteases . Like full-length TFIIB, the stable domain, which we refer to as TFIIBc, interacts with the TATA-binding protein (TBP) on DNA . However, TFIIBc is unable to substitute for TFIIB in an in vitro transcription assay . We show by gel mobility-shift experiments that TFIIBc arrests formation of the transcription complex after binding to TBP, and we conclude that the N-terminal region of TFIIB, which is missing from TFIIBc, is responsible for the recruitment of RNA polymerase II to the promoter . We also show that TFIIBc inhibits transcription by competing with full-length TFIIB for the interaction with TBP, either in the presence or in the absence of the TBP-associated factors . The acidic transcriptional activator GAL4-VP16 does not favor the assembly of the functional transcription complex over the nonfunctional complex containing TFIIBc . Thus, if the function of GAL4-VP16 is enhancement of the interaction between TFIIB and the TFIID-DNA complex, then this function can also be exerted on the protease-resistant domain TFIIBc. Proc Natl Acad Sci U S A, 1993 Jun 15, 90(12), 5593 - 7 Unusual charge configurations in transcription factors of the basic RNA polymerase II initiation complex; Karlin S; A systematic analysis of the primary sequences of the polymerase II initiation complex has revealed unusual charge features in the TFII family proteins . In particular, the proteins TFIIA alpha, TFIIE alpha, and TFIIF carry multiple charge clusters and hyper charge runs, sequence features occurring in < 4% of all (available) eukaryotic proteins . Possible implications for these charge structures are discussed in relation to the assembly and function of the polymerase II transcriptional complex. Proc Natl Acad Sci U S A, 1993 Jun 15, 90(12), 5484 - 8 Cytogenetic and molecular delineation of the smallest commonly deleted region of chromosome 5 in malignant myeloid diseases; Le Beau MM et al.; Loss of a whole chromosome 5 or a deletion of its long arm (5q) is a recurring abnormality in malignant myeloid neoplasms . To determine the location of genes on 5q that may be involved in leukemogenesis, we examined the deleted chromosome 5 homologs in a series of 135 patients with malignant myeloid diseases . By comparing the breakpoints, we identified a small segment of 5q, consisting of band 5q31, that was deleted in each patient . This segment has been termed the critical region . Distal 5q contains a number of genes encoding growth factors, hormone receptors, and proteins involved in signal transduction or transcriptional regulation . These include several genes that are good candidates for a tumor-suppressor gene, as well as the genes encoding five hematopoietic growth factors (CSF2, IL3, IL4, IL5, and IL9) . By using fluorescence in situ hybridization, we have refined the localization of these genes to 5q31.1 and have determined the order of these genes and of other markers within 5q31 . By hybridizing probes to metaphase cells with overlapping deletions involving 5q31, we have narrowed the critical region to a small segment of 5q31 containing the EGR1 gene . The five hematopoietic growth factor genes and seven other genes are excluded from this region . The EGR1 gene was not deleted in nine other patients with acute myeloid leukemia who did not have abnormalities of chromosome 5 . By physical mapping, the minimum size of the critical region was estimated to be 2.8 megabases . This cytogenetic map of 5q31, together with the molecular characterization of the critical region, will facilitate the identification of a putative tumor-suppressor gene in this band. Eur J Biochem, 1993 Jun 15, 214(3), 853 - 67 Myristoyl-CoA:protein N-myristoyltransferase activity in cancer cells . Purification and characterization of a cytosolic isoform from the murine leukemia cell line L1210; Boutin JA et al.; Myristoylation is a co-translational maturation process of proteins . It is extremely specific for the cosubstrate (myristoyl-CoA) and for the substrate protein that should bear a glycine at the N-terminus of the protein to be myristoylated . This acylation is catalyzed by the myristoyl-CoA:protein N-myristoyltransferase . Most of the molecular biochemistry and biology concerning this enzyme has been done on Saccharomyces cerevisiae . Because of the major importance of this pathway in several types of pathology, it is essential to study intensively the enzyme(s) isolated from mammalian tissue(s) to confirm that the enormous amount of work done on the yeast enzyme can be transposed to mammalian tissues . In earlier studies, we demonstrated the existence of a microsomal N-myristoyltransferase from the murine leukemia cell line L1210 {Boutin, J . A., Clarenc, J.-P., Ferry, G., Ernould, A . P., Remond, G., Vincent, M . & Atassi, G . (1991) Eur . J . Biochem . 201, 257-263}, a feature which is not shared by yeast, and examined the N-myristoyltransferase activities associated with L1210 cytosol . In the present work, we purified to homogeneity one of the isoforms (A) of the transferase from L1210 cytosol . The purified enzyme showed on SDS/PAGE an apparent molecular mass of 67.5 kDa, distinct from the 53-kDa yeast cytosolic enzyme . The purified enzyme from L1210 cytosol could be labeled with {14C}myristoyl-CoA . Rabbit antibodies were raised against the A isoform and used to immunoprecipitate the enzyme and immunoinhibit the activity from the same source . A survey of the specificity of the partially and completely purified isoforms was performed using peptides derived from the NH2-terminus of 42 proteins which are potential substrates for myristoylation, including oncogene products and virus structural proteins . We synthesized a series of compounds capable of inhibiting the cytosol activities of the enzyme . For example, a myristoyltetrahydroquinolein derivative showed an IC50 of about 0.1 microM . Based on both biophysical and biochemical evidence, the N-myristoyltransferases extracted from mammalian cell cytosols seem to be different from the extensively studied yeast enzyme. Proc Natl Acad Sci U S A, 1993 Jun 15, 90(12), 5853 - 7 Proline isomerases function during heat shock; Sykes K et al.; The cyclophilins (CYPs) and FK506 binding proteins (FKBPs) are two families of distinct proline isomerases that are targets for a number of clinically important immunosuppressive drugs . Members of both families catalyze cis/trans isomerization of peptidyl-prolyl bonds, which can be a rate-limiting step during protein folding in vitro and in vivo . We demonstrate in Saccharomyces cerevisiae that heat shock causes a 2- to 3-fold increase in the level of mRNA encoded by the major cytoplasmic CYP gene, CYP1 . The cloned CYP1 promoter confers heat-inducible expression upon a reporter gene, and transcriptional induction is mediated through sequences similar to the consensus heat shock response element . Disruption of CYP1 decreases survival of cells following exposure to high temperatures, indicating that CYP1 plays a role in the stress response . A second CYP gene, CYP2, encodes a cyclophilin that is located within the secretory pathway . Its expression is also stimulated by heat shock, and cells containing a disrupted CYP2 allele are more sensitive than wild-type cells to heat . By contrast, expression of the FKB1 gene, which encodes a cytoplasmic member of the yeast FKBP family, is neither heat responsive nor necessary for survival after exposure to heat stress. Proc Natl Acad Sci U S A, 1993 Jun 15, 90(12), 5446 - 9 Mapping of residues forming the voltage sensor of the voltage-dependent anion-selective channel; Thomas L et al.; Voltage-gated ion-channel proteins contain "voltage-sensing" domains that drive the conformational transitions between open and closed states in response to changes in transmembrane voltage . We have used site-directed mutagenesis to identify residues affecting the voltage sensitivity of a mitochondrial channel, the voltage-dependent anion-selective channel (VDAC) . Although charge changes at many sites had no effect, at other sites substitutions that increased positive charge also increased the steepness of voltage dependence and substitutions that decreased positive charge decreased voltage dependence by an appropriate amount . In contrast to the plasma membrane K+ and Na+ channels, these residues are distributed over large parts of the VDAC protein . These results have been used to define the conformational transitions that accompany voltage gating of an ion channel . This gating mechanism requires the movement of large portions of the VDAC protein through the membrane. Nature, 1993 Jun 10, 363(6429), 539 - 41 Honest signalling among gametes; Pagel M; The gametes of many lower eukaryotic organisms emit pheromones that attract gametes of the opposite mating type or sex . Gametes move or grow in the direction of the highest pheromone concentration, suggesting that the strength of the pheromonal signal is used to infer proximity, or that the strongest signal is most likely to be notice . Here I offer a new explanation of pheromonal signalling and chemotaxis in gametes . I show that pheromonal signals can be interpreted as sexually selected traits that honestly advertise variation in quality among gametes, given that signals are costly to produce and that gametes compete; by 'quality' I refer to some aspect of a gamete's fitness . A gamete's preference for a mating partner, then, is predicted to vary with the quality of a prospective partner as inferred from the strength of its signal . This view can explain characteristics of the signalling and mate selection behaviours of gametes that are not predicted by models of mate choice based on proximity or 'passive attraction' to the strongest signal . These include repeated partner exchanges, escalated exchanges of mating pheromones, and rejection of gametes that signal at low levels. Biochim Biophys Acta, 1993 Jun 6, 1177(2), 183 - 90 Coordinate induction of acyl-CoA binding protein, fatty acid binding protein and peroxisomal beta-oxidation by peroxisome proliferators; Vanden Heuvel JP et al.; Acyl-CoA binding protein (ACBP) and fatty acid binding protein (FABP) are important intracellular lipid binding proteins . The purpose of the present experiments was to test the hypothesis that peroxisome proliferators induce ACBP in rat hepatocytes as has been shown previously for FABP . The effects of two structurally dissimilar peroxisome proliferators perfluorodecanoic acid (PFDA) and clofibric acid (CPIB) were examined in primary rat hepatocyte cultures in a chemically defined media . Both compounds alter lipid metabolism in primary rat hepatocytes in a similar fashion, although PFDA is more potent than CPIB at inducing peroxisomal beta-oxidation . In addition, PFDA and CPIB compete with long-chain fatty acids for binding to FABP but do not compete with long-chain acyl-CoA esters for binding to ACBP . The concentration of ACBP and FABP was increased in peroxisome proliferator-treated hepatocytes relative to vehicle controls within 48 h of treatment . Evidence is given to support increases in ACBP and FABP mRNA being the cause of the increased protein levels by peroxisome proliferators . In addition, the peroxisome proliferators PFDA, perfluorooctanoic acid and ciprofibrate induced hepatic ACBP following in vivo administration to rats indicating that this phenomena is not exclusive to in vitro systems . Therefore, ACBP appears to be a member of the peroxisome proliferator loci, a group of lipid metabolizing proteins, including FABP, which are regulated by peroxisome proliferators such as fibric acids and perfluorinated fatty acids. J Biol Chem, 1993 Jun 5, 268(16), 12221 - 9 cDNA cloning and expression of the alpha and beta subunits of rat Rab geranylgeranyl transferase; Armstrong SA et al.; Rab geranylgeranyl transferase (Rab GG transferase) attaches 20-carbon geranylgeranyl groups to cysteine residues in Rab proteins that contain the COOH-terminal sequence Cys-X-Cys or Cys-Cys . Rab GG transferase consists of two components that are separable in high salt solutions . Component A is a 95-kDa protein, and Component B is a heterodimer consisting of a 60-kDa alpha subunit and a 38-kDa beta subunit . In the current paper, we have cloned cDNAs for the alpha and beta subunits of Component B . The cDNAs for the rat alpha and beta subunits predict proteins of 567 and 331 amino acids, respectively . The mRNAs for both subunits are expressed in many tissues . When transfected together in embryonic kidney 293 cells, the alpha and beta subunit cDNAs produced Rab GG transferase activity that was stimulated in vitro by the addition of purified Component A . Comparisons of the amino acid sequences suggest that the proteins encoded by the Saccharomyces cerevisiae genes MAD2 and BET2 are the yeast counterparts of the mammalian Rab GG transferase alpha and beta subunits, respectively. J Biol Chem, 1993 Jun 5, 268(16), 12046 - 54 N-recognin/Ubc2 interactions in the N-end rule pathway; Madura K et al.; The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue . In the yeast Saccharomyces cerevisiae, substrates of the N-end rule pathway are targeted for degradation by a complex that includes the 225-kDa N-recognin, encoded by UBR1, and the 20-kDa ubiquitin-conjugating enzyme encoded by UBC2 . We report that both physical stability and functional activity of the N-recognin.Ubc2 complex require the presence of a highly acidic 23-residue region at the C terminus of Ubc2 . Ubc2-C88A, an inactive variant of Ubc2 in which the active-site Cys-88 has been replaced by Ala, is shown to retain the affinity for N-recognin . Expression of Ubc2-C88A inhibits the N-end rule pathway, apparently as a result of competition between Ubc2 and Ubc2-C88A for binding to N-recognin . The two-hybrid (interaction cloning) technique was used to identify a approximately 170-residue C-terminal fragment of the 1,950-residue N-recognin as a Ubc2-interacting domain . We also show that the level of UBR1 mRNA decreases upon overexpression of UBC2 . This effect of UBC2 is observed with cells whose UBR1 is expressed from an unrelated promoter but is not observed if UBR1 contains a frameshift mutation, or if the Ubc2 protein lacks its C-terminal acidic region . The N-recognin.Ubc2 complex appears to regulate the expression of N-recognin through changes in the metabolic stability of its mRNA. J Mol Biol, 1993 Jun 5, 231(3), 634 - 45 DNA tridimensional context affects the reactivity of eukaryotic DNA topoisomerase I; Perini R et al.; Cleavage sites of eukaryotic DNA topoisomerase I on curved linear DNAs are clustered, map on the same side of the curve (the external one) and their distribution has the same period as the helical repeat, as observed on curved DNA tracts of Crithidia fasciculata, of Saccharomyces cerevisiae ARS1, of pT7CAT and on synthetic DNAs . The effects of the tridimensional context on both the cleavage and the topoisomerization reactions of DNA topoisomerase I were determined using serial DNA constructs made with inserts in which synthetic curves lie in a plane and in which the orientation of the planes of curvature is shifted by 72 degrees, 144 degrees, 216 degrees, 288 degrees and 360 degrees . The insertion of a curve markedly changes the reaction properties of the surrounding sequences. J Biol Chem, 1993 Jun 5, 268(16), 12143 - 9 A mitochondrial porin cDNA predicts the existence of multiple human porins; Ha H et al.; Pores formed in the outer membrane of mitochondria by mitochondrial porin make it permeable to water-soluble metabolites smaller than approximately 5 kDa . We have isolated a full-length cDNA encoding a human porin . This probe detects a single approximately 1.5-kilobase mRNA species on Northern blots, but multiple hybridizing bands on genomic Southern blots . The open reading frame predicts a 38.1-kDa protein with a pI of 6.59 that is homologous but not identical to a previously reported protein sequence of a 31-kDa porin isolated from human lymphocytes (porin 31HL) . The most striking difference between the two porins is that the sequence predicted by the cDNA is longer than the 31HL porin by 27 amino acids at the amino terminus and 38 amino acids at the carboxyl terminus . The porin cDNA directs the in vitro translation of two protein species of approximately 32 and approximately 36 kDa, which appear to result from alternate usage of AUG initiation codons . The 32-kDa protein is the predominant species imported into both rat and yeast mitochondria in vitro . Taken together, these results suggest that multiple porin proteins can be expressed in humans . Additionally, a porin consensus protein sequence has been identified that is conserved in eukaryotic organisms as diverse as yeast and man. EMBO J, 1993 Jun, 12(6), 2549 - 58 A U3 snoRNP protein with homology to splicing factor PRP4 and G beta domains is required for ribosomal RNA processing; Jansen R et al.; Yeast fibrillarin (NOP1) is an evolutionarily conserved, nucleolar protein necessary for multiple steps in ribosome biogenesis . Yeast mutants lacking a functional NOP1 gene can be complemented by human fibrillarin but are temperature sensitive for growth and impaired in pre-rRNA processing . In order to identify components which interact functionally with human fibrillarin in yeast, we isolated extragenic suppressors of this phenotype . One dominant suppressor, sof1-56, which is allele-specific for human fibrillarin and restores growth and pre-RNA processing at 35 degrees C, was cloned by in vivo complementation . The wild-type allele of SOF1 is essential for cell growth and encodes a novel 56 kDa protein . In its central domain, SOF1 contains a repeated sequence also found in beta-subunits of trimeric G-proteins and the splicing factor PRP4 . A single amino acid exchange in the G beta-like repeat domain is responsible for the suppressing activity of sof1-56 . Indirect immunofluorescence shows that SOF1 is located within the yeast nucleolus . Co-immunoprecipitation demonstrates the physical association of SOF1 with U3 small nucleolar RNA and NOP1 . In vivo depletion of SOF1 leads to impaired pre-rRNA processing and inhibition of 18S rRNA production . Thus, SOF1 is a new component of the nucleolar rRNA processing machinery. EMBO J, 1993 Jun, 12(6), 2295 - 302 Targeting of cytochrome b2 into the mitochondrial intermembrane space: specific recognition of the sorting signal; Schwarz E et al.; Cytochrome b2 contains 2-fold targeting information: an amino-terminal signal for targeting to the mitochondrial matrix, followed by a second cleavable sorting signal that functions in directing the precursor into the mitochondrial intermembrane space . The role of the second sorting sequence was analyzed by replacing one, two or all of the three positively charged amino acid residues which are present at the amino-terminal side of the hydrophobic core by uncharged residues or an acidic residue . With a number of these mutant precursor proteins, processing to the mature form was reduced or completely abolished and at the same time targeting to the matrix space occurred . The accumulation in the matrix depended on a high level of intramitochondrial ATP . At low levels of matrix ATP, the mutant proteins were sorted into the intermembrane space like the wild-type precursors . The results: (i) suggest the existence of one or more matrix components that specifically recognize the second sorting signal and thereby trigger the translocation into the intermembrane space; (ii) indicate that the mutant signals have reduced ability to interact with the recognition component(s) and then embark on the default pathway into the matrix by interacting with mitochondrial hsp70 in conjunction with matrix ATP; (iii) strongly argue against a mechanism by which the hydrophobic segment of the sorting sequence stops translocation in the hydrophobic phase of the inner membrane. Proc Natl Acad Sci U S A, 1993 Jun 1, 90(11), 5118 - 22 RNS2: a senescence-associated RNase of Arabidopsis that diverged from the S-RNases before speciation; Taylor CB et al.; Several self-compatible species of higher plants, such as Arabidopsis thaliana, have recently been found to contain S-like RNases . These S-like RNases are homologous to the S-RNases that have been hypothesized to control self-incompatibility in Solanaceous species . However, the relationship of the S-like RNases to the S-RNases is unknown, and their roles in self-compatible plants are not understood . To address these questions, we have investigated the RNS2 gene, which encodes an S-like RNase (RNS2) of Arabidopsis . Amino acid sequence comparisons indicate that RNS2 and other S-like RNases make up a subclass within an RNase superfamily, which is distinct from the subclass formed by the S-RNases . RNS2 is most similar to RNase LE {Jost, W., Bak, H., Glund, K., Terpstra, P., Beintema, J . J . (1991) Eur . J . Biochem . 198, 1-6.}, an S-like RNase from Lycopersicon esculentum, a Solanaceous species . The fact that RNase LE is more similar to RNS2 than to the S-RNases from other Solanaceous plants indicates that the S-like RNases diverged from the S-RNases prior to speciation . Like the S-RNase genes, RNS2 is most highly expressed in flowers, but unlike the S-RNase genes, RNS2 is also expressed in roots, stems, and leaves of Arabidopsis . Moreover, the expression of RNS2 is increased in both leaves and petals of Arabidopsis during senescence . Phosphate starvation can also induce the expression of RNS2 . On the basis of these observations, we suggest that one role of RNS2 in Arabidopsis may be to remobilize phosphate, particularly when cells senesce or when phosphate becomes limiting. Biochemistry, 1993 Jun 1, 32(21), 5622 - 8 Effect of mutations of ionic amino acids of cytochrome P450 1A2 on catalytic activities toward 7-ethoxycoumarin and methanol; Mayuzumi H et al.; Catalytic efficiencies, percentages of rates of product formation per NADPH oxidized, and rates of product formation per O2 consumed of ionic mutants of cytochrome P450 1A2 (P450 1A2) were studied . Efficiencies of Lys99Glu, Lys453Glu, and Arg455Glu mutants for the hydroxylation reaction toward 7-ethoxycoumarin in the reconstituted system were much lower than that of the wild type (less than 17%), which corresponds to lower turnover numbers for these mutants . In contrast, the catalytic efficiencies for the hydroxylation reaction toward methanol of the three mutants were more than 45% that of the wild type in spite of these mutants' lower turnover numbers . Turnover numbers and catalytic efficiencies of Arg137Leu and Lys401 Glu mutants toward both substrates were comparable to those of the wild type . The electron-transfer rate from the reductase to the heme of P450 1A2 was decreased by 30% upon addition of excess methanol, while it was not influenced by addition of excess 7-ethoxycoumarin . The turnover numbers toward both 7-ethoxycoumarin and methanol as well as the rate constant of electron transfer were decreased by 25-40% by raising the concentration of KCl from 0 to 300 mM in the reconstituted system containing 50 mM potassium phosphate buffer . The turnover numbers toward both substrates of the above-mentioned five ionic mutants caused by tert-butyl hydroperoxide in the absence of the reductase and NADPH were comparable to those of the wild type . The effect of phospholipid constituents on the catalytic activity toward 7-ethoxycoumarin of the wild type was also studied.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1993 Jun 1, 32(21), 5491 - 6 Thermodynamic characterization of the structural stability of the coiled-coil region of the bZIP transcription factor GCN4; Thompson KS et al.; The thermal stability of a 56 amino acid fragment of GCN4 has been studied by high-sensitivity differential scanning calorimetry and circular dichroism spectroscopy . This fragment contains the leucine zipper and part of the basic region . The thermal unfolding of GCN4-56 is a reversible process and can be well represented by a reaction of the form N2<-->2U, indicating that the unfolding of the leucine zipper is a two-state process in which the helices are only stable when they are in the coiled-coil conformation . As expected, the transition temperature is concentration dependent . At pH 7.06 and a protein concentration of 5 x 10(-4) M the transition temperature is close to 70 degrees C while at 5 x 10(-6) M it is close to 50 degrees C . The enthalpy change for unfolding is 31.5 kcal mol-1 at 70 degrees C . Since the isolated helices are unstable, interactions at the interface between the two helices play a key role in the stabilization of the native dimer . These interactions primarily involve the burial of apolar surface from the solvent (hydrophobic effect) and electrostatic interactions . Structural thermodynamic calculations have permitted a dissection of the magnitude of the various contributions to the total Gibbs free energy of stabilization. Biochem J, 1993 Jun 1, 292 ( Pt 2), 469 - 76 Indication of possible post-translational formation of disulphide bonds in the beta-sheet domain of human lysozyme; Kanaya E et al.; Lysozyme has two distinct folding domains, and in most molecules the alpha-helical domain folds more quickly than the beta-sheet domain in vitro {Radford, Dobson and Evans (1992) Nature (London) 358, 302-307} . In order to investigate the relationship between the formation of disulphide bonds and protein folding in vivo, we carried out cysteine scanning mutagenesis to shift positions of the disulphide bonds in both the alpha-helical and beta-sheet domains of human lysozyme . Of the constructed mutants (nine in the beta-sheet domain and 13 in the alpha-helical domain), the mutant L79CC81A, in which Leu-79 and Cys-81 in the beta-sheet domain were replaced by Cys and Ala respectively, was secreted by yeast . The rest of the mutants were retained in the insoluble fraction of the cell, probably because of a failure of folding . The distance between the two alpha-carbons at positions 79 and 95 in the wild-type protein is too far to form a disulphide bond, but analysis of the primary structure revealed that the major part of L79CC81A was secreted with a non-native disulphide bond Cys79-Cys95 and two free cysteine residues at positions 65 and 77 in the beta-sheet domain . These results suggest that the beta-sheet domain of human lysozyme can tolerate the shift of locations of disulphide bonds, and the non-native folding of mutated polypeptide chains in in vivo folding . The free residues Cys-65 and Cys-77 formed a disulphide bond in vitro by air oxidation, yielding two isomers . On the basis of our previous results and present study it is suggested that the formation of Cys6-Cys128 is the first step of the in vivo correct folding of human lysozyme, and disulphide bonds in the beta-sheet domain are post-translationally formed in vivo. Oncogene, 1993 Jun, 8(6), 1693 - 6 Use of the two-hybrid system to identify the domain of p53 involved in oligomerization; Iwabuchi K et al.; We used a yeast-based genetic assay, the two-hybrid system, to characterize the domain of the tumor-suppressor p53 involved in oligomerization . This assay relies on the reconstitution of the function of a transcriptional activator, the yeast GAL4 protein, via the interaction of a protein fused to the DNA-binding domain of GAL4 with a protein fused to the transcriptional activation domain of GAL4 . We show by a reconstruction experiment that this approach could detect the interaction of p53 deleted for its N-terminal activation domain with SV40 large T antigen . We then searched a library of human proteins present as activation domain hybrids for proteins that can interact with the hybrid of p53 with the DNA-binding domain . This search identified 36 plasmids containing the p53 gene, representing 10 different classes . These results provide an additional in vivo demonstration of p53 oligomerization . The smallest p53 fragment identified from screening the library contained only amino acids 331-393, indicating that this small C-terminal fragment is sufficient to mediate oligomerization . In addition, a mutant p53 protein could bind to the wild-type protein in this assay, providing support for the idea that mutant forms of p53 act in a dominant-negative manner through C-terminal oligomerization with the wild type. Mol Cell Biol, 1993 Jun, 13(6), 3792 - 801 Multiple SWI6-dependent cis-acting elements control SWI4 transcription through the cell cycle; Foster R et al.; The Saccharomyces cerevisiae SWI4 gene encodes an essential transcription factor which controls gene expression at the G1/S transition of the cell cycle . SWI4 transcription itself is cell cycle regulated, and this periodicity is crucial for the normal cell cycle regulation of HO and at least two of the G1 cyclins . Since the regulation of SWI4 is required for normal cell cycle progression, we have characterized cis- and trans-acting regulators of SWI4 transcription . Deletion analysis of the SWI4 promoter has defined a 140-bp region which is absolutely required for transcription and can function as a cell cycle-regulated upstream activating sequence (UAS) . The SWI4 UAS contains three potential MluI cell cycle boxes (MCBs), which are known cell cycle-regulated promoter elements . Deletion of all three MCBs in the SWI4 UAS decreases the level of SWI4 mRNA 10-fold in asynchronous cultures but does not abolish periodicity . These data suggest that MCBs are involved in SWI4 UAS activity, but at least one other periodically regulated element must be present . Since SWI6 is known to bind to MCBs and regulate their activity, the role of SWI6 in SWI4 expression was analyzed . Although the MCBs cannot account for the full cell cycle regulation of SWI4, mutations in SWI6 eliminate the normal periodicity of SWI4 transcription . This suggests that the novel cell cycle-regulated element within the SWI4 promoter is also SWI6 dependent . The constitutive transcription of SWI4 in SWI6 mutant cells occurs at an intermediate level, which indicates that SWI6 is required for the full activation and repression of SWI4 transcription through the cell cycle . It also suggests that there is another pathway which can activate SWI4 transcription in the absence of SWI6 . The second activator may also target MCB elements, since SWI4 transcription drops dramatically when they are deleted. Mol Cell Biol, 1993 Jun, 13(6), 3291 - 300 The p53 activation domain binds the TATA box-binding polypeptide in Holo-TFIID, and a neighboring p53 domain inhibits transcription; Liu X et al.; Antioncogene product p53 is a transcriptional transactivator . To investigate how p53 stimulates transcription, we examined the interaction of p53 with general transcription factors in vitro . We found that p53 binds directly to the human TATA box-binding polypeptide (TBP) . We also observed a direct interaction between p53 and purified holo-TFIID, a complex composed of TBP and a group of TBP-associated polypeptides known as TAFs . The p53 binding domain on TBP was mapped to the conserved region of TBP, including residues 220 to 271 . The TBP binding domain on p53 was mapped to the p53 activation domain between residues 20 and 57 . To analyze the significance of the p53-TBP interaction in p53 transactivation, we compared the ability of Gal4-p53 fusion proteins to bind to TBP in vitro and to activate transcription in transient transfection assays . Fusion proteins which bound to TBP activated transcription, and those that did not bind to TBP did not activate transcription to a detectable level, suggesting that a direct interaction between TBP and p53 is required for p53 transactivation . We also found that inclusion of residues 93 to 160 of p53 in a Gal4-p53 fusion repressed transcriptional activation 100-fold . Consequently, this region of p53 inhibits transcriptional activation by the minimal p53 activation domain . Highest levels of activation were observed with sequences 1 to 92 of p53 fused to Gal4, even though this construct bound to TBP in vitro with an affinity similar to that of other Gal4-p53 fusion proteins . We conclude that TBP binding is necessary for p53 transcriptional activation and that p53 sequences outside the TBP binding domain modulate the level of activation. Mol Cell Biol, 1993 Jun, 13(6), 3167 - 75 Mechanism of cleavage and ligation by FLP recombinase: classification of mutations in FLP protein by in vitro complementation analysis; Pan G et al.; The FLP recombinase of the 2 microns plasmid of Saccharomyces cerevisiae is a member of the integrase family of site-specific recombinases . Recombination catalyzed by members of this family proceeds via the ordered cleavage and religation of four strands of DNA . Although the amino acid sequences of integrase family members are quite different, each recombinase maintains an absolutely conserved tetrad of amino acids (R-191, H-305, R-308, Y-343; numbers are those of the FLP protein) . This tetrad is presumed to reflect a common chemical mechanism for cleavage and ligation that has evolved among all family members . The tyrosine is the nucleophile that causes phosphodiester bond cleavage and covalently attaches to the 3'-PO4 terminus, whereas the other three residues have been implicated in ligation of strands . It has recently been shown that cleavage by FLP takes place in trans; that is, a FLP molecule binds adjacent to the site of cleavage but receives the nucleophilic tyrosine from a molecule of FLP that is bound to another FLP-binding element (J.-W . Chen, J . Lee, and M . Jayaram, Cell 69:647-658, 1992) . These studies led us to examine whether the ligation step of the FLP reaction is performed by the FLP molecule bound adjacent to the cleavage site (ligation in cis) . We have found that FLP promotes ligation in cis . Furthermore, using in vitro complementation analysis, we have classified several mutant FLP proteins into one of two groups: those proteins that are cleavage competent but ligation deficient (group I) and those that are ligation competent but cleavage defective (group II) . This observation suggests that the active site of FLP is composed of several amino acid residues from each of two FLP molecules. Mol Cell Biol, 1993 Jun, 13(6), 3156 - 66 Genetic dissection of centromere function; Schulman IG et al.; A system to detect a minimal function of Saccharomyces cerevisiae centromeres in vivo has been developed . Centromere DNA mutants have been examined and found to be active in a plasmid copy number control assay in the absence of segregation . The experiments allow the identification of a minimal centromere unit, CDE III, independently of its ability to mediate chromosome segregation . Centromere-mediated plasmid copy number control correlates with the ability of CDE III to assemble a DNA-protein complex . Cells forced to maintain excess copies of CDE III exhibit increased loss of a nonessential artificial chromosome . Thus, segregationally impaired centromeres can have negative effects in trans on chromosome segregation . The use of a plasmid copy number control assay has allowed assembly steps preceding chromosome segregation to be defined. Gig Sanit, 1993 Jun, (6), 18 - 20 {Hygienic standardization of some biological pollutants in the water}; Kogai RE; Paper is devoted to problem of sanitary protection of water reservoirs from pollution by bioinsecticides and biosynthesis biologic waste . Experimental data on influence of biological pollutants on organoleptic water properties, water sanitary and toxicologic status are presented . Standards for open water bodies are recommended: dendrobacillin--6 x 10(4) org/l, trichodermin--2 x 10(4) org/l, grain moth pollen--0.05 mg/l, fodder yeast--0.08 mg/l, turingin--0.048 mg/l. J Bioenerg Biomembr, 1993 Jun, 25(3), 221 - 32 What information do inhibitors provide about the structure of the hydroquinone oxidation site of ubihydroquinone: cytochrome c oxidoreductase? Link TA, Haase U, Brandt U, von Jagow G. The Q cycle mechanism of the bc1 complex requires two quinone reaction centers, the hydroquinone oxidation (QP) and the quinone reduction (QN) center . These sites can be distinguished by the specific binding of inhibitors to either of them . A substantial body of information about the hydroquinone oxidation site has been provided by the analysis of the binding of QP site inhibitors to the bc1 complex in different redox states and to preparations depleted of lipid or protein components as well as by functional studies with mutant bc1 complexes selected for resistance toward the inhibitors . The reaction site is formed by at least five protein segments of cytochrome b and parts of the iron-sulfur protein . At least two different binding sites for QP site inhibitors could be detected, one for the methoxyacrylate-type inhibitors binding predominantly to cytochrome b, the other for the chromone-type inhibitors and hydroxyquinones binding predominantly to the iron-sulfur protein . The interactions with the protein segments, between different protein segments, and between protein and ligands (substrate, inhibitors) are discussed in detail and a working model of the QP pocket is proposed. EMBO J, 1993 Jun, 12(6), 2337 - 48 Characterization of the transcription activation function and the DNA binding domain of transcriptional enhancer factor-1; Hwang JJ et al.; The regions of transcriptional enhancer factor-1 (TEF-1) required for its activation function and sequence-specific DNA binding have been determined . Deletion analysis of a chimera between TEF-1 and the GAL4 DNA binding domain (DBD) indicated that at least three regions of TEF-1 were involved in transactivation . However, none of these regions functioned as independent activating domains . Moreover, none of the GAL4 chimeras containing individual TEF-1 regions interfered with the activity of endogenous HeLa cell TEF-1, while interference was observed with the GAL4-TEF-1 chimeras which functioned as transactivators . These results indicate that there is a general correlation between the abilities of a given GAL4-TEF-1 chimera to function in transcriptional activation and interference, thus supporting the idea that transactivation by TEF-1 is mediated by a limiting transcriptional intermediary factor . In addition, we show experimentally that the TEA/ATTS domain is a novel class of DBD involved in the sequence-specific DNA binding of TEF-1 and its Drosophila homologue scalloped . Two other regions of TEF-1 are also required for DNA binding . These regions are not part of the minimum DBD, but may function by antagonizing the effect of sequences which negatively regulate DNA binding mediated by both the TEF-1 TEA/ATTS domain and the GAL4 DBD . In addition, analysis of TEF-1 and scalloped derivatives in which their TEA/ATTS domains have been interchanged further indicates that the TEA/ATTS domain is not the only determinant of DNA binding specificity. Proc Natl Acad Sci U S A, 1993 Jun 1, 90(11), 4927 - 31 Sites of RNA polymerase III transcription initiation and Ty3 integration at the U6 gene are positioned by the TATA box; Chalker DL et al.; The function of a TATA element in RNA polymerase (EC 2.7.7.6) III transcription of a naturally TATA-containing U6 snRNA gene and a naturally TATA-less tRNA gene was probed by transcription and Ty3 transposition analyses . Deletion of the TATA box from a U6 minigene did not abolish transcription and Ty3 integration but changed the positions of initiation and insertion . Insertion of the U6 TATA box at three positions upstream of the TATA-less SUP2 tRNA(Tyr) gene resulted in novel transcription initiation and Ty3 integration patterns that depended upon position of the insertion . Nevertheless, the predominant tRNA gene initiation sites were not affected by insertion of the TATA sequence and remained at a fixed distance from the internal box A promoter element . Insertions of the TATA box upstream of a SUP2 box A mutant affected the level of transcription and restricted the use of upstream start sites, but they neither enhanced the use of TATA-dependent initiation sites nor restored expression to the level of the wild-type gene . We conclude that (i) the U6 TATA box is essential in vivo for correct initiation but not for transcription, (ii) a TATA box does not compensate for a weak box A sequence and so cannot perform equivalently, and (iii) the TATA-binding protein, and probably components of transcription factor IIIB, are present on the target at the time of Ty3 integration. Mol Cell Biol, 1993 Jun, 13(6), 3445 - 55 Meiosis-specific arrest revealed in DNA topoisomerase II mutants; Rose D et al.; Although the processes of mitosis and meiosis are similar, there is evidence for fundamental regulatory differences between the two . To examine these differences, we have compared the meiotic phenotype of DNA topoisomerase II mutants with their previously described mitotic phenotype (C . Holm, T . Goto, J . Wang, and D . Botstein, Cell 41:553-563, 1985) . top2 mutants in meiosis show no defects in the latest detectable stages of recombination, yet they arrest prior to spindle establishment at meiosis I . Fluorescence and electron microscopy reveal that top2 mutants exhibit wild-type levels of meiotic chromosome condensation and form morphologically normal synaptonemal complex but are delayed in the exit from pachytene . Arrested cells retain viability and form colonies if transferred to mitotic medium . Our results suggest that the top2 meiotic arrest is regulatory in nature . This arrest may have evolved to ensure the resolution of fortuitous tangles between nonhomologous chromosomes. J Nat Prod, 1993 Jun, 56(6), 921 - 5 Bioactive and other sesquiterpenoids from Porella cordeana; Harrigan GG et al.; Bioassay-directed fractionation of the MeCOEt extract of Porella cordeana yielded drimenin {1} and aristolone {4}, which were moderately toxic towards DNA-repair-deficient mutants of Saccharomyces cerevisiae . Three inactive sesquiterpenes, 7-ketoisodrimenin {2}, 7-ketoisodrimenin-5-ene {3}, and norpinguisanolide, were also obtained . Compounds 2 and 3 are new. Microbiol Rev, 1993 Jun, 57(2), 402 - 14 Heat shock proteins: molecular chaperones of protein biogenesis; Craig EA et al.; Heat shock proteins (Hsps) were first identified as proteins whose synthesis was enhanced by stresses such as an increase in temperature . Recently, several of the major Hsps have been shown to be intimately involved in protein biogenesis through a direct interaction with a wide variety of proteins . As a reflection of this role, these Hsps have been referred to as molecular chaperones . Hsp70s interact with incompletely folded proteins, such as nascent chains on ribosomes and proteins in the process of translocation from the cytosol into mitochondria and the endoplasmic reticulum . Hsp60 also binds to unfolded proteins, preventing aggregation and facilitating protein folding . Although less well defined, other Hsps such as Hsp90 also play important roles in modulating the activity of a number of proteins . The function of the proteolytic system is intertwined with that of molecular chaperones . Several components of this system, encoded by heat-inducible genes, are responsible for the degradation of abnormal or misfolded proteins . The budding yeast Saccharomyces cerevisiae has proven very useful in the analysis of the role of molecular chaperones in protein maturation, translocation, and degradation . In this review, results of experiments are discussed within the context of experiments with other organisms in an attempt to describe the current state of understanding of these ubiquitous and important proteins. Biotechniques, 1993 Jun, 14(6), 920 - 4 Elimination of false positives that arise in using the two-hybrid system; Bartel P et al.; The two-hybrid system is a genetic method to identify proteins that interact with a specific target protein, which is expressed in yeast as a hybrid with a DNA-binding domain . Use of this method entails transforming yeast, both with this DNA-binding domain hybrid and with a library of activation domain hybrids, followed by screening for transcriptional activation of a reporter gene . In addition to proteins that interact with the target protein, a number of false positives are identified . We provide possible explanations for these false positives along with rapid assays to eliminate them. Protein Sci, 1993 Jun, 2(6), 951 - 65 Structures of DNA-binding mutant zinc finger domains: implications for DNA binding; Hoffman RC et al.; Studies of Cys2-His2 zinc finger domains have revealed that the structures of individual finger domains in solution determined by NMR spectroscopy are strikingly similar to the structure of fingers bound to DNA determined by X-ray diffraction . Therefore, detailed structural analyses of single finger domains that contain amino acid substitutions known to affect DNA binding in the whole protein can yield information concerning the structural ramifications of such mutations . We have used this approach to study two mutants in the N-terminal finger domain of ADR1, a yeast transcription factor that contains two Cys2-His2 zinc finger sequences spanning residues 102-159 . Two point mutants at position 118 in the N-terminal zinc finger (ADR1b: 102-130) that adversely affect the DNA-binding activity of ADR1 have previously been identified: H118A and H118Y . The structures of wild-type ADR1b and the two mutant zinc finger domains were determined using two-dimensional nuclear magnetic resonance spectroscopy and distance geometry and were refined using a complete relaxation matrix method approach (REPENT) to improve agreement between the models and the nuclear Overhauser effect spectroscopy data from which they were generated . The molecular architecture of the refined wild-type ADR1b domain is presented in detail . Comparisons of wild-type ADR1b and the two mutants revealed that neither mutation causes a significant structural perturbation . The structures indicate that the DNA binding properties of the His 118 mutants are dependent on the identity of the side chain at position 118, which has been postulated to make a direct DNA contact in the wild-type ADR1 protein . The results suggest that the identity of the side chain at the middle DNA contact position in Cys2-His2 zinc fingers may be changed with impunity regarding the domain structure and can affect the affinity of the protein-DNA interaction. Cell Struct Funct, 1993 Jun, 18(3), 139 - 49 Folimycin (concanamycin A), a specific inhibitor of V-ATPase, blocks intracellular translocation of the glycoprotein of vesicular stomatitis virus before arrival to the Golgi apparatus; Muroi M et al.; Folimycin (concanamycin A) specifically inhibited vacuolar-type ATPase as far as examined . Folimycin blocked excretion of the glycoprotein (G protein) of vesicular stomatitis virus into the medium and, instead, G protein was accumulated intracellularly . The intracellularly accumulated G protein electrophoresed a little faster than mature one . The N-glycan of the G protein was endoglycosidase H-sensitive, and terminal galactose and N-acetylglucosamine were not detected essentially on sequential digestion with exoglycosidases, indicating that processings known to occur in the Golgi apparatus do not take place in the presence of folimycin . The oligosaccharide chain of the G protein was determined to have a composition of Man8GlcNAc2 as analyzed by Bio-Gel P-4 column chromatography and high-performance liquid chromatography following digestion with alpha- and then with beta-mannosidase . Activities of mannosidase I and glycosyltransferases prepared from baby hamster kidney cells were not inhibited as far as examined, indicating that the incompleteness of the N-glycosidic chain in folimycin-treated cells is not caused by inhibition of processing enzymes . Taken together these observations suggest that folimycin blocks the intracellular translocation of G protein before the step of trimming by mannosidase I which is confined to the cis compartment of the Golgi . The intracellular localization of G protein as revealed by fluorescence microscopy was in good accordance with this assumption. Plant Physiol, 1993 Jun, 102(2), 425 - 33 Studies of the role of the propeptides of the Arabidopsis thaliana 2S albumin; D'Hondt K et al.; To investigate the possible roles of the Arabidopsis thaliana 2S albumin propeptides with respect to sorting, processing, and stability of the protein in plant cells, five gene constructions deleting or modifying the propeptides were made based on one of the genes encoding the Arabidopsis 2S albumin . These constructions were introduced into tobacco (Nicotiana tabacum) plants . Using subcellular fractionation and immunocytochemistry on ripe seeds, it was demonstrated that none of the propeptides was necessary for the sorting of the protein . Detailed protein-chemical analysis of the mature gene products indicated that, for all of the modified 2S albumin precursors made, the proteins were stably folded and correctly processed . However, the latter is less efficient when the internal fragment between the small and the large subunit is missing or when this internal fragment is changed . In an attempt to establish a rapid assay system for modified 2S albumin precursors, yeast cells were transformed with the same gene constructs . It was demonstrated that the processing machinery in yeast cells differs from that in plants, and, in a perhaps related observation, differences in stability of a particular modified protein were observed. Proc Natl Acad Sci U S A, 1993 Jun 1, 90(11), 4882 - 6 Identification and cloning of a protein kinase-encoding mouse gene, Plk, related to the polo gene of Drosophila; Clay FJ et al.; We have determined the nucleotide sequence of a cDNA encoding a protein kinase that is closely related to the enzyme encoded by the Drosophila melanogaster mutant polo and that we have designated Plk (polo-like kinase) . Plk is also related to the products of the Saccharomyces cerevisiae cell cycle gene MSD2 (CDC5) and the recently described early growth response gene Snk . Together, Plk, polo, Snk, and MSD2 define a subfamily of serine/threonine protein kinases . Plk is expressed at high levels in a number of fetal and newborn mouse tissues but is not expressed in the corresponding adult organs . With the exception of adult hemopoietic tissues, the only adult tissues in which we could detect Plk expression were ovaries and testes . Taken together, the patterns of Plk expression suggest an association with proliferating cells . Since polo is required for mitosis in Drosophila it is possible that Plk is involved in some aspect of cell cycle regulation in mammalian cells. Mol Biol Rep, 1993 Jun, 18(1), 15 - 28 Murine monoclonal antibodies specific for conserved and non-conserved antigenic determinants of the human and murine Ku autoantigens; Wang J et al.; The Ku autoantigen is a DNA binding factor consisting of 70 and approximately 80 kDa proteins (p70 and p80, respectively) which form a heterodimer . The p70/p80 dimer appears to be crucial for the function of a 350 kDa DNA-dependent protein kinase (DNA-PK) that phosphorylates certain transcription factors in vitro . Previous studies have suggested that Ku is abundant in primate cells, but undetectable in most non-primate cells . However, it is unclear if this reflects low abundance of Ku (and possibly DNA-PK activity) in non-primate cells, a lack of antibodies crossreactive with non-primate Ku proteins, or both . Ku was first identified with human autoimmune sera, but the suitability of these sera for studying the distribution, abundance and function of Ku is limited by the polyclonal immune response to Ku and the presence of contaminating autoantibodies in most patients' sera . In the present studies, we determined the specificities of murine anti-Ku monoclonal antibodies (mAbs) using cellular Ku as well as recombinant human and murine Ku antigens . Immunofluorescence studies confirmed previous observations that Ku is undetectable in most nonprimate cells . However, small amounts of Ku could be detected in MOPC-315, but not L-929, cells by immunoprecipitating with mAb 162 . In addition, autoantibodies to Ku were identified in the sera of approximately 1/3 of MRL/lpr mice . The murine autoantibodies also immunoprecipitated a small amount of Ku (comparable to that seen with 162) from MOPC-315, but not L-929, cell lysates . Characterization of the mAb specificities by immunoblot analysis with Ku fusion proteins revealed that mAbs 111, S10B1, and N9C1 bound to distinct epitopes of human p80 (amino acids 610-705, 8-221, and 1-374, respectively) . All three mAbs were unreactive with murine p80 . MAbs N3H10 and S5C11 bound immediately adjacent to the DNA binding site of p70 (amino acids 506-541) . Only N3H10 displayed comparable reactivity with human and murine p70 on immunoblots, but it immunoprecipitated murine Ku poorly . S5C11 crossreacted more weakly with murine p70 on immunoblots, whereas 162 was completely unreactive with human or murine Ku on immunoblots, despite immunoprecipitating Ku efficiently . Studies with mAbs N3H10 and 162 suggest that the level of Ku is considerably lower in nonprimate cells than cells of primate origin, and that L-929 cells express little or no Ku protein.(ABSTRACT TRUNCATED AT 400 WORDS) Am J Dis Child, 1993 Jun, 147(6), 617 - 26 Peroxisomal disorders . Neurodevelopmental and biochemical aspects; Brown FR 3rd et al.; The peroxisomal disorders represent a group of inherited metabolic disorders that derive from defects of peroxisomal biogenesis and/or from dysfunction of single or multiple peroxisomal enzymes . Because peroxisomes are involved in the metabolism of lipids critical to the functioning of the nervous system, many of the peroxisomal disorders manifest with significant degrees of progressive psychomotor dysfunction . These disorders should be considered in the differential diagnosis of the infant with hypotonia and psychomotor delay (especially if accompanied by facial dysmorphisms, hepatomegaly, cataracts and/or retinitis, calcific stippling, short limbs, or combinations of these features), in the school-aged child with progressive neurologic dysfunction, and in adults with slowly progressive motor dysfunction . Current knowledge of peroxisomal biochemical and enzymatic processes permits precise identification of particular disorders within the peroxisomal disorder grouping . An effort should be made to identify the specific peroxisomal disorder to provide a precise explanation for neurodevelopmental deficits, to potentially prevent recurrence through genetic counseling, and to provide appropriate therapies when available. Virology, 1993 Jun, 194(2), 870 - 4 Characterization of B-cell epitopes in the envelope glycoproteins of simian immunodeficiency virus; Benichou S et al.; We identified previously a neutralizing epitope in the V2 domain of the simian immunodeficiency virus (SIVmac) external envelope protein . The present study reports identification of five additional linear epitopes of SIVmac (isolate 251) by immunological screening of a peptide library expressed in yeast, using SIVmac-infected macaque sera . Three epitopes were localized in the envelope glycoproteins and the two others in the reverse transcriptase and in the Rev regulatory protein . Antibody response against the four envelope epitopes was monitored for 2 years in 12 macaques experimentally infected by SIVmac251 . These four envelope regions represent major immunodominant epitopes of the SIVmac . Two epitopes are located in the V3 domain (a.a . 311-330) of the external gp130 and near the amino terminal part (a.a . 601-619) of the transmembrane gp36, in regions similar to those identified in HIVs, demonstrating immunological similarities between the envelopes of SIVs and HIVs . SIV-specific immunodominant epitopes were also identified in the V1 (a.a . 111-130) and V2 (a.a . 171-190) domains of the external gp130 . In particular, antibody response against the V2 neutralizing region seems to play some role in the control of disease progression in SIVmac-infected macaques. Mol Cell Biol, 1993 Jun, 13(6), 3598 - 610 Cloning of the gene encoding peptide-binding protein 74 shows that it is a new member of the heat shock protein 70 family; Domanico SZ et al.; We have previously described peptide-binding proteins of 72 and 74 kDa (PBP72/74), which have been implicated as playing a role in antigen processing and are serologically related to the 70-kDa heat shock protein (hsp70) family . Here we report the cloning and sequencing of the cDNA encoding PBP74 in mice and in humans, accomplished by using amino acid sequence information obtained from the purified protein . We show that PBP74 is highly homologous to members of the hsp70 family but, significantly, is not identical to any known member of this family . Inspection of the cDNA nucleotide sequence indicates that it encodes a 46-residue N-terminal peptide which is not present in the mature protein . Transcription and translation in vitro of the PBP74 cDNA verified that it encodes a form of PBP74 which is larger than the mature protein . The presequence does not conform to known motifs for organelle-targeting sequences, and at present, its function is not known . By confocal microscopy, PBP74 was localized to cytoplasmic vesicles but not to the nucleus, mitochondria, or plasma membrane by using antibodies specific for the N-terminal 16 residues of PBP74 . By RNA filter hybridization analysis, PBP74 mRNAs are detected in all cell types tested . Exposure of cells to heat shock does not result in an increase in the mRNA levels of PBP74, unlike the dramatic increase observed for the stress-inducible hsp70 mRNA . Thus, PBP74 appears to be a constitutive, new member of the hsp70 family. Gene, 1993 May 30, 127(2), 199 - 202 Construction of a UMP synthase expression cassette from Dictyostelium discoideum; Shi NQ et al.; We have prepared a DNA cassette containing the UMP synthase (UMPS)-encoding gene (PYR5-6) from Dictyostelium discoideum . This gene contains no introns and can be used for expression of the UMPS protein . Due to the high percentage of AT in the flanking regions, useful restriction sites were absent, therefore the PYR5-6 was subcloned as three separate parts, manipulated, and religated to make a full-length clone . After reconstructing the coding region, we examined its functionality by introducing this gene under the control of the yeast GAL1 promoter into several uracil-requiring mutants of Saccharomyces cerevisiae . These studies demonstrated that the reconstructed PYR5-6 gene was functional and could complement independent ura3 and ura5 mutations in yeast. Biochem Pharmacol, 1993 May 25, 45(10), 2129 - 34 Nitroxyl analogs as inhibitors of aldehyde dehydrogenase . C-nitroso compounds; Nagasawa HT et al.; We previously postulated that the catalase-mediated oxidation of cyanamide leads to the formation of the unstable intermediate, N-hydroxycyanamide, which spontaneously decomposes to nitroxyl, the putative inhibitor of aldehyde dehydrogenase (EC 1.2.1.3; AlDH) . Since it was not possible to provide direct evidence for the inhibition of AlDH by nitroxyl, we examined the activity of three representative substituted nitroxyls (C-nitroso compounds), viz . nitrosobenzene (NB), 1-nitrosoadamantane (NA), and 2-methyl-2-nitrosopropane (MNP), as direct inhibitors of yeast AlDH in vitro . While NB and NA were highly effective inhibitors in this system exhibiting IC50 values of 2.5 and 8.6 microM, respectively, MNP was considerably less effective with an IC50 of 0.15 mM . When tested in vivo, NA did not show any inhibitory activity on the hepatic AlDH, possibly due to the lack of site-specific delivery of the active monomeric form of this compound . However, NB at a low dose did inhibit hepatic AlDH as reflected by an increase in blood acetaldehyde levels . These results attest to the abilities of NB and NA to act as direct inhibitors of AlDH analogous to nitroxyl itself. Biochemistry, 1993 May 25, 32(20), 5466 - 71 Characterization of the quaternary structure and conformational properties of the human stem cell inhibitor protein LD78 in solution; Patel SR et al.; The human LD78 protein (sometimes referred to as human macrophage inflammatory protein-1 alpha) has been shown to protect multipotential hemopoietic stem cells from the effects of cytotoxic agents . Administration of the recombinant stem cell inhibitor molecule LD78 as an adjunct to chemotherapy has potential clinical benefit in reducing or preventing the neutropenia associated with this treatment . At physiological ionic strength, the 8-kDa LD78 molecule exists as soluble, heterogeneous, multimeric complexes of mass ranging from 100 to > 250 kDa . The hydrodynamic and structural properties of LD78 have been determined in various buffer solutions using analytical ultracentrifugation, circular dichroism, and fluorescence spectroscopy . The results demonstrate that defined, homogeneous monomer and tetramer forms of LD78 can be prepared which display distinct conformational properties . The combined use of hydrodynamic and spectroscopic analysis provides an insight into the pathway and molecular mechanics of LD78 self-association. J Biol Chem, 1993 May 25, 268(15), 11199 - 207 A novel RNA polymerase III transcription factor fraction that is not required for template commitment; Dieci G et al.; We have identified and partially characterized a novel class III transcription factor fraction (TFIIIE) from yeast nuclear extracts . TFIIIE is functionally distinct from the standard yeast transcription factor fractions, TFIIIB and TFIIIC . It is also different from either of the TFIIIB subfractions, B' and B" . TFIIIE is essential for specific transcription of both tRNA and 5 S RNA genes, its activity is sensitive to proteinase K, and it exhibits an apparent sedimentation coefficient of 4.0 S when analyzed on glycerol gradients . In the case of a tRNA gene, TFIIIE does not play a role in the formation of stable preinitiation complexes containing TFIIIB and TFIIIC . It is required for single as well as multiple rounds of transcription, however . Thus, TFIIIE is involved in the utilization of stable transcription complexes, but its action is not restricted to reinitiation events. J Biol Chem, 1993 May 25, 268(15), 11008 - 17 Functional interactions between SV40 T antigen and other replication proteins at the replication fork; Murakami Y et al.; The functional interaction of simian virus 40 (SV40) large tumor antigen (T antigen) with DNA polymerase alpha (pol alpha)-primase complex, human single-stranded DNA binding protein (HSSB), and DNA polymerase delta (pol delta) holoenzyme, which includes pol delta, activator I (also called replication factor C), and proliferating cell nuclear antigen, at the replication fork was examined using the purified components that support SV40 DNA replication . Dilution of reaction mixtures during RNA primer synthesis revealed that T antigen remained associated continuously with the fork, while the pol alpha-primase complex dissociated from the complex during oligoribonucleotide synthesis . T antigen unwound duplex DNA from the SV40 core origin at a rate of 200 base pairs/min . Pol alpha-primase complex inhibited the rate of the unwinding reaction, and HSSB, pol alpha, and primase were all required for this effect . These requirements are the same as those essential for DNA primase-catalyzed oligoribonucleotide synthesis (Matsumoto, T., Eki, T., and Hurwitz, J . (1990) Proc . Natl . Acad . Sci . U . S . A . 87, 9712-9716) . This result suggests that the pol alpha-primase complex interacts with T antigen and HSSB during the unwinding reaction to synthesize RNA primers and that the interaction decreases the rate of T antigen movement . While pol delta holoenzyme can elongate primed DNA chains at a rate of 400-600 nucleotides/min on singly primed phi X174 DNA, the rate of the leading strand synthesis catalyzed by pol delta holoenzyme in the SV40 replication system in vitro was about 200 nucleotides/min . This rate was similar to the unwinding rate catalyzed by T antigen . Thus, the rate of leading strand synthesis catalyzed by pol delta holoenzyme in vitro appears to be limited by the unwinding reaction catalyzed by T antigen. FEBS Lett, 1993 May 24, 323(1-2), 129 - 31 Processing of Kex2 pro-region at two interchangeable cleavage sites; Germain D et al.; In Saccharomyces cerevisiae, the Kex2 endoprotease (Kex2p) is required for the proteolytic maturation of alpha-pheromone and also for the removal of its own pro-region . Kex2p is specific for pairs of basic amino acid residues . Two putative processing sites are present in the pro-region of Kex2p . We have expressed processing site mutants of Kex2p and assayed the production of active Kex2p . Mutations affecting either putative cleavage site do not alter the activity . However, mutations affecting both sites led to a reduction in both Kex2 activity and the amount of protein . These results suggest that removal of Kex2p pro-peptide is required for the production of a stable enzyme and can occur at either processing site. Cell, 1993 May 21, 73(4), 735 - 45 Bos1p, an integral membrane protein of the endoplasmic reticulum to Golgi transport vesicles, is required for their fusion competence; Lian JP et al.; BOS1 encodes an integral endoplasmic reticulum (ER) membrane protein and genetically interacts with three other yeast genes (BET1, SEC22, and YPT1) whose products are required for membrane traffic between the ER and the Golgi apparatus . Using an assay that reconstitutes transport at this stage of the pathway, we find that anti-Bos1p antibody blocks protein export after vesicles bud from the ER but prior to fusion with the Golgi . Additionally, the depletion of Bos1p from the ER leads to the formation of transport-incompetent vesicles . Carrier vesicles, immunoisolated with anti-Bos1p antibody, are approximately 50 nm in size . These vesicles contain Bos1p, Sec22p, and Ypt1p, but not Bet1p . The functional interactions of Bos1p with Ypt1p and Sec22p may be necessary for the fusion competence of the ER to Golgi transport vesicles. J Mol Biol, 1993 May 20, 231(2), 293 - 310 Distortion of the DNA double helix by RAP1 at silencers and multiple telomeric binding sites; Gilson E et al.; Repressor Activator Protein 1 (RAP1) is an essential nuclear protein of the yeast Saccharomyces cerevisiae that recognizes a 13 base-pair (bp) consensus sequence found in numerous upstream activating sequences, at the silencers of transcriptionally repressed mating-type genes, and in telomeric tracts, called (C1-3 A) repeats . RAP1 has been shown to influence transcriptional activation, transcriptional repression, telomere length, circular plasmid segregation and meiotic recombination in vivo . We have studied the structure of the protein-DNA complex reconstituted in vitro with highly purified RAP1, by using DNase I and chemical footprinting . Both full-length RAP1 and its minimal DNA-binding domain of roughly 30 kDa, induce a distortion within the 13 bp recognition site, as demonstrated by reactivity to KMnO4 primarily at nucleotides 8 and 10 in the binding consensus Rc/AAYCCRYNCAYY . Dimethylsulphate reactivity shows that RAP1 binding does not create unpaired regions at its binding site, although the DNA may be locally underwound or aberrantly base-paired at the permanganate reactive nucleotides . In addition to the permanganate-sensitive distortion, the full-length RAP1, but not its DNA-binding domain, induces a bend in DNA 5' of the recognition sequence, altering the electrophoretic mobility of the protein-DNA complex . The KMnO4-reactivity has allowed a precise mapping of RAP1 molecules on telomeric DNA, revealing RAP1 sites as frequently as one per 18 bp of telomeric DNA, or potentially 20 RAP1 molecules bound per average telomeric tract of 370 bp . This suggests that RAP1 plays a major role in organizing yeast telomeres, and is consistent with recently published immunofluorescence studies showing a major fraction of RAP1 at the ends of meiotic chromosomes. Biochemistry, 1993 May 18, 32(19), 5127 - 31 Metallothionein detoxification function is impaired by replacement of both conserved lysines with glutamines in the hinge between the two domains; Cody CW et al.; Mammalian metallothioneins (MTs) possess eight highly conserved lysine residues, two of which constitute the hinge between two metal binding domains . By site-directed mutagenesis and recombinant DNA techniques, we replaced the interdomain lysines in Chinese hamster ovary MT2 with all possible combinations of glutamic acid and/or glutamine . The resultant MTs were expressed and assayed for detoxification function in a transformed yeast system . Results showed that these mutant MTs, like the native protein, bound seven atoms of divalent metal per molecule and conferred cadmium resistance to a metal-sensitive yeast host . Replacement of one or both of the lysines in the interdomain region was inconsequential to the structure and function of MT, unless both substituted residues were uncharged . When both lysines were replaced by glutamine (K30,31Q), a reduction in the ability of MT to protect yeast transformants against otherwise toxic levels of cadmium was observed . This diminished metal detoxification capacity was due to a decrease in the steady-state level of MT . These results suggest that at least one charged amino acid must be present in the hinge for the proper expression of MT. Proc Natl Acad Sci U S A, 1993 May 15, 90(10), 4513 - 7 Adaptability at the protein-DNA interface is an important aspect of sequence recognition by bZIP proteins; Kim J et al.; The related AP-1 and ATF/CREB families of transcriptional regulatory proteins bind as dimers to overlapping or adjacent DNA half-sites by using a bZIP structural motif . Using genetic selections, we isolated derivatives of yeast GCN4 that affect DNA-binding specificity at particular positions of the AP-1 target sequence . In general, altered DNA-binding specificity results from the substitution of larger hydrophobic amino acids for GCN4 residues that contact base pairs . However, in several cases, DNA binding by the mutant proteins cannot be simply explained in terms of the GCN4-AP-1 structure; movement of the protein and/or DNA structural changes are required to accommodate the amino acid substitutions . The quintet of GCN4 residues that make base-pair contacts do not entirely determine DNA-binding specificity because these residues are highly conserved in the bZIP family, yet many of the bZIP proteins bind to distinct DNA sites . The alpha-helical fork between the GCN4 DNA-binding and dimerization surfaces is important for half-site spacing preferences, because mutations in the fork alter the relative affinity for AP-1 and ATF/CREB sites . The basic region in the protein-DNA complex is a long isolated alpha-helix, with no constraints from other parts of a folded domain . From all of these considerations, we suggest that small shifts in position and orientation or local deformations in the alpha-helical backbone distinguish one bZIP complex from another. Proc Natl Acad Sci U S A, 1993 May 15, 90(10), 4364 - 8 An integrated approach for identifying and mapping human genes; Das Gupta R et al.; We have developed a method for generating expressed-sequence maps of human chromosomes . The method involves several steps that begin with libraries of highly representative short cDNAs prepared by using random oligomers as primers . The cDNA inserts are amplified by PCR with flanking vector primers . Chromosomal region-specific cDNA packets are prepared by hybridization of the cDNA inserts to DNA derived from yeast artificial chromosomes (YACs) assigned to defined regions of human chromosomes . The cDNA packets are cloned into yeast chromosome fragmentation vectors and used for transformation of yeast bearing the YAC used for affinity purification . Sequences in the cDNAs undergo homologous recombination with the corresponding exons in the genomic DNA yielding a set of truncated YACs . Each unique truncation specifies the location of an exon in the YAC . Since all of the truncation events end with the same vector sequence, it is possible to rescue and sequence these ends to generate expressed sequence tags . The method couples rapid purification of region-specific cDNAs with precise mapping of their genes on YACs . Appropriately truncated YACs also provide easy access to gene regulatory sequences . We describe the feasibility of individual steps of the method using the factor IX (F9) gene as a model system and we present the mapping of several expressed sequences corresponding to a 330-kb YAC containing DNA from human chromosome 6p21 . In addition, we obtained the sequence, including an intron-exon junction, flanking a particular truncation event. J Biol Chem, 1993 May 15, 268(14), 10564 - 72 Vacuolar ATPase mutants accumulate precursor proteins in a pre-vacuolar compartment; Yaver DS et al.; The vacuole of the yeast Saccharomyces cerevisiae contains a proton-translocating ATPase that acidifies the vacuolar lumen and generates an electrochemical potential across the vacuole membrane . Strains with chromosomal disruptions of the genes encoding the A, B, and c subunits of the vacuolar ATPase accumulate precursor forms of the vacuolar membrane protein alkaline phosphatase, and the soluble vacuolar hydrolases carboxypeptidase Y and proteinase A . We have found that the intracellular precursors in delta vat strains accumulate within the secretory pathway at some point before delivery to the vacuole but after transit to the Golgi complex . Purified vacuoles from delta vat cells do not contain the precursor forms of carboxypeptidase Y or alkaline phosphatase . In addition, vacuolar hydrolase-invertase hybrid proteins are inefficiently delivered to the vacuole in delta vat strains as demonstrated by vacuole isolation . Further subcellular fractionation to separate organelles indicate that significant amounts of the carboxypeptidase Y-invertase and alkaline phosphatase-invertase hybrid proteins are located in the late Golgi complex and/or post Golgi compartments. J Biol Chem, 1993 May 15, 268(14), 10546 - 52 EBP-80, a transcription factor closely resembling the human autoantigen Ku, recognizes single- to double-strand transitions in DNA; Falzon M et al.; We have previously reported the purification and characterization of the transcription factor EBP-80 (Falzon, M., and Kuff, E . L . (1989) J . Biol . Chem . 264, 21915-21922) . EBP-80 mediates the DNA methylation effect on transcription from an endogenous proviral long terminal repeat . Here we show that EBP-80 is very similar if not identical to the Ku autoantigen, a heterodimeric nuclear protein first detected by antibodies from autoimmune patients (Mimori, T., Akizuki, M., Yamagata, H., Inada, S., Yoshida, S., and Homma, M . (1981) J . Clin . Invest . 68, 611-620) . A number of laboratories have shown that the Ku protein complex binds to free double-stranded DNA ends . In this study, we have examined the binding properties of EBP-80 . EBP-80 binds single-stranded DNA with low affinity . Binding to random sequence double-stranded DNA depends on the length of the duplex and is optimal with oligomers of 30 and 32 base pairs; the protein complexes formed with these oligomers have Kd values of 15-20 pM . It binds with comparable high affinities to blunt-ended duplex DNA, to duplex DNA ending in hairpin loops, and to constructs in which an internal segment of duplex DNA is flanked by single-strand extensions . EBP-80 also interacts effectively with circular duplex molecules containing a 30-nucleotide single-stranded region (gap) or a double-stranded segment of nonhomology (bubble), but only weakly with the corresponding closed circular construct made up entirely of duplex DNA . EBP-80 prefers A/T to G/C ends . The binding properties of EBP-80 are consistent with the hypothesis that is recognizes single- to double-strand transitions in DNA . A model is presented for the interaction of EBP-80 with its target sequence in the proviral long terminal repeat. Nucleic Acids Res, 1993 May 11, 21(9), 2157 - 63 GAL4-I kappa B alpha and GAL4-I kappa B gamma activate transcription by different mechanisms; Morin PJ et al.; I kappa B proteins regulate Rel/NF-kappa B transcription complexes through a direct protein-protein interaction . In addition, we have previously shown that certain I kappa B proteins (I kappa B alpha and I kappa B gamma) can act as activators of transcription when fused to the DNA-binding domain of GAL4 . We now show that a mutant chicken I kappa B alpha protein that cannot interact with Rel proteins in vitro did not activate transcription when fused to GAL4 in chicken embryo fibroblasts (CEF) and Saccharomyces cerevisiae, and did not inhibit growth in yeast; in contrast, an I kappa B alpha mutant that can still interact in vitro with Rel proteins activated transcription in both CEF and yeast and inhibited growth in yeast . In CEF, GAL4-I kappa B alpha mediated transcription activation was inhibited by co-transfection with an expression vector for a RelA (p65) protein that contained sequences needed for interaction with I kappa B alpha but that was deleted of its transcription activation domain . Therefore, it appears that GAL4-I kappa B alpha activates transcription by interacting with an endogenous Rel family protein in CEF . In contrast, the activation domain from I kappa B gamma behaved as a genuine acidic activator of transcription and did not inhibit growth when expressed in yeast . Since transcription activation and growth inhibition by GAL4-I kappa B alpha mutants in yeast correlated with their ability to interact with vertebrate Rel proteins, our results suggest that these activities of GAL4-I kappa B alpha are mediated through interaction with a Rel-like protein in yeast, which is important for cell growth. Nucleic Acids Res, 1993 May 11, 21(9), 2057 - 63 The nucleolar transcription activator UBF relieves Ku antigen-mediated repression of mouse ribosomal gene transcription; Kuhn A et al.; Previously we have shown that the RNA polymerase I (Pol I)-specific transcription factor UBF stimulates transcription by both facilitating transcription complex formation and by relieving repression exerted by a negative-acting factor which competes for binding of the murine factor TIF-IB to the ribosomal gene promoter (1) . We have purified and functionally characterized this repressor protein from Ehrlich ascites cells . The final preparation contained two polypeptides with molecular masses of 75 and 90 kDa, respectively . Both polypeptides interact with the rDNA promoter as revealed by UV-crosslinking experiments . The specificity of binding to the ribosomal gene promoter was demonstrated in an electrophoretic mobility shift assay and by DNase footprinting . The biochemical properties of this negative-acting factor closely resemble those of the Ku antigen, a human nuclear DNA-binding heterodimer which is the target of autoantibodies in several autoimmune diseases . Anti-Ku antibodies precipitate the repressor activity and overcome transcription inhibition . The data demonstrate that regulation of Pol I gene transcription may involve an antirepression mechanism as already documented for Pol II genes and suggest that Ku protein may be causally involved in repressor-mediated down regulation of rRNA synthesis. Biochemistry, 1993 May 11, 32(18), 4698 - 701 Sequence-specific cleavage of DNA via nucleophilic attack of hydrogen peroxide, assisted by Flp recombinase; Kimball AS et al.; Hydrogen peroxide is capable of effecting the cleavage of a specific phosphodiester bond in DNA, when used in concert with the recombinase enzyme Flp from Saccharomyces cerevisiae . This cleavage is not caused by oxidative damage of the DNA backbone but instead is the result of nucleophilic attack by peroxide . A single phosphorus-oxygen bond is broken in the reaction . Cleavage of DNA by peroxide also occurs with an inactive mutant of Flp in which the active site nucleophile tyrosine has been replaced by phenylalanine . Besides providing information on the mechanism of strand cleavage by Flp, these results may contribute to the development of new synthetic DNA cleavage reagents that act by hydrolytic and not radical chemistry. FEBS Lett, 1993 May 10, 322(2), 101 - 4 Identification of phytanoyl-CoA ligase as a distinct acyl-CoA ligase in peroxisomes from cultured human skin fibroblasts; Pahan K et al.; Phytanic acid accumulates in excessive amounts in Refsum disease, a rare neurological disorder, due to a defect in its alpha-oxidation enzyme system in peroxisomes . The activation of phytanic acid to phytanoyl-CoA by phytanoyl-CoA ligase is a prerequisite for its alpha-oxidation . The studies described in this manuscript report that phytanoyl-CoA ligase in peroxisomes is an enzyme distinct from the previously reported acyl-CoA ligases. Cell, 1993 May 7, 73(3), 533 - 40 DNA topology and a minimal set of basal factors for transcription by RNA polymerase II; Parvin JD et al.; Immunoglobulin heavy chain (IgH) gene transcription in vitro can be reconstituted with a minimal reaction containing only TATA-binding protein (TBP), TFIIB, and RNA polymerase II (pol II) when the template is negatively supercoiled . Transcription from linear DNA templates containing either the IgH or the adenovirus major late promoters (MLPs) requires in addition TFIIF, TFIIE, TFIIH, and a fraction containing TFIIA and TFIIJ . Promoters vary in their activities in the minimal reaction . Initiation at the adenovirus MLP site was not observed in this reaction, even with templates containing negative superhelical density . When only TBP, TFIIB, and pol II were present in the reaction, the more negatively supercoiled the IgH template DNA was, the more active the transcription . It is suggested that the free energy of supercoiling promotes the formation of an open complex for initiation of transcription by the minimal set of transcription factors. Biochemistry, 1993 May 4, 32(17), 4515 - 31 Electron-transfer kinetics and electrostatic properties of the Rhodobacter sphaeroides reaction center and soluble c-cytochromes; Tiede DM et al.; The kinetics of electron transfer between the Rhodobacter sphaeroides R-26 reaction center and nine soluble c-cytochromes have been analyzed and compared to the patterns of the surface electrostatic potentials for each of the proteins . Characteristic first-order electron-transfer rates for 1:1 complexes formed at low ionic strength between the reaction center and the different c-cytochromes were identified and found to vary by a factor of almost 100, while second-order rates were found to differ by greater than 10(6) . A correlation was found between the location of likely electrostatic interaction domains on each cytochrome and its characteristic rate of electron transfer . The interaction domains were identified by mapping electrostatic potentials, calculated from the Poisson-Boltzmann equation, onto simulated "encounter surfaces" for each of the cytochromes and the reaction center . For the reaction center, the c-cytochrome binding domain was found to have almost exclusively net negative potential (< -3 kT) and to be shifted slightly toward the M-subunit side of the reaction center . The location of interaction domains of complementary, positive potential (> 3 kT) differed for each cytochrome . The correspondence between electrostatic, structural, and kinetic properties of 1:1 reaction center-cytochrome complexes leads to a proposed mechanism for formation of reaction center-cytochrome electron-transfer complexes that is primarily driven by the juxtaposition of regions of delocalized complementary potential . In this mechanism the clustering of charged residues is of primary importance and not the location of specific residues . A consequence of this mechanism is that many different sets of charge distributions are predicted to be capable of stabilizing a specific configuration for a reaction center-cytochrome complex . This mechanism for reaction center association with water-soluble c-cytochromes fits molecular recognition mechanisms proposed for c-cytochromes in nonphotosynthetic systems . In general, the kinetic scheme for reaction center driven cytochrome oxidation was found to vary between a simple two-state model, involving cytochrome in free and reaction center bound states, and a three-state model, that includes cytochrome binding in kinetically competent ("proximal") and incompetent ("distal") modes . The kinetically incompetent mode of cytochrome binding is suggested not to be an intrinsic feature of the reaction center-cytochrome association but is likely to be due to variation in the physical state of the reaction center. Genetics, 1993 May, 134(1), 63 - 80 Cell cycle arrest of cdc mutants and specificity of the RAD9 checkpoint; Weinert TA et al.; In eucaryotes a cell cycle control called a checkpoint ensures that mitosis occurs only after chromosomes are completely replicated and any damage is repaired . The function of this checkpoint in budding yeast requires the RAD9 gene . Here we examine the role of the RAD9 gene in the arrest of the 12 cell division cycle (cdc) mutants, temperature-sensitive lethal mutants that arrest in specific phases of the cell cycle at a restrictive temperature . We found that in four cdc mutants the cdc rad9 cells failed to arrest after a shift to the restrictive temperature, rather they continued cell division and died rapidly, whereas the cdc RAD cells arrested and remained viable . The cell cycle and genetic phenotypes of the 12 cdc RAD mutants indicate the function of the RAD9 checkpoint is phase-specific and signal-specific . First, the four cdc RAD mutants that required RAD9 each arrested in the late S/G2 phase after a shift to the restrictive temperature when DNA replication was complete or nearly complete, and second, each leaves DNA lesions when the CDC gene product is limiting for cell division . Three of the four CDC genes are known to encode DNA replication enzymes . We found that the RAD17 gene is also essential for the function of the RAD9 checkpoint because it is required for phase-specific arrest of the same four cdc mutants . We also show that both X- or UV-irradiated cells require the RAD9 and RAD17 genes for delay in the G2 phase . Together, these results indicate that the RAD9 checkpoint is apparently activated only by DNA lesions and arrests cell division only in the late S/G2 phase. Nat Genet, 1993 May, 4(1), 11 - 8 Genomic mismatch scanning: a new approach to genetic linkage mapping; Nelson SF et al.; Genomic mismatch scanning (GMS) is a new method of genetic linkage analysis that does not require conventional polymorphic markers or gel electrophoresis . GMS is ideally suited to affected-relative-pair mapping . DNA fragments from all regions of identity-by-descent between two relatives are isolated based on their ability to form extensive mismatch-free hybrid molecules . The genomic origin of this selected pool of DNA fragments is then mapped in a single hybridization step . Here we demonstrate the practicality of GMS in a model organism, Saccharomyces cerevisiae . GMS is likely to be applicable to other organisms, including humans, and may be of particular value in mapping complex genetic traits. Mol Pharmacol, 1993 May, 43(5), 741 - 8 Isolation of glucagon antagonists by random molecular mutagenesis and screening; Smith RA et al.; Glucagon has an important role in the regulation of glucose homeostasis, and glucagon antagonists may be effective therapeutic agents in the control of diabetes mellitus . We were able to identify a number of analogs with antagonist activity by creating libraries of mutant glucagon coding sequences, expressing them in a yeast (Saccharomyces cerevisiae) secretion system, and screening for clones that produce analogs that inhibit the glucagon stimulation of rat hepatocyte membrane adenylate cyclase . These libraries were constructed by allowing random misincorporation during the synthesis of oligonucleotides that contained the complete coding sequence for mammalian glucagon or for an analog (desHis1-glucagon) that had partial antagonist activity . We developed and used a simplified screening assay to test culture broths from > 3500 individual transformant yeast clones for their ability to inhibit glucagon-dependent adenylate cyclase activity . Ultimately, > 20 different analogs with antagonist activity were identified by recovering and sequencing plasmid DNA from yeast strains that were positive in the screening assay . Interestingly, several analogs were identified repeatedly in independent yeast clones and certain amino acid substitutions occurred in more than one analog . This clustering of randomly isolated mutations clearly delineates the regions of the glucagon molecule that are important for designing improved glucagon antagonists . A subset of the antagonists identified in yeast broth were produced by peptide synthesis to confirm their activities as pure compounds. Protein Sci, 1993 May, 2(5), 697 - 705 The curious case of protein splicing: mechanistic insights suggested by protein semisynthesis; Wallace CJ; The gradual accumulation of examples of protein splicing, in which a nested intervening sequence is spliced out of the interior of a polyprotein precursor, suggests that this curious phenomenon might prove to have universal phylogenetic distribution and biological significance . The known examples are reviewed, with the aim of establishing underlying patterns, and a generalized mechanism of autocatalytic protein splicing is proposed . The testable consequences of such a proposal and the possible evolutionary origins of the phenomenon are discussed. Genes Dev, 1993 May, 7(5), 857 - 69 GAL4 disrupts a repressing nucleosome during activation of GAL1 transcription in vivo; Axelrod JD et al.; Photofootprinting in vivo of GAL1 reveals an activation-dependent pattern between the UASG and the TATA box, in a sequence not required for transcriptional activation by GAL4 . The pattern results from a nucleosome whose position depends on sequences within the UASG . In the wild-type gene, activation by GAL4 and derivatives disrupts this nucleosome . This activity is independent of interactions with DNA-bound core transcription factors and is proportional to the strength of the activator . Presence of the nucleosome correlates with low basal transcription levels under various conditions, suggesting a role in limiting basal expression . We propose a role for the GAL4 activation domain in displacing a nucleosome and suggest that this is part of the mechanism by which GAL4 activates transcription in vivo. Mol Cell Biol, 1993 May, 13(5), 2635 - 43 Proto-oncogene FosB: the amino terminus encodes a regulatory function required for transformation; Wisdom R et al.; Overexpression of some members of the Fos gene family, including FosB, leads to transformation of established rodent fibroblasts . We have previously shown that transformation by FosB requires the presence of a C-terminal transcriptional activation domain . We now report that transformation by FosB also requires an intact DNA-binding domain composed of the functionally bipartite basic region and leucine zipper as well as sequences present in the N terminus that serve a regulatory function . Deletion of the N-terminal sequences results in proteins impaired in transcriptional activation and transformation . This region does not itself function as a transcriptional activation domain but instead regulates the transactivation functions present in the FosB-Jun complex . The requirement for this N-terminal region can be abolished by the presence of a strong constitutive activation domain . The primary sequence of the region that we have defined is highly conserved in the Fos family of proteins, suggesting functional conservation. J Virol, 1993 May, 67(5), 2496 - 502 Dominant negative mutants of human T-cell leukemia virus type I Rex and human immunodeficiency virus type 1 Rev fail to multimerize in vivo; Bogerd H et al.; Human T-cell leukemia virus type I (HTLV-I) Rex and human immunodeficiency virus type 1 (HIV-1) Rev are essential gene products required for the replication of these two pathogenic human retroviruses . Both Rex and Rev act at a posttranscriptional level by binding to highly structured RNA-response elements, the Rex-response element in HTLV-I and the Rev-response element in HIV-1 . Using a sensitive in vivo assay of protein-protein interaction, we now demonstrate that the HTLV-I Rex and HIV-1 Rev proteins readily form homomultimeric complexes in the absence of their cognate RNA-response elements yet fail to form heteromultimeric complexes with each other . Dominant negative mutations have been identified in both the rex and rev genes which presumably specify a critical activation or effector domain in each of these viral transactivators . Surprisingly, these dominant negative mutants of Rex and Rev fail to interact in vivo . These findings raise the possibility that the binding of nonfunctional monomers rather than functional multimers underlies the transdominant phenotype of these Rex and Rev mutants . Further, it seems likely that the assembly of functional and stable multimers of Rex and Rev in vivo may depend not only on the intrinsic multimerization domains of these proteins but also on the binding of a bridging cellular cofactor to the related activation domains present in each viral transactivator. J Eukaryot Microbiol, 1993 May-Jun, 40(3), 317 - 22 Characterization of kinetoplast DNA from Phytomonas serpens; Sa-Carvalho D et al.; The restriction enzyme digestion of kinetoplast DNA from four Phytomonas serpens isolates shows an overall similar band pattern . One minicircle from isolate 30T was cloned and sequenced, showing low levels of homology but the same general features and organization as described for minicircles of other trypanosomatids . Extensive regions of the minicircle are composed by G and T on the H strand . These regions are very repetitive and similar to regions in a minicircle of Crithidia oncopelti and to telomeric sequences of Saccharomyces cerevisiae . Conserved Sequence Block 3, present in all trypanosomatids, is one nucleotide different from the consensus in P . serpens and provides a basis to differentiate P . serpens from other trypanosomatids . Electron microscopy of kinetoplast DNA evidenced a network with organization similar to other trypanosomatids and the measurement of minicircles confirmed the size of about 1.45 kb of the sequenced minicircle. J Virol, 1993 May, 67(5), 2689 - 98 Vaccinia virus morphogenesis is blocked by a temperature-sensitive mutation in the I7 gene that encodes a virion component; Kane EM et al.; The ts16 mutation of vaccinia virus WR (R . C . Condit, A . Motyczka, and G . Spizz, Virology 128:429-443, 1983) has been mapped by marker rescue to the I7L open reading frame located within the genomic HindIII I DNA fragment . The I7 gene encodes a 423-amino-acid polypeptide . Thermolabile growth was attributed to an amino acid substitution, Pro-344-->Leu, in the predicted I7 protein . A normal temporal pattern of viral protein synthesis was elicited in cells infected with ts16 at the nonpermissive temperature (40 degrees C) . Electron microscopy revealed a defect in virion assembly at 40 degrees C . Morphogenesis was arrested at a stage subsequent to formation of spherical immature particles . Western immunoblot analysis with antiserum directed against the I7 polypeptide demonstrated an immunoreactive 47-kDa polypeptide accumulating during the late phase of synchronous vaccinia virus infection . Immunoblotting of extracts of wild-type virions showed that the I7 protein is encapsidated within the virus core . The I7 polypeptide displays amino acid sequence similarity to the type II DNA topoisomerase of Saccharomyces cerevisiae. Electrophoresis, 1993 May-Jun, 14(5-6), 523 - 30 Capillary zone electrophoresis of large DNA; Guszczynski T et al.; Capillary zone electrophoresis (CZE) of DNA 23.1 to 48.5 kb in length in polyacrylamide solutions of several concentrations provides evidence for polymer concentration and DNA length-dependent stretching and orientation of these species and suggests an effective separation at a polymer concentration of about 0.6% . Applying a 0.1% polyacrylamide concentration to the lambda-phage DNA ladder, at least 5 components are separated; separation improves with lowering of the field strength to 2 V/cm and, correspondingly, extended duration of CZE . Saccharomyces pombe chromosomal DNA separates into 3 major components on CZE at high field strength (270 V/cm) in 0.9% polyacrylamide solution, confirming a previous finding made on electrophoresis in a 1.1 mm ID tube at low field strength . However, the finding is limited to one source of the DNA plug, and the chromosomal identity of the components remains unknown . Methodological problems in the CZE of large DNA relate to the need for extended duration of pressure injection if absorbance detection is applied, the need to define the starting zone after extended pressure injection, the need to melt and digest agarose plugs prior to loading, and related needs for thermostating of the sample chamber and for software compatible with low voltage operation. Mol Biol Cell, 1993 May, 4(5), 495 - 510 FUS3 phosphorylates multiple components of the mating signal transduction cascade: evidence for STE12 and FAR1; Elion EA et al.; The mitogen-activated protein (MAP) kinase homologue FUS3 mediates both transcription and G1 arrest in a pheromone-induced signal transduction cascade in Saccharomyces cerevisiae . We report an in vitro kinase assay for FUS3 and its use in identifying candidate substrates . The assay requires catalytically active FUS3 and pheromone induction . STE7, a MAP kinase kinase homologue, is needed for maximal activity . At least seven proteins that specifically associate with FUS3 are phosphorylated in the assay . Many of these substrates are physiologically relevant and are affected by in vivo levels of numerous signal transduction components . One substrate is likely to be the transcription factor STE12 . A second is likely to be FAR1, a protein required for G1 arrest . FAR1 was isolated as a multicopy suppressor of a nonarresting fus3 mutant and interacts with FUS3 in a two hybrid system . Consistent with this FAR1 is a good substrate in vitro and generates a FUS3-associated substrate of expected size . These data support a model in which FUS3 mediates transcription and G1 arrest by direct activation of STE12 and FAR1 and phosphorylates many other proteins involved in the response to pheromone. Mutagenesis, 1993 May, 8(3), 175 - 8 Genotoxic and biochemical effects of dimethylamine; Galli A et al.; The genotoxicity of dimethylamine (DMA) was investigated in the D7 strain of Saccharomyces cerevisiae . DMA was able to induce mitotic gene conversion and point reverse mutation in the presence of metabolic activation (S9 fraction) . To study the co-mutagenicity/co-carcinogenicity or toxicity of DMA, changes in xenobiotic metabolizing enzymes were studied in purified microsomes from DMA-induced mice or rats receiving a single or three consecutive doses (25 or 50 mg/kg body wt) . Pentoxyresorufin O-dealkylase (class IIB1 P450, PROD), ethoxyresorufin O-deethylase (IA1, EROD) and p-nitrophenol hydroxylase (IIE1, pNPH) were all affected by DMA treatment with a loss of activity of 62, 55 and 54% in the mouse, or 64, 25 and 56% in the rat for PROD, EROD and pNPH activities, respectively, at the highest tested dose . No significant alteration of P450 IIIA-like activity was seen . Results indicated that the metabolites of DMA induced genetic activity in yeast . In addition, a clear non-specific hepatotoxic effect was recorded as a significant reduction in activity of the selected monooxygenases. Curr Genet, 1993 May-Jun, 23(5-6), 414 - 22 Donation of information to the unbroken chromosome in double-strand break repair; Roitgrund C et al.; We have used transformation of yeast with linearized plasmids to study the transfer of information to the unbroken chromosome during double-strand break repair . Using a strain which carried the wild-type HIS3 allele, and a linearized plasmid which carried a mutant his3 allele, we have obtained His- transformants . In these, double-strand break repair has resulted in precise transfer of genetic information from the plasmid to the chromosome . Such repair events, we suggest, are gene conversions which entail the formation of heteroduplex DNA on the (unbroken) chromosome . If this suggestion is correct, our results reflect the spatial distribution of such heteroduplex DNA . Transfer of information from the plasmid to the chromosome was obtained at a maximal frequency of 1.5% of the repair events, and showed a dependence with distance . Transformation to His- was also obtained with a 2-kbp insertion and with a deletion of 200 bp . The latter results suggest that gene conversion of large heterologies can occur via repair of a heteroduplex DNA intermediate. Chromosome Res, 1993 May, 1(1), 27 - 36 A protein homologous to human Ku p70-protein is required for reconstitution of Xenopus sperm pronuclei; Higashiura M et al.; The monoclonal antibody mAbH6, which recognizes one human Ku-protein (p70), cross-reacted with the counterpart protein from Xenopus eggs . The Xenopus antigen purified with a mAbH6-Sepharose column was a complex of 88 kDa and 72 kDa proteins . The role of Ku protein in nuclear structure formation was studied using a cell-free nuclear assembly extract derived from Xenopus interphase eggs . The protein was distributed on the surface of demembranated sperm chromatin and in the membrane vesicle fraction of the nuclear assembly extract . Addition of mAbH6 to the assembly extracts prevented the completion of reconstitution of pronuclei from demembranated sperm chromatins, although partial decondensation mediated by nucleoplasmin was not impeded . As a result, the sperm chromatin remained in partially swollen structures or formed round but small anomalous nuclei . Nuclear membranes were formed on the nuclei in mAbH6-inhibited extract systems, but DNA synthesis was largely decreased, suggesting the incomplete reconstitution of pronuclei . The incorporation of lamin to the nuclei was inhibited by mAbH6 . It is suggested that the Xenopus Ku-homologous protein has a role in the formation of the lamin layer after nuclear membrane reconstitution. Mol Cell Biol, 1993 May, 13(5), 3050 - 7 Loss of mitochondrial hsp60 function: nonequivalent effects on matrix-targeted and intermembrane-targeted proteins; Hallberg EM et al.; We have created yeast strains in which the mitochondrial chaperonin, hsp60, can be either physically depleted or functionally inactivated . Cells completely depleted of hsp60 stop growing but retain for awhile the capacity to reaccumulate hsp60 . While this newly made hsp60 is targeted to and processed correctly within the mitochondrion, assembly of a functional hsp60 complex does not occur . Rather, the hsp60 monomers are localized in different-size soluble complexes containing another mitochondrial chaperone, the mitochondrial form of hsp70 . A number of other mitochondrial matrix-targeted proteins synthesized in the absence of functional hsp60 are imported into mitochondria but often show some buildup of precursor forms and, unlike hsp60, accumulate as insoluble aggregates . By contrast, several mitochondrial proteins normally targeted to the intermembrane space show normal processing in the complete absence of a functional hsp60 complex . Similar and complementary results were obtained when we examined the metabolism of matrix- and intermembrane space-localized proteins in cells expressing three different temperature-sensitive alleles of HSP60 . In all cases, matrix-targeted proteins synthesized at nonpermissive (i.e., hsp60-inactivating) temperatures were correctly targeted to and processed within mitochondria but accumulated predominantly or totally as insoluble aggregates . The metabolism of two intermembrane space proteins, cytochrome b2 and cytochrome c1, was unaffected at the nonpermissive temperature, as judged by the correct processing and complete solubility of newly synthesized forms of both proteins . These findings are discussed with regard to current models of intermembrane targeting. J Biotechnol, 1993 May, 29(1-2), 75 - 89 Automatic bioprocess control . 5 . Biologically and technically caused effects during cultivation; Locher G et al.; Experimental programs are the basis for the development of new processes as well as for basic biological research . Hence, an unbiased approach is essential, otherwise the interpretation of results will be misleading . The complex chemical composition of cultivation media in combination with the impedded measuring under monoseptic conditions are ideal circumstances for misinterpretations and unsuited experimental approaches . In this article, practical examples for such pitfalls are given . They are undervalued in mass transfer and mixing effects, error propagation during RQ determination and possible influence of the medium preparation on the time evolution of the growth process . Further, the difficulty of sound interpretation is demonstrated by a batch cultivation carried out under sinusoidal changes of the stirrer speed . Summary conclusions close this series about equipment, methodology and benefits of bioprocess automation. Dev Comp Immunol, 1993 May-Jun, 17(3), 229 - 40 Phylogeny of immune recognition: fine specificity of fish immune repertoires to cytochrome C; Vallejo AN et al.; Using the structurally defined protein antigen cytochrome C, studies were conducted in an attempt to delineate the fine specificities of channel catfish immune repertoires . We have previously reported that species variants of cytochrome C were cross-stimulatory to peripheral blood leukocytes (PBL) from catfish immunized with the pigeon variant . Molecular database analyses revealed the existence of overlapping epitopes that appear to define the specificity of the immune response to a "family" of closely related antigens . To further explore these observations, studies were conducted to determine the contribution of peptide 81-104 to the immunogenicity of cytochrome C . Current data showed that peptide 81-104 and intact cytochrome C were stimulatory to PBL from fish previously immunized with the native molecule . In contrast, PBL from fish previously primed with the peptide 81-104 responded only to the immunizing peptide as well as to some, but not all, variants of the peptide 81-104 . The differences in the stimulatory capacities of the peptide variants appeared to correlate with amino acid substitutions at various positions of the peptide and changes in their predicted secondary structures. Anal Biochem, 1993 May 1, 210(2), 226 - 30 Synthesis and use of a chromogenic substrate analog for Ap4A catabolic enzymes; Garrison PN et al.; ATP was coupled with 5-bromo-4-chloro-3-indolyl phosphate using a water-soluble carbodiimide to yield 5-bromo-4-chloro-3-indolyl tetraphospho-5'-adenosine (BCIp4A) which is an analog of diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) . BCIp4A is a chromogenic substrate for three different types of Ap4A catabolic enzyme in alkaline phosphatase-coupled reactions . Ap4A phosphorylase I from Saccharomyces cerevisiae was used as a model enzyme to demonstrate that BCIp4A stains for enzymic activity in polyacrylamide gels under nondenaturing conditions . A yeast colony assay was developed to detect Ap4A phosphorylase I activity in situ using BCIp4A as a chromogenic substrate . Ap4A phosphorylase I was assayed in situ in yeast transformed with a multicopy plasmid containing APA1, the gene encoding Ap4A phosphorylase I . BCIp4A should facilitate screening of genomic or cDNA libraries for genes encoding Ap4A catabolic enzymes. Mutat Res, 1993 May, 287(1), 23 - 8 An evaluation of the use of in vitro tubulin polymerisation, fungal and wheat assays to detect the activity of potential chemical aneugens; Parry JM; The test chemicals included in the EC Aneuploidy Project were evaluated for their ability to induce aneuploidy or aneuploidy related endpoints in assays using in vitro tubulin polymerisation, fungi and wheat . The results obtained demonstrated considerable qualitative and quantitative differences between the responses of the assays to the 10 test chemicals . Fungal assays failed to respond to the potent mammalian spindle poisons colchicine and vinblastine and only three chemicals were positive in all three fungal test systems i.e . chloral hydrate, thimerosol and thiabendazole . The in vitro tubulin polymerisation assays produced unambiguous positive results with three chemicals i.e . colchicine, thimerosol and vinblastine sulphate . The hexaploid wheat assay produced a positive response with 8 of the test chemicals i.e . colchicine, econazole, thimerosol, pyrimethamine, thiabendazole, cadmium chloride, vinblastine and diazepam . However, the wheat assay was relatively insensitive to the potent spindle poison colchicine. Biochem Biophys Res Commun, 1993 Apr 30, 192(2), 590 - 6 The primary structure of L37--a rat ribosomal protein with a zinc finger-like motif; Chan YL et al.; The amino acid sequence of the rat 60S ribosomal subunit protein L37 was deduced from the sequence of nucleotides in a recombinant cDNA . Ribosomal protein L37 has 96 amino acids, the NH2-terminal methionine is removed after translation of the mRNA, and has a molecular weight of 10,939 . Ribosomal protein L37 has a single zinc finger-like motif of the C2-C2 type . Hybridization of the cDNA to digests of nuclear DNA suggests that there are 13 or 14 copies of the L37 gene . The mRNA for the protein is about 500 nucleotides in length . Rat L37 is related to Saccharomyces cerevisiae ribosomal protein YL35 and to Caenorhabditis elegans L37 . We have identified in the data base a DNA sequence that encodes the chicken homolog of rat L37. Biochim Biophys Acta, 1993 Apr 30, 1181(2), 141 - 7 Long chain fatty acid utilization of T-cells from autoimmune MRL-lpr/lpr mice; Ando S et al.; To test the hypothesis that T-cells which exhibit abnormal immunological behavior manifest derangements in the de novo synthesis of phospholipids, the utilization of {3H}palmitic acid in B220+ T-cells from autoimmune MRL-lpr/lpr mice was investigated . The rate of incorporation of {3H}palmitic acid into membrane phospholipids was markedly increased in intact B220+ T-cells compared to that in T-cells from immunologically normal mice . The activities of two key enzymes involved in the de novo synthesis of palmitoyl-phospholipids, acyl-coenzyme (CoA) ligase and acyl-CoA; sn-glycerol-3-phosphate acyl transferase, were significantly higher in homogenates from B220+ T-cell membranes compared with those in controls . Despite these findings, the molar concentration of individual palmitoyl glycerolipids was equivalent in the membranes of B220+ T-cells and control lymph node T-cells . The results indicate that T-cells from lupus mice exhibit complex defects in the biosynthesis and turnover of membrane phospholipids and suggest the possibility that these aberrations contribute to T-cell dysfunction in autoimmune diseases.
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