S. cerevisiae

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, while the number of transcription components has been increasing, fundamental similarities have emerged regarding the function of eukaryotic promoter elements and the factors that bind them to form preinitiation complexes . Among these, the ability of transcription factor IIIB (TFIIIB) and pol III to transcribe the Saccharomyces cerevisiae U6 gene suggests that the concept of a minimal pol II promoter comprising a TATA box and an initiator region has a parallel in the pol III system . Furthermore, for each of the three classes of eukaryotic RNA polymerase, the assembly of transcription preinitiation complexes and, to some extent, the nature of these complexes appears to be more similar than was previously anticipated . This work highlights the novel functions and transcriptional properties of newly identified pol III genes, discusses the diversity of pol III promoter structures and presents the notion that the exclusive use of extragenic promoters by some pol III genes (so-called type-3 genes) may have evolved since the divergence of yeast and higher eukaryotes . Additionally, recent progress is reviewed on the identification and cloning of subunits for TFIIIC and TFIIIB . Particular emphasis is given to two components of TFIIIB, the TATA-binding protein and a protein with TFIIB homology (PCF4), since the properties of these molecules suggest a model whereby the polymerase specificity of transcription complexes is determined.

Biochem Biophys Res Commun, 1993 Feb 15, 190(3), 747 - 53
Characterization of the factor E1BF from a rat hepatoma that modulates ribosomal RNA gene transcription and its relationship to the human Ku autoantigen; Hoff CM et al.; We have purified the DNA binding protein E1BF (Enhancer 1 Binding Factor) from the nuclei of Morris hepatoma 3924A . This protein, which consists of two polypeptides of 72 kDa and 85 kDa, binds to several DNA sequences involved in the regulation of rDNA transcription . UV-crosslinking studies show that interaction of the protein with DNA is primarily through the 72 kDa polypeptide . Purified E1BF can also modulate pol I-directed transcription in a cell-free systems derived from rat and mouse . E1BF is related to the human autoantigen Ku as demonstrated by immunological cross-reactivity with monoclonal antibodies directed against Ku p70 in Western blot and gel mobility shift assays.

J Biol Chem, 1993 Feb 15, 268(5), 3430 - 7
Protein-DNA interactions in the epsilon-globin gene silencer; Peters B et al.; The developmental control of expression of the human epsilon-globin gene appears to be mediated, at least in part, by a transcriptional silencer in the DNA 5' to the cap site of this gene . We have used site-directed mutagenesis and DNA-protein binding assays to define the active motifs of this epsilon-globin silencer . DNase I foot-printing of the silencer region with K562 cell nuclear extracts defined a sequence, which we designate as the epsilon-globin silencer motif or epsilon GSM (epsilon -278 to -258 base pairs (bp)) containing a region (epsilon -270 to -258) with 90% homology to the yeast mating type silencer, ABF-1 (autonomous replicating sequence binding factor one) and which also overlaps at (epsilon -269 to -262) with the human YY1 consensus sequence, an element which mediates transcription repression and activation of viral, mouse, and human genes . The DNase I footprint extended 5' in the silencer region to include an inverted repeat of a six-nucleotide motif (epsilon -267 to -278 bp) which shares 5 of 6 bases with the GATA-1 consensus sequence . In gel mobility shift assays, two specific proteins (A and B) in nuclear extracts from erythroleukemia K562 cells bound to the DNase I-footprinted region . Protein B, associated with epsilon-globin silencer activity in vitro, required an intact epsilon GSM sequence for binding . Mutation of 5 bases within the epsilon GSM in an epsilon-globin promoter-containing fragment extending upstream to 1400 bp in transient transfection assays increased activity by 3.0-fold compared with the native sequence, suggesting that the silencer activity was mediated by the epsilon GSM sequence . We found that protein A could be displaced by a competitor containing the GATA-1 consensus sequence, suggesting that protein A is a GATA-like protein . The region from -267 to -271 within the epsilon GSM and GATA-1 homology region was important for binding of both proteins A and B . These data suggest that protein binding to the epsilon GSM and GATA motifs mediate the negative effect of the silencer on transcription, possibly via direct competition for binding to this DNA region . Recombinant yeast ABF-1 and human YY1 bound to the epsilon GSM . Mutating three bases (epsilon -259, -262, -264) in the epsilon GSM decreased the binding affinity of protein B and recombinant human YY1 but increased the binding affinity of recombinant yeast ABF-1 . Furthermore, competitor containing the YY1 consensus sequence competed for protein B binding, whereas competitor containing a perfect yeast ABF-1 consensus sequence did not.(ABSTRACT TRUNCATED AT 400 WORDS)

J Biol Chem, 1993 Feb 15, 268(5), 3683 - 9
Ligation of synthetic activated DNA substrates by site-specific recombinases and topoisomerase I; Pan G et al.; The FLP protein of the 2-microns plasmid of Saccharomyces cerevisiae is a conservative site-specific recombinase that is involved in the amplification of the plasmid . This recombination reaction proceeds via the covalent attachment of the protein to the 3'-phosphoryl group at the site of the breaks through a phosphotyrosine linkage . We have recently developed an assay that measures FLP-mediated strand ligation independent of FLP-mediated cleavage and covalent attachment to the DNA . The substrate for ligation was produced by FLP-induced cleavage of the FLP recognition site followed by digestion with Pronase and was shown to contain (at least) a tyrosine residue at the 3'-PO4 terminus adjacent to the FLP cleavage sites . We have now synthesized artificial substrates that bear a tyrosine residue on the 3'-PO4 of an appropriate oligonucleotide and find that this substrate is ligated as efficiently as the previous ligation substrates that were isolated after FLP cleavage of the substrate . Analogous substrates for other members of the integrase family of recombinases (lambda integrase protein, P1-Cre protein) as well as for mammalian topoisomerase I are also active as ligation substrates with their cognate protein . This class of activated substrates should be useful in the study of breakage and reunion reactions involving DNA.

Nucleic Acids Res, 1993 Feb 11, 21(3), 439 - 46
The human hnRNP M proteins: identification of a methionine/arginine-rich repeat motif in ribonucleoproteins; Datar KV et al.; Recent reports indicate that proteins which directly bind to nascent RNA polymerase II transcripts, the heterogeneous nuclear ribonucleoproteins (hnRNPs), play an important role in both transcript-specific packaging and alternative splicing of pre-mRNAs . Here we describe the isolation and characterization of a group of abundant hnRNPs, the M1-M4 proteins, which appear as a cluster of four proteins of 64,000-68,000 daltons by two-dimensional electrophoresis . The M proteins are pre-mRNA binding proteins in vivo, and they bind avidly to poly(G) and poly(U) RNA homopolymers in vitro . Covalently associated polyadenylated RNA-protein complexes, generated by irradiating living HeLa cells with UV light, were purified and used to elicit antibodies in mice . The resulting antisera were then employed to isolate cDNA clones for the largest M protein, M4, by immunological screening . The deduced amino acid sequence of M4 indicates that the M proteins are members of the ribonucleoprotein consensus sequence family of RNA-binding proteins with greatest similarity to a hypothetical RNA-binding protein from Saccharomyces cerevisiae . The M proteins also possess an unusual hexapeptide-repeat region rich in methionine and arginine residues (MR repeat motif) that resembles a repeat in the 64,000 dalton subunit of cleavage stimulation factor, which is involved in 3'-end maturation of pre-mRNAs . Proteins immunologically related to M exist in divergent eukaryotes ranging from human to yeast.

Nucleic Acids Res, 1993 Feb 11, 21(3), 627 - 34
Kinetic analysis of the 5' splice junction hydrolysis of a group II intron promoted by domain 5; Franzen JS et al.; The 5' splice junction (5'SJ) of Group II intron transcripts is subject to a specific hydrolysis reaction (SJH) . This reaction occurs either within a single transcript containing intron sequences through domain 5 (D5) or by cooperation of two separate transcripts, one bearing the 5'SJ and another contributing D5 (1) . In this report we describe the latter reaction in terms of its kinetic parameters . A minimal D5 RNA of 36 nts (GGD5) was sufficient to promote SJH of a second transcript containing the 5' exon plus intron domains 1, 2, and 3 (E1:123) . Equimolar production of two RNAs, the 5' exon (E1) and an intron fragment containing domains 1, 2, and 3 (123) was observed . The kinetic coefficients were evaluated by an excess GGD5 approach . The apparent Km was complex, varying with GGD5 concentration . This behavior indicates heterogeneity in E1:123 with respect to GGD5 binding . The binding heterogeneity may result from formation of E1:123 dimers or from nicks in some molecules of each E1:123 preparation . The heterogeneity was always evident, but to a variable degree, regardless of the procedure by which E1:123 was isolated . The system may be described in terms of parameters analogous to kcat and Km . At infinite dilution of GGD5, the characterizing values were: k2 degrees (the analog of kcat) = 0.0055 min-1 and Km degrees = 0.22 microM . In the limit of GGD5 saturation, the values were: k2 infinity = 0.012 min-1 and Km infinity = 4.5 microM . A natural variant D5, representing the sequence from intron 1 of the yeast cytochrome-b gene, was also functional in SJH . This GGD5b1 was governed by similar Km degrees and Km infinity values, but was only one-third as active over the entire D5 concentration range . A different D5 isomer was entirely ineffective for SJH.

J Biol Chem, 1993 Feb 5, 268(4), 2284 - 7
Direct interaction between the transcriptional activation domain of human p53 and the TATA box-binding protein; Truant R et al.; The human p53 tumor suppressor gene product can activate transcription by RNA polymerase II in the yeast, Saccharomyces cerevisiae, as well as in human cells . Several viral transcriptional activator proteins have been shown to directly contact TBP, the TATA box-binding subunit of the general initiation factor, TFIID . In this report, we use protein affinity chromatography to show that the cellular transcription factor, p53, interacts directly and specifically with yeast TBP . The TBP binding domain of p53 was localized to its N-terminal 73 amino acids . This highly acidic portion of p53 functions as a transcriptional activation domain and is deleted in some tumors induced by the Friend leukemia virus . A human tumor-derived oncogenic point mutation of p53, which lies outside the activation domain of p53, but reduces its ability to activate transcription, greatly reduced the ability of p53 to bind yeast TBP in vitro . This mutation probably affects the overall conformation of the protein and indirectly interferes with the ability of p53 to contact TBP and activate transcription . In contrast, a mutated oncogenic form of p53 that is unaffected in its ability to activate transcription bound yeast TBP as well as wild type p53 . The human TBP activity in a HeLa extract also bound to the activation domain of p53 . Our data support a general model in which DNA-bound activator proteins activate transcription by interacting with TBP.

Nature, 1993 Feb 4, 361(6411), 460 - 3
DSS4-1 is a dominant suppressor of sec4-8 that encodes a nucleotide exchange protein that aids Sec4p function; Moya M et al.; The protein Sec4p plays an essential role at the final stage of the yeast secretory pathway and belongs to the ras superfamily of GTP-binding proteins, more specifically to a branch that includes Ypt1p in Saccharomyces cerevisiae and rab proteins in mammalian cells . GTP-binding proteins change conformation depending on whether GTP or GDP is bound and can thus act as a regulatory switch . The protein remains in its inactive, GDP-bound form until exchange of GTP for GDP allows it to stimulate a downstream effector . This interaction is curtailed by GTP hydrolysis . The rates of nucleotide exchange and GTP hydrolysis can be regulated by interaction with accessory proteins . Although GDP dissociation stimulators (GDS) have been identified that act on members of the ras and rho branches of the superfamily, less is known regarding GDSs that act on members of the Sec4/Ypt1/Rab subgroup . A preliminary characterization of a Rab3A GDP dissociation stimulating activity has been presented . We report here the use of suppressor analysis to clone a gene, dss4, encoding a 17K protein that aids Sec4p action in vivo by functioning as a GDP dissociation stimulator.

J Immunol Methods, 1993 Feb 3, 158(2), 267 - 76
Protein A antibody-capture ELISA (PACE): an ELISA format to avoid denaturation of surface-adsorbed antigens; Ngai PK et al.; Adsorption to a polymeric surface may severely alter the antigenic structure of proteins through unfolding . A conventional capture ELISA in which a protein antigen is adsorbed to the microtiter plate may be unsuitable for testing the specificity of antibodies directed against native proteins (C . Schwab and H.R . Bosshard (1992) J . Immunol . Methods 147, 125) . This problem can be overcome by PACE, a new ELISA procedure in which monoclonal or polyclonal antibodies are first allowed to equilibrate with biotinylated antigen in solution . Thereafter, the antigen-antibody complex (and free antibody) is bound to the microtiter plate through protein A . Captured antigen-antibody complex is detected by streptavidin-alkaline phosphatase and p-nitrophenylphosphate . A competition assay is accomplished by co-incubation of biotinylated and non-biotinylated antigens before capture to the protein A-coated plate . PACE combines the advantages of a solution-phase immunoassay (Farr assay) with the ease of a solid-phase ELISA . PACE has been used to test the conformational specificity of polyclonal and monoclonal antibodies against native and denatured cytochrome c, and of a polyclonal antiserum against a coiled coil leucine zipper peptide . Since a biotin group can be attached specifically to the N-terminal residue of synthetic peptides, PACE is also useful for assaying reactivity against peptide antigens which are difficult to adsorb to microtiter plates.

J Biochem (Tokyo), 1993 Feb, 113(2), 132 - 5
Identification and functional expression of a new member of the mammalian Kex2-like processing endoprotease family: its striking structural similarity to PACE4; Nakagawa T et al.; We used the polymerase chain reaction to identify a mouse cDNA which represented a new member of a growing class of mammalian endoproteases homologous to the yeast Kex2 protease involved in the processing of precursor proteins . This cDNA encoded a 915-residue protein, designated as PC6, containing a subtilisin-like catalytic domain closely related to those of other Kex2-like members (furin, PC2, PC1/3, PC4, and PACE4) . It exhibited striking sequence similarity to PACE4 and contained similar protein domains, such as the COOH-terminal Cys-rich region . Northern blot analysis revealed that PC6 mRNA, as with furin and PACE4 mRNAs, was expressed in various tissues and cell lines, with the highest level in the intestine . Transfection experiments revealed that PC6 was capable of cleaving precursors at dibasic sites . These observations suggest that PC6 is a candidate for a processing endoprotease responsible for the maturation of gastrointestinal peptides.

Mol Biol Cell, 1993 Feb, 4(2), 223 - 32
Specific cross-linking of the proline isomerase cyclophilin to a non-proline-containing peptide; McNew JA et al.; A peptide corresponding to an efficient peroxisomal targeting sequence, the carboxy terminal 12 amino acids of PMP20 from Candida boidinii, was employed as an affinity ligand to search for a peroxisomal targeting receptor . Two proteins from yeast extracts with apparent molecular masses of 20 and 80 kDa were detected by chemical cross-linking to radioiodinated peptide . Both proteins were present in cytosolic supernatants . The 20-kDa species did not cross-link to a control peptide with reversed sequence, whereas the 80-kDa protein cross-linked to both peptides . The cross-linking assay was used to purify the 20-kDa protein from Saccharomyces cerevisiae . Partial protein sequencing identified this protein as cyclophilin, the product of the CYP1 gene . This protein, a peptidyl-prolyl cis-trans isomerase, is the yeast homologue of the protein that mediates the immunosuppressant effects of the drug cyclosporin A (CsA) . Cross-linking of peptide to cyclophilin was inhibited by CsA . The cross-linking of cyclophilin to the PMP20-derived peptide was unanticipated because the peptide contains no prolines . The CYP1-encoded protein was not required to target proteins to peroxisomes because this organelle appeared to be assembled normally in a CYP1-disrupted strain . Furthermore, the final three amino acids of the peptide, which are critical for peroxisomal sorting, were not required for cross-linking to cyclophilin . We conclude that either cyclophilin is playing a nonessential facilitating role in peroxisomal targeting or that the interaction of the targeting peptide to cyclophilin is mimicking an interaction with an unidentified substrate or effector of cyclophilin.

Proteins, 1993 Feb, 15(2), 133 - 46
Successful prediction of the coiled coil geometry of the GCN4 leucine zipper domain by simulated annealing: comparison to the X-ray structure; Nilges M et al.; The recently solved X-ray structure of the dimerization region ("leucine zipper") of the yeast transcriptional activator GCN4 (O'Shea, E.K., Klemm, J.D., Kim, P.S., Alber, T . Science 254:539-544, 1991) is compared to previously predicted models which had been obtained by a conformational search procedure employing simulated annealing without any knowledge of the crystal coordinates (Nilges, M., Brunger, A.T . Protein Eng . 4:649-659, 1991) . During the course of the simulated annealing procedure, the models converged towards the X-ray structure . The averaged root mean square difference between the models and the X-ray structure is 1.26 and 1.75 A for backbone atoms and all nonhydrogen atoms at the dimerization interface, respectively . The local helix-helix crossing angle of the X-ray structure falls within the range predicted by the models; a slight unwinding of the coiled coil toward the N-terminal DNA-binding end of the dimerization region has been correctly predicted . Distance maps between the helices are largely identical . The region around asparagine 20 is asymmetric in the X-structure and in the models . Surface side chain dihedrals showed a large variation in the models although the chi 1, chi 2, chi 3, chi 4 3-fold dihedrals were correctly predicted in 69, 42, 43, and 44% of the cases, respectively . Phenomenological free energies of dimerization of the models show little correlation with the root mean square difference between the models and the X-ray structure.

Genes Dev, 1993 Feb, 7(2), 320 - 9
Stages in the second reaction of pre-mRNA splicing: the final step is ATP independent; Horowitz DS et al.; We have analyzed pre-mRNA splicing in yeast extracts immunodepleted of the PRP18 protein . We find that while the first step of splicing (cleavage at the 5' splice site, and generation of the exon 1 and lariat intermediates) is unaffected by the absence of PRP18, the second step of splicing (excision of the lariat intron and formation of mRNA) is substantially slower in the absence of PRP18 . The splicing intermediates that are formed in the absence of PRP18 can be rapidly chased into products by the addition of purified PRP18 protein . This chasing is not dependent on ATP, implying that ATP is not required during the second cleavage-and-ligation reaction . This result suggests that there are ordered stages within the second step of splicing and that PRP18 acts late in the second step, perhaps during the catalytic step . The ATP independence also supports the idea that this reaction proceeds by a transesterification mechanism.

Genes Dev, 1993 Feb, 7(2), 250 - 61
The extremely conserved amino terminus of RAD6 ubiquitin-conjugating enzyme is essential for amino-end rule-dependent protein degradation; Watkins JF et al.; The RAD6 gene of Saccharomyces cerevisiae encodes a ubiquitin-conjugating enzyme that is required for DNA repair, damage-induced mutagenesis, and sporulation . In addition, RAD6 mediates the multiubiquitination and degradation of amino-end rule protein substrates . The structure and function of RAD6 have been remarkably conserved during eukaryotic evolution . Here, we examine the role of the extremely conserved amino terminus, which has remained almost invariant among RAD6 homologs from yeast to human . We show that RAD6 is concentrated in the nucleus and that the amino-terminal deletion mutation, rad6 delta 1-9, does not alter the location of the protein . The amino-terminal domain, however, is essential for the multiubiquitination and degradation of amino-end rule substrates . In the rad6 delta 1-9 mutant, beta-galactosidase proteins bearing destabilizing amino-terminal residues become long lived, and purified rad6 delta 1-9 protein is ineffective in ubiquitin-protein ligase (E3)-dependent protein degradation in the proteolytic system derived from rabbit reticulocytes . The amino terminus is required for physical interaction of RAD6 with the yeast UBR1-encoded E3 enzyme, as the rad6 delta 1-9 protein is defective in this respect . The rad6 delta 1-9 mutant is defective in sporulation, shows reduced efficiency of DNA repair, but is proficient in UV mutagenesis . E3-dependent protein degradation by RAD6 could be essential for sporulation and could affect the efficiency of DNA repair.

Genetics, 1993 Feb, 133(2), 193 - 202
Insertional mutagenesis in Neurospora crassa: cloning and molecular analysis of the preg+ gene controlling the activity of the transcriptional activator NUC-1; Kang S et al.; The transcriptional activator NUC-1 controls the transcription of the genes for phosphorus acquisition enzymes, and its activity is regulated by the negative regulatory factors, PREG and PGOV In this report, we describe the cloning and molecular analysis of the preg+ gene . In Neurospora crassa, as in higher eukaryotes, transformation frequently results in nonhomologous integration of transforming DNA . Insertion of transforming DNA into host genes mutates the gene and provides a molecular tag for cloning it . We obtained two mutants that have an insertion in the preg+ and pgov+ genes, respectively, among 2 x 10(5) transformants . The preg+ gene was cloned by screening a Neurospora genomic DNA library with DNA sequences flanking the transforming DNA of the rescued plasmid . Northern analysis showed that the transcription of the preg+ gene is not regulated by phosphate . The carboxy-terminal half of PREG shows strong homology with Saccharomyces cerevisiae PHO80 whose function is analogous to that of PREG . The pregc mutations are located in the well conserved residues which may directly interact with the residues in the regulatory domain of NUC-1.

J Cell Biol, 1993 Feb, 120(4), 865 - 75
Cytosolic Sec13p complex is required for vesicle formation from the endoplasmic reticulum in vitro; Pryer NK et al.; The SEC13 gene of Saccharomyces cerevisiae is required in vesicle biogenesis at a step before or concurrent with the release of transport vesicles from the ER membrane . SEC13 encodes a 33-kD protein with sequence homology to a series of conserved internal repeat motifs found in beta subunits of heterotrimeric G proteins . The product of this gene, Sec13p, is a cytosolic protein peripherally associated with membranes . We developed a cell-free Sec13p-dependent vesicle formation reaction . Sec13p-depleted membranes and cytosol fractions were generated by urea treatment of membranes and affinity depletion of a Sec13p-dihydrofolate reductase fusion protein, respectively . These fractions were unable to support vesicle formation from the ER unless cytosol containing Sec13p was added . Cytosolic Sec13p fractionated by gel filtration as a large complex of about 700 kD . Fractions containing the Sec13p complex restored activity to the Sec13p- dependent vesicle formation reaction . Expression of SEC13 on a multicopy plasmid resulted in overproduction of a monomeric form of Sec13p, suggesting that another member of the complex becomes limiting when Sec13p is overproduced . Overproduced, monomeric Sec13p was inactive in the Sec13p-dependent vesicle formation assay.

Mol Cell Biol, 1993 Feb, 13(2), 961 - 9
Transcriptional activation by simian virus 40 large T antigen: interactions with multiple components of the transcription complex; Gruda MC et al.; Simian virus 40 (SV40) large T antigen is a potent transcriptional activator of both viral and cellular promoters . Within the SV40 late promoter, a specific upstream element necessary for T-antigen transcriptional activation is the binding site for transcription-enhancing factor 1 (TEF-1) . The promoter structure necessary for T-antigen-mediated transcriptional activation appears to be simple . For example, a promoter consisting of upstream TEF-1 binding sites (or other factor-binding sites) and a downstream TATA or initiator element is efficiently activated . It has been demonstrated that transcriptional activation by T antigen does not require direct binding to the DNA; thus, the most direct effect that T antigen could have on these simple promoters would be through protein-protein interactions with either upstream-bound transcription factors, the basal transcription complex, or both . To determine whether such interactions occur, full-length T antigen or segments of it was fused to the glutathione-binding site (GST fusions) or to the Gal4 DNA-binding domain (amino acids 1 to 147) (Gal4 fusions) . With the GST fusions, it was found that TEF-1 and the TATA-binding protein (TBP) bound different regions of T antigen . A GST fusion containing amino acids 5 to 172 (region T1) efficiently bound TBP . TEF-1 bound neither region T1 nor a region between amino acids 168 and 373 (region T2); however, it bound efficiently to the combined region (T5) containing amino acids 5 to 383.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Cell Biol, 1993 Feb, 13(2), 1130 - 6
B-myc inhibits neoplastic transformation and transcriptional activation by c-myc; Resar LM et al.; B-myc is a recently described myc gene whose product has not been functionally characterized . The predicted product of B-myc is a 168-amino-acid protein with extensive homology to the c-Myc amino-terminal region, previously shown to contain a transcriptional activation domain . We hypothesized that B-Myc might also function in transcriptional regulation, although its role in regulating gene expression is predicted to be unique, because B-Myc lacks the specific DNA-binding motif found in other Myc proteins . To determine whether B-Myc could interact with the transcriptional machinery, we studied the transcriptional activation properties of a chimeric protein containing B-Myc sequences fused to the DNA-binding domain of the yeast transcriptional activator GAL4 (GAL4-B-Myc) . We found that GAL4-B-Myc strongly activated expression of a GAL4-regulated reporter gene in mammalian cells . In addition, full-length B-Myc was able to inhibit or squelch reporter gene activation by a GAL4 chimeric protein containing the c-Myc transcriptional activation domain . We also observed that B-Myc dramatically inhibited the neoplastic cotransforming activity of c-Myc and activated Ras in rat embryo cells . Because B-Myc inhibits both neoplastic transformation and transcriptional activation by c-Myc, we suggest that the transforming activity of c-Myc is related to its ability to regulate transcription . Whether B-Myc functions biologically to squelch transcription and/or to regulate transcription through a specific DNA-binding protein remains unestablished.

Mol Cell Biol, 1993 Feb, 13(2), 1013 - 22
FAR1 is required for posttranscriptional regulation of CLN2 gene expression in response to mating pheromone; Valdivieso MH et al.; Yeast cells arrest during the G1 interval of the cell cycle in response to peptide mating pheromones . The FAR1 gene is required for cell cycle arrest but not for a number of other aspects of the pheromone response . Genetic evidence suggests that FAR1 is required specifically for inactivation of the G1 cyclin CLN2 . From these observations, the FAR1 gene has been proposed to encode an element of the interface between the mating pheromone signal transduction pathway and the cell cycle regulatory apparatus . We show here that FAR1 is necessary for the decrease in CLN1 and CLN2 transcript accumulation observed in response to mating pheromone but is unnecessary for regulation of the same transcripts during vegetative growth . However, the defect in regulation of CLN1 expression is dependent upon CLN2 . We show that pheromone regulates the abundance of Cln2 at a posttranscriptional level and that FAR1 is required for that regulation . From these observations, we suggest that FAR1 function is limited to posttranscriptional regulation of CLN2 expression by mating pheromone . The failure of mating pheromone to repress CLN2 transcript levels in far1 mutants can be explained by the stimulatory effect of the persistent Cln2 protein on CLN2 transcription via the CLN/CDC28-dependent feedback pathway.

Cell Immunol, 1993 Feb, 146(2), 284 - 99
Invertebrate cytokines . III: Invertebrate interleukin-1-like molecules stimulate phagocytosis by tunicate and echinoderm cells; Beck G et al.; Phagocytosis is the predominant defense mechanism of invertebrates . Here we show that phagocytosis by echinoderm bladder amoebocytes and tunicate granular amoebocytes can be enhanced by invertebrate interleukin-1-like molecules . As little as 5 ng/ml of invertebrate interleukin-1 produced a significant stimulation of echinoderm and tunicate amoebocyte phagocytosis . Stimulation of phagocytosis by echinoderm interleukin-1-like molecules was inhibited by antisera to vertebrate interleukin-1 . Invertebrate interleukin-1 also acted as an opsonin when preincubated with erythrocytes or yeast . In addition, the cellular mechanisms of invertebrate phagocytosis were studied using pharmacologic agents to inhibit echinoderm amoebocyte phagocytosis . The energy requirements and involvement of cellular cytoskeletal elements in phagocytosis by bladder amoebocytes were similar to those of mammalian macrophages . These results demonstrate a role for interleukin-1 in invertebrate host defense mechanisms.

Genes Chromosomes Cancer, 1993 Feb, 6(2), 124 - 31
Regulation of transcription by the retinoblastoma protein; Horowitz JM; The product of the retinoblastoma gene (RB1) is believed to function as a negative regulator of cell growth . Recent experimental results suggest that RB1 may exert its growth-suppressing activity by regulating the transcription of a variety of growth-related genes, including FOS, MYC, and TGFBI . A series of biochemical and molecular analyses suggest that RB1 indirectly affects gene expression via cell-cycle-regulated interactions with transcription factors, such as E2F and SPI . Determination of the mechanisms regulating such protein-protein interactions and the identification of additional targets of RB1 function will provide vital insights into the role of this tumor-suppressor gene in mammalian cell proliferation.

Protein Sci, 1993 Feb, 2(2), 175 - 82
Mapping antibody binding sites on cytochrome c with synthetic peptides: are results representative of the antigenic structure of proteins?
Schwab C, Twardek A, Lo TP, Brayer GD, Bosshard HR.
Crystallographic work on antigen-antibody complexes has revealed that extensive surface areas of proteins may interact with antibodies . On the other hand, most experimental approaches to locate and define antigenic determinants of protein antigens rely on the linear sequence of the polypeptide chain . Hence the question arises whether mapping of antibody binding sites by analysis of the reactivity of anti-protein antibodies with synthetic peptides can provide a representative picture of the antigenic structure of a protein antigen . We have addressed this question using yeast iso-1 cytochrome c as a protein antigen against which antisera were raised in rabbits . The reaction of the antisera with 103 synthetic hexapeptides covering the entire sequence of cytochrome c was tested by the pepscan procedure in which peptides are coupled to polyethylene rods and tested by ELISA . For the assay, anti-cytochrome c antibodies were fractionated by affinity chromatography on native yeast iso-1 cytochrome c and on apo-cytochrome c; the latter is a random coil . It was found that only antibodies retained by the apo-cytochrome c affinity column react with synthetic peptides . These antibodies comprise a small fraction, probably less than 2%, of all cytochrome c-specific antibodies . The majority of antigenic determinants, which seem to consist of strongly conformation-dependent topographic epitopes, could not be uncovered by the peptide approach . Epitope mapping with short peptides seems of limited usefulness in the case of small, globular, and conformationally stable proteins like cytochrome c.

Science, 1993 Jan 29, 259(5095), 683 - 6
The effect of posttranslational modifications on the interaction of Ras2 with adenylyl cyclase; Kuroda Y et al.; Ras proteins undergo a series of posttranslational modifications that are critical for their cellular function . These modifications are necessary to anchor Ras proteins to the membrane . Yeast Ras2 proteins were purified with various degrees of modification and examined for their ability to activate their effector, adenylyl cyclase . The farnesylated intermediate form of Ras2 had more than 100 times higher affinity for adenylyl cyclase than for the unprocessed form . The subsequent palmitoylation reaction had little effect . In contrast, palmitoylation was required for efficient membrane localization of the Ras2 protein . These results indicate the importance of farnesylation in the interaction of Ras2 with its effector.

Cell, 1993 Jan 29, 72(2), 233 - 45
Oncogenic activity of the c-Myc protein requires dimerization with Max; Amati B et al.; c-Myc (Myc) and Max proteins dimerize and bind DNA through basic-helix-loop-helix-leucine zipper motifs (b-HLH-LZ) . Using a genetic approach, we demonstrate that binding to Max is essential for Myc transforming activity and that Myc homodimers are inactive . Mutants of Myc and Max that bind efficiently to each other but not to their wild-type partners were generated by either exchanging the HLH-LZ domains or reciprocally modifying LZ dimerization specificities . While transformation defective on their own, complementary mutants restore Myc transforming activity when coexpressed in cells . The HLH-LZ exchange mutants also have dominant negative activity on wild-type Myc function . In addition, wild-type max antagonizes myc function in a dose-dependent manner, presumably through competition of Max-Max and Myc-Max dimers for common target DNA sites . Therefore, Max can function as both suppressor and activator of Myc . A general model for the role of Myc and Max in growth control is discussed.

Cell, 1993 Jan 29, 72(2), 247 - 60
Molecular cloning and functional analysis of Drosophila TAF110 reveal properties expected of coactivators; Hoey T et al.; The general transcription factor TFIID is a multiprotein complex containing the TATA-binding protein and several associated factors (TAFs), some of which may function as coactivators that are essential for activated, but not basal, transcription . Here we describe the isolation and characterization of the first gene encoding a TAF protein . The deduced amino acid sequence of TAF110 revealed the presence of several glutamine- and serine/threonine-rich regions reminiscent of the protein-protein interaction domains of the regulatory transcription factor Sp1 that are involved in transcription activation and multimerization . In both Drosophila cells and yeast, TAF110 specifically interacts with the glutamine-rich activation domains of Sp1 . Moreover, purified Sp1 selectively binds recombinant TAF110 in vitro . These findings taken together suggest that TAF110 may function as a coactivator by serving as a site of protein-protein contact between activators like Sp1 and the TFIID complex.

Nature, 1993 Jan 28, 361(6410), 369 - 71
Resistance to cadmium mediated by ubiquitin-dependent proteolysis; Jungmann J et al.; Cadmium is a potent poison for living cells . In man, chronic exposure to low levels of cadmium results in damage to kidneys and has been linked to neoplastic disease and ageing, and acute exposure can cause damage to a variety of organs and tissues . Cadmium reacts with thiol groups and can substitute for zinc in certain proteins, but the reason for its toxicity in vivo remains uncertain . In eukaryotes, an important selective proteolysis pathway for the elimination of abnormal proteins that are generated under normal or stress conditions is ATP-dependent and mediated by the ubiquitin system . Substrates of this pathway are first recognized by ubiquitin-conjugating enzymes (or auxiliary factors) which covalently attach ubiquitin, a small and highly conserved protein, to specific internal lysine residues of proteolytic substrates . Ubiquitinated substrates are then degraded by the proteasome, a multisubunit protease complex . Here we show that expression of this ubiquitin-dependent proteolysis pathway in yeast is activated in response to cadmium exposure and that mutants deficient in specific ubiquitin-conjugating enzymes are hypersensitive to cadmium . Moreover, mutants in the proteasome are hypersensitive to cadmium, suggesting that cadmium resistance is mediated in part by degradation of abnormal proteins . This indicates that a major reason for cadmium toxicity may be cadmium-induced formation of abnormal proteins.

Biochemistry, 1993 Jan 26, 32(3), 908 - 17
Conformational analysis of {D-Ala9}alpha-factor and {L-Ala9}alpha-factor in solution and in the presence of lipid; Gounarides JS et al.; The conformations in solution and in the presence of lipid vesicles of {D-Ala9} and {L-Ala9} analogues of the alpha-factor (WHWLQLKPGQPMY) from the yeast Saccharomyces cerevisiae were examined by NMR spectroscopy . Although both peptides are flexible molecules, NOE and NH d delta/dT data indicate that the {D-Ala9}alpha-factor analogue in DMSO and aqueous solution adopts a type II beta-turn about residues 8 and 9 . In contrast, various NMR parameters for the less active {L-Ala9} analogue do not provide evidence for a regular secondary structure in solution . Transfer NOE data indicate that for both peptides binding to the lipid is strongest for the N-terminal residues . The C-terminus of the {D-Ala9} analogue appears to be more constrained in the bound state than the C-terminus of the {L-Ala9} analogue . This result is consistent with transfer NOE evidence that the type II beta-turn conformation of the {D-Ala9}alpha-factor is maintained in the lipid bound state.

Biochemistry, 1993 Jan 26, 32(3), 929 - 36
Exploring the interface between the N- and C-terminal helices of cytochrome c by random mutagenesis within the C-terminal helix; Fredericks ZL et al.; Buried within cytochrome c lies a highly-conserved helix-helix interface formed by the perpendicular packing of the C-terminal helix against the N-terminal helix . This interface involves a peg-in-hole interaction between Gly-6 and Leu-94 and an aromatic-aromatic interaction between Phe-10 and Tyr-97 . To gain insight into protein design, we investigated the relationship between the sequence of the interface and the physiological function of yeast iso-1-cytochrome c . A library of mutants at positions 94 and 97 of the C-terminal helix was created to examine the effect of novel amino acid combinations . We isolated 45 of the 400 possible amino acid combinations, 32 of which result in a functional cytochrome c . Contrary to evolutionary conservation of the peg-in-hole and aromatic-aromatic interactions, we find that side-chain volume and conservation of aromatic residues do not play an essential role in determining function . Additionally, we find negatively-charged residues within the interface that result in a functional cytochrome c . Examination of the 45 missense mutants indicates that approximately 120 unique combinations are compatible with function . These results show that the interface is flexible . However, truncation of the C-terminal helix at position 94 abolishes function, suggesting that the interface is essential . The correlation observed between our library of mutants and the mutation matrix compiled by Gonnet et al . {Gonnet, G . H., Cohen, M . A., & Benner, S . A . (1992) Science 256, 1443-1445} demonstrates the potential use of the matrix to predict the effect of sequence changes on natural proteins and to optimize the design of novel proteins.

J Biol Chem, 1993 Jan 25, 268(3), 2113 - 9
Site-directed mutagenesis of HIV-1 integrase demonstrates differential effects on integrase functions in vitro; Leavitt AD et al.; The retroviral integrase (IN) protein is essential for integration of retroviral DNA into the host cell genome . To identify functional domains within the protein and to assess the importance of conserved residues, we performed site-directed mutagenesis of HIV-1 IN and analyzed the mutants in vitro for IN-mediated activities: 3' processing (att site-specific nuclease activity), strand transfer (the joining of att site oligonucleotides to target DNA), disintegration (the reverse of strand transfer), and integration site selection . Changing the conserved residue His-16 either to Cys or to Val in a proposed zinc-finger region had minimal effect on IN activities . Alteration of two highly conserved amino acid residues, Asp-116-->Ile and Glu-152-->Gly, each resulted in complete or nearly complete loss of 3' processing, strand transfer, and disintegration, whereas alteration of another conserved residue, Trp-235-->Glu, had no demonstrable effect on any of the activities in vitro . Two mutants, Asp-64-->Val and Arg-199-->Cys delta, each demonstrated differential effects on IN activities . Asp-64-->Val has no demonstrable strand transfer or disintegration activity yet maintains 3' processing activity at a diminished level . Arg-199-->Cys delta, which lacks part of the carboxyl terminus of IN, has impaired strand transfer activity without loss of disintegration activity . Use of a target site selection assay showed that all of our mutants with strand transfer activity maintain the same integration pattern as wild type IN . We conclude that not all highly conserved IN residues are essential for IN activities in vitro, zinc coordination by the proposed zinc-finger domain may not be required for the activities assayed, alteration of single residues can yield differential effects on IN activities, and target site selection into naked DNA is not necessarily altered by changes in strand transfer activity.

Nucleic Acids Res, 1993 Jan 25, 21(2), 267 - 72
Transcription regulation by murine B-myb is distinct from that by c-myb; Watson RJ et al.; The transcription regulatory properties of murine B-myb protein were compared to those of c-myb . Whereas c-Myb trans-activated an SV40 early promoter containing multiple copies of an upstream c-Myb DNA-binding site (MBS-1), and similarly the human c-myc promoter, B-Myb was unable to do so . Full-length B-Myb translated in vitro did not bind MBS-1; however, truncation of the B-Myb C-terminus or fusion of the B-Myb DNA-binding domain to the c-Myb C-terminus showed that it was inherently competent to interact with this motif . Further evidence from co-transfection experiments, demonstrating that B-Myb inhibited trans-activation by c-Myb, suggested that failure of B-Myb to trans-activate these promoters did not simply occur through lack of binding to MBS-1 . Moreover, using GAL4/B-Myb fusions, it was found that an acidic region of B-Myb, which by comparison to c-Myb was expected to contain a transcription activation domain, actually had no inherent trans-activation activity and indeed appeared to trans-inhibit c-Myb . In contrast to the above findings, both B-Myb and c-Myb were able to weakly trans-activate the DNA polymerase alpha promoter . Results obtained here demonstrate that the activities of B-Myb and c-Myb are clearly distinct and suggest that these related proteins may have different functions in regulation of target gene expression.

J Biol Chem, 1993 Jan 25, 268(3), 1735 - 41
Hydrolysis of short acyl chain inositol lipids by phospholipase C-delta 1; Rebecchi MJ et al.; We investigated the relationship between substrate aggregation and activation of phosphoinositide-specific phospholipase C-delta 1 (PLC-delta 1), isolated from bovine brain cytosol . The inositol lipids 1,2-dibutyryl-sn-glycero-3-phosphoinositol (di-C4-PI), 1,2-dihexanoyl-sn-glycero-3-phosphoinositol (di-C6-PI), and 1,2-dioctanoyl-sn-glycero-3-phosphoinositol (di-C8-PI) were prepared from synthetic cytidine diphosphate diglyceride analogs in a reaction with myo-inositol catalyzed by yeast phosphatidylinositol synthase . All three lipids served as substrates for PLC-delta 1 at concentrations significantly below their critical micelle concentration (cmc) . Under these conditions, steps that might limit the reaction rate, such as membrane adsorption or penetration into the phospholipid surface, were eliminated . Below the cmc, the concentration of lipid substrate required to produce hydrolysis followed the order: di-C8-PI < di-C6-PI << di-C4-PI . Calcium was essential for hydrolysis of the short chain substrates at all lipid concentrations tested . The dependence of the reaction on calcium suggests that this ion activates PLC-delta 1 at a step other than adsorption to or penetration of the membrane surface . As the concentration of di-C8-PI was raised above the cmc, the reaction velocity increased 2-3-fold . These results are consistent with the idea that micellar or bilayer aggregates of phosphoinositol are not required for PLC-catalyzed hydrolysis, although the reaction rate is enhanced by micelle formation.

Science, 1993 Jan 22, 259(5094), 510 - 3
Altered specificity of DNA-binding proteins with transition metal dimerization domains; Cuenoud B et al.; The bZIP motif is characterized by a leucine zipper domain that mediates dimerization and a basic domain that contacts DNA . A series of transition metal dimerization domains were used to alter systematically the relative orientation of basic domain peptides . Both the affinity and the specificity of the peptide-DNA interaction depend on domain orientation . These results indicate that the precise configuration linking the domains is important; dimerization is not always sufficient for DNA binding . This approach to studying the effect of orientation on protein function complements mutagenesis and could be used in many systems.

Nature, 1993 Jan 21, 361(6409), 226 - 33
The gene involved in X-linked agammaglobulinaemia is a member of the src family of protein-tyrosine kinases; Vetrie D et al.; X-linked agammaglobulinaemia (XLA) is a human immunodeficiency caused by failure of pre-B cells in the bone marrow to develop into circulating mature B cells . A novel gene has been isolated which maps to the XLA locus, is expressed in B cells, and shows mutations in families with the disorder . The gene is a member of the src family of proto-oncogenes which encode protein-tyrosine kinases . This is, to our knowledge, the first evidence that mutations in a src-related gene are involved in human genetic disease.

J Mol Biol, 1993 Jan 20, 229(2), 566 - 9
Crystallographic characterization of a PHO4-DNA complex; Hakoshima T et al.; Crystals have been obtained of the DNA-binding domain of the yeast transcription factor PHO4 in complexes with several synthetic fragments of DNA with appropriate cognate sequences . Crystals suitable for X-ray diffraction studies were produced in the case of a complex of the protein with a 17 base-pair fragment of DNA from a solution in polyethylene glycol and calcium chloride . The crystals have the space group of P4(1)2(1)2 or P4(3)2(1)2 with unit cell dimensions a = b = 56.7 A, c = 447.8 A . The diffraction data at 3 A resolution were collected using synchrotron radiation with a Weissenberg camera for macromolecular crystallography.

Biochemistry, 1993 Jan 19, 32(2), 708 - 14
Characterization of an intermediate in the folding pathway of phosphoglycerate kinase: chemical reactivity of genetically introduced cysteinyl residues during the folding process; Ballery N et al.; The unfolding-refolding kinetics of yeast phosphoglycerate kinase were studied using the chemical reactivity of genetically introduced cysteinyl residues as conformational probes and far-ultraviolet circular dichroism . A unique internal cysteinyl residue was introduced in several mutants at selected positions in the N- and C-domains . The cysteinyl residues were at positions 97 (the unique cysteinyl residue of the wild-type enzyme), 183 in the N-domain, 285 and 324 in the C-domain . A similar strategy has been used to study the unfolding-refolding transition under equilibrium conditions {Ballery et al . (1990) Protein Eng . 3, 199-204} . Except for the mutant C97A,A183C, whose cysteinyl residue is located at the domain interface, three labeling phases were observed during the refolding process, indicating the presence of three species, the unfolded, intermediate, and folded proteins . The comparison of the data obtained following the accessibility of the thiol group to 5,5'-dithiobis(2-nitrobenzoate) and ellipticity at 218 nm indicated that all mutants have the same folding pathway and allowed us to characterize the intermediate . In this species, each domain appeared to have a high content of secondary structure but a flexible tertiary structure; this intermediate, which had the characteristics of a molten globule, remained in fluctuating equilibrium with a widely unfolded form . The same folding intermediate was detected for mutant C97A,A183C; however, the cysteinyl residue being totally accessible to the reagent, it is likely that in this intermediate the interdomain interactions are not established . Domain pairing and formation of the native tertiary structure occur simultaneously in the slow phase of refolding . The validity and limitations of the methodology are discussed.

FEBS Lett, 1993 Jan 18, 316(1), 12 - 6
Localization of Kex2-like processing endoproteases, furin and PC4, within mouse testis by in situ hybridization; Torii S et al.; By in situ hybridization analysis, we show here the localization of furin and PC4, which are both members of a growing family of endoproteases structurally related to the yeast precursor processing protease Kex2, within mouse testis . Furin transcript was detected in both germ and somatic cells, while PC4 transcript was found only in round spermatids . Proenkephalin transcript was also localized in round spermatids . These observations suggest that, within testis, PC4 is involved in processing of peptide precursors such as proenkephalin and may play a role in regulation of sperm maturation, while furin may serve as a more general processing endoprotease.

Biochim Biophys Acta, 1993 Jan 18, 1145(1), 180 - 2
Misuse of graphical analysis in nonlinear sugar transport kinetics by Eadie-Hofstee plots; Fuhrmann GF et al.; It has become common practice to analyse the sugar transport kinetics from initial uptake rates in Saccharomyces cerevisiae cells with Eadie-Hofstee plots . These plots often demonstrate a nonlinear behaviour . They have been resolved incorrectly into two quasilinear components indicating the presence of (at least) two uptake systems or components, with Km values differing by a factor of about 10 . This graphical analysis neglects the obvious additivity of the two hypothetical systems and is therefore in error . A more efficient way to determine kinetic parameters from initial uptake experiments is to use computer-assisted nonlinear regression analysis.

Biochem J, 1993 Jan 15, 289 ( Pt 2), 599 - 604
The competition plot: a simple test of whether two reactions occur at the same active site; Chevillard C et al.; The competition plot is a method for determining whether or not two enzyme-catalysed reactions occur at the same active site . It is a plot of total rate against p, where p varies from 0 to 1 and specifies the concentrations (1-p)a0 and pb0 of two substrates in terms of reference concentrations a0 and b0 chosen so as to give the same rates at p = 0 and p = 1 . If the two substrates react at the same site, the competition plot gives a horizontal straight line, i.e . the total rate is independent of p . Independent reactions at two separate sites give a curve with a maximum; separate reactions with cross-inhibition generate curves with either maxima or minima according to whether the Michaelis constants of the two substrates are smaller or larger than their inhibition constants in the other reactions . Although ambiguous results can sometimes arise, experimental strategies exist for avoiding them, for example working as close as possible to the lower of the two limiting rates . When tested with yeast hexokinase, the plot indicated phosphorylation of glucose and fructose at the same site . Conversely, with a mixture of yeast hexokinase and galactokinase it indicated phosphorylation of glucose and galactose at different sites . In both cases the observed behaviour agreed with the known properties of the enzymes . A slight modification to the definition of this plot allows it to be applied also to enzymes that deviate from Michaelis-Menten kinetics.

Cell, 1993 Jan 15, 72(1), 131 - 42
The DNA-dependent protein kinase: requirement for DNA ends and association with Ku antigen; Gottlieb TM et al.; The DNA-dependent protein kinase (DNA-PK) phosphorylates Sp1 and several other nuclear proteins . Here, we show that Sp1 and the DNA-PK must be colocalized on the same DNA molecule for efficient phosphorylation to occur . Interestingly, we find that the DNA-PK binds to and is activated by the ends of DNA molecules . Furthermore, we show that the DNA binding properties of the DNA-PK are identical to those of Ku, a well-characterized human autoimmune antigen . We demonstrate that the DNA-PK can be fractionated into two components, one of which is Ku and the other of which is a polypeptide of approximately 350 kd . DNA cross-linking and coimmunoprecipitation studies indicate that the catalytic 350 kd DNA-PK component is directed to DNA by protein-protein interactions with Ku . The implications of the unusual DNA binding mode and multicomponent nature of the DNA-PK are discussed.

Biochem Biophys Res Commun, 1993 Jan 15, 190(1), 289 - 93
Molecular cloning of the smaller subunit(P52) of rat liver mitochondrial processing protease; Kitada S et al.; A cDNA encoding the smaller subunit (P52) of mitochondrial processing protease was isolated from a rat liver cDNA library, using cDNA fragment for yeast MAS1 as the probe . The deduced amino acid sequence is highly homologous to those of PEP from Neurospora crassa and MAS1 from Saccharomyces cerevisiae . After in vitro transcription and translation, the precursor peptide was imported into isolated rat liver mitochondria and processed to its mature form.

J Biol Chem, 1993 Jan 15, 268(2), 873 - 9
Purification and characterization of SAR1p, a small GTP-binding protein required for transport vesicle formation from the endoplasmic reticulum; Barlowe C et al.; SEC12 encodes an integral membrane glycoprotein essential for vesicle formation from the endoplasmic reticulum (ER) in yeast . The SAR1 gene was discovered as a multicopy suppressor of a sec12ts strain and encodes a 21-kDa GTP-binding protein also required for protein transport from the ER to the Golgi apparatus (Nakano, A., and Muramatsu, M . (1989) J . Cell Biol . 109, 2677-2691) . We have purified Sar1p to apparent homogeneity from cells harboring a galactose-regulated recombinant SAR1 . Purified Sar1p binds guanine nucleotides specifically and exhibits GTPase activity (0.001 min-1) . Nucleotide exchange and hydrolysis rates are greatly increased in the presence of Mg2+ and nonionic detergents or phospholipids . An assay that measures the formation of a vesicle intermediate in ER to Golgi transport was devised that is dependent on the addition of purified Sar1p . This assay employs membranes prepared from wild-type cells and cytosol fractions depleted of Sar1p due to overproduction of Sec12p or by gel filtration chromatography . The gel-filtered cytosol requires the addition of Sar1p and GTP to support vesicle budding . Sar1p prebound with GTP gamma S inhibits Sar1p function in the vesicle formation assay . The results indicate a role for Sar1p in vesicle budding from the ER and suggest that GTP hydrolysis by Sar1p is required for this event.

J Biol Chem, 1993 Jan 15, 268(2), 1383 - 90
Mutational analysis of alpha-subunit of protein farnesyltransferase . Evidence for a catalytic role; Andres DA et al.; Protein farnesyltransferase from rat brain is composed of tightly associated alpha- and beta-subunits of 377 and 437 amino acids that migrate on SDS-polyacrylamide gels with apparent molecular masses of 49 and 46 kDa, respectively . The enzyme attaches farnesyl groups to cysteines in p21ras and other proteins that contain cysteine residues at the fourth position from the COOH terminus . Production of stable enzyme in animal cells requires the simultaneous synthesis of both subunits, and all activity is lost when the subunits are dissociated chemically . The beta-subunit functions in the Zn(2+)-dependent binding of the protein substrate . The role of the alpha-subunit is unknown . In the current studies we used in vitro mutagenesis and transfection of cloned cDNAs to define the parts of the alpha-subunit that are necessary to stabilize the beta-subunit and to support farnesyl transfer . Deletion of 51 amino acids at the NH2 terminus of the alpha-subunit allowed normal stabilization of the beta-subunit and production of normal enzyme activity, but deletion of 106 amino acids abolished both of these properties . A proline-rich region at residues 12-34 of the alpha-subunit is not required for activity, but its presence explains the anomalously slow migration of the polypeptide on SDS-polyacrylamide gels . Deletion of only 5 amino acids at the COOH terminus of the alpha-subunit reduced activity appreciably . Substitution of asparagine for a conserved lysine at position 164 produced an alpha-subunit that stabilized the beta-subunit normally and permitted normal binding of the two substrates, farnesyl pyrophosphate and p21H-ras . Nevertheless, the rate of transfer of the bound farnesyl group to p21H-ras was markedly reduced . The latter finding suggests that the alpha-subunit plays a direct role in the catalytic reaction in addition to its role in the stabilization of the beta-subunit.

J Biol Chem, 1993 Jan 15, 268(2), 1101 - 8
Mutations of the FLP recombinase gene that cause a deficiency in DNA bending and strand cleavage; Kulpa J et al.; We have used site-directed mutagenesis to change several amino acids of the C-terminal portion of the FLP recombinase of Saccharomyces cerevisiae . These residues are absolutely conserved among the six FLP-like proteins from various yeast strains . We have examined the ability of the altered proteins to catalyze recombination in vivo and in vitro and to perform various partial steps of the reaction in vitro . Two of the mutations produced a partial defect in DNA binding but the remainder resulted in normal binding . All of these mutations caused impairment of the ability of the protein to induce the type II bend of the FRT site, and some of these proteins were also defective in DNA strand cleavage . None of the mutations affected the ability of the proteins to perform synapsis between two FRT sites, but some were defective in strand ligation . Interestingly, some mutant proteins showed impairment of the initial stages of the recombination reaction on a linear substrate and yet they maintained the ability to resolve a Holliday intermediate in the reaction . We conclude that this conserved region of the FLP protein is important for the early stage(s) of the recombination reaction.

Nature, 1993 Jan 14, 361(6408), 170 - 3
A role for reverse transcripts in gene conversion; Derr LK et al.; Recombination between a diffusible reverse transcript and its homologous chromosomal allele has been proposed as a mechanism for the precise removal of introns from DNA and gene conversion of dispersed repeated sequences . We have reported that RNA-mediated recombination occurs in the yeast Saccharomyces cerevisiae . This recombination requires expression of the retrotransposon Ty, and results in intron loss from a plasmid-borne marker gene and the formation of pseudogenes . Because the pseudogenes are embedded in Ty sequences, chromosomal insertion could have been mediated by Ty integrase or by homologous recombination with endogenous Ty sequences . The structure of the chromosomal recombinants and the fact that plasmid and chromosomal recombination can have different requirements demanded a direct demonstration of RNA-mediated gene conversion of a chromosomal allele . Here we report the first demonstration, to our knowledge, of recombination between a reverse transcript and its chromosomal homologue and describe an assay that specifically detects this novel recombination pathway.

Biochemistry, 1993 Jan 12, 32(1), 298 - 309
NMR studies of the dynamics of the multidomain protein urokinase-type plasminogen activator; Nowak UK et al.; u-PA (urokinase-type plasminogen activator or urokinase) has been studied under a variety of solution conditions by 1-D and 2-D NMR spectroscopy . Very high quality spectra could be obtained from the recombinant protein despite the high molecular mass (46 kDa) by appropriate choice of solution conditions; mildly acidic pH and low ionic strength were found to be optimal . Comparison of spectra of u-PA with spectra of the isolated kringle and protease domains, the EGF-kringle pair, and a synthetic peptide from the kringle-protease linker region, enabled sequential assignments in the u-PA spectrum to be made for kringle resonances, and domain-specific assignments for many others . Simulations of line shapes in both 1-D and 2-D spectra enabled effective correlation times for the different domains, both isolated and in the intact protein, to be determined . These have permitted a model of the u-PA dynamics to be put forward involving extensive, but not unrestricted, motion between the different domains.

Biochemistry, 1993 Jan 12, 32(1), 183 - 90
Destabilizing effects of replacing a surface lysine of cytochrome c with aromatic amino acids: implications for the denatured state; Bowler BE et al.; A series of mutations at the highly solvent-exposed lysine 73 of iso-1-cytochrome c have been prepared by site-directed mutagenesis . These mutations were designed to probe denatured-state effects on the unfolding equilibrium of this protein . The hydrophilic amino acid Lys was replaced with the hydrophobic amino acids Met, Tyr, Phe, and Trp . The idea was to induce stabilizing hydrophobic interactions in the unfolded state, while having little effect on the folded-state energy due to the high solvent exposure of this site . Fourier transform infrared spectral analyses indicate that none of these mutations significantly affect the native fold of the protein . The stability of each protein to guanidine hydrochloride denaturation was monitored at 25 degrees C by circular dichroism spectroscopy . All four hydrophobic mutants decreased the value of delta Go uH2O, the free energy of unfolding of the protein in the absence of denaturant, by 1.0-1.5 kcal/mol . The delta Go uH2O values for these proteins correlate linearly (correlation coefficient of 0.98) with the hydrophobicity of the amino acid at position 73 of the sequence . These data are consistent with the idea that the position-73 mutants are more buried in the denatured state than in the native state, suggestive of a compact denatured state where such interactions would be possible.

J Biol Chem, 1993 Jan 5, 268(1), 305 - 14
Studies of the DNA binding properties of histone H4 amino terminus . Thermal denaturation studies reveal that acetylation markedly reduces the binding constant of the H4 "tail" to DNA; Hong L et al.; The effect of acetylation on the DNA binding properties of the rigidly conserved histone H4 amino-terminal tail has been studied in detail using the technique of thermal denaturation . The quantitative DNA-binding parameters for both the non- and fully acetylated H4 amino terminus have been determined from thermal denaturation data for complexes of the peptides bound to mixed sequence 146-base pair DNA . We find that under dilute buffer conditions (5 mM Tris-HCl) the binding constant for the non-acetylated peptide to double-stranded DNA is 5 x 10(11) M-1 and that acetylation of lysine residues in the peptide reduces the binding constant to 1 x 10(5) M-1 . The dramatic differences observed in the binding constants for the non- and fully acetylated peptides are probably due to the effect of acetylation on the even distribution of positively charged residues in the H4 amino terminus . In other experiments, the binding of both peptides to a 30-base pair oligonucleotide has been studied in solution with varying concentrations of sodium, magnesium, and phosphate ions . These experiments demonstrate that both magnesium and phosphate ions have strong effects on the binding of the H4 tail to DNA, especially weakening the binding of the acetylated peptide . For instance, the dissociation of the non-acetylated peptide from DNA requires 6 mM magnesium, yet the binding of the acetylated peptide is abolished in only 30 microM magnesium . The modulation of the DNA binding interactions of the H4 amino terminus by physiologically relevant ionic conditions, in addition to the effect of acetylation, can be important in the regulation of chromatin structure and function.

J Biol Chem, 1993 Jan 5, 268(1), 5 - 8
Detection and modulation in vivo of helix-loop-helix protein-protein interactions; Finkel T et al.; Studies are described that allow for the in vivo detection of helix-loop-helix (HLH) protein-protein interaction . The assay used requires HLH protein-protein interaction to reconstitute a functional GAL4 transcriptional activator, which in turn activates a reporter gene placed downstream of GAL4 DNA binding sequences . Using this assay, we are able to detect intracellular heterodimerization but not homodimerization of the MyoD, E12, and Id gene products . In addition, using this system we are unable to detect stable heterodimerization between MyoD and c-Jun . We also show that expression of activated rasH gene product does not inhibit and may stabilize HLH protein-protein interaction . This system may be of general utility in studying the modulation of transcription factor interactions.

J Biol Chem, 1993 Jan 5, 268(1), 235 - 40
Characterization of cathepsin B specificity by site-directed mutagenesis . Importance of Glu245 in the S2-P2 specificity for arginine and its role in transition state stabilization; Hasnain S et al.; The pH dependence of cathepsin B-catalyzed hydrolyzes is very complex . At least seven dissociable groups are involved in the binding and hydrolysis of 7-amido-4-methyl coumarin and p-nitroaniline (pNA)-based substrates containing a P1 Arg and either a Phe or Arg at the P2 position . By site-directed mutagenesis we show that a previous suggestion, that Arg202 is one of the groups which influences the pH dependence of cathepsin B-catalyzed hydrolysis of the Z-Arg-Arg-pNA substrate, is not valid . However, it was found that Glu245, which has a pKa of 5.1 in rat cathepsin B, is responsible for the S2-P2 specificity for Arg-containing substrates and controls the pH dependence of their hydrolysis . Furthermore, the data indicate that Glu245, which forms a hydrogen bond with the guanidinium group of the substrate's P2 Arg, contributes about 1.8 kcal/mol to transition state stabilization in the protonated state and about 0.6 kcal/mol in the deprotonated state . Mutation of Glu245 to Gln results in a 16-fold decrease in kcat but does not affect Km . While cathepsin B has a 7-fold preference for Phe over Arg at the P2 position of a substrate, binding of the aromatic side chain does not appear to be influenced by Glu245.

Minerva Stomatol, 1993 Jan-Feb, 42(1-2), 15 - 8
{DNA extraction from hard dental tissues}; Avitabile M et al.; Two different standard ways of DNA extraction (salting out and phenol-chloroform methods) were assayed in order to recovery nucleic acids from dental tissues . The DNA extracted was tested for purity by means of transverse alternating field electrophoresis (TAFE) using Saccharomyces cerevisiae chromosomes as markers . Both extraction methods give similar qualitative and quantitative results being a DNA yield from hard dental tissues approximately 30% of those extracted from the whole tooth . Our results indicate salting out as a preferable method due to its rapidity and usefulness.

Hum Mol Genet, 1993 Jan, 2(1), 55 - 60
Localization of seven new genes around the HLA-A locus; el Kahloun A et al.; A yeast artificial chromosome (YAC B30) with a 320 kb insert of genomic DNA which includes the HLA-A gene was used to screen a cDNA library of human duodenal mucosa . Seven cDNA clones were isolated which correspond to seven new non-HLA class I structural genes . These new genes are located within a region that may well contain the gene responsible for hemochromatosis and have therefore been named HCG I-VII (Hemochromatosis Candidate Gene) . HCG I, III, V and VI are probably single copy genes, situated at 180, 155, 140 and 230 kb centromeric to HLA-A, respectively . HCG II, IV and VII present several copies: one copy of HCG II, one of HCG IV and one of HCG VII are centromeric to HLA-A (at 30, 70 and 100 kb respectively) . Another copy of HCG IV is 20 kb telomeric to HLA-A . Each of the genes localized on the YAC B30 is associated with an CpG/HTF island.

Hum Mol Genet, 1993 Jan, 2(1), 35 - 8
Characterisation of the exon structure of the Fanconi anaemia group C gene by vectorette PCR; Gibson RA et al.; A cDNA for Fanconi anaemia complementation group C (FACC) has recently been cloned . We have now isolated a yeast artificial chromosome clone containing the FACC gene, and used vectorette PCR to determine its exon structure . The 1674-nucleotide coding sequence of the gene is highly interrupted, and contains 14 exons ranging in size from 53-204 bp . All exon donor and acceptor splice sites fit well with consensus sequences . Knowledge of the FACC exon boundaries and adjacent intron sequences was used to design polymerase chain reactions for amplification of all 14 exons from genomic DNA . Characterisation of splice site mutations in Fanconi anaemia patients with abnormal FACC transcripts and screening of large numbers of patients for mutations by amplification of the coding sequence from genomic DNA will now be possible.

Biofizika, 1993 Jan-Feb, 38(1), 108 - 16
{Analysis of the conformational mobility of DNA in transcriptionally-active chromatin}; Kraevskii VA et al.; An attempt is made to estimate the degree of conformational flexibility of DNA in different fractions of nucleosomes of transcriptionally active chromatin of higher eukaryotes, and in yeast nucleosomes as well . We have used the circular dichroism method to estimate conformational changes in DNA induced by temperature shift . The DNA conformational potential was shown to be approximately the same in all the fractions of nucleosomes under study.

Vaccine, 1993, 11(4), 449 - 56
An experimental vaccine cocktail for Plasmodium falciparum malaria; Bathurst IC et al.; Surface proteins from several different life-cycle stages of the malaria parasite Plasmodium falciparum were expressed at high levels in the yeast Saccharomyces cerevisiae . Purified proteins, both individually and in cocktails, were used to immunize mice and goats in conjunction with either Freund's adjuvant or a muramyl tripeptide-based adjuvant . Immune responses were measured by enzyme-linked immunosorbent assays and by the ability of antisera to inhibit (1) the invasion of hepatocytes by live sporozoites, (2) in vitro invasion of human erythrocytes by live merozoites, and (3) the development of oocytes in the mosquito vector . These results suggest that cocktails of different stage-specific antigens can provide the components necessary to block the development of the malaria parasite at multiple stages of its life cycle.

Mech Ageing Dev, 1993 Jan, 66(3), 283 - 98
Gene expression and aging; Thakur MK et al.; Considerable amount of data has accumulated during the past few years showing several changes in gene expression as a function of age . However, the basic mechanism of aging still remains poorly understood . In this review, we have mainly analysed the data pertaining to the hypothesis that aging is associated with genetic instability and have attempted further to highlight the gaps that need to be bridged in order to have a clear picture of the aging phenomenon . Extensive investigations employing new and novel approaches are needed in future to elucidate the intricately interwoven patterns of molecular control that underlie the various aspects of gene expression during aging.

Mol Carcinog, 1993, 7(2), 76 - 82
Creation of an active estrogen-responsive element by a single base change in the flanking sequence of a cellular oncogene: a possible mechanism for hormonal carcinogenesis?
Nawaz Z, Stancel GM, McDonnell DP, Hyder SM.
Estrogens are considered to act as promoters in a multistep process of hormonal carcinogenesis, although the molecular mechanisms by which these hormones act in tumorigenesis are unclear at present . Estradiol is known to induce expression of certain proto-oncogenes, and this led us to examine potential regulatory regions of the cellular c-fos oncogene . The 5'-flanking region of the murine c-fos contains a 13-bp palindromic sequence (GGTCTnnnAGACC) with striking homology to the consensus estrogen-responsive element (ERE) GGTCAnnnTGACC . However, the c-fos sequence did not bind the human estrogen receptor or confer hormonal responsiveness in a yeast-based transcriptional test system . Importantly, a single base change in the fifth position of the c-fos sequence (GGTCTnnnAGACC to GGTCA/GnnnAGACC) produced an element that bound the estrogen receptor and conferred estrogen-dependent transcriptional activation of a reporter gene . This suggests a specific hypothesis by which estrogens could act as tumor promoters . In this paradigm, the regulatory region of the cellular oncogenes, tumor suppressor genes, and growth-factor genes contain inactive sequences with close homologies to hormone-responsive elements . Initiation occurs when some agent (e.g., a chemical carcinogen) causes a mutation in such a sequence to create a functional hormone-responsive element . Estrogens, acting through their receptors and the mutated element, can then activate the target gene to stimulate cell proliferation and increase the population of initiated cells.

Dev Comp Immunol, 1993 Jan-Feb, 17(1), 29 - 39
A humoral opsonin from the solitary urochordate Styela clava; Kelly KL et al.; Opsonins play a key role in invertebrate humoral immune systems . An opsonin for yeast was identified in the plasma of the tunicate, Styela clava . In vitro cultures of hemocytes with homologous plasma-incubated yeast exhibited significantly higher levels of phagocytosis than controls . Studies indicated that the opsonic activity of Styela clava plasma increased the overall capacity for phagocytosis . Opsonization was inhibited by the carbohydrates mannan, N-acetyl-D-galactosamine, and galactose, and by the divalent cation chelator, EDTA . These data suggest that the Styela clava opsonin may share some functional similarities with a C-type lectin . Incubation of yeast with Styela clava and Styela plicata plasma prior to phagocytosis by hemocytes from both species indicated the Styela clava opsonin is species specific.

Mol Biol Cell, 1993 Jan, 4(1), 79 - 92
Phosphorylation independent activation of human cyclin-dependent kinase 2 by cyclin A in vitro; Connell-Crowley L et al.; p33cdk2 is a serine-threonine protein kinase that associates with cyclins A, D, and E and has been implicated in the control of the G1/S transition in mammalian cells . Recent evidence indicates that cyclin-dependent kinase 2 (Cdk2), like its homolog Cdc2, requires cyclin binding and phosphorylation (of threonine-160) for activation in vivo . However, the extent to which mechanistic details of the activation process are conserved between Cdc2 and Cdk2 is unknown . We have developed bacterial expression and purification systems for Cdk2 and cyclin A that allow mechanistic studies of the activation process to be performed in the absence of cell extracts . Recombinant Cdk2 is essentially inactive as a histone H1 kinase (< 4 x 10(-5) pmol phosphate transferred.min-1 x microgram-1 Cdk2) . However, in the presence of equimolar cyclin A, the specific activity is approximately 16 pmol.mon-1 x microgram-1, 4 x 10(5)-fold higher than Cdk2 alone . Mutation of T160 in Cdk2 to either alanine or glutamic acid had little impact on the specific activity of the Cdk2/cyclin A complex: the activity of Cdk2T160E was indistinguishable from Cdk2, whereas that of Cdk2T160A was reduced by five-fold . To determine if the Cdk2/cyclin A complex could be activated further by phosphorylation of T160, complexes were treated with Cdc2 activating kinase (CAK), purified approximately 12,000-fold from Xenopus eggs . This treatment resulted in an 80-fold increase in specific activity . This specific activity is comparable with that of the Cdc2/cyclin B complex after complete activation by CAK (approximately 1600 pmol.mon-1 x microgram-1) . Neither Cdk2T160A/cyclin A nor Cdk2T160E/cyclin A complexes were activated further by treatment with CAK . In striking contrast with cyclin A, cyclin B did not directly activate Cdk2 . However, both Cdk2/cyclin A and Cdk2/cyclin B complexes display similar activity after activation by CAK . For the Cdk2/cyclin A complex, both cyclin binding and phosphorylation contribute significantly to activation, although the energetic contribution of cyclin A binding is greater than that of T160 phosphorylation by approximately 5 kcal/mol . The potential significance of direct activation of Cdk2 by cyclins with respect to regulation of cell cycle progression is discussed.

Yeast, 1993 Jan, 9(1), 21 - 32
The SCH9 protein kinase mRNA contains a long 5' leader with a small open reading frame; di Blasi F et al.; The SCH9 yeast gene, that was previously identified as a suppressor of cdc25 and ras1- ras2-ts temperature-sensitive mutants, encodes a putative protein kinase that positively regulates the progression of yeast cells through the G1 phase of the cell cycle . We have determined the structure of the SCH9 transcription unit, using primer extension and S1 mapping techniques . The corresponding mRNA included an unusually long 5' region of more than 600 nucleotides preceding the major open reading frame (ORF) . While the latter corresponded to a protein of 824 amino acids, an upstream open reading frame (uORF) within the 5' leader could potentially encode a 54 amino acid peptide . To investigate the role of the AUGs within the uORF, we engineered chimaeric plasmid vectors in which SCH9 sequences including the promoter, the mRNA leader and the first 514 nucleotides of the major ORF were fused in-frame with beta-galactosidase-coding sequences . Upon introduction into yeast cells, the fusion protein was efficiently expressed . However, mutational disruption of the uORF using oligonucleotide-directed mutagenesis did not affect the level of expression of the fusion protein . This indicates that regulatory mechanisms in Saccharomyces cerevisiae prevent upstream AUGs within the SCH9 mRNA leader sequence from influencing translation from downstream initiation codons.

Trends Biochem Sci, 1993 Jan, 18(1), 31 - 4
Pre-tRNA splicing: variation on a theme or exception to the rule?
Phizicky EM, Greer CL.
There has been a growing recognition that there are many conserved features among apparently diverse RNA splicing systems, suggesting that they may have a common origin . However, pre-tRNA splicing is an apparent exception in nearly all respects . Features of this unique class should be considered in any comprehensive discussion of the origin(s) of splicing and its implications for the evolution of gene structure.

Protein Eng, 1993 Jan, 6(1), 51 - 8
Molecular modeling of coiled-coil alpha-tropomyosin: analysis of staggered and in register helix-helix interactions; Cregut D et al.; In register and staggered models of tropomyosin coiled-coil were built from X-ray C alpha coordinates and refined via molecular dynamics . The two models show similar structural features with the X-ray structure of GCN4 leucine zipper . Empirical energetic methods used to compare the in register and staggered models indicate that both are equally probable . The two models have similar profiles of solvation free energy of folding for residues at positions a and d of the repeating heptad, indicating that residues at these positions are as well buried in an in register structure as in a staggered one . Neither the in register nor the 14 residues staggered structure can be ruled out based on hydrophobic or e-g' (g-e') electrostatic interactions which are not able to distinguish between the two models and are therefore not selective . However, the eg-b'c' electrostatic interactions, although smaller in magnitude, are in favor of the in register model . Furthermore, analysis of hydrophobic and electrostatic interactions along the tropomyosin sequence shows that bulky residues in positions a and d prevent the formation of inter-chain salt bridges.

EMBO J, 1993 Jan, 12(1), 177 - 86
CDC39, an essential nuclear protein that negatively regulates transcription and differentially affects the constitutive and inducible HIS3 promoters; Collart MA et al.; The yeast HIS3 promoter region contains two functionally distinct TATA elements, TC and TR, that are responsible respectively for initiation from the +1 and +13 sites . Both TC and TR support basal HIS3 transcription and require the TATA binding protein TFIID, but only TR responds to transcriptional activation by GCN4 and GAL4 . By selecting for yeast strains that increase transcription by a GCN4 derivative with a defective activation domain, we have isolated a temperature-sensitive mutation in CDC39, a previously defined gene implicated in cell-cycle control and the pheromone response . This cdc39-2 mutation causes increased basal transcription of many, but not all genes, as well as increased transcriptional activation by GCN4 and GAL4 . Surprisingly, basal HIS3 transcription from the +1 initiation site is strongly increased, while initiation from the +13 site is barely affected . Thus, unlike acidic activator proteins that function through TR, CDC39 preferentially affects transcription mediated by TC . CDC39 is an essential gene that encodes a very large nuclear protein (2108 amino acids) containing two glutamine-rich regions . These observations suggest that CDC39 negatively regulates transcription either by affecting the general RNA polymerase II machinery or by altering chromatin structure.

Mol Biochem Parasitol, 1993 Jan, 57(1), 1 - 14
Developmental gene expression in Eimeria bovis; Abrahamsen MS et al.; By differential screening of stage-specific cDNA libraries of Eimeria bovis, we have identified and isolated a large set of genes that are regulated during development of the sporozoites and merozoites . Duplicate lifts of cDNA libraries constructed from partially sporulated oocysts and merozoites were probed with radioactively labeled first-strand cDNA prepared from partially sporulated oocyst and merozoite mRNA . Out of 60,000 plaques screened in each case, over 250 plaques from the partially sporulated oocyst library preferentially hybridized with the oocyst cDNA probe and 67 plaques from the merozoite library preferentially hybridized with the merozoite cDNA probe . Three of the oocyst phage and 7 of the merozoite phage were selected for further characterization . Northern analysis revealed a common pattern of mRNA expression for the oocyst cDNA clones . Consistent with the results of the differential screen, no hybridization to merozoite RNA was detected with any of these 3 oocyst cDNA clones . The expression of the merozoite cDNA clones was more complex, with 3 different classes of merozoite genes being identified based on their pattern of developmental regulation . Although each of the merozoite clones was expressed to some extent during sporulation, in all cases, expression was higher in merozoites than in partially sporulated oocysts, consistent with the restriction of expression defined by the differential screen . Sequence analysis revealed that 2 of the merozoite cDNA clones encode elongation factor 1 alpha and the ubiquitin/ribosomal protein fusion, and 1 of the sporozoite cDNAs displays a significant identity to insulin-degrading enzyme . The developmental expression of E . bovis genes involved in protein synthesis and degradation provides additional evidence for the importance of regulation of protein metabolism during parasite development.

Mol Pharmacol, 1993 Jan, 43(1), 120 - 6
Identification and characterization of the cytochrome P450 enzymes involved in N-dealkylation of propafenone: molecular base for interaction potential and variable disposition of active metabolites; Botsch S et al.; The activity of metabolizing enzymes determines plasma concentrations and hence effects of drugs . Identification of these enzymes may allow the prediction of both the interaction potential of drugs and the variability deriving from certain pathways . The antiarrhythmic propafenone is extensively biotransformed to the active metabolites 5-hydroxypropafenone and N-desalkylpropafenone . Whereas 5-hydroxylation is catalyzed by CYP2D6, the enzyme involved in N-dealkylation has not been identified . We, therefore, characterized the enzyme involved in the formation of N-desalkylpropafenone by using both in vitro {human liver microsomes, specific antibodies or inhibitors, and stably expressed cytochrome P450 (P450) enzymes} and in vivo (formation of N-desalkylpropafenone in patients under conditions of chronic therapy) approaches . Formation of N-desalkylpropafenone can be described by Michaelis-Menten kinetics . A strong correlation was observed between maximum rate of formation (Vmax) of N-desalkylpropafenone and the amount of CYP1A2 (r = 0.83, p < 0.001) and CYP3A (r = 0.54, p < 0.05) in the microsomal fraction of 20 human livers . In vitro intrinsic clearances (derived from Vmax/Km) indicated a wide interindividual variability in seven human livers (from 0.01 to 0.1 ml/hr/mg of protein) . Antibodies directed against CYP3A and CYP1A2 inhibited formation of N-desalkylpropafenone by 54 +/- 10% and 24 +/- 16%, respectively . The CYP2D6-mediated formation of 5-hydroxypropafenone was unaffected by these antibodies . Verapamil (substrate of CYP3A4 and CYP1A2) and midazolam (substrate of CYP3A4) were competitive inhibitors of N-desalkylpropafenone formation (Ki = 70 microM and 25 microM for verapamil and midazolam, respectively) . Coding sequences for CYP1A2 and CYP3A4 were inserted in a yeast expression vector and introduced into Saccharomyces cerevisiae strain W(R) . Both CYP1A2 and CYP3A4 catalyzed N-dealkylation of propafenone, with specific activities of 0.32 pmol/min/pmol of P450 and 0.16 pmol/min/pmol of P450, respectively . Our data indicate that N-dealkylation of propafenone is mediated via CYP3A4 and CYP1A2 . From experiments on the molecular level interactions of propafenone with other drugs that are metabolized by CYP3A4 and CYP1A2 can be predicted . Such interactions have been reported for cyclosporin, rifampicin, warfarin, and theophylline . Moreover, in vitro intrinsic clearances showed a wide interindividual variability . Therefore, variable plasma concentrations of the active metabolite N-desalkylpropafenone are expected in vivo . We tested this hypothesis in 14 patients (dose of 150 mg of propafenone three times per day) during chronic oral therapy and observed steady state plasma concentrations of N-desalkylpropafenone ranging from 4 to 293 ng/ml.(ABSTRACT TRUNCATED AT 400 WORDS)

Mol Pharmacol, 1993 Jan, 43(1), 115 - 9
4-Methylpyrazole inhibits fatty acyl coenzyme synthetase and diminishes catalase-dependent alcohol metabolism: has the contribution of alcohol dehydrogenase to alcohol metabolism been previously overestimated?
Bradford BU, Forman DT, Thurman RG.
Alcohol dehydrogenase (ADH)-deficient deer mice were used as an animal model to investigate the effect of 4-methylpyrazole on alcohol metabolism . After intraperitoneal dosing of these mutant mice with 4-methylpyrazole, rates of ethanol and methanol metabolism in vivo were decreased significantly, by 41% and 35%, respectively . In perfused liver, rates of ethanol metabolism were also decreased up to 61% by 100 microM 4-methylpyrazole . Further, when livers were perfused with methanol, a selective substrate for catalase, rates of methanol metabolism were decreased by 64% by 4-methylpyrazole . It was further determined that 4-methylpyrazole administration caused negligible changes in total hepatic catalase activity and in rates of oxidation of ethanol by isolated microsomes; rather, it acts on catalase-dependent alcohol metabolism by limiting the supply of H2O2 . In this study, 4-methylpyrazole inhibited fatty acyl CoA synthetase competitively in liver homogenates . Fatty acyl CoA synthetase is a key enzyme involved in the supply of substrate for peroxisomal oxidation of alcohols via catalase-H2O2 . When palmitate was studied, rates of formaldehyde production from methanol were reduced competitively by 4-methylpyrazole; however, when the product palmitoyl CoA was used, the addition of 4-methylpyrazole did not alter activity . 4-Methylpyrazole also inhibited fatty acyl CoA synthetase activity measured directly from CoA disappearance . These data indicate that fatty acyl CoA synthetase is inhibited by 4-methylpyrazole, thus reducing the availability of H2O2 for catalase-dependent alcohol metabolism . Inhibition of methanol metabolism in deer mice expressing ADH indicates that this phenomenon also occurs in species with ADH . Taken together, these data support the hypothesis that the contribution of ADH to alcohol metabolism may have been previously overestimated.

Crit Rev Oncog, 1993, 4(2), 115 - 36
Structure and function of eukaryotic proprotein processing enzymes of the subtilisin family of serine proteases; Van de Ven WJ et al.; Production of a broad spectrum of regulatory proteins in eukaryotes occurs via an intricate cascade of biosynthetic and secretory processes . Often these proteins initially are synthesized as parts of higher molecular weight, but inactive, precursor proteins . Specific endoproteolytic processing of these proproteins is required to generate the regulatory proteins in a mature and biologically active form . Such endoproteolysis generally occurs at cleavage sites consisting of particular sequence motifs of basic amino acids, often paired basic residues . This phenomenon, first observed almost 25 years ago, has intrigued scientists ever since then . Nevertheless, the responsible enzymes remained elusive for long . The first known eukaryotic enzyme with the exquisite cleavage specificity for paired basic amino acid residues was the prohormone processing enzyme kexin (EC 3.4.21.61), a subtilisin-like serine protease that is encoded by the KEX2 gene of yeast Saccharomyces cerevisiae . Recently, a number of kexin-like mammalian proprotein-processing enzymes were discovered . The enzyme furin, which is encoded by the fur gene, was the first and can be considered the prototype of a mammalian subclass of subtilisin-like serine proteases . It is predicted to contain a "prepro" domain, a subtilisin-like catalytic domain, a middle domain, a cysteine-rich region, a transmembrane anchor, and a cytoplasmic domain . Furin is expressed in a wide variety of tissues, perhaps even in all tissues . In all likelihood, it is the enzyme responsible for the proteolytic bioactivation of a wide variety of precursor proteins . Two other novel mammalian proprotein-processing enzymes are PC1 (also known as PC3) and PC2 . Some of the protein domains of these enzymes resemble those in kexin and furin, however, there are also differences . The PC1/PC3 and PC2 enzymes exhibit a more restricted expression pattern than furin . It has been suggested that PC1/PC3 and PC2 are involved primarily in the processing of prohormones within the regulated secretory pathway of cells of endocrine and neural tissue . Recently, the coding sequences for two other candidate mammalian proprotein-processing enzymes were identified . They were called PACE4 and PC4 . Like that of furin, the tissue distribution of PACE4 is widespread . PC4, however, may represent a candidate for a precursor-processing endoprotease that is specifically expressed in the testicular germ cells . Finally, DNA sequences encoding kexin- and furin-like candidate pro-protein-processing enzymes have been identified in Drosophila melanogaster, Dfur1 and Dfur2 genes; in Xenopus laevis, Xen-14 gene; and in Caenorhabditis elegans, bli-4 gene.(ABSTRACT TRUNCATED AT 400 WORDS)

Proc Natl Acad Sci U S A, 1993 Jan 1, 90(1), 138 - 42
The type 1 human immunodeficiency virus Tat binding protein is a transcriptional activator belonging to an additional family of evolutionarily conserved genes; Ohana B et al.; The type 1 human immunodeficiency virus Tat protein is a powerful transcriptional activator when bound to an RNA structure (TAR) present at the extreme 5' terminus of viral mRNA . Since transcriptional activation requires binding of Tat to RNA, it has been suggested that Tat enhances initiation or elongation through a direct interaction with cellular transcription factors . Here we show through protein fusion experiments that the previously identified cellular Tat binding protein, TBP-1, although unable to bind DNA, is a strong transcriptional activator when brought into proximity of several promoter elements . Transcriptional activity depends upon the integrity of at least two highly conserved domains: one resembling a nucleotide-binding motif and the other motif common to proteins with helicase activity . Our studies further reveal that TBP-1 represents one member of a large, highly conserved gene family that encodes proteins demonstrating strong amino acid conservation across species . Finally, we identified a second family member that, although 77% similar to TBP-1, does not activate transcription from the promoters examined . This finding, together with the observation that TBP-1 does not activate each promoter examined, suggests that this gene family may encode promoter-specific transcriptional activators.

Mol Cell Biol, 1993 Jan, 13(1), 543 - 50
Concerted action of the transcriptional activators REB1, RAP1, and GCR1 in the high-level expression of the glycolytic gene TPI; Scott EW et al.; In Saccharomyces cerevisiae, the TPI gene product, triosephosphate isomerase, makes up about 2% of the soluble cellular protein . Using in vitro and in vivo footprinting techniques, we have identified four binding sites for three factors in the 5' noncoding region of TPI: a REB1-binding site located at positions -401 to -392, two GCR1-binding sites located at positions -381 to -366 and -341 to -326, and a RAP1-binding site located at positions -358 to -346 . We tested the effects of mutations at each of these binding sites on the expression of a TPI::lacZ gene fusion which carried 853 bp of the TPI 5' noncoding region integrated at the URA3 locus . The REB1-binding site is dispensable when material 5' to it is deleted; however, if the sequence 5' to the REB1-binding site is from the TPI locus, expression is reduced fivefold when the site is mutated . Because REB1 blocks nucleosome formation, the most likely function of its binding site in the TPI controlling region is to prevent the formation of nucleosomes over the TPI upstream activation sequence . Mutations in the RAP1-binding site resulted in a 10-fold reduction in expression of the reporter gene . Mutating either GCR1-binding site alone had a modest effect on expression of the fusion . However, mutating both GCR1-binding sites resulted in a 68-fold reduction in the level of expression of the reporter gene . A LexA-GCR1 fusion protein containing the DNA-binding domain of LexA fused to the amino terminus of GCR1 was able to activate expression of a lex operator::GAL1::lacZ reporter gene 116-fold over background levels . From this experiment, we conclude that GCR1 is able to activate gene expression in the absence of REB1 or RAP1 bound at adjacent binding sites . On the basis of these results, we suggest that GCR1 binding is required for activation of TPI and other GCR1-dependent genes and that the primary role of other factors which bind adjacent to GCR1-binding sites is to facilitate of modulate GCR1 binding in vivo.

Mol Cell Biol, 1993 Jan, 13(1), 487 - 95
A conserved alternative splice in the von Recklinghausen neurofibromatosis (NF1) gene produces two neurofibromin isoforms, both of which have GTPase-activating protein activity; Andersen LB et al.; Sequence analysis has shown significant homology between the catalytic regions of the mammalian ras GTPase-activating protein (GAP), yeast Ira1p and Ira2p (inhibitory regulators of the RAS-cyclic AMP pathway), and neurofibromin, the protein encoded by the NF1 gene . Yeast expression experiments have confirmed that a 381-amino-acid segment of neurofibromin, dubbed the GAP-related domain (GRD), can function as a GAP . Using the RNA polymerase chain reaction with primers flanking the NF1-GRD, we have identified evidence for alternative splicing in this region of the NF1 gene . In addition to the already published sequence (type I), an alternative RNA carrying a 63-nucleotide insertion (type II) is present in all tissues examined, although the relative amounts of types I and II vary . The insertion is conserved across species but is not present in GAP, IRA1, or IRA2 . GenBank searches have failed to identify significant similarity between the inserted sequence and known DNA or protein sequences, although the basic amino acid composition of the insertion shares features with nuclear targeting sequences . Expression studies in yeasts show that despite the partial disruption of the neurofibromin-IRA-GAP homology by this insertion, both forms of the NF1-GRD can complement loss of IRA function . In vivo assays designed to compare the GAP activity of the two alternatively spliced forms of the NF1-GRD show that both can increase the conversion of GTP-bound ras to its GDP-bound form, although the insertion of the 21 amino acids weakens this effect . The strong conservation of this alternative splicing suggests that both type I and II isoforms mediate important biological functions of neurofibromin.

Mol Cell Biol, 1993 Jan, 13(1), 399 - 407
Direct interaction of the tau 1 transactivation domain of the human glucocorticoid receptor with the basal transcriptional machinery; McEwan IJ et al.; We have used a yeast (Saccharomyces cerevisiae) cell free transcription system to study protein-protein interactions involving the tau 1 transactivation domain of the human glucocorticoid receptor that are important for transcriptional transactivation by the receptor . Purified tau 1 specifically inhibited transcription from a basal promoter derived from the CYC1 gene and from the adenovirus 2 major late core promoter in a concentration-dependent manner . This inhibition or squelching was correlated with the transactivation activity of tau 1 . Recombinant yeast TATA-binding protein (yTFIID), although active in vitro, did not specifically reverse the inhibitory effect of tau 1 . In addition, no specific interaction between tau 1 and yTFIID could be shown in vitro by affinity chromatography . Taken together, these results indicate that the tau 1 transactivation domain of the human glucocorticoid receptor interacts directly with the general transcriptional apparatus through some target protein(s) that is distinct from the TATA-binding factor . Furthermore, this assay can be used to identify interacting factors, since after phosphocellulose chromatography of a whole-cell yeast extract, a fraction that contained an activity which selectively counteracted the squelching effect of tau 1 was found.

Mol Cell Biol, 1993 Jan, 13(1), 391 - 8
The effect on chromosome stability of deleting replication origins; Dershowitz A et al.; The observed spacing between chromosomal DNA replication origins in Saccharomyces cerevisiae is at least four times shorter than should be necessary to ensure complete replication of chromosomal DNA during the S phase . To test whether all replication origins are required for normal chromosome stability, the loss rates of derivatives of chromosome III from which one or more origins had been deleted were measured . In the case of a 61-kb circular derivative of the chromosome that has two highly active origins and one origin that initiates only 10 to 20% of the time, deletion of either highly active origin increased its rate of loss two- to fourfold . Deletion of both highly active origins caused the ring chromosome to be lost in approximately 20% of cell divisions . This very high rate of loss demonstrates that there are no efficient cryptic origins on the ring chromosome that are capable of ensuring its replication in the absence of the origins that are normally used . Deletion of the same two origins from the full-length chromosome III, which contains more than six replication origins, had no effect on its rate of loss . These results suggest that the increase in the rate of loss of the small circular chromosome from which a single highly active origin was deleted was caused by the failure of the remaining highly active origin to initiate replication in a small fraction (approximately 0.003) of cell cycles.

J Virol, 1993 Jan, 67(1), 204 - 12
DNA strand exchange catalyzed by proteins from vaccinia virus-infected cells; Zhang W et al.; Vaccinia virus infection induces expression of a protein which can catalyze joint molecule formation between a single-stranded circular DNA and a homologous linear duplex . The kinetics of appearance of the enzyme parallels that of vaccinia virus DNA polymerase and suggests it is an early viral gene product . Extracts were prepared from vaccinia virus-infected HeLa cells, and the strand exchange assay was used to follow purification of this activity through five chromatographic steps . The most highly purified fraction contained three major polypeptides of 110 +/- 10, 52 +/- 5, and 32 +/- 3 kDa . The purified protein requires Mg2+ for activity, and this requirement cannot be satisfied by Mn2+ or Ca2+ . One end of the linear duplex substrate must share homology with the single-stranded circle, although this homology requirement is not very high, as 10% base substitutions had no effect on the overall efficiency of pairing . As with many other eukaryotic strand exchange proteins, there was no requirement for ATP, and ATP analogs were not inhibitors . Electron microscopy was used to show that the joint molecules formed in these reactions were composed of a partially duplex circle of DNA bearing a displaced single-strand and a duplex linear tail . The recovery of these structures shows that the enzyme catalyzes true strand exchange . There is also a unique polarity to the strand exchange reaction . The enzyme pairs the 3' end of the duplex minus strand with the plus-stranded homolog, thus extending hybrid DNA in a 3'-to-5' direction with respect to the minus strand . Which viral gene (if any) encodes the enzyme is not yet known, but analysis of temperature-sensitive mutants shows that activity does not require the D5R gene product . Curiously, v-SEP appears to copurify with vaccinia virus DNA polymerase, although the activities can be partially resolved on phosphocellulose columns.

DNA Seq, 1993, 3(5), 323 - 6
Sequence and expression of a Drosophila melanogaster cDNA encoding a putative ribosomal protein; Zhang N et al.; An abundant ovarian cDNA from Drosophila melanogaster has been cloned and sequenced . The predicted protein sequence is similar to that of the ribosomal protein 1024 of Dictyostelium discoideum, the 40S ribosomal protein ys11 of Saccharomyces cerevisiae and a 22 kd protein from Trypanosoma brucei . It seems, therefore, that the Drosophila cDNA also encodes a ribosomal protein . Transcripts are found at all stages of the life cycle but are especially abundant in the ovary, suggesting that this mRNA is maternally stored for utilization in embryogenesis to enable the rapid production of ribosomal proteins and assembly of ribosomes.

DNA Seq, 1993, 3(5), 319 - 22
Sequence of mouse glucose-6-phosphate dehydrogenase cDNA; Zollo M et al.; A full-length mouse glucose-6-phosphate dehydrogenase (G6PD) cDNA has been isolated and sequenced, and the evolutionary conservation of many portions of the sequence has been verified by comparison with that of human and other sources.

Proc Natl Acad Sci U S A, 1993 Jan 1, 90(1), 30 - 4
Retinoic acid receptors and retinoid X receptors: interactions with endogenous retinoic acids; Allenby G et al.; The binding of endogenous retinoids and stereoisomers of retinoic acid (RA) to the retinoid nuclear receptors, RA receptor (RARs) and retinoid X receptors (RXRs), was characterized using nucleosol preparations from transiently transfected COS-1 cells . Among several stereoisomers of RA tested, including 7-cis-, 9-cis-, 11-cis-, 13-cis-, and all-trans-RA, only 9-cis-RA effectively competes with 9-cis-{3H}RA binding to the RXRs . Additionally, the endogenous retinoid trans-didehydro-RA (t-ddRA) does not interact with RXRs, whereas the 9-cis form of ddRA competes effectively . RXRs (alpha, beta, and gamma) bind 9-cis-RA with dissociation constants (Kd) of 15.7, 18.3, and 14.1 nM, respectively . In contrast to the selectivity of RXRs for 9-cis-RA, RARs bind both t-RA and 9-cis-RA with high affinity, exhibiting Kd values in the 0.2-0.7 nM range for both ligands . Unlike RARs, the cellular RA binding proteins CRABPI or CRABPII bind t-RA but do not bind 9-cis-RA . Consistent with the binding data, 9-cis-RA and 9-cis-ddRA transcriptionally activate both GAL4-RXR and GAL4-RAR chimeric receptors with EC50 values of 3-20 nM for 9-cis-RA and 9-cis-ddRA, whereas t-RA and t-ddRA efficiently activate only GAL4-RAR chimeric receptors . Thus, 9-cis forms of endogenous retinoids can contribute to the pleiotropic effects of retinoids by interacting with both the RARs and RXRs.

Mol Cell Biol, 1993 Jan, 13(1), 57 - 62
MCM1 binds to a transcriptional control element in Ty1; Errede B; Some Ty1 transposable-element insertion mutations of Saccharomyces cerevisiae activate adjacent-gene expression . These Ty1-activated genes are regulated similarly to certain mating genes . This report shows that the MCM1 protein, which binds to several mating genes, also binds to a transcriptional regulatory sequence in Ty1 . The binding of MCM1 to Ty1 correlates with the ability of its binding site to function as a component of the Ty1 transcriptional activator . This correlation supports the idea that MCM1 is important for Ty1-activated gene expression . At mating-gene promoters, MCM1 binds with coactivators or repressors such as STE12, alpha 1, or alpha 2 . In contrast, MCM1 binds without these associated DNA-binding proteins at its site in Ty1 . This finding suggests that its role in Ty1-mediated transcription is different from that at mating genes.

Cancer Res, 1993 Jan 1, 53(1), 89 - 93
A temperature sensitive topoisomerase II allele confers temperature dependent drug resistance on amsacrine and etoposide: a genetic system for determining the targets of topoisomerase II inhibitors; Nitiss JL et al.; We have developed a simple system for determining the specific contribution of topoisomerase II targeting to the cytotoxic activity of a drug . We have constructed yeast strains that are permeable to anti-topoisomerase II drugs, carry a DNA repair mutation, rad52, and also have a temperature sensitive topoisomerase II mutation, top2-1 . Strains carrying the top2-1 mutation have nearly normal topoisomerase II activity at 25 degrees C but less than 10% of wild type activity at 36 degrees C . We find that at a semi-permissive temperature (30 degrees C), there is sufficient topoisomerase II activity for viability; but since the topoisomerase II activity is greatly reduced, the cells are very resistant to anti-topoisomerase II drugs . Conversely, such cells are hypersensitive to the topoisomerase I inhibitor camptothecin . These results provide strong support for the model that drug stabilized DNA cleavage, rather than a lack of enzyme activity, is responsible for cell killing by eukaryotic anti-topoisomerase II agents . They also show that there is a minimum level of topoisomerase II activity in yeast that is consistent with viability but also allows a high degree of resistance to anti-topoisomerase II agents.

J Virol, 1993 Jan, 67(1), 543 - 7
Mutations in the C terminus of herpes simplex virus type 1 DNA polymerase can affect binding and stimulation by its accessory protein UL42 without affecting basal polymerase activity; Tenney DJ et al.; We have analyzed the effects of mutations in the herpes simplex virus type 1 DNA polymerase (Pol) C-terminal UL42 binding domain on the activity of Pol and its ability to form complexes with and be stimulated by UL42 in vitro . Wild-type Pol expressed in Saccharomyces cerevisiae was both bound and stimulated by UL42 in vitro . C-terminal truncations of 19 and 40 amino acids (aa) did not affect the ability of Pol to be stimulated by UL42 in vitro . This stimulation as well as basal Pol activity in the presence of UL42 was inhibited by polyclonal anti-UL42 antiserum, thus indicating a physical interaction between Pol and UL42 . Removal of the C-terminal 59 aa of Pol and internal deletions of 72 aa within the Pol C terminus eliminated stimulation by UL42 . None of the truncations or deletions within Pol affected basal polymerase activity . In contrast with their ability to be stimulated by UL42, only wild-type Pol and Pol lacking the C-terminal 19 aa bound UL42 in a coimmunoprecipitation assay . These results demonstrate that a functional UL42 binding domain of Pol is separable from sequences necessary for basal polymerase activity and that the C-terminal 40 aa of Pol appear to contain a region which modulates the stability of the Pol-UL42 interaction.

Mamm Genome, 1993, 4(8), 445 - 50
Ribosomal DNA clusters in pulsed-field gel electrophoretic analysis of human acrocentric chromosomes; Srivastava AK et al.; For determination of the extent to which ribosomal DNA (rDNA) is organized in tandemly repeated arrays, cellular DNA was digested with a restriction enzyme (EcoRV) that does not cut within the single 44-kb rDNA unit, and fragments separated by PFGE were hybridized to specific rDNA probes . A series of bands large enough to contain 15 to more than 30 rDNA repeat units was observed . In YACs containing cloned rDNA, however, such clusters were not observed, presumably because, as shown here for a clone starting with 1.5 tandem repeat units, there is a tendency for repeat units to delete out of the insert . By comparative gel electrophoretic analyses of DNAs from rodent hybrid cells containing singly isolated human chromosomes, most of the bands seen in total human DNA were assigned to at least one of the acrocentric chromosomes . Thus, large characteristic assemblies of DNA containing rDNA and lacking EcoRV sites were stable enough to be conserved in some human/rodent hybrid lines . When further digested with HindIII, which cuts rDNA at several points, the rDNA in each band yielded the expected fragments . If the large species consist completely of clusters of tandemly repeated rDNA units, they account for about half of the total cellular rDNA content estimated by saturation hybridization measurements.

Ukr Biokhim Zh, 1993 Jan-Feb, 65(1), 97 - 100
{Inactivation of glutathione reductase by quinones and nitrosocarbamides}; Bironaite DA et al.; Quinones inactivate oxidized glutathione reductase (EC 1.6.4.2) from yeast with rate constants (ki) ranging from 0.03 M-1s-1 (p-benzoquinone) to 10(3) M-1s-1 (bromanyl) . It is glutathione, but not NADP+ that protects the enzyme from inactivation, which shows that quinones interact with a glutathione-binding centre, cysteine-2, most probably . The mechanism of inactivation by quinones differs from that by nitrosoureas which inactivate only the reduced enzyme, modifying the reduced catalytic disulphide . 1,3-bis-(2-chloroethyl)-1-nitrosourea acts as the most rapid inactivator of the enzyme, possessing ki of 0.77 M-1s-1.

Agents Actions, 1993, 38 Spec No, C59 - 60
Chromosomal mapping of the human interleukin-1 receptor antagonist gene (IL-1RN) and isolation of specific YAC clones; Steinkasserer A et al.; Using a panel of somatic rodent-human cell hybrids, we show that the interleukin-1 receptor antagonist gene (IL-1RN) maps to the long arm of human chromosome 2 . Linkage studies permitted the regional localization of this gene to band q14-21 . This is the same region in which the IL-1 alpha and IL-1 beta genes are localized . Three yeast artificial chromosome (YAC) clones containing the IL-1RN gene were isolated, and these will be used for further characterization of this chromosome 2 region.

Cell Mol Biol Res, 1993, 39(4), 355 - 60
How does the GAL4 transcription factor recognize the appropriate DNA binding sites in vivo?
Kodadek T.
The GAL4 protein of yeast activates the transcription of several genes involved in galactose metabolism . This event requires that GAL4 bind to upstream activation sites with the consensus sequence 5'-CGGN5(T/A)N5CCG-3' . We review the general requirements that must be met for a protein such as GAL4 to find and remain bound to its target site in vivo . Evidence is presented that the GAL4 DNA-binding domain itself has insufficient intrinsic sequence selectivity to fulfill the task of targeting GAL4 to the appropriate sites in a yeast genome . Possible mechanisms by which this selectivity is enhanced in vivo are discussed.

J Comp Physiol {B}, 1993, 163(6), 463 - 9
Seasonal changes in critical enzymes of lipogenesis and triacylglycerol synthesis in the marmot (Marmota flaviventris); Mostafa N et al.; Fatty acid metabolism and triacylglycerol synthesis are critical processes for the survival of hibernating mammals that undergo a prolonged fasting period . Fatty acid synthase, fatty-acid-CoA ligase, diacylglycerol acyltransferase, and monoacylglycerol acyltransferase activities were measured in liver and in white and brown adipose tissue, in order to determine whether enzymes of lipogenesis and triacylglycerol synthesis vary seasonally during hibernation in the yellow-bellied marmot (Marmota flaviventris) . Compared with mid-winter hibernation, fatty acid synthase activity was higher in all three tissues during early spring when marmots emerged from hibernation and in mid-summer when they were feeding, consistent with the synthesis of fatty acids from the carbohydrate-rich summer diet . Fatty-acid-CoA ligase and diacylglycerol acyltransferase activities were highest in summer in white adipose tissue when triacylglycerol synthesis would be expected to be high; diacylglycerol acyltransferase activity was also high in brown adipose tissue during spring and summer . In liver, however, diacylglycerol acyltransferase specific activity was highest during hibernation, suggesting that triacylglycerol synthesis may be prominent in liver in winter . Monoacylglycerol acyltransferase activity, which may aid in the retention of essential fatty-acids, was 80-fold higher in liver than in white or brown adipose tissue, but did not vary seasonally . Its dependence on palmitoyl-CoA suggests that a divalent cation might play a role in enzyme activation . The high hepatic diacylglycerol acyltransferase activity during hibernation suggests that the metabolism of very low density lipoprotein may be important in the movement of adipose fatty acids to brown adipose tissue and muscle during the rewarming that occurs periodically during hibernation.(ABSTRACT TRUNCATED AT 250 WORDS)

Ciba Found Symp, 1993, 176, 218 - 28; discussion 229-32
The cycle of SEC4 function in vesicular transport; Novick P et al.; Sec4 is a Ras-like GTP-binding protein required for exocytosis in yeast . Unlike Ras, it is the ability of Sec4 to cycle between the GTP- and GDP-bound forms rather than the absolute levels of the GTP-bound form that is critical for function . This cycle may be coupled to an observed cycle of Sec4 localization within the cell . Sec4 binds to secretory vesicles which then fuse with the plasma membrane in exocytosis . Sec4 can recycle from the plasma membrane through a soluble pool to rebind to a new round of vesicles . We have found an activity in yeast (Saccharomyces cerevisiae) comparable to that of the GDP dissociation inhibitor protein isolated from mammalian cells that releases GDP-bound Sec4 from membranes . DSS4-1, a dominant suppressor of the sec4-8 temperature-sensitive mutation, encodes a nucleotide exchange protein . The cycle of Sec4 may function to allow the assembly and subsequent disassembly of a set of proteins necessary for exocytosis . Candidates for members of this set of proteins are encoded by sec genes which show strong genetic interactions with sec4-8 . Two of these (SEC8 and SEC15) encode large proteins which form a complex that is peripherally associated with the plasma membrane.

Acta Biochim Pol, 1993, 40(3), 421 - 8
Comparative studies on O-acetylhomoserine sulfhydrylase: physiological role and characterization of the Aspergillus nidulans enzyme; Brzywczy J et al.; O-acetylhomoserine sulfhydrylase (OAH SHLase) from Aspergillus nidulans is an oligomeric protein with a broad substrate specificity with regard to sulfhydryl compounds . As its Saccharomyces cerevisiae counterpart the enzyme also reacts with O-acetylserine and is inhibited by carbonyl reagents but not by antiserum raised against the yeast enzyme . In contrast to Saccharomyces cerevisiae the enzyme is not essential for Aspergillus nidulans as indicated by the completely prototrophic phenotype of OAH SHLase-negative mutants . Its major physiological role in Aspergillus nidulans seems to be recycling of the thiomethyl group of methylthio-adenosine but it is also a constituent of the alternative pathway of cysteine synthesis.

Nucleic Acids Symp Ser, 1993, (29), 75 - 6
Sequence analysis of low-molecular-weight RNAs by the use of non-radioactive labeling; Matsuki T et al.; A new procedure for non-radioactive labeling of the 3'-terminal -OH was developed to facilitate the sequencing works on low-molecular-weight RNAs . At first, biotinylated pCp (cytidine 3',5'-bisphosphate) was synthesized and ligated to the 3'-terminal of yeast tRNA(Phe) with the aid of RNA ligase . Although the labeling efficiency was high enough, this labeling resulted in a lack of the sequence ladders around the 3'-terminal 10 nucleotides . A longer oligonucleotide, p(dC)(dT)16-Biotin was designed to overcome this defect and proved to be a better non-radioactive 3'-labeling probe.

Skin Pharmacol, 1993, 6(4), 282 - 91
Evaluation of phototoxic and photogenotoxic risk associated with the use of photosensitizers in suntan preparations: application to tanning preparations containing bergamot oil; Moysan A et al.; Bases for the elaboration of a standardized protocol are proposed for studying phototoxic effects of skin tanning preparations containing photosensitizing agents . The experimental procedure includes in vivo phototoxicity tests, evaluation of the photogenotoxic risk and determination of the photosensitizer concentration in plasma after topical application . This procedure was carried out with tanning preparations containing a well-known photosensitizer, 5-methoxypsoralen, as a component of bergamot oil . The whole study has been performed using topical application of the commercial suntan product, i.e . containing the sunscreens and all other components . Whereas the exposure to solar simulated radiation never triggered any phototoxic response, a photosensitizing effect was observed for skin type I volunteers exposed to high doses of ultraviolet A . The transepidermal penetration resulted in a 5-methoxypsoralen concentration of 1-4 ng/ml in the suction blister fluid . The photogenotoxicity of this suction blister fluid containing 5-methoxypsoralen and also other ingredients of the tanning preparation was assayed on yeast cells and was found to be rather low . 5-Methoxypsoralen was also detected in plasma after repeated applications but at low concentrations (about 1 ng/ml) which do not present a potential risk for systemic ocular effects.

Receptors Channels, 1993, 1(1), 1 - 9
Functional carboxyl terminal deletion map of protein kinase C alpha; Su L et al.; The phorbol ester receptor protein kinase C (PKC) gene family encodes essential mediators of various eukaryotic cellular signals . Based on the predicted amino acid (aa) sequence homology of more than ten distinct PKC gene coding sequences, four highly conserved regions C1-C4 and five variable regions V1-V5 have been defined for the different PKC subtypes . Some of these regions, such as C1 and C3/V4/C4, have been correlated with specific PKC functions, such as activator binding and enzymatic activity, respectively, while the biological role of others is unknown . The biological significance of the PKC carboxyl terminus is unclear and the predicted boundary of the catalytic C4 region is controversial due to different interpretations of aa sequence comparisons . We explored the PKC alpha carboxyl terminal requirement for basic PKC function and mapped the boundary of the sequences essential for enzymatic activity based on functional criteria . cDNAs encoding normal and random carboxyl terminal truncations of bovine PKC alpha were introduced into Saccharomyces cerevisiae, allowing its rapid functional expression and characterization for catalytic as well as biological activity . We found that deletion of up to 11 carboxyl terminal aa still results in a phorbol ester-responsive, biologically active enzyme in vivo which is dependent on calcium and phospholipids for catalytic activation in vitro . Deletion of 15 and 23 aa results in marginal and total loss of catalytic activity, respectively, and in complete loss of biological activity for both truncations.(ABSTRACT TRUNCATED AT 250 WORDS)

J Cell Sci, 1993 Jan, 104 ( Pt 1), 89 - 95
Analysis of conserved binding proteins for nuclear localization sequences; Stochaj U et al.; Correct targeting of nuclear proteins is mediated by nuclear localization sequences (NLS) which permit specific binding to the nucleus and subsequent translocation across the nuclear envelope via the nuclear pore complex . It is proposed that nuclear import is facilitated by NLS-receptors which reside in the cytoplasm and at the nuclear pore . These NLS-receptors could facilitate an early step of nuclear protein import, i.e . targeting and binding of nuclear proteins at the nuclear pore . We have generated anti-idiotype antibodies against the SV40 T-antigen nuclear localization sequence that allowed us to study NLS-binding proteins in a variety of different organisms . Proteins of similar size are recognized by these antibodies in yeast, Drosophila, rat and human cells . Cytological analysis indicates that the NLS-binding proteins reside in part at nuclear pores . One of the proteins recognized by anti-idiotype antibodies is identical to a previously identified NLS-binding protein . Using isolated yeast nuclei we demonstrate that the anti-idiotype antibodies compete for binding of nuclear proteins in vitro . We show that the yeast mutant npl3, which is defective in nuclear protein localization, has an altered distribution of antigens recognized by these anti-idiotype antibodies, at the semi-permissive temperature . Our results suggest that a set of proteins common to various eukaryotes recognizes nuclear localization sequences.

EMBO J, 1993 Jan, 12(1), 233 - 41
Nuclear PRP20 protein is required for mRNA export; Amberg DC et al.; The yeast PRP20 protein is highly homologous in structure and function to the RCC1 protein of higher eukaryotes . The RCC1 protein is involved in the regulation of the onset of mitosis, whereas the PRP20 protein was shown to be required for accurate and efficient mRNA metabolism . The first observable phenotype in mutant prp20 cells when shifted from permissive to non-permissive temperature is a loss of nuclear PRP20 protein . Concomitantly, an accumulation of poly(A)+ RNA in the nucleus is observed . The temperature-sensitive RCC1 allele in the mutant hamster cell line tsBN2 leads to a similar accumulation of mRNA in the nucleus.

Oncogene, 1993 Jan, 8(1), 117 - 24
Phosphorylation of Src mutants at Tyr 527 in fibroblasts does not correlate with in vitro phosphorylation by CSK; MacAuley A et al.; In normal fibroblasts, the product of the cellular src gene, p60c-src or Src, is repressed by phosphorylation at its C-terminal tyrosine residue, Tyr 527 . Mutations in Src that prevent phosphorylation cause enzymatic activation and malignant transformation . The tyrosine kinases that phosphorylate Src at Tyr 527 in vivo have not been identified, but a tyrosine kinase known as CSK is an excellent candidate . CSK has the unusual ability to phosphorylate Src in vitro only at Tyr 527 . To examine whether CSK has the appropriate sequence specificy to explain the phosphorylation of Src at Tyr 527 in fibroblasts, we have made use of a set of C-terminal substitution mutants of Src . These mutants were previously characterized for their levels of Tyr 527 phosphorylation when expressed in Rat2 fibroblasts . The ability of CSK to phosphorylate selected mutants has now been tested, using both in vitro phosphorylation assays and co-expression of CSK with the Src mutants in a heterologous organism, Saccharomyces cerevisiae . We also tested whether the mutant Src molecules could autophosphorylate at Try 527, by examining the phosphorylation state of catalytically active forms expressed in the absence of CSK in yeast cells . The results show that CSK has strict sequence specificity for the normal Src sequence, although it can also phosphorylate the Lck sequence . The other mutant Src molecules tested were not phophorylated by CSK, even though some of these mutants are highly phosphorylated at Tyr 527 in Rat 2 cells . All the mutants that are phosphorylated at Tyr 527 in Rat2 cells are also able to autophosphorylate at Tyr 527 . The results suggest that CSK, autophosphorylation, and phosphorylation by kinases other than CSK, may all contribution to repressing Src catalytic activity in fibroblasts.

J Virol, 1993 Jan, 67(1), 19 - 28
Proteolytic processing of Ty3 proteins is required for transposition; Kirchner J et al.; Ty3 is a retroviruslike element found in Saccharomyces cerevisiae . It encodes GAG3 and GAG3-POL3 polyproteins which are processed into mature proteins found in the Ty3 viruslike particle . In this study, the region encoding a protease that is homologous to retroviral aspartyl proteases was identified and shown to be required for production of mature Ty3 proteins and transposition . The Ty3 protease has the Asp-Ser-Gly consensus sequence found in copia, Ty1, and Rous sarcoma virus proteases, rather than the Asp-Thr-Gly found in most retroviral proteases . The Asp-Ser-Gly consensus is flanked by residues similar to those which flank the active sites of cellular aspartyl proteases . Mutations were made in the Ty3 active-site sequence to examine the role of the protease in Ty3 particle maturation and to test the functional significance of the Ser active-site variant in the consensus sequence . Mutation of the active-site Asp blocked processing of Gag3 and Gag3-Pol3 and allowed identification of a GAG3-POL3 polyprotein . This protein was turned over rapidly in cells expressing the mutant Ty3 . Changing the active-site Ser to Thr caused only a modest reduction in the levels of certain Ty3 proteins . Five putative cleavage sites of this protease in Ty3 GAG3 and GAG3-POL3 polyproteins were defined by amino-terminal sequence analysis . The existence of an additional protein(s) of unknown function, encoded downstream of the protease-coding region, was deduced from the positions of these amino termini and the sizes of known Ty3 proteins . Although Ty3 protease cleavage sites do not correspond exactly to known retroviral protease cleavage sites, there are similarities . Residues P3 through P2' in the regions encompassing each of the five sites are uncharged, and no P1 position is occupied by an amino acid with a branched beta carbon.

Biochemistry, 1992 Dec 29, 31(51), 12847 - 54
Protein conformational perturbations affect the photoreduction of native cytochrome c peroxidase (III) at alkaline pH; Wang J et al.; Ferric cytochrome c peroxidase (CCP) undergoes a ligation-state transition from a pentacoordinate, high-spin (5c/hs) heme to a hexacoordinate, low-spin (6c/1s) heme when titrated over a pH range of 7.30-9.70 . This behavior is similar to that exhibited by the ferrous form of the enzyme . However, the photodissociation of the low-spin, axial ligand, exhibited by ferrous CCP at alkaline pH, is not observed for ferric CCP . Instead, a photoinduced reduction of the ferric heme is apparent in the pH range 7.90-9.70 . In the absence of O2 and redox mediators such as methyl viologen (MV2+), the reoxidation of the photoreduced enzyme is very slow (tau 1/2 approximately 3 min) . F(-)-bound CCP(III) (6c/hs) displays similar pH-dependent photoreduction . Horseradish peroxidase, however, does not . The formation of 6c/1s heme coincides with the onset of appreciable photoreduction (between laser pulses, > 60 ms) of CCP (III) at alkaline pH, suggesting a global protein conformational rearrangement within or around its heme pocket . Photoreduction of alkaline CCP(III) most likely involves intramolecular electron transfer (ET) from the aromatic residue in the proximal heme pocket to the photoexcited heme . We speculate that the kinetics of electron transfer are affected by changes in the orientation of Trp-191.

Nucleic Acids Res, 1992 Dec 25, 20(24), 6565 - 73
Solid-phase synthesis of branched oligoribonucleotides related to messenger RNA splicing intermediates; Damha MJ et al.; The chemical synthesis of oligoribonucleotides containing vicinal (2'-5')- and (3'-5')-phosphodiester linkages is described . The solid-phase method, based on silyl-phosphoramidite chemistry, was applied to the synthesis of a series of branched RNA {(Xp)nA2' (pN)n3'(pN)n} related to the splicing intermediates derived from Saccharomyces cerevisiae rp51a pre-messenger RNA . The branched oligonucleotides have been thoroughly characterized by nucleoside and branched nucleotide composition analysis . Branched oligoribonucleotides will be useful in the study of messenger RNA splicing and in determining the biological role of RNA 'lariats' and 'forks' in vivo.

Nucleic Acids Res, 1992 Dec 25, 20(24), 6451 - 4
The TATA-binding protein participates in TFIIIB assembly on tRNA genes; Huet J et al.; The TATA-binding protein TBP has been recently recognized as a general class III transcription factor . Using the gel shift assay to monitor initiation complex assembly on a yeast tRNA gene, we show that TBP is required for the TFIIIC-dependent assembly of TFIIIB . TFIIIB depleted of TBP by a simple chromatographic step does not bind stably to the TFIIIC-tDNA complex . Addition of yeast or human recombinant TBP allows the formation of a TFIIIB-TBP-TFIIIC-tDNA complex . The presence of TBP in the complex was inferred from the effect of anti-TBP antibodies and from the different migration properties of TFIIIB-TBP-tDNA complexes formed with yeast or human TBP.

Cell, 1992 Dec 24, 71(7), 1223 - 37
The GCN4 basic region leucine zipper binds DNA as a dimer of uninterrupted alpha helices: crystal structure of the protein-DNA complex; Ellenberger TE et al.; The yeast transcriptional activator GCN4 is 1 of over 30 identified eukaryotic proteins containing the basic region leucine zipper (bZIP) DNA-binding motif . We have determined the crystal structure of the GCN4 bZIP element complexed with DNA at 2.9 A resolution . The bZIP dimer is a pair of continuous alpha helices that form a parallel coiled coil over their carboxy-terminal 30 residues and gradually diverge toward their amino termini to pass through the major groove of the DNA-binding site . The coiled-coil dimerization interface is oriented almost perpendicular to the DNA axis, giving the complex the appearance of the letter T . There are no kinks or sharp bends in either bZIP monomer . Numerous contacts to DNA bases and phosphate oxygens are made by basic region residues that are conserved in the bZIP protein family . The details of the bZIP dimer interaction with DNA can explain recognition of the AP-1 site by the GCN4 protein.

Eur J Biochem, 1992 Dec 15, 210(3), 671 - 81
Assignment of the magnetic resonances of the imino protons and methyl protons of Bombyx mori tRNA(GlyGCC) and the effect of ion binding on its structure; Amano M et al.; The magnetic resonances in the low-field H-NMR spectra of Bombyx mori tRNA(GlyGCC), corresponding to the hydrogen-bonded imino protons of the helical stems and tertiary base pairs, could be tentatively assigned by means of the sequential nuclear Overhauser effects . While B . mori tRNA(GlyGCC) does not contain the G19C56 tertiary base pair, the D20G57 base pair exists between the D and T loops, which was not found in the X-ray crystal structure of yeast tRNA(Phe) . The effects of Mg2+, spermine and temperature on the conformation of this tRNA have also been examined based on the behavior of the assigned resonance signals . Mg2+ stabilize the D and T stems and the tertiary structure between the D and T loops . Spermine affects the resonances of the D and anticodon stems, and A23G9, but does not stabilize them . While the acceptor stem melts sequentially from both ends (G7C66 and G1C72) with increasing temperature, the anticodon stem melts from only one end (G39C31) and the G26C44 base pair is the most stable . In the tertiary structure between the variable loop and D stem, G10G45 melts first and G22G46 last . Yeast tRNA(Phe) has also been examined, and the results were compared with those for B . mori tRNA(Gly).

Biochem Biophys Res Commun, 1992 Dec 15, 189(2), 1150 - 6
Dissociation of complexes between 70 kDa stress proteins and presecretory proteins is facilitated by a cytosolic factor; Chirico WJ; Members of the 70 kDa stress protein family were shown previously to facilitate the posttranslational translocation of presecretory proteins into the endoplasmic reticulum and protein precursors into mitochondria . To identify proteins that interact with 70 kDa stress proteins during the early steps of posttranslational translocation, polyclonal antibodies were raised against purified yeast cytosolic stress proteins . They were used to immunoprecipitate complexes between 70 kDa stress proteins and a radiolabeled presecretory protein, prepro-alpha-factor, that was translated in vitro . Complexes between prepro-alpha-factor and 70 kDa stress proteins were stable, but could be dissociated in the presence of ATP and crude cytosolic extracts from yeast . These results are consistent with the idea that 70 kDa stress proteins act as molecular chaperones in translocation by binding to precursor proteins before or during their passage across membranes.

Proc Natl Acad Sci U S A, 1992 Dec 15, 89(24), 12078 - 82
Organization of the human skeletal myosin heavy chain gene cluster; Yoon SJ et al.; Myosin is an important structural and enzymatic component of skeletal muscle . Multiple myosin isoforms are encoded by a multigene family and are expressed in different developmental stages and fiber types . In humans and mice, skeletal myosin heavy chain (MYH) genes are clustered on a single chromosome (17p and 11, respectively) . Since the structural organization of the gene cluster may affect its expression as well as shed light on MYH genetic alterations, a physical map of the human MYH gene cluster was constructed . Nine yeast artificial chromosomes containing MYH genes were isolated and used to construct a contiguous set (contig) of overlapping yeast artificial chromosomes . This contig encompasses a genetic marker mapped to 17p13.1 . Six MYH genes were located within a 500-kilobase segment of human DNA . The order of the genes within this cluster does not correspond to the developmental pattern of expression of individual members.

Proc Natl Acad Sci U S A, 1992 Dec 15, 89(24), 11920 - 4
Ku autoantigen is the regulatory component of a template-associated protein kinase that phosphorylates RNA polymerase II; Dvir A et al.; The carboxyl-terminal domain of RNA polymerase II contains a tandemly repeated heptapeptide sequence . Previous work has shown that this sequence is phosphorylated at multiple sites by a template-associated protein kinase, in a reaction that is closely associated with the initiation of RNA synthesis . We have purified this kinase to apparent homogeneity from human (HeLa) cells . The purified kinase phosphorylates native RNA polymerase II only in the presence of DNA and the general transcription factors TFIID (TBP), TFIIB, and TFIIF . Two kinase components are required for full activity: a catalytic component and a DNA-binding regulatory component . The regulatory component has been identified as Ku autoantigen, based on the molecular weights of its component polypeptides, its DNA-binding properties, and its reactivity with anti-Ku monoclonal antibodies . The Ku autoantigen recruits the catalytic component of the kinase to the template . Ku autoantigen has been previously proposed to interact with DNA by a characteristic bind-and-slide mechanism . This mode of interaction may provide a mechanism for targeting the kinase to the transcription complex and other DNA-bound substrates.

Proc Natl Acad Sci U S A, 1992 Dec 15, 89(24), 11764 - 8
Domain motions in phosphoglycerate kinase: determination of interdomain distance distributions by site-specific labeling and time-resolved fluorescence energy transfer; Haran G et al.; 3-Phosphoglycerate kinase is composed of two globular domains separated by a wide cleft . The substrate binding sites are situated on the inner surfaces of the two domains . By analogy to other kinases, it has been postulated that the catalytic mechanism of phosphoglycerate kinase involves a hinge bending domain motion that brings the substrates together to allow phosphoryl transfer . To characterize this large-scale conformational change, as well as the dynamics of the unliganded enzyme in solution, we have applied site-directed mutagenesis and time-resolved nonradiative energy transfer techniques . Two genetically engineered cysteines (Cys-135 and Cys-290), one in each of the two domains, were covalently labeled with a donor and acceptor pair of fluorescent probes . Analysis of subnanosecond fluorescence decay curves yielded the equilibrium distribution of interdomain distances . In the absence of substrates, the distribution of distances between the two labeled sites was very broad, with a full width at half maximum estimated as 20 A or broader, indicative of a large number of conformational substates in solution . The mean distance, 31.5 +/- 1 A, was 8 A smaller than in the crystal structure . Upon addition of ATP alone or of ATP and 3-phosphoglycerate, the average distance increased to 38 +/- 1 A and the width of the distribution decreased . Addition of 3-phosphoglycerate alone induced a similar but smaller change . The rate of conformational state fluctuations (interconversion between states) was found to be slow on the nanosecond time scale, as expected for a protein with a relatively large interdomain contact area.

J Biol Chem, 1992 Dec 15, 267(35), 25032 - 8
Characterization of a novel, potent, and specific inhibitor of serine palmitoyltransferase; Zweerink MM et al.; We have examined the mechanism of action of two natural products identified as broad spectrum antifungal agents (VanMiddlesworth, F., Dufresne, C., Wincott, F . E., Mosley, R . T., and Wilson, K . E . (1992) Tetrahedron Lett., in press; VanMiddlesworth, F., Giacobbe, R . A., Lopez, M . Garrity, G., Bland, J . A., Bartizal, K., Fromtling, R . A., Polishook, J., Zweerink, M . M., Edison, A . M., Rozdilsky, W., Wilson, K . E., and Monaghan, R . L . (1992) J . Antibiot . (Tokyo) 45, 861-867), designated sphingofungin B (2S-amino-3R,4R,5S,14-tetrahydroxyeicos-6-enoic acid) and sphingofungin C (2S-amino-5S-acetoxy-3R,4R,14-trihydroxyeicos-6-enoic acid), and find they are potent specific inhibitors of serine palmitoyltransferase, which catalyze the committed step of sphingolipid biosynthesis . We used Saccharomyces cerevisiae as a model to investigate the mechanism of the antifungal activity of these compounds . Macromolecular synthesis was not immediately affected by either sphingofungin B or C, synthesis continued for 60-90 min following the addition of drug to growing cultures . Significant loss of viability with sphingofungins required growing cultures and began only after several hours, with greater than 99.9% of drug-treated cells non-viable after 24 h . No lysis or other gross changes in cell morphology were observed in drug-treated cells . The structural similarity of sphingofungin B and C to sphingosine and phytosphingosine prompted us to investigate their effects on sphingolipid synthesis . Nanomolar levels of the drugs inhibited the incorporation of {3H}inositol into sphingolipid before incorporation into the sphingolipid precursor, phosphatidylinositol was affected, suggesting specific inhibition of sphingolipid synthesis . This hypothesis was confirmed by experiments in which the growth inhibitory activity of both drugs was completely ablated by the addition of phytosphingosine, dihydrosphingosine, or ketodihydrosphingosine to the culture medium . Reversal of antifungal activity by ketodihydrosphingosine suggested that serine palmitoyltransferase could be the actual target of these compounds . Direct evidence for this hypothesis was the observation of inhibition of serine palmitoyltransferase activity in crude membrane preparations at nanomolar concentrations of each drug . The potent inhibition of serine palmitoyltransferase coupled with the apparent lack of effect of these compounds on other cellular functions suggests that sphingofungin B and C will prove to be important new tools for studying the role of sphingolipids in yeast and perhaps in other organisms.

Biochem Biophys Res Commun, 1992 Dec 15, 189(2), 749 - 58
Cloning and nucleotide sequence of human liver cDNA encoding for cystathionine gamma-lyase; Lu Y et al.; We have cloned and sequenced a full-length cDNA (1083 bp) encoding the human liver cystathionine-gamma-lyase enzyme (cystathionase) . The human cystathionase sequence presented a substantial deletion of 132 bases (44 amino acids) compared to that reported for rat cystathionase, and of 135 bases (45 amino acids) compared to that reported for yeast cystathionase . After re-alignment for the missing nucleotides, the human cDNA sequence shows significant amino acid homology to that for the rat enzyme (85%) and the yeast enzyme (50%) . A search for an undeleted cDNA, by the polymerase chain reaction, yielded a second clone which contained the missing 132 bases . Flanking nucleotides in the latter clone were identical to those in the cDNA clone containing the deletion . The two forms of human cystathionase deduced from the two cDNA clones may be derived from two different genes or may be splice variants.

Biochemistry, 1992 Dec 15, 31(49), 12337 - 44
Introduction of a disulfide bond into cytochrome c stabilizes a compact denatured state; Betz SF et al.; We introduced a novel disulfide bond, modeled on that of bullfrog cytochrome c, into yeast iso-1-cytochrome c . The disulfide spontaneously forms upon purification . A variety of techniques were used to examine the denaturation of this variant and several non-cross-linked controls . Denaturation is reversible and, with the exception of the protein in which the two cysteines are blocked, consistent with a two-state process . Comparison of the calorimetric and van't Hoff enthalpy changes indicates that denaturation is two-state at pH 4.6 . Calorimetric and fluorescence-monitored guanidine hydrochloride (GdnHCl) denaturation data indicate that the free energy of denaturation for the cross-linked protein (delta Gd at 300 K) is decreased relative to non-cross-linked controls . The dependence of delta Gd on GdnHCl concentration, the GdnHCl concentration that denatures half the protein, as well as the enthalpy, entropy, and heat capacity changes (mGdnHCl, Cm, delta Hd, delta Sd, and delta Cp, respectively), all decrease in magnitude upon introduction of the cross-link . The decrease in delta Hd and delta Sd were confirmed by monitoring absorbance at several wavelengths as a function of temperature . The cross-link also decreases the pH dependence of these observables . Circular dichroism studies indicate the denatured state of the cross-linked protein possesses more structure than non-cross-linked proteins, and this structure is refractory to increases in temperature and chemical denaturant . We conclude that the diminished values of delta Gd, delta Hd, delta Sd, delta Cp, and mGdnHCl result from the denatured state of the cross-linked variant being more compact and possessing more structure than non-cross-linked controls.

Science, 1992 Dec 11, 258(5089), 1784 - 7
Identification of a second pseudoautosomal region near the Xq and Yq telomeres; Freije D et al.; The telomeres of Xq and Yq have been observed to associate during meiosis, and in rare cases a short synaptonemal complex is present . Molecular cloning of loci from Xqter and Yqter has revealed that their sequence homology extends over 400 kilobases, which suggests the possibility of genetic exchange . This hypothesis was tested by the development of two highly informative microsatellite markers from yeast artificial chromosome clones that carried Xqter sequences and the following of their inheritance in a set of reference pedigrees from the Centre d'Etude du Polymorphisme Humain in Paris, France . From a total of 195 informative male meioses, four recombination events between these loci were observed . In three cases, paternal X alleles were inherited by male offspring, and in one case a female offspring inherited her father's Y allele . These data support the existence of genetic exchange at Xq-Yq, which defines a second pseudoautosomal region between the sex chromosomes.

Science, 1992 Dec 11, 258(5089), 1780 - 4
Nucleosome core displacement in vitro via a metastable transcription factor-nucleosome complex; Workman JL et al.; In order to function, transcription factors must compete for DNA binding with structural components of chromatin, including nucleosomes . Mechanisms that could be used in this competition have been characterized with the use of the DNA binding domain of the yeast GAL4 protein . The binding of GAL4 to a nucleosome core resulted in a ternary complex containing GAL4, the core histone proteins, and DNA . This ternary complex was unstable; upon the addition of nonspecific competitor DNA, it dissociated into either the original nucleosome core particle or GAL4 bound to naked DNA . Nucleosome core destabilization by GAL4 did not require a transcriptional activation domain . These data demonstrate the displacement of nucleosome cores as a direct result of binding by a regulatory factor . Similar mechanisms might affect the establishment of factor occupancy of promoters and enhancers in vivo.

Science, 1992 Dec 11, 258(5089), 1748 - 55
Crystal structure of a complex between electron transfer partners, cytochrome c peroxidase and cytochrome c; Pelletier H et al.; The crystal structure of a 1:1 complex between yeast cytochrome c peroxidase and yeast iso-1-cytochrome c was determined at 2.3 A resolution . This structure reveals a possible electron transfer pathway unlike any previously proposed for this extensively studied redox pair . The shortest straight line between the two hemes closely follows the peroxidase backbone chain of residues Ala194, Ala193, Gly192, and finally Trp191, the indole ring of which is perpendicular to, and in van der Waals contact with, the peroxidase heme . The crystal structure at 2.8 A of a complex between yeast cytochrome c peroxidase and horse heart cytochrome c was also determined . Although crystals of the two complexes (one with cytochrome c from yeast and the other with cytochrome c from horse) grew under very different conditions and belong to different space groups, the two complex structures are closely similar, suggesting that cytochrome c interacts with its redox partners in a highly specific manner.

Biochim Biophys Acta, 1992 Dec 10, 1180(2), 221 - 4
Phytanic acid alpha-oxidation in human cultured skin fibroblasts; Singh I et al.; We studied the oxidation of {1-14C}phytanic acid, 3-methyl substituted fatty acid, to pristanic acid and 14CO2 in human skin fibroblasts . The specific activity for alpha-oxidation of phytanic acid in peroxisomes was 29- and 124-fold higher than mitochondria and endoplasmic reticulum . This finding demonstrates for the first time the presence of fatty acid alpha-oxidation enzyme system in peroxisomes.

Biochemistry, 1992 Dec 8, 31(48), 12155 - 61
Orotidylate decarboxylase: insights into the catalytic mechanism from substrate specificity studies; Shostak K et al.; Pyrimidine nucleotides were tested as substrates for pure yeast orotidylate decarboxylase in an attempt to gain insight into the nature of the catalytic mechanism of the enzyme . Substitutions of the 5-position in the pyrimidine ring of the orotidylate substrate resulted in compounds that are either excellent inhibitors or substrates of the enzyme . The 5-bromo- and 5-chloroorotidylates are potent inhibitors while the 5-fluoro derivative is a good substrate with a turnover number 30 times that observed with orotidylate . When carbon 5 of the pyrimidine ring is replaced by nitrogen in 5-azaorotidylate, the resulting compound is unstable in solution with a half-life of 25 min at pH 6 . However, studies with freshly generated 5-azaorotidylate show that an enzyme-dependent reaction occurs, presumably decarboxylation . This enzyme reaction follows simple Michaelis-Menten kinetics . Because the 5-aza group is not electrophilic, an enzyme mechanism utilizing a nucleophilic addition of the enzyme at the 5-position is ruled out . We also present studies that are not compatible with a mechanism requiring the formation of a Schiff's base prior to decarboxylation . The enzyme is tolerant of modest substitution at the 4-position, for the 4-keto group can be replaced with a thioketone . However, no catalysis is observed when the same substitution is made at the 2-position . Similarities in the substrate specificity of orotate phosphoribosyltransferase and orotidylate decarboxylase led us to compare the amino acid sequences of the two enzymes; significant (20%) sequence homology was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

J Biol Chem, 1992 Dec 5, 267(34), 24655 - 60
Inactivation of ribonuclease inhibitor by thiol-disulfide exchange; Fominaya JM et al.; Porcine ribonuclease inhibitor (RI) contains 30 1/2-cystinyl residues, all of which occur in the reduced form . Reaction of the native protein with 5,5'-dithiobis (2-nitrobenzoic acid) resulted in the release of 30 mol of the product 5-mercapto-2-nitrobenzoate, and the loss of the RNase inhibitory activity . A linear relationship between the degree of modification and inactivation was observed . The rate of modification was greatly increased in the presence of 6 M guanidinium HCl . Reaction with substoichiometric amounts of 5,5'-dithiobis(2-nitrobenzoic acid) was found to yield a mixture of fully reduced active molecules, and fully oxidized inactive ones, but no partially oxidized forms were detected . This suggests that an "all-or-none" type of modification and inactivation took place . All 1/2-cystinyl residues in the inactive, monomeric inhibitor had formed disulfide bridges, judged by the absence of either free thiol groups or mixed disulfides with 5-mercapto-2-nitrobenzoate . This fully disulfide-cross-linked molecule had an open conformation compared to the native one, as shown by gel filtration and limited proteolysis . Reaction of phenylarsinoxide with vicinal dithiols yields products that are much more stable than those with monothiols . Titration of RI with this reagent yielded complete inactivation at a reagent/thiol ratio of 0.5 . Taken together, these observations suggest that the thiol groups in RI have a diminished reactivity due to three-dimensional constraints . After the initial modification of a small number of thiol groups, a conformational change occurs which causes an increase in reactivity of the remaining thiols . The thiol groups are situated close enough together to permit the formation of 15 disulfide bridges in the inactive molecule.

J Biol Chem, 1992 Dec 5, 267(34), 24471 - 5
Cleavage of tRNA by Fe(II)-bleomycin; Huttenhofer A et al.; We have investigated the action of the chemotherapeutic agent Fe(II)-bleomycin on yeast tRNA(Phe), an RNA of known three-dimensional structure . In the absence of Mg2+ ions, the RNA is cleaved preferentially at two major positions, A31 and G53, both of which are located at the terminal base pairs of hairpin loops, and coincide with the location of tight Mg2+ binding sites . A fragment of the tRNA (residues 47-76) containing the T stem-loop is also cleaved specifically at G53 . Cleavage of both the intact tRNA and the tRNA fragment is abolished in the presence of physiological concentrations of Mg2+ (> 0.5 mM) . Since Fe(II) is not displaced from bleomycin under these conditions, we infer that tight binding of Mg2+ to tRNA excludes productive interactions between Fe(II)-bleomycin and the RNA . These results also show that loss of cleavage is not due to Mg(2+)-dependent formation of tertiary interactions between the D and T loops . In contrast, cleavage of synthetic DNA analogs of the anticodon and T stem-loops is not detectably inhibited by Mg2+, even at concentrations as high as 50 mM . In addition, the site specificities observed in cleavage of RNA and DNA differ significantly . From these results, and from similar findings with other representative RNA molecules, we suggest that the cleavage of RNA by Fe(II)-bleomycin is unlikely to be important for its therapeutic action.

Science, 1992 Dec 4, 258(5088), 1598 - 604
Roles of SWI1, SWI2, and SWI3 proteins for transcriptional enhancement by steroid receptors; Yoshinaga SK et al.; The SWI1, SWI2, and SWI3 proteins, which are required for regulated transcription of numerous yeast genes, were found also to be essential for rat glucocorticoid receptor function in yeast; the receptor failed to activate transcription in strains with mutations in the SWI1, SWI2, or SWI3 genes . Certain mutations in genes encoding components of chromatin, identified as suppressors of swi mutations, partially relieved the SWI- requirement for receptor function . Immunoprecipitation of glucocorticoid receptor derivatives from wild-type (SWI+) yeast extracts coprecipitated the SWI3 protein; such receptor-SWI3 complexes were not detected in swi1- or swi2- mutant strains, implying that a complex of multiple SWI proteins may associate with the receptor . Prior incubation of a Drosophila embryo transcription extract with the yeast SWI3-specific antibody inhibited receptor function in vitro whereas the antibody had no effect if added after initiation complex formation . Thus, positive regulation by the glucocorticoid receptor in vivo and in vitro appears to require its interaction, at an early step, with one or more SWI proteins.

Curr Opin Cell Biol, 1992 Dec, 4(6), 992 - 9
The mitogen-activated protein kinase activator; Ahn NG et al.; The mitogen-activated protein kinase appears to be regulated by another growth factor regulated kinase, the mitogen-activated protein kinase activator . In the past year, much progress has been made in purifying and characterizing the mitogen-activated protein kinase activator, in determining its primary structure, and in identifying another protein kinase that may function upstream to regulate its activity.

Naturwissenschaften, 1992 Dec, 79(12), 551 - 9
Mechanisms of electromagnetic interaction with cellular systems; Grundler W et al.; The question of how electromagnetic fields--static or low to high frequency--interact with biological systems is of great interest . The current discussion among biologists, chemists, and physicists emphasizes aspects of experimental verification and of defining microscopic and macroscopic mechanisms . Both aspects are reviewed here . We emphasize that in certain situations nonthermal interactions of electromagnetic fields occur with cellular systems.

Biotechniques, 1992 Dec, 13(6), 870 - 4
Quantitation of radioactively labeled RNA by hybrid selection using biotinylated oligonucleotides; Min J et al.; We describe a procedure to quantify specific, radioactively labeled RNA sequences . This procedure combines hybrid selection of an RNA using biotinylated oligonucleotides with gel electrophoretic analysis of the selected RNA . We show that the hybrid selection procedure is specific and quantitative . It enriches a specific RNA sequence at least 600-fold . Specificity and sensitivity are increased to at least 10,000-fold enrichment by a combination of RNase T1 digestion of the RNA:oligonucleotide hybrid prior to selection, followed by gel electrophoretic fractionation of the selected RNA fragment . Furthermore, this modification allows one to quantify specific regions of an RNA transcript, as well as to monitor several different RNA sequences in one experiment . It is estimated that the sensitivity of this procedure is high enough to detect specific RNA sequences present at 1 part in 100,000.

Experientia, 1992 Dec 1, 48(11-12), 1041 - 58
Stress-induced photon emission from perturbed organisms; Slawinski J et al.; This paper reviews an ultraweak luminescent response of selected biological systems (lower and higher plants, insects and spermatozoa) to certain kinds of detrimental mechanical, temperature, chemical and photochemical stress and to lethal factors . The enhancing effect of white light and formaldehyde on the ultraweak luminescence of yeast and spermatozoa cells is described for the first time . An increase in the percentage of long wavelengths (lambda > 600 nm) with an increase in reaction time, and a significant influence of the suspending medium on the ultraweak luminescence, were observed . The vitality and motility of bull spermatozoa and the vitality of yeast cells were drastically decreased by treatment with white light, water, formaldehyde and iron-ions . Successive irradiation of intact bull spermatozoa cells with white light caused an increase in the intensity of delayed luminescence . An attempt has been undertaken to find stochastic models of non-stationary photon emission . The quasi-relaxation descending stage of non-stationary processes can be modeled as the Integrated Moving Average process IMA (0, 1, 1), and memory and transfer functions can describe the degree of perturbation in the yeast Saccharomyces cerevisiae . The relation of the ultraweak luminescence response to perturbations of homeostasis is discussed in the framework of biochemical and physical models.

J Cell Biol, 1992 Dec, 119(6), 1541 - 57
Primary structure and cellular localization of chicken brain myosin-V (p190), an unconventional myosin with calmodulin light chains; Espreafico EM et al.; Recent biochemical studies of p190, a calmodulin (CM)-binding protein purified from vertebrate brain, have demonstrated that this protein, purified as a complex with bound CM, shares a number of properties with myosins (Espindola, F . S., E . M . Espreafico, M . V . Coelho, A . R . Martins, F . R . C . Costa, M . S . Mooseker, and R . E . Larson . 1992 . J . Cell Biol . 118:359-368) . To determine whether or not p190 was a member of the myosin family of proteins, a set of overlapping cDNAs encoding the full-length protein sequence of chicken brain p190 was isolated and sequenced . Verification that the deduced primary structure was that of p190 was demonstrated through microsequence analysis of a cyanogen bromide peptide generated from chick brain p190 . The deduced primary structure of chicken brain p190 revealed that this 1,830-amino acid (aa) 212,509-D) protein is a member of a novel structural class of unconventional myosins that includes the gene products encoded by the dilute locus of mouse and the MYO2 gene of Saccharomyces cerevisiae . We have named the p190-CM complex "myosin-V" based on the results of a detailed sequence comparison of the head domains of 29 myosin heavy chains (hc), which has revealed that this myosin, based on head structure, is the fifth of six distinct structural classes of myosin to be described thus far . Like the presumed products of the mouse dilute and yeast MYO2 genes, the head domain of chicken myosin-V hc (aa 1-764) is linked to a "neck" domain (aa 765-909) consisting of six tandem repeats of an approximately 23-aa "IQ-motif." All known myosins contain at least one such motif at their head-tail junctions; these IQ-motifs may function as calmodulin or light chain binding sites . The tail domain of chicken myosin-V consists of an initial 511 aa predicted to form several segments of coiled-coil alpha helix followed by a terminal 410-aa globular domain (aa, 1,421-1,830) . Interestingly, a portion of the tail domain (aa, 1,094-1,830) shares 58% amino acid sequence identity with a 723-aa protein from mouse brain reported to be a glutamic acid decarboxylase . The neck region of chicken myosin-V, which contains the IQ-motifs, was demonstrated to contain the binding sites for CM by analyzing CM binding to bacterially expressed fusion proteins containing the head, neck, and tail domains . Immunolocalization of myosin-V in brain and in cultured cells revealed an unusual distribution for this myosin in both neurons and nonneuronal cells.(ABSTRACT TRUNCATED AT 400 WORDS)

EMBO J, 1992 Dec, 11(13), 5051 - 61
A new subclass of nucleoporins that functionally interact with nuclear pore protein NSP1; Wimmer C et al.; NSP1 is a nuclear pore protein (nucleoporin) essential for cell growth . To identify the components that functionally interact with NSP1 in the living cell, we developed a genetic screen for mutants that are lethal in a genetic background of mutated, but not wild type NSP1 . Fourteen synthetic lethal mutants were obtained, belonging to at least four different complementation groups . The genes of two complementation groups, NSP116 and NSP49, were cloned . Like the previously described nucleoporins, these genes encode proteins with many repeat sequences . NSP116 and NSP49, however, contain a new repetitive sequence motif 'GLFG', which classifies them as a subclass of nucleoporins . NSP116 and NSP49, tagged with the IgG binding domain of protein A and expressed in yeast, are located at the nuclear envelope . These data provide in vivo evidence that distinct subclasses of nucleoporins physically interact or share overlapping function in nuclear pore complexes.

EMBO J, 1992 Dec, 11(13), 5033 - 9
A conformational rearrangement in the spliceosome is dependent on PRP16 and ATP hydrolysis; Schwer B et al.; PRP16 is an RNA-dependent ATPase that is required for the second catalytic step of pre-mRNA splicing . We have previously shown that PRP16 protein binds stably to spliceosomes that have completed 5' splice site cleavage and lariat formation . PRP16 then promotes 3' splice site cleavage and exon ligation in an ATP-dependent fashion . We now demonstrate that PRP16 can hydrolyse all nucleoside triphosphates and corresponding deoxynucleotides; complementation of the second catalytic step shows the same broad nucleotide specificity . These results link the nucleotide requirement of step 2 to PRP16 . Interestingly, we find that PRP16 promotes a conformational change in the spliceosome which results in the protection of the 3' splice site against oligo-directed RNase H cleavage . This structural rearrangement is dependent on the hydrolysis of ATP, since ATP gamma S, a competitive inhibitor of the PRP16 ATPase activity, does not promote the protection of the 3' splice site and formation of mRNA.

Eur J Biochem, 1992 Dec 1, 210(2), 467 - 73
Mechanism of carboxypeptidase-Y-catalysed peptide semisynthesis; Christensen U et al.; The initial rate steady-state kinetics of carboxypeptidase-Y-catalyzed hydrolysis and aminolysis reactions with some alpha-N-benzoyl-L-tyrosinyl compounds has been investigated using L-valinamide as the nucleophile in aminolysis . Hydrolysis of alpha-N-benzoyl-L-tyrosinyl ethyl ester, 4-nitroanilide, and -amide has been studied in the pH range 4-9 . The results are interpreted in terms of the classical serine proteinase mechanism, which involves enzyme-substrate complex formation, followed by acylation and deacylation of the enzyme . The three reactions share the same deacylation step . It is rate-determining with the ester substrate, but with the 4-nitroaniline acylation is and this is even more pronounced with the amide . From the pH dependencies, no change of rate-determining step is apparent in the range pH 4-9 . For the 4-nitroanilide and the amide substrates, the kinetic parameter, Kc/Km, is influenced by an ionizing group with a pK value of 6 . Probably this is the active-site histidine residue, which thus is active in acylation in its deprotonated form . That group affects the deacylation reaction similarly as seen from the kinetics of the ester substrate . Aminolysis occurs in parallel to hydrolysis in the presence of reactive nucleophiles . Here L-valinamide was used as model nucleophile . The analysis of the observed kinetic effects of L-valinamide on the initial rate behaviour of carboxypeptidase-Y-catalyzed hydrolysis reactions suggests a reaction mechanism which involves (a) the binding of the free nucleophile to the free enzyme and (b) reaction of the free nucleophile with the acyl-enzyme complex forming an enzyme-aminolysis product complex, which dissociates into the free enzyme and the aminolysis product . The reactions are characterized by a number of kinetic parameters, the values of which are determined . The results of aminolysis progress reactions indicate that the formation of the product in high yields is strongly dependent on the leaving group of the substrate . The initial production of aminolysis product, however, is the same for the three substrates . But the fact that their Kc/Km values differ by several orders of magnitude leads to significantly different progresses of the aminolysis . The ester substrate is the only one that efficiently competes with and hinders the hydrolysis of the aminolysis product.

Proc Natl Acad Sci U S A, 1992 Dec 1, 89(23), 11156 - 60
At least three distinct proteins are necessary for the reconstitution of a specific multiprotein complex at a eukaryotic chromosomal origin of replication; Estes HG et al.; We have reconstituted in vitro a multistage assembly of a protein complex that specifically recognizes a yeast genomic origin of replication, the autonomously replicating sequence ARS121 . The first step in the assembly was the interaction of the known origin-binding factor OBF1 and another factor, OBF2, with the ARS121 origin of replication to form the OBF1-OBF2-origin complex . This complex was the substrate for the ATP-dependent binding of a third DNA-binding activity, the core binding factor, CBF . Binding of CBF to the origin, identified by the retarded mobility of the origin DNA fragment in agarose gels, required, in addition to ATP and the OBF1-OBF2-origin complex, a functional essential core nucleotide sequence . ARS121 DNA containing mutations in the core, which inactivate the origin in vivo, did not sustain stable CBF binding, whereas ARS121 DNA mutated outside the boundaries of the essential core, which has normal origin function, bound CBF as wild type . This tight, direct correlation between the ability of the origin to bind CBF and its function as an origin of replication in vivo strongly suggest that the multiprotein complex reconstituted in vitro has a key role in the initiation of DNA replication.

Mol Cell Biol, 1992 Dec, 12(12), 5758 - 67
A mutation outside the two zinc fingers of ADR1 can suppress defects in either finger; Camier S et al.; A second-site mutation that restored DNA binding to ADR1 mutants altered at different positions in the two zinc fingers was identified . This mutation (called IS1) was a conservative change of arginine 91 to lysine in a region amino terminal to the two zinc fingers and known from previous experiments to be necessary for DNA binding . IS1 increased binding to the UAS1 sequence two- to sevenfold for various ADR1 mutants and twofold for wild-type ADR1 . The change of arginine 91 to glycine decreased binding twofold, suggesting that this arginine is involved in DNA binding in the wild-type protein . The increase in binding by IS1 did not involve protein-protein interactions between the two ADR1 monomers, nor did it require the presence of the sequences flanking UAS1 . However, the effect of IS1 was influenced by the sequence of the first finger, suggesting that interactions between the region amino terminal to the fingers and the fingers themselves could exist . A model for the role of the amino-terminal region based on these results and sequence homologies with other DNA-binding motifs is proposed.

Mol Cell Biol, 1992 Dec, 12(12), 5652 - 8
Separate information required for nuclear and subnuclear localization: additional complexity in localizing an enzyme shared by mitochondria and nuclei; Rose AM et al.; The TRM1 gene of Saccharomyces cerevisiae codes for a tRNA modification enzyme, N2,N2-dimethylguanosine-specific tRNA methyltransferase (m2(2)Gtase), shared by mitochondria and nuclei . Immunofluorescent staining at the nuclear periphery demonstrates that m2(2)Gtase localizes at or near the nuclear membrane . In determining sequences necessary for targeting the enzyme to nuclei and mitochondria, we found that information required to deliver the enzyme to the nucleus is not sufficient for its correct subnuclear localization . We also determined that mislocalizing the enzyme from the nucleus to the cytoplasm does not destroy its biological function . This change in location was caused by altering a sequence similar to other known nuclear targeting signals (KKSKKKRC), suggesting that shared enzymes are likely to use the same import pathway as proteins that localize only to the nucleus . As with other well-characterized mitochondrial proteins, the mitochondrial import of the shared methyltransferase depends on amino-terminal amino acids, and removal of the first 48 amino acids prevents its import into mitochondria . While this truncated protein is still imported into nuclei, the immunofluorescent staining is uniform throughout rather than at the nuclear periphery, a staining pattern identical to that described for a fusion protein consisting of the first 213 amino acids of m2(2)Gtase in frame with beta-galactosidase . As both of these proteins together contain the entire m2(2)Gtase coding region, the information necessary for association with the nuclear periphery must be more complex than the short linear sequence necessary for nuclear localization.

Mol Cell Biol, 1992 Dec, 12(12), 5516 - 26
A REB1-binding site is required for GCN4-independent ILV1 basal level transcription and can be functionally replaced by an ABF1-binding site; Remacle JE et al.; The ILV1 gene of Saccharomyces cerevisiae encodes the first committed step in isoleucine biosynthesis and is regulated by general control of amino acid biosynthesis . Deletion analysis of the ILV1 promoter revealed a GC-rich element important for the basal level expression . This cis-acting element, called ILV1BAS, is functional independently of whether GCN4 protein is present . Furthermore, unlike the situation at HIS4, the magnitude of GCN4-mediated derepression is independent of ILV1BAS . The element has homology to the consensus REB1-binding sequence CGGGTARNNR . Gel retardation assays showed that REB1 binds specifically to this element . We show that REB1-binding sites normally situated in the SIN3 promoter and in the 35S rRNA promoter can substitute for the ILV1 REB1 site . Furthermore, a SIN3 REB1 site containing a point mutation that abolishes REB1 binding does not support ILV1 basal level expression, suggesting that binding of REB1 is important for the control of ILV1 basal level expression . Interestingly, an ABF1-binding site can also functionally replace the ILV1 REB1-binding site . A mutated ABF1 site that displays a very low affinity for ABF1 does not functionally replace the ILV1 REB1 site . This suggests that ABF1 and REB1 may have related functions within the cell . Although the REB1-binding site is required for the ILV1 basal level expression, the site on its own stimulates transcription only slightly when combined with the CYC1 downstream promoter elements, indicating that another ILV1 promoter element functions in combination with the REB1 site to control high basal level expression.

Mol Cell Biol, 1992 Dec, 12(12), 5464 - 73
Multiple functional domains of human U2 small nuclear RNA: strengthening conserved stem I can block splicing; Wu J et al.; We showed previously that a branch site mutation in simian virus 40 early pre-mRNA that prevented small t antigen mRNA splicing could be efficiently suppressed by a compensatory mutation in a coexpressed U2 small nuclear (sn) RNA gene . We have now generated second-site mutations in this suppressor gene to investigate regions of U2 RNA required for function . A number of mutations in a putative stem at the 5' end of the molecule inhibited splicing, indicating that bases in this region are important for activity . However, several lines of evidence suggested that formation of the entire stem is not essential for splicing . Indeed, mutations that strengthen the stem actually inhibited splicing, and evidence that this prevents a required base-pairing interaction with U6 snRNA is presented . These results suggest that the relative stabilities of competing intra- and intermolecular base-pairing interactions play an important role in the splicing reaction . Mutations in a conserved single-stranded region immediately 3' to the branch site recognition sequence all inhibited splicing, indicating that this region is required for U2 function, although its exact role remains unknown . Finally, two mutations in the loop of stem IV at the 3' end of the molecule, which destroy the binding site of U2 sn ribonucleoprotein B", prevented small t splicing; this finding contrasts with previous studies which utilized different assay systems . Analysis of the accumulation and subcellular localization of all of the mutant RNAs showed that they were similar to those of the parental suppressor U2 RNA, indicating that the effects observed indeed reflect defects in splicing.

J Cell Biol, 1992 Dec, 119(5), 1097 - 116
Morphological analysis of protein transport from the ER to Golgi membranes in digitonin-permeabilized cells: role of the P58 containing compartment; Plutner H et al.; The glycoside digitonin was used to selectively permeabilize the plasma membrane exposing functionally and morphologically intact ER and Golgi compartments . Permeabilized cells efficiently transported vesicular stomatitis virus glycoprotein (VSV-G) through sealed, membrane-bound compartments in an ATP and cytosol dependent fashion . Transport was vectorial . VSV-G protein was first transported to punctate structures which colocalized with p58 (a putative marker for peripheral punctate pre-Golgi intermediates and the cis-Golgi network) before delivery to the medial Golgi compartments containing alpha-1,2-mannosidase II and processing of VSV-G to endoglycosidase H resistant forms . Exit from the ER was inhibited by an antibody recognizing the carboxyl-terminus of VSV-G . In contrast, VSV-G protein colocalized with p58 in the absence of Ca2+ or the presence of an antibody which inhibits the transport component NSF (SEC18) . These studies demonstrate that digitonin permeabilized cells can be used to efficiently reconstitute the early secretory pathway in vitro, allowing a direct comparison of the morphological and biochemical events involved in vesicular tafficking, and identifying a key role for the p58 containing compartment in ER to Golgi transport.

Dev Biol, 1992 Dec, 154(2), 231 - 44
Genetic approaches to understanding muscle development; Epstein HF et al.; The analysis of both naturally occurring and experimentally induced mutants has greatly advanced our understanding of muscle development . Molecular biological techniques have led to the isolation of genes associated with inherited human diseases that affect muscle tissues . Analysis of the encoded proteins in conjunction with the mutant phenotypes can provide powerful insights into the function of the protein in normal muscle development . Systematic searches for muscle mutations have been made in experimental systems, most notably the fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans . In addition, known muscle protein genes from other organisms have been used to isolate homologs from genetically manipulatable organisms, allowing mutant analysis and the study of protein function in vivo . Mutations in transcription factor genes that affect mesoderm development have been isolated and genetic lesions affecting myofibril assembly have been identified . Genetic experiments inducing mutations and rescuing them by transgenic methods have uncovered functions of myofibrillar protein isoforms . Some isoforms perform muscle-specific functions, whereas others appear to be replaceable by alternative isoforms . Mutant analysis has also uncovered a relationship between proteins at the cell membrane and the assembly and alignment of the myofibrillar apparatus . We discuss examples of each of these genetic approaches as well as the developmental and evolutionary implications of the results.

Cell Struct Funct, 1992 Dec, 17(6), 335 - 9
Changes in intracellular cAMP level and activities of adenylcyclase and phosphodiesterase during meiosis of lily microsporocytes; Sato S et al.; In the yeasts, Saccharomyces cerevisiae and Schyzosaccharomyces pombe, reduction of intracellular cyclic adenosine monophosphate (cAMP) is known to trigger the sporulation processes by activating various meiosis specific genes . In order to ascertain whether a similar mechanism is operative in higher plants, we carried out preliminary studies on lily microsporocytes . Measurement of cAMP levels as well as the activities of adenyl cyclase and phosphodiesterase in somatic cells and different stages of meiosis, and arrest of its in protoplasts cultured under conditions of high cAMP provided direct evidence that similar phenomena occur in plant meiocytes as earlier documented in yeasts.

J Cell Biol, 1992 Dec, 119(6), 1451 - 7
A signal-anchor sequence selective for the mitochondrial outer membrane; McBride HM et al.; pOMD29 is a hybrid protein containing the NH2-terminal topogenic sequence of a bitopic, integral protein of the outer mitochondrial membrane in yeast, OMM70, fused to dihydrofolate reductase . The topogenic sequence consists of two structural domains: an NH2-terminal basic region (amino acids 1-10) and an apolar region which is the predicted transmembrane segment (amino acids 11-29) . The transmembrane segment alone was capable of targeting and inserting the hybrid protein into the outer membrane of intact mitochondria from rat heart in vitro . The presence of amino acids 1-10 enhanced the rate of import, and this increased rate depended, in part, on the basic amino acids located at positions 2, 7, and 9 . Deletion of a large portion of the transmembrane segment (amino acids 16-29) resulted in a protein that exhibited negligible import in vitro . Insertion of pOMD29 into the outer membrane was not competed by import of excess precursor protein destined for the mitochondrial matrix, indicating that the two proteins may have different rate-limiting steps during import . We propose that the structural domains within amino acids 1-29 of pOMD29 cooperate to form a signal-anchor sequence, the characteristics of which suggest a model for proper sorting to the mitochondrial outer membrane.

EMBO J, 1992 Dec, 11(13), 4787 - 94
Role of ATP in the intramitochondrial sorting of cytochrome c1 and the adenine nucleotide translocator; Wachter C et al.; Import of precursor proteins across the mitochondrial inner membrane requires ATP in the matrix . However, some precursors can still cross the outer membrane in ATP-depleted mitochondria . Here we show that the adenine nucleotide translocator is imported normally into the inner membrane after the matrix has been depleted of ATP . This result supports the earlier suggestion that the translocator inserts into the inner membrane without passing through the matrix . Depletion of matrix ATP also has no detectable effect on the import and maturation of cytochrome c1, which is targeted to the intermembrane space . It thus seems probable that cytochrome c1 does not completely cross the inner membrane during its import pathway.

Genes Dev, 1992 Dec, 6(12A), 2417 - 28
SIT4 protein phosphatase is required for the normal accumulation of SWI4, CLN1, CLN2, and HCS26 RNAs during late G1; Fernandez-Sarabia MJ et al.; In Saccharomyces cerevisiae, the RNA levels of the G1 cyclins CLN1, CLN2, and HCS26 increase dramatically during the late G1 phase of the cell cycle . The SIT4 gene, which encodes a serine/threonine protein phosphatase, is required for the normal accumulation of CLN1, CLN2, and HCS26 RNAs during late G1 . This requirement for SIT4 in normal G1 cyclin RNA accumulation is at least partly via SWI4 . Strains containing mutations in SIT4 are sensitive to the loss of either CLN2 or CLN3 function . At the nonpermissive temperature, temperature-sensitive sit4 strains are blocked for both bud emergence and DNA synthesis . Heterologous expression of CLN2 in the absence of SIT4 function results in DNA synthesis, but most of the cells are still blocked for bud emergence . Therefore, SIT4 is required for at least two late G1 or G1/S functions: the normal accumulation of G1 cyclin RNAs (which is required for DNA synthesis) and some additional function that is required for bud emergence or cell cycle progression through late G1 or G1/S.

Yeast, 1992 Dec, 8(12), 1101 - 3
Genetic and physical mapping of the WBP1 locus close to CENV; te Heesen S et al.; The WBP1 locus, encoding an essential component of the N-oligosaccharyl transferase, was mapped both genetically and physically . The gene is located on chromosome V between CENV and gcn4 . The distance from CENV sequences is 2 kb.

Mol Immunol, 1992 Dec, 29(12), 1427 - 35
Non-linear epitopes of the large subunit of Ku autoantigen recognized by monoclonal and autoantibodies; Wen J et al.; Sera from certain patients with SLE, scleroderma and other autoimmune diseases react with the two subunits of the Ku protein: 86 and 70 kDa . Previous experiments indicated that a region of 40 amino acids near the C-terminus of the 86 kDa subunit between amino acids 667 and 708 was critical for binding of monoclonal and some autoimmune antibodies . In the present study, a series of additional 5' deletions and site-specific mutations in the critical region were produced and the immunoreactivities of the recombinant proteins were examined . ELISA and immunoblot analyses showed that three non-competing monoclonal antibodies specific for the 86 kDa subunit require stretches of amino acids significantly longer than 40 amino acids for reactivity, suggesting that the antigen is recognized in a folded state with perhaps more than one contact point . The reactivities of 12 of 24 anti-Ku positive autoimmune sera screened depended on the same amino acid sequences required for binding of the monoclonal antibodies, site-specific mutations reduced the reactivities of monoclonal and autoantibodies in a similar way . Preincubation of native Ku protein with the monoclonal antibodies shifted the electrophoretic mobility of Ku protein-DNA complex, suggesting that these monoclonal antibodies bind to epitopes on the surface of the native Ku protein . Taken together, the results from the deletion and site-directed mutagenesis demonstrate that both monoclonal and autoantibodies recognize non-linear epitopes of the 86 kDa polypeptide . These findings indicate that in a large portion of patients the anti-Ku autoimmune response is similar to the normal immune response to the Ku antigen in mice.

FEBS Lett, 1992 Nov 30, 313(3), 229 - 31
Three-dimensional structure of apotransketolase . Flexible loops at the active site enable cofactor binding; Sundstrom M et al.; The structure determination of apotransketolase and the comparison of its three-dimensional structure with that of the holoenzyme has revealed that no large conformational changes are associated with cofactor binding . Two loops at the active site are flexible in the apoenzyme which enables ThDP to reach its binding site . Binding of the cofactor induces defined conformations for these two loops at the active site . One of these loops is directly involving in binding of the cofactors, Ca2+ and ThDP . This loop acts like a flap which closes off the diphosphate binding site . After binding of the cofactor, residues of this loop form interactions to residues of loop 383-398 from the second subunit . These interactions stabilize the conformation of the two loops from a flexible to a 'closed' conformation.

Cell, 1992 Nov 27, 71(5), 803 - 17
A novel base-pairing interaction between U2 and U6 snRNAs suggests a mechanism for the catalytic activation of the spliceosome; Madhani HD et al.; Prior to the chemical steps of mRNA splicing, the extensive base-pairing interaction between the U4 and U6 spliceosomal snRNAs is disrupted . Here, we use a mutational analysis in yeast to demonstrate a conserved base-pairing interaction between the U6 and U2 snRNAs that is mutually exclusive with the U4-U6 interaction . In this novel pairing, conserved sequences in U6 interact with a sequence in U2 that is immediately upstream of the branch point recognition region . Remarkably, the residues in U6 that can be consequently juxtaposed with the intron substrate include those that have been proposed previously to be catalytic . Both the first and second steps of splicing are inhibited when this base-paired structure is mutated . These observations, together with the high conservation of the U2-U6 structure, lead us to propose that it might be a component of the spliceosomal active site.

Cell, 1992 Nov 27, 71(5), 833 - 40
The capture of a DNA double helix by an ATP-dependent protein clamp: a key step in DNA transport by type II DNA topoisomerases; Roca J et al.; The binding of linear and circular forms of DNA to yeast DNA topoisomerase II or its complex with AMPPNP, the nonhydrolyzable beta,gamma-imido analog of ATP, was carried out to probe the ATP analog-induced conformational change of the enzyme . Binding of the ATP analog is shown to convert the enzyme to a circular clamp with an annulet, through which only a linear DNA can pass; subsequent circularization of the bound linear DNA forms a salt-stable catenane between the protein circular clamp and the DNA ring . Analysis of catenane formation between a small DNA ring originally bound to the topoisomerase and a large DNA ring subsequently added, under conditions such that the two do not exchange, supports a model in which a second DNA double-helix can enter the open jaws of a DNA-bound protein clamp, and the closure of the jaws upon ATP-binding traps the second duplex and transports it through an enzyme-operated gate in the first DNA duplex.

Nucleic Acids Res, 1992 Nov 25, 20(22), 5985 - 9
TGA cysteine codons and intron sequences in conserved and nonconserved positions are found in macronuclear RNA polymerase genes of Euplotes octocarinatus; Kaufmann J et al.; The gene sequences of the second largest subunits of RNA polymerases I and II of Euplotes octocarinatus, RPA2 and RPB2, were determined and compared to the respective known sequences of Saccharomyces cerevisiae . The similarity of the derived polypeptide sequences permitted their assignment to the respective polymerases and allowed the comparison of the zinc binding regions . In frame TGA codons were detected, which are likely to encode conserved cysteinyl residues in the putative zinc-finger region of the RPA2 gene . They were also found in other positions in both the RPA2 and RPB2 genes . The RPB2 gene contains a 30 bp intron close to the 5'-end of its coding region . The 5'-ends of the coding regions of all three genes encoding the largest subunits of the three different polymerases were also analyzed . The zinc finger structures again show the use of TGA codons for conserved cysteinyl residues in two of the genes . An N-terminal intron is located in the RPB1 gene at a conserved position as compared to the respective genes of several other eucarya.

Nucleic Acids Res, 1992 Nov 25, 20(22), 5927 - 35
The FLP protein contacts both major and minor grooves of its recognition target sequence; Panigrahi GB et al.; The FLP protein of the 2 microns plasmid of Saccharomyces cerevisiae promotes conservative site-specific recombination between DNA sequences that contain the FLP recognition target (FRT) . FLP binds to each of the three 13 base pair symmetry elements in the FRT site in a site-specific manner . We have probed both major and minor groove contacts of FLP using dimethyl sulphate, monoacetyl-4-hydroxyaminoquinoline 1-oxide and potassium permanganate and find that the protein displays extensive interactions with residues of both the major and minor grooves of 10 base pairs of each symmetry element . We find no evidence that the FRT site assumes a single-stranded conformation upon FLP binding.

Biochemistry, 1992 Nov 24, 31(46), 11489 - 99
Role of internal thermodynamics in determining hydrogen tunneling in enzyme-catalyzed hydrogen transfer reactions; Rucker J et al.; Previous investigations have indicated a role for hydrogen tunneling in the yeast alcohol dehydrogenase catalyzed oxidation of benzyl alcohol {Cha, Y., Murray, C . J., & Klinman, J . P . (1989) Science 243, 1325} and the bovine plasma amine oxidase catalyzed oxidation of benzylamine {Grant, K.L., & Klinman, J . P . (1989) Biochemistry 28,6597} . In the present studies, values of protium to tritium and deuterium to tritium isotope effects and their temperature dependencies have been measured using ring-substituted substrates for yeast alcohol dehydrogenase and bovine plasma amine oxidase, revealing tunneling in each case . The results of these studies indicate that hydrogen tunneling is a general phenomenon and is not limited to enzyme reactions with degenerate energy levels for bound substrates and products . An analysis of internal thermodynamics in the yeast alcohol dehydrogenase reaction shows that tunneling occurs when delta H degrees is endothermic and that the degree of tunneling appears to increase as delta H degrees decreases toward zero.

J Mol Biol, 1992 Nov 20, 228(2), 637 - 51
Solution structures of two zinc-finger domains from SWI5 obtained using two-dimensional 1H nuclear magnetic resonance spectroscopy . A zinc-finger structure with a third strand of beta-sheet; Neuhaus D et al.; This paper describes the detailed three-dimensional structures of two zinc-finger domains from the yeast transcription factor SWI5, calculated using the results of the n.m.r . experiments described in the accompanying paper . The structure of finger 2 is essentially similar to those previously obtained by others for isolated, synthetic single zinc-finger domains in solution, and for the three zinc-finger peptide Zif268 in its crystalline complex with DNA . The N-terminal half of the sequence forms a two-stranded, irregular beta-sheet containing both of the metal-binding cysteine residues, while the remainder of the structure forms a helix . Approximately the first half of this helix is alpha-helical, whereas the C-terminal portion, including the two metal-binding histidine residues, is 3(10) helical . Four invariant hydrophobic residues form a core to the structure . In contrast to all previously described structures of zinc-finger domains, finger 1 has an additional strand in the beta-sheet, formed by residues N-terminal to the formal start of the finger motif . This additional strand plays a role in stabilising the folded form of finger 1, since a two-finger peptide lacking the N-terminal residues showed folded structure in finger 2 but not in finger 1.

J Mol Biol, 1992 Nov 20, 228(2), 315 - 21
Numerous group I introns with variable distributions in the ribosomal DNA of a lichen fungus; DePriest PT et al.; The length of the small subunit ribosomal DNA (SSU rDNA) differs significantly among individuals from natural populations of the ascomycetous lichen complex Cladonia chlorophaea . The sequence of the 3' region of the SSU rDNA from two individuals, chosen to represent the shortest and longest sequences, revealed multiple insertions within a region that otherwise aligned with a 520-nucleotide sequence of the SSU rDNA in Saccharomyces cerevisiae . The high degree of variability in SSU rDNA size can be accounted for by different numbers of insertions; one individual had two group I introns and the second had five introns, two of which were clearly related to introns at identical positions in the other individual . Yet, introns in different positions, whether within an individual or between individuals, were not similar in sequence . The distribution of introns at three of the positions is consistent with either intron loss or acquisition, and clearly indicates the dynamic variability in this region of the nuclear genome . All seven insertions, which ranged in size from 210 to 228 nucleotides, had the conserved sequence and secondary structural elements of group I introns . The variation in distribution and sequence of group I introns within a short highly conserved region of rDNA presents a unique opportunity for examining the molecular evolution and mobility of group I introns within a systematics framework.

Biochem Biophys Res Commun, 1992 Nov 16, 188(3), 1280 - 5
Enhanced production of platelet-activating factor in stimulated rat leukocytes pretreated with triacsin C, A novel acyl-coA synthetase inhibitor; Hayashi M et al.; Triacsin C, a product of Streptmyces sp . SK-1894, was previously reported as an inhibitor of long chain acyl-CoA synthetase . Pretreatment with triacsin C (500 nM) for 1h enhanced production of platelet-activating factor in rat neutrophils, followed by stimulation with A23187 or fMLP . Amount of lyso-PAF was also augumented . Triacsin C alone did not increase PAF content and did not modulate enzymatic activities of acytransferase, cholinephosphotransferase, acetylhydrolase, acetyltransferase or phospholipase A2 . These results suggest that triacsin C might enhance supply of substrate for PAF synthesis, i.e . accumulation of lyso-PAF by interfering reacylation pathway.

Anal Biochem, 1992 Nov 15, 207(1), 24 - 31
Multiple nucleic acid labeling and rainbow detection; Hoeltke HJ et al.; A method which allows discrete nucleic acid sequences to be detected with differently colored hybridization signals on the same blot involving only a single hybridization step is described . Nucleic acid probes labeled with digoxigenin, fluorescein, or biotin are hybridized simultaneously to immobilized target nucleic acids . Differential colorimetric detection is carried out in consecutive alkaline phosphatase-based immunoassays with one of three 3-hydroxy-2-naphthoic acid anilide phosphate/diazonium salt combinations as substrate . Each label is visualized by a different color precipitate (green, red, and blue) directly on the membrane . We demonstrate the use of this method in multicolor plasmid mapping, detection of different genomic sequences on a single Southern blot, discrimination of transcription levels in a Northern blot, and colony screening . Advantages and limitations of the method, as well as further applications, are discussed.

Anal Biochem, 1992 Nov 15, 207(1), 106 - 8
A preparation and purification of {1-14C}acetylcarnitine; Roughan G et al.; {1-14C}Acetylcarnitine was prepared from {1-14C}acetate and L-carnitine using acetyl-CoA synthetase and carnitine acetyltransferase . The product was purified by ion-exchange and thin-layer chromatography . Conversion of {1-14C}acetate to {1-14C}acetylcarnitine was better than 90% and overall recovery of the pure product was greater that 80%.

Proc Natl Acad Sci U S A, 1992 Nov 15, 89(22), 10930 - 4
Induction of aP2 gene expression by nonmetabolized long-chain fatty acids; Grimaldi PA et al.; Long-chain fatty acids (FA) have been shown to regulate expression of the gene for the adipocyte FA-binding protein aP2 . We examined whether this effect was exerted by FA themselves or by a FA metabolite . The alpha-bromo derivative of palmitate, an inhibitor of FA oxidation, was synthesized in the radioactive form, and its metabolism was investigated and correlated with its ability to induce aP2 in Ob1771 preadipocytes . alpha-Bromopalmitate was not utilized by preadipocytes . It was not cleared from the medium over a 24-hr period and was not incorporated into cellular lipids . Short incubations indicated that alpha-bromopalmitate exchanged across the preadipocyte membrane but remained in the free form inside the cell . In line with this, preadipocyte homogenates did not activate alpha-bromopalmitate to the acyl form . However, although it was not metabolized, bromopalmitate was much more potent than native FA in inducing aP2 gene expression . Induction exhibited the characteristics previously described for native FA, indicating that a similar if not identical mechanism was involved . The data indicated that induction of aP2 was exerted by unprocessed FA . Finally, in contrast to preadipocytes, adipocytes metabolized bromopalmitate . This reflected increased activity with cell differentiation of a palmitoyl-CoA synthase that could activate palmitate and bromopalmitate at about one-fifth the rate for palmitate . In preadipocytes, the predominant fatty-acyl-CoA synthase, arachidonyl-CoA synthase, had very low affinity for both FA . Increased activity of the palmitoyl-CoA synthase, which has a wider substrate range, is likely to be important for initiation of lipid deposition.

Proc Natl Acad Sci U S A, 1992 Nov 15, 89(22), 10563 - 7
Identification of a negative regulatory function for steroid receptors; McDonnell DP et al.; This report describes the identification of a negative regulator of estrogen and progesterone receptor function . Using a reconstituted estrogen-responsive transcription system in Saccharomyces cerevisiae, we have identified a "repressor function," which when mutated, increases the transcriptional activity of the estrogen and progesterone receptors . In the case of the estrogen receptor this mutation increases the sensitivity of estrogen-mediated activation by at least four orders of magnitude . Analysis of derivatives of the estrogen receptor indicated that this repressor specifically affects the transcription activity of the TAF1 activation domain of the estrogen receptor . The repressor was cloned by complementation and identified as SSN6, a previously described mediator of glucose repression in yeast . Our results indicate that SSN6 is likely to be involved also in the repression of other cellular activators . Interestingly, deletion of the SSN6 protein allows the antiestrogens ICI 164384 and nafoxidine to behave as more potent agonists of estrogen receptor function, while RU486 also becomes a more potent activator of progesterone receptor function . These data suggest that in wild-type cells the role of hormone is twofold: it promotes DNA binding of the receptor and it also induces a conformational change in the receptor which overcomes the effects of this repressor function.

J Biol Chem, 1992 Nov 15, 267(32), 23232 - 6
Isolation and sequence of the cDNAs encoding the subunits of the isozyme form of wheat protein synthesis initiation factor 4F; Allen ML et al.; The nucleotide sequences of the cDNAs for the two subunits, p82 and p28, of the isozyme form of wheat germ eukaryotic initiation factor 4F (eIF-(iso)4F) were determined . The cDNA for the p82 subunit encodes a polypeptide of 86,514 Da . The deduced amino acid sequence of p82 contains possible motifs for ATP binding, metal binding, and phosphorylation . The cDNA sequence for the small subunit, p28, which is a m7G cap-binding protein, encodes a polypeptide of 23,524 Da . The deduced amino acid sequence of p28 is similar (approximately 38%) to cap-binding proteins from yeast and mammals . The p28 of wheat eIF-(iso)4F does not contain a serine or threonine in the vicinity of the serine (Ser53) of mammalian cap-binding protein which is phosphorylated and shown to affect activity in mammalian cells.

Arch Biochem Biophys, 1992 Nov 15, 299(1), 15 - 22
The topology of the glutamine and ATP binding sites of human asparagine synthetase; Larsen MC et al.; Human asparagine synthetase was examined using a combination of chemical modifiers and specific monoclonal antibodies . The studies were designed to determine the topological relation between the nucleotide binding site and the glutamine binding site of the human asparagine synthetase . The purified recombinant enzyme was chemically modified at the glutamine binding site by 6-diazo-5-oxo-L-norleucine (DON), and at the ATP binding site by 8-azidoadenosine 5'-triphosphate (8-N3ATP) . The effects of chemical modification with DON included a loss of glutamine-dependent reactions, but no effect on ATP binding as measured during ammonia-dependent asparagine synthesis . Similarly, modification with 8-N3ATP resulted in a loss of ammonia-dependent asparagine synthesis, but no effect on the glutaminase activity . A series of monoclonal antibodies was also examined in relation to their epitopes and the sites modified by the two covalent chemical modifiers . It was found that several antibodies were prevented from binding by specific chemical modification, and that the antibodies could be classified into groups correlating to their relative binding domains . These results are discussed in terms of relative positions of the glutamine and ATP binding sites on asparagine synthetase.

Eur J Biochem, 1992 Nov 15, 210(1), 193 - 203
Intron excision from tRNA precursors by plant splicing endonuclease requires unique features of the mature tRNA domain; Stange N et al.; It has been proposed that yeast and Xenopus splicing endonucleases initially recognize features in the mature tRNA domain common to all tRNA species and that the sequence and structure of the intron are only minor determinants of splice-site selection . In accordance with this postulation, we show that yeast endonuclease splices heterologous pre-tRNA(Tyr) species from vertebrates and plants which differ in their mature domains and intron secondary structures . In contrast, wheat germ splicing endonuclease displays a pronounced preference for homologous pre-tRNA species; an extensive study of heterologous substrates revealed that neither yeast pre-tRNA species specific for leucine, serine, phenylalanine and tyrosine nor human and Xenopus pre-tRNA(Tyr) species were spliced . In order to identify the elements essential for pre-tRNA splicing in plants, we constructed chimeric genes coding for tRNA precursors with a plant intron secondary structure and with mature tRNA(Tyr) domains from yeast and Xenopus, respectively . The chimeric pre-tRNA comprising the mature tRNA(Tyr) domain from Xenopus was spliced efficiently in wheat germ extract, whereas the chimeric construct containing the mature tRNA(Tyr) domain from yeast was not spliced at all . These data indicate that intron secondary structure contributes to the specificity of plant splicing endonuclease and that unique features of the mature tRNA domain play a dominant role in enzyme-substrate recognition . We further investigated the influence of specific nucleotides in the mature domain on splicing by generating a number of mutated pre-tRNA species . Our results suggest that nucleotides located in the D stem, i.e . in the center of the pre-tRNA molecule, are recognition points for plant splicing endonuclease.

Science, 1992 Nov 13, 258(5085), 1143 - 5
In vitro transcriptional activation by a metabolic intermediate: activation by Leu3 depends on alpha-isopropylmalate; Sze JY et al.; In the absence of the leucine biosynthetic precursor alpha-isopropylmalate (alpha-IPM), the yeast LEU3 protein (Leu3p) binds DNA and acts as a transcriptional repressor in an in vitro extract . Addition of alpha-IPM resulted in a dramatic increase in Leu3p-dependent transcription . The presence of alpha-IPM was also required for Leu3p to compete effectively with another transcriptional activator, GAL4/VP16, for limiting transcription factors . Therefore, the addition of alpha-IPM appears to convert a transcriptional repressor into an activator . This represents an example in eukaryotes of direct transcriptional regulation by a small effector molecule.

Nucleic Acids Res, 1992 Nov 11, 20(21), 5831 - 6
RecA-AC: single-site cleavage of plasmids and chromosomes at any predetermined restriction site; Koob M et al.; We have developed a novel version of the Achilles' Cleavage (AC) reaction in which virtually any restriction site on DNA of any size can be converted to a unique cleavage site . We first polymerized RecA protein on a synthetic oligodeoxyribonucleotide (oligo) in the presence of a nonhydrolyzable ATP analogue to generate oligo:RecA nucleoprotein filaments . These filament were then incubated with plasmid or intact chromosomal DNA from Saccharomyces cerevisiae to form stable complexes in the yeast LEU2 gene at the target sequence identical (or complementary) to that of the oligo . When HhaII (HinfI) methyltransferase (M.HhaII) was added, all of the recognition sites for HhaII with the exception of the one protected by the RecA filament were methylated and thus no longer cleaved by the cognate restriction endonuclease (HinfI) . After inactivation of the RecA and the M.HhaII, HinfI was used to efficiently cleave the plasmid or chromosome specifically at the targeted restriction site . Since oligos specific for any sequence can be easily synthesized and the other reagents necessary to perform RecA-mediated AC (RecA-AC) reactions on both plasmids and intact chromosomes are readily available, this procedure can be applied immediately to the precise dissection and analysis of genomic DNA from any source and to any other research problem requiring efficient, highly specific cleavage of DNA at predetermined sites.

Science, 1992 Nov 6, 258(5084), 924 - 31
The annexins and exocytosis; Creutz CE; The annexins are a group of homologous proteins that bind phospholipids in the presence of calcium . They may provide a major pathway for communication between cellular membranes and their cytoplasmic environment . Annexins have a characteristic "bivalent" activity in the sense that they can draw two membranes together when activated by calcium . This has led to the hypothesis that certain members of this protein family may initiate contact and fusion between a secretory vesicle membrane and the plasma membrane during the process of exocytosis.

Science, 1992 Nov 6, 258(5084), 995 - 8
Prevention of protein denaturation under heat stress by the chaperonin Hsp60; Martin J et al.; The increased synthesis of heat shock proteins is a ubiquitous physiological response of cells to environmental stress . How these proteins function in protecting cellular structures is not yet understood . The mitochondrial heat shock protein 60 (Hsp60) has now been shown to form complexes with a variety of polypeptides in organelles exposed to heat stress . The Hsp60 was required to prevent the thermal inactivation in vivo of native dihydrofolate reductase (DHFR) imported into mitochondria . In vitro, Hsp60 bound to DHFR in the course of thermal denaturation, preventing its aggregation, and mediated its adenosine triphosphate-dependent refolding at increased temperatures . These results suggest a general mechanism by which heat shock proteins of the Hsp60 family stabilize preexisting proteins under stress conditions.

J Mol Biol, 1992 Nov 5, 228(1), 7 - 12
Protein folding within the cell is influenced by controlled rates of polypeptide elongation; Crombie T et al.; Previous studies have proposed that specific translational pauses have evolved to promote protein folding inside the cell by temporally separating the folding of specific regions of some polypeptide chains during their synthesis . Here we show that this is the case for a bifunctional protein in Saccharomyces cerevisiae . The yeast TRP3 gene contains a translational pause comprising ten contiguous non-preferred codons within its second functional domain (indoleglycerol phosphate synthase) . Site-directed mutagenesis was used to remove this translational pause by increasing the codon bias of the region without changing the amino acid sequence of the protein (to create the gene TRP3pr: pause replaced) . The TRP3pr gene was able to complement a trp3:: URA3 null mutation in yeast . No significant differences in the doubling times of TRP3 or TRP3pr yeast transformants were observed during growth at 25 degrees C, 30 degrees C or 37 degrees C, or in the presence of sublethal concentrations of the analogue, 5-methyltryptophan . However, further analysis of TRP3 and TRP3pr transformants revealed that the removal of the translational pause causes a 1.5-fold decrease in indoleglycerol phosphate synthase activity per TRP3 mRNA . This observation which is statistically significant (P < 0.05) and reproducible, suggests that translational pausing promotes the correct intracellular folding of the TRP3 protein.

J Exp Biol, 1992 Nov, 172, 105 - 12
Evidence for a conserved 95-120 kDa subunit associated with and essential for activity of V-ATPases; Manolson MF et al.; Vacuoles purified from Saccharomyces cerevisiae bearing the vph1-1 mutation had no detectable bafilomycin-sensitive ATPase activity or ATP-dependent proton pumping . Furthermore, the vacuolar H(+)-ATPase (V-ATPase) nucleotide binding subunits were no longer associated with vacuolar membranes yet were present at wild-type levels in yeast whole-cell extracts . The VPH1 gene was cloned by screening a lambda gt11 expression library with antibodies directed against a 95 kDa vacuolar integral membrane protein and independently cloned by complementation of the vph1-1 mutation . Deletion disruption of the VPH1 gene revealed that the VPH1 gene is required for vacuolar H(+)-ATPase assembly and vacuolar acidification but is not essential for cell viability or for targeting and maturation of vacuolar proteases . VPH1 encodes a predicted polypeptide of 840 amino acid residues (95.6 kDa) with putative membrane-spanning regions . Cell fractionation and immunodetection demonstrate that Vph1p is a vacuolar integral membrane protein that co-purifies with V-ATPase activity . Vph1p has 42% identity to the 116 kDa polypeptide of the rat clathrin-coated vesicles/synaptic vesicle proton pump, 42% identity to the TJ6 mouse immune suppressor factor, 42% identity to the Caenorhabditis elegans proton pump homologue and 54% identity to the predicted polypeptide encoded by the yeast gene STV1 (Similar To VPH1, identified as an open reading frame next to the BUB2 gene.

Yeast, 1992 Nov, 8(11), 973 - 5
Mapping the putative RNA helicase genes by sequence overlapping; Chang TH et al.; An 'electronic' gene mapping procedure based on computer-aided search for overlapping gene sequences was used to identify adjacent genes and localize several putative RNA helicase genes to different chromosomes . PRP28 and AMD1 genes map to the right arm of chromosome IV next to sup2, which encodes a tyrosine tRNA . PRP16, previously mapped to chromosome XI, is tightly linked to MRP-L20 . PRP22 is adjacent to PRE1, whose chromosomal location is currently unknown . The utility of this approach in yeast gene mapping is evaluated.

J Nat Prod, 1992 Nov, 55(11), 1648 - 54
Bioactive ergost-5-ene-3 beta, 7 alpha-diol derivatives from Pseudobersama mossambicensis; Gunatilaka AA et al.; Bioactivity-directed fractionation of the methyl ethyl ketone extract of Pseudobersama mossambicensis resulted in the isolation of ergosta-5,24(28)-diene-3 beta,7 alpha-diol {1}, 24,28-epoxyergost-5-ene-3 beta,7 alpha-diol {2}, and ergost-5-ene-3 beta,7 alpha,24,28-tetraol {3} . All three sterols showed selective activity towards DNA repair-deficient yeast mutants . The sterol 1 also showed cytotoxicity towards wild-type P-388 murine leukemia cells . The isolation, structural elucidation, and biological activities of these sterols are reported . The sterol 3 is most probably an artifact formed from 2 during the isolation process.

J Microsc, 1992 Nov, 168 ( Pt 2), 169 - 80
High-resolution scanning electron microscopy of frozen-hydrated cells; Walther P et al.; Cryo-fixed yeast Paramecia and sea urchin embryos were investigated with an in-lens type field-emission SEM using a cold stage . The goal was to further develop and investigate the processing of frozen samples for the low-temperature scanning electron microscope (LTSEM) . Uncoated frozen-hydrated samples were imaged with the low-voltage backscattered electron signal (BSE) . Resolution and contrast were sufficient to visualize cross-fractured membranes, nuclear pores and small vesicles in the cytoplasm . It is assumed that the resolution of this approach is limited by the extraction depth of the BSE which depends upon the accelerating voltage of the primary beam (V0) . In this study, the lowest possible V0 was 2.6 kV because below this value the sensitivity of the BSE detector is insufficient . It is concluded that the resolution of the uncoated specimen could be improved if equipment were available for high-resolution BSE imaging at 0.5-2 kV . Higher resolution was obtained with platinum cryo-coated samples, on which intramembranous particles were easily imaged . These images even show the ring-like appearance of the hexagonally arranged intramembranous particles known from high-resolution replica studies . On fully hydrated samples at high magnification, the observation time for a particular area is limited by mass loss caused by electron irradiation . Other potential sources of artefacts are the deposition of water vapour contamination and shrinkage caused by the sublimation of ice . Imaging of partially dehydrated (partially freeze-dried) samples, e.g . high-pressure frozen Paramecium and sea urchin embryos, will probably become the main application in cell biology . In spite of possible shrinkage problems, this approach has a number of advantages compared with any other electron microscopy preparation method: no chemical fixation is necessary, eliminating this source of artefacts; due to partial removal of the water additional structures in the cytoplasm can be investigated; and finally, the mass loss due to electron beam irradiation is greatly reduced compared to fully frozen-hydrated specimens.

Scand J Clin Lab Invest, 1992 Nov, 52(7), 717 - 24
Enzymatic microdetermination of plasma and serum free fatty acids; Jebens E et al.; A simple and sensitive enzymatic method for determination of plasma and serum fatty acids (FAs) is described . The method is based on acylation of long chain FAs by a bacterial acyl-CoA synthetase (ACS) producing equivalent amounts of acyl-CoA and AMP . AMP production was measured using the coupled reaction of myokinase (MK), pyruvate kinase (PK) and lactate dehydrogenase (LDH) allowing fluorinate detection of NADH . Two moles of NAD were produced per mole of FA acylated . Concentrations of substrates and enzymes were kept as low as possible maintaining the ACS reaction as rate limiting . Addition of fat-free human serum albumin (HSA) to standards reduced initial reaction rates but did not affect end-point fluorescence levels . Triton X-100 partly counteracted the inhibition by HSA . To keep albumin concentration low, plasma or serum samples were diluted by 1:400 . Duplicate measurements of plasma or serum FA concentrations between 0 and 2 mmol l-1 can then be performed on 5 microliters samples with intra- and inter-assay variation coefficients of 1.7 and 4% respectively.

Trends Genet, 1992 Nov, 8(11), 376 - 81
Eukaryotic replication origins as promoters of bidirectional DNA synthesis; Heintz NH et al.; Recent work in yeast shows that eukaryotic origins of DNA replication are multipartite regulatory elements resembling promoters of transcription . As for the regulation of transcription, accessory transcription factors appear to function in concert with basic origin recognition factors to regulate initiation of DNA synthesis at specific subsets of origins . The participation of transcription factors in the regulation of DNA replication may facilitate temporal control of transcription and replication during the cell cycle, as well as providing a mechanism for integrating origin selection with the cellular transcriptional programPublication Types:

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