S. cerevisiae

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, Plasmodium yoelii and Plasmodium chabaudi . When the Sal-1 Pv200 sequence was compared with the corresponding sequence from the Belem strain of P . vivax, it was found that the two merozoite surface antigens were relatively well conserved with an overall amino acid sequence identity of 81% . A region of 23 repeated glutamine residues, found in the sequence of the Belem isolate was not found, however, in the Sal-1 sequence . Amino- and carboxy-terminal domains of the Pv200 protein were expressed in the yeast Saccharomyces cerevisiae . Each recombinant protein was shown to react with antibodies in sera from splenectomized Bolivian Saimiri monkeys that had been infected previously with P . vivax, and in human sera from individuals with a history of exposure to vivax malaria . The availability of recombinant DNA-derived Pv200 proteins will now allow a full assessment of their utility in the diagnosis and immunoprophylaxis of the benign tertian malaria associated with P . vivax infection.

Chem Pharm Bull (Tokyo), 1992 Feb, 40(2), 446 - 8
Study on stereospecificity of enzyme reaction related to peroxisomal bile acid synthesis in rat liver; Koibuchi Y et al.; We examined stereoselectivities of enzymes related to bile acid formation in hepatic peroxisomes using two stereoisomers of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid (THCA) and its coenzyme A (CoA) derivatives . The activity of acyl-CoA synthetase for 25 S-THCA was 1.4-times higher than that for 25 R-THCA . The difference was also observed after clofibrate-treatment . This activity was located in microsomes, differing from palmitoyl-CoA synthetase located in mitochondria, peroxisomes and microsomes . There was no stereoselectivity in the reaction of peroxisomal fatty acyl-CoA oxidase for THCA isomers, and the activity was one tenth of that for acyl-CoA synthetase . Considering the overall reaction of peroxisomal bile acid formation, the stereoselective difference observed in THCA-CoA synthesis should be denied . Thus, the previous finding that the overall formation of bile acid from THCA was not stereoselective was further confirmed . Furthermore, the activity for THCA oxidation was not induced by clofibric acid, suggesting that there would be different isozymes of peroxisomal acyl-CoA oxidase against THCA-CoA and palmitoyl-CoA.

Genomics, 1992 Feb, 12(2), 394 - 400
Physical mapping of a 950-kb region surrounding a locus (D10S102) tightly linked to the MEN2A gene; Tokino T et al.; We have constructed a long-range contig of cosmid and YAC clones around D10S102, a locus that is tightly linked to the gene responsible for multiple endocrine neoplasia type 2A (MEN2A) . With D10S102 as a starting point, a 360-kb cosmid contig was constructed by bidirectional genomic walking, and at least six fragments from these cosmids showed high sequence homology to other species . Five YAC clones were also isolated at the D10S102 locus, and they formed a contig covering 950 kb of genomic DNA . Furthermore, we obtained six RFLP systems from the contig, which will serve as new resources for fine-scale genetic linkage mapping of the MEN2A locus.

J Clin Invest, 1992 Feb, 89(2), 385 - 91
Cloning and expression of a mutant methylmalonyl coenzyme A mutase with altered cobalamin affinity that causes mut- methylmalonic aciduria; Crane AM et al.; Distinct genotypic and phenotypic forms of methylmalonyl CoA mutase (MCM) apoenzyme deficiency can be delineated by biochemical analysis of mutant fibroblasts . One form, designated mut-, expresses a phenotype in which residual enzyme activity is evident in cultured cells exposed to high concentrations of hydroxycobalamin . We describe cloning of an MCM cDNA from cells exhibiting a mut- phenotype and characterization of the mutant gene product overexpressed in primary muto human fibroblasts and Saccharomyces cerevisiae . Three novel base changes were observed . Recombinant clones containing one of these base changes (G717V) express four characteristics of the mut- phenotype: failure to constitute {14C}propionate incorporation activity in fibroblasts assayed under basal cell culture conditions, constitution of {14C}propionate incorporation activity in fibroblasts stimulated with 0.1-1.0 micrograms/ml hydroxycobalamin, interallelic complementation with alleles bearing an R93H mutation, and an apparent Km (adenosylcobalamin) 1,000-fold higher than normal . These results demonstrate that the G717V mutation produces the mut- phenotype and localizes determinants for adenosylcobalamin binding near the carboxyl terminus of MCM.

Chem Pharm Bull (Tokyo), 1992 Feb, 40(2), 381 - 6
Synthesis and glutamate-agonistic activity of (S)-2-amino-3-(2,5-dihydro-5-oxo-3-isoxazolyl)-propanoic acid derivatives; Tamura N et al.; (S)-2-Amino-3-(2,5-dihydro-5-oxo-3-isoxazolyl)propanoic acid, (3a) and its analogs (3b--h) were prepared and evaluated for glutamate receptor-agonistic and antifungal activities . Several (S)- and (R)-2-amino-3-isoxazolylpropanoic acid derivatives (3a--d) were synthesized starting with (S)- and (R)-N-tert-butoxycarbonyaspartic acid alpha-methyl esters (4, n = 1) by means of Masamune's chain extension reaction followed by isoxazolone formation with hydroxylamine and subsequent deprotection reactions . Furthermore, (S)- and (R)-N-tert-butoxycarbonylglutamic acid alpha-methyl esters (4, n = 2) were converted to (S)- and (R)-2-amino-4-isoxazolylbutyric acid derivatives (3e-h) via the same sequence of reactions.

Oncogene, 1992 Feb, 7(2), 229 - 35
Induction of Xenopus oocyte meiotic maturation by the dbl oncogene product; Graziani G et al.; The dbl oncogene was originally identified by transfection of NIH3T3 cells with DNA from a human diffuse B-cell lymphoma . The dbl oncogene product is a cytoplasmic phosphoprotein distributed between the cytosolic and cytoskeletal matrix-associated membrane fractions . Nucleotide sequence analysis has indicated that the predicted dbl product is very hydrophilic with no detectable similarity to known oncogene products . We have more recently discovered that a region of dbl essential for its transforming activity shows significant sequence similarity to a yeast cell cycle gene, CDC24, which is involved in cell polarity and bud formation in the division cycle of Saccharomyces cerevisiae . This sequence similarity suggests a possible role for dbl in cell division . We report here that the dbl oncogene product is able to induce maturation of Xenopus oocytes . Germinal vesicle breakdown (GVBD) was observed when oocytes were microinjected with the soluble fraction of SF9 insect cells infected with a dbl recombinant baculovirus as well as with the in vitro-transcribed dbl mRNA . Moreover, extracts of oocytes microinjected with dbl mRNA showed activation of H1 histone kinase activity . These findings define a new biologic activity of the dbl product and provide the opportunity to analyse dbl interactions with other components of signaling pathways involved in oocyte maturation.

EMBO J, 1992 Feb, 11(2), 725 - 32
Interstitial telomeres are hotspots for illegitimate recombination with DNA molecules injected into the macronucleus of Paramecium primaurelia; Katinka MD et al.; DNA molecules injected into the macronucleus of Paramecium primaurelia replicate either as free linear telomerized or chromosome integrated molecules . In the present study we show that when a 1.77 kb BamHI DNA fragment harbouring the his3 gene of Saccharomyces cerevisiae was microinjected into the macronucleus, a fraction of the molecules are integrated into the chromosome via an illegitimate recombination process . The injected molecules were mostly inserted at their extremities at multiple points in the genome by replacing the Paramecium sequences . However, insertion sites were not totally at random . Roughly 30% of the molecules were integrated next to or in telomeric repeats . These telomeric repeats were not at the extremities of chromosomes but occupy an internal or interstitial position . We argue that such sites are hotspots for integration as the probability of random insertion near or in an interstitial telomeric site, of which there are 25-60 in a macronucleus is between 5 x 10(-4) and 3 x 10(-5).

Mol Cell Biol, 1992 Feb, 12(2), 734 - 46
Three novel functional variants of human U5 small nuclear RNA; Sontheimer EJ et al.; We have identified and characterized three new variants of U5 small nuclear RNA (snRNA) from HeLa cells, called U5D, U5E, and U5F . Each variant has a 2,2,7-trimethylguanosine cap and is packaged into an Sm-precipitable small nuclear ribonucleoprotein (snRNP) particle . All retain the evolutionarily invariant 9-base loop at the top of stem 1; however, numerous base changes relative to the abundant forms of U5 snRNA are present in other regions of the RNAs, including a loop that is part of the yeast U5 minimal domain required for viability and has been shown to bind a protein in HeLa extracts . U5E and U5F each constitute 7% of the total U5 population in HeLa cells and are slightly longer than the previously characterized human U5 (A, B, and C) species . U5D, which composes 5% of HeLa cell U5 snRNAs, is present in two forms: a full-length species, U5DL, and a shorter species, U5DS, which is truncated by 15 nucleotides at its 3' end and therefore resembles the short form of U5 (snR7S) in Saccharomyces cerevisiae . We have established conditions that allow specific detection of the individual U5 variants by either Northern blotting (RNA blotting) or primer extension; likewise, U5E and U5F can be specifically and completely degraded in splicing extracts by oligonucleotide-directed RNase H cleavage . All variant U5 snRNAs are assembled into functional particles, as indicated by their immunoprecipitability with anti-(U5) RNP antibodies, their incorporation into the U4/U5/U6 tri-snRNP complex, and their presence in affinity-purified spliceosomes . The higher abundance of these U5 variants in 293 cells compared with that in HeLa cells suggests possible roles in alternative splicing.

J Virol, 1992 Feb, 66(2), 1261 - 6
Induction of polyomavirus DNA replication by carcinogens in polyomavirus-transformed rat cells: evidence that the viral enhancer is not the primary target in the induction pathway; Baru M et al.; In the polyomavirus (Py)-transformed rat cell line designated LPT, replication of the integrated Py DNA can be induced by exposure of the cells to carcinogens . In view of the observation that enhancer elements are essential components of the Py origin of replication, it appeared plausible that the induction is triggered by synthesis or modification of an enhancer-binding protein which is required for activation of the viral origin . To test this hypothesis, we have used a plasmid containing a modified Py origin (test plasmid), in which the Py enhancer has been replaced with five repeats of the yeast GAL4 upstream activating sequence, and a plasmid encoding the GAL4 transcriptional activator protein . Previous studies in which these two plasmids were cotransfected into mouse cells that are permissive for Py showed that the GAL4 protein can transactivate the modified Py origin and cause replication of the test plasmid . When similar cotransfection assays were performed in LPT cells, no replication of the test plasmid was observed unless the cells were exposed to the carcinogen mitomycin C subsequent to the transfection, in which case replication of the test plasmid was induced . Control experiments showed that even though the GAL4 protein was required for the induction, its concentration was not affected by the exposure to mitomycin C . These results indicate that the primary target in the induction pathway is not an enhancer-binding protein; instead, the induction appears to be triggered by changes in other components of the replication initiation complex which may be associated with the origin core.

Science, 1992 Jan 31, 255(5044), 603 - 6
The Son of sevenless gene product: a putative activator of Ras; Bonfini L et al.; The Son of sevenless (Sos) gene functions in signaling pathways initiated by the sevenless and epidermal growth factor receptor tyrosine kinases . The Sos gene has now been isolated and sequenced . Its product is a 1595-amino acid protein similar to the CDC25 protein in Saccharomyces cerevisiae, a guanine nucleotide exchange factor that activates Ras . These results imply a role for the ras pathway in Drosophila neuronal development.

Nature, 1992 Jan 30, 355(6359), 409 - 15
Molecular dissection of the secretory pathway; Rothman JE et al.; A combination of biochemistry in animal cell-free systems and genetics in yeast is revealing the molecular machinery of the secretory pathway of eukaryotes . Transporting vesicles have a simple coat structure and employ a general mechanism for fusion that is conserved in evolution.

Biochemistry, 1992 Jan 28, 31(3), 867 - 78
Formation and photolability of low-spin ferrous cytochrome c peroxidase at alkaline pH; Wang JL et al.; The ferrous form of native cytochrome c peroxidase (CCP) is known to undergo a reversible transition when titrated over the pH range of 7.00-9.70 . This transition produces a conversion from a pentacoordinate high-spin to a hexacoordinate low-spin heme active site and is clearly apparent in the heme optical absorption spectra . Here, we report the characterization of this transition and its effect upon the local heme environment using various optical spectroscopies . The formation of hexacoordinate low-spin heme is interpreted to involve the binding of His-52 at the distal site after the perturbation of the extensive H-bonded network within and around the heme pocket of CCP(II) at alkaline pH . Interestingly, CD investigations of CCP(II) in the far-UV and Soret regions indicate the dissappearance of a single high-spin species and the existence of at least two low-spin species of CCP(II) as the pH is raised above 7.90 . Furthermore, transient resonance Raman experiments demonstrate that the hexacoordinate low-spin species can be photolyzed within 10-ns laser pulses, producing a species similar to the low-pH (high-spin) form of CCP(II) at alkaline pH . However, the extent of photolysis is quite pH dependent, with a maximum photodissociation yield at pH = 8.50.

J Biol Chem, 1992 Jan 25, 267(3), 2032 - 7
A single amino acid mutation (Ser180----Cys) determines the polymorphism in cytochrome P450g (P4502C13) by altering protein stability; Faletto MB et al.; Cytochrome P450g is polymorphic in the male rat . This polymorphism is characterized by a 20-40-fold difference in the hepatic content of P450g in the two phenotypes . Sequencing of cDNAs from high (+g) and low (-g) phenotype rats has shown that the low phenotype is due to a defective mRNA containing nine base mutations encoding 7 amino acid substitutions . To determine the role of these structural changes in the phenotypic expression of P450g, we altered each of these residues by site-directed mutagenesis in the present studies and expressed the normal and mutant cDNAs in Saccharomyces cerevisiae . P450+g protein was expressed at a level 4-6-fold higher than that of P450-g in yeast cells, despite the presence of identical mRNA levels . This difference in protein expression approaches the difference seen in the rat . A single amino acid change from Ser180 in P450+g to Cys in P450-g, in a highly conserved region in the P4502C subfamily, was found to be solely responsible for the phenotypic differences in expression of P450g . Protein half-life studies demonstrated that this mutation increases the degradation of P450g . This is the first example of a single amino acid substitution which alters the phenotypic expression of a P450 protein by affecting its stability.

Science, 1992 Jan 24, 255(5043), 450 - 3
Polymerase II promoter activation: closed complex formation and ATP-driven start site opening; Wang W et al.; Studies on bacterial RNA polymerases have divided the initiation pathway into three steps, namely (i) promoter binding to form the closed complex; (ii) DNA melting to form an open complex, and (iii) messenger RNA initiation . Potassium permanganate was used to detect DNA melting by mammalian RNA polymerase II in vitro . Closed complexes formed in a rate-limiting step that was stimulated by the activator GAL4-VP16 . Adenosine triphosphate was then hydrolyzed to rapidly melt the DNA within the closed complex to form an open complex . Addition of nucleoside triphosphates resulted in the melted bubble moving away from the start site, completing initiation.

Cell, 1992 Jan 24, 68(2), 353 - 64
Ligand-induced redistribution of a human KDEL receptor from the Golgi complex to the endoplasmic reticulum; Lewis MJ et al.; Resident luminal endoplasmic reticulum (ER) proteins carry a targeting signal (usually KDEL in animal cells) that allows their retrieval from later stages of the secretory pathway . In yeast, the receptor that promotes this selective retrograde transport has been identified as the product of the ERD2 gene . We describe here the properties of a human homolog of this protein (hERD2) . Overproduction of hERD2 improves retention of a protein with a weakly recognized variant signal (DDEL) . Moreover, overexpression of KDEL or DDEL ligands causes a redistribution of hERD2 from the Golgi apparatus to the ER . Mutation of hERD2 alters the ligand specificity of this effect, implying that it interacts directly with the retained proteins . Ligand control of receptor movement may limit retrograde flow and thus minimize fruitless recycling of secretory proteins.

Nature, 1992 Jan 16, 355(6357), 219 - 24
Chromatin as an essential part of the transcriptional mechanism; Felsenfeld G; The increasingly detailed biochemical definition of the protein complexes that regulate gene transcription has led to the re-emergence of questions about the role of histones . Much recent evidence suggests that transcriptional activation requires that transcription factors successfully compete with histones for binding to promoters, and that there may be more than one mechanism by which this is achieved.

Proc Natl Acad Sci U S A, 1992 Jan 15, 89(2), 599 - 602
Discrimination between related DNA sites by a single amino acid residue of Myc-related basic-helix-loop-helix proteins; Dang CV et al.; A yeast genetic system was developed to study how the basic regions of basic-helix-loop-helix (bHLH) proteins distinguish between related consensus bHLH binding sites, with nucleotide sequence CANNTG . The yeast bHLH protein CBF1 binds to the sequence CAC(A/G)TG found in the yeast centromere element CDE1 and in promoter regions of several yeast genes involved in methionine biosynthesis . Using a functional assay to rescue a mutant cbf1 yeast strain from methionine auxotrophy, we determined that the basic region of CBF1 could be replaced by the homologous region of either the vertebrate USF transcription factor or c-Myc, both of which bind CACGTG . The homologous region of the AP4 transcription factor, which recognizes the sequence CAGCTG, could not functionally replace the CBF1 basic region . However, only a single substitution, Met----Arg, in the AP4 basic region of the inactive chimera CBF-AP4 was sufficient to restore CBF1 function . In randomization experiments, only arginine or lysine provided functional substitutions at the AP4 methionine position . The results suggest that this conserved arginine residue in the basic regions of Myc-related bHLH proteins discriminates between CAC(A/G)TG and related sites.

Proc Natl Acad Sci U S A, 1992 Jan 15, 89(2), 589 - 93
Regulation of DNA replication in vitro by the transcriptional activation domain of GAL4-VP16; Cheng LZ et al.; Studies of DNA viruses have provided evidence that eukaryotic transcriptional activator proteins can enhance the efficiency of DNA replication as well as transcription . The mechanism of this effect was studied in vitro using the chimeric transcription factor GAL4-VP16 and a DNA template containing GAL4 binding sites adjacent to the simian virus 40 origin of DNA replication . The binding of GAL4-VP16 prevented the repression of DNA replication which otherwise occurred when the template was assembled into chromatin . Relief of repression by GAL4-VP16 required both its DNA-binding and transcriptional activation domains but did not require RNA synthesis . The results are consistent with a general model in which transcriptional activators stimulate eukaryotic DNA replication by modifying the outcome of the competition between initiation factors and histones for occupancy of the origin.

FEBS Lett, 1992 Jan 13, 296(1), 82 - 6
Enzymes involved in the dynamic equilibrium of core histone acetylation of Physarum polycephalum; Lopez-Rodas G et al.; DEAE-Sepharose chromatography of extracts from plasmodia of the myxomycete Physarum polycephalum revealed the presence of multiple histone acetyltransferases and histone deacetylases . A cytoplasmic histone acetyltransferase B, specific for histone H4, and two nuclear acetyltransferases A1 and A2 were identified; A1 acetylates all core histones with a preference for H3 and H2A, whereas A2 is specific for H3 and also slightly for H2B . Two histone deacetylases, HD1 and HD2, could be discriminated . They differ with respect to substrate specificity and pH dependence . For the first time the substrate specificity of histone deacetylases was determined using HPLC-purified individual core histone species . The order of acetylated substrate preference is H2A much greater than H3 greater than or equal to H4 greater than H2B for HD1 and H3 greater than H2A greater than H4 for HD2, respectively; HD2 is inactive with H2B as substrate . Moreover histone deacetylases are very sensitive to butyrate, since 2 mM butyrate leads to more than 50% inhibition of enzyme activity.

Nucleic Acids Res, 1992 Jan 11, 20(1), 97 - 104
A new method for the isolation of recombinant baculovirus; Patel G et al.; An improved method for the isolation of baculovirus recombinants is described . The method involves the replication and maintenance of the baculovirus genome in the yeast Saccharomyces cerevisiae which was accomplished by the isolation of a baculovirus recombinant containing yeast ARS and CEN sequences ensuring stable replication in yeast and a URA3 selectable marker . The viral DNA maintained its ability to replicate in insect cells . An efficient and rapid selection system was set up, to isolate viral recombinants in yeast; DNA from selected yeast colonies was transfected into insect cells to obtain recombinant virus . We demonstrate the utility of this system by isolating recombinant viruses that express two different members of the CREB/ATF family of transcription factors.

FASEB J, 1992 Jan 6, 6(2), 749 - 51
Role of Glu318 and Thr319 in the catalytic function of cytochrome P450d (P4501A2): effects of mutations on the methanol hydroxylation; Hiroya K et al.; Polar amino acids in the (putative) distal site are well conserved in P450s . For example, Glu318 for P450d is well conserved as either Glu or Asp for P450s, and Thr319 for P450d is also conserved for P450s . We have studied how mutations at Glu318 and Thr319 of P450d influence the catalytic activity toward methanol associated with the activation of O2 . Catalytic activities of Glu318Asp, Glu318Ala, and Thr319Ala mutants toward methanol were 60, 25, and 38%, respectively, compared with that of the wild type . O2 consumption and NADPH oxidation rates of each mutants varied corresponding to the catalytic activities . However, surprisingly, efficiency (16-40%) of incorporated O to the substrate vs . consumed O2 for the Glu318Ala and Thr319Ala mutants were higher than that (9%) of the wild type . In addition, H2O2, which is produced from uncoupling for the wild-type P450d, was not observed for reaction of the Glu318Ala and Thr319Ala mutants . It seemed that consumed O2 was partially reduced to 2 mol of H2O by 4-electron transfer from NADPH for the wild-type and Thr319Ala mutant . However, for the two Glu318 mutants, it appeared that the consumed O2 was not reduced in the same way . It was thus suggested that the conserved Glu318 and Thr319 of P450d are not essential for the activation of O2 in the methanol oxidation . Role of the water molecule or the methanol molecule in the catalytic function was implied.

J Mol Biol, 1992 Jan 5, 223(1), 337 - 42
Evolutionary conservativeness of electric field in the Cu,Zn superoxide dismutase active site . Evidence for co-ordinated mutation of charged amino acid residues; Desideri A et al.; Equipotential lines were calculated, using the Poisson-Boltzmann equation, for six Cu,Zn superoxide dismutases with different protein electric charge and various degrees of sequence homology, namely those from ox, pig, sheep, yeast, and the isoenzymes A and B from the amphibian Xenopus laevis . The three-dimensional structures of the porcine and ovine superoxide dismutases were obtained by molecular modelling reconstruction using the structure of the highly homologous bovine enzyme as a template . The three-dimensional structure of the evolutionary distant yeast Cu,Zn superoxide dismutase was recently resolved by us, while computer-modelled structures are available for X . laevis isoenzymes . The six proteins display large differences in the net protein charge and distribution of electrically charged surface residues but the trend of the equipotential lines in the proximity of the active sites was found to be constant in all cases . These results are in line with the very similar catlytic rate constants experimentally measured for the corresponding enzyme activities . This analysis shows that electrostatic guidance for the enzyme-substrate interaction in Cu,Zn superoxide dismutases is related to a spatial distribution of charges, arranged so as to maintain, in the area surrounding the active sites, an identical electrostatic potential distribution, which is conserved in the evolution of this protein family.

J Biol Chem, 1992 Jan 5, 267(1), 618 - 23
Molecular characterization of VAC1, a gene required for vacuole inheritance and vacuole protein sorting; Weisman LS et al.; Cell division requires an accurate partitioning of cytoplasmic organelles . The segregation of vacuoles in the budding yeast Saccharomyces cerevisaie occurs at a specific time in the cell cycle and is spatially targeted to the small bud . Several yeast vac mutants have been isolated which are defective in this process . We have now cloned the VAC1 gene, corresponding to the first of these mutants, vac1-1 . This gene encodes a protein of 515 amino acids, without homolog in the current data bases . It contains neither long hydrophobic stretches nor a classical leader peptide . The most notable aspect of the sequence is the presence of three zinc fingers . Yeast in which the VAC1 gene has been entirely deleted are viable . However, they grow more slowly than wild-type cells and only form microcolonies when grown on glycerol at 37 degrees C . These yeast are defective in vacuole segregation at both the permissive and nonpermissive temperatures . The vac1 mutant was previously shown to mislocalize carboxypeptidase Y to the cell surface, suggesting that Vac1p is involved in more than one vesicular traffic pathway.

J Biol Chem, 1992 Jan 5, 267(1), 331 - 8
Ku polypeptides synthesized in vitro assemble into complexes which recognize ends of double-stranded DNA; Griffith AJ et al.; The Ku protein is composed of two polypeptide subunits, p70 and p80, and binds DNA ends in vitro . Previous studies suggested that p70 and p80 are physically associated in vivo, although such an association may have been mediated by DNA . We have now utilized full-length Ku polypeptides synthesized in vitro to examine the association of p70, p80, and linear DNA to form a complex . In gel filtration chromatography, p70 migrates as a 70-kDa structure, whereas p80 migrates at 150 kDa . Co-translation of the two cDNAs yields complexes which migrate at 300 kDa and contain equimolar quantities of the p70 and p80 polypeptides, providing direct evidence that p70 and p80 assemble into a complex in the absence of DNA . To demonstrate that this recombinant protein complex binds DNA, we developed a radiolabeled protein electrophoretic mobility shift assay . When radiolabeled proteins synthesized in vitro were incubated with linear DNA and fractionated in a nonreducing, nondenaturing gel, a band representing a complex of p70, p80, and the DNA was seen . Formation of this Ku-DNA complex required free DNA ends, and binding to DNA ends was not observed with individual p70 or p80 subunits . DNA binding was not reconstituted by mixing the individual subunits together . These studies thus demonstrate that it is the complex of p70 and p80, not individual p70 or p80, which possesses the DNA binding properties previously described for native Ku protein . These results provide new information about the assembly, structure, and DNA binding properties of the Ku protein.

EMBO J, 1992 Jan, 11(1), 335 - 43
Roles of U4 and U6 snRNAs in the assembly of splicing complexes; Vankan P et al.; A series of U4 and U6 snRNA mutants was analysed in Xenopus oocytes to determine whether they block splicing complex assembly or splicing itself . All the U4 and U6 mutants found to be inactive in splicing complementation resulted in defects in assembly of either U4/U6 snRNP or of splicing complexes . No mutants were found to separate the entry of U5 and U6 snRNAs into splicing complexes and neither of these RNAs was able to associate with the pre-mRNA in the absence of U4 . In the absence of U6 snRNA, however, U4 entered a complex containing pre-mRNA as well as the U1 and U2 snRNAs . U6 nucleotides whose mutation resulted in specific blockage of the second step of splicing in Saccharomyces cerevisiae are shown not to be essential for splicing in the oocyte assay . The results are discussed in terms of the roles of U4 and U6 in the assembly and catalytic steps of the splicing process.

Genes Dev, 1992 Jan, 6(1), 93 - 104
Parallel pathways of gene regulation: homologous regulators SWI5 and ACE2 differentially control transcription of HO and chitinase; Dohrmann PR et al.; Two independent pathways of transcriptional regulation that show functional homology have been identified in yeast . It has been demonstrated previously that SWI5 encodes a zinc finger DNA-binding protein whose transcription and cellular localization both are cell cycle regulated . We show that ACE2, whose zinc finger region is nearly identical to that of SWI5, shows patterns of cell cycle-regulated transcription and nuclear localization similar to those seen previously for SWI5 . Despite their similarities, SWI5 and ACE2 function in separate pathways of transcriptional regulation . SWI5 is a transcriptional activator of the HO endonuclease gene, whereas ACE2 is not . In contrast, ACE2 is a transcriptional activator of the CTS1 gene (which encodes chitinase), whereas SWI5 is not . An additional parallel between the SWI5/HO pathway and the ACE2/CTS1 pathway is that HO and CTS1 both are cell cycle regulated in the same way, and HO and CTS1 both require the SWI4 and SWI6 transcriptional activators . Overproduction of either SWI5 or ACE2 permits transcriptional activation of the target gene from the other pathway, suggesting that the DNA-binding proteins are capable of binding in vivo to promoters that they do not usually activate . Chimeric SWI5/ACE2 protein fusion experiments suggest that promoter specificity resides in a domain distinct from the zinc finger domain.

Genes Dev, 1992 Jan, 6(1), 81 - 92
Max: functional domains and interaction with c-Myc; Kato GJ et al.; The product of the c-myc proto-oncogene is a DNA-binding protein, the deregulated expression of which is associated with a variety of malignant neoplasms . The cDNA for the max gene was recently cloned as a result of the ability of its protein product to interact with the c-Myc protein . We studied bacterially produced Max, c-Myc, and a series of truncated c-Myc proteins . Full-length c-Myc alone cannot bind DNA . However, a truncated c-Myc protein comprising the basic, helix-loop-helix, and leucine zipper regions can bind specifically to DNA bearing the sequence GGGCAC(G/A)TGCCC . Max protein, either alone or in a heteromeric complex with full-length c-Myc, binds to the same core sequence . Using a novel combination of chemical and photo-cross-linking analysis, we demonstrate that either Max or a c-Myc/Max heteromeric complex binds to DNA virtually exclusively in a dimeric structure . Using fusion proteins in cultured cells, we establish a number of functional characteristics of Max . First, we show that Max can interact with c-Myc intracellularly in a manner dependent on the integrity of the helix-loop-helix and leucine zipper motifs . Second, a nuclear localization domain that contains the sequence PQSRKKLR is mapped to the carboxy-terminal region of Max . Third, Max lacks a transcriptional activation domain that is functional in Chinese hamster ovary cells when fused to a heterologous DNA-binding domain . These data suggest that Max may serve as a cofactor for c-Myc in transcriptional activation or, by itself, as a transcriptional repressor.

Proc Natl Acad Sci U S A, 1992 Jan 1, 89(1), 407 - 10
Component H of the DNA-dependent RNA polymerases of Archaea is homologous to a subunit shared by the three eucaryal nuclear RNA polymerases; Klenk HP et al.; The gene encoding component H of the DNA-dependent RNA polymerase (RNAP, EC 2.7.7.6) of Sulfolobus acidocaldarius has been identified by comparison of the amino acid sequence with the derived amino acid sequence of an open reading frame (ORF88) in the RNAP operon . Corresponding genes were identified in Halobacterium halobium and were cloned and sequenced from Thermococcus celer and Methanococcus vannielii . All these rpoH genes are situated between the promoters of the RNAP operons and the corresponding rpoB and rpoB2 genes . The archaeal H subunits show high sequence similarity to each other and to the C-terminal portions of the largest of four subunits shared by all three specialized nuclear RNAPs . These correlations are further evidence for the striking similarity between archaeal and eucaryal RNAP structures and transcription systems.

Mol Cell Biol, 1992 Jan, 12(1), 413 - 21
Factors involved in specific transcription by mammalian RNA polymerase II: purification and analysis of transcription factor IIA and identification of transcription factor IIJ; Cortes P et al.; The previously described transcription factor IIA (TFIIA) protein fraction was separated into two factors that affect transcription, TFIIA and TFIIJ . TFIIA was found to have a stimulatory effect, and TFIIJ was found to be required for transcription . The requirement of TFIIJ was observed when bacterially produced purified human or yeast (Saccharomyces cerevisiae) TATA-binding protein (TBP) was used in lieu of the endogenous HeLa cell TFIID complex, suggesting that TFIIJ may be part of the TFIID complex . The stimulatory activity of TFIIA was found also to be dependent on the source of the TBP . Transcription reactions reconstituted with TFIID were stimulated by TFIIA; however, when human or yeast TBP was used instead of TFIID, TFIIA had no effect . TFIIA was found to interact with the TBP and was extensively purified by the use of affinity chromatography on columns containing immobilized recombinant yeast TBP . TFIIA is a heterotrimer composed of polypeptides of 34, 19, and 14 kDa . These three polypeptides were required to isolate, by using the gel mobility shift assay, a stable complex between TBP and the TATA box sequence.

J Bacteriol, 1992 Jan, 174(2), 362 - 6
Developmental regulation of CUP gene expression through DNA methylation in Mucor spp; Cano-Canchola C et al.; Inserts which carried the CUP gene from Saccharomyces cerevisiae or Mucor racemosus were used as hybridization probes to measure the methylation state and expression of the CUP gene from Mucor rouxii at different stages of growth . It was observed that the fungus contains a CUP multigene family . All the CUP genes were present in a hypermethylated DNA region in nongrowing and isodiametrically growing spores and were not transcribed at these stages . After germ tube emergence, CUP genes became demethylated and transcriptionally active . Development, demethylation, and transcription of CUP genes were blocked by the ornithine decarboxylase inhibitor 1,4-diaminobutanone . These results suggest that genes that are activated during development became demethylated in this fungus.

Dev Biol, 1992 Jan, 149(1), 27 - 40
Tissue-restricted accumulation of a ribosomal protein mRNA is not coordinated with rRNA transcription and precedes growth of the sea urchin pluteus larva; Angerer LM et al.; We have identified an mRNA that encodes a protein, SpS24, of the small ribosomal subunit in the sea urchin, Strongylocentrotus purpuratus . RNA blot and in situ hybridization analyses show that the SpS24 gene is active during early oogenesis, downregulated in the mature egg and during cleavage, and reactivated in the early blastula . The mRNA then increases in abundance at least 100-fold . Later in development, expression of SpS24 mRNA becomes restricted primarily to cells in the oral ectoderm and endoderm of the pluteus larva, and the message is undetectable in aboral ectoderm cells and most mesenchyme cells . To determine whether transcription of the ribosomal RNA genes occurs at a higher rate in oral ectoderm and endoderm tissues, a probe for the transcribed spacer was used in RNase protection and in situ hybridization assays . High concentrations of rRNA-processing intermediates were observed in unfertilized eggs and shown to reside primarily, if not exclusively, in the cytoplasm . The spatial and temporal distributions of these sequences strongly suggest that they are associated with heavy bodies . New embryonic rRNA transcripts are first detectable at the very early blastula stage . In later embryos, the content of this transcribed spacer sequence is similar in all but a few cells, which implies that they synthesize rRNA at a similar low rate . Comparison of available estimates of rRNA transcription rate with the potential rate of SpS24 protein synthesis, calculated from SpS24 mRNA prevalence, shows that oral ectoderm and endoderm cells have the capacity to synthesize 15- to 30-fold more SpS24 protein than is required to keep pace with rRNA synthesis in these cells . Because the sea urchin embryo develops from an egg to a pluteus larva in the absence of growth, this stockpiling of SpS24 mRNA anticipates rather than accompanies the onset of growth, which does not begin until after feeding . Upregulation of this gene is therefore part of the developmental program, rather than a physiological response to nutrient availability.

Gene Expr, 1992, 2(1), 39 - 47
Identification of novel steroid-response elements; Nawaz Z et al.; A rapid method for defining novel steroid-responsive elements has been developed . Large libraries of degenerate oligonucleotides were analyzed using a yeast-based screen to identify estrogen-responsive DNA sequences . From a library of 40,000 recombinants, seven estrogen-responsive clones were identified . When sequenced, these elements showed remarkable diversity and were different from the consensus vitellogenin A2 ERE . One surprising result was the presence of the two half sites as direct repeats in some of the clones . This implies that in vivo estrogen receptor can bind and transactivate yeast genes through response elements in which the two half sites align as direct repeats . This protocol requires no purified protein and specifically selects for functional response elements . It has a wide application in the study of any transcription factor/DNA interaction.

Prikl Biokhim Mikrobiol, 1992 Jan-Feb, 28(1), 44 - 9
{A comparative study of the effect of pyrazole on the activity of enzymes in the alcohol/polyol dehydrogenase family}; Sudovtsov VE; A comparative study of the effect of pyrazol, an inhibitor of the coenzyme-binding site of alcohol dehydrogenases, on the activity of enzymes of the alcohol/polyol dehydrogenase group has been carried out . Commercial preparations of alcohol dehydrogenases from the cytoplasm of horse liver cells and yeast cells, as well as the enzyme from the cytoplasm of Trichosporon pullulans cells was completely inhibited by 1 mM pyrazol, while alcohol dehydrogenases from Candida utilis and Saccharomyces carlsbergensis were inhibited only by 25% and the enzymes from Saccharomyces cerevisiae and Torulopsis candida by 30 and 38%, respectively . The inhibition degree of alcohol dehydrogenases from the cytoplasm of liver cells of various mammals (bull, calf, rat, gopher) and birds (hen, pheasant, duck) varied from 12 to 42% in the presence of 1 mM pyrazol . The activity of sorbitol dehydrogenase from the liver cytoplasm of these mammals and birds changed neither in the presence of 1 mM pyrazol, nor in the case of a 15-fold increase of the inhibitor concentration . Possible structural differences in the coenzyme-binding site of the active center of the enzymes under study are discussed.

Electrophoresis, 1992 Jan-Feb, 13(1-2), 1 - 6
Irreversible trapping of DNA during crossed-field gel electrophoresis; Viovy JL et al.; Using an original protocol with a rotating gel electrophoresis apparatus, it is shown that duplex DNA undergoing crossed-field electrophoresis in agarose gets trapped in the gel when the field is increased above a threshold value which decreases with the chain length and depends on the angle between the fields in a non-monotonous manner . This trapping is irreversible, i.e . once trapped at a high field strength, chains are unable to resume their motion when the field is returned to a lower value at which they moved prior to trapping . A model of trapping by "tight knots" is proposed . It predicts a trapping threshold proportional to the inverse square of the electric field, in qualitative agreement with the data . The implications of our results for the separation of large DNA molecules are discussed.

Cytogenet Cell Genet, 1992, 59(1), 17 - 9
Chromosomal localization of the human gene for palmitoyl-CoA ligase (FACL1); Stanczak H et al.; We have localized the gene (FACL1) encoding human long chain fatty acid-coenzyme A ligase, E.C.6.2.1.3, also known as palmitoyl-CoA ligase . Using in-situ hybridization, we have mapped this gene to chromosome region 3q13 in the human karyotype.

J Bacteriol, 1992 Jan, 174(2), 434 - 40
Phosphoglycerate mutase from Streptomyces coelicolor A3(2): purification and characterization of the enzyme and cloning and sequence analysis of the gene; White PJ et al.; The enzyme 3-phosphoglycerate mutase was purified 192-fold from Streptomyces coelicolor, and its N-terminal sequence was determined . The enzyme is tetrameric with a subunit Mr of 29,000 . It is 2,3-bisphosphoglycerate dependent and inhibited by vanadate . The gene encoding the enzyme was cloned by using a synthetic oligonucleotide probe designed from the N-terminal peptide sequence, and the complete coding sequence was determined . The deduced amino acid sequence is 64% identical to that of the phosphoglycerate mutase of Saccharomyces cerevisiae and has substantial identity to those of other phosphoglycerate mutases.

Protein Seq Data Anal, 1992, 5(1), 33 - 8
Hydrophobic zippers and hook-and-eye: evolutionarily conserved protein sequence motifs in eukaryotic acidic ribosomal proteins which are assumed to be involved in the association of the protein family; Tsurugi K; The acidic ribosomal protein family of eukaryotic cells is thought to form a complex on ribosomes mainly by hydrophobic forces . To investigate the structural basis of how they associate with one another, the primary sequences of the related proteins accumulated from various organisms were analyzed searching for evolutionarily conserved hydrophobic motifs . Initially it is shown that all the P1-type 13-kDa proteins contain a bilateral hydrophobic zipper on a putative alpha-helix, which consists of two periodic arrays of hydrophobic amino acid residues arranged on the opposite sides of an alpha-helix . The P2-type 13-kDa proteins, except for those from the yeast Saccharomyces cerevisiae, are shown to contain two kinds of hydrophobic areas on putative alpha-helices, which can sterically bind to each other in a hook-and-eye fashion . On the other hand, the 38-kDa proteins contain a hydrophobic zipper and a hydrophobic hook in different helical regions . Thus, it is proposed that the 13-kDa proteins associate with the 38-kDa proteins via the hydrophobic zipper or hydrophobic hook-and-eye, and associate with one another with these hydrophobic elements.

Methods Enzymol, 1992, 219, 267 - 86
Use of sec mutants to define intermediates in protein transport from endoplasmic reticulum; Rexach MF et al.; In this chapter we have discussed the methodology used to identify and characterize three intermediates in protein transport from the ER that represent stages of transport vesicle budding, targeting, and fusion . The intermediates are obtained using a variety of transport inhibitors: low-temperature incubations, addition of chemicals, or inactivation of Sec protein function using temperature-sensitive mutants or specific antibodies . In all cases, the transport block imposed by the inhibitor is reversible, permitting assessment of the requirements for transport from each intermediate stage . Based on the differential requirements for Sec protein function, as well as the distinct kinetics and efficiency of transport from each intermediate, we demonstrate that each transport intermediate is functionally distinct.

Eur Biophys J, 1992, 21(5), 357 - 62
Application of dynamic light scattering to studies of protein folding kinetics; Gast K et al.; The applicability of dynamic light scattering to studies of the kinetics of unfolding and refolding reactions of proteins is discussed and demonstrated experimentally . The experimental set-up and the data acquisition and data evaluation schemes that have been optimized for kinetic experiments are described . The relationship of the signal-to-noise ratio to the minimum data acquisition time that is needed to obtain results of sufficiently high precision is discussed . It turns out that the attainable time resolution is of the order of a few seconds for proteins with molar masses of about 50,000 g.mol(-1) and concentrations of 1 g.1(-1) . Thus, DLS is too slow to follow conformational changes in the subsecond region, but it is useful for studies of unfolding-refolding reactions of proteins that proceed with time constants in the range of seconds or minutes . This is demonstrated by investigations of the kinetics of the cold denaturation of 3-phosphoglycerate kinase from yeast.

Eur Biophys J, 1992, 21(5), 337 - 44
Interaction of recombinant human epidermal growth factor with phospholipid vesicles . A steady-state and time-resolved fluorescence study of the bis-tryptophan sequence (Trp49-Trp50); Li De La Sierra IM et al.; The interaction of recombinant human epidermal growth factor with small unilamellar phospholipid vesicles was studied by steady-state and time-resolved fluorescence of the bis-tryptophan sequence (Trp49-Trp50) . Steady-state anisotropy measurements demonstrate that strong binding occurred with small unilamellar vesicles made up of acidic phospholipids at acidic pH only (pH < or = 4.7) . An apparent stoichiometry for 1,2-dimyristoyl-sn-phosphoglycerol of about 12 phospholipid molecules per molecule of human epidermal growth factor was estimated . The binding appears to be more efficient at temperatures above the gel to liquid-crystalline phase transition . The conformation and the environment of the Trp-Trp sequence are not greatly modified after binding, as judged from the invariance of the excited state lifetime distribution and from that of the fast processes affecting the anisotropy decay . This suggests that the Trp-Trp sequence is not embedded within the bilayer, in contrast to the situation in surfactant micelles (Mayo et al . 1987; Kohda and Inigaki 1992).

Ciba Found Symp, 1992, 170, 20 - 5; discussion 25-9
Is START a switch?
Cross F, McKinney J.
The cell cycle in Saccharomyces cerevisiae is controlled by regulation of START in late G1 . The CLN1, CLN2 and CLN3 family of cyclin homologues is required for cells to pass START . They probably act by activating the CDC28 protein kinase . Expression of CLN1 or CLN3 under the control of an inducible promoter shows that transcription of either gene is sufficient for cyclin-deficient strains arrested in G1 to traverse START . A model of START regulation involves activation of CDC28 kinase by any CLN protein, leading to activation of CLN1 and CLN2 transcription in a positive feedback loop and passage through START . The cell cycle-dependent transcriptional regulators SWI4 and SWI6 may be components of the feedback loop . Cell cycle commitment entails resistance to the inhibitory action of mating factor, which correlates with peak levels of CLN1 and CLN2 mRNAs . FAR1 encodes an alpha-factor-dependent inhibitor of CLN function whose expression is markedly reduced at the time of START . The interplay of all these factors may sharpen the START transition such that it is close to an all-or-nothing switch event . This may be important for several START-dependent events to be activated at the same time, leading to coordinated cell cycle progression.

Autoimmunity, 1992, 13(4), 265 - 7
The nuclear autoimmune antigen Ku is also present on the cell surface; Dalziel RG et al.; Polyclonal antibodies were raised against the individual 85 and 70 kDa subunits of the Ku complex purified from nuclear extract prepared from the T cell line MLA144 . They specifically recognise the appropriate subunits of the Ku complex from whole cell extract of HeLa cells using Western blot analysis . They are also able to identify the Ku proteins present in the cell membrane using FACS analysis.

Vaccine, 1992, 10(13), 900 - 3
Contribution to hepatitis B prevention; Stephenne J; Hepatitis B is widely recognized as an important public health problem . The only effective way to control hepatitis B is by vaccination . First generation plasma-derived hepatitis B vaccines have limitations . Advances in recombinant DNA technology have led to the development of yeast-derived recombinant hepatitis B vaccine which is now used extensively in the developed world . This article reviews the development of this new generation vaccine and the efforts to facilitate universal vaccination programmes particularly in the developing world . The issue of the cost of new generation vaccines, in relation to the major investment required for research and development and also the quality of the final product, is discussed.

Gene Expr, 1992, 2(3), 297 - 309
Mutation analysis of the Cys-X2-Cys-X19-Cys-X2-Cys motif in the beta subunit of eukaryotic translation initiation factor 2; Castilho-Valavicius B et al.; Recessive lethal mutations in the beta subunit of eIF-2 that restore HIS4 expression in the absence of an AUG start codon were isolated from diploid Saccharomyces cerevisiae strains . DNA sequence analysis of these alleles and of eIF-2 beta suppressor alleles isolated from haploid strains, identified point mutations that altered one of six amino acids that map to a Cys-X2-Cys-X19-Cys-X2-Cys "zinc finger" motif and immediately adjacent residues . Five of the affected amino acids are identical in the human and yeast eIF-2 beta protein . Together with earlier studies (Donahue et al., 1988), these point mutations implicate the zinc finger domain of eIF-2 beta in start-site selection during the scanning process . We have supplemented the mutations obtained by genetic selection with an additional set of constructed mutations in this region . Our studies indicate that the cysteine residues and the intervening amino acids of this motif are essential for eIF-2 beta function in translation initiation in vivo . However, the effects observed in cells containing a copy of eIF-2 beta with a deletion of this motif suggest that this mutated form is still able to associate with other components of the initiation complex, imparting defects on translation initiation . Thus, this motif may be required only for later events that lead to initiator codon recognition . Alterations in defined positions, as found in our suppressor alleles, could lead to recognition of non-AUG codons.

Gene Expr, 1992, 2(3), 273 - 83
Differential modes of activation define orphan subclasses within the steroid/thyroid receptor superfamily; Lydon JP et al.; We report that three orphan receptors, hERR1, hERR2, and hTR2, members of the steroid/thyroid receptor (SR/TR) superfamily, can be activated by different ligand-independent pathways . hERR1 and hERR2 exhibit constitutive activity in the absence of exogenously added ligands . Furthermore, this constitutive activity is localized in the carboxy terminal domain of both receptors and can be transferred to other members of this superfamily using domain switch strategies . In addition, we show that hERR1 can remain constitutively active in the less evolved eukaryotic cell Saccharomyces cerevisiae . In contrast, hTR2 is not constitutively active . However, a chimera of hTR2 can be activated in a ligand-independent manner through a signal transduction pathway initiated at the cell membrane by the neurotransmitter dopamine . Like hERR1 and hERR2, hTR2 is ligand-independently activated through its carboxy terminal domain . Together, these results suggest the existence of emerging subgroups within the SR/TR superfamily that can regulate gene expression through different modes of activation.

Acta Biochim Pol, 1992, 39(2), 205 - 13
The 45 kDa and 27 kDa yeast's protein kinases are not immunologically related; Szyszka R et al.; Two yeast casein kinase type-1 species of 45 kDa and 27 kDa (CK1) were purified to apparent homogeneity and used for investigation of their immunological affinity . Antisera against the two kinases were isolated; the antibody against the 45 kDa kinase did not react with the 27 kDa enzyme . The 27 kDa casein kinase was recognized only by its own antibody . The obtained data strongly suggest that the low molecular mass CK-1 is not a proteolytic product of the 45 kDa kinase species.

Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi, 1992, 10(1), 43 - 4
{An electrophoretic karyotype of Acanthamoeba polyphaga}; Liu JY; An electrophoretic karyotype of Acanthamoeba polyphaga has been preliminarily analysed by means of pulse field gel electrophoresis (PFGE) . Ten chromosomal DNA bands are distinguishable on the gel . Using yeast (Saccharomyces cerevisiae) chromosomal DNA as size standard, we estimate the size of the chromosomes to be between about 200 kilobase pairs (kb) and 2 megabase pairs (mb).

Digestion, 1992, 51(1), 42 - 50
Presence and properties of acyl coenzyme A synthetase for medium-chain fatty acids in rat intestinal mucosa; Ohkubo Y et al.; A system was developed for assay of acyl coenzyme A (acyl CoA) activity on medium-chain fatty acids in rat intestinal mucosa . Using this system, we compared the characteristics of octanoyl (C8:0) CoA synthetase activity and palmitoyl (C16:0) CoA synthetase activity . Palmitoyl CoA synthetase activity as a function of palmitate concentration followed Michaelis-Menten kinetics, but octanoyl CoA synthetase activity as a function of octanoate concentration showed a biphasic reaction curve . The distributions of octanoyl CoA synthetase activity and palmitoyl CoA synthetase activity along the gastrointestinal tract were similar, both activities being present mainly in the middle portion of the small intestine . Incubation of octanoate with a homogenate of intestinal mucosa revealed that octnoate, like palmitate, is incorporated into phospholipids and triglycerides after its CoA activation . Oral administration of medium chain triglycerides induced a nearly 2-fold increase in octanoyl CoA synthetase activity in rat intestinal mucosa, whereas oral administration of long chain triglycerides did not affect the palmitoyl CoA synthetase activity . These results indicate that acyl CoA synthetase for medium-chain fatty acids in rat intestinal mucosa plays a key role in the utilization of medium-chain fatty acids for lipid synthesis, and that it is regulated in a different manner from acyl CoA synthetase for long-chain fatty acids.

Bioseparation, 1992, 2(6), 363 - 73
Synthesis of dye conjugates of ethylene oxide-propylene oxide copolymers and application in temperature-induced phase partitioning; Alred PA et al.; Synthesis of conjugates of the ethylene oxide/propylene oxide copolymer UCON 50-HB-5100 and the triazine dyes Cibacron Blue F3G-A and Procion Yellow HE-3G is described . The UCON-dye conjugate of Procion Yellow HE-3G is used as a ligand for affinity partitioning of glucose-6-phosphate dehydrogenase from bakers' yeast . The enzyme is first partitioned in a two-phase system composed of UCON, UCON-ligand and dextran, and the two phases isolated in separate containers . A small amount of salt is then added to the upper phase, which contains the UCON-ligand-enzyme complex, and the temperature increased above the cloud point of the UCON polymer to give a new two-phase system . The new two-phase system consists of an upper salt/water phase containing free enzyme and a lower UCON/water phase containing free UCON-ligand . Temperature-induced phase partitioning is thus seen to be of much assistance in dissociating enzyme-ligand complex, recovering enzyme and recycling UCON-ligand.

J Ind Microbiol, 1992 Jan, 9(1), 53 - 61
Detoxification of polycyclic aromatic hydrocarbons by fungi; Sutherland JB; The polycyclic aromatic hydrocarbons (PAHs) are a group of hazardous environmental pollutants, many of which are acutely toxic, mutagenic, or carcinogenic . A diverse group of fungi, including Aspergillus ochraceus, Cunninghamella elegans, Phanerochaete chrysosporium, Saccharomyces cerevisiae, and Syncephalastrum racemosum, have the ability to oxidize PAHs . The PAHs anthracene, benz{a}anthracene, benzo{a}pyrene, fluoranthene, fluorene, naphthalene, phenanthrene, and pyrene, as well as several methyl-, nitro-, and fluoro-substituted PAHs, are metabolized by one or more of these fungi . Unsubstituted PAHs are oxidized initially to arene oxides, trans-dihydrodiols, phenols, quinones, and tetralones . Phenols and trans-dihydrodiols may be further metabolized, and thus detoxified, by conjugation with sulfate, glucuronic acid, glucose, or xylose . Although dihydrodiol epoxides and other mutagenic and carcinogenic compounds have been detected as minor fungal metabolites of a few PAHs, most transformations performed by fungi reduce the mutagenicity and thus detoxify the PAHs.

Nucleic Acids Symp Ser, 1992, (27), 177 - 8
Sequence-specific DNA recognition by basic peptides; Morii T et al.; A chiral template with C2 symmetry has been used for modeling a dimeric interface of DNA binding protein . An oligopeptide derived from the basic region of MyoD, a recently described "helix-loop-helix" class of DNA binding protein, has been tethered to the template . Among the four models which differ in chirality and polarity with respect to the arrangement of two subunits, only one dimer model with right-handed and C-terminus to C-terminus arrangement of the peptide subunits binds DNA containing native MyoD binding sequence.

Genetica, 1992, 86(1-3), 203 - 14
Ty1-copia group retrotransposons and the evolution of retroelements in the eukaryotes; Flavell AJ; Ty1-copia group retrotransposons are among the best studied transposable elements in the eukaryotes . This review discusses the extent of these transposons in the eukaryote kingdoms and compares models for the evolution of these genetic elements in the light of recent phylogenetic data . These data show that the Ty1-copia group is widespread among invertebrate eukaryotes, especially in the higher plant kingdom, where these genetic elements are unusually common and heterogeneous in their sequence . The phylogenetic data also suggest that the present day spectrum of Ty1-copia group retrotransposons has been influenced both by divergence during vertical transmission down evolving lineages and by horizontal transmission between distantly related species . Lastly, the factors affecting Ty1-copia group retrotransposon copy number and sequence heterogeneity in eukaryotic genomes and the effects of transpositional quiescence and defective retrotransposons upon evolution of Ty1-copia group retrotransposons are discussed.

Acta Physiol Scand Suppl, 1992, 607, 41 - 7
The membrane sector of vacuolar H(+)-ATPase by itself is impermeable to protons; Beltran C et al.; The sensitivity for vanadate of proton uptake activities of isolated chromaffin granules and yeast vacuoles was investigated . About 0.5 mM vanadate caused 50% inhibition of ATP-dependent proton uptake activity in both membranes . The proton conductivity across chromaffin granule membranes and yeast vacuoles was assayed in the presence and absence of the catalytic sectors of their respective V-ATPases . Removal of the catalytic sectors by cold treatment in the presence of MgATP did not change the proton conductivity of the membranes . Similar results were obtained with yeast vacuoles of mutants that did not assemble the catalytic sector of the enzyme . These results are in contrast to the effect of removing the catalytic sectors of F-ATPases from various sources . The mechanistic and biological significance of the difference in the behavior of the two families of proton pumps is discussed.

Int Rev Immunol, 1992, 8(4), 337 - 55
Expression of the mouse mammary tumor virus ORF gene in cultured cells; Blochlinger K et al.; We have recently shown that expression vectors harboring the open reading frame of the long terminal repeat region of mouse mammary tumor virus direct the synthesis of a product which acts as a superantigen in transgenic mice . The detection of the ORF protein has been hampered by the extremely low levels of expression observed in these mice, as estimated from the low levels of specific mRNA . To study the properties of the ORF protein, we attempted its expression in different cell types in culture . The experiments performed in yeast show that the ORF gene product is a glycoprotein of approximately 45 kDA . As expected from the derived primary sequence, the unglycosylated product made in the presence of tunicamycin has a molecular weight of 36 kDA . No secretion of the glycosylated protein was observed . Curiously, the full-length molecule was made in lower amounts than a truncated version which contains only the C-terminal half of the protein . Transfection experiments in different mammalian cells suggest that high expression of the ORF protein might have an adverse effect on survival of cells in culture.

Bioessays, 1992 Jan, 14(1), 9 - 16
Reversible histone modifications and the chromosome cell cycle; Bradbury EM; During the eukaryotic cell cycle, chromosomes undergo large structural transitions and spatial rearrangements that are associated with the major cell functions of genome replication, transcription and chromosome condensation to metaphase chromosomes . Eukaryotic cells have evolved cell cycle dependent processes that modulate histone:DNA interactions in chromosomes . These are; i) acetylations of lysines; ii) phosphorylations of serines and threonines and iii) ubiquitinations of lysines . All of these reversible modifications are contained in the well-defined very basic N- and C-terminal domains of histones . Acetylations and phosphorylations markedly affect the charge densities of these domains whereas ubiquitination adds a bulky globular protein, ubiquitin, to lysines in the C-terminal tails of H2A and H2B . Histone acetylations are strictly associated with genome replication and transcription; histone H1 and H3 phosphorylations correlate with the process of chromosome condensation . The subunits of histone H1 kinase have now been shown to be cyclins and the p34CDC2 kinase product of the cell cycle control gene CDC2 . It is probable that all of the processes that control chromosome structure:function relationships are also involved in the control of the cell cycle.

EMBO J, 1992 Jan, 11(1), 367 - 72
Drosophila UbcD1 encodes a highly conserved ubiquitin-conjugating enzyme involved in selective protein degradation; Treier M et al.; Ubiquitin-dependent selective protein degradation serves to eliminate abnormal proteins and provides controlled short half-lives to certain cellular proteins, including proteins of regulatory function such as phytochrome, yeast MAT alpha 2 repressor, p53 and cyclin . Moreover, ubiquitin-dependent proteolysis is thought to play an essential role during development and in programmed cell death . We have cloned a gene from Drosophila melanogaster, UbcD1, coding for a protein with striking sequence similarity to the yeast ubiquitin-conjugating enzymes UBC4 and UBC5 . These closely related yeast enzymes are known to be central components of a major proteolytic pathway of Saccharomyces cerevisiae . By doing a precise open reading frame replacement in the yeast genome we could show that the Drosophila UbcD1 enzyme can functionally substitute for yeast UBC4 . UbcD1 driven by the UBC4 promoter rescues growth defects and temperature sensitivity of yeast ubc4 ubc5 double mutant cells . Moreover, expression of UbcD1 restores proteolysis proficiency in the ubc4 ubc5 double mutant, indicating that the Drosophila enzyme also mediates protein degradation . This structural and functional conservation suggests that the UbcD1-UBC4-UBC5 class of enzymes defines a major proteolytic pathway in probably all eukaryotes.

Mol Cell Biol, 1992 Jan, 12(1), 266 - 75
The basic region of myogenin cooperates with two transcription activation domains to induce muscle-specific transcription; Schwarz JJ et al.; Myogenin is a skeletal muscle-specific transcription factor that can activate myogenesis when introduced into a variety of nonmuscle cell types . Activation of the myogenic program by myogenin is dependent on its binding to a DNA sequence known as an E box, which is associated with numerous muscle-specific genes . Myogenin shares homology with MyoD and other myogenic regulatory factors within a basic region and a helix-loop-helix (HLH) motif that mediate DNA binding and dimerization, respectively . Here we show that the basic region-HLH motif of myogenin alone lacks transcriptional activity and is dependent on domains in the amino and carboxyl termini to activate transcription . Analysis of these N- and C-terminal domains through creation of chimeras with the DNA-binding domain of the Saccharomyces cerevisiae transcription factor GAL4 revealed that they act as strong transcriptional activators . These transcription activation domains are dependent for activity on a specific amino acid sequence within the basic region, referred to as the myogenic recognition motif (MRM), when an E box is the target for DNA binding . However, the activation domains function independent of the MRM when DNA binding is mediated through a heterologous DNA-binding domain . The activation domain of the acidic coactivator VP16 can substitute for the myogenin activation domains and restore strong myogenic activity to the basic region-HLH motif . Within a myogenin-VP16 chimera, however, the VP16 activation domain also relies on the MRM for activation of the myogenic program . These findings reveal that DNA binding and transcriptional activation are separable functions, encoded by different domains of myogenin, but that the activity of the transcriptional activation domains is influenced by the DNA-binding domain . Activation of muscle-specific transcription requires collaboration between the DNA-binding and activation domains of myogenin and is dependent on events in addition to DNA binding.

Biotechnol Ther, 1992, 3(1-2), 15 - 34
Muramyl peptide adjuvants for Plasmodium falciparum and Plasmodium vivax circumsporozoite vaccines in rodent model systems; Bathurst IC et al.; Circumsporozoite proteins from the malaria parasites Plasmodium falciparum and Plasmodium vivax were expressed at high levels in the yeast Saccharomyces cerevisiae . Recombinant proteins varied both in length and in number of the natural amino acid repeat motifs . The proteins were purified and used to immunize mice, guinea pigs, and rabbits . Novel muramyl peptide adjuvants were used that increased the immune response as measured by ELISA assays, indirect immunofluorescence of fixed sporozoites, and the invasion of cultured liver cells by live sporozoites . These results suggest that an improved humoral response to recombinant circumsporozoite vaccines might be achieved by varying the design of the recombinant protein and by the use of novel adjuvant systems.

Dev Genet, 1992, 13(6), 440 - 67
There are two mechanisms of achiasmate segregation in Drosophila females, one of which requires heterochromatic homology; Hawley RS et al.; There are numerous examples of the regular segregation of achiasmate chromosomes at meiosis I in Drosophila melanogaster females . Classically, the choice of achiasmate segregational partners has been thought to be independent of homology, but rather made on the basis of availability or similarities in size and shape . To the contrary, we show here that heterochromatic homology plays a primary role in ensuring the proper segregation of achiasmate homologs . We observe that the heterochromatin of chromosome 4 functions as, or contains, a meiotic pairing site . We show that free duplications carrying the 4th chromosome pericentric heterochromatin induce high frequencies of 4th chromosome nondisjunction regardless of their size . Moreover, a duplication from which some of the 4th chromosome heterochromatin has been removed is unable to induce 4th chromosome nondisjunction . Similarly, in the absence of either euchromatic homology or a size similarity, duplications bearing the X chromosome heterochromatin also disrupt the segregation of two achiasmate X chromosome centromeres . Although heterochromatic regions are sufficient to conjoin nonexchange homologues, we confirm that the segregation of heterologous chromosomes is determined by size, shape, and availability . The meiotic mutation Axs differentiates between these two processes of achiasmate centromere coorientation by disrupting only the homology-dependent mechanism . Thus there are two different mechanisms by which achiasmate segregational partners are chosen . We propose that the absence of diplotene-diakinesis during female meiosis allows heterochromatic pairings to persist until prometaphase and thus to co-orient homologous centromeres . We also propose that heterologous disjunctions result from a separate and homology-independent process that likely occurs during prometaphase . The latter process, which may not require the physical association of segregational partners, is similar to those observed in many insects, in Saccharomyces cerevisiae and in C . elegans males . We also suggest that the physical basis of this process may reflect known properties of the Drosophila meiotic spindle.

J Enzyme Inhib, 1992, 6(2), 175 - 80
Purification and characterization of invertase inhibitors from Dioscorea rotundata tuber; Nok AJ et al.; Three invertase inhibitors (A), (B) and (C) from Dioscorea rotundata tuber were resolved on DEAE-cellulose ion exchanger . Two of the inhibitors, (B) and (C), were proteins and homogenous on polyacrylamide gels Mr 21,000 +/- 85 and 26,982 +/- 40/36,307 +/- 50 respectively . The inhibitors (B) and (C) were inactivated at 60 degrees C and had activity-pH optima at 5.2 and 6.4 respectively . (B) and (C) were non competitive inhibitors of invertase from yam and other sources.

Biochem Biophys Res Commun, 1991 Dec 31, 181(3), 1125 - 30
The conformation of a-factor is not influenced by the S-prenylation of Cys12; Gounarides JS et al.; Two-Dimensional NMR was used to examine the solution conformation of the lipopeptide a-factor, YIIKGVFWDPAC (S-farnesyl) OCH3, from the yeast Saccharomyces cerevisiae and five analogues containing various S-alkylated cysteines in DMSO-d6 . NOESY data, NH temperature coefficients, and 3J alpha NH coupling constants indicate that the a-factor is a predominantly unstructured peptide in DMSO . Similar results were obtained for the other peptides indicating that S-prenylation of Cys12 does not affect the conformation of these peptides.

Science, 1991 Dec 20, 254(5039), 1808 - 10
Reverse transcriptase encoded by a human transposable element; Mathias SL et al.; L1 elements are highly repeated mammalian DNA sequences whose structure suggests dispersal by retrotransposition . A consensus L1 element encodes a protein with sequence similarity to known reverse transcriptases . The second open reading frame from the human L1 element L1.2A was expressed as a fusion protein targeted to Ty1 virus-like particles in Saccharomyces cerevisiae and shown to have reverse transcriptase activity . This activity was eliminated by a missense mutation in the highly conserved amino acid motif Y/F-X-D-D . Thus, L1 represents a potential source of the reverse transcriptase activity necessary for dispersion of the many classes of mammalian retroelements.

J Chromatogr, 1991 Dec 27, 588(1-2), 157 - 64
High-performance affinity chromatography of messenger RNA; Goss TA et al.; A 50-mer of thymidylic acid, (dT)50, was coupled to silica inside prepacked columns using the N-hydroxysuccinimide chemistry . The resulting (dT)50-silica columns were used to resolve oligomers of adenylic acid, (dA)19-24, and to separate poly(A) mRNA (messenger RNA) from Saccharomyces . Oligomers which differed in length by a single nucleotide base were readily resolved . Using either (dT)50- or (dT)18-silica, poly(A) mRNA could be purified in as little as 8 min . The poly(A) mRNA isolated appeared to be full length and could be used directly for T4 RNA ligase and RNAse A and T1 enzymatic reactions . The (dT)50-silica column was used to fractionate total poly(A) mRNA by tail length . While the separation was primarily due to poly(A) tail length, most fractions appeared to contain multiple tail lengths . Whether this represents an intrinsic feature of the RNA or a limitation of the method is discussed . These studies show that polynucleotides in the kilobase size range can be separated rapidly and with good resolution on DNA-silica.

J Biol Chem, 1991 Dec 25, 266(36), 24670 - 5
Mitochondrial and peroxisomal beta oxidation of the branched chain fatty acid 2-methylpalmitate in rat liver; Vanhove G et al.; A number of isoprenoids (e.g . pristanic acid and the side chains of fat soluble-vitamins) is degraded or shortened via beta oxidation . We synthesized 2-methyl-palmitate and 2-methyl{1-14C} palmitate as a model substrate for the study of the beta oxidation of branched (isoprenoid) fatty acids in rat liver . 2-Methylpalmitate was well oxidized by isolated hepatocytes and its oxidation was stimulated after treatment of the animals with a peroxisome proliferator . Subcellular fractionation of rat liver demonstrated that 2-methylpalmitate is activated to its CoA ester in endoplasmic reticulum, mitochondria, and peroxisomes and that mitochondria and peroxisomes are capable of beta-oxidizing 2-methylpalmitate . At low unbound 2-methylpalmitate concentrations and in the presence of competing straight chain fatty acids, a condition encountered in vivo, peroxisomal 2-methyl-palmitate oxidation was 2- to 4-fold more active than mitochondrial oxidation . Treatment of rats with a peroxisome proliferator markedly stimulated mitochondrial but only slightly peroxisomal 2-methylpalmitate oxidation . The same treatment dramatically induced palmitoyl-CoA oxidase but did not change 2-methyl-palmitoyl-CoA oxidase activity . Our results indicate 1) that in untreated rats peroxisomes contribute for an important part to the oxidation of 2-methylpalmitate; 2) that treatment with a peroxisome proliferator stimulates mainly the mitochondrial component of 2-methylpalmitate oxidation; and 3) that palmitoyl-CoA and 2-methylpalmitoyl-CoA are oxidized by different peroxisomal oxidases.

Nucleic Acids Res, 1991 Dec 25, 19(24), 6819 - 21
Requirement of protein factors and ATP for the disassembly of the spliceosome after mRNA splicing reaction; Sawa H et al.; Pre-mRNA splicing reaction occurs in a large ribonucleoprotein complex called the spliceosome . After the splicing reaction, the spliceosome is disassembled to release the splicing products including spliced mRNA . Here we show that protein factors in a HeLa nuclear extract or a DEAE-cellulose fraction as well as ATP are required to release the splicing products form the spliceosome in which the splicing reaction has already completed.

J Biol Chem, 1991 Dec 25, 266(36), 24390 - 7
Mutation of invariant cysteines of mammalian metallothionein alters metal binding capacity, cadmium resistance, and 113Cd NMR spectrum; Cismowski MJ et al.; Using a yeast expression vector system, we have expressed both wild type and six mutated Chinese hamster metallothionein coding sequences in a metal-sensitive yeast strain in which the endogenous metallothionein gene has been deleted . The mutant proteins have single or double cysteine to tyrosine replacements (C13Y, C50Y, and C13,50Y), single cysteine to serine replacements (C13S and C50S), or a single cysteine to alanine replacement (C50A) . These proteins function in their yeast host in cadmium detoxification to differing extents . Metallothioneins which contain a cysteine mutation at position 50 (C50Y, C50S, C50A, and C13,50Y) conferred markedly less cadmium resistance than wild type metallothionein, or metallothionein with a single cysteine mutation at position 13 (C13Y and C13S) . Wild type and three of the mutant Chinese hamster metallothioneins (C13Y, C50Y, and C13,50Y) were purified from yeast grown in subtoxic levels of either CdCl2 or 113CdCl2 . All three of the mutant proteins bound less cadmium than the wild type protein when metal-binding stoichiometries were determined . The one-dimensional 113Cd NMR spectrum of the recombinant wild type Chinese hamster metallothionein was compared to the spectra of native rat and rabbit liver metallothioneins . The close correspondence between the 113Cd chemical shifts in these metallothioneins is consistent with the presence of two separate metal clusters, A and B, corresponding, respectively, to the alpha- and beta-domains, in the recombinant metallothionein . The one-dimensional 113Cd NMR spectra recorded on each of the three mutant metallothioneins, on the other hand, provide some indication as to the structural basis for the reduced, by one, metal stoichiometry of each of the mutant metallothioneins . For the C13Y mutant, it appears that the beta-domain now binds a total of two metal ions whereas with the C50Y mutant, the alpha-domain appears metal-deficient . For the double mutant, C13,50Y, the 113Cd resonances are indicative of major structural reorganizations in both domains.

Cell, 1991 Dec 20, 67(6), 1231 - 40
TFIID binds in the minor groove of the TATA box; Starr DB et al.; We have analyzed the interaction of the general RNA polymerase II transcription factor TFIID with its DNA-binding site, the TATA box (consensus sequence TATAAAA) . We have demonstrated that TFIID, unlike most sequence-specific DNA-binding proteins, interacts primarily within the minor groove of the DNA helix . This was established by a novel approach involving complete replacement of the thymines and adenines in the TATA box with cytosines and inosines, respectively . This substitution exchanged the major groove of TATAAAA for that of the sequence CGCGGGG, without altering the surface of the minor groove . The unusual DNA-binding properties of TFIID revealed by this study have important implications for TFIID specificity and function and, more generally, for sequence-specific recognition by DNA-binding proteins.

Biochemistry, 1991 Dec 17, 30(50), 11615 - 20
Periodicity of amide proton exchange rates in a coiled-coil leucine zipper peptide; Goodman EM et al.; The two-stranded coiled-coil motif, which includes leucine zippers, is a simple protein structure that is well suited for studies of helix-helix interactions . The interaction between helices in a coiled coil involves packing of "knobs" into "holes", as predicted by Crick in 1953 and confirmed recently by X-ray crystallography for the GCN4 leucine zipper {O'Shea, E.K., Klemm, J.D., Kim, P.S., & Alber, T . (1991) Science 254, 539} . A striking periodicity, extending over six helical turns, is observed in the rates of hydrogen-deuterium exchange for amide protons in a peptide corresponding to the leucine zipper of GCN4 . Protons at the hydrophobic interface show the most protection from exchange . The NMR chemical shifts of amide protons in the helices also show a pronounced periodicity which predicts a short H-bond followed by a long H-bond every seven residues . This variation was anticipated in 1953 by Pauling and is sufficient to give rise to a local left-handed superhelical twist characteristic of coiled coils . The amide protons that lie at the base of the "hole" in the "knobs-into-holes" packing show slow amide proton exchange rates and are predicted to have short H-bond lengths . These results suggest that tertiary interactions can lead to highly localized, but substantial, differences in stability and dynamics within a secondary structure element and emphasize the dominant nature of packing interactions in determining protein structure.

FEBS Lett, 1991 Dec 16, 295(1-3), 189 - 94
A single activity carboxyl methylates both farnesyl and geranylgeranyl cysteine residues; Volker C et al.; Members of the Ras superfamily of small GTP-binding proteins, gamma-subunits of heterotrimeric G proteins and nuclear lamin B are subject to a series of post-translational modifications that produce prenylcysteine methylester groups at their carboxyl termini . The thioether-linked polyisoprenoid substituent can be either farnesyl (C15) or geranylgeranyl (C20) . Small molecule prenylcysteine derivatives with either the C15 or C20 modification, such as N-acetyl-S-trans,trans-farnesyl-L-cysteine (AFC), S-trans,trans-farnesylthiopropionate (FTP), as well as the corresponding geranylgeranyl derivatives (AGGC and GGTP) are substrates for the carboxyl methyltransferase . Saccharomyces cerevisiae ste14 mutants that lack RAS and a-factor carboxyl methyltransferase activity are also unable to methylate farnesyl and geranylgeranylcysteine derivatives . Moreover, C20-substituted cysteine analogs directly compete for carboxyl methylation with the C15-substituted cysteine analogs and vice versa . Finally, AGGC is even more effective than AFC as an inhibitor of Ras carboxyl methylation, despite the fact that Ras is methylated at a farnesylcysteine rather than a geranylgeranylcysteine residue.

Biochem Biophys Res Commun, 1991 Dec 16, 181(2), 657 - 63
Enzyme chemistry of dithiohemiacetals: synthesis and characterization of S-D-dithiomandeloylglutathione as an alternate substrate for glyoxalase I; Li J et al.; Both the D- and L-forms of S-dithiomandeloylglutathione (1) have been synthesized by a dithioester-interchange reaction between GSH and S-carboxy-methyl(D,L)-dithiomandelate . Kinetic and product analysis studies indicate that yeast glyoxalase I efficiently catalyzes the stereoselective conversion of D-1 to GSH-phenylglyoxal dithiohemiacetal (2), isolated as a disulfide adduct between 2 and a second molecule of GSH . This observation suggests that dithioester substrate analogues should be generally useful as mechanistic probes of enzyme catalyzed reactions involving thiohemiacetal intermediates.

Biochem Biophys Res Commun, 1991 Dec 16, 181(2), 524 - 8
The phosphotransferase activity of casein kinase II is required for its physiological function in vivo; Birnbaum MJ et al.; We have previously shown that the inviability associated with disruption of both catalytic subunits of casein kinase II in Saccharomyces cerevisiae can be rescued by plasmids expressing the catalytic subunit of the Drosophila enzyme (Padmanabha et al., 1990, Mol . Cell . Biol . 10, 4089) . Here we describe the construction of mutant forms of the Drosophila catalytic subunit in which residues known to be crucial for catalytic activity in other protein kinases have been altered by site-directed mutagenesis . Mutation of either Lys66 or Asp173, which correspond to Lys72 and Asp184 of cAMP-dependent protein kinase, respectively, yields a casein kinase II catalytic subunit which fails to rescue a yeast strain lacking both endogenous catalytic subunit genes . The data indicate that the phosphotransferase activity of casein kinase II is required for its physiological function in vivo.

Biochem J, 1991 Dec 15, 280 ( Pt 3), 777 - 81
Possible role of histone acetylation and histone H1(0) replacement for the initiation of replication in regenerating rat liver; Weiss G et al.; The role of histone acetylation and DNA synthesis has been investigated extensively in the regenerating rat liver system in the presence and absence of the cyclophosphamide derivative mafosfamide . We demonstrate a mafosfamide-induced inhibition of maximum histone acetyltransferase activity followed by a second elevation of enzyme activity and an accompanying total suppression of DNA synthesis for 7-8 h . The maximum of histone acetyltransferase activity, in parallel with an elevated acetylation in vivo, the consecutive replacement of histone H1(0) amd initiation of replication occur sequentially in the presence and absence of mafosfamide, but with a temporary delay of 7-8 h . Our data indicate that modifications of histone acetyltransferase (EC 2.3.1.48) activity do not significantly influence the acetylation patterns of histones H3 and H4 . The mafosfamide-induced change of histone acetyltransferase activity and acetylation in vivo, the shift of histone H1(0) exchange and the consecutive transition of initiation of replication suggest that these three events might be functionally related.

Proc Natl Acad Sci U S A, 1991 Dec 15, 88(24), 11091 - 4
Uncoupling thermotolerance from the induction of heat shock proteins; Smith BJ et al.; Exposure of cells to elevated temperatures causes a rapid increase in the synthesis of heat shock proteins (hsps) and induces thermotolerance, the increased ability of cells to survive exposure to lethal temperatures; however, the connection between hsp induction and the acquisition of thermotolerance is unclear . hsp induction in the yeast Saccharomyces cerevisiae is mediated by the activation of heat-shock transcription factor, and recently we have described a mutation, hsf1-m3, in heat-shock transcription factor that prevents the factor's activation . We now demonstrate that this mutation results in a general block in heat-shock induction but does not affect the acquisition of thermotolerance . Our results indicate that high-level induction of the major hsps is not required for cells to acquire thermotolerance.

J Biol Chem, 1991 Dec 15, 266(35), 24116 - 20
A site-directed approach for constructing temperature-sensitive ubiquitin-conjugating enzymes reveals a cell cycle function and growth function for RAD6; Ellison KS et al.; We have determined the gene sequence of a temperature-sensitive allele of the cell cycle-related ubiquitin-conjugating enzyme CDC34 (UBC 3) from Saccharomyces cerevisiae . The basis of temperature sensitivity is a missense mutation resulting in a proline to serine substitution at a residue that is conserved in all ubiquitin-conjugating enzymes identified thus far . This observation raised the possibility that other temperature-sensitive ubiquitin-conjugating enzymes could be generated in the same way . We therefore created the corresponding substitution in the DNA repair-related ubiquitin-conjugating enzyme, RAD6 (UBC2), and examined the effect of temperature on the cell proliferation and DNA repair-related functions of this altered polypeptide . Yeast strains carrying this mutation proved to be temperature-sensitive with respect to cell proliferation but not with respect to the DNA damage-processing phenotypes exhibited by other rad6 mutants . Upon further investigation of the proliferation defect exhibited by this mutant, we discovered that other rad6 gene mutants deleted for the gene undergo cell cycle arrest at the nonpermissive temperature, whereas the engineered temperature-sensitive allele showed no evidence of a cell cycle defect . From these findings, we conclude that the proliferation function of RAD6 can be subdivided into a growth component and a cell division cycle component and that the growth component is unrelated to the DNA repair functions of RAD6 . A reasonable interpretation of these results is that different proteins are targeted for ubiquitination in each case . The conserved proline residue of RAD6 and CDC34 is part of a turn motif common to all ubiquitin-conjugating enzymes . It is therefore likely that site-directed substitution of prolines located in turns can be generally applied for the creation of other temperature-sensitive ubiquitin-conjugating enzymes and possibly other proteins as well.

J Biol Chem, 1991 Dec 15, 266(35), 24044 - 7
Glucose increases the expression of the ATP/ADP translocator and the glyceraldehyde-3-phosphate dehydrogenase genes in Chlorella; Hilgarth C et al.; A presumably full-length cDNA clone of the mitochondrial ATP/ADP translocator (AAT) of Chlorella kessleri has been isolated and sequenced . The expression of the AAT gene is highly increased in the presence of D-glucose (14 mM) . At least nine more genes are activated when autotrophically grown Chlorella cells switch to heterotrophic growth . Among these is the HUP1 gene coding for the hexose transporter (Sauer, N., Caspari, T., Klebl, F., and Tanner, W . (1990) Proc . Natl . Acad . Sci . U.S.A . 87, 7949-7952) and, as also shown in this paper, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene . When glucose or the nonmetabolizable analogue 6-deoxyglucose is added to the cells, an increased expression of GAPDH or AAT is observed after 10 or 30 min, respectively . Hexose uptake mutants (HUP1-) do not respond to sugars in this way, which indicates that either the inducer has to be internalized or that the HUP1 translocator is part of the signal transduction mechanism.

Proc Natl Acad Sci U S A, 1991 Dec 15, 88(24), 11197 - 201
Primary structure of the catalytic subunit of human DNA polymerase delta and chromosomal location of the gene; Chung DW et al.; The catalytic subunit (Mr approximately 124,000) of human DNA polymerase delta has been cloned by PCR using poly(A)+ RNA from HepG2 cells and primers designed from the amino acid sequence of regions highly conserved between bovine and yeast DNA polymerase delta . The human cDNA was 3443 nucleotides in length and coded for a polypeptide of 1107 amino acids . The enzyme was 94% identical to bovine DNA polymerase delta and contained the numerous highly conserved regions previously observed in the bovine and yeast enzymes . The human enzyme also contained two putative zinc-finger domains in the carboxyl end of the molecule, as well as a putative nuclear localization signal at the amino-terminal end . The gene coding for human DNA polymerase delta was localized to chromosome 19.

J Biol Chem, 1991 Dec 15, 266(35), 23525 - 8
Regulation of gene expression by insulin and tumor necrosis factor alpha in 3T3-L1 cells . Modulation of the transcription of genes encoding acyl-CoA synthetase and stearoyl-CoA desaturase-1; Weiner FR et al.; Insulin and tumor necrosis factor alpha (TNF alpha) produce potent and opposing physiological signals in adipocytes . However, genes that are co-regulated by the hormone and cytokine during and after adipocyte differentiation have not been characterized . Using 3T3-L1 cells, we have studied the regulation of the expression of genes encoding acyl-CoA synthetase (ACS), and stearoyl CoA desaturase-1 (SCD-1), two enzymes that play key roles in the metabolism of long chain fatty acids . Insulin is required for triggering the transcriptional activation of the ACS and SCD-1 genes at an early stage in adipocyte differentiation . In mature adipocytes insulin elicits a 4-fold increase in the rates of transcription of the two genes . However, when 3T3-L1 adipocytes are treated with TNF alpha the cytokine causes a 75-90% decrease in the levels of ACS and SCD-1 mRNAs . The decline in mRNA content is associated with similar decrements in the rates of transcription of the ACS and SCD-1 genes . Thus, the ACS and SCD-1 genes are subject to stimulation and counter-regulation (at the transcriptional level) by insulin and TNF alpha, respectively . The opposing effects of insulin and TNF alpha are observed in developing and terminally differentiated adipocytes . Unlike the ACS and SCD-1 genes, the genes that encode the lipogenic enzymes lipoprotein lipase and malic enzyme are not subject to counter-regulation by insulin and TNF alpha at the transcriptional level in 3T3-L1 adipocytes . These observations on the control of ACS and SCD-1 expression suggest possible mechanisms by which adipocytes can markedly adjust their capacity for long chain fatty acid metabolism in response to external stimuli.

Genes Dev, 1991 Dec, 5(12B), 2521 - 33
Efficient association of U2 snRNPs with pre-mRNA requires an essential U2 RNA structural element; Zavanelli MI et al.; To understand the role of U2 RNA structure in pre-mRNA splicing we have characterized several cold-sensitive mutations in an essential stem-loop of yeast U2 . Although mutant U2 is stable in vivo after a shift to restrictive temperature, splicing is rapidly inhibited, suggesting a direct effect on U2 function rather than U2 synthesis or snRNP assembly . Splicing complexes form at 23 degrees C in both mutant and wild-type extracts; however, stable association of mutant U2 snRNPs with pre-mRNA in vitro is inefficient at 15 degrees C, a temperature permissive for spliceosome assembly in wild-type extracts, indicating that the cold-sensitive defect is in U2 snRNP association with the assembling spliceosome . In vivo RNA structure probing reveals that the bulk of U2 RNA is misfolded in the mutants, even at permissive temperature . We propose that U2 stem-loop IIa is recognized by an assembly factor that assists U2 snRNP binding to pre-mRNA and that the cold sensitivity is due to a critical deficiency of correctly folded U2 for spliceosome assembly at low temperatures . Evolutionary conservation of the potential to form an interfering alternative RNA structure suggests the possibility that splicing could be regulated negatively at an early step by control of U2 snRNA conformation.

Biochim Biophys Acta, 1991 Dec 2, 1129(1), 73 - 82
The conformation of constitutive DNA interaction sites for eukaryotic DNA topoisomerase I on intrinsically curved DNAs; Camilloni G et al.; The analysis of the sites which are cleaved constitutively and preferentially by eukaryotic DNA topoisomerase I on two intrinsically curved DNAs reveals the conformational features that provoke the cleavage reaction on the curve-inducing sequence elements in the absence of supercoiling . This analysis is based on the observation (Caserta et al . (1989) Nucleic Acids Res . 17, 8521-8532 and (1990) Biochemistry 29, 8152-8157) that the reaction of eukaryotic DNA topoisomerase I occurs on two types of DNA sites: sites S (Supercoiled induced) and sites C (Constitutive, whose presence is topology-independent) . We report that sites C are abundant on the intrinsically curved DNAs analyzed . The DNAs studied were two intrinsically curved segments of different origin: the Crithidia fasciculata kinetoplast DNA and the bent-containing domain B of the Saccharomyces cerevisiae ARS1 . On these DNA segments DNA topoisomerase I cleaves at the junctions between the poly(A) tracts and mixed-sequence DNA . Analysis of the conformation of the double helix around the cleavage sites has revealed that the reaction occurs in correspondence of a defined DNA conformational motif . This motif is described by the set of Eulerian angular values that define the axial path of DNA (helical twist, deflection angle, direction) and of the orthogonal components of wedge (roll and tilt).

Radiat Res, 1991 Dec, 128(3), 243 - 50
Different oxygen enhancement ratios for induced and unrejoined DNA double-strand breaks in eukaryotic cells; Frankenberg-Schwager M et al.; DNA double-strand breaks (DSBs) are 2.9 times more frequently induced in yeast cells exposed to sparsely ionizing 30-MeV electrons under oxic compared to anoxic conditions . The rejoining of DSBs induced under anoxic conditions was investigated under conditions allowing repair of potentially lethal damage and compared to the rejoining of DSBs induced in oxic cells . In contrast to the biphasic rejoining kinetics of DSBs induced in oxic cells, the rejoining kinetics of DSBs induced in anoxic cells is complicated by the formation of secondary DSBs . These arise during postirradiation incubation of cells, presumably as a consequence of repair processes acting on radiation-induced lesions other than DSBs . These secondary DSBs may at least partially explain the finding that a greater fraction of unrejoinable DSBs is present in cells irradiated under anoxic compared to oxic conditions . As a consequence, the oxygen enhancement ratio of the yield of the remaining DSBs is decreasing in the course of DSB rejoining.

Proc Natl Acad Sci U S A, 1991 Dec 1, 88(23), 10510 - 4
GAL4 is phosphorylated as a consequence of transcriptional activation; Sadowski I et al.; GAL4 protein isolated from yeast in which it is active is phosphorylated predominantly on two different serine residues . One of these was identified as Ser-837; substitution of this residue for alanine has no detectable effect on transcriptional activation by GAL4 . Phosphorylation at Ser-837 requires that both the DNA binding and transcriptional activation functions be intact . We propose that some phosphorylations of GAL4, including that at Ser-837, occur concomitantly with activation of transcription.

J Cell Biol, 1991 Dec, 115(5), 1419 - 25
G1/S control of anchorage-independent growth in the fibroblast cell cycle; Guadagno TM et al.; We have developed methodology to identify the block to anchorage-independent growth and position it within the fibroblast cell cycle . Results with NRK fibroblasts show that mitogen stimulation of the G0/G1 transition and G1-associated increases in cell size are minimally affected by loss of cell anchorage . In contrast, the induction of G1/S cell cycle genes and DNA synthesis is markedly inhibited when anchorage is blocked . Moreover, we demonstrate that the anchorage-dependent transition maps to late G1 and shortly before activation of the G1/S p34cdc2-like kinase . The G1/S block was also detectable in NIH-3T3 cells . Our results: (a) distinguish control of cell cycle progression by growth factors and anchorage; (b) indicate that anchorage mediates G1/S control in fibroblasts; and (c) identify a physiologic circumstance in which the phenotype of mammalian cell cycle arrest would closely resemble Saccharomyces cerevisiae START . The close correlation between anchorage independence in vitro and tumorigenicity in vivo emphasizes the key regulatory role for G1/S control in mammalian cells.

J Cell Biol, 1991 Dec, 115(5), 1249 - 57
A role for unsaturated fatty acids in mitochondrial movement and inheritance; Stewart LC et al.; Yeast cells with the mdm2 mutation display temperature-sensitive growth and defective intracellular mitochondrial movement at the non-permissive temperature . The latter phenotype includes both an absence of mitochondrial transfer into daughter buds of mitotically growing cells and an aberrant mitochondrial distribution in cells exposed to mating pheromone . The wild-type MDM2 gene was cloned by complementation, and DNA sequence analysis revealed a large open reading frame encoding a putative protein of 58.4 kD . The predicted protein sequence is identical to that reported for the yeast OLE1 gene encoding fatty acid desaturase . Unsaturated fatty acid levels are substantially decreased in mdm2 cells after a prolonged incubation at the non-permissive temperature . The addition of oleic acid complements the temperature-sensitive growth and mitochondrial distribution defects of the mutant cells . These results indicate that mdm2 is a temperature-sensitive allele of OLE1 and demonstrate an essential role for unsaturated fatty acids in mitochondrial movement and inheritance.

Endocrinology, 1991 Dec, 129(6), 3053 - 63
Isolation of two complementary deoxyribonucleic acid clones from a rat insulinoma cell line based on similarities to Kex2 and furin sequences and the specific localization of each transcript to endocrine and neuroendocrine tissues in rats; Hakes DJ et al.; We have identified two rat insulinoma cDNAs that code for proteins homologous to the Kex2 dibasic protease of yeast and the mammalian furin gene product . A 5.0-kilobase (kb) cDNA, termed BDP, coding for a 752-amino acid protein and a 2.5-kb cDNA coding for a 636-amino acid protein, which was found to be the rat equivalent of the human insulinoma PC2 protein, were isolated . The proteins encoded by these clones contain a specific N-terminal signal sequence, indicating that both enter the secretory pathway . Neither protein contains a C-terminal transmembrane domain as is found in kex2 and furin, suggesting that the proteins may be soluble . Both proteins contain regions surrounding the active site residues which show amino acid identities to both kex2 (43% for BDP and 41% for RPC2) and furin (57% for BDP and 53% for RPC2) . Probes specific for the mRNAs of each protein were used to localize the expression of each protein in endocrine and neuroendocrine tissues.

EMBO J, 1991 Dec, 10(12), 3805 - 17
The p65 subunit is responsible for the strong transcription activating potential of NF-kappa B; Schmitz ML et al.; The nuclear form of the NF-kappa B transcription factor binds to DNA as a heterodimer of a 50 kDa (p50) and 65 kDa (p65) polypeptide . The two polypeptides are encoded by different genes but share a long region of homology, the NRD motif, encompassing domains required for DNA binding and dimerization . In this study we have analysed the contribution of the two subunits to the strong transactivating potential of NF-kappa B . Transient expression of the p65 subunit alone resulted in a potent transactivation of a CAT reporter construct under the control of two NF-kappa B binding sites in monkey COS and mouse L cells . The strongly DNA binding p50 subunit showed only very weak, if any, induction of gene expression . Co-expression of p50 suppressed the transactivation by p65 presumably by competitive DNA binding of transcriptionally inactive p50 dimers (KBF1) . Fusion of p65 sequences to DNA binding domain of the yeast GAL4 transcription factor allowed detection of the principal transactivation domain of p65 (TA1) in the C-terminal 30 amino acid sequence . TA1 is likely to adopt an amphipathic alpha-helical structure which clusters serine residues on the hydrophilic surface, a structural feature conserved between human, mouse and Xenopus p65 . The unique C-terminal third of p65 contained at least one more activation domain, TA2, within a 90 amino acid sequence directly adjacent to TA1 . In two mammalian cell lines, TA1 and TA2 acted separately, while in an insect cell line, the two domains were inactive after their separation . Our study suggests that the p50 subunit in NF-kappa B might only serve a helper function in DNA binding whereas the p65 subunit is responsible for initiating transcription . Homodimers of p50 seem to have the potential of down-regulating kappa B-specific gene expression.

Genes Dev, 1991 Dec, 5(12B), 2534 - 46
An ATP-independent complex commits pre-mRNA to the mammalian spliceosome assembly pathway; Michaud S et al.; Previous studies have identified five distinct mammalian splicing complexes that assemble on pre-mRNA in vitro . Of these complexes, which include H, E, A, B, and C, only the B and C complexes have been isolated and shown directly to be functional intermediates in the splicing pathway . In this report we carried out a systematic analysis of the temporal and functional relationships among the H, E, A, and B complexes . Using gel filtration to isolate each complex, we show that H complex, which consists primarily of hnRNP proteins, assembles first in either the presence or absence of ATP . Subsequently, E complex, which contains stably bound U1 snRNP, is detected in reactions lacking ATP, whereas A complex, which contains stably bound U1 and U2 snRNPs, is detected in reactions containing ATP . We show that E complex can be chased into A and B complexes and that A complex can be chased into B complex . Both E and A complexes can also be chased into spliced products . In contrast, H complex cannot be chased into A or B complexes or spliced products under the same conditions . We conclude that in addition to the two spliceosome complexes (B and C), two distinct pre-splicesome complexes (E and A) are functional intermediates in the splicing pathway . Comparison of the efficiency of splicesome assembly on different pre-mRNAs has revealed dramatic differences . We show that these differences are first apparent at the time of E complex assembly . Thus, we conclude that E complex commits pre-mRNA to the splicing pathway and that this step is critical in determining the efficiency of mammalian spliceosome assembly.

Curr Opin Genet Dev, 1991 Dec, 1(4), 538 - 43
Telomeres--what's new at the end?
Henderson ER, Larson DD.
Telomeres are specialized chromatin domains located at the ends of chromosomes . They are involved in chromosome replication, stability and localization in the nucleus . In addition to these functions, recent work suggests that telomeres are involved in such superficially diverse cellular phenomena as ageing, cancer, nuclear architecture and nuclear/cellular division.

Biol Mass Spectrom, 1991 Dec, 20(12), 796 - 800
Assessing the multimeric states of proteins: studies using laser desorption mass spectrometry; Farmer TB et al.; We have developed a technique which utilizes matrix-assisted laser desorption mass spectrometry to study the subunit association of proteins . Aqueous protein samples are treated with a dilute solution of glutaraldehyde, a cross-linking agent which reacts with free amino groups on proteins . This agent effectively traps the multimeric form, preventing it from dissociating in the sample preparation and desorption process . Proteins measured include lysozyme, carbonic anhydrase, apomyoglobin, glucose 6-phosphate dehydrogenase, ovine lutropin, yeast alcohol dehydrogenase, avidin and pyruvate kinase . Dimeric and tetrameric complexes up to 250,000 Da have been measured in this manner.

Biotechniques, 1991 Dec, 11(6), 778 - 83
SOFI: a bidimensional detector for fast direct on-line quantification of beta particles on blots; Mastrippolito R et al.; We present a high-speed, high-resolution beta imager developed to replace autoradiographic films currently used in molecular biology experiments . It allows the user to locate and make quantitative analyses of 32P-labeled molecules on a 25.6 x 25.6-cm flat surface . Combining new techniques--scintillating optical fibers and multianode photomultipliers--this fast imager offers several advantages when compared with recent gas detectors and flexibility for further improvements . Several biological applications will be discussed.

J Biochem (Tokyo), 1991 Dec, 110(6), 884 - 8
Nitrogenous ligation at the sixth coordination position of the Thr-301 to Lys-mutated P450IIC2 heme iron; Imai Y et al.; Threonine-301 of P450IIC2 was replaced by lysine via site-directed mutagenesis . The Lys-mutated P450 exhibited absorption spectra that were characteristic of the nitrogenous-ligand-bound form of P450 . In the oxidized form, the Soret band was red-shifted as compared with the typical ferric low-spin form of P450 and the beta band was more intense than the alpha band . In the reduced form, two Soret peaks were observable at 447 and 423 nm and their relative heights were dependent on pH, indicating the existence of two interconvertible states of ferrous Lys-mutated P450 which are in equilibrium . In addition, the interaction of external ligands with the P450 heme iron was profoundly inhibited both in the oxidized and reduced forms . These findings suggest that epsilon-amino nitrogen of Lys-301, which was introduced by amino acid substitution, occupies the 6th coordination position with the heme iron of the Lys-mutated P450, because, owing to conformation of the P450 protein, the epsilon-amino group may be located at just the right position for coordination as the internal 6th ligand.

J Biochem (Tokyo), 1991 Dec, 110(6), 1022 - 9
1H and 15N NMR study of human lysozyme; Ohkubo T et al.; The 15N signal assignment of human lysozyme was carried out by using 1H-1H and 1H-15N two dimensional experiments . To solve the severe overlap problem of the NH signals, uniform labeling of the protein with 15N was introduced . The uniformly 15N labeled protein was prepared using a high-expression system of Saccharomyces cerevisiae . From the analyses of 1H and 15N NMR spectra, all of the backbone 15N signals of the molecule were assigned to each specific residue in the amino acid sequence . Recently published proton signal assignments {Redfield & Dobson (1990) Biochemistry, 29, 7201-7214} were confirmed by these complementary data . In addition, assignments were extended to side chain 15NH2 groups of asparagine and glutamine . Elements of secondary structure were deduced from the pattern of sequential and medium-range NOE connectivities . Two beta-sheets and four alpha-helices could be identified in the protein, which were in good agreement with those determined by X-ray crystallography . The interaction between human lysozyme and its inhibitor N-acetyl-chitotriose was investigated by 15N-1H HMQC spectra . Most of the 15N-NH cross-peaks in the spectra were separated well enough to be followed during the titration experiment . Residues whose NH proton signals decrease in intensity upon complex formation, are located mainly around subsites B, C, and D . Local conformational changes were observed around the fourth helix adjacent to the cleft of human lysozyme.

Mol Endocrinol, 1991 Dec, 5(12), 2014 - 24
Prohormone-converting enzymes: regulation and evaluation of function using antisense RNA; Bloomquist BT et al.; Several putative peptide-processing endoproteases have been identified by homology to the yeast Kex2 endoprotease, including furin, PC2, and PC1 . However, the question is still open as to which might be involved in peptide posttranslational processing . To enable detailed comparisons of physiological changes in peptide processing with biochemical and molecular biological studies, we cloned rat pituitary cDNAs for PC1 and PC2 . The amino acid sequence homologies among rat, human, and mouse PC1, PC2, and furin are consistent with each being a highly conserved but distinct member of a larger family of mammalian subtilisin-like proteases . PC1 and PC2 mRNAs show a restricted distribution among rat tissues and cultured cell lines, consistent with a role in tissue-specific peptide processing; the occurrence of furin mRNA among these tissues and cell lines is much more widespread, being high in many nonneuroendocrine tissues . In the neurointermediate pituitary, PC1 and PC2 mRNAs are strikingly regulated in response to dopaminergic agents, in parallel with mRNAs for POMC, peptidylglycine alpha-amidating monooxygenase, and carboxypeptidase-H . In AtT-20 cells, PC1 mRNA is coregulated with POMC and peptidylglycine alpha-amidating monooxygenase mRNAs in response to CRH and glucocorticoids . When the endogenous PC1 mRNA level in AtT-20 cells is significantly and specifically decreased by stable expression of antisense RNA to PC1, biosynthetic labeling of newly synthesized POMC-derived peptides shows a substantial blockade of normal POMC processing . These data are consistent with a role for PC1 protein in endoproteolysis, either as a processing endoprotease or as the activator of the actual processing endoprotease(s).

Genetics, 1991 Dec, 129(4), 1085 - 98
Chromosome rearrangement by ectopic recombination in Drosophila melanogaster: genome structure and evolution; Montgomery EA et al.; Ectopic recombination between interspersed repeat sequences generates chromosomal rearrangements that have a major impact on genome structure . A survey of ectopic recombination in the region flanking the white locus of Drosophila melanogaster identified 25 transposon-mediated rearrangements from four parallel experiments . Eighteen of the 25 were generated from females carrying X chromosomes heterozygous for interspersed repeat sequences . The cytogenetic and molecular analyses of the rearrangements and the parental chromosomes show: (1) interchromosomal and intrachromosomal recombinants are generated in about equal numbers; (2) ectopic recombination appears to be a meiotic process that is stimulated by the interchromosomal effect to about the same degree as regular crossing over; (3) copies of the retrotransposon roo were involved in all of the interchromosomal exchanges; some copies were involved much more frequently than others in the target region; (4) homozygosis for interspersed repeat sequences and other sequence variations significantly reduced ectopic recombination.

Oncogene, 1991 Dec, 6(12), 2349 - 52
HTLV-1 Tax has distinct but overlapping domains for transcriptional activation and for enhancer specificity; Fujii M et al.; Tax1 of human T-cell leukemia virus type 1 (HTLV-1) activates viral transcription dependent upon three 21-bp enhancer elements in the long terminal repeat . Difficulties in detecting any association of Tax1 with the viral enhancer have hampered elucidation of the molecular mechanisms of Tax1-mediated transcriptional activation . By constructing a fusion protein with the heterologous DNA-binding domain of yeast GAL4, Tax1 was shown to be a potent transcriptional activator dependent on the presence of GAL4-binding sites . Deletions of the Tax1 portion of the fusion protein revealed that almost the entire region of Tax1 (amino acids 2-337) is required for activation, and the activity correlated well with that of the viral enhancer . The GAL/Tax1 mutant lacking 41 residues of the C-terminus of Tax1, GAL/Tax1(2-312), was inactive for the viral enhancer, but activity was recovered by adding the heterologous activation domain of herpes simplex virus VP16 . These results indicate that Tax1 has two distinct but overlapping functional domains for transcriptional activation and for enhancer specificity . Thus, Tax1 is thought to be a transcription factor acting in the enhancer complex rather than as a catalytic or allosteric modifier of pre-existing cellular transcription factors.

J Cell Biol, 1991 Dec, 115(6), 1601 - 9
Protein folding causes an arrest of preprotein translocation into mitochondria in vivo; Wienhues U et al.; With vital yeast cells, a hybrid protein consisting of the amino-terminal third of the precursor to cytochrome b2 and of the entire dihydrofolate reductase was arrested on the import pathway into mitochondria . Accumulation of the protein in the mitochondrial membranes was achieved by inducing a stable tertiary structure of the dihydrofolate reductase domain . Thereby, three salient features of mitochondrial protein uptake in vivo were demonstrated: its posttranslational character; the requirement for unfolding of precursors; and import through translocation contact sites . The permanent occupation of translocation sites by the fusion protein inhibited the import of other precursors; it did, however, not lead to leakage of mitochondrial ions, implying the existence of a channel that is sealed around the membrane spanning polypeptide segment.

J Cell Biol, 1991 Dec, 115(6), 1521 - 34
Isolation, characterization, and expression of cDNAs encoding murine alpha-mannosidase II, a Golgi enzyme that controls conversion of high mannose to complex N-glycans; Moremen KW et al.; Golgi alpha-mannosidase II (GlcNAc transferase I-dependent alpha 1,3{alpha 1,6} mannosidase, EC 3.2.1.114) catalyzes the final hydrolytic step in the N-glycan maturation pathway acting as the committed step in the conversion of high mannose to complex type structures . We have isolated overlapping clones from a murine cDNA library encoding the full length alpha-mannosidase II open reading frame and most of the 5' and 3' untranslated region . The coding sequence predicts a type II transmembrane protein with a short cytoplasmic tail (five amino acids), a single transmembrane domain (21 amino acids), and a large COOH-terminal catalytic domain (1,124 amino acids) . This domain organization which is shared with the Golgi glycosyl-transferases suggests that the common structural motifs may have a functional role in Golgi enzyme function or localization . Three sets of polyadenylated clones were isolated extending 3' beyond the open reading frame by as much as 2,543 bp . Northern blots suggest that these polyadenylated clones totaling 6.1 kb in length correspond to minor message species smaller than the full length message . The largest and predominant message on Northern blots (7.5 kb) presumably extends another approximately 1.4-kb downstream beyond the longest of the isolated clones . Transient expression of the alpha-mannosidase II cDNA in COS cells resulted in 8-12-fold overexpression of enzyme activity, and the appearance of cross-reactive material in a perinuclear membrane array consistent with a Golgi localization . A region within the catalytic domain of the alpha-mannosidase II open reading frame bears a strong similarity to a corresponding sequence in the rat liver endoplasmic reticulum alpha-mannosidase and the vacuolar alpha-mannosidase of Saccharomyces cerevisiae . Partial human alpha-mannosidase II cDNA clones were also isolated and the gene was localized to human chromosome 5.

EMBO J, 1991 Dec, 10(13), 4089 - 95
Identification of a novel recognition sequence for the integrin alpha 4 beta 1 in the COOH-terminal heparin-binding domain of fibronectin; Mould AP et al.; The type III connecting segment of fibronectin contains two cell binding sites, represented by the peptides CS1 and CS5, that are recognized by the integrin receptor alpha 4 beta 1 . Using assays measuring the spreading of A375-SM human melanoma cells, we now report that the adhesion promoting activity of a 29 kDa protease fragment of fibronectin containing the COOH-terminal heparin-binding domain (HepII), but lacking CS1 and CS5, is completely sensitive to anti-alpha 4 and anti-beta 1 antibodies, suggesting that HepII contains a third alpha 4 beta 1-binding sequence . Examination of the primary structure of HepII revealed a sequence with homology to CS1 . A 19mer peptide spanning this region (designated H1) was found to support cell spreading to the same level as the 29 kDa fragment . H1-dependent adhesion was completely sensitive to anti-alpha 4 and anti-beta 1 antibodies . When soluble peptides were tested for their ability to block cell spreading on the 29 kDa fragment, a 13mer peptide comprising the central core of H1 was found to be completely inhibitory . The active region of H1 was localized to the pentapeptide IDAPS, which is homologous to LDVPS from the active site of CS1 . Taken together, these results identify a novel peptide sequence in the HepII region of fibronectin that supports alpha 4 beta 1-dependent cell adhesion.

Genes Dev, 1991 Dec, 5(12B), 2431 - 40
RNA polymerase II carboxy-terminal domain contributes to the response to multiple acidic activators in vitro; Liao SM et al.; The largest subunit of RNA polymerase II contains a unique carboxy-terminal domain (CTD) that consists of repeats of the heptapeptide YSPTSPS . RNA polymerase II CTD truncation mutations affect the ability to induce transcription of a subset of yeast genes in vivo, and the lack of response to induction maps to the upstream activating sequences of these genes . Here, we report that progressive truncation of the yeast RNA polymerase II CTD causes progressive loss of trans-activator-dependent transcription in nuclear extracts but has little effect on elongation or termination . Specific transcription, which is reduced by up to 50-fold in these assays, can be restored in the defective nuclear extracts by adding purified wild-type RNA polymerase II . The defects in factor-dependent transcription are observed with templates that are assembled into nucleosomes as well as with templates that are not so assembled . Defects in factor-independent transcription are also observed, but these are not as profound as those observed in the presence of trans-activators . These results indicate that the RNA polymerase II CTD functions during transcription initiation and is required for normal levels of activated transcription in vitro.

Genes Dev, 1991 Dec, 5(12B), 2392 - 404
A meiosis-specific protein kinase homolog required for chromosome synapsis and recombination; Rockmill B et al.; The mek1 (meiotic kinase) mutant of Saccharomyces cerevisiae was isolated in a screen for sporulation-proficient, meiotic-lethal mutants . Diploids homozygous for a mek1 null mutation produce only 13% viable spores . mek1 spore inviability is rescued by a spo13 mutation, which causes cells to bypass the meiosis I division . In a mek1 null mutant, meiotic recombination is reduced but not completely eliminated . Nuclear spreads of meiotic chromosomes from mek1 diploids reveal numerous stretches of synaptonemal complex (SC) that are shorter than wild-type SCs . Analysis of a mek1::lacZ fusion gene and Northern blot hybridization demonstrate that the MEK1 transcript is present only in meiosis . The sequence of the MEK1 gene predicts a 56.8-kD protein with homology to serine-threonine protein kinases . The MEK1 gene maps to chromosome XV, 13 cM proximal to CDC64 . Models for the function of the MEK1 gene product are proposed.

Genes Dev, 1991 Dec, 5(12A), 2212 - 24
Coactivators for a proline-rich activator purified from the multisubunit human TFIID complex; Tanese N et al.; The mechanisms of transcriptional activation directed by sequence-specific regulators is central to understanding gene regulation . Here, we report the isolation of coactivators responsible for mediating transcriptional activation by Gal4-Pro, a hybrid regulator containing the proline-rich activation domain of human CTF/NFI . Chromatographic studies indicate that endogenous human TFIID consists of a multisubunit complex containing the TATA-binding protein (TBP), coactivators, and other associated factors . A fraction containing the coactivator activity was separated from the endogenous TBP after disrupting the tightly associated complex with urea . The urea-purified TBP was active for basal level transcription but no longer could support activation by Gal4-Pro . However, when the two separated components were added together, activated levels of transcription were restored in the presence of Gal4-Pro . Immunoaffinity purification of the TFIID complex identifies several polypeptides specifically associated with the endogenous TBP, some or all of which function as coactivators when reconstituted with Gal4-Pro . The isolated coactivators also mediate activation by a chimeric glutamine-rich activator derived from Sp1 but not the Gal4-VP16 activator, suggesting distinct factor requirements for different types of transcriptional regulators.

FASEB J, 1991 Dec, 5(15), 3108 - 13
Detecting lacZ gene expression in living cells with new lipophilic, fluorogenic beta-galactosidase substrates; Zhang YZ et al.; Current methods for detecting lacZ expression in transformed cells are limited because they require such harsh conditions that viability of the cells after detection is drastically reduced . To overcome this problem, we developed a series of new substrates for detection of lacZ expression in living cells under standard culture or physiological conditions . After incubation with these fluorogenic substrates, cultured lacZ-positive mammalian cells appear morphologically normal, continue to divide, and retain the fluorescent product . Because the product is so well retained, fluorescence intensity can be quantitatively related to the level of gene expression . We have demonstrated this correlation using transformed yeast cells bearing various plasmids, each containing the lacZ gene and a unique promoter sequence with known capabilities for promoting gene expression in yeast.

Glycobiology, 1991 Dec, 1(6), 553 - 62
Biosynthesis of N-acetylglucosamine-P-P-dolichol, the committed step of asparagine-linked oligosaccharide assembly; Lehrman MA; Asparagine-linked glycosylation is initiated by the synthesis of N-acetylglucosaminylpyrophosphoryl dolichol (GlcNAc-P-P-dolichol), which is extended by a series of glycosyltransferases to yield Glc3Man9GlcNAc2-P-P-dolichol (where Glc is glucose and Man is mannose) . The oligosaccharide unit is then transferred en bloc to asparagine residues of nascent polypeptides in the lumen of the rough endoplasmic reticulum . The question here is whether GlcNAc-P-P-dolichol biosynthesis is a fixed process unaffected by cellular events, or a regulated reaction responsive to cellular requirements for glycoprotein biosynthesis . Several lines of evidence indicate that the latter is the case and that GlcNAc-P-P-dolichol biosynthesis may be subject to multiple forms of regulation . Recent information about the N-acetylglucosamine-1-P transferase (GPT) responsible for this reaction and the cloning of cDNA candidates for this enzyme have provided further insight into these mechanisms . This review will examine current hypotheses dealing with GPT and its role in the committed step of asparagine-linked glycosylation.

Biotechniques, 1991 Dec, 11(6), 711 - 2, 714, 716 passim
Accurate evaluation of the sizes of DNA fragments (from 30 to 4700 kb) in pulse field gel electrophoresis; Crete N et al.; Construction of long-range genomic maps by pulse field gel electrophoresis requires optimum resolution of large DNA fragments . Using the transverse-alternating field electrophoresis system, we describe a method to accurately evaluate the sizes of fragments generated by rare-cutter digestions within the 30-4700-kb range . A protocol generating large (greater than 1000 kb) molecules by partial digestion is also reported.

J Mol Evol, 1991 Dec, 33(6), 483 - 94
Evidence for selective evolution in codon usage in conserved amino acid segments of human alphaherpesvirus proteins; Schachtel GA et al.; The genomes of human viruses herpes simplex 1 (HSV1) and varicella zoster (VZV), although similar in biology, largely concordant in gene order, and identical in many amino acid segments, differ widely in their genomic G + C (abbreviated S) content, which is high in HSV1 (68%) and low in VZV (46%) . This paper analyzes several striking codon usage contrasts . The S difference in coding regions is dramatically large in codon site 3, S3, about 42% . The large difference in S3 is maintained at the same level in a subset of closely similar genes and even in corresponding identical amino acid blocks . A similar difference in S levels in silent site 1 (S1) is found in leucine and arginine . The difference in S3 levels occurs in every gene and in every multicodon amino acid form . The S difference also exists in amino acid usage, with HSV1 using significantly more codon types SSN, while VZV uses more codon types WWN (where W stands for A or T) . The nonoverlapping and narrow histograms of S3 gene frequencies in both viruses suggest that the difference has arisen and been maintained by a process of selective rather than nonselective effects . This is in sharp contrast to the relatively large variance seen for highly similar genes in the human versus yeast analysis . Interpretations and hypotheses to explain the HSV1 vs VZV codon usage disparity relate to virus-host interactions, to the role of viral genes in DNA metabolism, to availability of molecular resources (molecular Gause exclusion principle), and to differences in genomic structure.

Proc Natl Acad Sci U S A, 1991 Dec 1, 88(23), 10686 - 90
A contingent replication assay for the detection of protein-protein interactions in animal cells; Vasavada HA et al.; We have developed a sensitive and rapid assay system termed the contingent replication assay (CRA) for selecting cDNAs with desired functional properties from a cDNA library . The system functions in animal cells and permits enrichment of the desired cDNA in small-scale and convenient experiments . The assay can be used for the enrichment of proteins that activate transcription from conditional enhancers, bind to specific DNA sequences, or interact with target proteins of interest . In this communication we report the application of this assay to study protein-protein interactions in animal cells.

Plant Mol Biol, 1991 Dec, 17(6), 1189 - 201
Isolation and characterization of tomato cDNA and genomic clones encoding the ubiquitin gene ubi3; Hoffman NE et al.; We report here the isolation and nucleotide sequence of tomato cDNA and genomic clones encoding a ubiquitin extension protein homologous to the yeast gene ubi3 . Sites similar to upstream activating sites commonly found in the promoters of yeast ribosomal genes were observed in the tomato promoter . The tomato ubi3 promoter also contained elements found in the rbcS promoter from pea . The transcription initiation site was determined to occur 66 bp upstream of the initiating Met . RFLP mapping revealed that the gene was located on chromosome 1, 23 cM from marker TG301 . A ubi3 gene-specific probe hybridized to a single 800 nt transcript . Expression was reduced in heat-shocked plants and plants kept in the dark . Expression was highest in young leaves and immature green fruit and lowest in mature leaves and petals . We isolated the original cDNA clone using an antibody prepared against chloroplast polypeptides . Immunological studies did not detect ubiquitin or ubiquitin extension proteins in the chloroplast . However, higher-molecular-weight chloroplast proteins were detected with ubiquitin antisera suggesting that ubiquitin conjugates are transported into the chloroplast.

Science, 1991 Nov 29, 254(5036), 1361 - 4
Folding of circularly permuted transfer RNAs; Pan T et al.; All of the ribose-phosphate linkages in yeast tRNA(Phe) that could be cleaved without affecting the folding of the molecule have been determined in a single experiment . Circular permutation analysis subjects circular tRNA molecules to limited alkaline hydrolysis in order to generate one random break per molecule . Correctly folded tRNAs were identified by lead cleavage at neutral pH, a well-characterized reaction that requires proper folding of tRNA(Phe) . Surprisingly, most of the circularly permuted tRNA molecules folded correctly . This result suggests that the tRNA folding motif could occur internally within other RNA sequences, and a computer search of Genbank entries has identified many examples of such motifs.

Biochem Biophys Res Commun, 1991 Nov 27, 181(1), 474 - 80
Thermostabilization of recombinant human and bovine CuZn superoxide dismutases by replacement of free cysteines; Hallewell RA et al.; Human CuZn superoxide dismutase (HSOD) has two free cysteines: a buried cysteine (Cys6) located in a beta-strand, and a solvent accessible cysteine (Cys111) located in a loop region . The highly homologous bovine enzyme (BSOD) has a single buried Cys6 residue . Cys6 residues in HSOD and BSOD were replaced by alanine and Cys111 residues in HSOD by serine . The mutant enzymes were expressed and purified from yeast and had normal specific activities . The relative resistance of the purified proteins to irreversible inactivation of enzymatic activity by heating at 70 degrees C was HSOD Ala6 Ser111 greater than BSOD Ala6 Ser109 greater than BSOD Cys6 Ser109 (wild type) greater than HSOD Ala6 Cys111 greater than HSOD Cys6 Ser111 greater than HSOD Cys111 (wild type) . In all cases, removal of a free cysteine residue increased thermostability.

Biochemistry, 1991 Nov 26, 30(47), 11206 - 11
Absorption spectral study of cytochrome P450d-phenyl isocyanide complexes: effects of mutations at the putative distal site on the conformational stability; Krainev AG et al.; Interactions of phenyl isocyanide (PheNC) with purified engineered cytochrome P450d wild type and putative distal mutants, Glu318Asp and Glu318Ala, were studied with optical absorption spectra . The wild type and the mutant Glu318Asp were purified as the high-spin state, while the mutant Glu318Ala was purified as the oxygen-bound low-spin form . Thus, it is suggested that Glu318 is important to make the appropriate heme environment of P450d . Spectral dissociation constants (0.19-0.39 mM) of the ligand for the ferric mutants were lower than that (0.74 mM) of the wild type . These dissociation constants were changed by adding a substrate, 7-ethoxycoumarin . The reduced wild type-PheNC complex showed a Soret peak at 451 nm, while the reduced mutant-PheNC complexes showed two peaks at 451 and 423 nm . The 451-nm peak of the complexes decreased with the concomitant increase of a new peak at 433 nm at room temperature . Thus, it was suggested that P450d can take two conformationally different forms from the characteristic spectral features . The Soret spectral conversions which followed the first-order kinetics were analyzed by changing the temperature . The activation energy (69 kcal/mol) for the conversion for the wild type was higher than those (37-50 kcal/mol) for the mutants . The activation energy for the wild type further increased (by 55%) by adding the substrate, while those for the mutants were essentially unchanged by adding the substrate . We discuss the important role of Glu318 at the putative distal site of P450d in the packing or the conformational stability of the putative distal site of the P450d molecule.

J Biol Chem, 1991 Nov 25, 266(33), 22707 - 17
Synthesis of DNA by DNA polymerase epsilon in vitro; Lee SH et al.; The isolation of DNA polymerase (Pol) epsilon from extracts of HeLa cells is described . The final fractions contained two major subunits of 210 and 50 kDa which cosedimented with Pol epsilon activity, similar to those described previously (Syvaoja, J., and Linn, S . (1989) J . Biol . Chem . 264, 2489-2497) . The properties of the human Pol epsilon and the yeast Pol epsilon were compared . Both enzymes elongated singly primed single-stranded circular DNA templates . Yeast Pol epsilon required the presence of a DNA binding protein (SSB) whereas human Pol epsilon required the addition of SSB, Activator 1 and proliferating cell nuclear antigen (PCNA) for maximal activity . Both enzymes were totally unable to elongate primed DNA templates in the presence of salt; however, activity could be restored by the addition of Activator 1 and PCNA . Like Pol delta, Pol epsilon formed complexes with SSB-coated primed DNA templates in the presence of Activator 1 and PCNA which could be isolated by filtration through Bio-Gel A-5m columns . Unlike Pol delta, Pol epsilon bound to SSB-coated primed DNA in the absence of the auxiliary factors . In the presence of salt, Pol epsilon complexes were less stable than they were in the absence of salt . In the in vitro simian virus 40 (SV40) T antigen-dependent synthesis of DNA containing the SV40 origin of replication, yeast Pol epsilon but not human Pol epsilon could substitute for yeast or human Pol delta in the generation of long DNA products . However, human Pol epsilon did increase slightly the length of DNA chains formed by the DNA polymerase alpha-primase complex in SV40 DNA synthesis . The bearing of this observation on the requirement for a PCNA-dependent DNA polymerase in the synthesis and maturation of Okazaki fragments is discussed . However, no unique role for human Pol epsilon in the in vitro SV40 DNA replication system was detected.

J Mol Biol, 1991 Nov 20, 222(2), 179 - 87
Restoration of the self-splicing activity of a defective group II intron by a small trans-acting RNA; Suchy M et al.; The yeast mitochondrial group II intron bI1 is self-splicing in vitro . We have introduced a deletion of hairpin C1 within the structural domain 1 that abolishes catalytic activity of the intron in the normal splicing reaction in cis, but does less severely affect a reaction in trans, the reopening of ligated exons . Since exon reopening is supposed to correspond to a reverse 3' cleavage this suggests that the deletion specifically blocks the first reaction step . The intron regains its activity to self-splice in cis by intermolecular complementation with a small RNA harbouring sequences lacking in the mutant intron . These results demonstrate the feasibility to reconstitute a functionally active structure of the truncated intron by intermolecular complementation in vitro . Furthermore, the data support the hypothesis that group II introns are predecessors of nuclear pre-mRNA introns and that the small nuclear RNAs of the spliceosome arose by segregation from the original intron.

J Mol Biol, 1991 Nov 20, 222(2), 133 - 7
Topoisomer heterogeneity of plasmid chromatin in living cells; Morse RH; Previous investigations of topoisomer distributions of simian virus 40 (SV40) DNA from monkey cells have revealed that these circular mini-chromosomes, like relaxed, naked, closed circular DNA, exist as a Gaussian distribution of topoisomers . I have extended this comparison by measuring topoisomer distributions for a variety of plasmid episomes that are stably propagated in cells of the yeast Saccharomyces cerevisiae . The breadth of the topoisomer distributions for plasmid chromatin, including SV40, is approximately constant when normalized for DNA length, as is the breadth of distribution for naked DNA . However, the distributions for plasmid chromatin are substantially broader than those for the corresponding relaxed, naked DNAs . The breath is constant for plasmids differing in transcriptional activity, and varies only slightly between synchronized and unsynchronized populations of yeast cells, suggesting that variation in plasmid linking number with transcription or replication does not account for the observed heterogeneity in linking number . Topoisomer heterogeneity for plasmid chromatin in vivo may be due to heterogeneity in the number of nucleosomes on each plasmid, which could reflect either the nature of the assembly process or the dynamics of nucleosomes within the cell.

FEBS Lett, 1991 Nov 18, 293(1-2), 85 - 8
Mitochondrial preproteins en route from the outer membrane to the inner membrane are exposed to the intermembrane space; Rassow J et al.; Mitochondrial precursor proteins are known to be imported at sites of close contact between mitochondrial outer and inner membranes . We have identified translocation intermediates exposed to the intermembrane space, including the precursor of the ADP/ATP carrier accumulated at the general insertion site GIP, and the precursor of F1-ATPase subunit beta accumulated on its import pathway at low levels of ATP . These results suggest that mitochondrial contact sites are not sealed structures, but that polypeptides pass (at least partly) through the intermembrane space on their route from the outer membrane to the inner membrane.

Biochim Biophys Acta, 1991 Nov 18, 1070(1), 246 - 52
Proteins and peptides bound to long-circulating liposomes; Maruyama K et al.; Liposome formulations with prolonged circulation time have recently been developed as a potential sustained-release drug delivery system . Data shown in this report indicate that such formulations can also be used to prolong the circulation time of proteins and peptides by conjugating them to the surface of liposomes . Increase of the circulation halflife ranged from 2- to 150-fold depending on the protein/lipid ratio of the liposomal formulation, liposome size, and the lipid composition of liposomes . Since the proteins/peptides localize on the liposome surface, instead of being entrapped inside the liposomes, they are directly available for binding to its receptor molecules and express the biological activity . This strategy has been successfully applied to two proteins with known fast clearance rate, i.e . asialofetuin and ricin A-chain . The biological activities of both proteins are preserved when they are formulated in liposomes . Incorporation of a peptide, i.e . a-factor of the yeast Saccharomyces cerevisiae, into the liposome membrane also significantly enhanced the circulation time of the peptide.

Anal Biochem, 1991 Nov 15, 199(1), 29 - 34
Ultraviolet nicking of large DNA molecules from pulsed-field gels for southern transfer and hybridization; Lee H et al.; Large DNA molecules separated in pulsed-field gels are not efficiently transferred from the gel for Southern hybridization . Various procedures for fragmenting the DNA prior to transfer are in use, but quantitative details that permit reproducible application have not been reported . We have determined the optimum level of energy for uv nicking of large DNA needed to promote efficient Southern transfer and detection by hybridization . To ensure consistent results we have used a uv oven equipped with a detector that measures only 200-400 nm wavelengths, and we report the total energy delivered . Using uv nicking and the transfer techniques described, we can obtain hybridization signals overnight with single-copy DNA probes on Southern blots of large DNA fragments separated by pulsed-field gel electrophoresis.

Anal Biochem, 1991 Nov 15, 199(1), 137 - 41
Quantitation of DNA and RNA in crude tissue extracts by flow injection analysis; Caldarone EM et al.; An automated two-dye flow injection analysis system to quantitate DNA and RNA in crude extracts of tissues is described . The method uses the fluorochrome dyes ethidium bromide and Hoechst 33258 . DNA concentration is determined directly from its fluorescence in Hoechst dye . RNA is estimated from fluorescence in ethidium bromide after subtraction of the fluorescence due to DNA . This method has several advantages: a simple extraction procedure, a low detection limit (0.01 micrograms DNA and 0.10 micrograms RNA), automation, and a high sample throughput.

Anal Biochem, 1991 Nov 15, 199(1), 142 - 6
On-line detection of nitric oxide formation in liquid aqueous phase by electron paramagnetic resonance spectroscopy; Mordvintcev P et al.; A method for the detection of the nitric oxide radical (NO) in oxygen-containing aqueous solution by means of electron paramagnetic resonance spectroscopy (EPR) is described . NO evolving from the spontaneous decomposition of 3-morpholinosydnonimine (SIN-1) was trapped by Fe(2+)-diethyldithiocarbamate (DETC) complex dissolved in yeast cell membranes . The resulting mononitrosyl-Fe(2+)-(DETC)2 complex was stable and exhibited a characteristic EPR signal at g perpendicular = 2.04 and g parallel = 2.02 with an unresolved triplet hyperfine structure at g perpendicular in frozen solution and an isotropic triplet signal at gav = 2.03 at 37 degrees C . The amount of NO trapped was calculated from the amplitude of one of the triplet lines calibrated by means of a dinitrosyl-Fe(2+)-thiosulfate standard . The lower detection limit of NO was 0.5 nmol/(ml x h) due to a low background NO signal . The upper detection limit was about 10 nmol NO/40 mg traps (DETC-loaded yeast cells), because of saturation of traps . The trapping efficiency approached 60% under anaerobic conditions and with low concentrations of SIN-1, but decreased progressively with higher concentrations and in the presence of oxygen . Nitrite (up to 0.1 mM) did not increase the background NO level . The sensitivity was sufficient to follow the rate of NO release from SIN-1 on-line at 37 degrees C in a flat quartz cuvette . The time course of NO release detected by EPR spectrometry correlated with the time course of nitrite accumulation measured by diazotation . In conclusion, this method will permit the on-line detection of NO formation from endogenous and pharmacological sources in oxygen-containing aqueous media.

J Biol Chem, 1991 Nov 15, 266(32), 21355 - 7
Enhancing protein engineering capabilities by combining mutagenesis and semisynthesis; Wallace CJ et al.; If site-directed mutagenesis could be used to facilitate protein semisynthesis, then structural engineering goals should be achieved that are unattainable by either technique alone . We tested this possibility by mutating Ser65 of yeast cytochrome c to methionine, creating a new site for CNBr cleavage . Fragments obtained by cleaving there were found to refold cooperatively, bringing together the breakpoint termini and leading to efficient autocatalytic peptide bond synthesis . Structurally modified fragments may be substituted for natural ones . Generally, naturally occurring sites are unsuitable for autocatalytic religation, for reasons briefly discussed, and thus the power of this new approach lies in the freedom to choose sites, including enzymatic ones, that are appropriate to the semisynthetic goals.

Nucleic Acids Res, 1991 Nov 11, 19(21), 5895 - 900
FLP-mediated recombination in the vector mosquito, Aedes aegypti; Morris AC et al.; The activity of a yeast recombinase, FLP, on specific target DNA sequences, FRT, has been demonstrated in embryos of the vector mosquito, Aedes aegypti . In a series of experiments, plasmids containing the FLP recombinase under control of a heterologous heat-shock gene promoter were co-injected with target plasmids containing FRT sites into preblastoderm stage mosquito embryos . FLP-mediated recombination was detected between (i) tandem repeats of FRT sites leading to the excision of specific DNA sequences and (ii) FRT sites located on separate plasmids resulting in the formation of heterodimeric or higher order multimeric plasmids . In addition to FRT sites originally isolated from the yeast 2 microns plasmid, a number of synthetic FRT sites were also used . The synthetic sites were fully functional as target sites for recombination and gave results similar to those derived from the yeast 2 microns plasmid . This successful demonstration of yeast FLP recombinase activity in the mosquito embryo suggests a possible future application of this system in establishing transformed lines of mosquitoes for use in vector control strategies and basic studies.

J Biol Chem, 1991 Nov 5, 266(31), 21150 - 7
Epitope-tagged ubiquitin . A new probe for analyzing ubiquitin function; Ellison MJ et al.; In the present work, a method based on an epitope-tagged ubiquitin derivative is described that allows for the unambiguous detection of ubiquitin-protein conjugates formed in vivo or in vitro . Expression in the yeast Saccharomyces cerevisiae of ubiquitin that has been tagged at its amino terminus with a peptide epitope results in the formation of tagged ubiquitin-protein conjugates that are detectable by immunoblotting with a monoclonal antibody that recognizes the tag . The expression of tagged ubiquitin has no adverse effect on vegetative growth and, moreover, can suppress the stress-hypersensitive phenotype of yeast lacking the polyubiquitin gene UBI4 . We also show that tagged ubiquitin is correctly conjugated in vivo and in vitro to a short-lived test protein and can be covalently extended into the multimeric ubiquitin chain that is normally required for the degradation of this protein . Surprisingly, however, conjugation of tagged ubiquitin inhibits proteolysis . These and related results suggest that the amino-terminal region of ubiquitin is important in protease-substrate recognition and that the multiubiquitin chain is a dynamic transient structure . The potential of tagged ubiquitin for the identification and isolation of ubiquitin-protein conjugates and ubiquitin-related enzymes, and as a tool in mechanistic studies is discussed.

Proc Natl Acad Sci U S A, 1991 Nov 1, 88(21), 9578 - 82
The two-hybrid system: a method to identify and clone genes for proteins that interact with a protein of interest; Chien CT et al.; We describe a method that detects proteins capable of interacting with a known protein and that results in the immediate availability of the cloned genes for these interacting proteins . Plasmids are constructed to encode two hybrid proteins . One hybrid consists of the DNA-binding domain of the yeast transcriptional activator protein GAL4 fused to the known protein; the other hybrid consists of the GAL4 activation domain fused to protein sequences encoded by a library of yeast genomic DNA fragments . Interaction between the known protein and a protein encoded by one of the library plasmids leads to transcriptional activation of a reporter gene containing a binding site for GAL4 . We used this method with the yeast SIR4 protein, which is involved in the transcriptional repression of yeast mating type information . (i) We used the two-hybrid system to demonstrate that SIR4 can form homodimers . (ii) A small domain consisting of the C terminus of SIR4 was shown to be sufficient to mediate this interaction . (iii) We screened a library to detect hybrid proteins that could interact with the SIR4 C-terminal domain and identified SIR4 from this library . This approach could be readily extended to mammalian proteins by the construction of appropriate cDNA libraries in the activation domain plasmid.

Proc Natl Acad Sci U S A, 1991 Nov 1, 88(21), 9488 - 92
Do interhelical side chain-backbone hydrogen bonds participate in formation of leucine zipper coiled coils?
Tropsha A, Bowen JP, Brown FK, Kizer JS.
The leucine zipper proteins are a group of transcriptional regulators that dimerize to form a DNA binding domain . It has been proposed that this dimerization results from the hydrophobic association of the alpha-helices of two leucine zipper monomers into a coiled coil . We propose a model for a coiled coil based on a periodic hydrophobic-hydrophilic amino acid motif found in the leucine zipper regions of 11 transcriptional regulatory proteins . This model predicts the symmetrical formation of secondary hydrogen bonds between the polar side chains of one helix and the peptide carbonyls of the opposite chain, supplementing the interactions between hydrophobic side chains . Physical modeling (CPK) and in vacuo molecular mechanics calculations of the stability of the GCN4 leucine zipper coiled coil configured in accordance with this model demonstrate a greater stability for this conformer than for a conformer configured according to a current hydrophobic model . Molecular dynamics simulations show similar stability of the two models in vacuo but a higher stability of the hydrophobic model in water.

Proc Natl Acad Sci U S A, 1991 Nov 1, 88(21), 9392 - 6
FUS3 represses CLN1 and CLN2 and in concert with KSS1 promotes signal transduction; Elion EA et al.; FUS3 is functionally redundant with KSS1, a homologous yeast protein kinase, for a step(s) in signal transduction between the beta subunit of the guanine nucleotide binding protein (G protein), STE4, and the mating type-specific transcriptional activator, STE12 . Either FUS3 or KSS1 can execute this function; when neither gene encoding these protein kinases is present, signal transduction is blocked, causing sterility . This functional redundancy is strain dependent; some standard laboratory strains (S288C) are kss1- . FUS3 has additional functions required for cell cycle arrest and vegetative growth that do not overlap with KSS1 functions . FUS3 mediates cell cycle arrest during mating through transcriptional repression of two G1 cyclins (CLN1 and CLN2) and through posttranscriptional inhibition of a third G1 cyclin (CLN3) . FUS3 is also required for vegetative growth in haploid strains dependent upon CLN3 for cell cycle progression but is not required in strains dependent upon either CLN1 or CLN2, suggesting a functional divergence among the three G1 cyclins . The diverse roles for FUS3 suggest that the FUS3 protein kinase has multiple substrates, some of which may be shared with KSS1.

Cell, 1991 Nov 1, 67(3), 557 - 67
Family of proteins that interact with TFIID and regulate promoter activity; Meisterernst M et al.; A family of proteins was shown to bind cooperatively with TFIID to core promoters, as previously demonstrated for the general initiation factor TFIIA . These factors form distinct complexes with TFIID, fail to bind DNA in the absence of TFIID, differ chromatographically from TFIIA, and compete with TFIIA for binding to TFIID . Our results suggest the formation of heterogeneous preinitiation complexes at the step involving TFIIA interactions . This establishes a molecular switch that regulates basal level transcription in vitro and has consequences for transcriptional activation by gene-specific activators.

Mol Cell Biol, 1991 Nov, 11(11), 5781 - 91
Mutations in a conserved region of RNA polymerase II influence the accuracy of mRNA start site selection; Hekmatpanah DS et al.; A sensitive phenotypic assay has been used to identify mutations affecting transcription initiation in the genes encoding the two large subunits of Saccharomyces cerevisiae RNA polymerase II (RPB1 and RPB2) . The rpb1 and rpb2 mutations alter the ratio of transcripts initiated at two adjacent start sites of a delta-insertion promoter . Of a large number of rpb1 and rpb2 mutations screened, only a few affect transcription initiation patterns at delta-insertion promoters, and these mutations are in close proximity to each other within both RPB1 and RPB2 . The two rpb1 mutations alter amino acid residues within homology block G, a region conserved in the large subunits of all RNA polymerases . The three strong rpb2 mutations alter adjacent amino acids . At a wild-type promoter, the rpb1 mutations affect the accuracy of mRNA start site selection by producing a small but detectable increase in the 5'-end heterogeneity of transcripts . These RNA polymerase II mutations implicate specific portions of the enzyme in aspects of transcription initiation.

Mol Cell Biol, 1991 Nov, 11(11), 5746 - 55
nirA, the pathway-specific regulatory gene of nitrate assimilation in Aspergillus nidulans, encodes a putative GAL4-type zinc finger protein and contains four introns in highly conserved regions; Burger G et al.; The nucleotide sequence of nirA, mediating nitrate induction in Aspergillus nidulans, has been determined . Alignment of the cDNA and the genomic DNA sequence indicates that the gene contains four introns and encodes a protein of 892 amino acids . The deduced NIRA protein displays all characteristics of a transcriptional activator . A putative double-stranded DNA-binding domain in the amino-terminal part comprises six cysteine residues, characteristic for the GAL4 family of zinc finger proteins . An amino-terminal highly acidic region and two proline-rich regions are also present . The nucleotide sequences of two mutations were determined after they were mapped by transformation with overlapping DNA fragments, amplified by the polymerase chain reaction . nirA87, a mutation conferring noninducibility by nitrate and nitrite, has a -1 frameshift at triplet 340, which eliminates 549 C-terminal amino acids from the polypeptide . Under the assumption that the truncated polypeptide is stable, it comprises the zinc finger domain and the acidic region, which seem not sufficient for transcriptional activation . nirAd-106, an allele conferring nitrogen metabolite derepression of nitrate and nitrite reductase activity, includes two transitions, changing a glutamic acid to a lysine and a valine to an alanine, situated between a basic and a proline-rich region of the protein . Northern (RNA) analysis of the wild type and of constitutive (nirAc) and derepressed (nirAd) mutants show that the nirA transcript does not vary between these strains, being in all cases constitutively expressed . On the other hand, transcript levels of structural genes (niaD and niiA) do vary, being highly inducible in the wild type but constitutively expressed in the nirAc mutant . The nirAd mutant appears phenotypically derepressed, because the niaD and niiA transcript levels are overinduced in the presence of nitrate but are still partially repressed in the presence of ammonium.

Mol Cell Biol, 1991 Nov, 11(11), 5727 - 34
Structural and functional dissection of a membrane glycoprotein required for vesicle budding from the endoplasmic reticulum; d'Enfert C et al.; Sec12p is a membrane glycoprotein required for the formation of a vesicular intermediate in protein transport from the endoplasmic reticulum to the Golgi apparatus in Saccharomyces cerevisiae . Comparison of the N-linked glycosylation of Sec12p, a Sec12p-invertase hybrid protein, and a derivative of Sec12p lacking 71 carboxy-terminal amino acids showed that Sec12p is a type II membrane protein . Analysis of two truncated forms of Sec12p and of a temperature-sensitive mutant indicated that the C-terminal domain of Sec12p is not essential for protein transport, whereas the integrity and membrane attachment of the cytoplasmic N-terminal domain are essential . Expression of a soluble cytoplasmic domain dramatically inhibited the growth of a sec12 temperature-sensitive strain by increasing the transport defect at a normally permissive temperature . This growth inhibition as well as the sec12 temperature-sensitive defect were suppressed by the overproduction of Sar1p, a small GTP-binding protein that participates in protein transport . Sar1p membrane association was enhanced by elevated levels of Sec12p . These results suggest that the cytoplasmic domain of Sec12p interacts with Sar1p and that the complex may function to promote vesicle formation.

Mol Cell Biol, 1991 Nov, 11(11), 5624 - 30
The intron-containing hsp82 gene of the dimorphic pathogenic fungus Histoplasma capsulatum is properly spliced in severe heat shock conditions; Minchiotti G et al.; We have isolated and characterized a heat-inducible gene, hsp82, from the dimorphic pathogenic fungus Histoplasma capsulatum, which is a filamentous mold at 25 degrees C and a unicellular yeast at 37 degrees C . This gene, which has a high degree of homology with other members of the hsp82 gene family, is split into three exons and two introns of 122 and 86 nucleotides, respectively . Contrary to what has been demonstrated in Drosophila melanogaster, Saccharomyces cerevisiae, and other organisms, hsp82 mRNA in H . capsulatum is properly spliced during the severe heat conditions of 37 to 40 degrees C in the temperature-sensitive Downs strain . Splicing accuracy was also observed at 42 degrees C in the temperature-tolerant G222B strain, which showed no evidence of accumulation of primary transcripts . Furthermore, the intron containing the beta-tubulin gene is also properly spliced at the upper temperature range, suggesting that the lack of a block in splicing may be a general phenomenon in this organism.

Mol Cell Biol, 1991 Nov, 11(11), 5410 - 6
Conserved mechanism of tRNA splicing in eukaryotes; Zillmann M et al.; The ligation steps of tRNA splicing in yeast and vertebrate cells have been thought to proceed by fundamentally different mechanisms . Ligation in yeast cells occurs by incorporation of an exogenous phosphate from ATP into the splice junction, with concomitant formation of a 2' phosphate at the 5' junction nucleotide . This phosphate is removed in a subsequent step which, in vitro, is catalyzed by an NAD-dependent dephosphorylating activity . In contrast, tRNA ligation in vertebrates has been reported to occur without incorporation of exogenous phosphate or formation of a 2' phosphate . We demonstrate in this study the existence of a yeast tRNA ligase-like activity in HeLa cells . Furthermore, in extracts from these cells, the entire yeastlike tRNA splicing machinery is intact, including that for cleavage, ligation, and removal of the 2' phosphate in an NAD-dependent fashion to give mature tRNA . These results argue that the mechanism of tRNA splicing is conserved among eukaryotes.

Mol Cell Biol, 1991 Nov, 11(11), 5735 - 45
nit-4, a pathway-specific regulatory gene of Neurospora crassa, encodes a protein with a putative binuclear zinc DNA-binding domain; Yuan GF et al.; nit-4, a pathway-specific regulatory gene in the nitrogen circuit of Neurospora crassa, is required for the expression of nit-3 and nit-6, the structural genes which encode nitrate and nitrite reductase, respectively . The complete nucleotide sequence of the nit-4 gene has been determined . The predicted NIT4 protein contains 1,090 amino acids and appears to possess a single Zn(II)2Cys6 binuclear-type zinc finger, which may mediate DNA binding . Site-directed mutagenesis studies demonstrated that cysteine and other conserved amino acid residues in this possible DNA-binding domain are necessary for nit-4 function . A stretch of 27 glutamines, encoded by a CAGCAA repeating sequence, occurs in the C terminus of the NIT4 protein, and a second glutamine-rich domain occurs further upstream . A NIT4 protein deleted for the polyglutamine region was still functional in vivo . However, nit-4 function was abolished when both the polyglutamine region and the glutamine-rich domain were deleted, suggesting that the glutamine-rich domain might function in transcriptional activation . The homologous regulatory gene from Aspergillus nidulans, nirA, encodes a protein whose amino-terminal half has approximately 60% amino acid identity with NIT4 but whose carboxy terminus is completely different . A hybrid nit-4-nirA gene was constructed and found to function in N . crassa.

Trends Genet, 1991 Nov-Dec, 7(11-12), 346 - 51
Regulation of p21ras activity; Lowy DR et al.; The ras genes encode GTP/GDP-binding proteins that participate in mediating mitogenic signals from membrane tyrosine kinases to downstream targets . The activity of p21ras is determined by the concentration of GTP-p21ras, which is tightly regulated by a complex array of positive and negative control mechanisms . GAP and NF1 can negatively regulate p21ras activity by stimulating hydrolysis of GTP bound to p21ras . Other cellular factors can positively regulate p21ras by stimulating GDP/GTP exchange.

Bioconjug Chem, 1991 Nov-Dec, 2(6), 447 - 51
Generation of 5-fluorouracil from 5-fluorocytosine by monoclonal antibody-cytosine deaminase conjugates; Senter PD et al.; Cytosine deaminase (CDase) catalyzes the conversion of cytosine to uracil and is also able to convert the clinically used antifungal agent 5-fluorocytosine (5FC) into the anticancer drug 5-fluorouracil (5FU) . The enzyme was purified from bakers' yeast in a six-step procedure . Studies indicated that bakers' yeast CDase had a molecular weight of approximately 32 kDa and was composed of two subunits of equal molecular weights . Monoclonal antibodies were covalently attached to CDase, forming conjugates that could bind to antigens on tumor cell surfaces . The combination of L6-CDase and 5FC was equivalent in cytotoxic activity to 5FU when tested against the H2981 human lung adenocarcinoma cell line (L6 positive, 1F5 negative) . 5FC alone was noncytotoxic . The activation of 5FC was immunologically specific since 1F5-CDase did not enhance 5FC activity.

Yeast, 1991 Nov, 7(8), 881 - 8
The MAT locus revisited within a 9.8 kb fragment of chromosome III containing BUD5 and two new open reading frames; Jacquet M et al.; This paper reports the DNA sequence of a segment of 9.8 kb of the chromosome III . The sequenced DNA contains the MAT alpha locus . The new sequence of the MAT alpha locus differs from the previously reported sequence by six modifications in the W segment . We have found the same modifications in the HML locus . The corrected sequence contains, in HML, an open reading frame (ORF) of 190 codons which ends at the border between the W segment and the flanking DNA . In the MAT locus, this ORF extends in the flanking DNA up to 538 codons . This ORF corresponds to a gene independently identified as BUD5 (Chant et al., 1991) . This gene presents homologies with the exchange factors SDC25 and CDC25 . A large ORF of 1399 codons is found on the opposite side of MAT alpha (toward the telomere) . This ORF corresponds to a new gene YCR724 . Next to this gene is a small ORF, YCR725, of 127 codons . The localization of this fragment on chromosome III, originally supposed to be distal from the MAT locus based on genetic distance, illustrates variation in recombination frequency along the chromosome and suggests the existence of hot spots of recombination between MAT and the THR4 locus.

Trends Biochem Sci, 1991 Nov, 16(11), 412 - 6
RNA polymerase III (C) and its transcription factors; Gabrielsen OS et al.; Transcription of small genes by RNA polymerase III or C (pol III) involves many of the strategies that are used for transcription complex formation and occasionally the same components as those used by RNA polymerase II or B (pol II) . Transcription complex formation is a multistep process that leads to the binding of a single initiation factor, TFIIIB, which in turn directs the selection of pol III . The general transcription factor TFIID can be involved in both pol II and pol III transcription . These and other similarities point towards a unifying mechanism for eukaryotic transcription initiation.

Genes Dev, 1991 Nov, 5(11), 2108 - 16
The cap and poly(A) tail function synergistically to regulate mRNA translational efficiency; Gallie DR; The cap structure and the poly(A) tail are important regulatory determinants in establishing the translational efficiency of a messenger RNA . Although the mechanism by which either determinant functions remains poorly characterized, the interaction between the poly(A) tail-poly(A)-binding protein complex and events occurring at the 5' terminus during translation initiation has been an intriguing possibility . In this report, the mutual dependence of the cap and the poly(A) tail was studied . Poly(A)+ and poly(A)- luciferase (Luc) mRNAs generated in vitro containing or lacking a cap were translated in vivo in tobacco protoplasts, Chinese hamster ovary cells, and yeast following delivery by electroporation . The poly(A) tail-mediated regulation of translational efficiency was wholly dependent on the cap for function . Moreover, cap function was enhanced over an order of magnitude by the presence of a poly(A) tail . The relative differences in stability between the mRNAs could not account for the synergism . The synergism between the cap and poly(A) tail was not observed in yeast cells in which active translation had been disrupted . In addition, the synergism was not observed in in vitro translation lysates . These data demonstrate that the cap and the poly(A) tail are interdependent for optimal function in vivo and suggest that communication between the two regulatory determinants may be important in establishing efficient translation.

Cell, 1991 Nov 1, 67(3), 517 - 28
Crystal structure of a MAT alpha 2 homeodomain-operator complex suggests a general model for homeodomain-DNA interactions; Wolberger C et al.; The MAT alpha 2 homeodomain regulates the expression of cell type-specific genes in yeast . We have determined the 2.7 A resolution crystal structure of the alpha 2 homeodomain bound to a biologically relevant DNA sequence . The DNA in this complex is contacted primarily by the third of three alpha-helices, with additional contacts coming from an N-terminal arm . Comparison of the yeast alpha 2 and the Drosophila engrailed homeodomain-DNA complexes shows that the protein fold is highly conserved, despite a 3-residue insertion in alpha 2 and only 27% sequence identity between the two homeodomains . Moreover, the orientation of the recognition helix on the DNA is also conserved . This docking arrangement is maintained by side chain contacts with the DNA--primarily the sugar-phosphate backbone--that are identical in alpha 2 and engrailed . Since these residues are conserved among all homeodomains, we propose that the contacts with the DNA are also conserved and suggest a general model for homeodomain-DNA interactions.

J Biomol NMR, 1991 Nov, 1(4), 457 - 71
The value of chemical shift parameters in the description of protein solution structures; Gao Y et al.; An increasing number of protein solution structures, calculated on the basis of nuclear Overhauser enhancement cross-peak intensities observed in two- or higher dimensional NOESY experiments, are becoming available . Among these structures regions of uncertainty are frequently observed particularly with respect to loops and surface side chains . These are commonly ascribed to either a lack of NOE constraints or to some intrinsic mobility within the protein . A powerful method of structural analysis which may resolve this problem is based on the information content of the chemical shift . The value of such an analysis is illustrated here with cytochromes b5 and c, proteins for which high-quality crystallographic and NMR data are available . Comparison of these using a pseudocontact shift-based analysis indicates that NOE data should be combined with the chemical shift data in order to uncover fully the ensemble of protein states and their dynamics in solution.

Oncogene, 1991 Nov, 6(11), 2019 - 26
Differential regulation of three members of the ATF/CREB family of DNA-binding proteins; Flint KJ et al.; The ATF/CRE binding site can mediate transcriptional activation by cAMP, the adenovirus E1A protein and the human T-cell leukaemia virus 1 (HTLV1) tax protein . A large number of different proteins bind specifically to this element either as homodimers or as heterodimers . Using GAL4-ATF/CREB fusions, we have investigated the regulatory functions of three members of this family . CREB1 (CREB) is strongly activated by cAMP and weakly activated by the E1A protein . In contrast, CREB2 (CRE-BP1, ATF2) is strongly activated by E1A but is insensitive to cAMP stimulation . ATF1 is weakly activated by cAMP but is not activated by E1A . All three proteins are insensitive to activation by the HTLV1 tax protein . The N-terminal region of CREB2, from amino acid residues 19 to 112, is both necessary and sufficient for E1A activation . This region contains a putative C2H2 metal-binding finger, and single amino acid substitutions of the cysteine residues severely decreased CREB2 activity . In contrast, mutations affecting a potential protein kinase A and casein kinase II phosphorylation site within this region had little effect.

Virology, 1991 Nov, 185(1), 513 - 8
Characterization of a nuclear localization sequence in the polyomavirus capsid protein VP1; Moreland RB et al.; Expression of VP1-beta-galactosidase fusion proteins in the yeast Saccharomyces cerevisiae was used to identify a domain of the polyomavirus VP1 capsid protein which targets this protein to the nucleus . Fusion of the first 17 amino acids of VP1 to beta-galactosidase was sufficient for nuclear localization, whereas fusion of the first 12 amino acids gave a "mixed" cytoplasmic-nuclear phenotype . Mutation of a putative targeting sequence MAPKR(5)K from R to S changed the localization of a 21 amino acid fusion protein from the nucleus to cytoplasm . These results define a nuclear location signal in the amino terminus of polyomavirus VP1 and separate this function from the high-affinity DNA binding function previously defined for this region.

Clin Chim Acta, 1991 Oct 31, 202(3), 123 - 32
Accumulation and impaired in vivo metabolism of di- and trihydroxycholestanoic acid in two patients; Wanders RJ et al.; Two patients with a suspected peroxisomal disorder on the basis of neurological, craniofacial, hepatological and other abnormalities were studied . The phenotype of both girls was remarkably similar from birth until age 1.5 yr . Detailed studies in plasma revealed normal plasma very-long-chain fatty acids but the presence of di- and trihydroxycholestanoic acids and the C29-dicarboxylic bile acid, all known to occur in plasma from Zellweger patients . These results suggest an isolated defect in the peroxisomal beta-oxidation of the side chains of the cholestanoic acids . Activation of trihydroxycholestanoic acid and beta-oxidation of trihydroxycholestanoyl-CoA, measured in a liver biopsy, were normal, however, as was the peroxisomal beta-oxidation of palmitate . Although the molecular defect remains unknown, the results stress the importance of performing multiple analyses in any patient suspected to suffer from a peroxisomal disorder and indicate that screening for peroxisomal disorders based upon analysis of only plasma very long chain fatty acids with or without analysis of erythrocyte plasmalogen levels, may be inadequate.

Science, 1991 Oct 25, 254(5031), 539 - 44
X-ray structure of the GCN4 leucine zipper, a two-stranded, parallel coiled coil; O'Shea EK et al.; The x-ray crystal structure of a peptide corresponding to the leucine zipper of the yeast transcriptional activator GCN4 has been determined at 1.8 angstrom resolution . The peptide forms a parallel, two-stranded coiled coil of alpha helices packed as in the "knobs-into-holes" model proposed by Crick in 1953 . Contacts between the helices include ion pairs and an extensive hydrophobic interface that contains a distinctive hydrogen bond . The conserved leucines, like the residues in the alternate hydrophobic repeat, make side-to-side interactions (as in a handshake) in every other layer of the dimer interface . The crystal structure of the GCN4 leucine zipper suggests a key role for the leucine repeat, but also shows how other features of the coiled coil contribute to dimer formation.

Nucleic Acids Res, 1991 Oct 25, 19(20), 5619 - 24
Analysis of the mechanism of interaction of simian Ku protein with DNA; Paillard S et al.; Ku protein is a relatively abundant DNA-binding protein which was first detected as the autoantigen in a patient with scleroderma-polymyositis overlap syndrome (hence the name 'Ku') . It is a heterodimer of two polypeptide chains of molecular weights 85,000 and 72,000, and it characteristically binds, in vitro, to the ends of DNA fragments, and translocates to form regular multimeric complexes, with one protein bound per 30 bp of DNA . We have studied the mechanism of interaction of Ku protein with DNA in vitro, using protein extracted from cultured monkey cells . We find that the precise structure of the DNA ends is not important for binding, as Ku protein can bind to hairpin loops and to mononucleosomes . Bound protein also does not require DNA ends for continued binding, since complexes formed with linear DNAs can be circularized by DNA ligase . Dissociation of the complex also appears to require DNA ends, since ligase closed circular complexes were found to be extremely stable even in the presence of 2 M NaCl . We also found that Ku molecules slide along DNA, with no preferential binding to specific sequences . Thus, Ku protein behaves like a bead threaded on a DNA string, a binding mechanism which allows us to make a new hypothesis concerning the function of this protein in the nucleus.

J Mol Biol, 1991 Oct 20, 221(4), 1153 - 64
Function of P11, a tertiary base pairing in self-splicing introns of subgroup IA; Jaeger L et al.; There is phylogenetic evidence for the existence of a new pairing in subgroup IA1 self-splicing introns . This tertiary interaction, called P11, which is extraneous to the catalytic centre of these ribozymes was modelled after a "pseudoknot" and grafted by computer modelling on the common core structure of group I introns that was recently proposed by Michel & Westhof . In order to probe the function of the P11 pairing, we mutated the P11 helix in the intron of the large ribosomal precursor of Saccharomyces cerevisiae mitochondria (Sc.LSU) . Our experimental data show that the P11 pairing plays a role in stabilizing the overall fold of the RNA molecule . While P11 is not essential for self-splicing activity in vitro, mutants with disrupted P11 require higher concentration of MgCl2 for self-splicing . By contrast, mutants with a reinforced P11 pairing (via introduction of several G.C base-pairs) self-splice more efficiently than the wild-type at 55 degrees C . Based on this work, the possible engineering of new stable versions of the ribozyme is discussed.

Biochem J, 1991 Oct 15, 279 ( Pt 2), 587 - 93
The interaction of 1-fluoro-D-glucopyranosyl fluoride with glucosidases; Konstantinidis A et al.; 1 . 1-Fluoro-D-glucopyranosyl fluoride undergoes pH-independent loss of F- ion at a rate of 1 x 10(-8) s-1 at 50.0 degrees C, some 10(3)-fold slower than alpha-D-glucopyranosyl fluoride and 4 x 10(4)-fold slower than beta-D-glucopyranosyl fluoride . 2 . The (inverting) amyloglucosidase II of Aspergillus niger hydrolyses the difluoride according to Michaelis-Menten kinetics (Km 34 mM and kcat . 0.27 s-1), by apparently the same (simple) mechanism by which it hydrolyses alpha-D-glucopyranosyl fluoride (Km 38 mM and kcat . 730 s-1), rather than by the Hehre resynthesis-hydrolysis mechanism used to transform beta-D-glucopyranosyl fluoride . 3 . The difluoride is also a substrate for the (inverting) trehalase of pig kidney {Km 17.3 mM and Vmax . 6.2 x 10(-4) relative to alpha-D-glucopyranosyl fluoride (Km 38 mM}) . 4 . The quantitatively similar effect of fluorine substitution on the one-step enzymic reactions and on the non-enzymic reactions suggests that they go through similar (oxocarbonium-ion-like) transition states . 5 . The difluoride is a substrate for the (retaining) beta-glucosidases from Aspergillus wentii (A3 enzyme) and sweet-almond meal (B isoenzyme) and for the retaining alpha-glucosidase from rice: comparison with the appropriate monofluoride reveals a variable rate-retarding effect of the second fluorine atom on kcat./Km that correlates with other measures of oxocarbonium ion character in the transition state . 6 . The difluoride is a substrate for the (retaining) alpha-glucosidase from yeast, but also gives an insidious mimicry of active-site-directed irreversible inhibition, which we tentatively attribute either to formation of the non-covalent complex or to the fluoroglucosyl-enzyme increasing the well-known tendency of this enzyme to come out of solution by adsorption on the walls of the vessel.

Proc Natl Acad Sci U S A, 1991 Oct 15, 88(20), 9330 - 4
Propeptide of carboxypeptidase Y provides a chaperone-like function as well as inhibition of the enzymatic activity; Winther JR et al.; The zymogen of the vacuolar carboxypeptidase Y from Saccharomyces cerevisiae was purified and characterized with respect to activation as well as refolding in vitro . The purified procarboxypeptidase Y has no detectable activity but can be efficiently activated by proteinase K from Tritirachium album . We used this method of activation as a tool for the investigation of refolding procarboxypeptidase Y in vitro . The proenzyme, denatured in 6 M guanidinium chloride, is renatured efficiently after dilution of the denaturant, whereas the mature enzyme regains little activity in the same procedure . Changes in intrinsic fluorescence reveal the mature enzyme to be considerably more stable than the proenzyme toward denaturation with guanidinium chloride . This suggests that the propeptide induces a metastable structure important for overcoming energy barriers that might otherwise obstruct a productive folding pathway . The relatively large number of charged amino acid residues and a high theoretical potential for alpha-helix formation in the carboxypeptidase Y propeptide suggest a structural similarity to a number of other propeptides and heat shock proteins.

Proc Natl Acad Sci U S A, 1991 Oct 15, 88(20), 9056 - 60
RNase-like domain in DNA-directed RNA polymerase II; Shirai T et al.; DNA-directed RNA polymerase is responsible for gene expression . Despite its importance, many details of its function and higher-order structure still remain unknown . We report here a local sequence similarity between the second largest subunit of RNA polymerase II and bacterial RNases Ba (barnase), Bi, and St . The most remarkable similarity is that the catalytic sites of the RNases are shared with the eukaryotic RNA polymerase II subunits of Drosophila melanogaster and Saccharomyces cerevisiae . Several amino acids conserved among the RNases and the RNase-like domains of the RNA polymerase subunits are located in the neighborhood of the catalytic sites of barnase, whose three-dimensional structure has been resolved . This observation suggests the functional importance of the RNase-like domain of the RNA polymerase subunits and indicates that the RNase-like domain may have RNase activity . The location of the RNase-like domain relative to the region necessary for RNA polymerization is similar to the relative proximity of 5'----3' or 3'----5' exonuclease and the region of polymerase activity of DNA polymerase I . The RNase-like domain might work in proofreading, as in RNA-directed RNA polymerase of influenza virus, or it may contribute to RNA binding through an unknown function.

J Biol Chem, 1991 Oct 15, 266(29), 19342 - 50
Peroxide modification of monoalkylated glutathione reductase . Stabilization of an active-site cysteine-sulfenic acid; Miller H et al.; Hydrogen peroxide reacts with two-electron reduced glutathione reductase (GR EH2 species) to give the native oxidized enzyme (E) without detectable intermediates . Prior alkylation of the EH2 interchange thiol with iodoacetamide, however, dramatically changes both the course and overall rate of the peroxide reaction . This oxidation, monitored spectrally, is characterized by an intermediate (EHRint) with enhanced long wavelength absorbance extending to 800 nm . This species decays in a second peroxide-dependent phase to an enzyme form (EHRox) easily distinguished from E . Quenching experiments with catalase allow the isolation of a stable mixture consisting of 36% monoalkylated GR (EHR), 60% EHRint, and 4% EHRox; NADPH titration and anaerobic dithiothreitol addition lead to quantitative reduction of EHRint to EHR, and there is an increase in thiol titer of 0.8-SH/FAD on NADPH reduction . Of the four titratable thiols present in EHR, 2.7 are lost on oxidation to EHRox and 0.7-0.8 mol of cysteic acid/FAD is formed . On the basis of these and other observations, we conclude that alkylation of the EH2 interchange thiol, which blocks disulfide formation, allows peroxide reaction at the remaining charge-transfer thiol to proceed via a stabilized cysteine-sulfenic acid intermediate (EHRint), which undergoes further oxidation to the corresponding cysteic acid (EHRox).

J Biol Chem, 1991 Oct 15, 266(29), 19158 - 61
Mammalian seryl-tRNA synthetase associates with mRNA in vivo and has homology to elongation factor 1 alpha; Miseta A et al.; Previous work in our laboratories (Slobin, L . I., and Greenberg, J.R . (1988) Eur . J . Biochem . 173, 305-310) showed that messenger ribonucleoprotein (mRNP) particles possess a polypeptide component of approximately 62 kDa that appears to share a common epitope with eucaryotic elongation factor 1 alpha (EF-1 alpha) . We report here that the previously unidentified mRNP constituent corresponds to seryl-tRNA synthetase (SerRS) . Furthermore, we show that SerRS contains a sequence motif that is shared by both EF-1 alpha and glutaminyl-tRNA synthetase . We also find that the association of SerRS with mRNA depends on the functional state of the latter . Our data suggest that SerRS may participate directly in the initiation phase of protein synthesis.

NMR Biomed, 1991 Oct, 4(5), 227 - 45
The molecular environment of intracellular sodium: 23Na NMR relaxation; Rooney WD et al.; The comprehensive approach described in the accompanying paper is illustrated here with the 23Na signal of a concentrated solution of bovine serum albumin (BSA) in saline and the intracellular (Nai) 23Na resonance of a dense suspension of Na(+)-loaded yeast cells . We use frequency shift reagents to discriminate the latter from the extracellular resonance . We find that the Nai signal corresponds to that of an effective single population of Na+ ions exhibiting a single type c spectrum . This is true despite the fact that the yeast protoplasm is too large and too compartmentalized for a given Na+ ion to sample its entirety on the relevant NMR timescale . Our results show clearly that, in addition to the decay of transverse magnetization, the recovery of longitudinal magnetization is biexponential . This is required for a type c spectrum but has not often been detected . The temperature dependence of the relaxation rate constants of the Nai resonance is not consistent with either a simple Debye process or a discrete exchange mechanism connecting two sites in the fast limit . We have fitted the data using an asymmetric continuous distribution of correlation times for the fluctuations of electric field gradients sensed by the Nai nuclei . The analogous distribution function for the Na+ in a 44% (w/w) BSA solution is quite similar to that of the Nai at the same temperature . This suggests that while the macromolecular environment of the Nai ions is quite congested, it is also isotropic on quite a small spatial scale . Also, one can use the correlation time distribution function, obtained from fitting the relaxation data, to calculate a relaxometry curve . This is useful because experimental 23Na relaxometry is difficult . The calculated curve may be a reasonable model for the mostly extracellular 23Na resonance encountered in vivo.

Biotechnology (N Y), 1991 Oct, 9(10), 994 - 5
Protein stabilization by engineered metal chelation; Kellis JT Jr et al.; A ligand can shift a protein's folding/unfolding equilibrium by binding with higher affinity to the native state . A metal-chelating site consisting of two histidines separated by three residues (His-X3-His) engineered into an alpha-helix provides a general and easily-implemented means for protein stabilization by this mechanism . We have tested this approach with the iso-1-cytochrome c of Saccharomyces cerevisiae substituted with histidine at positions 4 and 8 in its N-terminal alpha-helix . One mM Cu(II) complexed to iminodiacetate stabilizes the cytochrome c variant by ca . 1 kcal/mol, as determined by guanidinium chloride-induced unfolding . The protein's folding/unfolding equilibrium is shifted by a free energy equal to that calculated from the metal ion's preferential binding to the native protein . Given the ubiquity of surface alpha-helices and the additional possibility of inserting di-histidine chelating sites into turns and beta-structures, we conclude that this is a useful method for protein stabilization.

Nucleic Acids Res, 1991 Oct 11, 19(19), 5425 - 31
Common sets of nuclear factors binding to the conserved promoter sequence motif of two coordinately regulated ER protein genes, GRP78 and GRP94; Liu ES et al.; The GRP78 and GRP94 are two constitutively expressed ER resident proteins that are coordinately induced in response to stress conditions . The control of their induction is at the transcriptional level . We have previously demonstrated that the GRP78 and the GRP94 promoters share a common regulatory domain which is highly conserved . We report here that within this 36 bp promoter region is a CG/CAAT and a GC-rich sequence motif which are important for basal level and induced expression of the gene . Gel mobility shift assays with HeLa nuclear extracts and the conserved element from GRP78 and GRP94 show two shared, specific protein-DNA complexes . By ultraviolet cross-linking, the sizes of the proteins labeled in the slower-migrating complex are 210-, 110-, a doublet at 90/92- and 70 kD, and in the faster-migrating complex, protein species of about 55 kD . The formation of the second complex can be inhibited by competition with the coding strand of the conserved GRP element in a sequence specific manner . In addition, the Ku autoantigen which is abundant in HeLa cell extracts also binds . The sizes of the nuclear factors binding to the GRP78 and GRP94 conserved promoter elements are strikingly similar, providing further evidence that the two genes are coordinately regulated by common trans-acting factors.

Biochim Biophys Acta, 1991 Oct 8, 1090(2), 181 - 7
Expression of the Ku protein during cell proliferation; Yaneva M et al.; The Ku protein is a DNA-binding nuclear protein complex composed of 86 kDa and 70 kDa subunits . Recently, in vitro studies suggested a role of the Ku protein in the activation of gene transcription . We studied the expression of these proteins during cell proliferation by Northern blot hybridizations using specific cDNA probes and by immunofluorescence and immunoblot analysis using specific monoclonal antibodies . The genes coding for both subunits were activated during late G1-phase in the transition of human PHA-stimulated lymphocytes from quiescent (G0 phase) to proliferative (S phase) state . These genes were inactivated when human leukemia cells HL60 were differentiated into monocytes upon treatment with the phorbol ester TPA . Changes at the protein level were significantly smaller than changes at the mRNA levels in both cell systems, suggesting a high stability of the Ku protein . Immunofluorescence studies demonstrated nucleolar as well as nuclear localization of the Ku protein in quiescent lymphocytes and during the early G1-phase; during the late G1, S and G2 phases, the Ku protein was only localized in discrete structures in the nucleoplasm . These results demonstrate that the gene expression for the Ku protein is associated with the proliferative state of the cells and that the nucleolar localization of the Ku protein is cell-cycle-dependent.

J Biol Chem, 1991 Oct 5, 266(28), 18751 - 60
Histone acetylation in Zea mays . II . Biological significance of post-translational histone acetylation during embryo germination; Georgieva EI et al.; Multiple forms of histone acetyltransferases and histone deacetylases, which have been separated and characterized in the accompanying manuscript (Lopez-Rodas, G., Georgieva, E . I., Sendra, R., and Loidl, P . (1991) J . Biol . Chem . 266, 18745-18750), together with in vivo acetate incorporation, were studied during the germination of Zea mays embryos . Total histone acetyltransferase activity increases during germination with two maxima at 40 and 72 h after start of germination . This fluctuation is mainly due to the cytoplasmic B-enzyme which predominantly acetylates histone H4 up to the diacetylated form . The nuclear histone acetyltransferase A2, specific for H3, is low throughout germination, except at 24 h, when it transiently becomes the main activity . Both enzymes are also present in the dry embryo, whereas the second nuclear enzyme A1, specific for H3 and H4, is absent in the initial stage of differentiation . The two histone deacetylases, HD1 and HD2, exhibit entirely different patterns . Whereas HD1 activity is low in the dry embryo and increases during germination, HD2 is the predominant enzyme at the start of differentiation, but almost disappears at later stages . Analysis of the in vivo acetate incorporation reveals that H4 is present in up to tetraacetylated subspecies . The pattern of acetate incorporation into core histones closely resembles the fluctuations of histone acetyltransferase B . Based on the analysis of thymidine kinase activity a close correlation was established between histone acetyltransferase B and DNA replication, whereas the A2 enzyme is associated with transcriptional activity . Histone deacetylase HD1 obviously serves a specific function in the dry embryo and could be a prerequisite for DNA repair processes . The study confirms the idea of DNA repair processes . The study confirms the idea of multiple functions of histone acetylation and assigns distinct enzymes, involved in this modification, to certain nuclear processes.

J Biol Chem, 1991 Oct 5, 266(28), 18745 - 50
Histone acetylation in Zea mays.I . Activities of histone acetyltransferases and histone deacetylases; Lopez-Rodas G et al.; DEAE-Sepharose chromatography of extracts from Zea mays meristematic cells revealed multiple histone acetyltransferase and histone deacetylase enzyme forms . An improved method for nuclear isolation allowed us to discriminate nuclear and cytoplasmic enzymes . Two nuclear histone acetyltransferases, A1 and A2, a cytoplasmic B-enzyme and two nuclear histone deacetylases, HD1 and HD2, have been identified . The histone specificity of the different enzyme forms has been studied in an in vitro system, using chicken erythrocyte histones as substrate . The cytoplasmic histone acetyltransferase B is the predominant enzyme, which acetylates mainly histone H4 and to a lesser extent H2A . The nuclear histone acetyltransferase A1 preferentially acetylates H3 and also H4, whereas enzyme A2 is specific for H3 . This substrate specificity was confirmed with homologous Z . mays histones . The two histone deacetylases differ from each other with respect to ionic strength dependence, inhibition by acetate and butyrate, and substrate specificity . The strong inhibitory effect of acetate on histone deacetylases was exploited to distinguish different histone acetyltransferase forms.

Proc Natl Acad Sci U S A, 1991 Oct 1, 88(19), 8784 - 6
In vivo catalysis of a metabolically essential reaction by an antibody; Tang Y et al.; We have established a growth selection requirement for a catalytic antibody with modest chorismate mutase activity . Conversion of (-)-chorismate into prephenate is the key step in the biosynthesis of the aromatic amino acids tyrosine and phenylalanine . Strains of the yeast Saccharomyces cerevisiae containing an insertion mutation in the structural gene for the enzyme chorismate mutase (EC 5.4.99.5) require exogenous supplements of these two amino acids for efficient growth . Intracellular expression of the heterologous antibody catalyst in one such strain, identified by random mutagenesis and genetic selection, provides a substantial growth advantage under auxotrophic conditions; complementation was not observed with an unrelated esterolytic antibody . In addition to demonstrating that tailored immunoglobulin catalysts can carry out vital biochemical reactions in vivo, these experiments provide a powerful selection assay for identifying genetic changes within the antibody molecule itself that augment chemical efficiency.

Proc Natl Acad Sci U S A, 1991 Oct 1, 88(19), 8611 - 5
Mammalian Sec23p homologue is restricted to the endoplasmic reticulum transitional cytoplasm; Orci L et al.; The yeast Sec23 protein is required in vivo and in vitro for transport of proteins from the endoplasmic reticulum (ER) to the Golgi apparatus . Ultrastructural localization of the Sec23p mammalian homologue (detected by antibody cross-reaction) in exocrine and endocrine pancreatic cells shows a specific distribution to the cytoplasmic zone between the transitional ER cisternae and Golgi apparatus where it appears associated with the tubular protuberances of the transitional ER cisternae, as well as with a population of vesicles, and surrounding cytoplasm . When ER-Golgi transport is interrupted with an energy poison, protuberances and transfer vesicles markedly decrease but Sec23p immunoreactive sites remain in the transitional cytoplasm not apparently tethered by membrane attachment . This unanticipated degree of organization suggests that cytosolic proteins, such as Sec23p, may be retained in specialized areas of the cytoplasm . A structure within the transitional zone may organize the flux of transport vesicles and Sec proteins so as to ensure efficient protein traffic in this limb of the secretory pathway.

Proc Natl Acad Sci U S A, 1991 Oct 1, 88(19), 8510 - 4
Sp1-dependent activation of a synthetic promoter by human immunodeficiency virus type 1 Tat protein; Kamine J et al.; The Tat protein coded by human immunodeficiency virus (HIV) is a strong activator of viral gene expression from the long terminal repeat (LTR) . It appears that Tat-mediated trans-activation of the HIV LTR is predominantly a transcriptional event . It has been reported that Tat acts at the level of both transcriptional initiation and elongation through interaction with a nascent RNA target sequence termed TAR (for trans-activation response element) . However, the precise mechanism(s) by which Tat mediates TAR-dependent transcriptional activity is not known . To determine whether Tat functions similarly to other eukaryotic transcriptional activators through any of the conventional promoter elements, we tested Tat activity on synthetic promoters containing consensus sequences required for binding transcription factor Sp1 and a TATA box . Here, we report that a chimeric Tat protein targeted to the promoter region by the DNA-binding domain of yeast transcription factor GAL4 activates the synthetic promoter . Because this trans-activation depends on Sp1-binding sites, Tat can apparently mediate transcriptional activation through its interaction with Sp1 . Mutational analysis of the gal4-tat chimeric gene reveals that the N-terminal 48-amino acid region of Tat constitutes the activation region for Sp1-dependent trans-activation . This region of Tat exhibits substantially more activity than the N-terminal 58 amino acids of Tat, which includes the arginine-rich basic region . Effects of specific mutations in the 48-amino acid Tat region of GAL4-Tat on trans-activation of the synthetic promoter mimic the effects of these specific mutations on Tat-mediated trans-activation of the HIV-1 LTR, suggesting that trans-activation of both the synthetic promoter and the intact LTR occurs by a common mechanism.

Proc Natl Acad Sci U S A, 1991 Oct 1, 88(19), 8293 - 6
E1BF is an essential RNA polymerase I transcription factor with an intrinsic protein kinase activity that can modulate rRNA gene transcription; Zhang J et al.; We previously described the purification and characterization of E1BF, a rat rRNA gene core promoter-binding factor that consists of two polypeptides of 89 and 79 kDa . When this factor was incubated in the absence of any exogenous protein kinase under conditions optimal for protein phosphorylation, the 79-kDa polypeptide of E1BF was selectively phosphorylated . The labeled phosphate could be removed from the E1BF polypeptide by treatment with calf intestinal alkaline phosphatase or potato acid phosphatase . Elution of the protein from the E1BF-promoter complex formed in an electrophoretic mobility-shift assay followed by incubation of the concentrated eluent with {gamma-32P} ATP resulted in the selective labeling of the 79-kDa band . The E1BF-associated protein kinase did not phosphorylate casein or histone H1 . Fraction DE-B, a preparation containing RNA polymerase I and all polymerase I transcription factors (including E1BF), lost polymerase I transcriptional activity when treated with phosphatase . The phosphatase-induced inactivation of polymerase I activity associated with fraction DE-B could be reversed by the addition of purified E1BF . Treatment of purified E1BF with heat, SDS, or an ATP affinity analog eliminated its capacity to reactivate dephosphorylated fraction DE-B . These data demonstrate that (i) polymerase I promoter-binding factor E1BF contains an intrinsic substrate-specific protein kinase and (ii) E1BF is an essential polymerase I transcription factor that can modulate rRNA gene transcription by protein phosphorylation . Further, these studies have provided a direct means to identify a protein kinase or any other enzyme that can interact with a specific DNA sequence.

Indian J Biochem Biophys, 1991 Oct-Dec, 28(5-6), 389 - 94
Kinetic analyses of the pyruvate dehydrogenase complex from Candida 107 (NCYC 911); Rajalakshmi KJ et al.; Candida 107 (NCYC 911) accumulates up to 45% of the biomass as triglycerides under conditions of nitrogenous substrate limitation in the medium . In oilseeds and adipocytes, lipid accumulation is preceded and accompanied by increased activity of key enzymes such as pyruvate dehydrogenase . However, in Candida 107, the activity of this complex was greatly reduced during lipogenesis . The initial velocity patterns were in accordance with a Hexa Uni Ping Pong mechanism . The Km values for the various substrates were similar to those found for the yeast Saccharomyces cerevisiae, but much higher than those reported for the mammalian enzyme . Product inhibition studies indicated that the Ki for acetyl coenzyme A and NADH were higher than those reported for other yeasts . The values for Ki were similar to those found for the liver enzyme, whereas the enzyme complex from heart had much lower Ki values for products . It has been suggested that in the heart and kidney, pyruvate dehydrogenase is regulated by product inhibition whereas in the liver this does not appear to be the mechanism . Therefore, it is probable, that like the liver enzyme, pyruvate dehydrogenase from Candida 107 may not be regulated by product inhibition.

Yeast, 1991 Oct, 7(7), 699 - 716
Functional analysis of a conserved amino-terminal region of HSP70 by site-directed mutagenesis; Nicolet CM et al.; Hsp70 proteins have been highly conserved throughout evolution . As a first step in a structure-function analysis of hsp70, we constructed and analysed the consequences of mutations in a portion of the SSA1 gene, a member of the Saccharomyces cerevisiae HSP70 multigene family, that encodes a nearly invariant region near the amino terminus . Analysis of strains expressing SSA1 proteins with alterations at positions 8, 11 and 15 showed that these conserved residues within this region are critical for normal functioning of the protein . SSA1 protein containing either of two changes at position 15 was able to slightly complement the inviability of an ssa1ssa2ssa4 strain, but was inactive in other complementation assays . The other mutant proteins tested were unable to complement any tested phenotype . Effective interallelic complementation of several phenotypes was observed when a mutant protein substituted at position 8 was expressed in the same cell with either of two proteins carrying substitutions at position 15, suggesting that hsp70 acts as a multimer . Evidence from previous studies suggests that hsp70 proteins engage in ATP-driven cycles of binding and release from peptides . The ability of the mutant proteins to bind ATP and a peptide was tested . The Ssa1p carrying a substitution at position 8, which inhibits growth of cells carrying wild-type SSA proteins, showed a defect in release from a peptide relative to wild type . Two mutations, one each at position 8 and 15, resulted in accumulation of phosphorylated isoforms which may be a normal, transient hsp70 intermediate.

Mol Endocrinol, 1991 Oct, 5(10), 1498 - 503
Multiple domains of the glucocorticoid receptor involved in synergism with the CACCC box factor(s); Muller M et al.; Steroid induction of responsive genes functions through the synergistic activity of steroid receptor-binding sequences with adjacent transcription factor-binding sites . To analyze the mechanism of synergy we tested different human glucocorticoid receptor mutants for synergistic function with another transcription factor in comparison with intrinsic trans-activation obtained with a single receptor binding site (glucocorticoid response element) . Multiple domains were found to be involved in synergistic activity of the glucocorticoid receptor with the CACCC box factor . Deletions within the N-terminal receptor half affected simultaneously intrinsic trans-activation and synergism . However, deletion of the hormone-binding domain mainly impaired synergism rather than intrinsic trans-activation, clearly showing that this domain synergizes by a mechanism independent of intrinsic activation . A chimeric protein where the DNA-binding domain of the glucocorticoid receptor was replaced by that of the yeast GAL4 protein also showed functional synergism . These data suggest that some of the receptor domains outside the DNA-binding domain synergize by their intrinsic trans-activating property, but the hormone-binding domain contributes to synergism by a different mechanism.

Biochem Genet, 1991 Oct, 29(9-10), 461 - 75
High-mobility group and other nonhistone substrates for nuclear histone N-acetyltransferase; Wong LC et al.; A variety of nonhistone proteins and polyamines has been studied for their substrate activity for nuclear histone N-acetyltransferase . Nonhistone chromatin high-mobility group (HMG) proteins are found to be as good a substrate for the enzyme as histones . The enzyme also acetylates spermidine and spermine . However, protamine, bovine serum albumin, and ubiquitin are not substrates . Chymotryptic peptides of histone and HMGs retained about 64% of the substrate activity, but trypsin treatment reduced the substrate activity by more than 85% . Both N-acetyltransferase activities for HMGs and histones are copurified through salt extraction, polyethylene glycol fractionation, and chromatography on DEAE-cellulose, phosphocellulose columns, and a HPLC anionic-exchange column . The highly purified nuclear histone acetyltransferase shows similar optimal pH and ping-pong kinetics for both HMGs and histones . The Km for HMG is 0.25 mg/ml . HMGs are able to accept the acetyl group from isolated acetyl-enzyme intermediate . Denatured gel analysis shows that HMG 1 and HMG 2 are the major proteins acetylated . High salt concentrations, mononucleotides, and DNA, which inhibit histone substrate activity of the enzyme, also inhibit HMG substrate activity . These observations suggest that there is a major nuclear N-acetyltransferase which is responsible for the acetylation of both histones and HMGs and perhaps also of spermine and spermidine . Thus the regulation of the structure and function of chromatin through postsynthetic acetylation can be achieved by a single nuclear N-acetyltransferase.

Genetics, 1991 Oct, 129(2), 597 - 602
A mathematical model of interference for use in constructing linkage maps from tetrad data; King JS et al.; In determining genetic map distances it is necessary to infer crossover frequencies from the ratios of recombinant and parental progeny . To do this accurately, in intervals where multiple crossovers may occur, a mathematical model of chiasma interference must be assumed when mapping in organisms displaying such interference . In Saccharomyces cerevisiae the model most frequently used is that of R.W . Barratt . An alternative to this model is presented . This new model is implemented using a microcomputer and standard numerical methods . It is demonstrated to fit ranked tetrad data from Saccharomyces more closely than the Barratt model and thus generates more accurate estimates of map distances when used with two-point data . A computer program implementing the model has been developed for use in calculating map distances from tetrad data in Saccharomyces.

Jpn J Pharmacol, 1991 Oct, 57(2), 187 - 96
Modificational changes in function and morphology of cultured macrophages by geraniin; Ushio Y et al.; Treatment of peritoneal macrophages with geraniin, isolated from Geranium funbergii, markedly induced the phagocytosis of living yeasts . Marked increases in phagocytosis and acid phosphatase activity in macrophage lysates were observed 24 hr after the beginning of geraniin treatment . As observed by electron microscopy, macrophages that had been treated for 24 hr with geraniin had a markedly thickened surface layer which was positive to ruthenium red, compared to the control cells . In addition, geraniin treatment of macrophages appeared to induce remarkably large mitochondria, more coated pits and prominent lysosomal granules . In conclusion, the stimulation of phagocytosis and acid phosphatase activity of macrophages by geraniin treatment may involve alterations of the plasma membrane and cytoplasmic reorganization.

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