Nature, 1993 Apr 29, 362(6423), 857 - 60 Oncoprotein MDM2 conceals the activation domain of tumour suppressor p53; Oliner JD et al.; The tumour-suppressor gene p53 is inactivated in most human malignancies either by missense mutations or by binding to oncogenic proteins . In human soft tissue sarcomas, inactivation apparently results from MDM2 gene amplification . MDM2 is an oncogene product that may function by binding to p53 and inhibiting its ability to activate transcription . Here we show that, when expressed in Saccharomyces cerevisiae, human MDM2 inhibits human p53's ability to stimulate transcription by binding to a region that nearly coincides with the p53 acidic activation domain . The isolated p53 activation domain fused to another DNA-binding protein is also inactivated by MDM2, confirming that MDM2 can inhibit p53 function by concealing the activation domain of p53 from the cellular transcription machinery. Nucleic Acids Res, 1993 Apr 25, 21(8), 1775 - 81 A repetitive DNA sequence associated with the centromeres of Chironomus pallidivittatus; Rovira C et al.; A clone containing centromere-associated DNA from Chironomus pallidivittatus was obtained by microdissection-microcloning . It hybridizes to the centromeric end of one chromosome and exclusively to regions in the three remaining, metacentric chromosomes to which centromeres have previously been localized on cytological grounds . In the metacentric positions the hybridization can be assigned to thin bands . The clone contains 155bp tandem repeats and short flanking regions represented in all of the centromeres . Titration experiments show that the four centromeres together contain 200kb of 155bp repeat per genome . In a line of tissue culture cells the amounts are increased by a factor 1.5-2, resulting in proportionately extended arrays of tandem repeats . Each repeat contains two invertrepeats surrounding a region containing only AT base pairs, a feature with some similarity to functionally essential elements in the Saccharomyces cerevisiae centromere. Cell, 1993 Apr 23, 73(2), 407 - 15 Mutations in U1 snRNA bypass the requirement for a cell type-specific RNA splicing factor; Nandabalan K et al.; Previous studies have demonstrated that efficient splicing of the primary transcript of the yeast MER2 gene requires the MER1 protein, which is produced only in meiotic cells . A genetic selection was devised to recover second-site mutations that bypass the requirement for MER1 in MER2 RNA-splicing . This selection identified a mutation in SNR19, the gene for U1 snRNA . The suppressor mutation affects the first residue in U1 snRNA, allowing this nucleotide to base pair with the eighth nucleotide in the MER2 intron . This base in MER2 lies outside the conserved hexanucleotide that defines the 5' splice site in yeast . The MER2 5' splice site (GUUCGU) differs from the consensus in yeast (GUAYGU) at the third position . When this nucleotide is mutated to restore the consensus, base pairing with U1 snRNA is increased and the requirement for MER1 is alleviated. Biochem J, 1993 Apr 15, 291 ( Pt 2), 569 - 73 Site-directed mutagenesis of mouse steroid 7 alpha-hydroxylase (cytochrome P-450(7) alpha): role of residue-209 in determining steroid-cytochrome P-450 interaction; Iwasaki M et al.; We have cloned a cDNA encoding mouse steroid 7 alpha-hydroxylase P450(7) alpha (cytochrome P-450(7) alpha) and expressed it in Saccharomyces cerevisiae . Mouse P450(7) alpha is 70% identical in its amino acid sequence with the mouse steroid 15 alpha-hydroxylase P450(15) alpha (2A4) . The Leu at position 209 of P450(15) alpha is the most important residue to determine the steroid hydroxylase activity of the P450 {Lindberg and Negishi (1989) Nature (London) 339, 632-634} . The P450(7) alpha contains Asn at the position corresponding to the Leu-209 of P450(15) alpha, although both P450s hydroxylate testosterone . The CO-reduced P450(7) alpha complex is unstable, so that it is quickly converted into the inactive P420, whereas the P450(15) alpha is very stable . The P450(7) alpha, however, is stabilized either by addition of testosterone or by a mutation of Asn-209 to Leu . The mutant P450(7) alpha displays a 17-fold lower Vmax . value than the wild-type enzyme . Unexpectedly, it also has 3-fold lower Km and Kd values . Residue 209 in P450(7) alpha, therefore, appears to be located at a critical site of the haem-substrate-binding pocket . Corticosterone inhibits the testosterone 7 alpha-hydroxylase activity of the wild-type P450(7) alpha, whereas it does not inhibit the mutant P450(7) alpha . Conversely, the P450(15) alpha activity becomes inhibited by corticosterone upon the replacement of Leu-209 by Asn . In addition, this mutation increases the corticosterone 15 alpha-hydroxylase activity of P450(15) alpha at least 20-fold . Whereas the inhibition by corticosterone depends on the presence of Asn at position 209, deoxycorticosterone inhibits the activities of the P450s regardless of the type of residue at 209 . The results indicate, therefore, that the identity of residue 209 determines the affinity as well as specificity of steroid binding to both P450(7) alpha and P450(15) alpha. Eur J Biochem, 1993 Apr 15, 213(2), 849 - 57 Two different genes from Schwanniomyces occidentalis determine ribosomal resistance to cycloheximide; Del Pozo L et al.; Two genes (SCR1 and SCR2) encoding natural cycloheximide resistance in the budding yeast Schwanniomyces occidentalis have been cloned by expression in Saccharomyces cerevisiae . Both genes determine resistance to the inhibitory action of cycloheximide on the ribosome, SCR1 and SCR2 are present as single copies in Schwanniomyces occidentalis, where they map on chromosomes II and V, respectively . The nucleotide sequence of SCR2 contains an open reading frame of 321 nucleotides which is interrupted by an intron of 452 nucleotides . It encodes a polypeptide of 106 amino acids of molecular mass 12.25 kDa and pI 11.19 . The deduced amino acid sequence shows a high degree of similarity to the L41 protein of the 60S ribosomal subunit from several eukaryotic organisms . The intron and the 5' non-coding region of SCR2 possess conserved elements which are typical of yeast ribosomal protein genes . A single amino acid change determines the resistance or sensitive phenotype to cycloheximide of the 80S ribosome since replacement of Gln56 in L41 from Schwanniomyces with Pro, by site-directed mutagenesis, confers cycloheximide sensitivity . SCR2 may serve as a practical yeast cloning marker if integrated in a multicopy plasmid. Proc Natl Acad Sci U S A, 1993 Apr 15, 90(8), 3157 - 61 Identification and characterization of a nuclease activity specific for G4 tetrastranded DNA; Liu Z et al.; We have identified a nuclease activity that is specific for G4 tetrastranded DNA . This activity, found in a partially purified fraction for a yeast telomere-binding protein, binds to DNA molecules with G4 tetrastranded structure, regardless of their nucleotide sequences, and cleaves the DNA in a neighboring single-stranded region 5' to the G4 structure . Competition with various G4-DNA molecules inhibits the cleavage reaction, suggesting that this nuclease activity is specific for G4 tetrastranded DNA . The existence of this enzymatic activity that reacts with G4 DNAs but not with single-stranded or Watson-Crick duplex DNAs suggests that tetrastranded DNA may have a distinct biological function in vivo. Biochem J, 1993 Apr 15, 291 ( Pt 2), 649 - 56 Phosphorylation and redistribution of the phosphatidylinositol-transfer protein in phorbol 12-myristate 13-acetate- and bombesin-stimulated Swiss mouse 3T3 fibroblasts; Snoek GT et al.; By immunofluorescence microscopy it was shown that the phosphatidylinositol-transfer protein (PI-TP) becomes associated with the Golgi membranes when confluent (quiescent) Swiss mouse 3T3 fibroblast cells are stimulated with phorbol 12-myristate 13-acetate (PMA) and bombesin . Dibutyryl cyclic AMP or dexamethasone had no effect on the intracellular redistribution of PI-TP . In exponentially growing cells and in serum-starved (semi-quiescent) cells, PI-TP is already associated with Golgi structures . Stimulation of semi-quiescent cells by PMA resulted in a rapid redistribution of PI-TP . A similar yet slower response was observed after stimulation with bombesin . Stimulation of semi-quiescent 3T3 cells by PMA significantly increased the phosphorylation of PI-TP, as shown by immunoprecipitation of PI-TP from pre-labelled cells . No significant increase in phosphorylation of PI-TP was observed after stimulation of these cells by bombesin . Purified PI-TP was shown to be a substrate for protein kinase C in vitro . The possibility that the phosphorylation of PI-TP after activation of protein kinase C is involved in the observed redistribution of PI-TP is discussed. Biochem Biophys Res Commun, 1993 Apr 15, 192(1), 273 - 80 Removals of hydrogen peroxide and hydroxyl radical by thiol-specific antioxidant protein as a possible role in vivo; Lim YS et al.; Thiol-specific antioxidant protein (Protector Protein; PRP) from Saccharomyces cerevisiae was found to remove hydrogen peroxide and hydroxyl radical in the presence of dithiothreitol (DTT) . Without DTT as a reducing equivalent, the antioxidant protein did not show the activities for destroying hydrogen peroxide and hydroxyl radical . N-ethylmaleimide (NEM) was observed to prevent the PRP from both removing hydrogen peroxide and protecting the cleavage of DNA . These observations suggest that the sulfhydryl of cysteine in PRP could function as a strong nucleophile to attack and destroy H2O2 and .OH. Nature, 1993 Apr 15, 362(6421), 630 - 2 Homeotic genes autonomously specify one aspect of pattern in the Drosophila mesoderm; Greig S et al.; Transplantation and ablation experiments have led to the generalization that in insects the mesoderm is naive, and that pattern is imposed upon it by the ectoderm . This has been demonstrated directly by mosaic analysis for the case of one muscle in Drosophila . The unique character of this muscle depends on the activity of sex-determining and homeotic genes, not in the muscle itself, but in the nerve that innervates it . Indirect evidence suggests, however, that homeotic genes specify some aspects of mesoderm patterning autonomously . Homeotic genes are expressed in the mesoderm, and are regulated in a segment-specific pattern analogous to, but different from, that seen in the ectoderm . Moreover, the effects of homeotic mutations on the muscles do not always mirror transformations seen in the epidermis . Here we examine this problem directly, by expressing homeotic genes ectopically in the mesoderm without altering their expression in the overlying ectoderm . We find that the pattern of adult muscle precursor cells characteristic of the thorax can be converted to that seen in the abdomen by expressing the homeotic gene abdominal-A specifically in the mesoderm. Nature, 1993 Apr 8, 362(6420), 563 - 5 Specificity domains distinguish the Ras-related GTPases Ypt1 and Sec4; Dunn B et al.; The essential Ras-related GTPases Ypt1 and Sec4 act at distinct stages of the secretion pathway in the yeast Saccharomyces cerevisiae: Ypt1 is required for vesicular transport from the endoplasmic reticulum to the Golgi apparatus, whereas Sec4 is required for fusion of secretory vesicles to the plasma membrane . Here we use chimaeras of the two proteins to identify a 9-residue segment of Ypt1 that, when substituted for the analogous segment of Sec4, allows the chimaera to perform the minimal functions of both proteins in vivo . This segment corresponds to loop L7 of the p21ras crystal structure . Substitution of a 24-residue Ypt1 segment, including the residues just mentioned, together with 12 residues of Ypt1 corresponding to the 'effector region' of p21ras (loop L2; refs 7,8), transforms Sec4 into a fully functional Ypt1 protein without residual Sec4 function. Nature, 1993 Apr 8, 362(6420), 560 - 3 Interactions of three domains distinguishing the Ras-related GTP-binding proteins Ypt1 and Sec4; Brennwald P et al.; The genes SEC4 and YPT1 encode Ras-related GTP-binding proteins in the yeast Saccharomyces cerevisiae . Ypt1 is necessary for vesicular transport from the endoplasmic reticulum to the Golgi, whereas Sec4 is required for fusion of post-Golgi secretory vesicles to the plasma membrane . Recently, three structural domains have been proposed to specify the stage in cellular transport at which members of the Sec4/Ypt1/Rab family act: the effector domain, the C-terminal hypervariable region, and a region corresponding to loop 7 in the structure of p21ras (ref . 8) . Here we use Sec4/Ypt1 chimaeras to show that these three regions cooperate to specify Ypt1 function and that the C-terminal hypervariable region is needed for Ypt1 localization to the Golgi . Unexpectedly, we found that a single chimaera can function as either Ypt1 or Sec4 without missorting carboxypeptidase Y or invertase. J Biol Chem, 1993 Apr 5, 268(10), 7594 - 601 Binding of Ku protein to DNA . Measurement of affinity for ends and demonstration of binding to nicks; Blier PR et al.; Ku, also known as nuclear Factor IV, is an abundant nuclear DNA-binding protein which requires free DNA ends for the initial interaction with double-stranded DNA (dsDNA) and can bind at multiple sites along dsDNA in an energy-independent manner . Its function in vivo is unknown, but it has been implicated in both DNA replication and repair and in transcriptional control . We have used an electrophoretic mobility shift assay to further define the DNA binding properties of the Ku protein . Titration of Ku to a fixed amount of any of several target linear dsDNA fragments produced ladders of shifted bands proportional to the length of DNA, confirming the multiple binding activity of Ku and demonstrating its sequence-independent nature . Using a short DNA fragment with one Ku binding site, the binding constant of Ku for dsDNA ends was calculated to be 2.4 x 10(9) M-1 . Competitive inhibition experiments confirmed the requirement of a free DNA end for binding by Ku and demonstrated that Ku binds isolated nicks in dsDNA . Nick binding was also observed directly using radiolabeled singly nicked circular DNA . The relative affinities of Ku for specific nick sites and free DNA ends were approximately equal, and nick binding was sequence-independent . Finally, in a study of a possible role for Ku in protecting or repairing damaged DNA, Ku was shown to inhibit the ability of T4 DNA ligase to circularize linear dsDNA molecules, demonstrating that some Ku molecules remain at the DNA terminus rather than translocate . A similar inhibition was not observed at nicks . These experiments document a new DNA binding specificity for Ku and further suggest that the high affinity end and nick binding activity is biologically relevant to its functions in vivo. J Biol Chem, 1993 Apr 5, 268(10), 7064 - 8 Effect of myristoylation on GTP-dependent binding of ADP-ribosylation factor to Golgi; Haun RS et al.; ADP-ribosylation factors (ARFs), a family of approximately 20-kDa guanine nucleotide-binding proteins that activate cholera toxin ADP-ribosyltransferase in vitro, have been implicated in intracellular protein trafficking and are thought to cycle between cytosolic and membrane compartments . Although isolated predominantly as soluble proteins, ARFs associate with membranes and phospholipids in a GTP-dependent manner . In contrast to other small GTP-binding proteins, ARFs are NH2 terminally myristoylated . Using a bacterial expression system, recombinant myristoylated and non-myristoylated human ARF5 were produced to investigate the role of myristoylation in its association with Golgi . The recombinant ARFs (myristoylated and non-myristoylated) exhibited similar biochemical activity as measured by GTP binding and in vitro activation of cholera toxin . Myristoylated ARF5, however, demonstrated a temperature- and GTP-dependent association with Golgi membranes, whereas non-myristoylated ARF did not bind to Golgi under any of the experimental conditions . These data indicate that myristoylation is necessary, although not sufficient, for membrane attachment, but is not necessary for activation of cholera toxin. Genome, 1993 Apr, 36(2), 224 - 9 Direct end labelling of telomeres; Kolchinsky A et al.; A novel approach of direct end labelling of telomeres is presented . Chromosome-sized, agarose-embedded DNA was treated with T4 DNA polymerase to remove protruding 3' end of telomeres and to generate single-stranded 5' ends . The DNA was then labelled by the same enzyme in the presence of {alpha-32P}dGTP and cold dATP and dTTP . Labelled yeast chromosomes separated by pulsed field gel electrophoresis maintained their integrity . Digestion of yeast chromosomes separated in pulsed field gels with a restriction nuclease (HinfI), followed by conventional electrophoresis in the second dimension, resulted in a fingerprint-like pattern of labelled telomeres . This was very similar to the hybridization pattern of a similar two-dimensional gel probed with cloned yeast telomeric sequence . The same approach enabled us to label telomeres in soybean, determine their size, and to reveal polymorphisms in the length of telomeres between the closely related subspecies Glycine max (soybean) and Glycine soja. Curr Opin Genet Dev, 1993 Apr, 3(2), 278 - 85 Interactions of coiled coils in transcription factors: where is the specificity? Baxevanis AD, Vinson CR. Amphipathic alpha-helices create the dimerization interface in the bZIP and bHLH classes of DNA-binding proteins . These amphipathic helices have been shown to enter into a wide variety of specific dimerization interactions, and this large array of possible combinatorial interactions may provide for fine control of biological function . In bHLH-ZIP proteins, the addition of a leucine-zipper region immediately carboxyl-terminal to the helix-loop-helix region provides for an additional level of both dimerization specificity and control, again through the interaction of amphipathic alpha-helices . Interhelical electrostatic interactions have been implicated in regulating dimerization specificity. Trends Biochem Sci, 1993 Apr, 18(4), 131 - 5 The nucleolar snRNAs: catching up with the spliceosomal snRNAs; Fournier MJ et al.; Despite their early discovery, research into the small RNAs associated with the eukaryotic nucleolus (snoRNAs) has lagged behind that of their cousins, the small nuclear RNAs which are known to function in mRNA splicing (spliceosomal snRNAs) . Recent progress has now shown that the snoRNAs also occupy a vital niche in the RNA world, participating in the processing of ribosomal RNA . Like the spliceosomal snRNAs, the snoRNAs exist as ribonucleoprotein (RNP) particles which appear to assemble into a large multi-RNA RNP complex for pre-rRNA maturation. Mol Gen Genet, 1993 Apr, 238(1-2), 43 - 8 Electrophoretic karyotypes of the elm tree pathogen Ophiostoma ulmi (sensu lato); Dewar K et al.; Pulsed field gel electrophoresis using OFAGE, TAFE, and CHEF systems has been used to more fully characterize karyotypic variation within the two closely related fungal species of Ophiostoma ulmi sensu lato . Twelve wild-type and laboratory strains, representing the less aggressive species O . ulmi and both of the biotypes of the more aggressive species O . novo-ulmi were studied and their karyotypes determined . Depending on the strain, a minimum of four to a minimum of eight chromosomal DNA bands were present that fall into three distinct size classes, with one exception . Strain CESS16K (O . novo-ulmi, North American aggressive subgroup) contains a unique chromosomal DNA band which comigrated near a Saccharomyces cerevisiae chromosome of 0.95 Mb . This unique band was the smallest O . ulmi s . l . chromosomal DNA observed . Seven of the twelve strains shared a common chromosomal DNA banding pattern, whereas each of the other five had a unique karyotype . There was no correlation between chromosome profile and species, as some O . novo-ulmi and O . ulmi strains shared common electrophoretic karyotypes. Mol Gen Genet, 1993 Apr, 238(1-2), 185 - 92 At least four regulatory genes control sulphur metabolite repression in Aspergillus nidulans; Natorff R et al.; Mutations in four genes: sconA (formerly suA25meth, mapA25), sconB (formerly mapB1), sconC and sconD, the last two identified in this work, relieve a group of sulphur amino acid biosynthetic enzymes from methionine-mediated sulphur metabolite repression . Exogenous methionine has no effect on sulphate assimilation in the mutant strains, whereas in the wild type it causes almost complete elimination of sulphate incorporation . In both mutant and wild-type strains methionine is efficiently taken up and metabolized to S-adenosylmethionine, homocysteine and other compounds, scon mutants also show elevated levels of folate-metabolizing enzymes which results from the large pool of homocysteine found in these strains . The folate enzymes appear to be inducible by homocysteine and repressible by methionine (or S-adenosylmethionine). Hepatology, 1993 Apr, 17(4), 628 - 37 Decreased mitochondrial oxidation of fatty acids in pregnant mice: possible relevance to development of acute fatty liver of pregnancy; Grimbert S et al.; Severe impairment of the beta-oxidation of fatty acids, as a consequence of a single factor or a combination of different causes, leads to microvesicular steatosis of the liver . In an effort to understand the mechanism(s) leading to the development of acute fatty liver of pregnancy in some women, we determined the effects of pregnancy on the mitochondrial oxidation of fatty acids in mice . In vivo, the rate of oxidation of the whole fatty-acid chain length was determined by measuring the rate of exhalation of {14C}CO2 after intragastric administration of a tracer dose of {U-14C}palmitic acid . {14C}CO2 exhalation was not significantly decreased at 14 days of gestation, but it had declined by 40% at 18 days of gestation (i.e., 24 to 48 hr before delivery) . The rate of first beta-oxidation cycle was assessed by measuring the rate of {14C}CO2 exhalation after administration of {1-14C}octanoic acid, {1-14C}butyric acid or {1-14C}palmitic acid . {14C}CO2 exhalation had declined by 60%, 46%, and 24% after administration of {1-14C}octanoic acid, {1-14C}butyric acid and {1-14C}palmitic acid, respectively, in 18-day-pregnant mice . Total hepatic lipids and triglycerides, expressed per gram of liver, remained unchanged in 18-day-pregnant mice . In vitro, the rate of mitochondrial beta-oxidation (expressed per milligram of protein) had decreased by 47% at 18 days' gestation with {U-14C}palmitic acid as substrate and by 33% with {1-14C}octanoic acid but remained unchanged with {1-14C}palmitic acid . The activity of the tricarboxylic acid cycle, assessed by the formation of {14C}CO2 from {1-14C}acetic acid, had decreased by 24% . We conclude that the mitochondrial oxidation of fatty acids decreased during late-term pregnancy in mice as a consequence of both decreased mitochondrial beta-oxidation of medium-chain fatty acids, and decreased activity of the tricarboxylic acid cycle . We suggest that this effect, in combination with other factors, may contribute to the development of fatty liver of pregnancy in some pregnant women. EMBO J, 1993 Apr, 12(4), 1475 - 85 Conserved features in the mode of replication of eukaryotic ribosomal RNA genes; Hernandez P et al.; It was previously shown that a 1.5 kb fragment located in the non-transcribed spacer (NTS) is the earliest replicating region of pea (Pisum sativum) rDNA in synchronized root cells . In the present report the structure of this region was characterized . It contains a cluster of four 11 bp near matches to the Saccharomyces cerevisiae ARS consensus sequence (ACS) . These near matches are embedded in an A+T rich domain located upstream from the transcription initiation site . We identified and mapped an intrinsic DNA bending locus 5' to the cluster of near matches . Several eukaryotic origins including the ARS from the budding yeast show very similar structural features . This observation strengthens the notion that pea rDNA replication initiates at or near this region . Replication of the entire pea rDNA repeat was analysed by two-dimensional (2D) agarose gel electrophoresis . The results obtained indicate that only a small fraction of the potential origins is used in each replication round . Forks moving in the direction opposite to rRNA transcription are stalled at a polar replication fork barrier (RFB), which mapped near the 3' end of the transcription unit . Consequently, most of pea rDNA appears to replicate in a unidirectional manner . These results show that the strategy used to replicate pea and yeast rRNA genes is very similar, suggesting that it has been conserved and might be common to most eukaryotes. EMBO J, 1993 Apr, 12(4), 1375 - 85 GAL4 is regulated by a glucose-responsive functional domain; Stone G et al.; The Saccharomyces cerevisiae transcriptional activator GAL4 is regulated by the presence of available carbon sources . Galactose induces activity by inhibiting the negative regulator GAL80, while glucose, the preferred carbon source, antagonizes GAL4 function by several mechanisms . In the present study we present evidence that one mechanisms for inhibition of GAL transcription by glucose involves direct inhibition of the GAL4 protein . We demonstrate that a large, previously uncharacterized, central region of GAL4 contains at least three 'inhibitory domains' and a 'glucose response domain' (GRD) . Deletion of the entire central region eliminates direct inhibition of GAL4 by glucose, and furthermore, fusion of the central region to a heterologous transcriptional activator confers inhibition by glucose . The central region inhibitory domains constitutively inhibit transcriptional activation when the GRD is absent . Direct inhibition of GAL4 activity can be detected within 30 min following glucose addition and may represent an early mechanism promoting a switch from galactose to glucose utilization . A model for the regulatory role of the central region is presented, involving interaction with an additional protein that inhibits GAL4 activity when glucose is present. DNA Cell Biol, 1993 Apr, 12(3), 265 - 73 Characterization of the DNA-binding properties of the early growth response-1 (Egr-1) transcription factor: evidence for modulation by a redox mechanism; Huang RP et al.; The binding of the transcription factor early growth response-1 (Egr-1) to its specific DNA-binding sequence GCGGGGGCG occurs through the interaction of three zinc finger motifs . DNA binding by Egr-1 can be modified by alteration of reduction-oxidation (redox) state . Using gel retardation assays, we show that binding of Egr-1 protein is specific and is dependent on the presence of reducing agents in a dose-dependent manner . The zinc finger region is the domain subject to conformation changes by redox . Oxidized or metal-free Egr-1 does not bind . Nuclear extracts of several cell types contain a heat-sensitive factor(s) that induces the ability of Egr-1 protein to bind to DNA in otherwise suboptimal conditions containing insufficient reducing agent . This inducing activity may be replaced by Ref-1, a protein identified and characterized by Curran and co-workers (Xanthoudakis and Curran, 1992) . The possibility arises that the transcription-regulating activity of Egr-1 may be regulated by the redox state in the cell via factors such as Ref-1 that modulate its DNA-binding activity. Proc Natl Acad Sci U S A, 1993 Apr 1, 90(7), 2685 - 9 p70 lupus autoantigen binds the enhancer of the T-cell receptor beta-chain gene; Messier H et al.; The p70 (Ku) autoantigen has been described as a nonhistone nuclear protein recognized by antibodies from lupus patients . In our studies on the regulation of T-cell receptor (TCR) beta-chain gene expression we have identified the p70 lupus autoantigen as a DNA-binding protein that binds the enhancer of the TCR beta-chain gene . This enhancer is essential for expression of the TCR beta gene . The core TCR beta enhancer contains the E3 motif, which we show here is essential for enhancer activity . The protection of the E3 motif in T cells and the marked reduction in enhancer activity when the E3 motif is mutated underline its physiological importance in regulating beta enhancer activity . The p70 lupus autoantigen gene was identified by screening T-cell lambda gt11 libraries with an E3 probe . The gene encodes a protein which binds the E3 motif in a sequence-specific manner . The identification of a 70-kDa protein as a major E3-binding protein by UV crosslinking is consistent with the conclusion that the p70 lupus autoantigen binds the beta enhancer . Finally, we have shown that T-cell nuclear proteins which bind the E3 motif bear p70 (Ku) lupus autoantigenic determinants . Together these data suggest that the p70 autoantigen binds a critical motif in the beta enhancer and probably regulates TCR beta gene expression. Nature, 1993 Apr 1, 362(6419), 475 - 7 TFIIIC relieves repression of U6 snRNA transcription by chromatin; Burnol AF et al.; The U6 small nuclear (sn)RNA gene (SNR6) from the yeast Saccharomyces cerevisiae is transcribed by RNA polymerase III in vivo . This gene is unusual in having a TATA box at position -30, and an essential B-block element located downstream of the T-rich termination signal . The B block is one of the two intragenic promoter elements of transfer RNA genes that are recognized by transcription factor (TF)IIIC (ref . 4) . But accurate in vitro transcription of yeast U6 snRNA gene by PolIII in a purified system requires only TFIIIB components, including the TATA-box binding protein TBP . Here we report that, after nucleosome reconstitution or chromatin assembly, U6 snRNA synthesis becomes dependent on TFIIIC and on the integrity of the B-block element . This observation resolves an apparent paradox between in vitro and in vivo results concerning the necessity of the downstream B-block element and sheds light on a new role of TFIIIC in gene activation. Photochem Photobiol, 1993 Apr, 57(4), 702 - 6 Photosensory transduction in ciliates . II . Possible role of G-protein and cGMP in Stentor coeruleus; Fabczak H et al.; The heterotrichous ciliate, Stentor coeruleus, exhibits a well-defined photophobic response to a sudden increase in the intensity of visible light . The phobic reactions usually appear with a latency period (i.e . a time delay between the onset of the stimulus and the stop response) . This latency of phobic response was significantly increased when the cells were incubated with 8-bromo-guanosine 3',5'-cyclic monophosphate . In the presence of this nucleotide, a reduction of cell responsiveness (i.e . the number of photophobically responding cells) was also observed . Similar effects were observed when cells were treated with pertussis toxin, a G-protein activity modulator, and 3'-isobutyl-methylxanthine, an inhibitor of guanosine 3',5'-cyclic monophosphate (cGMP) phosphodiesterase . The G-protein activator fluoroaluminate and 6-anilino-5,8-quinolinedione (LY 83583) (an effective agent for lowering cellular cGMP levels) showed opposite effects on the cell photophobic response . These results indirectly suggest that the level of cytoplasmic cGMP, possibly modulated by a G-protein-coupled cGMP phosphodiesterase, plays a phototransducing role in Stentor . In addition, using an antiserum raised against bovine transducin, a cross-reacting protein with an apparent molecular mass of 39 kDa was detected on immunoblots . The alpha-subunit of a Stentor G-protein has also been partially cloned and sequenced . However, the possible coupling between the G-protein and the putative phosphodiesterase remains to be established. Curr Opin Genet Dev, 1993 Apr, 3(2), 219 - 25 Histones, nucleosomes and transcription; Svaren J et al.; It is becoming increasingly clear that nucleosome structure is integrally involved in gene regulation . In particular, the study of inducible genes has shown that nucleosomes not only contribute to a repressed basal state, but can also be rearranged in response to induction . The mechanism of this process is just beginning to be elucidated, and genetic studies have implicated several proteins in the modulation of nucleosome structure. Mol Endocrinol, 1993 Apr, 7(4), 616 - 27 The retinoic acid receptor-beta 2 contains two separate cell-specific transactivation domains, at the N-terminus and in the ligand-binding domain; Folkers GE et al.; In contrast to other members of the steroid/thyroid hormone superfamily, not much is known about the regions involved in transactivation of the receptors for retinoic acid . To determine the transactivation function of RARs, fusion proteins between the DNA-binding domain of the yeast transcription factor GAL4 and retinoic acid receptor-alpha (RAR alpha) or RAR beta were made . Transfection of these constructs resulted in RA-induced activation of a GAL4-responsive element-containing promoter . Deletion analysis revealed that RAR beta-2 has two transcription activation functions (TAFs) . TAF-1 activates transcription constitutively and was mapped to the first 32 amino acids of the A-region . TAF-2 is located in the ligand-binding domain between amino acids 137 and 410 and activated transcription only in the presence of RA . The presence of two TAFs was confirmed by cotransfection of RAR beta deletion constructs with the human RAR beta-2 promoter as reporter, showing that the absence of RAR beta TAF-1 causes a decrease in transactivation, whereas truncation of TAF-2 completely blocks this function . Internal deletions in the ligand-binding domain in both GAL-RAR beta and RAR beta expression constructs resulted in a nonfunctional receptor, indicating that the complete ligand-binding domain is required for its transactivation function . Furthermore, we have shown that the contribution of the two TAFs in transcription activation varies among different cell lines, suggesting that they act in a cell-specific manner. Plant Mol Biol, 1993 Apr, 22(1), 83 - 90 Molecular cloning and expression of a MAP kinase homologue from pea; Stafstrom JP et al.; The cdc2 kinases are important cell cycle regulators in all eukaryotes . MAP kinases, a closely related family of protein kinases, are involved in cell cycle regulation in yeasts and vertebrates, but previously have not been documented in plants . We used PCR to amplify Brassica napus DNA sequences using primers corresponding to amino sequences that are common to all known protein kinases . One sequence was highly similar to KSS1, a MAP kinase from Saccharomyces cerevisiae . This sequence was used to isolate a full-length MAP kinase-like clone from a pea cDNA library . The pea clone, called D5, shared approximately 50% amino acid identity with MAP kinases from yeasts and vertebrates and about 41% identity with plant cdc2 kinases . An expression protein encoded by D5 was recognized by an antiserum specific to human MAP kinases (ERKs) . Messenger RNA corresponding to D5 was present at similar levels in all tissues examined, without regard to whether cell division or elongation were occurring in those tissues. Trends Biochem Sci, 1993 Apr, 18(4), 128 - 31 The MAP kinase cascade is essential for diverse signal transduction pathways; Nishida E et al.; Mitogen-activated protein (MAP) kinases are activated by combined tyrosine and threonine phosphorylation catalysed by MAP kinase kinase, a novel class of protein kinases with dual specificity for both tyrosine and serine/threonine . MAP kinase kinase is turned on by serine/threonine phosphorylation catalysed by an immediate upstream kinase . The MAP kinase cascade appears to be conserved during evolution and thus might play an essential role in diverse intracellular signaling processes from yeasts to vertebrates. J Pharmacol Exp Ther, 1993 Apr, 265(1), 366 - 72 Metabolic activation of the nitroaromatic antiandrogen flutamide by rat and human cytochromes P-450, including forms belonging to the 3A and 1A subfamilies; Berson A et al.; The in vitro metabolic activation of flutamide, a nitroaromatic antiandrogen which produces hepatitis in a few recipients, was first studied with male rat liver microsomes . There was no electron spin resonance evidence for the reduction of flutamide by reduced nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P-450 reductase into a nitro anion free radical . In contrast, flutamide was oxidatively transformed by cytochrome P-450 into reactive metabolite(s) that covalently bound to microsomal proteins . Covalent binding required oxygen and NADPH, and was decreased by the nucleophile glutathione and by the cytochrome P-450 inhibitors SKF 525-A, piperonyl butoxide and troleandomycin (an inhibitor of the cytochrome P-450 3A subfamily) . Covalent binding was increased markedly by pretreatment with dexamethasone (an inducer of the cytochrome P-450 3A subfamily) and moderately by pretreatment with beta-naphthoflavone (an inducer of the 1A family) . Covalent binding was immunoinhibited markedly by anticytochrome P-450 3A immunoglobulin G and moderately by anticytochrome P-450 1A immunoglobulin G . Covalent binding was much lower with liver microsomes from female rats (not expressing P-450 3A2) . Covalent binding of flutamide also occurred with human liver microsomes (where it was inhibited by troleandomycin), and with yeast microsomes expressing human liver cytochromes P-450 1A1, 1A2 or 3A4 . We concluded that flutamide was oxidatively transformed into chemically reactive metabolite(s) by rat and human cytochromes P-450, including forms belonging to the 3A and 1A subfamilies. Proc Natl Acad Sci U S A, 1993 Apr 1, 90(7), 2979 - 83 The sluggish-A gene of Drosophila melanogaster is expressed in the nervous system and encodes proline oxidase, a mitochondrial enzyme involved in glutamate biosynthesis; Hayward DC et al.; Certain gene mutations in Drosophila melanogaster cause sluggish motor activity . We have localized the transcription unit of the sluggish-A gene to a 14.7-kb region at the base of the X chromosome and have cloned corresponding cDNAs . The predicted protein product has significant sequence similarity to Saccharomyces cerevisiae proline oxidase (EC 1.5.99.8), a mitochondrial enzyme which catalyzes the first step in the conversion of proline to glutamate . In the mutant fly, mitochondrial proline oxidase activity is reduced and has kinetic properties different from those of the wild type, providing further evidence that the gene encodes proline oxidase . Indeed, the free proline level in mutant flies is elevated . When the mutant is rescued by transformation, the proline oxidase and free proline levels, as well as the motor and phototactic behavior, are restored to normal . During embryonic development the sluggish-A transcript is predominantly expressed in the nervous system . Significantly, it has previously been reported that a mouse mutant, PRO/Re, which has reduced proline oxidase activity and elevated free proline levels, also exhibits sluggish behavior. Mol Cell Biol, 1993 Apr, 13(4), 2366 - 76 Nuclear hormone receptors involved in neoplasia: erb A exhibits a novel DNA sequence specificity determined by amino acids outside of the zinc-finger domain; Chen H et al.; The erb A oncogene is a dominant negative allele of a thyroid hormone receptor gene and acts in the cancer cell by encoding a transcriptional repressor . We demonstrate here that the DNA sequence recognition properties of the oncogenic form of the erb A protein are significantly altered from those of the normal thyroid hormone receptors and more closely resemble those of the retinoic acid receptors; this alteration appears to play an important role in defining the targets of erb A action in neoplasia . Unexpectedly, the novel DNA recognition properties of erb A are encoded by an N-terminal region not previously implicated as playing this function in current models of receptor-DNA interaction . Two N-terminal erb A amino acids in particular, histidine 12 and cysteine 32, contribute to this phenomenon, acting in conjunction with amino acids in the zinc finger domain . The effects of the N-terminal domain can be observed at the level of both DNA binding and transcriptional modulation . Our results indicate that unanticipated determinants within the nuclear hormone receptors participate in DNA sequence recognition and may contribute to the differential target gene specificity displayed by different receptor forms. Eur J Biochem, 1993 Apr 1, 213(1), 21 - 30 Evidence for a common structure for a class of membrane channels; Holzenburg A et al.; Electron microscopic analysis of gap-junction-like structures isolated from an anthropod (Nephrops norvegicus) and composed of a 16-kDa polypeptide, show the functional unit to be a star-shaped hexamer of protein arranged around a central channel which runs perpendicular to the plane of the membrane . Estimations of the molecular volume carried out on an averaged projection are consistent with a subunit mass of 16-18 kDa . Fourier transform infrared spectroscopy indicates a high alpha-helical content for the protein, supporting secondary-structure predictions of four transmembrane alpha helices/monomer . The averaged projection shows a close resemblance to a hexamer of the 16-kDa protein built on the basis of a four alpha-helical bundle {Finbow, M . E., Eliopoulos, E . E., Jackson, P . J., Keen, J . N., Meagher, L., Thompson, P., Jones, P . C . & Findlay, J . B . C . (1992) Protein Eng . 5, 7-15} . The reconstructed image is also similar to that obtained for gap-junction-like channels isolated from a related arthropod {Homarus americanus; Sikerwar, S . S., Downing, K . H . & Glaeser, R . M . (1991) J . Struct . Biol . 106, 255-263} whose protein content was unknown but which we demonstrate may be composed of a related 16-kDa protein . Previous studies have shown a high sequence identity of the Nephrops 16-kDa protein with the 16-kDa proteolipid subunit c of the vascular H(+)-ATPase, both of which in turn bear similarity to the 8-kDa proteolipid subunit of the F1F0-ATP synthase . Expression of cDNA coding for the Nephrops 16-kDa protein in Saccharomyces cerevisiae, in which the endogenous gene coding for the V-ATPase proteolipid has been inactivated, restores V-ATPase activity and cell growth. Gene, 1993 Mar 30, 125(2), 217 - 22 Characterization of the 130-bp repeat enhancer element of the rat ribosomal gene: functional interaction with transcription factor E1BF; Ghosh AK et al.; The 130-bp repetitive element (RE) of the rat rDNA (ribosomal RNA-encoding gene) intergenic spacer stimulated the synthesis of rRNA four- to sixfold, in comparison with that of the promoter alone, both in vivo and in vitro, when ligated to the rat rDNA promoter . The addition of increasing amounts of highly purified E1BF (enhancer-1 binding factor), which binds to the rat rDNA promoter and an upstream nonrepetitive enhancer element {Zhang and Jacob, Mol . Cell . Biol . 10 (1990) 5177-5186}, to an in vitro transcription system resulted in enhancement of rDNA transcription from the recombinant plasmids containing the promoter or promoter-RE . However, E1BF-mediated stimulation of transcription under the influence of the RE continued at higher concentrations of E1BF than did the control transcription from the promoter alone . The binding affinity of E1BF for the RE was comparable to its affinity for the nonrepetitive far upstream enhancer element previously characterized in our laboratory . The sequences protected by E1BF in the RE differed from those protected by UBF (upstream control element-binding factor), a well characterized pol I transcription factor . These data suggest that E1BF belongs to a class of transcription factors which interact with the promoter and spacer cis-acting RE to modulate rDNA transcription. Biochemistry, 1993 Mar 30, 32(12), 3178 - 87 Disulfide bond contribution to protein stability: positional effects of substitution in the hydrophobic core of the two-stranded alpha-helical coiled-coil; Zhou NE et al.; To investigate the positional effect of the disulfide bond on the structure and stability of a two-stranded alpha-helical coiled-coil, an interchain disulfide bond was systematically introduced into the hydrophobic core of a de novo designed model coiled-coil at the N-terminus (position 2), C-terminus (position 33), and nonterminal positions a (positions 9, 16, 23, and 30) and d (positions 5, 12, 19, and 26) . The rate of formation of a disulfide bond is faster at position d compared to at the corresponding position a under nondenaturing conditions, suggesting that position d is more suitable for engineering a disulfide bond . The structure and stability of the reduced and oxidized coiled-coils were determined by circular dichroism studies in the absence and presence of guanidine hydrochloride . Our results demonstrate that the improvement of protein stability by introduction of a disulfide bond is very relevant to its location and the most effective disulfide bonds are those that can be introduced in the hydrophobic core without any disruption of the protein structure . The disulfide bond at position d with near-optimal geometry does not perturb the coiled-coil structure and makes the largest contribution to coiled-coil stability . In contrast, the inappropriate geometry of the disulfide bond at nonterminal position a introduces a high strain energy on the disulfide bond which disrupts the coiled-coil structure . At positions a, the closer the disulfide bridge is to the center of the coiled-coil, the larger the disruption on the coiled-coil structure and the smaller the contribution the disulfide bond makes to coiled-coil stability . The computer modeling results also suggest that an insertion of an interchain disulfide bond at position a in the GCN4 leucine zipper X-ray structure has a higher potential energy than insertion at position d . The energy-minimized coiled-coil structure with an interchain disulfide bond at position a has a larger root mean square difference from the X-ray structure of GCN4 than the coiled-coil with a disulfide bond at position d . Because interhelical interactions are common in globular proteins as well as coiled-coils, the results obtained in this study will have general utility for selecting the sites for engineering disulfide bonds between alpha-helices. Gene, 1993 Mar 30, 125(2), 211 - 6 Cloning of the mouse cDNA encoding DNA topoisomerase I and chromosomal location of the gene; Koiwai O et al.; The mouse cDNA encoding DNA topoisomerase I (TopoI) was cloned and the nucleotide sequence of 3512 bp was determined . The cDNA clone contained an open reading frame encoding a protein of 767 amino acids (aa), which is 2 aa longer than its human counterpart . Overall aa sequence homology between the mouse and human, and between the mouse and yeast (Saccharomyces cerevisiae) sequences was 96% and 42%, respectively . The mouse TopI gene was mapped at position 54.5 on chromosome 2 from linkage analyses of a three-point cross test with Geg, Ada, and a as marker genes. Gene, 1993 Mar 30, 125(2), 111 - 4 Circularly permuted DNA, RNA and proteins--a review; Pan T et al.; Circular permutation represents a form of macromolecular isomerization when the normal termini are covalently linked and new termini introduced by breaking the backbone elsewhere . Here, we describe implications of circular permutation on the folding and function of biologically relevant macromolecules . A method permitting the analysis of the folding of all circularly permuted isomers of RNA is presented that has been successfully applied for a tRNA and the binding site of the coliphage R17 coat protein. Philos Trans R Soc Lond B Biol Sci, 1993 Mar 29, 339(1289), 279 - 85; discussion 285-6 The role of heat-shock proteins in thermotolerance; Parsell DA et al.; The role of heat-shock proteins (hsps) in thermotolerance was examined in the budding yeast Saccharomyces cerevisiae and in the fruit fly Drosophila melanogaster . In yeast cells, the major protein responsible for thermotolerance is hsp 100 . In cells carrying mutations in the hsp 100 gene, HSP 104, growth is normal at both high and low temperatures, but the ability of cells to survive extreme temperatures is severely impaired . The loss of thermotolerance is apparently due to the absence of the hsp 104 protein itself because, with the exception of the hsp 104 protein, no differences in protein profiles were observed between mutant and wild-type cells . Aggregates found in mutant cells at high temperatures suggest that the cause of death may be the accumulation of denatured proteins . No differences in the rates of protein degradation were observed between mutant and wild-type cells . This, and genetic analysis of cells carrying multiple hsp 70 and hsp 104 mutations, suggests that the primary function of hsp 104 is to rescue proteins from denaturation rather than to degrade them once they have been denatured . Drosophila cells do not produce a protein in the hsp 100 class in response to high temperatures . In this organism, hsp 70 appears to be the primary protein involved in thermotolerance . Thus, the relative importance of different hsps in thermotolerance changes from organism to organism. J Biol Chem, 1993 Mar 25, 268(9), 6470 - 6 A low-Km, rolipram-sensitive, cAMP-specific phosphodiesterase from human brain . Cloning and expression of cDNA, biochemical characterization of recombinant protein, and tissue distribution of mRNA; McLaughlin MM et al.; We have isolated cDNA clones from human frontal cortex cDNA libraries that encode a unique subtype of the low-Km, cAMP-specific phosphodiesterases (PDEs IV) . The 564-amino acid sequence of the protein (human brain PDE IV (hPDE IVB)) shows significant homology to a PDE IV subtype expressed in human monocytes (hPDE IVA), particularly within the approximately 300-amino acid PDE IV catalytic domain . The degree of protein sequence identity is much greater between hPDE IVB and a homolog derived from rat brain (92% over 562 amino acids) than between hPDE IVB and hPDE IVA (76% over 538 amino acids), suggesting a greater subtype-specific versus species-specific conservation of protein sequence . Analysis of the distribution of hPDE IVB mRNA expression revealed a restricted pattern, with an approximately 4-kilobase mRNA detected in brain, heart, lung, and skeletal muscle and not in placenta, liver, kidney, or pancreas . An additional approximately 5-kilobase hPDE IVB-related mRNA species was detected in brain tissue . Recombinant hPDE IVB displayed all of the expected kinetic characteristics for a PDE IV, including sensitivity to the isozyme-selective inhibitor rolipram (Ki = 0.085 microM) . Scatchard analysis of (R)-{3H}rolipram binding data suggested the presence of two noninteracting high affinity rolipram-binding sites (Kd = 0.4 and 6 nM) or a negatively cooperative interaction among multiple binding sites. Biochemistry, 1993 Mar 23, 32(11), 2756 - 62 Effects of surface amino acid replacements in cytochrome c peroxidase on intracomplex electron transfer from cytochrome c; Corin AF et al.; Transient absorption techniques were used to measure the intracomplex electron transfer rates between four recombinant yeast cytochrome c peroxidases and iso-1 cytochrome c (cytc) . The binding affinities and catalytic activities with cytc were previously examined {Corin et al . (1991) Biochemistry 30, 11585} . The four include a wild-type peroxidase (ECcP) and three others, each of which has one surface aspartic acid converted to lysine at position 37, 79, or 217 . These sites have been suggested to be within or proximal to the recognition site for cytc . These mutants conduct electron transfer with cytc but differ with respect to the ionic strength profiles of their limiting rate constants . At pH and mu = 114 mM, ECcP and D217K show similar limiting rate constants for electron transfer with cytc, k(lim), of ca . 2000 s-1 . In the same peroxidase concentration range, the D37K mutant exhibits a k(obs) of ca . 100 s-1 . Instability of the compound I form of D79K prevented a complete study of the intracomplex kinetics of this mutant by this technique . At pH 6 and low ionic strength (8 mM), D37K exhibits a dramatic increase in k(obs) to ca . 800 s-1 while the other two recombinants show a marked decrease to values < 150 s-1 . D37K displays much lower affinity for cytc than do the other peroxidases at higher ionic strengths {Hake et al . (1992) J . Am . Chem . Soc . 114, 5442}, thus preventing adequate complexation necessary for efficient electron transfer . Variations in binding affinity do not explain the more subtle ionic strength kinetic profile observed for D217K.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1993 Mar 16, 32(10), 2548 - 53 The cytochrome P450 1A2 active site: topology and perturbations caused by glutamic acid-318 and threonine-319 mutations; Tuck SF et al.; Phenyldiazene reacts with rat liver CYP1A2 expressed in Saccharomyces cerevisiae to give a phenyl-iron complex that rearranges to a mixture (NB:NA:NC:ND = 12:54:14:20, subscript indicates pyrrole ring) of N-phenyl-PPIX (PPIX = protoporphyrin) regioisomers . The same isomer pattern is obtained in each instance when the purified or microsomal enzyme reacts with phenyldiazene, indicating that the active site topology is not altered by removal of the protein from the membrane . Reaction of the enzyme with biphenylhydrazine gives a similar distribution of N-biphenyl-PPIX isomers, but reaction with (2-naphthyl)-hydrazine only gives the NC and ND regioisomers and a trace of the NA isomer of N-(2-naphthyl)-PPIX . The mutations E318D, E318A, and E318V cause relatively minor changes in the observed regioisomer ratios . In contrast, the mutations T319A, T319V, and T319S suppress formation of the NC and ND isomers of N-phenyl-PPIX . The reaction of T319A with biphenylhydrazine yields major amounts of the NB adduct rather than the small amounts observed with CYP1A2 and the Glu-318 mutants, but does not give the NC and ND regioisomers . Other, less dramatic, changes in the isomer ratios are also observed . The results indicate that the active site of CYP1A2 is open above all four quadrants of the heme group including, to some extent, the region above pyrrole ring B . Pyrrole ring B is completely inaccessible in most cytochrome P450 enzymes.(ABSTRACT TRUNCATED AT 250 WORDS) J Biol Chem, 1993 Mar 15, 268(8), 5615 - 23 Purification and characterization of the prohormone convertase PC1(PC3); Zhou Y et al.; The prohormone convertases PC1 (also known as PC3) and PC2 have been implicated in the biosynthesis of several polypeptide hormones and neuropeptides . In order to understand the regulation and the cell biology of prohormone cleavage, we have purified recombinant mouse PC1 from the conditioned medium of overexpressing Chinese hamster ovary cells . Recombinant PC1 was found to be an 87-kDa calcium-dependent proteinase with an inhibitor profile similar to that of Kex2 and furin . However, unlike furin, the optimum pH for PC1 activity is between pH 5.5 and 6.5 . Like furin, the enzyme is activated at millimolar rather than at micromolar concentrations of calcium . Chinese hamster ovary/PC1 cells secrete the mature form of PC1, converted by a proteolytic cleavage on the carboxyl side of the RSKR motif located at residues 80-83 . This conversion occurs very early in biosynthesis, suggesting that, like Kex2 and furin, PC1 may be activated autocatalytically . Specificity studies with fluorogenic substrates showed that the enzyme prefers substrates with an arginine 4 amino acids amino-terminal to the cleavage site; synthetic tripeptide substrates containing only pairs of basic amino acids are not well cleaved . However, the neuropeptide precursor proenkephalin is cleaved by PC1 to yield a peptide B-sized peptide; since peptide B represents the naturally occurring carboxyl-terminal fragment of proenkephalin, these data suggest a role for PC1 in the processing of this precursor. J Biol Chem, 1993 Mar 15, 268(8), 5450 - 6 Cloning of the cDNA and expression of moubatin, an inhibitor of platelet aggregation; Keller PM et al.; Moubatin, a new type of specific inhibitor of collagen-induced platelet aggregation, has been isolated from the soft tick Ornithodoros moubata (Waxman, L., and Connolly, T . M . (1993) J . Biol . Chem . 268, 5445-5449) . A polymerase chain reaction-generated hybridization probe, produced using primers based on moubatin protein sequence, identified phage containing the entire cDNA sequence of moubatin . Analysis of the predicted amino acid sequence yielded a mature protein of 156 amino acids with a putative prepeptide of 15 amino acids . Comparison of the sequence of moubatin to that of other proteins in the Swiss PROT data base revealed no significant homology . The cDNA sequence was cloned into the yeast expression vector pKH4 alpha 2, producing a biologically active protein which inhibited collagen-stimulated aggregation of washed human platelets with an IC50 of about 100 nM, which is similar to the potency of native tick moubatin . A concentration of recombinant moubatin that fully inhibited collagen-stimulated aggregation did not inhibit aggregation induced by a variety of other platelet agonists, again demonstrating comparable properties of the recombinant and native proteins . Moubatin did not inhibit platelet adhesion to collagen even at a concentration up to 16 times its IC50 for the inhibition of aggregation . This specificity for inhibiting collagen-stimulated aggregation and not adhesion to collagen indicates that moubatin is unique among the natural product inhibitors of collagen stimulation of platelets . Further examination of the mechanism of moubatin-mediated inhibition of collagen-stimulated aggregation revealed that 1-6 microM moubatin diminished the second phase of aggregation induced by ADP, inhibited aggregation in response to submaximal concentrations of the thromboxane A2 mimetic U46619, and competed for the binding of a thromboxane A2 receptor antagonist to platelet membranes . Therefore, at higher concentrations, moubatin may affect more than one aspect of platelet signal transduction including the thromboxane A2 receptor . The availability of recombinant moubatin will allow further investigation of its unique activities in vitro and in vivo. Gene, 1993 Mar 15, 125(1), 57 - 64 Cloning and characterisation of pepC, a gene encoding a serine protease from Aspergillus niger; Frederick GD et al.; We have cloned a gene, pepC, encoding a serine proteinase, PEPC, from Aspergillus niger by screening a phage lambda genomic DNA library with a gene (PRB1) from Saccharomyces cerevisiae which codes for proteinase YscB . The nucleotide (nt) sequence of pepC revealed that the gene is composed of two exons of 369 nt and 1230 nt separated by a single 70-nt intron . The deduced protein of 533 amino acids (aa) has a putative signal sequence for transport into the endoplasmic reticulum . Based on the extensive homology shown with serine proteinases (SerP) of the subtilisin family, which includes the active site triad, we hypothesise that the protein is made as a larger precursor which is matured by the cleavage of 130-140 aa from its N terminus and possibly by the removal of approx . 70 aa from its C terminus. Proc Natl Acad Sci U S A, 1993 Mar 15, 90(6), 2370 - 4 Male-sterility induction in transgenic tobacco plants with an unedited atp9 mitochondrial gene from wheat; Hernould M et al.; Cytoplasmic male sterility in plants is associated with mitochondrial dysfunction . We have proposed that a nuclear-encoded chimeric peptide formed by mitochondrial sequences when imported into the mitochondria may impair organelle function and induce male sterility in plants . A model developed to test this hypothesis is reported here . Assuming that the editing process in higher plant mitochondria reflects a requirement for producing active proteins, we have used edited and unedited coding sequences of wheat ATP synthase subunit 9 (atp9) fused to the coding sequence of a yeast coxIV transit peptide . Transgenic plants containing unedited atp9 exhibited either fertile, semifertile, or male-sterile phenotypes; controls containing edited atp9 or only the selectable marker gave fertile plants . Pollen fertility ranged from 31% to 75% in fertile plants, 10% to 20% in semifertile plants, and < 2% in male-sterile plants . Genetic and molecular data showed that the chimeric plasmid containing the transgene is inherited as a Mendelian trait . The transgenic protein is imported into the mitochondria . The production and frequency of semifertile or male-sterile transgenic plants conform to the proposed hypothesis. Nucleic Acids Res, 1993 Mar 11, 21(5), 1193 - 8 The pre-mRNA binding K protein contains a novel evolutionarily conserved motif; Siomi H et al.; The K protein is among the major pre-mRNA-binding proteins (hnRNPs) in vertebrate cell nuclei . It binds tenaciously to cytidine-rich sequences and is the major oligo(rC/dC)-binding protein in vertebrate cells . We have cloned a cDNA of the Xenopus laevis hnRNP K and determined its sequence . The X.laevis hnRNP K is a 47 kD protein that is remarkably similar to its human 66 kD counterpart except for two large internal deletions . The sequence of hnRNP K contains a 45 amino acid repeated motif which is almost completely conserved between the X.laevis and human proteins . We found that this repeated motif, the KH motif (for K homology), shows significant homology to several proteins some of which are known nucleic acids binding proteins . The homology is particularly strong with the archeabacterial ribosomal protein S3 and with the saccharomyces cerevisiae protein MER1 which is required for meiosis-specific splicing of the MER 2 transcript . As several of the proteins that contain the KH motif are known to bind RNA, this domain may be involved in RNA binding. Biochemistry, 1993 Mar 9, 32(9), 2144 - 53 Assignment of 1H, 15N, and 13C resonances, identification of elements of secondary structure and determination of the global fold of the DNA-binding domain of GAL4; Shirakawa M et al.; Almost complete assignments of the 1H, 15N, and aliphatic 13C resonances of the 62-residue N-terminal DNA-binding domain of GAL4 {GAL4(62)} have been obtained using a combination of two-dimensional homonuclear and two- and three-dimensional double- and triple-resonance heteronuclear NMR methods . The sequential NOE connectivities, amide proton exchange measurements, and 13C alpha chemical shift data indicate the presence of two short alpha-helices in the N-terminal half of the polypeptide . Residues 1-9 and 41-62 appear to be unstructured and flexible in solution . Analysis of the 13C alpha chemical shifts also revealed a significant downfield shift of approximately +3 ppm, relative to random-coil values, for the four nonbridging Zn(II) ligands, Cys 14, 21, 31, and 38 . Interestingly, no such correlation was observed for the two bridging ligands, Cys 11 and 28 . Preliminary structure calculations using a subset of distance restraints derived from three-dimensional 1H-15N and 1H-13C NOESY-HSQC spectra are consistent with the recently reported solution structures of Zn(II)2GAL4(7-49) {Kraulis, P., et al . (1992) Nature 356, 448-450} and of Cd(II)2GAL4(65) {Baleja, J . D., et al . (1992) Nature 356, 450-453}. Science, 1993 Mar 5, 259(5100), 1466 - 8 Requirement for a GTPase-activating protein in vesicle budding from the endoplasmic reticulum; Yoshihisa T et al.; The binding and hydrolysis of guanosine triphosphate (GTP) by the small GTP-binding protein Sar1p is required to form transport vesicles from the endoplasmic reticulum (ER) in Saccharomyces cerevisiae . Experiments revealed that an interaction between Sar1p and the Sec23p subunit of an oligomeric protein is also required for vesicle budding . The isolated Sec23p subunit and the oligomeric complex stimulated guanosine triphosphatase (GTPase) activity of Sar1p 10- to 15-fold but did not activate two other small GTP-binding proteins involved in vesicle traffic (Ypt1p and ARF) . Activation of GTPase was inhibited by an antibody to Sec23p but not by an antibody that inhibits the budding activity of the other subunit of the Sec23p complex . Also, activation was thermolabile in pure samples of Sec23p that were isolated from two independent sec23 mutant strains . It appears that Sec23p represents a new class of GTPase-activating protein because its sequence shows no similarity to any known member of this family. J Biol Chem, 1993 Mar 5, 268(7), 4889 - 902 Comparison of the acyl chain specificities of human myristoyl-CoA synthetase and human myristoyl-CoA:protein N-myristoyltransferase; Kishore NS et al.; Human myristoyl-CoA synthetase and myristoyl-CoA:protein N-myristoyltransferase (hNmt) have been partially purified from an erythroleukemia cell line . Their substrate specificities were examined using two in vitro assays of enzyme activity together with a panel of C7-C17 saturated fatty acids plus 72 myristic acid analogs containing oxygen, sulfur, ketocarbonyl, ester, amide, cis and trans double bonds, triple bonds, and para-substituted phenyl groups . There is an inverse relationship between the polarity and the activity of C14 fatty acid substrates of myristoyl-CoA synthetase . Surveys of tetradecenoic and tetradecynoic acids suggest that myristate is bound to the synthetase in a bent conformation with a principal bend occurring in the vicinity of C5-C6 . The synthetase can tolerate a somewhat wider range of physical chemical properties in acyl chains than can the monomeric hNmt . However, like myristoyl-CoA synthetase, there is an inverse relationship between acyl chain polarity and the activities of hNmt's acyl-CoA substrates . Moreover, the acyl chain of myristoyl-CoA appears to be bound to hNmt in a bent conformation with bends located in the vicinity of C5 and C8 . The acyl chain specificities of both enzymes make them well suited to utilize efficiently any cellular pools of 5Z-tetradecenoic and 5Z,8Z-tetradecadienoic acids and their CoA derivatives . This feature may account for the recent observation that in some mammalian cell lineages, certain N-myristoyl-proteins are heterogeneously acylated with these C14 fatty acids . Finally, the acyl-CoA binding sites of human and Saccharomyces cerevisiae Nmts appear to have been highly conserved . Given their overlapping yet distinct peptide substrate specificities, development of species-specific inhibitors of Nmts should probably focus on structural features recognized in the enzymes' peptide substrates rather than in the acyl chain of their acyl-CoA substrates. Biochemistry, 1993 Mar 2, 32(8), 1951 - 7 Chiral recognition at cytochrome P450 1A2 active site: effects of mutations at the putative distal site on the bindings of asymmetrical axial ligands; Krainev AG et al.; Effects of mutations at the putative distal site of cytochrome P450 1A2 on chiral discrimination for binding (R)-(+)- and (S)-(-)-1-(1-naphthyl)ethylamine (ligand I), (R)-(-)- and (S)-(+)-1-cyclohexylethylamine (ligand II), and (R)-(+)- and (S)-(-)-1-(4-pyridyl)ethanol (ligand III) were studied by optical absorption spectra . The wild-type P450 1A2 exhibited different dissociation constants (Kd) for the R- and S-enantiomers of these ligands . The R/S ratios of the Kd values for ligands I and II were 5.2 and 2.9, respectively, and the S/R ratio for ligand III was 6.0 . Mutations at the putative distal site, such as Glu318Asp and Glu318Ala, remarkably enhanced the discrimination: the R/S ratio of the Kd values for ligand I increased from 5.2 to 20-60, while the R/S ratio for ligand II decreased from 2.9 to 0.8-0.9 . These remarkable changes in the R/S ratios were not observed with Glu318Asp mutation for ligand III binding, whereas affinities for both enantiomers of ligand III were markedly decreased by the Glu318Ala mutation . Mutation Thr319Ala increased the R/S ratio of the Kd values for ligand I slightly but markedly decreased the R/S ratio of ligand II (from 2.9 to 0.8) and the S/R ratio of ligand III (from 6.0 to 1.0) . Similar enhancements of the chiral discriminations were observed with the mutation Lys250Leu at another putative substrate-recognition site . Differences between the R- and S-enantiomers of the standard enthalpy and entropy of ligand III binding were changed most remarkably by the Thr319Ser mutation.(ABSTRACT TRUNCATED AT 250 WORDS) Biokhimiia, 1993 Mar, 58(3), 348 - 56 {Modification of RNA ligase histidine residues by diethylpyrocarbonate}; Sabaliauskene V et al.; The practicality of Tris-HCl buffer for modification of histidine residues by diethylpyrocarbonate (DEPC) was studied using a model protein-hexokinase . It was found that modification was selective at pH 7.5 . Conditions for modification of one histidine residue in the protein molecule were specified . In 30 mM Tris-HCl buffer pH 7.5, 10-min interaction of RNA-ligase with 0.3 mM DEPC was accompanied by modification of one histidine residue, as a result of which the ability to form a covalent AMP-RNA-ligase complex decreased 3 times . Modification of two histidine residues of RNA-ligase resulted in a complete loss of the enzyme activity . At increasing DEPC concentration modification affected all of the seven histidine residues of RNA-ligase . The kinetic parameters (Km and V) for the native and modified enzymes were determined and compared. Mol Endocrinol, 1993 Mar, 7(3), 331 - 40 Human estrogen receptor bound to an estrogen response element bends DNA; Nardulli AM et al.; We have used gel mobility shift assays to examine changes in DNA bending induced by binding of human estrogen receptor (hER) to a series of estrogen response element (ERE) containing DNA fragments . Competition experiments with ERE-containing DNA fragments and antibody supershift experiments demonstrated that ER in crude extracts from MCF-7 human breast cancer cells exhibited specific interaction with the ERE . Using DNA bending standards, we found that binding of ER to a single ERE induced a reproducible DNA bend of 56 degrees . This was 1.65-fold greater than the 34 degrees bending angle we recently reported for binding of bacterially expressed ER DNA binding domain . The DNA bending angle induced was the same whether the salt-extracted receptor was unoccupied, occupied by 17 beta-estradiol, or occupied by trans-hydroxytamoxifen . To determine if proteins associated with ER in MCF-7 cells affect the degree of bending, we examined the ability of partially purified hER expressed in yeast to bend DNA . The degree of bending induced by the partially purified yeast ER was the same as the bending induced by crude MCF-7 cell ER . More highly purified ER from yeast extracts did not bind to an ERE-containing DNA fragment, suggesting that additional proteins may play an important role in the interaction of the ER with the ERE . When two EREs were present in the DNA fragment, a small but reproducible increase in bending was observed . Our demonstration that binding of hER to the ERE induces DNA bending suggests a possible role for DNA bending in ER-induced transcription activation. Trends Biochem Sci, 1993 Mar, 18(3), 90 - 5 Multiple functions of nucleosomes and regulatory factors in transcription; Workman JL et al.; The in vivo packaging of DNA with histone proteins to form chromatin makes its transcription a difficult process . Biochemical and genetic studies are beginning to reveal mechanistic details of how transcriptional regulatory factors confront at least two hurdles created by nucleosomes, the primary structural unit of chromatin . Regulatory factors must gain access to their respective binding sites and activate the formation of transcription complexes at core promoter elements . Distinct regulatory factors may be specialized to perform these functions. Trends Biochem Sci, 1993 Mar, 18(3), 84 - 9 A template for the protein kinase family; Taylor SS et al.; The crystal structure of the catalytic subunit of cAMP-dependent protein kinase, complexed with ATP and a 20-residue inhibitor peptide, is reviewed and correlated with chemical and genetic data . The striking convergence of the structure with the biochemistry and genetics provides for the first time a molecular basis for understanding how this enzyme functions, as well as an explanation for the highly conserved residues that are scattered throughout the molecule . Because these residues probably serve a common role in all eukaryotic protein kinases, this first protein kinase structure serves as a general template for the entire family of enzymes. Trends Biochem Sci, 1993 Mar, 18(3), 102 - 6 The metabolic role of leucovorin; Stover P et al.; Interest in determining if leucovorin, known chemically as 5-formyltetra-hydrofolate, plays a role in one-carbon metabolism is reemerging . While investigations in the 1940s suggested it was an important donor of one-carbon units in folate-mediated biosynthetic reactions, studies between the 1950s and 1980s disproved this hypothesis and dismissed its presence in biological systems as artifactual . Recently, new data has focused attention on the possible biological function of this compound that is widely used in cancer chemotherapy. Biophys J, 1993 Mar, 64(3), 749 - 53 Metal binding properties of single amino acid deletion mutants of zinc finger peptides: studies using cobalt(II) as a spectroscopic probe; Shi Y et al.; Peptides corresponding to Cys2His2 zinc finger domains from which one amino acid has been deleted have been synthesized and their metal-binding properties characterized . In contrast to earlier reports (Parraga, G., S . Horvath, L . Hood, E . T . Young, and R . E . Klevit . 1990 . Proc . Natl . Acad . Sci . USA . 87:137-141.), such peptides do bind metal ions such as cobalt(II) . A peptide with the sequence ProTyrLysCysProGluCysLysSerPheSerGlnLysSerAspLeuValLysHisGlnArgThrHis ThrGly (which corresponds to a previously characterized consensus zinc finger sequence from which a Gly residue immediately following the second Cys residue has been deleted) was found to form a 1:1 peptide to cobalt(II) complex with an absorption spectrum quite similar to those previously observed for zinc finger peptide-cobalt(II) complexes . The dissociation constant for this complex is 6 x 10(-6)M, a factor of 100 times higher than that for the parent peptide . A peptide with the sequence LysProTyrProCysGlyLeuCysArgCysPheThrArgArgAspLeuLeulleArgHisAlaGln - LyslleHisSerGlyAsnLeu corresponding to a similar mutation of the peptide ADR1 was also characterized . Spectroscopic studies with cobalt(II) revealed that this peptide forms both 1:1 and 2:1 peptide to cobalt(II) complexes . The absorption spectra of the two forms and the dissociation constants were determined via deconvolution methods . In contrast, the parent peptide ADR1a was found to form only a 1:1 complex under comparable conditions and this 1:1 complex was found to be more stable than that for the mutant . These results reveal that deletion mutations do adversely affect the stability of zinc finger peptide-metal complexes but that the effects are not as drastic as had been previously described. Mol Biochem Parasitol, 1993 Mar, 58(1), 177 - 80 Molecular cloning of a rho family gene of Entamoeba histolytica; Lohia A et al.; An Entamoeba histolytica gene (Eh rho1) was cloned that encodes a putative low-molecular-mass GTP-binding protein, most similar to the ras homologue rho . The Eh rho1 open reading frame was 208 amino acids long and encoded a 23-kDa protein similar to Saccharomyces cerevisiae RHO1-RHO4 and CDC42 and human rhoA, rac1, and G25K gene products . This similarity was greatest at the NH2 terminus of Eh rho1 where two GTP-binding sites and a possible effector site were conserved . A cysteine residue at the COOH terminus of Eh rho1 was followed by eight hydrophobic amino acids rather than the three hydrophobic amino acids present in other ras family proteins. Proteins, 1993 Mar, 15(3), 330 - 7 Site-specific proteolytic cleavage of Ku protein bound to DNA; Paillard S et al.; Ku protein, a relatively abundant nuclear protein associated with DNA of mammalian cells, is known to be a heterodimer with subunits of 85 and 72 kDa which binds in vitro to DNA ends and subsequently translocates along the molecule . The functional role played by this protein in the cell, however, remains to be elucidated . We have observed here that Ku protein, purified from cultured monkey cells, is the target of specific endoproteolysis in vitro, by which the 85 kDa subunit is cleaved at a precise site while the 72 kDa subunit remains intact . This cleavage releases an 18 kDa polypeptide and converts Ku protein into a heterodimer composed of the 72 kDa subunit associated with a 69 kDa fragment from the 85 kDa subunit . The proteolyzed form of Ku protein, denoted Ku', has DNA binding properties similar to those of Ku protein . The proteolytic mechanism, which is inhibited by leupeptin and chymostatin, is extremely sensitive to ionic conditions, in particular to pH, being very active at pH 7.0 and completely inhibited at pH 8.0 . In addition, cleavage occurs only when Ku protein is bound to DNA, not free in solution . We suggest that in vivo, such proteolysis might be necessary for Ku protein function at some stage of the cell cycle. Proteins, 1993 Mar, 15(3), 223 - 34 Pitch diversity in alpha-helical coiled coils; Seo J et al.; Two complementary methods for measuring local pitch based on heptad position in alpha-helical coiled coils are described and applied to six crystal structures . The results reveal a diversity of pitch values: two-stranded coiled coils appear to have pitch values near 150 A; the values for three- and four-stranded coiled coils range closer to 200 A . The methods also provide a rapid and sensitive gauge of local coiled-coil conformation . Polar or charged residues in the apolar interface between coiled-coil helices markedly affect local pitch values, suggesting a connection between pitch uniformity and coiled-coil stability . Moreover, the identification of a skip residue (heptad frame shift) in the hemagglutinin glycoprotein of influenza virus (HA) allows interpretation of local pitch changes . These results on relatively short coiled-coil structures have relevance for the much longer fibrous proteins (many of which have skip residues) whose detailed structures are not yet established . We also show that local pitch values from molecular dynamics predictions of the GCN4 leucine zipper are in striking agreement with the high-resolution crystal structure--a result not readily discerned by direct comparison of atomic coordinates . Taken together, these methods reveal specific aspects of coiled-coil structure which may escape detection by global analyses of pitch. Arthritis Rheum, 1993 Mar, 36(3), 410 - 5 Localized scleroderma progressing to systemic disease . Case report and review of the literature; Birdi N et al.; We describe a 15-year-old girl with biopsy-proven morphea who developed progression to systemic disease 2 years after initial presentation . In contrast to other reported patients with localized scleroderma, some of whom have had mild, nonprogressive systemic involvement, this patient developed severe, debilitating disease, with skin tightness, sclerodactyly, esophageal involvement, restrictive pulmonary disease, and myopathy . From the time of her initial evaluation, the patient was positive for antinuclear antibodies (ANA), which were shown to be primarily directed against the Ku antigens . This observation suggests that ANA may be a prognostic indicator for progression to systemic disease. Proc Natl Acad Sci U S A, 1993 Mar 1, 90(5), 1657 - 61 A selective transcriptional induction system for mammalian cells based on Gal4-estrogen receptor fusion proteins; Braselmann S et al.; Most mammalian cells neither express any Gal4-like activity nor endogenous estrogen receptor, thus rendering estrogen an inert signal for them . For these two reasons we have developed a selective induction system based on the estrogen-regulable transcription factor Gal-ER . Gal-ER consists of the DNA-binding domain of the yeast Gal4 protein fused to the hormone-binding domain of the human estrogen receptor and hence should exclusively regulate a transfected gene under the control of a Gal4-responsive promoter in mammalian cells . Two major improvements of this induction system were made . First, a synthetic Gal4-responsive promoter was constructed which consisted of four Gal4-binding sites, an inverted CCAAT element, a TATA box, and the adenovirus major late initiation region . This promoter showed extremely low basal activity in the absence and high inducibility in the presence of ligand-activated Gal-ER . Second, the transcription factor Gal-ER was rendered more potent and less susceptible to cell type-specific variation by fusing the strong activating domain of the herpesvirus protein VP16 onto its C terminus . In response to estrogen, Gal-ER-VP16 induced the Gal4-responsive promoter at least 100-fold in transiently transfected NIH 3T3 and P19 cells . Rat fibroblast cell lines expressing integrated Gal-ER and Gal4-responsive fos genes were transformed in a strictly estrogen-dependent manner . The exogenous fos gene was rapidly induced to maximal levels within 1-2 hr of estrogen addition . Elevated Fos activity in turn stimulated transcription of the endogenous fra-1 gene . These data demonstrate the utility of the Gal-ER induction system as a powerful genetic switch for regulating heterologous genes and, in particular, for identifying Fos targets in mammalian cells. Proc Natl Acad Sci U S A, 1993 Mar 1, 90(5), 1639 - 41 Strategies for the identification of interacting proteins; Guarente L; Many problems in modern biology involve complex arrays of interacting protein and, in some cases, RNA molecules . The initial challenge facing investigators is to identify the important players that drive the process under study . This difficult task is ameliorated somewhat by the development of methods designed to keep pace with the magnitude of this challenge . I have outlined a few of these approaches at the cutting edge of cloning interacting proteins . A perhaps more daunting prospect is to dissect the important molecules once they are in hand, to identify key interactions, and, ultimately, to move to an understanding of function in cells . For this, of course, all of the tools of genetics, biochemistry, and molecular biology, extant and yet to be developed, will have to be tapped. Mol Cell Biol, 1993 Mar, 13(3), 1876 - 82 PRP19: a novel spliceosomal component; Cheng SC et al.; We have isolated the gene of a splicing factor, PRP19, by complementation of the temperature-sensitive growth defect of the prp19 mutant of Saccharomyces cerevisiae . The gene encodes a protein of 502 amino acid residues of molecular weight 56,500, with no homology to sequences in the data base . Unlike other PRP proteins or mammalian splicing factors, the sequence of PRP19 has no discernible motif . Immunoprecipitation studies showed that PRP19 is associated with the spliceosome during the splicing reaction . Although the exact function of PRP19 remains unknown, PRP19 appears to be distinct from the other PRP proteins or other spliceosomal components. Mol Cell Biol, 1993 Mar, 13(3), 1779 - 87 A dosage-dependent suppressor of a temperature-sensitive calmodulin mutant encodes a protein related to the fork head family of DNA-binding proteins; Zhu G et al.; The cmd1-1 mutation of calmodulin causes temperature-sensitive growth in Saccharomyces cerevisiae . We have isolated a dosage-dependent suppressor of cmd1-1, designated HCM1 . Twentyfold overexpression of HCM1 permits strains carrying cmd1-1 to grow at temperatures up to and including 34 degrees C but does not suppress the lethality of either cmd1-1 at higher temperatures or the deletion of CMD1 . Thus, overexpression of HCM1 does not bypass the requirement for calmodulin but enhances the ability of the mutant calmodulin to function . HCM1 is not essential for growth, but deletion of HCM1 exacerbates the phenotype of a strain carrying cmd1-1 . HCM1 is located on chromosome III, which was recently sequenced . Our results correct errors in the published DNA sequence . The putative polypeptide encoded by HCM1 is 564 amino acids long and has a predicted molecular weight of 63,622 . Antisera prepared against Hcm1p detect a protein that is overproduced in yeast strains overexpressing HCM1 and has an apparent molecular mass of 65 kDa . Eighty-six amino acid residues in the N terminus of Hcm1p show 50% identity with a DNA-binding region of the fork head family of DNA-binding proteins . When fused to the DNA-binding domain of Gal4p, residues 139 to 511 of Hcm1p can act as a strong activator of transcription . However, overexpression of HCM1 does not affect the expression of calmodulin . Furthermore, Hcm1p does not bind to calmodulin in a gel overlay assay . Thus, overexpression of HCM1 enhances calmodulin function by an apparently indirect mechanism. Mol Cell Biol, 1993 Mar, 13(3), 1371 - 7 The ubc-2 gene of Caenorhabditis elegans encodes a ubiquitin-conjugating enzyme involved in selective protein degradation; Zhen M et al.; The ubiquitin-protein conjugation system is involved in a variety of eukaryotic cell functions, including the degradation of abnormal and short-lived proteins, chromatin structure, cell cycle progression, and DNA repair . The ubiquitination of target proteins is catalyzed by a ubiquitin-activating enzyme (E1) and ubiquitin-conjugating enzymes (E2s) and in some cases also requires auxiliary substrate recognition proteins (E3s) . Multiple E2s have been found, and these likely possess specificity for different classes of target proteins . Here we report the cloning and characterization of a gene (ubc-2) encoding a ubiquitin-conjugating enzyme which is involved in the selective degradation of abnormal and short-lived proteins in the nematode Caenorhabditis elegans . The nematode ubc-2 gene encodes a 16.7-kDa protein with striking amino acid sequence similarity to Saccharomyces cerevisiae UBC4 and UBC5 and Drosophila UbcD1 . When driven by the UBC4 promoter, ubc-2 can functionally substitute for UBC4 in yeast cells; it rescues the slow-growth phenotype of ubc4 ubc5 mutants at normal temperature and restores their ability to grow at elevated temperatures . Western blots (immunoblots) of ubc4 ubc5 yeast cells transformed with ubc-2 reveal a protein of the expected size, which cross-reacts with anti-Drosophila UbcD1 antibody . C . elegans ubc-2 is constitutively expressed at all life cycle stages and, unlike yeast UBC4 and UBC5, is not induced by heat shock . Both trans and cis splicing are involved in the maturation of the ubc-2 transcript . These data suggest that yeast UBC4 and UBC5, Drosophila UbcD1, and C . elegans ubc-2 define a highly conserved gene family which plays fundamental roles in all eukaryotic cells. Curr Genet, 1993 Mar, 23(3), 205 - 10 Cloning of the C-URA3 gene and construction of a triple auxotroph (his5, ade1, ura3) as a useful host for the genetic engineering of Candida maltosa; Ohkuma M et al.; The C-URA3 gene of the n-alkane assimilating-yeast Candida maltosa was cloned by complementation of the ura3 mutation of Saccharomyces cerevisiae . The nucleotide sequence of C-URA3 and its deduced amino-acid sequence showed significant homology to those of the orotidine 5'-phosphate decarboxylases of other fungal species . To construct a useful host for genetic engineering of C . maltosa using C-URA3 as a marker, one allele of C-URA3 in a double auxotroph (his5, ade1) was disrupted by C-ADE1, and subsequently two kinds of ura3 mutants were isolated by selecting for spontaneous 5-fluoro-orotic acid (5FOA) resistance . One of the mutants was homozygous for the disruption (ura3::C-ADE1/ura3::C-ADE1); the other was heterozygous (ura3::C-ADE1/ura3) . The ura3::C-ADE1 allele in the latter strain was re-substituted by C-URA3 to rescue the adenine auxotroph (his5, ade1, C-URA3/ura3) . Finally, by selecting a 5FOA-resistant mutant, a triple auxotroph (his5, ade1, ura3/ura3) was isolated. Biochem Mol Biol Int, 1993 Mar, 29(4), 661 - 72 T2G4 telomere repeats does not provide telomere function to a BPV-1 containing DNA fragment in mouse C127 cells; Shervington A; The ability of the (T2G4)n telomeric repeats to maintain a linear structure in an extrachromosomal replicating plasmid in mouse C127 cells was tested in a vector based on Bovine papillomavirus type-1, ARS, HIS3 and the neo genes . Digestion with BamHI releases a BPV-1 containing fragment with a (T2G4)n repeats at each end which was introduced to yeast and microinjected into mouse C127 cells . While the linear construct was maintained as extrachromosomal structure in yeast cells, none of the resulting G418-resistant mouse cells transformants were found to have extrachromosomally replicating linear plasmids . Analysis of transformed mouse C127 DNA suggested that in some, the linear fragment had integrated into mouse chromosomes, whereas in other cell lines the fragment may have circularised and possibly been replicating extrachromosomally as a high molecular weight structure . In some of the mouse transformants the (T2G4)n repeats had been deleted from retained plasmid sequences. J Biochem (Tokyo), 1993 Mar, 113(3), 350 - 4 Binding of an intrinsic ATPase inhibitor to the interface between alpha- and beta-subunits of F1FoATPase upon de-energization of mitochondria; Mimura H et al.; Yeast mitochondrial F1FoATPase has three regulatory subunit proteins: ATPase inhibitor, 9K protein, and 15K protein . Mutant yeasts lacking one or more of these protein factors were constructed by gene disruption {Ichikawa, N . et al . (1990) J . Biol . Chem . 265, 6274-6278; Yoshida, Y . et al . (1990) Eur . J . Biochem . 193, 49-53} . Dissipation of the electrochemical potential of protons of the mitochondrial inner membrane by an uncoupler or by a combination of valinomycin and potassium ions induced ATP-hydrolyzing activity of F1FoATPase in mitochondria of all the mutants, as in those of wild-type cells . However, the ATPase activity was inactivated within a minute in normal mitochondria, but was not suppressed in inhibitor-deficient mitochondria, and in mitochondria lacking either 9K or 15K protein, the inactivation of ATPase was slow and incomplete . Covalent binding of inhibitor protein to the enzyme was achieved with a zero length cross-linker, EEDQ, in uncoupled normal mitochondria, in which the inhibitor linked directly to both the alpha- and beta-subunits . This result strongly suggests that the binding site of the inhibitor protein is located at the interface between the two subunits. Genetics, 1993 Mar, 133(3), 499 - 508 A ubiquitin-conjugating enzyme, RAD6, affects the distribution of Ty1 retrotransposon integration positions; Liebman SW et al.; A galactose-inducible Ty1 element was used to generate 59 independent Ty1 inserts that inactivate the CAN1 gene . As found in previous studies, the distribution of these elements shows a gradient of insertion frequency from highest to lowest between the 5' and 3' ends of the gene . However, 53 independent Ty1 and Ty2 insertions isolated by an identical procedure in an isogenic rad6 deletion strain do not show this bias . In this strain, the Ty elements insert randomly throughout CAN1 . These results show that the ubiquitin-conjugating enzyme, RAD6, alters the integration site preferences of Ty1 retrotransposons. Mol Cell Biol, 1993 Mar, 13(3), 1933 - 42 Kid-1, a putative renal transcription factor: regulation during ontogeny and in response to ischemia and toxic injury; Witzgall R et al.; We have identified a new putative transcription factor from the rat kidney, termed Kid-1 (for kidney, ischemia and developmentally regulated gene 1) . Kid-1 belongs to the C2H2 class of zinc finger genes . Its mRNA accumulates with age in postnatal renal development and is detected predominantly in the kidney . Kid-1 mRNA levels decline after renal injury secondary to ischemia or folic acid administration, two insults which result in epithelial cell dedifferentiation, followed by regenerative hyperplasia and differentiation . The low expression of Kid-1 early in postnatal development, and when renal tissue is recovering after injury, suggests that the gene product is involved in establishment of a differentiated phenotype and/or regulation of the proliferative response . The deduced protein contains 13 C2H2 zinc fingers at the COOH end in groups of 4 and 9 separated by a 32-amino-acid spacer . There are consensus sites for phosphorylation in the NH2 terminus non-zinc finger region as well as in the spacer region between zinc fingers 4 and 5 . A region of the deduced protein shares extensive homology with a catalytic region of Raf kinases, a feature shared only with TFIIE among transcription factors . To determine whether Kid-1 can modulate transcription, a chimeric construct encoding the Kid-1 non-zinc finger region (sense or antisense) and the DNA-binding region of GAL4 was transfected into COS and LLC-PK1 cells together with a chloramphenicol acetyltransferase (CAT) reporter plasmid containing GAL4 binding sites, driven by either a minimal promoter or a simian virus 40 enhancer . CAT activity was markedly inhibited in cells transfected with the sense construct compared with the activity in cells transfected with the antisense construct . To our knowledge, this pattern of developmental regulation, kidney expression, and regulation of transcription is unique among the C2H2 class of zinc finger-containing DNA-binding proteins. Scanning Microsc, 1993 Mar, 7(1), 343 - 9; discussion 349-50 Progress in scanning electron microscopy of frozen-hydrated biological specimens; Hermann R et al.; Modern scanning electron microscopy yields structural information down to 2 to 5 nm from thin, beam transparent biological specimens . This paper examines the possibilities of garnering this level of structural information from bulk, frozen-hydrated samples . Freeze-fractured, frozen-hydrated yeast cells, frequently taken as a yardstick to monitor progress in low-temperature scanning electron microscopy, have been used to optimize both metal shadowing methods and observation parameters (e.g . accelerating voltage, electron beam irradiation of the specimen) . Uncoated frozen-hydrated yeast cells do not change electrically at an accelerating voltage of 30 kV . Increasing charging effects are however observed with decreasing accelerating voltages . Very thin metal films are therefore used for specimen coating to localize and enhance the specific secondary electron signal . Planar-magnetron sputtering of a 1 nm metal layer provides high resolution secondary electron images, at 30 kV, of freeze-fractured, frozen-hydrated yeast cells in an in-lens field-emission scanning electron microscope . Structural information comparable to that of transmission electron microscopy of freeze-fractures is attained . Planar-magnetron sputtering of either chromium, tungsten or platinum results in essentially the same information density (smallest visible significant structural detail) . Frozen-hydrated samples are very beam sensitive and have to be observed under minimum dose conditions. Plant Physiol, 1993 Mar, 101(3), 915 - 24 Analysis of genes negatively regulated by phytochrome action in Lemna gibba and identification of a promoter region required for phytochrome responsiveness; Okubara PA et al.; As a step to understanding how the photoreceptor phytochrome acts to change the transcription of specific nuclear genes in Lemna gibba, we wish to compare promoter elements involved in negative regulation by phytochrome with those involved in positive regulation . We have isolated three genes negatively regulated by phytochrome, designated NR (negatively phytochrome regulated) genes (P.A . Okubara, E.M . Tobin {1991} Plant Physiol 96:1237-1245), and we have now sequenced two of these . The promoters of both contain some sequence motifs that are identical with motifs from other genes . We used a transient assay in L . gibba to demonstrate that approximately 1.7 kb pairs of the NPR1 promoter and 1.1 kb pairs of the NPR2 promoter could confer negative phytochrome regulation to a luciferase reporter gene . Deletion analysis of the NPR2 promoter showed that sequences between -208 and -82 from the transcription start were necessary for negative phytochrome regulation . However, this region was not sufficient to confer negative regulation by phytochrome to another promoter . Additionally, we noted that this region showed no similarity to a region identified as important for the negative regulation of the oat phyA promoter (W.B . Bruce, X.-W . Deng, P.H . Quail {1991} EMBO J 10:3015-3024), but it does contain a sequence element found in several other kinds of genes, including ones positively regulated by phytochrome . The deduced amino acid sequences of NPR1 and NPR2 were found to share similarities with many abscisic acid-induced or seed-abundant proteins . Thus, these genes, like other phytochrome-regulated genes, might respond to multiple regulatory signals. Ukr Biokhim Zh, 1993 Mar-Apr, 65(2), 42 - 7 {Paracatalytic inactivation of pyruvate decarboxylase in the presence of quinones}; Vovk AI et al.; Pyruvate promotes the yeast pyruvate decarboxylase inactivation under the influence of substituted p-benzoquinones . Pyruvate decarboxylase activity is not renewed after the removal of low-molecular impurities by gel filtration and subsequent addition of dithiothreitol, thiamine diphosphate, magnesium chloride . The inactivation rate under joint action of 2-methyl-5-isopropyl-p-benzoquinone and pyruvate is regulated by the pseudo-first-order equation . The relationship between pseudo-first-order rate constant and pyruvate concentration takes the shape of hyperbola . The inactivation order with respect to quinone is determined by oxidant concentration and pH value . Maximum pseudo-first-order rate constant values in the presence of the excess substrate and 2-methyl-5-isopropyl-p-benzoquinone are observed at pH 5.9-6.0 . The data obtained evidence for the fact that during inactivation quinone interacts with "active acetaldehyde" being the intermediate in the process of catalysis with pyruvate decarboxylase. Mol Biochem Parasitol, 1993 Mar, 58(1), 7 - 15 Selective toxicity to malaria parasites by non-intercalating DNA-binding ligands; Ginsburg H et al.; The DNA of malarial parasites is significantly richer in A and T than that of mammalian cells . Antibiotics which bind to the minor groove of B-DNA with a preference for AT-rich sequences, such as distamycin A, netropsin, 4'-6-diamidino-2-phenylindole (DAPI) and bis-benzimide (Hoechst 33258) were found to inhibit the growth and propagation of Plasmodium falciparum in culture . Distamycin A readily inhibited nucleic acid and protein synthesis and was more toxic to the ring stage than to the trophozoite stage in various parasite strains, irrespective of their susceptibility to chloroquine . Distamycin A, netropsin, DAPI and Hoechst 33258 were considerably more toxic to parasites than to mammalian cells, while chromomycin A3 and mithramycin A, which bind preferentially to GC-rich sequences, were either equally toxic or more harmful to mammalian cells . These results suggest that the mere difference in DNA base composition of parasites and host cells may account for the selective toxicity of minor groove ligands . Distamycin A, DAPI and Hoechst 33258 were also found to be more toxic to Saccharomyces cerevisiae grown on glycerol than to yeast cells grown on glucose, consistent with the preferential binding of these ligands to the relatively AT-rich mitochondrial DNA of yeast cell . These results underscore the generality of selective toxicity of minor groove binders endowed by the DNA base composition. J Cell Biol, 1993 Mar, 120(5), 1083 - 91 Mapping DNA within the mammalian kinetochore; Cooke CA et al.; The location of the cis-acting DNA sequences that direct the assembly of the mammalian kinetochore is not known . A variety of circumstantial evidence, however, has led to the widespread belief that they are present throughout the kinetochore including the kinetochore outer plate . To investigate this question directly, we have used two independent methods to localize DNA in and around the mammalian kinetochore . Both methods fail to reveal DNA in the outer kinetochore plate, finding instead that the outer-most detectable DNA in the centromere is located in the inner kinetochore plate . Our results imply that the outer kinetochore plate is primarily a proteinaceous structure . It is thus unlikely that fibers observed in the outer plate correspond to chromatin, as previously assumed . Our observations suggest that current models of kinetochore structure may need to be reconsidered. Biochem Biophys Res Commun, 1993 Feb 26, 191(1), 95 - 102 A provisional mechanism for regulating the aminoacyl-tRNA synthetases; Black S; A mechanism is outlined for regulating aminoacyl-tRNA synthetases with a nonheme iron-containing protein serving as the key regulator . This mechanism is formulated from experiments with a complex-bound valyl-tRNA synthetase from yeast that is activated by thiols, by tRNA, and by an iron-containing protein preparation with characteristic spectral properties. Science, 1993 Feb 26, 259(5099), 1288 - 93 Crystal structure of a synthetic triple-stranded alpha-helical bundle; Lovejoy B et al.; The x-ray crystal structure of a peptide designed to form a double-stranded parallel coiled coil shows that it is actually a triple-stranded coiled coil formed by three alpha-helices . Unlike the designed parallel coiled coil, the helices run up-up-down . The structure is stabilized by a distinctive hydrophobic interface consisting of eight layers . As in the design, each alpha-helix in the coiled coil contributes one leucine side chain to each layer . The structure suggests that hydrophobic interactions are a dominant factor in the stabilization of coiled coils . The stoichiometry and geometry of coiled coils are primarily determined by side chain packing in the solvent-inaccessible interior, but electrostatic interactions also contribute. Cell, 1993 Feb 26, 72(4), 587 - 94 The acidic activation domains of the GCN4 and GAL4 proteins are not alpha helical but form beta sheets; Van Hoy M et al.; The most common class of activation domains, the so-called acidic activators, has been proposed either to adopt an amphipathic alpha-helical structure or to exist as unstructured "acid blobs." However, genetic analysis of an acidic activation domain in the yeast GAL4 protein has suggested that the structure of the activation region is a beta sheet . To distinguish between these models, we conducted a biophysical analysis of peptides corresponding to the yeast GAL4 and GCN4 acidic activation domains . Circular dichroism spectroscopy shows that the peptides are not alpha helical, but that they can undergo a transition to a structure that is almost 100% beta sheet in character in slightly acidic solution . We also show that the artificial acidic activator AH has structural properties that are markedly different from the natural GAL4 and GCN4 domains and does not adopt a beta-rich structure at reduced pH. Cell, 1993 Feb 26, 72(4), 575 - 85 Genetic evidence that an activation domain of GAL4 does not require acidity and may form a beta sheet; Leuther KK et al.; Regulation of gene expression in eukaryotes relies on intricate protein-protein interactions . Transcription of the galactose genes in yeast has been a productive model for this type of interaction . The positive activator in this system, GAL4, has a bifunctional C-terminus . It contains both a prototypic acidic activation domain and a region that binds the negative regulator, GAL80 . We have taken advantage of this colocalization of functions to subject the region to a constrained mutagenesis analysis: one function was maintained, while the other one was altered . This analysis and the experiments it suggested have led us to two conclusions: first, the acidic amino acids are not, as commonly thought, required for activation; second, this region is not unstructured or alpha helical, but its function may require a beta sheet. Biochem Biophys Res Commun, 1993 Feb 26, 191(1), 196 - 200 Identification of the Epstein-Barr virus nuclear antigen 2 transactivation domain; Horvath GC et al.; The Epstein-Barr virus nuclear antigen 2 (EBNA2) protein activates the expression of viral and cellular genes . A set of seven yeast GAL4-EBNA2 fusion proteins were constructed in order to identify the transactivation domain of EBNA2 . These fusion proteins were tested for their ability to transactivate a target gene in Hela and BJAB cells . This analysis has demonstrated that the EBNA2 polyproline domain is dispensable for transactivation while the acidic carboxy terminus defined by amino acids 337-467 is essential . This result is consistent with the analysis of a variety of viral and cellular eucaryotic transactivators in which an acidic domain of the protein has been shown to be indispensible for function. Nucleic Acids Res, 1993 Feb 25, 21(4), 1013 - 8 Transcription factor IIA stimulates the expression of classical polIII-genes; Meissner W et al.; Protein fractions containing TFIIA, a transcription factor known to be involved in transcription initiation by RNA polymerase II and 5'-regulated polymerase III genes (e.g . U6), were tested for their role in in vitro transcription of classical pol III genes . These fractions were shown to stimulate a basal transcription system, reconstituted from highly purified fractions hTFIIIB and hTFIIIC . We demonstrate that this stimulating activity isolated from HeLa cells coelutes over at least six chromatographic steps with hTFIIA . Moreover the native molecular mass and the stability of this activity against heat treatment are comparable to those of hTFIIA . Finally we show that recombinant TFIIA from Saccharomyces cerevisiae can substitute for the human factor in pol III transcription in vitro which proves that TFIIA is also involved in the efficient expression of classical pol III genes. Nature, 1993 Feb 25, 361(6414), 751 - 3 Magnetic bead capture of expressed sequences encoded within large genomic segments; Tagle DA et al.; Magnetic bead capture utilizes biotin-streptavidin magnetic bead technology to isolate cDNAs rapidly from large genomic intervals, giving several thousand-fold enrichment of the selected cDNAs . The technique can allow parallel analysis of several large genomic segments of varying complexities and can be applied to the isolation of expressed sequences from various tissue sources. Nature, 1993 Feb 25, 361(6414), 726 - 30 Putative X-linked adrenoleukodystrophy gene shares unexpected homology with ABC transporters; Mosser J et al.; Adrenoleukodystrophy (ALD) is an X-linked disease affecting 1/20,000 males either as cerebral ALD in childhood or as adrenomyeloneuropathy (AMN) in adults . Childhood ALD is the more severe form, with onset of neurological symptoms between 5-12 years of age . Central nervous system demyelination progresses rapidly and death occurs within a few years . AMN is a milder form of the disease with onset at 15-30 years of age and a more progressive course . Adrenal insufficiency (Addison's disease) may remain the only clinical manifestation of ALD . The principal biochemical abnormality of ALD is the accumulation of very-long-chain fatty acids (VLCFA) because of impaired beta-oxidation in peroxisomes . The normal oxidation of VLCFA-CoA in patients' fibroblasts suggested that the gene coding for the VLCFA-CoA synthetase could be a candidate gene for ALD . Here we use positional cloning to identify a gene partially deleted in 6 of 85 independent patients with ALD . In familial cases, the deletions segregated with the disease . An identical deletion was detected in two brothers presenting with different clinical ALD phenotypes . Candidate exons were identified by computer analysis of genomic sequences and used to isolate complementary DNAs by exon connection and screening of cDNA libraries . The deduced protein sequence shows significant sequence identity to a peroxisomal membrane protein of M(r) 70K that is involved in peroxisome biogenesis and belongs to the 'ATP-binding cassette' superfamily of transporters. Nucleic Acids Res, 1993 Feb 25, 21(4), 941 - 7 Rye nuclease I as a tool for structural studies of tRNAs with large variable arms; el Adlouni C et al.; A single-strand-specific nuclease from rye germ (Rn nuclease I) was used for secondary and tertiary structure investigations of tRNAs with large variable arms (class II tRNAs) . We have studied the structure in solution of two recently sequenced tRNA(Leu): yeast tRNA(Leu)(ncm5UmAA) and bovine tRNA(Leu)(XmAA) as well as yeast tRNA(Leu)(UAG), tRNA(Leu)(m5CAA) and tRNA(Ser)(IGA) . The latter is the only tRNA with a long variable arm for which the secondary and tertiary structure has already been studied by use of chemical probes and computer modelling . The data obtained in this work showed that the general model of class II tRNAs proposed by others for tRNA(Ser) can be extended to tRNAs(Leu) as well . However interesting differences in the structure of tRNAs(Leu) versus tRNA(Ser)(IGA) were also noticed . The main difference was observed in the accessibility of the variable loops to nucleolytic attack of Rn nuclease I: variable loops of all studied tRNA(Leu) species were cut by Rn nuclease I, while that of yeast tRNA(Ser)(IGA) was not . This could be due to differences in stability of the variable arms and the lengths of their loops which are 3 and 4 nucleotides in tRNA(Ser)(IGA) and tRNAs(Leu) respectively. Nature, 1993 Feb 25, 361(6414), 749 - 50 Study of allosteric communication between protomers by immunotagging; Lindsley JE et al.; Ligand-induced allosteric changes in proteins are important in their cellular functions and regulation, and both concerted and sequential examples are known . The distinction has entailed elaborate analysis, however, and only a few systems have been unequivocally analysed . We have investigated the coupling between ATP usage and DNA transport by type II DNA topoisomerases, and one key question concerning allostery in these dyadic enzymes is whether ATP binding to one protomer can induce a concerted conformational change in the entire enzyme . Here we use an enzyme with one immunotagged subunit defective in ATP binding and one wild-type subunit to show that it can . Our approach should be generally applicable in the study of allostery and communication between members of a macromolecular assembly. Biochemistry, 1993 Feb 23, 32(7), 1795 - 802 Product dependence of deuterium isotope effects in enzyme-catalyzed reactions; Cook PF et al.; Theory for enzyme-catalyzed reactions is developed for the dependence on product concentration of deuterium isotope effects on V and V/K . Generally, a product that decreases the off-rate for a second product to zero causes the isotope effect on V/K to decrease to DKeq and that on V to decrease to a value between 1 and DKeq . If the second product off-rate is decreased to a finite value, DV and D(V/K) will decrease to a value greater than DKeq, while if there is no effect on the off-rate for the second product, DV and D(V/K) will not change . Interestingly, for a ping-pong mechanism, the presence of the product that provides a reversible connection between the isotope-sensitive step and the isotope-insensitive half-reaction will give an isotope effect on V/K for the latter . (In the absence of the product, the isotope effect on V/K for the isotope-insensitive half-reaction will be unity.) Theory is supported with data for alcohol and lactate dehydrogenases . For lactate dehydrogenase, D(V/Kpyruvate) decreases from 1.93 +/- 0.02 at zero to 1.16 +/- 0.02 at infinite lactate concentration, while DV decreases from a value of 1.75 +/- 0.03 at zero to a value of 0.93 +/- 0.05 at infinite lactate concentration . Thus, it appears that the pathway in which lactate is released first is greatly preferred, but the pathway in which NAD+ is released before lactate is observed at high lactate concentration.(ABSTRACT TRUNCATED AT 250 WORDS) FEBS Lett, 1993 Feb 22, 318(1), 80 - 2 Around the growth phase transition S . cerevisiae's make-up favours sustained oscillations of intracellular metabolites; Richard P et al.; Under a limited set of hitherto incompletely defined conditions, inhibition of respiration has been shown to cause transient oscillations in NAD(P)H fluorescence of yeast cells . In this paper, we apply a new method {1992, Anal . Biochem . 204, 118-132} for extraction of intracellular metabolites . This method involves spraying the cells into -40 degrees C methanol; the neutral pH allows extraction of nearly all intracellular metabolites, including NADH . Close to the shift from glucose to ethanol as a growth substrate, the cells acquire a make-up amenable to sustained oscillations in intracellular concentrations of NADH and glycolytic intermediates such as glucose-6-phosphate . NADH was found to oscillate between 200 microM and 400 microM intracellular concentration . The cellular make-up determining the tendency to oscillate is 'remembered' by the cells after three hours of starvation. Nature, 1993 Feb 18, 361(6413), 660 - 2 Evidence for an essential non-Watson-Crick interaction between the first and last nucleotides of a nuclear pre-mRNA intron; Parker R et al.; Nuclear pre-messenger RNA splicing requires the action of five small nuclear (sn) RNAs, U1, U2, U4, U5 and U6, and more than 50 proteins . The mechanistic similarity of nuclear pre-mRNA splicing and group II self-splicing suggests that many of the central processes of nuclear pre-mRNA splicing are based on RNA-RNA interaction . To understand the mechanism of pre-mRNA splicing, the interactions, and their temporal relationships, that occur between the snRNAs and the pre-mRNA during splicing must be identified . Several snRNA-snRNA and snRNA-intron interactions have been demonstrated but the putative RNA-based interactions that recognize the AG dinucleotide at the 3' splice site during 3' cleavage and exon ligation are unknown . We report here the reciprocal suppression between 5' and 3' splice site mutations in the yeast actin intron, and propose that the 3' splice site is positioned for 3' cleavage and exon ligation, at least in part, through a non-Watson-Crick interaction between the guanosines at the 5' and 3' splice sites. Eur J Biochem, 1993 Feb 15, 212(1), 1 - 11 RNA polymerase III . Genes, factors and transcriptional specificity; Willis IM; Recent studies on RNA polymerase III (pol III) gene transcription have provided a new awareness of the molecular complexity of this process . Fortunately, w