Mol Cell Biol, 1992 Apr, 12(4), 1568 - 77 Production of the CYS3 regulator, a bZIP DNA-binding protein, is sufficient to induce sulfur gene expression in Neurospora crassa; Paietta JV; The cys-3+ gene of Neurospora crassa encodes a bZIP (basic region-leucine zipper) regulatory protein that is essential for sulfur structural gene expression (e.g., ars-1+) . Nuclear transcription assays confirmed that cys-3+ was under sulfur-regulated transcriptional control and that cys-3+ transcription was constitutive in sulfur controller (scon)-negative regulator mutants . Given these results, I have tested whether expression of cys-3+ under high-sulfur (repressing) conditions was sufficient to induce sulfur gene expression . The N . crassa beta-tubulin (tub) promoter was fused to the cys-3+ coding segment and used to transform a cys-3 deletion mutant . Function of the tub::cys-3 fusion in homokaryotic transformants grown under high-sulfur conditions was confirmed by Northern (RNA) and Western immunoblot analysis . The tub::cys-3 transformants showed arylsulfatase gene expression under normally repressing high-sulfur conditions . A tub::cys-3ts fusion encoding a temperature-sensitive CYS3 protein was used to confirm that the induced structural gene expression was due to CYS3 protein function . Constitutive CYS3 production did not induce scon-2+ expression under repressing conditions . In addition, a cys-3 promoter fusion to lacZ showed that CYS3 production was sufficient to induce its own expression and provides in vivo evidence for autoregulation . Finally, an apparent inhibitory effect observed with a strain carrying a point mutation at the cys-3 locus was examined by in vitro heterodimerization studies . These results support an interpretation of CYS3 as a transcriptional activator whose regulation is a crucial control point in the signal response pathway triggered by sulfur limitation. Mol Cell Biol, 1992 Apr, 12(4), 1412 - 21 Characterization of the formate (for) locus, which encodes the cytosolic serine hydroxymethyltransferase of Neurospora crassa; McClung CR et al.; Serine hydroxymethyltransferase (SHMT) occupies a central position in one-carbon (C1) metabolism, catalyzing the reaction of serine and tetrahydrofolate to yield glycine and 5,10-methylenetetrahydrofolate . Methylenetetrahydrofolate serves as a donor of C1 units for the synthesis of numerous compounds, including purines, thymidylate, lipids, and methionine . We provide evidence that the formate (for) locus of Neurospora crassa encodes cytosolic SHMT . The for+ gene was localized to a 2.8-kb BglII fragment by complementation (restoration to formate-independent growth) of a strain carrying a recessive for allele, which confers a growth requirement for formate . The for+ gene encodes a polypeptide of 479 amino acids which shows significant similarity to amino acid sequences of SHMT from bacterial and mammalian sources (47 and 60% amino acid identity, respectively) . The for+ mRNA has several different start and stop sites . The abundance of for+ mRNA increased in response to amino acid imbalance induced by glycine supplementation, suggesting regulation by the N . crassa cross-pathway control system, which is analogous to general amino acid control in Saccharomyces cerevisiae . This was confirmed by documenting that for+ expression increased in response to histidine limitation (induced by 3-amino-1,2,4-triazole) and that this response was dependent on the presence of a functional cross-pathway control-1 (cpc-1) gene, which encodes CPC1, a positively acting transcription factor . There are at least five potential CPC1 binding sites upstream of the for+ transcriptional start, as well as one that exactly matches the consensus CPC1 binding site in the first intron of the for+ gene. Behring Inst Mitt, 1992 Apr, (91), 13 - 20 The CEPH YAC library; Dausset J et al.; Because of their large size, YACs provide is a powerful tool for physical mapping studies of complex genomes . As it will be advantageous to have genomic libraries of clones with large inserts for analyzing megabase sized regions of the human genome, we have investigated a number of parameters in order to increase the insert size of the YACs . We constructed a genomic library currently containing more than 85,000 YAC clones . Mean sizes of YACs produced at several stages of construction of the library range from 430 kb to 1,200 kb, representing 13 haploid equivalents of the human genome . This library was organized in order to allow rapid screen of YACs for large scale physical mapping of the human genome. Yeast, 1992 Apr, 8(4), 315 - 23 A Ser/Thr-rich multicopy suppressor of a cdc24 bud emergence defect; Bender A et al.; MSB2 was identified previously as a multicopy suppressor of a temperature-sensitive mutation in CDC24, a gene required for polarity establishment and bud formation in Saccharomyces cerevisiae . The inferred MSB2 product contains 1306 amino acids, 42% of which are Ser or Thr . Its Ser+Thr-richness and hydrophobicity profile suggest that Msb2p may be an integral membrane protein containing a long, periplasmic, N-terminal domain and a short, cytoplasmic, C-terminal domain . Cells that lack MSB2 display no obvious mutant phenotypes . MSB2 is located between the centromere and KSS1 on the right arm of chromosome VII . Although physical mapping suggests that MSB2 and LEU1 (on the left arm of chromosome VII) are approximately 40 kb apart, the genetic map distance observed between leu1 and an msb2::URA3 marker was only 2.3 cM. Yeast, 1992 Apr, 8(4), 253 - 9 Molecular cloning and analysis of autonomous replicating sequence of Candida maltosa; Sasnauskas K et al.; A Candida maltosa chromosomal DNA fragment which confers high frequency transformation of C . maltosa and autonomous replication of recombinant plasmids was cloned and sequenced . Analysis of the nucleotide sequence of the cloned DNA revealed a sequence homologous for C . maltosa autonomously replicating sequence (ARS) elements . Vector pRJ1 for C . maltosa was constructed, which contained a 1.3 kb ARS sequence, pICEM-19H and the ADE1 gene of C . maltosa . Southern blot analysis suggested that the copy number of pRJ1 in C . maltosa was approximately 20 per genome . The sequence analysis also revealed an open reading frame, encoding a polypeptide with high homology (70%) to the RS15 protein of Brugia pagangi . This open reading frame has an intron with canonical sites for correct splicing in Saccharomyces cerevisiae. Biotechniques, 1992 Apr, 12(4), 574 - 9 Rapid separation of DNA molecules by agarose gel electrophoresis: use of a new agarose matrix and a survey of running buffer effects; White HW; This report describes the use of a new type of agarose (FastLane agarose) for faster separation of DNA by agarose gel electrophoresis . DNA molecules separated in this agarose exhibited electrophoretic mobilities up to 30% higher than similar separations in standard analytical grade agarose . DNA molecules of all sizes examined showed higher mobilities in FastLane agarose . The mobility increase was predominantly due to the low electroendosmosis of FastLane agarose and was most pronounced in pulsed field gel electrophoresis separations . The magnitude of mobility increase varied depending on the conditions used for electrophoresis. Mol Biol Cell, 1992 Apr, 3(4), 445 - 50 Phosphorylation of FAR1 in response to alpha-factor: a possible requirement for cell-cycle arrest; Chang F et al.; Exposure of yeast a cells to alpha-factor causes cells to arrest in the G1 phase of the cell cycle . The FAR1 gene is required for this cell-cycle arrest; its product is necessary for the inhibition of a G1 cyclin, CLN2 . Earlier work demonstrated that alpha-factor caused an increase in the transcription of FAR1 severalfold over a measurable basal level . We now show that transcriptional induction of FAR1 from a heterologous promoter is not sufficient to inhibit CLN2 in the absence of alpha-factor . We also show that FAR1 is phosphorylated in response to alpha-factor and propose that this phosphorylation may be required for FAR1 activity. J Autoimmun, 1992 Apr, 5 Suppl A, 67 - 72 Molecular mechanisms of immunosuppression; Baumann G et al.; The immunosuppressive drug cyclosporin A (CsA, Sandimmun, SIM) is currently being evaluated in a variety of autoimmune disorders with some remarkable successes . Despite the wide empiric application of CsA, the precise mechanism of action of this drug remains elusive . To identify the molecular mode of action of CsA in the process of T cell activation, we have compared the biological profile of cyclophilin-binding cyclosporin analogues (CBCA), which lack immunosuppressive properties, with CsA . We have found that CsA binding to its intracellular receptor (cyclophilin) is required but not sufficient for immunosuppression . Moreover, inhibition of the peptidyl-prolyl cis-trans isomerase activity of cyclophilin does not seem to be relevant for the inhibitory effects of CsA . In analogy to the immunosuppressants FK506 and rapamycin, a specific structure at the 'effector' domain of the CsA molecule different from the immunophilin 'binding' domain determines the biological activity . Overall, a significant understanding of the structure-activity relationship of CsA has emerged . This will have a major impact on the identification of the precise mechanism of action of CsA and its side effects in the process of immunosuppression. Mutat Res, 1992 Apr, 266(2), 135 - 41 On spontaneous mutagenesis and cell cultivation conditions; Lyubimova KA et al.; The leu2 revertant content of a Saccharomyces cerevisiae cell culture increases as the leucine concentration in the nutrient solid medium decreases . Reversions form in the S-phase of the cell cycle . If a cell culture from a medium with a low concentration of leucine containing the revertants which have just formed is transferred on a medium with a normal or higher than normal leucine content, these 'newborn' revertants disappear at the end of the G1-phase or at the beginning of the S-phase of the next cell cycle . These data can be explained either by a difference in the ability of revertants formed in the culture to compete with the cells of the initial strain on different media, or on the basis of the intermediate heteroduplex model proposed by F.W . Stahl (1988). Proc Natl Acad Sci U S A, 1992 Apr 1, 89(7), 2907 - 11 CDK2 encodes a 33-kDa cyclin A-associated protein kinase and is expressed before CDC2 in the cell cycle; Elledge SJ et al.; Critical cell cycle transitions are controlled by the coordinate actions of the p34cdc2 protein kinase and its regulatory subunits, cyclins . Recently we identified another human p34 homolog, cyclin-dependent kinase 2 (CDK2) by complementation of a cdc28-4 mutation in Saccharomyces cerevisiae using a lambda YES human cDNA expression library . CDK2 is 66% identical to CDC2Hs and 89% identical to the Xenopus Eg1 gene, forming a distinct subfamily of CDC2-related protein kinases . We have found that CDK2 encodes a 33-kDa cyclin A-associated protein kinase that contains phosphotyrosine, two characteristics it shares with CDC2Hs . However, we show that the subunit composition of these two protein kinase complexes can vary in different cell types, that they have different in vitro substrate preferences, and that CDK2 mRNA is observed much earlier than CDC2Hs mRNA when lymphocytes are stimulated to enter the cell cycle . We suggest that cells in different developmental or transformed states may have different mechanisms of cell cycle regulation. Genes Dev, 1992 Apr, 6(4), 568 - 77 The combination of dissimilar alleles of the A alpha and A beta gene complexes, whose proteins contain homeo domain motifs, determines sexual development in the mushroom Coprinus cinereus; Kues U et al.; The A mating-type factor is one of two gene complexes that allows mating cells of the mushroom Coprinus cinereus to recognize self from nonself and to regulate a pathway of sexual development that leads to meiosis and sporulation . We have identified seven A genes separated into two subcomplexes corresponding to the classical A alpha and A beta loci . Four genes, one alpha and three beta, all coding for proteins with a homeo domain-related motif, determine A-factor specificity; their allelic forms are so different in sequence that they do not cross-hybridize . It requires only one of these four genes to be heteroallelic in a cell to trigger A-regulated sexual development, and it is the different combinations of their alleles that generate the multiple A factors found in nature . The other three genes cause no change in cell morphology and may regulate the activity of the four specificity genes. Inflammation, 1992 Apr, 16(2), 117 - 33 Bovine neutrophils recruited by endotoxin to a teat cistern continuously produce oxygen radicals and show increased phagocytosis and extracellular chemiluminescence; Sandgren CH et al.; Bovine neutrophils were harvested from a teat cistern following endotoxin infusion and were compared with blood neutrophils by measurements of chemiluminescent and phagocytic activity towards C3- and IgG-opsonized and unopsonized yeast particles . Both phagocytosis and luminol-dependent chemiluminescence elicited by all three particles were enhanced in the teat cells . The increase in the luminol-dependent chemiluminescence towards C3- and IgG-opsonized particles was due to an enhanced extracellular release of myeloperoxidase . The observed increase in phagocytosis of unopsonized yeast was shown to reflect the interaction between up-regulated CR3 receptors on the surface of the teat neutrophils and the yeast particles . A high chemiluminescent activity of the teat neutrophils in both the luminol- and lucigenin-dependent systems in the absence of a phagocytic prey indicated that the NADPH oxidase was permanently active and that myeloperoxidase was continuously released by the cells . Treatment of neutrophils with cytochalasin B showed that the chemiluminescence and phagocytosis of teat neutrophils were less sensitive to this drug than that of blood neutrophils . These results indicate that the teat neutrophils have up-regulated their receptors for IgG- and C3-opsonized and unopsonized yeast on the cell surface by the action of actin . The cells also have a permanently active NADPH oxidase dependent on the association with actin and show a higher tendency than blood neutrophils to secrete the content of their primary granules during phagocytosis. J Mol Evol, 1992 Apr, 34(4), 331 - 5 Planarian mitochondria . II . The unique genetic code as deduced from cytochrome c oxidase subunit I gene sequences; Bessho Y et al.; The cytochrome c oxidase subunit I (COI) gene sequences from planarian (Dugesia japonica) DNA, most probably of mitochondrial origin, are heterogeneous . Taking advantage of the heterogeneity that occurs primarily in silent sites of the COI DNA sequences, amino acid assignments of several codons have been deduced as nonuniversal: UGA = Trp, AAA = Asp, and AGR (R: A or G) = Ser . In addition, UAA, a stop codon in the universal genetic code, is tentatively assumed to be a tyrosine codon, because three of the sequences examined have UAA at the well-conserved tyrosine site of UAY (Y: U or C) in other planarian sequences as well as in the mitochondria of human, Xenopus, sea urchin, Drosophila, Trypanosoma, and Saccharomyces cerevisiae . AUA would most probably be an isoleucine codon in these mitochondria, whereas it is a methionine codon in the majority of nonplant mitochondria. EMBO J, 1992 Apr, 11(4), 1383 - 90 p53: a transdominant regulator of transcription whose function is ablated by mutations occurring in human cancer; Unger T et al.; Gal4-p53 fusion constructs demonstrate that wild type p53 is a potent transactivator in human lung cancer cells with the transactivation domain for p53 residing in amino acids 1-42 . Strikingly, a variety of lung cancer derived p53 mutations occurring outside this domain disrupt this activity . Temperature sensitive conformational shifts of p53 mutant proteins to the wild type form exist and, with a temperature downshift, several mutants become transcriptionally active . Wild type p53 protein is known to form oligomers with mutant p53 and cotransfection of wild type and mutant genes shows that p53 acts in a transdominant manner that is independent of the DNA binding specificity . Transcription is either increased or decreased depending on whether the wild type is more or less abundant than the mutant form . Finally, lung cancers differ in their ability to support the transactivation related functions, providing evidence of other abnormalities of the p53 system in human cancer. Genes Dev, 1992 Apr, 6(4), 557 - 67 Direct induction of G1-specific transcripts following reactivation of the Cdc28 kinase in the absence of de novo protein synthesis; Marini NJ et al.; In Saccharomyces cerevisiae, the genes encoding the HO endonuclease, G1-specific cyclins CLN1 and CLN2, as well as most proteins involved in DNA synthesis, are periodically transcribed with maximal levels reached in late G1 . For HO and the DNA replication genes, cell cycle stage-specific expression has been shown to be dependent on the Cdc28 kinase and passage through START . Here, we show that cells released from cdc28ts arrest in the presence of cycloheximide show wild-type levels of induction for HO, CLN1, and CDC9 (DNA ligase) . Induction is gradual with a significant lag not seen in untreated cells where transcript levels fluctuate coordinately with the cell cycle . This lag may be due, at least in part, to association of the Cdc28 peptide with G1 cyclins to form an active kinase complex because overexpression of CLN2 prior to release in cycloheximide increases the rate of induction for CDC9 and HO . Consistent with this, release from pheromone arrest (where CLN1 and CLN2 are not expressed) in cycloheximide shows no induction at all . Transcriptional activation of CDC9 is likely to be mediated through a conserved promoter element also present in genes for other DNA synthesis enzymes similarly cell cycle regulated . The element contains an intact MluI restriction enzyme recognition site (consensus approximately 5'-A/TPuACGCGTNA/T-3') . Insertion of a 20-bp fragment from the CDC9 promoter (containing a MluI element) upstream of LacZ confers both periodic expression and transcriptional induction in cycloheximide following release from cdc28ts arrest . High levels of induction depended on both the MluI element and CDC28 . These results suggest that the activity of trans-acting factors that operate through the MluI element may be governed by phosphorylation by the Cdc28 kinase. Proc Natl Acad Sci U S A, 1992 Apr 1, 89(7), 3025 - 9 Suramin is an inhibitor of DNA topoisomerase II in vitro and in Chinese hamster fibrosarcoma cells; Bojanowski K et al.; The antitrypanosomal and antifiliarial drug suramin is currently under investigation for treatment of advanced malignancies including prostatic cancer, adrenocortical cancer, and some lymphomas and sarcomas . Here we show that suramin is a potent inhibitor of the nuclear enzyme DNA topoisomerase II . Suramin inhibited purified yeast topoisomerase II with an IC50 of about 5 microM, as measured by decatenation or relaxation assays . Suramin did not stabilize the covalent DNA-topoisomerase II reaction intermediate ("cleavable complex"), whereas other inhibitors of this enzyme, such as amsacrine, etoposide, and the ellipticines, are known to stabilize the intermediate . In contrast, the presence of suramin strongly inhibited the cleavable-complex formation induced by amsacrine or etoposide . Accumulation of the endogenous cleavable complex was also inhibited . Suramin entered the nucleus of DC-3F Chinese hamster fibrosarcoma cells exposed to radiolabeled suramin for 24 hr as shown by both optic and electron microscopy . The suramin present in the nucleus seemed to interact with topoisomerase II, since suramin reduced the number of amsacrine-induced protein-associated DNA strand breaks in DC-3F cells and protected these cells from the cytotoxic action of amsacrine . Cells resistant to 9-hydroxyellipticine, which have been shown to have an altered topoisomerase II activity, are about 7-fold more resistant to suramin than the sensitive parental cells as shown by 72-hr growth inhibition assay . Our results suggest that DNA topoisomerase II is a target of suramin action and that this action may play a role in the cytotoxic activity of suramin. J Virol, 1992 Apr, 66(4), 2594 - 9 Mouse mammary tumor gene int-3: a member of the notch gene family transforms mammary epithelial cells; Robbins J et al.; Expression of a 2.3-kb RNA species is induced in mammary tumors as a consequence of insertional mutagenesis at the int-3 locus by the mouse mammary tumor virus . The nucleotide sequence and biological activity of this mammary tumor-specific int-3 RNA species were determined . It contains an open reading frame which encodes a 57-kDa protein . The translated protein possesses six nearly contiguous 32-amino-acid repeats which are related to a similar motif in the Saccharomyces cerevisiae cdc-10-encoded cell cycle protein . In addition, the int-3 cdc-10 repeats are bounded by the PEST amino acid sequence motif which is commonly found in proteins having a rapid turnover and may represent sites for phosphorylation . The int-3 cdc-10 repeat sequences are 50% identical to a portion of the intracellular domain of the neurogenic Drosophila notch gene product . Activation of expression of a recombinant int-3 genomic DNA fragment encoding the 2.3-kb RNA species in HC11 mouse mammary epithelial cells in vitro induces anchorage-independent growth in soft agar. Hum Mol Genet, 1992 Apr, 1(1), 19 - 28 Reconstruction of the 2.4 Mb human DMD-gene by homologous YAC recombination; Den Dunnen JT et al.; The human dystrophin gene, mutations of which cause Duchenne and Becker muscular dystrophy, measures 2.4 Mb . This size seriously limits its cloning as a single DNA fragment and subsequent in-vitro expression studies . We have used stepwise in-vivo recombination between overlapping yeast artificial chromosomes (YACs) to reconstruct the dystrophin gene . The recombinant YACs are mitotically stable upon propagation in haploid yeast cells . In contrast, specific combinations of YACs display a remarkable mitotic and meiotic instability in diploid cells . Non-disjunction is rare for overlapping YACs, but increases upon sporulation of diploid cells containing non-overlapping molecules . We have exploited this feature in a three-point recombination to bridge a 280 kb gap between two non-overlapping YACs for which no YAC of proper polarity existed . Our largest recombinant YAC measures 2.3 Mb and contains the entire muscle specific DMD-gene with the exception of a 100 kb region containing the in-frame exon 60 . The latter segment has a high tendency to undergo deletions in multi-molecular interactions, probably due to the presence of as yet unidentified instability-enhancing sequences . Fluorescent in situ hybridizations confirmed that the 2.3 Mb DMD YAC contained Xp21-sequences only and indicated a compact tertiary structure of the DMD-gene in interphase lymphocyte nuclei . We conclude that the yeast system is a flexible, efficient and generally applicable tool to reconstruct or build genomic regions from overlapping YAC constituents . Its application to the human dystrophin gene has provided many possibilities for future studies. Hum Mol Genet, 1992 Apr, 1(1), 47 - 52 Isolation of a new gene from the distal short arm of the human X chromosome that escapes X-inactivation; Yen PH et al.; A gene, designated GS1, was identified by its association with a CpG island approximately 100 kb telomeric to the steroid sulfatase (STS) locus on the distal short arm of the human X chromosome . Both cDNA and genomic clones of the GS1 gene have been isolated and characterized . The cDNA clone detects a 2.3 kb transcript in human placenta and fibroblasts, and may encode a protein of 214 amino acid residues . Although sequences homologous to GS1 cDNA are present on chromosomes 1, 20, X, and Y, the functional GS1 gene is on the X chromosome . The GS1 gene appears to be non-essential, as there are no obvious clinical differences between STS deficient patients with point mutations in the STS gene, and patients with a deletion of the STS and GS1 genes . The GS1 gene is expressed from mouse-human cell hybrids containing active or inactive human X chromosomes, indicating that it escapes X inactivation . Characterization of GS1 genomic clones revealed that the gene consists of 4 exons spanning over 105 kb, with its transcriptional direction opposite to that of the STS gene . The isolation and characterization of a new gene which escapes X inactivation from distal Xp is of interest as it adds to our understanding of the structural organization of the human X chromosome and may help in providing clues regarding the mechanism of X-inactivation. Biochemistry, 1992 Mar 31, 31(12), 3197 - 203 The DNA-binding domain of the Cys-3 regulatory protein of Neurospora crassa is bipartite; Kanaan MN et al.; Cys-3, the major sulfur regulatory gene of Neurospora crassa, encodes a regulatory protein that is capable of sequence-specific interaction with DNA . The interaction is mediated by a region within the CYS3 protein (the bzip region) which contains a potential dimer-forming surface, the leucine zipper, and an adjacent basic DNA contact region, NH2-terminal to the leucine zipper . To investigate the bipartite nature of the bzip region, a series of cys-3 mutants obtained by oligonucleotide-directed mutagenesis were expressed and tested for dimer formation as well as DNA binding and in vivo function . The results demonstrate that CYS3 protein exists as a dimer in the presence and absence of the target DNA and that dimerization of CYS3 is mediated strictly by the leucine zipper, which is required for both cys-3 function in vivo and DNA-binding activity in vitro . Furthermore, a truncated CYS3 protein corresponding to just the bzip region was found to mediate dimer formation and to possess DNA-binding activity . A CYS3 mutant protein with a pure methionine zipper showed significant, although reduced, function in vivo and in vitro. Nature, 1992 Mar 26, 356(6367), 358 - 61 Suppression of a myosin defect by a kinesin-related gene; Lillie SH et al.; Motor proteins in cells include myosin, which is actin-based, and kinesin, dynein and dynamin, which are microtubule-based . Several proteins have recently been identified that have amino-acid sequences with similarity to the motor domains of either myosin or kinesin, but are otherwise dissimilar . This has led to the suggestion that these may all be motor proteins, but that they are specialized for moving different cargos . Genetic analysis can address the question of the different functions of these new proteins . Studies of a temperature-sensitive mutation (myo2-66) in a gene of the myosin superfamily (MYO2) have implicated the Myo2 protein (Myo2p) in the process of polarized secretion in yeast (Saccharomyces cerevisiae) . To understand more about the role of Myo2p, we have looked for 'multicopy suppressors' (heterologous genes that, when overexpressed, can correct the temperature sensitivity of the myo2-66 mutant) . Here we report the identification of such a suppressor (SMY1) that (surprisingly) encodes a predicted polypeptide sharing sequence similarity with the motor portion of proteins in the kinesin superfamily. J Biol Chem, 1992 Mar 25, 267(9), 5942 - 8 A sequence in beta-hexosaminidase from Dictyostelium discoideum required for sorting of proteins to a compartment involved in developmentally induced secretion; Lacoste CH et al.; To study the sorting of proteins in Dictyostelium discoideum, we used vector constructs that contain cDNA coding for the entire beta-hexosaminidase protein to prepare transformants of a mutant that lacks this enzyme activity . These transformants overexpressed active, normally processed beta-hexosaminidase . The overexpressed enzyme colocalized with other acid hydrolases in the soluble fraction of vesicles in the lysosomal region of Percoll gradients . The sorting of other hydrolases was unaltered . We also prepared transformants with constructs that contain 22 (Hex 22-Inv), 70 (Hex 70-Inv), and 532 (Hex 532-Inv) amino-terminal amino acids from beta-hexosaminidase fused in frame with the coding sequence for the yeast SUC2 gene product, invertase . Fusion molecular masses were those expected for fully N-glycosylated proteins . Hex 22-Inv was rapidly (t1/2 less than 30 min) and quantitatively secreted . The others were slowly (t1/2 greater than 5 h) and partially secreted . Each expressed invertase activity . During growth, the invertase activity of Hex 70-Inv and Hex 532-Inv was retained to the same extent as that of endogenous lysosomal enzymes . Most of the Hex 70-Inv migrated in Percoll gradients with vesicles of intermediate density (d = 1.055), but a portion co-migrated with lysosomal enzymes at d = 1.08 . Hex 70-Inv was sulfated, and its N-glycosides were resistant to endoglycosidase H, indicating Golgi processing . Hex 70-Inv and Hex 532-Inv, like endogenous lysosomal enzymes, were subject to developmentally induced secretion. Cell, 1992 Mar 20, 68(6), 1163 - 75 Antifolding activity of hsp60 couples protein import into the mitochondrial matrix with export to the intermembrane space; Koll H et al.; Cytochrome b2 reaches the intermembrane space of mitochondria by transport into the matrix followed by export across the inner membrane . While in the matrix, the protein interacts with hsp60, which arrests its folding prior to export . The bacterial-type export sequence in pre-cytochrome b2 functions by inhibiting the ATP-dependent release of the protein from hsp60 . Release for export apparently requires, in addition to ATP, the interaction of the signal sequence with a component of the export machinery in the inner membrane . Export can occur before import is complete provided that a critical length of the polypeptide chain has been translocated into the matrix . Thus, hsp60 combines two activities: catalysis of folding of proteins destined for the matrix, and maintaining proteins in an unfolded state to facilitate their channeling between the machineries for import and export across the inner membrane . Anti-folding signals such as the hydrophobic export sequence in cytochrome b2 may act as switches between these two activities. Biochemistry, 1992 Mar 17, 31(10), 2798 - 805 Laser flash photolysis study of the kinetics of electron transfer reactions of flavocytochrome b2 from Hansenula anomala: further evidence for intramolecular electron transfer mediated by ligand binding; Walker MC et al.; Intramolecular electron transfer between the heme and flavin cofactors of flavocytochrome b2 is an obligatory step during the enzymatic oxidation of L-lactate and subsequent reduction of cytochrome c . Previous kinetic studies using both steady-state and transient methods have suggested that such intramolecular electron transfer is inhibited when pyruvate, the two-electron oxidation product of L-lactate, is bound at the active site of Hansenula anomala flavocytochrome b2 . In contrast to this, we have recently demonstrated using laser flash photolysis that intramolecular electron transfer could be observed in the flavocytochrome b2 from Saccharomyces cerevisiae only when pyruvate was present {Walker, M., & Tollin, G . (1991) Biochemistry 30, 5546-5555}, despite a large thermodynamic driving force of 100 mV and apparently favorable cofactor geometry as indicated by crystallographic studies . In the present study, we have utilized laser flash photolysis to investigate intramolecular electron transfer in the flavocytochrome b2 from H . anomala in an effort to address these apparently conflicting interpretations with respect to the influence of pyruvate on enzyme properties . The results obtained are closely comparable to those we reported using the protein from Saccharomyces . Thus, in the absence of pyruvate, bimolecular reduction of both the heme and FMN cofactors by deazaflavin semiquinone occurs (k approximately 10(9) M-1 s-1), followed by a protein concentration dependent intermolecular electron transfer from the semiquinone form of the FMN cofactor to the heme (k approximately 10(7) M-1 s-1).(ABSTRACT TRUNCATED AT 250 WORDS) FEBS Lett, 1992 Mar 16, 299(3), 205 - 8 Determination of the DNA binding site of the GAL4 protein . A photo-CIDNP study; Serikawa Y et al.; After the assignment of 1H NMR signals of aromatic side chains by means of specific deuteration, we analyzed the DNA binding site of GAL4 by measuring photo-CIDNP spectra . The results showed that Trp36 is involved in both the specific interaction with UASG and non-specific DNA binding . This residue is located inside the Cys-rich region, but outside the putative Zn-finger . The photo-CIDNP spectrum also showed that the side chains of Tyr40 and His53 are not exposed on the surface of the protein. Proc Natl Acad Sci U S A, 1992 Mar 15, 89(6), 2355 - 9 A gene encoding a putative tyrosine phosphatase suppresses lethality of an N-end rule-dependent mutant; Ota IM 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, mutational inactivation of the N-end rule pathway is neither lethal nor phenotypically conspicuous . We have used a "synthetic lethal" screen to isolate a mutant that requires the N-end rule pathway for viability . An extragenic suppressor of this mutation was cloned and found to encode a 750-residue protein with strong sequence similarities to protein phosphotyrosine phosphatases . This heat-inducible gene was named PTP2 . Null ptp2 mutants grow slowly, are hypersensitive to heat, and are viable in either the presence or absence of the N-end rule pathway . We discuss possible connections between dephosphorylation of phosphotyrosine in proteins and the N-end rule pathway of protein degradation. Proc Natl Acad Sci U S A, 1992 Mar 15, 89(6), 2185 - 9 RNA-dependent RNA polymerase consensus sequence of the L-A double-stranded RNA virus: definition of essential domains; Ribas JC et al.; The L-A double-stranded RNA virus of Saccharomyces cerevisiae makes a gag-pol fusion protein by a -1 ribosomal frameshift . The pol amino acid sequence includes consensus patterns typical of the RNA-dependent RNA polymerases (EC 2.7.7.48) of (+) strand and double-stranded RNA viruses of animals and plants . We have carried out "alanine-scanning mutagenesis" of the region of L-A including the two most conserved polymerase motifs, SG...T...NT..N ( . = any amino acid) and GDD . By constructing and analyzing 46 different mutations in and around the RNA polymerase consensus regions, we have precisely defined the extent of domains and specific residues essential for viral replication . Assuming that this highly conserved region has a common secondary structure among different viruses, we predict a largely beta-sheet structure. Arch Biochem Biophys, 1992 Mar, 293(2), 342 - 8 17 beta-estradiol hydroxylation catalyzed by human cytochrome P450 1A1: a comparison of the activities induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin in MCF-7 cells with those from heterologous expression of the cDNA; Spink DC et al.; Exposure of MCF-7 breast cancer cells to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes an elevated cytochrome P450 content and a marked increase in the microsomal hydroxylation of 17 beta-estradiol (E2) at the C-2, C-4, C-15 alpha, and C-6 alpha positions . In this study we investigated the involvement of cytochromes P450 of the 1A gene subfamily in this metabolism of E2 . Hydroxylation at each of these four positions of E2 was inhibited by P450 1A-subfamily inhibitors, alpha-naphthoflavone, benzo{a}pyrene, and 7-ethoxyresorufin . Northern blots showed that treatment of MCF-7 cells with TCDD resulted in production of the 2.6-kb CYP1A1 mRNA, but not the 3.0-kb CYP1A2 mRNA . Immunoblot analyses with anti-P450 1A antibodies confirmed the production of P450 1A1 protein in TCDD-treated MCF-7 cells . Anti-rat P450 1A IgG inhibited the hydroxylation of E2 at C-2, C-15 alpha, and C-6 alpha, but not hydroxylation at C-4 . E2 hydroxylation by human cytochromes P450 1A1 and P450 1A2 was assessed in experiments with microsomes from Saccharomyces cerevisiae after transformation with cDNAs encoding the two cytochromes . The major hydroxylase activities of expressed human P450 1A1 were at the C-2, C-15 alpha, and C-6 alpha positions of E2; expressed human P450 1A2 catalyzed hydroxylation predominately at C-2 . While both expressed P450s 1A1 and 1A2 had minor hydroxylase activities at the C-4 position, neither catalyzed a low-Km hydroxylation at C-4 similar to that observed with microsomes from TCDD-treated MCF-7 cells . These results provide strong evidence that P450 1A1 catalyzes the hydroxylations of E2 at the C-2, C-15 alpha, and C-6 alpha in incubations with microsomes from TCDD-treated MCF-7 cells, but suggest TCDD may also induce a cytochrome P450 E2 4-hydroxylase that is distinct from P450 1A1 or P450 1A2. FEBS Lett, 1992 Mar 9, 299(2), 163 - 5 Kinetic mechanism of pyruvate decarboxylase . Evidence for a specific protonation of the enzymic intermediate; Ermer J et al.; Decarboxylation of pyruvate by pyruvate decarboxylase (EC 4.1.1.1) was performed in a reaction mixture containing 50% deuterium . The isolated product, acetaldehyde, was investigated directly by 1H NMR and by mass spectrometry after conversion to the 2,4-dinitrophenyl hydrazone . The protium content of 56% at acetaldehyde C1 demonstrates a specific protonation of the corresponding intermediate by the enzyme . Proton inventory studies and enzyme modification indicate the 4' amino group of the coenzyme, thiamine pyrophosphate, in an immonium structure being a possible proton donor . A 'partially concerted' mechanism is suggested for the reaction steps following the decarboxylation. J Biol Chem, 1992 Mar 5, 267(7), 4806 - 14 Systematic mutational analysis of cAMP-dependent protein kinase identifies unregulated catalytic subunits and defines regions important for the recognition of the regulatory subunit; Gibbs CS et al.; A library of mutants of the catalytic subunit of the Saccharomyces cerevisiae cAMP-dependent protein kinase was screened in vitro for mutants defective in the recognition of the regulatory subunit . The mutations identified were mapped onto the three-dimensional structure of the mouse catalytic subunit with a peptide inhibitor . Mutations defective in the recognition of both the regulatory subunit and the peptide substrate Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) mapped to the peptide-binding site shared by all substrates and inhibitors of the catalytic subunit and functionally define the binding site for the autoinhibitor sequence in the hinge region of the regulatory subunit . Mutants defective only in the recognition of the regulatory subunit identified residues that comprise additional binding sites for the regulatory subunit . The majority of these residues are clustered on the surface of the catalytic subunit in a region flanking the distal portion of the autoinhibitor/peptide-binding site . The simultaneous substitution of Lys233, Asp237, Lys257, and Lys261 in this region caused a 260-fold decrease in affinity for the regulatory subunit, whereas the catalytic efficiency toward Kemptide decreased by only 1.8-fold . The substitution of autophosphorylated Thr241, also in this region, and the 3 residues interacting with the phosphate also caused an unregulated phenotype. J Biol Chem, 1992 Mar 5, 267(7), 4322 - 6 Adenosine phosphorothioates (ATP alpha S and ATP tau S) differentially affect the two steps of mammalian pre-mRNA splicing; Tazi J et al.; We have investigated the function of ATP hydrolysis in mammalian pre-mRNA in vitro splicing using adenosine phosphorothioates (ATP alpha S and ATP tau S) known to affect the activity of a number of ATP-requiring enzymes . Spliceosome assembly, but neither one of the two transesterification reactions involved in splicing, occurs with ATP alpha S suggesting that at least two types of ATP-requiring factors are brought into play . ATP alpha S has no effect in the presence of normal ATP and, therefore, spliceosomes assembled in the presence of ATP alpha S remain competent for splicing when supplied with normal ATP . ATP tau S noticeably and irreversibly inhibits the second transesterification reaction, i.e . at a time when most of the analog has been hydrolyzed and regenerated to normal ATP by creatine phosphate . This indicates that the inhibition results from an earlier event, most likely the thiophosphorylation of spliceosomal proteins . Under this assumption, the inhibition could be due to the failure of the thiophosphorylated proteins to be dephosphorylated . Indeed, okadaic acid, a potent inhibitor of protein phosphatases, inhibits the second step of a reaction in the presence of normal ATP . We propose that some splicing factors undergo phosphorylation-dephosphorylation cycles during spliceosome assembly and splicing, while others that could be the mammalian equivalents of the RNA helicase-like proteins recently discovered in yeast most likely bind and hydrolyze ATP. Biochim Biophys Acta, 1992 Mar 4, 1124(2), 112 - 8 Marked enhancement of acyl-CoA synthetase activity and mRNA, paralleled to lipoprotein lipase mRNA, in adipose tissues of Zucker obese rats (fa/fa); Shimomura I et al.; To clarify the role of acyl-CoA synthetase in development of obesity, the mRNA levels and activities were studied in Zucker fatty rats (fa/fa) . In Zucker fatty rats compared with their lean littermates, marked enhancement of ACS were observed in adipose tissues . Obese/lean rats ratio of ACS activity and mRNA in abdominal subcutaneous fat (3.3- and 3.9-fold, respectively) were greater than in mesenteric fat (2.0- and 2.2-fold) . The enhancement of ACS activity and mRNA in the liver of fatty rats (1.2- and 1.8-fold) were less than those in the adipose tissues . There were no enhancement of ACS activities and mRNA levels in heart tissue of the obese rats . LPL mRNA levels were also enhanced in adipose tissue of fatty rats and obese/lean ratio of LPL mRNA was also higher in abdominal subcutaneous fat than mesenteric fat (6.2- vs 3.1-fold) . The larger obese/lean rats ratio of LPL and ACS parameters in abdominal subcutaneous fat than mesenteric fat may be related to the observation that the increase of subcutaneous fat weight was larger than that of mesenteric fat weight in fatty rats (21.1- vs 4.9-fold) . Integrated enhancement of LPL and ACS gene expression in adipose tissue may play an important role in the development of obesity. Biochemistry, 1992 Mar 3, 31(8), 2384 - 92 Cytochrome c and cytochrome c peroxidase complex as studied by resonance Raman spectroscopy; Hildebrandt P et al.; Complex formation between ferricytochrome c peroxidase (CCP) and ferricytochrome c from yeast {cyt(Y)} and horse heart {cyt(H)} was studied by resonance Raman spectroscopy . On the basis of a detailed spectral analysis of the free proteins, it was possible to attribute changes in the spectra of the complexes to the individual proteins . At pH 7.0 both cyt(Y) and cyt(H) binding induces an increase in the six-coordinate low-spin configuration of CCP from 9% to 19% at the expense of the five-coordinate high-spin state, which drops from 84% to 74% . In the free and complexed state, CCP exhibits a constant fraction of the six-coordinate high-spin form (approximately 7%) . In addition to affecting the coordination state, there is also a cyt-specific structural response of CCP to complexation . In the cyt(Y)-CCP complex, the peripheral vinyl and propionate substituents of CCP are more rigidly fixed in the protein matrix, whereas binding of cyt(H) only slightly perturbs the conformations of these side chains . The biological significance of the conformational changes in CCP are discussed . In contrast to CCP, there are no detectable structural changes in either cyt(Y) or cyt(H) upon complex formation. Berl Munch Tierarztl Wochenschr, 1992 Mar 1, 105(3), 95 - 6 {Adjuvant effects of glucan on antibody formation in swine}; Benda V et al.; The effect of glucan, a yeast product isolated from Saccharomyces cerevisiae, on the antibody production to the bovine serum albumin and tetanus toxoid was investigated in miniature pigs . A significant stimulation of the immune response to both antigens differing in their thymus dependency was demonstrated during three weeks after the glucan application, by ELISA, in comparison to the untreated controls. Yeast, 1992 Mar, 8(3), 205 - 13 The complete sequence of K3B, a 7.9 kb fragment between PGK1 and CRY1 on chromosome III, reveals the presence of seven open reading frames; Bolle PA et al.; We have determined the nucleotide sequence of a segment from chromosome III of Saccharomyces cerevisiae extending over 7.9 kb between the PGK1 and CRY1 loci . The fragment contains seven open reading frames, YCR241, YCR242, YCR243, YCR244, YCR245, YCR246 and YCR247, of more than 70 codons . The study of the effects of a global disruption of YCR242, YCR243, YCR244, YCR245 and YCR247 shows that they are not essential for growth and division. Mol Microbiol, 1992 Mar, 6(6), 677 - 81 Microtubule assembly and phage morphogenesis: new results and classical paradigms; Weinstein B et al.; The classical analyses of phage morphogenesis provide experimental paradigms for dissecting other assembly pathways . A new set of results, derived from examination of the quantitative controls of tubulin levels and microtubule assembly in Saccharomyces cerevisiae, evokes the 'balance of components' hypothesis . These results show that balanced levels of the tubulin proteins are crucial for microtubule assembly . Imbalances leading to excess beta tubulin have far more deleterious consequences than those leading to excess alpha tubulin, including dramatic cellular toxicity, quantitative depolymerization of cellular microtubules, and self-aggregation of the excess beta tubulin . These and other results suggest that beta tubulin may possess a unique ability to interact with a component of microtubule nucleating sites, and provide a rationale for the universal polarity of nucleated microtubules. Mol Cell Biol, 1992 Mar, 12(3), 928 - 35 The RNA polymerase II 15-kilodalton subunit is essential for viability in Drosophila melanogaster; Harrison DA et al.; A small, divergently transcribed gene is located 500 bp upstream of the suppressor of Hairy-wing locus of Drosophila melanogaster . Sequencing of a full-length cDNA clone of the predominant 850-nucleotide transcript reveals that this gene encodes a 15,100-Da protein with high homology to a subunit of RNA polymerase II . The RpII15 protein is 46% identical to the RPB9 protein of Saccharomyces cerevisiae, one of the smallest subunits of RNA polymerase II from that species . Among those identical residues are four pairs of cysteines whose spacing is suggestive of two metal-binding "finger" domains . The gene is expressed at all developmental stages and in all tissues . Two deletions within the RpII15 gene are multiphasic lethal deletions, with accumulation of dead animals commencing at the second larval instar . Ovary transplantation experiments indicate that survival of mutant animals to this stage is due to the persistence of maternal gene product throughout embryogenesis and early larval development . The RpII15 gene product is thus necessary for viability of D . melanogaster. Mol Cell Biol, 1992 Mar, 12(3), 1292 - 303 Two alternative pathways of double-strand break repair that are kinetically separable and independently modulated; Fishman-Lobell J et al.; HO endonuclease-induced double-strand breaks in Saccharomyces cerevisiae can undergo recombination by two distinct and competing pathways . In a plasmid containing a direct repeat, in which one repeat is interrupted by an HO endonuclease cut site, gap repair yields gene conversions while single-strand annealing produces deletions . Consistent with predictions of the single-strand annealing mechanism, deletion formation is not accompanied by the formation of a reciprocal recombination product . Deletions are delayed 60 min when the distance separating the repeats is increased by 4.4 kb . Moreover, the rate of deletion formation corresponds to the time at which complementary regions become single stranded . Gap repair processes are independent of distance but are reduced in rad52 mutants and in G1-arrested cells. Mol Cell Biol, 1992 Mar, 12(3), 1218 - 25 A gene from the variant surface glycoprotein expression site encodes one of several transmembrane adenylate cyclases located on the flagellum of Trypanosoma brucei; Paindavoine P et al.; The bloodstream form of Trypanosoma brucei contains transcripts of at least four genes showing partial sequence homology to the genes for eucaryotic adenylate and guanylate cyclases (S . Alexandre, P . Paindavoine, P . Tebabi, A . Pays, S . Halleux, M . Steinert, and E . Pays, Mol . Biochem . Parasitol . 43:279-288, 1990) . One of these genes, termed ESAG 4, belongs to the polycistronic transcription unit of the variant surface glycoprotein (VSG) gene . Whereas ESAG 4 is transcribed only in the bloodstream form of the parasite, the three other genes, GRESAG 4.1, 4.2, and 4.3, are also expressed in procyclic (insect) forms . These genes differ primarily in a region presumed to encode a large extracellular domain . We show here that ESAG 4-related glycoproteins of about 150 kDa can be found in the trypanosome membrane, that they are detected, by light and electron gold immunocytochemistry, only at the surface of the flagellum, and that the products of at least two of these genes, ESAG 4 and GRESAG 4.1, can complement a Saccharomyces cerevisiae mutant for adenylate cyclase . The recombinant cyclases are associated with the yeast membrane fraction and differ with respect to their activation by calcium: while the GRESAG 4.1 and yeast cyclases are inhibited by calcium, the ESAG 4 cyclase is stimulated . ESAG 4 thus most probably encodes the calcium-activated cyclase that has been found to be expressed only in the bloodstream form of T . brucei (S . Rolin, S . Halleux, J . Van Sande, J . E . Dumont, E . Pays, and M . Steinert . Exp . Parasitol . 71:350-352, 1990) . Our data suggest that the trypanosome cyclases are not properly regulated in yeast cells. J Neurosci, 1992 Mar, 12(3), 705 - 17 A unique Kex2-like endoprotease from Drosophila melanogaster is expressed in the central nervous system during early embryogenesis; Hayflick JS et al.; Complementary DNA sequences were cloned from a Drosophila library encoding a 1,101 amino acid polypeptide that we have named dKLIP-1 . The deduced protein is structurally similar to the yeast KEX2 prohormone endoprotease including the conserved Asp, His, and Ser catalytic triad residues characteristic of the subtilisin family . When coexpressed in vivo with pro-beta-NGF, dKLIP-1 greatly enhanced the endoproteolytic conversion of the precursor to mature beta-NGF by cleavage at a -Lys-Arg- doublet . In adults, dKLIP-1 transcripts were detected in cortical regions of the CNS and fat body . Most striking, however, was the high level of maternal transcripts deposited into developing oocytes . The temporal and spatial expression of dKLIP-1 mRNAs during embryonic development indicates a potential role for this novel Kex2p-like endoprotease in early embryogenesis and neurogenesis. Proc Natl Acad Sci U S A, 1992 Mar 1, 89(5), 1695 - 9 Highly conserved repetitive DNA sequences are present at human centromeres; Grady DL et al.; Highly conserved repetitive DNA sequence clones, largely consisting of (GGAAT)n repeats, have been isolated from a human recombinant repetitive DNA library by high-stringency hybridization with rodent repetitive DNA . This sequence, the predominant repetitive sequence in human satellites II and III, is similar to the essential core DNA of the Saccharomyces cerevisiae centromere, centromere DNA element (CDE) III . In situ hybridization to human telophase and Drosophila polytene chromosomes shows localization of the (GGAAT)n sequence to centromeric regions . Hyperchromicity studies indicate that the (GGAAT)n sequence exhibits unusual hydrogen bonding properties . The purine-rich strand alone has the same thermal stability as the duplex . Hyperchromicity studies of synthetic DNA variants indicate that all sequences with the composition (AATGN)n exhibit this unusual thermal stability . DNA-mobility-shift assays indicate that specific HeLa-cell nuclear proteins recognize this sequence with a relative affinity greater than 10(5) . The extreme evolutionary conservation of this DNA sequence, its centromeric location, its unusual hydrogen bonding properties, its high affinity for specific nuclear proteins, and its similarity to functional centromeres isolated from yeast suggest that this sequence may be a component of the functional human centromere. Genomics, 1992 Mar, 12(3), 510 - 6 Localization of the D5 dopamine receptor gene to human chromosome 4p15.1-p15.3, centromeric to the Huntington's disease locus; Eubanks JH et al.; Genes encoding G-protein-coupled receptors, including dopamine, serotonin, muscarinic cholinergic, and adrenergic receptors, play an important role in neurotransmission and may be involved in the pathophysiology of diseases such as Alzheimer's disease, Parkinson's disease, or Huntington's disease (HD) . We mapped the gene encoding the D5 dopamine receptor (DRD5) to human chromosome 4p, an area implicated in HD and the Wolf-Hirschhorn syndrome, using gene-specific amplification with the polymerase chain reaction on a panel of somatic cell hybrids carrying different human chromosomes . Further localization of the DRD5 gene was carried out through the isolation and analysis of yeast artificial chromosomes, fluorescence in situ suppression hybridization to human metaphase chromosomes, and analysis of a panel of somatic cell hybrids subdividing human chromosome 4 into nine regions . The human DRD5 gene is located at 4p15.1-p15.33, centromeric to the location of the Huntington's disease locus although not in the obligate area containing the HD gene . The localization of the DRD5 gene to 4p15.1-p15.33 suggests the possibility that cis-position effects could be responsible for the altered D1-type dopamine receptor number observed in HD tissues or that the DRD5 gene could be a candidate for some of the abnormalities associated with the Wolf-Hirschhorn syndrome. Trends Biochem Sci, 1992 Mar, 17(3), 119 - 23 Ribonucleotide reductase: regulation, regulation, regulation; Elledge SJ et al.; Ribonucleotide reductase (RNR) catalyses the rate limiting step in the production of deoxyribonucleotides needed for DNA synthesis . It is composed of two dissimilar subunits, R1, the large subunit containing the allosteric regulatory sites, and R2, the small subunit containing a binuclear iron center and a tyrosyl free radical . Recent isolation of the mammalian and yeast RNR genes has shown that, in addition to the well documented allosteric regulation, the synthesis of the enzyme is also tightly regulated at the level of transcription . The mRNAs for both subunits are cell-cycle regulated and, in yeast, inducible by DNA damage . Yeast encode a second large subunit gene, RNR3, that is expressed only in the presence of DNA damage . This regulation is thought to provide a metabolic state that facilitates DNA replicational repair processes. Trends Biochem Sci, 1992 Mar, 17(3), 114 - 9 Dual-specificity protein kinases: will any hydroxyl do? Lindberg RA, Quinn AM, Hunter T. Protein kinases are classified by the target amino acid in their substrates . Those protein kinases that phosphorylate hydroxyamino acids comprise two groups, the protein-tyrosine and protein-serine/threonine kinases, which, until recently, had been thought to be mutually exclusive . However, several new protein kinases have been discovered that, by the criterion of primary structure, would be classified as protein-serine/threonine kinases but which, surprisingly, are able to phosphorylate tyrosine residues . Even more surprising, there are reports of protein kinases that are capable of phosphorylating both tyrosine and serine/threonine residues . We review and discuss recent developments concerning these 'dal-specificity' protein kinases. J Biotechnol, 1992 Mar, 23(1), 71 - 82 Identification of polymerized-albumin receptor domain in the pre-S2 region of hepatitis B virus surface antigen M protein; Itoh Y et al.; The pre-S2-coding region in the hepatitis B virus surface antigen M (P31; pre-S2 + S) protein gene was modified to identify a polymerized-albumin receptor (PAR) domain by deleting restriction fragments or performing site-directed mutagenesis . The modified M protein genes (M-P31x; x = d, e, f, h and i) were cloned into the yeast generalized-expression vector pGLD 906-1 and expressed in Saccharomyces cerevisiae under the control of yeast glyceraldehyde-3-phosphate dehydrogenase gene promoter . The PAR activities of these gene products suggested that the PAR domain is located in the hydrophilic and highly conserved domain in the pre-S2 region (around Leu12 approximately Tyr21) . Antibodies specific for a pre-S2 peptide (Phe8 approximately Pro34, subtype adr), which covers the PAR domain, were purified from sera of rabbits immunized with yeast-derived M protein particles having a natural PAR domain . Immune electron microscopy showed that the purified antibodies could aggregate HBV particles . Therefore, it was speculated that the PAR domain overlapped with the dominant virus-neutralizing and virus-protecting epitopes. Science, 1992 Feb 28, 255(5048), 1130 - 2 Transcription factor IID mutants defective for interaction with transcription factor IIA; Buratowski S et al.; Transcription factor IID (TFIID) recognizes the TATA element of promoters transcribed by RNA polymerase II (RNAPII) and serves as the base for subsequent association by other general transcription factors and RNAPII . The carboxyl-terminal domain of TFIID is highly conserved and contains an imperfect repetition of a 60-amino acid sequence . These repeats are separated by a region rich in basic amino acids . Mutagenesis of the lysines in this region resulted in a conditioned phenotype in vivo, and the mutant proteins were defective for interactions with transcription factor IIA in vitro . Binding of TFIID to DNA was unaffected . These results suggest that the basic domain of TFIID is important for protein-protein interactions. J Biol Chem, 1992 Feb 25, 267(6), 4128 - 36 COQ2 is a candidate for the structural gene encoding para-hydroxybenzoate:polyprenyltransferase; Ashby MN et al.; Coenzyme Q functions as a lipid-soluble electron carrier in eukaryotes . In Saccharomyces cerevisiae, the enzymes responsible for the assembly of the polyisoprenoid side chain and subsequent transfer to para-hydroxybenzoate (PHB) are encoded by the nuclear genes COQ1 and COQ2, respectively . Yeast mutants defective in coenzyme Q biosynthesis are respiratory defective and provide a useful tool to study this non-sterol branch of the isoprenoid biosynthetic pathway . We isolated a 5.5-kilobase genomic DNA fragment that was able to functionally complement a coq2 strain . Additional complementation analyses located the COQ2 gene within a 2.1-kilobase HindIII-BglII restriction fragment . Sequence analyses revealed the presence of a 1,116-base pair open reading frame coding for a predicted protein of 372 amino acids and a molecular mass of 41,001 daltons . The amino acid sequence exhibits a typical amino-terminal mitochondrial leader sequence and six potential membrane-spanning domains . Primer extension and Northern analyses indicate the gene is transcriptionally active . Transformation of a coq2 strain with the 2.1-kilobase HindIII-BglII genomic restriction fragment on a multicopy plasmid restores PHB:polyprenyltransferase activity to wild-type levels . Disruption of the chromosomal COQ2 gene indicates the gene is not essential for viability, yet is required for PHB:polyprenyltransferase activity and respiratory function . In addition, the deduced amino acid sequence of PHB:polyprenyltransferase contains a putative allylic polyprenyl diphosphate-binding site . The presence of this aspartate-rich domain in a number of functionally distinct proteins which utilize polyprenyl diphosphate substrates is reported. J Biol Chem, 1992 Feb 25, 267(6), 3750 - 7 Shuffling of amino acid sequence: an important control in synthetic peptide studies of nucleic acid-binding domains . Binding properties of fragments of a conserved eukaryotic RNA binding motif; Nadler SG et al.; We have used synthetic peptides to study a conserved RNA binding motif in yeast poly(A)-binding protein . Two peptides, 45 and 44 amino acids in length, corresponding to amino and carboxyl halves of a 90-amino acid RNA-binding domain in the protein were synthesized . While the amino-terminal peptide had no significant affinity for nucleic acids, the carboxyl-terminal peptide-bound nucleic acids with similar characteristics to that for the entire 577 residue yeast poly(A)-binding protein . In 100 mM NaCl, the latter peptide retained over 50% of the intrinsic binding free energy of the protein, as well as, similar RNA versus DNA binding specificity . However, shuffling of the sequence of this 44 residue peptide had surprisingly little effect on its nucleic acid binding properties suggesting the overriding importance of amino acid composition as opposed to primary sequence . Deletion studies on the 44 residue peptide with the "correct" sequence succeeded in identifying amino acids important for conferring RNA specificity and for increasing our understanding of the molecular basis for nucleic acid binding by synthetic peptides . The shuffled peptide study, however, clearly indicates that considerable caution must be exercised before extrapolating results of structure/function studies on synthetic peptide analogues to the parent protein. J Biol Chem, 1992 Feb 25, 267(6), 3718 - 24 In vivo mutational analysis of the NGFI-A zinc fingers; Wilson TE et al.; NGFI-A is a mammalian transcription factor that contains zinc fingers similar to those observed in several other proteins, including NGFI-C and Krox-20 . To define precisely the DNA binding domain of NGFI-A, we selected mutants using a chimeric transcriptional activator that contains the NGFI-A zinc finger domain sandwiched between the lexA DNA binding domain and the GAL4 transcriptional activating domain . Expression of this lexA-NGFI-A-GAL4 (LAG) trimeric protein in yeast significantly retarded their growth, unlike an activator containing only the lexA and GAL4 components . This suggested that LAG inappropriately regulates genes in yeast that contain NGFI-A binding sites . Yeast that contained LAG reverted to wild-type growth at high frequency by inactivation of LAG . The mutations recovered from these revertants were specifically limited to the 83-residue NGFI-A zinc finger domain by requiring that the lexA and GAL4 portions of the LAG chimera remain functional . Nearly all of the 93 mutants obtained contained single missense mutations that mapped within the zinc fingers to residues thought to be important for zinc finger function . Deletion analysis of native NGFI-A verified that residues distant from the zinc fingers do not influence DNA binding, thus establishing the minimal functional DNA binding domain . Interestingly, many zinc finger residues ascribed specific functions by x-ray crystallography were never mutated in yeast, implying that the identity of these residues is not critical . Surprisingly, not all of the mutations tested significantly impaired NGFI-A-specific DNA binding, suggesting that the function of these zinc fingers is more diverse than previously recognized. Nucleic Acids Res, 1992 Feb 25, 20(4), 771 - 5 Uracil interference, a rapid and general method for defining protein-DNA interactions involving the 5-methyl group of thymines: the GCN4-DNA complex; Pu WT et al.; We describe a novel uracil interference method for examining protein contacts with the 5-methyl group of thymines . The protein of interest is incubated with target DNA containing randomly distributed deoxyuracil substitutions that is generated by carrying out the polymerase chain reaction in the presence of a mixture of TTP and dUTP . After separating DNA-protein complexes away from unbound DNA, the locations of deoxyuracil residues that either do or do not interfere with DNA-binding are determined by cleavage with uracil-N-glycosylase followed by piperidine . Using this uracil interference assay, we show that the methyl groups of the four core thymines, but not the two peripheral thymines, of the optimal binding site (ATG-ACTCAT) are important for high affinity binding of GCN4 . Similar, but not identical, results are obtained using KMnO4 interference, another method used for studying protein-DNA interactions involving thymine residues . These observations strongly suggest that GCN4 directly contacts the 5-methyl groups of the four core thymines that lie in the major groove of the target DNA . Besides providing specific structural information about protein-DNA complexes, uracil interference should also be useful for identifying DNA-binding proteins and their target sites in eukaryotic promoter regions. Biochemistry, 1992 Feb 25, 31(7), 2107 - 13 Synthesis and biochemical characterization of the new sulfhydryl-reactive ATP analogue 8-thiocyano-ATP . Its interaction with Na,K-ATPase and kinases; Scheiner-Bobis G et al.; The synthesis of 8-thiocyano-ATP (CNS8-ATP) is described . At 37 degrees C the ATP analogue inactivates Na,K-ATPase, hexokinase, and pyruvate kinase . In all three cases, inactivation can be prevented by the addition of ATP, thus indicating that CNS8-ATP is recognized within the ATP binding site of the above enzymes . Incubation of the inactivated enzymes with dithiothreitol restores the catalytic activities . Therefore, it is likely that in these enzymes a mixed disulfide (E-S-S8-ATP) is formed between a sulfhydryl in the ATP binding site (E-SH) and the ATP analogue: {formula: see text} From the pseudo-first-order inactivation kinetics, a KD = 2.7 microM with k2 = 0.142 min-1 is calculated for the hexokinase and a KD = 40 microM with k2 = 0.347 min-1 is calculated for the pyruvate kinase interactions with the ATP analogue . At 4 degrees C, Na,K-ATPase recognizes CNS8-ATP with a KD = 8.3 microM . At 37 degrees C, the enzyme becomes inactivated by the ATP analogue in a biphasic manner . Inactivation results in the incorporation of {alpha-32P}8-CNS8-ATP into the catalytic alpha-subunit of the enzyme . Limited tryptic digestion in the presence of 150 mM KCl results in the formation of a radioactive peptide of Mr = 56,000, known to bear the purine binding domain of Na,K-ATPase . The results described in this article verify CNS8-ATP as a sulfhydryl-reactive ATP analogue and characterize this new ATP analogue as a useful tool for structure/function studies on ATP-recognizing enzymes. Biochemistry, 1992 Feb 25, 31(7), 2068 - 73 1H NMR assignment and secondary structure of the cell adhesion type III module of fibronectin; Baron M et al.; The secondary structure of the tenth type III module from human fibronectin has been determined using NMR . This type of module appears many times in a wide variety of proteins . The type III module described here contains an Arg-Gly-Asp sequence known to be involved in cell-cell adhesion . The module was expressed in yeast and characterized by amino acid sequencing and mass spectrometry . 2D and 3D NMR spectroscopy of 15N-labeled protein was used to perform sequence-specific assignment of the spectrum . The secondary structure was defined by patterns of nuclear Overhauser effects, 3JNH-alpha CH spin-spin coupling constants, and amide proton solvent exchange rates . The molecule consists of seven beta-strands in two antiparallel beta-sheets with an immunoglobulin-like fold similar to that predicted for homologous modules in the cytokine receptor super family {Bazan, J . F . (1990) Proc . Natl . Acad . Sci . U.S.A . 87, 6934-6938} . The Arg-Gly-Asp sequence is located on a loop between the beta-strands F and G. Cell, 1992 Feb 21, 68(4), 743 - 54 U5 snRNA interacts with exon sequences at 5' and 3' splice sites; Newman AJ et al.; U5 snRNA is an essential pre-mRNA splicing factor whose function remains enigmatic . Specific mutations in a conserved single-stranded loop sequence in yeast U5 snRNA can activate cleavage of G1----A mutant pre-mRNAs at aberrant 5' splice sites and facilitate processing of dead-end lariat intermediates to mRNA . Activation of aberrant 5' cleavage sites involves base pairing between U5 snRNA and nucleotides upstream of the cleavage site . Processing of dead-end lariat intermediates to mRNA correlates with base pairing between U5 and the first two bases in exon 2 . The loop sequence in U5 snRNA may therefore by intimately involved in the transesterification reactions at 5' and 3' splice sites . This pattern of interactions is strikingly reminiscent of exon recognition events in group II self-splicing introns and is consistent with the notion that U5 snRNA may be related to a specific functional domain from a group II-like self-splicing ancestral intron. Biochim Biophys Acta, 1992 Feb 21, 1099(2), 123 - 30 Antipeptide antibodies to the carboxy terminal and the DCCD binding region of the human mitochondrial ATP synthase beta-subunit; Noer AS et al.; Antibodies to defined epitopes on the human ATP synthase would provide a powerful tool in the definition of the subunit composition of the enzyme complex and in the characterization of any defect in its assembly in diseases associated with mitochondrial disorders . Antibodies have been thus raised against synthetic peptides, corresponding to two regions on the human ATP synthase beta-subunit: the C-terminal region, and a region which includes the two dicyclohexylcarbodiimide (DCCD)-reactive glutamic acid residues suggested to be involved in the enzyme catalytic activity . The antibodies to the C-terminal peptide reacted with the ATP synthase beta-subunit in ELISA, in Western immunoblotting and in immunohistochemical experiments, and had the ability to immunoprecipitate the enzyme complex . The antibodies to the DCCD-binding region peptide did not react to the ATP synthase beta-subunit in its native configuration, although reacted well under Western immunoblotting conditions. J Mol Biol, 1992 Feb 20, 223(4), 959 - 76 Oxidation state-dependent conformational changes in cytochrome c; Berghuis AM et al.; High-resolution three-dimensional structural analyses of yeast iso-1-cytochrome c have now been completed in both oxidation states using isomorphous crystalline material and similar structure determination methodologies . This approach has allowed a comprehensive comparison to be made between these structures and the elucidation of the subtle conformational changes occurring between oxidation states . The structure solution of reduced yeast iso-1-cytochrome c has been published and the determination of the oxidized protein and a comparison of these structures are reported herein . Our data show that oxidation state-dependent changes are expressed for the most part in terms of adjustments to heme structure, movement of internally bound water molecules and segmental thermal parameter changes along the polypeptide chain, rather than as explicit polypeptide chain positional shifts, which are found to be minimal . This result is emphasized by the retention of all main-chain to main-chain hydrogen bond interactions in both oxidation states . Observed thermal factor changes primarily affect four segments of polypeptide chain . Residues 37-39 show less mobility in the oxidized state, with Arg38 and its side-chain being most affected . In contrast, residues 47-59, 65-72 and 81-85 have significantly higher thermal factors, with maximal increases being observed for Asn52, Tyr67 and Phe82 . The side-chains of two of these residues are hydrogen bonded to the internally bound water molecule, Wat166, which shows a large 1.7 A displacement towards the positively charged heme iron atom in the oxidized protein . Further analyses suggest that Wat166 is a major factor in stabilizing both oxidation states of the heme through differential orientation of dipole moment, shift in distance to the heme iron atom and alterations in the surrounding hydrogen bonding network . It also seems likely that Wat166 movement leads to the disruption of the hydrogen bond from the side-chain of Tyr67 to the Met80 heme ligand, thereby further stabilizing the positively charged heme iron atom in oxidized cytochrome c . In total, there appear to be three regions about which oxidation state-dependent structural changes are focussed . These include the pyrrole ring A propionate group, Wat166 and the Met80 heme ligand . All three of these foci are linked together by a network of intermediary interactions and are localized to the Met80 ligand side of the heme group . Associated with each is a corresponding nearby segment of polypeptide chain having a substantially higher mobility in the oxidized protein.(ABSTRACT TRUNCATED AT 400 WORDS) Experientia, 1992 Feb 15, 48(2), 172 - 8 Genetic analysis of ubiquitin-dependent protein degradation; Sommer T et al.; Selective degradation of cellular proteins serves to eliminate abnormal proteins and to mediate the turnover of certain short-lived proteins, many of which have regulatory functions . In eukaryotes a major pathway for selective protein degradation is ATP-dependent and is mediated by the ubiquitin system . This pathway involves substrate recognition by components of a ubiquitin-protein ligase system, covalent attachment of ubiquitin moieties to proteolytic substrates, and subsequent degradation of these conjugates by a multicatalytic protease complex . Recent genetic evidence suggests that the remarkable selectivity of this process is largely controlled at the level of substrate recognition by the ubiquitin ligase system . In Saccharomyces cerevisiae, ubiquitin-conjugating enzymes UBC1, UBC4 and UBC5 have been identified as key components of this highly conserved degradation pathway . Genetic analysis indicates that ubiquitin-dependent proteolysis is essential for cell viability and that UBC4 and UBC5 enzymes are essential components of the eukaryotic stress response. J Biol Chem, 1992 Feb 15, 267(5), 3358 - 67 DNA wrapping and bending by a mitochondrial high mobility group-like transcriptional activator protein; Fisher RP et al.; Mitochondrial transcription factor 1 (mtTF1) is the only accessory protein known to be required for accurate and efficient promoter recognition by mammalian mitochondrial RNA polymerase . It activates transcription by binding immediately upstream of transcriptional start sites and shows an inherent flexibility in primary DNA sequence requirement . By application of a purification strategy designed for human and mouse mtTF1, a protein resembling mtTF1 was recently isolated from yeast mitochondria; its size (19 kDa), DNA-binding properties, and amino acid composition suggest identity to HM, a previously described abundant protein of yeast mitochondria . Both human and yeast proteins show a general ability to wrap or condense and unwind DNA in vitro and bend DNA at specific sequences . Recent determinations of the amino acid sequences of the human and yeast proteins reveal that both contain domains homologous to the nuclear high mobility group (HMG) proteins which have been implicated in diverse functions such as chromatin compaction and transcription stimulation . The ability to unwind and bend DNA may be fundamental to the documented roles of the mammalian protein in mitochondrial DNA transcription and replication priming and suggests a similar function for the yeast protein in yeast mitochondria. J Biol Chem, 1992 Feb 15, 267(5), 3302 - 7 Ornithine delta-aminotransferase mutations in gyrate atrophy . Allelic heterogeneity and functional consequences; Brody LC et al.; Ornithine delta-aminotransferase is a nuclear-encoded mitochondrial matrix enzyme which catalyzes the reversible interconversion of ornithine and alpha-ketoglutarate to glutamate semialdehyde and glutamate . Inherited deficiency of ornithine delta-aminotransferase results in ornithine accumulation and a characteristic chorioretinal degeneration, gyrate atrophy of the choroid and retina . We have surveyed the ornithine delta-aminotransferase genes of gyrate atrophy patients for mutations . Using a variety of techniques, we discovered and molecularly characterized 21 newly recognized ornithine delta-aminotransferase alleles . We determined the consequences of these and three previously described mutations on ornithine delta-aminotransferase mRNA, antigen, and enzyme activity in cultured fibroblasts . The majority (20/24) of these alleles produce normal amounts of normally sized ornithine delta-aminotransferase mRNA . By contrast, only 2/24 had normal amounts of ornithine delta-aminotransferase antigen . Reproducing these mutations by site-directed mutagenesis and expressing the mutant ornithine delta-aminotransferase in Chinese hamster ovary cells confirms that several of these mutations inactivate ornithine delta-aminotransferase and cause gyrate atrophy in these patients. Eur J Biochem, 1992 Feb 15, 204(1), 337 - 52 Electron-proton coupling in cytochrome c studied using protein variants; Gao Y et al.; An NMR study of the cytochrome c variant Asn52Ile is used to show how the redox state change in native cytochrome c is coupled to a rearrangement of a proton network which runs through the cytochrome c molecule . The substitution breaks the H-bond network and removes the coupling . The uncovering of this putative proton channel and the connection of changes to it with redox state changes of the iron centre of the protein allows a possible description of the way in which redox energy state changes can be coupled to energization and gating of protons in membranes. J Biol Chem, 1992 Feb 15, 267(5), 3316 - 24 PPIase catalysis by human FK506-binding protein proceeds through a conformational twist mechanism; Park ST et al.; FK506-binding protein (FKBP) catalyzes the cis-trans isomerization of the peptidyl-prolyl amide bond (the PPIase reaction) and is the major intracellular receptor for the immunosuppressive drugs FK506 and rapamycin . One mechanism proposed for catalysis of the PPIase reaction requires attack of an enzyme nucleophile on the carbonyl carbon of the isomerized peptide bond . An alternative mechanism requires conformational distortion of the peptide bond with or without assistance by an enzyme hydrogen bond donor . We have determined the kinetic parameters of the human FKBP-catalyzed PPIase reaction . At 5 degrees C, the isomerization of Suc-Ala-Leu-Pro-Phe-pNA proceeds in 2.5% trifluorethanol with kcat = 600 s-1, Km = 0.5 mM and kcat/Km = 1.2 x 10(6) M-1s-1 . The kcat/Km shows little pH dependence between 5 and 10 . A normal secondary deuterium isotope effect is observed on both kcat and kcat/Km . To investigate dependence on enzyme nucleophiles and proton donors, we have replaced eight potential catalytic residues with alanine by site-directed mutagenesis . Each FKBP variant efficiently catalyzes the PPIase reaction . Taken together, these data support an unassisted conformational twist mechanism with rate enhancement due in part to desolvation of the peptide bond at the active site . Fluorescence quenching of the buried tryptophan 59 residue by peptide substrate suggests that isomerization occurs in a hydrophobic environment. Proc Natl Acad Sci U S A, 1992 Feb 15, 89(4), 1249 - 52 A strategy for the generation of conditional mutations by protein destabilization; Park EC et al.; Conditional mutations such as temperature-sensitive (ts) mutations are important for the analysis of protein function but are often difficult, or impossible, to obtain . Here we present a simple method for generating conditional mutations based on the use of a protein-destabilizing genetic element in combination with systems allowing the induction and repression of gene expression . This genetic cassette can be fused to other protein-coding sequences, and once transcription is turned off and synthesis of the gene product ceases, the preexisting protein is rapidly degraded . We have applied this method to the analysis of the yeast ARD1 gene product, a subunit of an N-terminal acetyltransferase, and show that a complete loss of ARD1 product can be achieved in less than one generation . Despite the rapid loss of ARD1 protein, there is a prolonged delay in the expression of the ard1 mutant phenotype, suggesting that the acetylated substrates of ARD1 are metabolically stable and/or exert a long-lasting effect on processes such as the repression of the silent mating type cassettes. J Chromatogr, 1992 Feb 14, 574(2), 225 - 35 Large-scale purification and characterization of recombinant tick anticoagulant peptide; Lehman ED et al.; Recombinant tick anticoagulant peptide (r-TAP), a potent and specific inhibitor of blood coagulation factor Xa, was purified to greater than 99% homogeneity at the multi-gram scale . Genetically engineered yeast secreted 200-250 mg/l of the heterologous protein into the medium . Cells were separated from broth by diafiltration and purification was done by two chromatographic steps, both conducive to operation on a large scale . Analysis of the purified protein by several methods indicated that it was greater than 99% homogeneous and no incompletely processed or truncated proteins were detected . Physico-chemical characterization data of r-TAP show that it exists as a monomer in solution and no evidence of post-translational modification was observed . The purified protein was fully active in inhibiting human coagulation factor Xa. Biochim Biophys Acta, 1992 Feb 11, 1129(3), 273 - 7 Use of a dot blot hybridization method for identification of pure tRNA species on different membranes; Heitzler J et al.; The characterization of a tRNA in purification procedures usually involves aminoacylation assays but recently, the hybridization by dot blot with specific oligonucleotides as probes has been used for the tRNA identification . We present here an optimization of a dot blot hybridization method for the tRNA detection by comparing the efficiency of eight different nylon membranes . Neutral 0.22 microns porosity membranes (Nytran, Biodine A) give the best detection efficiency when small quantities of material (less than 40 ng of tRNA) are dotted on filter; by contrast, neutral 0.45 microns porosity membranes (such as Hybond N) are the most efficient when larger quantities of tRNA are dotted on the filter . The described technique allows to detect less than 20 pg of a pure tRNA species . Its use in the identification of Saccharomyces cerevisiae initiator tRNA(Met) in counter-current distribution fractions is shown. J Chromatogr, 1992 Feb 7, 574(1), 35 - 40 Sensitive determination of cystathionine and assays for cystathionine beta- and gamma-lyase, as well as cystathionine beta-synthase, using high-performance liquid chromatography; Ohmori S et al.; Cystathionine was cleaved into 2-ketobutyric acid, cysteine and ammonia by cystathionase . 2-Ketobutyric acid was converted into 3-ethyl-2-hydroxy-6,7-dimethoxyquinoxaline (EHDQ) by reaction with 1,2-diamino-4,5-dimethoxybenzene . When EHDQ was measured in a mobile phase of pH 2.1 using high-performance liquid chromatography with ultraviolet detection, 250 pmol of L-cystathionine in 250 microliters of the reaction mixture could be determined . Because EHDQ has a strong fluorescence in a mobile phase of pH 6.5 at 447 nm, on excitation at 365 nm, as little as 2.5 pmol of cystathionine in 250 microliters of the reaction mixture could be determined by high-performance liquid chromatography with fluorimetric detection . Cystathionase activity was assayed on the basis of the same principle by determining cystathionine in as little as 63 ng of rat liver by fluorimetric detection . Cystathionine beta-synthase activity was measured by the same method by determining cystathionine formed in only 113 ng of wet weight of rat liver . Using these methods, both cystathionine beta- and gamma-lyase activities in Saccharomyces cerevisiae were determined, because quinoxaline derivatives from pyruvate and 2-ketobutyrate could be measured simultaneously by high-performance liquid chromatography. Cell, 1992 Feb 7, 68(3), 597 - 612 Characterization of SAP-1, a protein recruited by serum response factor to the c-fos serum response element; Dalton S et al.; We used a yeast genetic screen to isolate cDNAs that encode a protein, SRF accessory protein-1 (SAP-1), that is recruited to the c-fos serum response element (SRE) as part of a ternary complex that includes serum response factor (SRF) . SAP-1 requires DNA-bound SRF for ternary complex formation and makes extensive DNA contacts to the 5' side of SRF, but does not bind DNA autonomously . Ternary complex formation by SAP-1 requires only the DNA-binding domain of SRF, which can be replaced by that of the related yeast protein MCM1 . We isolated cDNAs encoding two forms of SAP-1 protein, SAP-1a and SAP-1b, which differ at their C termini . Both SAP-1 proteins contain three regions of striking homology with the elk-1 protein, including an N-terminal ets domain . Ternary complex formation by SAP-1 requires both the ets domain and a second conserved region 50 amino acids to its C-terminal side . SAP-1 has similar DNA binding properties to the previously characterized HeLa cell protein p62/TCF. Biochemistry, 1992 Feb 4, 31(4), 972 - 82 Recognition of tertiary structure in tRNAs by Rh(phen)2phi3+, a new reagent for RNA structure-function mapping; Chow CS et al.; With photoactivation Rh(phen)2phi3+ promotes strand cleavage at sites of tertiary interaction in tRNA . The rhodium complex, which binds double-helical DNA by intercalation in the major groove, yields no cleavage in double-helical regions of the RNA or in unstructured single-stranded regions . Instead, Rh(phen)2phi3+ appears to target regions which are structured so that the major groove is open and accessible for stacking with the complex, as occurs where bases are triply bonded . So as to examine the specificity of this novel reagent and to evaluate its use in probing structural changes in RNAs, cleavage studies have been conducted on two structurally characterized tRNAs, tRNA(Phe) and tRNA(Asp) from yeast, the unmodified yeast tRNA(Phe) transcript, and a chemically modified tRNA(Phe), as well as on a series of tRNA(Phe) mutants . On tRNA(Phe) strong cleavage is observed at residues G22, G45, U47, psi 55, and U59; weaker cleavage is observed at A44, m7G46, and C48 . On tRNA(Asp) cleavage is found at residues A21 through G26, psi 32, and U48, with minor cleavage apparent at A44, G45, A46, psi 55, U59, and U60 . There is a striking similarity in cleavage observed on these tRNAs, and the sites of cleavage mark regions of tertiary folding . Cleavage on the unmodified tRNA(Phe) transcript resembles closely that found on native yeast tRNA(Phe), but additional sites, primarily in the anticodon loop and stem, are evident . The results indicate that globally the structures containing or lacking the modified bases appear to be the same; the differences in cleavage observed may reflect a loosening or alteration in the structure due to the absence of the modified bases . Cleavage results on mutants of tRNA(Phe) illustrate Rh(phen)2phi3+ as a sensitive probe in characterizing tRNA tertiary structure . Results are consistent with other assays for structural or functional changes . Uniquely, Rh(phen)2phi3+ appears to target directly sites of tertiary interaction . Cleavage results on mutants which involve base changes within the triply bounded region of the molecule indicate that it is the structure of the triply bonded array rather than the individual nucleotides which are being targeted . Chemical modification to promote selective depurination of the third base (m7G46) involved in the triple in the folded, native tRNA leads to the reduction of cleavage by the metal complex; this result shows directly the importance of the stacked triple base structure for recognition by the metal complex.(ABSTRACT TRUNCATED AT 400 WORDS) Biochemistry, 1992 Feb 4, 31(4), 964 - 72 Primary structure of the Thermoplasma proteasome and its implications for the structure, function, and evolution of the multicatalytic proteinase; Zwickl P et al.; The proteasome or multicatalytic proteinase is a high molecular mass multisubunit complex ubiquitous in eukaryotes but also found in the archaebacterial proteasome is made of two different subunits only, and yet the complexes are almost identical in size and shape . Cloning and sequencing the gene encoding the small (beta) subunit of the T . acidophilum complex completes the primary structure of the archaebacterial proteasome . The similarity of the derived amino acid sequences of 233 (alpha) and 211 (beta) residues, respectively, indicates that they arose from a common ancestral gene . All the sequences of proteasomal subunits from eukaryotes available to date can be related to either the alpha-subunit or beta-subunit of the T . acidophilum "Urproteasome", and they can be distinguished by means of a highly conserved N-terminal extension, which is characteristic for alpha-type subunits . On the basis of circumstantial evidence we suggest that the alpha-subunits have regulatory and targeting functions, while the beta-subunits carry the active sites. Genomics, 1992 Feb, 12(2), 264 - 75 Distribution of moderately repetitive sequences pTR5 and LF1 in Xq24-q28 human DNA and their use in assembling YAC contigs; Zucchi I et al.; Xq24-q28 DNA, from a hamster/human hybrid cell containing only that portion of the human X chromosome, was found to contain 56 TaqI restriction fragments that hybridized to the moderately repetitive sequence pTR5 . Using the pTR5 sequence as a probe in colony hybridization, 136 cognate yeast artificial chromosome (YAC) clones were detected among a collection of 820 containing about three genomic equivalents of the Xq24-q28 DNA . The YACs were then grouped into 48 contigs and single clones containing one or more of the TaqI fragments . Overlaps were confirmed both by fingerprinting YACs with AluI and L1 probes and by additional information . A less complete analysis was also carried out with a second moderately repetitive sequence, LF1, and some smaller contigs were merged into larger ones . Moderately repetitive sequences can thus be used as probes for multiple loci in single hybridization experiments and can help to organize and confirm YAC overlaps during the development of maps with long-range contiguity. Proc Natl Acad Sci U S A, 1992 Feb 1, 89(3), 957 - 61 Analysis of CpG suppression in methylated and nonmethylated species; Schorderet DF et al.; The development of nearest-neighbor analysis led to the finding that the frequency of the dinucleotide CpG is markedly depressed in vertebrates . One explanation of this suppression is that methylation of CpG found in vertebrates represents a mutational hot spot through deamination of methylcytidine to thymidine . We have examined the role of methylated CpG as a factor in CpG suppression by comparing CpG distributions in coding regions of 121 genes from six species, three with methylated DNA and three with nonmethylated DNA . Overall base composition shows that all species exhibit CpG suppression, with the methylated forms showing significantly greater suppression than nonmethylated forms . When the data are analyzed by CpG position, the mean values of the methylated forms exhibit greater suppression than nonmethylated forms at positions I-II and II-III, but there is considerable overlap of suppression scores for individual species . At position III-I, CpG suppression is marked in all methylated species, and it is reversed in all nonmethylated species . Our analysis supports the hypothesis that CpG patterns at positions II-III and III-I in methylated forms are affected by mutation acting through deamination of methylcytidine to thymidine . We speculate that the excess of CpGs at position III-I in nonmethylated forms may be related to a requirement for minimal thermal stability of the DNA. Proc Natl Acad Sci U S A, 1992 Feb 1, 89(3), 922 - 6 Structural and enzymatic characterization of a purified prohormone-processing enzyme: secreted, soluble Kex2 protease; Brenner C et al.; The prohormone-processing Kex2 protease of the budding yeast Saccharomyces cerevisiae can be converted from an intracellular membrane protein to a soluble, secreted, and active form by deletion of the transmembrane domain and C-terminal tail . One such molecule was purified to near homogeneity from the culture medium of an overexpressing yeast strain . Amino acid sequence analysis revealed that the N terminus of mature Kex2 protease is created by a potentially autoproteolytic cleavage at Lys108-Arg109, prior to the domain homologous to subtilisin, followed by trimming of Leu-Pro and Val-Pro dipeptides by the Ste13 dipeptidyl aminopeptidase . Kinetic parameters were examined using fluorogenic peptidyl-methylcoumarin amide substrates . Initial burst titration indicated that the preparation was entirely active . Measurements of dependence of activity on pH yielded a simple curve suggesting titration of a single ionizable group . Activity was half-maximal at pH 5.7 and nearly constant from pH 6.5 to 9.5 . Discrimination between substrates was as great as 360-fold in Km and 130-fold in kcat . Substrates with a Lys-Arg dipeptide preceding the cleaved bond were preferred, having kcat/Km values up to 1.1 x 10(7) sec-1.M-1 . The enzyme cleaved substrates having Arg-Arg, Pro-Arg, Ala-Arg, and Thr-Arg with increased Km but with unchanged kcat . In contrast, the enzyme displayed a dramatically lower kcat for a Lys-Lys substrate with a smaller increase in Km . Thus the two residues preceding the cleaved bond may play distinct roles in the selectivity of binding and cleavage of prohormone substrates. Proc Natl Acad Sci U S A, 1992 Feb 1, 89(3), 1060 - 4 Transcription factor TFIID induces DNA bending upon binding to the TATA element; Horikoshi M et al.; The TATA box-binding factor TFIID plays a primary role in the process of transcription initiation by RNA polymerase II and its regulation by various gene-specific factors . Here we employ a permuted binding site/gel retardation assay with recombinant yeast and human TFIID to show that this factor induces DNA bending around the TATA element . These results are consistent with the presence of G + C-rich sequence elements flanking the consensus TATA element and led to the recently confirmed suggestion that TFIID interacts with the TATA element via the minor groove . They also raise the possibility that TFIID-induced bending might facilitate promoter interactions of other general factors in the preinitiation complex or interactions between general transcription factors and regulatory factors bound at upstream sites. Radiat Res, 1992 Feb, 129(2), 177 - 83 Protein synthesis in irradiated cells . I . Ultraviolet radiation; Straaten H et al.; Excision-deficient haploid yeast cells (Saccharomyces cerevisiae) were exposed to 254-nm UV radiation and protein synthesis inhibition was measured for a large number of different proteins resolved by two-dimensional gel electrophoresis . The derived UV-radiation sensitivities exhibited an overall increase with protein molar mass . Quantitatively, this behavior is compatible with a well known mechanism of transcription inactivation--termination of RNA chains at UV-radiation-induced pyrimidine dimers--if the respective target sizes are inferred from protein molar mass . The observed deviations from the predicted response suggest that (i) UV-radiation damage may also interfere with recognition/binding of RNA polymerase to regulatory sequences and (ii) the frequency of photolesions for a specific protein encoding gene may differ markedly from the mean induction rate for the total yeast genome. Mol Cell Biol, 1992 Feb, 12(2), 716 - 23 Complex recognition site for the group I intron-encoded endonuclease I-SceII; Wernette C et al.; We have characterized features of the site recognized by a double-stranded DNA endonuclease, I-SceII, encoded by intron 4 alpha of the yeast mitochondrial COX1 gene . We determined the effects of 36 point mutations on the cleavage efficiency of natural and synthetic substrates containing the Saccharomyces capensis I-SceII site . Most mutations of the 18-bp I-SceII recognition site are tolerated by the enzyme, and those mutant sites are cleaved between 42 and 100% as well as the wild-type substrate is . Nine mutants blocked cleavage to less than or equal to 33% of the wild-type, whereas only three point mutations, G-4----C, G-12----T, and G-15----C, block cleavage completely . Competition experiments indicate that these three substrates are not cleaved, at least in part because of a marked reduction in the affinity of the enzyme for those mutant DNAs . About 90% of the DNAs derived from randomization of the nucleotide sequence of the 4-bp staggered I-SceII cleavage site are not cleaved by the enzyme . I-SceII cleaves cloned DNA derived from human chromosome 3 about once every 110 kbp . The I-SceII recognition sites in four randomly chosen human DNA clones have 56 to 78% identity with the 18-bp site in yeast mitochondrial DNA; they are cleaved at least 50% as well as the wild-type mitochondrial substrate despite the presence of some substitutions that individually compromise cleavage of the mitochondrial substrate . Analysis of these data suggests that the effect of a given base substitution in I-SceII cleavage may depend on the sequence at other positions. Mol Cell Biol, 1992 Feb, 12(2), 674 - 84 RelB, a new Rel family transcription activator that can interact with p50-NF-kappa B; Ryseck RP et al.; We have identified a serum-inducible gene, relB, which encodes a protein of 558 amino acids containing a region with high similarity to c-Rel and other members of the Rel family . Transcriptional activation analysis of GAL4-RelB fusion proteins in yeast cells reveals that RelB contains in its C-terminal 180 amino acids a transcriptional activation domain . The N-terminal part including the region of similarity with the Rel family shows no detectable transcriptional activity . RelB does not bind with high affinity to NF-kappa B sites, but heterodimers between RelB and p50-NF-kappa B do bind to different NF-kappa B-binding sites with a similar affinity to that shown by p50-NF-kappa B homodimers . However, RelB/p50-NF-kappa B heterodimers, in contrast to p50-NF-kappa B homodimers, transactivate transcription of a promoter containing a kappa B-binding site. Mol Cell Biol, 1992 Feb, 12(2), 631 - 7 A dominant activating mutation in the effector region of RAS abolishes IRA2 sensitivity; Tanaka K et al.; Previously described mutations in RAS genes that cause a dominant activated phenotype affect the intrinsic biochemical properties of RAS proteins, either decreasing the intrinsic GTPase or reducing the affinity for guanine nucleotides . In this report, we describe a novel activating mutation in the RAS2 gene of Saccharomyces cerevisiae that does not alter intrinsic biochemical properties of the mutant RAS2 protein . Rather, this mutation, RAS2-P41S (proline 41 to serine), which lies in the effector region of RAS, is shown to abolish the ability of the IRA2 protein to stimulate the GTPase activity of the mutant RAS protein . This mutation also modestly reduced the ability of the mutant protein to stimulate the target adenylate cyclase in an in vitro assay, although in vivo the phenotypes it induced suggest that it retains potency in stimulation of adenylate cyclase . Our results demonstrate that although the effector region of RAS appears to be important for interaction with both target effector and negative regulators of RAS, it is possible to eliminate negative regulator responsiveness and retain potency in effector stimulation. Mol Cell Biol, 1992 Feb, 12(2), 563 - 75 Characterization of double-strand break-induced recombination: homology requirements and single-stranded DNA formation; Sugawara N et al.; In the yeast Saccharomyces cerevisiae, a double-strand chromosome break created by the HO endonuclease is frequently repaired in mitotically growing cells by recombination between flanking homologous regions, producing a deletion . We showed that single-stranded regions were formed on both sides of the double-strand break prior to the formation of the product . The kinetics of the single-stranded DNA were monitored in strains with the recombination-deficient mutations rad52 and rad50 as well as in the wild-type strain . In rad50 mutants, single-stranded DNA was generated at a slower rate than in the wild type, whereas rad52 mutants generated single-stranded DNA at a faster rate . Product formation was largely blocked in the rad52 mutant . In the rad50 rad52 double mutant, the effects were superimposed in that the exonucleolytic activity was slowed but product formation was blocked . rad50 appears to act before or at the same stage as rad52 . We constructed strains containing two ura3 segments on one side of the HO cut site and one ura3 region on the other side to characterize how flanking repeats find each other . Deletions formed preterentially between the homologous regions closest to the double-strand break . By varying the size of the middle ura3 segment, we determined that recombination initiated by a double-strand break requires a minimum homologous length between 63 and 89 bp . In these competition experiments, the frequency of recombination was dependent on the length of homology in an approximately linear manner. Mol Cell Biol, 1992 Feb, 12(2), 444 - 54 Functional characterization of the NF-kappa B p65 transcriptional activator and an alternatively spliced derivative; Ruben SM et al.; The NF-kappa B transcription factor complex is composed of two proteins, designated p50 and p65, both having considerable homology to the product of the rel oncogene . We present evidence that the p65 subunit is a potent transcriptional activator in the apparent absence of the p50 subunit, consistent with in vitro results demonstrating that p65 can interact with DNA on its own . To identify the minimal activation domain, chimeric fusion proteins between the DNA binding domain of the yeast transcriptional activator protein GAL4 and regions of the carboxy terminus of p65 were constructed, and their transcriptional activity was assessed by using a GAL4 upstream activation sequence-driven promoter-chloramphenicol acetyltransferase fusion . This analysis suggests that the boundaries of the activation domain lie between amino acids 415 and 550 . Moreover, single amino acid changes within residues 435 to 459 greatly diminished activation . Similar to other activation domains, this region contains a leucine zipper-like motif as well as an overall net negative charge . To identify those residues essential for DNA binding, we made use of a naturally occurring derivative of p65, lacking residues 222 to 231 (hereafter referred to as p65 delta), and produced via an alternative splice site . Gel mobility shift analysis using bacterially expressed p65, p65 delta, and various mutants indicates that residues 222 to 231 are important for binding to kappa B DNA . Coimmunoprecipitation analysis suggests that these residues likely contribute to the multimerization function required for homomeric complex formation or heteromeric complex formation with p50 in that no association of p65 delta with itself or with p50 was evident . However, p65 delta was able to form weak heteromeric complexes with p65 that were greatly reduced in their ability to bind DNA . On the basis of these findings, we suggest that subtle changes within the proposed multimerization domain can elicit different effects with the individual Rel-related proteins and that a potential role of p65 delta may be to negatively regulate NF-kappa B function through formation of nonfunctional heteromeric complexes. Anal Biochem, 1992 Feb 1, 200(2), 239 - 43 A gel electrophoresis assay for phytochelatins; Abrahamson SL et al.; Phytochelatins are metal-binding peptides produced by higher plants and some fungi in response to heavy metal exposure . Established methods for analyzing cell-free extracts for the presence of phytochelatins include gel-filtration chromatography and HPLC . We have developed a nondenaturing polyacrylamide gel electrophoresis assay for phytochelatins that combines a small sample size with detection via metal binding . This assay can be used for the measurement of the relative affinity of phytochelatins for a variety of metal and semimetal ions. Anal Biochem, 1992 Feb 1, 200(2), 230 - 4 Assay of thiols and disulfides based on the reversibility of N-ethylmaleimide alkylation of thiols combined with electrolysis; Nishiyama J et al.; A simple and specific method for analyzing thiols and disulfides on the basis of the reversibility of N-ethylmaleimide (NEM) alkylation of thiols is described . When the adduct of NEM and glutathione (GSH) was electrolyzed at neutral pH, all of the GSH was recovered . When the adduct was exposed to pH 11.0 for 15 min at 30 degrees C before electrolysis, GSH was not detected . The same behavior was observed after protein thiols reacted with NEM . This pH-dependent production of thiol from the adduct was used to assay GSH and oxidized glutathione in yeast cells, to assay sulfhydryl groups and disulfide bonds in authentic proteins, and to protect thiols from oxidation during enzymatic digestion of protein . This method is useful for assay of thiols and disulfides of both small and large molecules and can be used to identify labile thiols in biological samples that are oxidized during extraction procedures. J Biomol Struct Dyn, 1992 Feb, 9(4), 791 - 805 The affinity of DNA-microtubule protein complexes and their disruption by tubulin binding drugs; Mello CM et al.; Using the gel shift assay system, we have measured the apparent affinity constant for the interaction of two different DNAs with MAP proteins found in both total calf brain microtubules and heat stable brain preparations . Both DNAs studied contained centromere/kinetochore sequences- one was enriched in the calf satellite DNA; the other was a large restriction fragment containing the yeast CEN11 DNA sequence . Complexes formed using both DNAs had similar Kapp values in the range of 2.1 x 10(7) M-1 to 2.0 x 10(8) M-1 . CEN11 DNA-MTP complexes had by far the highest Kapp value of 2.0 x 10(8) M-1 . The CEN11 DNA sequence is where the yeast kinetochore of chromosome 11 is formed and where the single yeast microtubule is bound in vivo . The CEN11 conserved region II known binding sites-(dA/dT)n runs- for mammalian MAP2 protein, are in good agreement with this higher Kapp value . The effects of the classical tubulin binding drugs colchicine, podophyllotoxin and vinblastine on the DNA-MAP protein complex stability were investigated by determining the drug concentrations where the complexes were destabilized . Only the complexes formed from total microtubule protein (tubulin containing) were destabilized over a wide drug concentration range . Heat stable brain protein complexes (no tubulin) were largely unaffected . Furthermore, it took 10-100 fold higher drug concentrations to disrupt the CEN11 DNA complexes compared to the calf thymus satellite DNA enriched complexes . These data support our previous results suggesting that there is a DNA sequence dependent interaction with MAP proteins that appears to be conserved in evolution (Marx et . al., Biochim . Biophys . Acta . 783, 383-392, 1984; Marx and Denial, Molecular Basis of Cancer 172B, 65-75 1985) . In addition, these results imply that the classical tubulin binding drugs may exert their biological effects in cells at least in part by disrupting DNA-Protein complexes of the type we have studied here. Bioessays, 1992 Feb, 14(2), 113 - 8 The end of the message: 3'-end processing leading to polyadenylated messenger RNA; Wahle E; Almost all messenger RNAs carry a polyadenylate tail that is added in a post-transcriptional reaction . In the nuclei of animal cells, the 3'-end of the RNA is formed by endonucleolytic cleavage of the primary transcript at the site of poly(A) addition, followed by the polymerisation of the tail . The reaction depends on specific RNA sequences upstream as well as downstream of the polyadenylation site . Cleavage and polyadenylation can be uncoupled in vitro . Polyadenylation is carried out by poly(A) polymerase with the aid of a specificity factor that binds the polyadenylation signal AAUAAA . Several additional factors are required for the initial cleavage . A newly discovered poly(A)-binding protein stimulates poly(A) tail synthesis and may be involved in the control of tail length . Polyadenylation reactions different from this scheme, either in other organisms or under special physiological circumstances, are discussed. Trends Biochem Sci, 1992 Feb, 17(2), 55 - 8 DNA polymerase epsilon: in search of a function; Hubscher U et al.; The current model of eukaryotic DNA replication involves the two DNA polymerases delta and alpha as the leading and lagging strand enzymes, respectively . A DNA polymerase first discovered in yeast has now been found in all eukaryotic cells and is termed DNA polymerase epsilon . In yeast, the gene for DNA polymerase epsilon has recently been found to be essential for viability, raising new questions about its functions. Mol Biol Cell, 1992 Feb, 3(2), 129 - 42 Protein translocation mutants defective in the insertion of integral membrane proteins into the endoplasmic reticulum; Stirling CJ et al.; Yeast mutants defective in the translocation of soluble secretory proteins into the lumen of the endoplasmic reticulum (sec61, sec62, sec63) are not impaired in the assembly and glycosylation of the type II membrane protein dipeptidylaminopeptidase B (DPAPB) or of a chimeric membrane protein consisting of the multiple membrane-spanning domain of yeast hydroxymethylglutaryl CoA reductase (HMG1) fused to yeast histidinol dehydrogenase (HIS4C) . This chimera is assembled in wild-type or mutant cells such that the His4c protein is oriented to the ER lumen and thus is not available for conversion of cytosolic histidinol to histidine . Cells harboring the chimera have been used to select new translocation defective sec mutants . Temperature-sensitive lethal mutations defining two complementation groups have been isolated: a new allele of sec61 and a single isolate of a new gene sec65 . The new isolates are defective in the assembly of DPAPB, as well as the secretory protein alpha-factor precursor . Thus, the chimeric membrane protein allows the selection of more restrictive sec mutations rather than defining genes that are required only for membrane protein assembly . The SEC61 gene was cloned, sequenced, and used to raise polyclonal antiserum that detected the Sec61 protein . The gene encodes a 53-kDa protein with five to eight potential membrane-spanning domains, and Sec61p antiserum detects an integral protein localized to the endoplasmic reticulum membrane . Sec61p appears to play a crucial role in the insertion of secretory and membrane polypeptides into the endoplasmic reticulum. EMBO J, 1992 Feb, 11(2), 653 - 65 Identification and characterization of Ref-1, a nuclear protein that facilitates AP-1 DNA-binding activity; Xanthoudakis S et al.; Fos and Jun form a heterodimeric complex that regulates gene transcription by binding to the activator protein-1 (AP-1) DNA sequence motif . Previously, we demonstrated that the DNA-binding activity of Fos and Jun is regulated in vitro by a novel redox (reduction-oxidation) mechanism . Reduction of a conserved cysteine (cys) residue in the DNA-binding domains of Fos and Jun by chemical reducing agents or by a nuclear redox factor stimulates DNA-binding activity . Here, we describe purification and characterization of a 37 kDa protein (Ref-1) corresponding to the redox factor . Although Ref-1 does not bind to the AP-1 site in association with Fos and Jun, it partially copurifies with a subset of AP-1 proteins . Purified Ref-1 protein stimulates AP-1 DNA-binding activity through the conserved Cys residues in Fos and Jun, but it does not alter the DNA-binding specificity of Fos and Jun . Ref-1 may represent a novel redox component of the signal transduction processes that regulate eukaryotic gene expression. EMBO J, 1992 Feb, 11(2), 691 - 7 Biochemical demonstration of complex formation of histone pre-mRNA with U7 small nuclear ribonucleoprotein and hairpin binding factors; Melin L et al.; Histone RNA 3' end formation occurs through a specific cleavage reaction that requires, among other things, base-pairing interactions between a conserved spacer element in the pre-mRNA and the minor U7 snRNA present as U7 snRNP . An oligonucleotide complementary to the first 16 nucleotides of U7 RNA can be used to characterize U7 snRNPs from nuclear extracts by native gel electrophoresis . Using similar native gel techniques, we present direct biochemical evidence for a stable association between histone pre-mRNA and U7 snRNPs . Other complexes formed in the nuclear extract are dependent on the 5' cap structure and on the conserved hairpin element of histone pre-mRNA, respectively . However, in contrast to the U7-specific complex, their formation is not required for processing . Comparison of several authentic and mutant histone pre-mRNAs with different spacer sequences demonstrates that the formation and stability of the U7-specific complex closely follows the predicted stability of the potential RNA-RNA hybrid . However, this does not exclude a stabilization of the complex by U7 snRNP structural proteins. Mol Biochem Parasitol, 1992 Feb, 50(2), 325 - 33 Structure and expression of the gene for Pv200, a major blood-stage surface antigen of Plasmodium vivax; Gibson HL et al.; Molecular cloning and structure analysis of the gene encoding the Pv200 protein of the Sal-1 strain of Plasmodium vivax revealed an overall identity of 34-37% when the deduced amino acid sequence was compared with the sequences of various major merozoite surface antigens of Plasmodium falciparum,