Mol Cell Biol, 1994 Aug, 14(8), 5175 - 81 A critical role for chromatin in mounting a synergistic transcriptional response to GAL4-VP16; Chang C et al.; The role of chromatin in mounting a synergistic transcriptional response to GAL4-VP16 was investigated . Strong synergy was observed when chromatin templates were used in vitro . The synergy was severely reduced when naked DNA templates were transcribed . In vivo synergy was strong when nonreplicating templates were used . However, the use of replicating templates, which involved transient disruptions of chromatin, led to strong reductions in synergy . In both of these low-synergy responses, transcription levels were high . We infer that strong synergy has a requirement for chromatin that may be understood in terms of the competition between multiple activator molecules and histone cores for promoter DNA. Proc Natl Acad Sci U S A, 1994 Jul 5, 91(14), 6403 - 7 Specific protein-protein interactions between the essential mammalian spliceosome-associated proteins SAP 61 and SAP 114; Chiara MD et al.; Spliceosome-associated proteins (SAPs) 61, 62, and 114 can be UV-crosslinked to pre-mRNA in purified spliceosomal complexes and are associated with U2 small nuclear ribonucleoproteins (snRNP) . These proteins also compose the essential heterotrimeric splicing factor SF3a, and products of yeast pre-mRNA processing genes PRP9, PRP11, and PRP21 are their likely yeast counterparts . We report the isolation of a cDNA encoding SAP 61 and find that it is 30% identical in amino acid sequence to PRP9 . A C-terminal Cys2His2 zinc-finger-like motif, which could be involved in the pre-mRNA binding, is the most highly conserved region of the protein . We also demonstrate specific protein-protein interactions between SAPs 61 and 114 and show that the N terminus of SAP 61 is required for this interaction . Significantly, the corresponding proteins are also known to interact in yeast: PRP9 interacts with PRP21, and the N-terminal portion of PRP9 is required . Previous work showed that direct interactions also occur between SAPs 62 and 114 and between the corresponding PRPs 11 and 21 . These observations indicate that the specific protein-protein interactions that occur between the three prespliceosomal factors have been conserved between yeast and mammals. Proc Natl Acad Sci U S A, 1994 Jul 5, 91(14), 6388 - 92 Sak, a murine protein-serine/threonine kinase that is related to the Drosophila polo kinase and involved in cell proliferation; Fode C et al.; We have isolated murine cDNAs encoding two isoforms of a putative protein-serine/threonine kinase, designated Sak-a and Sak-b, which differ in their noncatalytic C-terminal ends . The kinase domain of Sak is related to the catalytic domains of the Drosophila polo, Saccharomyces cerevisiae CDC5, and murine Snk and Plk kinases, a family of proteins for which a role in controlling cell proliferation has been established (polo, CDC5) or implicated (Snk, Plk) . Northern and in situ RNA analyses of Sak gene expression in mouse embryos and adult tissues revealed that expression was associated with mitotic and meiotic cell division . In addition, during embryogenesis, Sak expression was prominent in the respiratory and olfactory mucosa . The pattern of Sak expression and its sequence homology with the polo gene family suggest that the Sak kinase may play a role in cell proliferation . In support of this, cell growth was suppressed by expression of a Sak-a-antisense fragment in CHO cells. FEBS Lett, 1994 Jul 4, 348(1), 70 - 4 Cloning and expression of a novel form of leukotriene B4 omega-hydroxylase from human liver; Kikuta Y et al.; We have isolated and sequenced a cDNA for human liver LTB4 omega-hydroxylase . The cDNA encoded a protein of 520 amino acids with a molecular weight of 59,853 Da . The cDNA-deduced amino acid sequence showed 87.3% homology to that of human polymorphonuclear leukocytes (PMN) LTB4 omega-hydroxylase (CYP4F3) . Northern blot analysis revealed that the mRNA hybridized to the specific cDNA fragment is expressed in human liver, but not in human PMN . The microsomes from yeast cells transfected with the cDNA catalyzed the omega-hydroxylation of LTB4 with a Km of 44.8 microM . These results clearly show that a new form of the CYP4F LTB4 omega-hydroxylase exists in human liver. FEBS Lett, 1994 Jul 4, 348(1), 27 - 32 Involvement of cell wall beta-glucan in the action of HM-1 killer toxin; Kasahara S et al.; HM-1 killer toxin secreted from Hansenula mrakii inhibits the growth of Saccharomyces cerevisiae cells by interfering with beta-1,3-glucan synthesis . We found that HM-1 killer toxin killed intact cells but not protoplasts . In addition, cells lacking the functional KRE6 allele (kre6 delta) became resistant to higher concentration of HM-1 killer toxin . As reported by Roemer and Bussey {(1991) Proc . Natl . Acad . Sci . 88 11295-11299}, cells lacking functional KRE6 had a reduced level of the cell wall beta-1,6-glucan compared to that in cells harboring the normal KRE6 . These results suggest that the cell wall beta-glucan is involved in the action of HM-1 killer toxin . Addition of HM-1 killer toxin with several kinds of oligosaccharides revealed that either beta-1,3- or beta-1,6-glucan blocked the cytocidal action of HM-1 killer toxin whereas alpha-1,4-glucan and chitin did not . Mannan also interfered with HM-1 killer toxin action, but this inhibitory effect was much weaker than that observed with beta-1,3- or beta-1,6-glucans . Thus, it appears that the cell wall beta-glucan interacts with HM-1 killer toxin, and that this toxin-beta-glucan commitment is required for the action of HM-1 killer toxin. J Virol, 1994 Jul, 68(7), 4213 - 9 Interacting domains of E2F1, DP1, and the adenovirus E4 protein; Cress WD et al.; Recent experiments demonstrate that a family of related proteins constitute the E2F transcription factor activity and that the interaction of two of these gene products, E2F1 and DP1, generates a heterodimer with DNA binding and transcriptional activating capacity . Previous experiments have shown that the adenovirus E4 19-kDa protein facilitates the formation of a stable E2F dimer on the adenovirus E2 promoter . We now show that coexpression of the E2F1 and DP1 products in transfected SAOS-2 cells, together with the E4 product, generates a multicomponent complex with specificity to the adenovirus E2 promoter . Using a yeast two-hybrid assay system, we find that the E2F1 hydrophobic heptad repeat (E2F1 amino acid residues 206 to 283) allows interaction with a corresponding domain of the DP1 protein (amino acids 196 to 245) . We also find that the adenovirus E4 protein interacts with the DP1 hydrophobic heptad repeat domain, but we could not detect a direct interaction between E2F1 and E4 . Additional assays demonstrate that the E4 protein can dimerize . Since our previous experiments have shown that mutations within the E2F1 hydrophobic heptad repeat element abolish the E4-mediated transcription enhancement in transfection assays, we conclude that the E4 protein likely interacts with the E2F1-DP1 heterodimer by directly binding to the DP1 product . As a consequence of the ability of E4 to dimerize, we propose that the stable complex formed on the two E2F sites within the E2 promoter is composed of two E2F1-DP1 heterodimers held together by an E4 dimer. Genetics, 1994 Jul, 137(3), 689 - 700 SIP1 is a catabolite repression-specific negative regulator of GAL gene expression; Mylin LM et al.; The yeast Snf1p kinase is required for normal expression of many genes involved in utilization of non-glucose carbon . Snf1p is known to associate with several proteins . One is Sip1p, a protein that becomes phosphorylated in the presence of Snf1p and thus is a candidate Snf1p kinase substrate . We have isolated the SIP1 gene as a multicopy suppressor of the gal83-associated defect in glucose repression of GAL gene expression . Multicopy SIP1 also suppressed the gal82-associated defect in glucose repression, suggesting that SIP1, GAL83 and GAL82 function interdependently . Multicopy SIP1 gene reduces GAL1, GAL2, GAL7 and GAL10 gene expression three- to fourfold in cells grown in the presence of glucose but has no effect in cells grown on nonrepressing carbon . Sip1-deletion cells exhibited a two- to threefold increase in GAL gene expression compared to wild-type cells when grown on glucose . These studies show that SIP1 is a catabolite repression-specific negative regulator of GAL gene expression . Northern analysis revealed two SIP1 transcripts whose relative abundance changed with carbon source . Western blots revealed that Sip1p abundance is not markedly affected by carbon source, suggesting that Sip1p may be regulated post-translationally. J Mol Evol, 1994 Jul, 39(1), 112 - 4 Constant and variable parts of the 155-bp centromeric repeat in Camptochironomus; Liao C et al.; Centromeres in dipteran insects belonging to the subgenus Camptochironomus (C . tentans and C . pallidivittatus) contain tandem repeats of a 155-bp repeat . A special structural feature of the 155-bp unit is a region with two palindromes connected by a short piece of DNA with only AT base pairs, which has at least a superficial similarity to a functionally important part of the Saccharomyces cerevisiae centromere . As a parameter for functional importance we have measured frequencies of mutations along the unit in samples of repeats from the two species . We find that it consists of about equal parts of highly conserved and considerably less-conserved DNA . The palindromes are localized in the conserved part of the repeat. Plant Mol Biol, 1994 Jul, 25(4), 705 - 19 A dioxygenase gene (Ids2) expressed under iron deficiency conditions in the roots of Hordeum vulgare; Okumura N et al.; A lambda zapII cDNA library was constructed from mRNA isolated from Fe-deficient barley roots and screened with cDNA probes made from mRNA of Fe-deficient and Fe-sufficient (control) barley roots . Seven clones were selected . Among them a clone having the putative full-length mRNA of dioxygenase as judged by northern hybridization was selected and named Ids2 (iron deficiency-specific clone 2) . Using a cDNA fragment as probe, two clones from the genomic library (lambda EMBL-III) were isolated and one was sequenced . The predicted amino acid sequence of Ids2 resembled that of 2-oxoglutarate-dependent dioxygenase . Ids2 is expressed in the Fe-deficient barley roots but is not in the leaves . The expression is repressed by the availability of Fe . Ids2 was also strongly expressed under Mn deficiency and weakly under Zn deficiency or excess NaCl (0.5%) . The upstream 5'-flanking region of Ids2 has a root-specific cis element of the CaMV 35S promoter and a nodule-specific element of leghemoglobin, a metal regulatory element (MRE) and several Cu regulatory elements (UAS) of yeast metallothionein (CUP1). Biophys Chem, 1994 Jul, 51(1), 21 - 35 Glycolytic pH oscillations in a flow reactor; Hocker CG et al.; A new type of flow reactor (UCSTR) has been developed that uses anisotropic ultrafiltration membranes in a continuous flow stirred tank reactor (CSTR) to facilitate the study of nonlinear enzyme catalyzed reactions . The design allows the study of enzymes with subunit molecular weights > or = 9000 dalton and protein concentrations up to at least 2 mg/ml under flow conditions with a residence time of 3 min or more, in a reactor of volume 1.67 ml . The UCSTR allows continuous potentiometric or spectrophotometric measurement without design change . Calibration of reactor performance was carried out by reproducing pH oscillations in the ferrocyanide-hydrogen peroxide reaction . Experimental verification of oscillatory glycolysis in the UCSTR was carried out with extract of rat skeletal muscle . Input feeds were fructose-6-phosphate and ATP with low concentrations of phosphate as buffer . Oscillations in pH, sustained for over eight hours, were observed . A six-step mechanism, including product activation and substrate inhibition, seven concentration variables, and four enzymes sufficed simulate the pH oscillations observed in the UCSTR. FEMS Microbiol Lett, 1994 Jul 1, 120(1-2), 163 - 7 Amino- and aminomethylcholesterol derivatives with fungicidal activity; Elkihel L et al.; Among a series of aminocholesterol derivatives synthesized, 7-aminocholesterol is the strongest inhibitor of yeast cell growth . Using sterol auxotrophic mutant strains, we showed that this compound inhibits cell proliferation by interfering with ergosterol biosynthesis . The sterol pattern of treated cells revealed that 7-aminocholesterol inhibits delta 8-->delta 7-sterol isomerase and delta 14-sterol reductase as morpholine inhibitors . However, the novel feature of this compound is a strong cytotoxicity to yeast. J Steroid Biochem Mol Biol, 1994 Jul, 50(1-2), 31 - 40 Effect of tumour progression on the androgenic regulation of the androgen receptor, TRPM-2 and YPT1 genes in the Shionogi carcinoma; Rennie PS et al.; Progression of an androgen-dependent tumour to an androgen-independent state is characterized by the loss of apoptotic potential, a property of cells which have differentiated under the influence of androgens . In an attempt to relate progression to mechanisms of apoptotic failure, we compared the relative levels of expression of androgen receptor and TRPM-2 (clusterin) genes in androgen-dependent and -independent tumours derived from the Shionogi carcinoma . The amount of 10 kb androgen receptor mRNA in androgen-dependent and -independent cells was similar thus showing no relationship to progression . Owing to cross-hybridization of androgen receptor cDNA with non-receptor transcripts, two new androgen-repressed mRNAs (ADS31 and ADS39) were cloned . Each was found to have a 20/21 bp GC-rich region of sequence homology with the androgen receptor, implying selective conservation of a domain whose function is unknown . Sequencing results also revealed that ADS31 cDNA encodes a polypeptide identical to mouse YPT1, a ras-related GTP-binding protein . Expression of the ADS31/YPT1, ADS39 and TRPM-2 genes was sensitive to androgen withdrawal and replacement both in the parent androgen-dependent and the recurrent androgen-independent carcinomas . The uncoupling of TRPM-2 expression and apoptosis observed in androgen-independent tumour cells implies that the function of androgen receptor becomes more restricted with tumour progression . Furthermore, the fact that the expression of ADS31/YPT1 transcript becomes dominant in the advanced stages of androgen-independent growth, suggests that the mechanism of progression is subserved by duplication and possibly redundancy of alternative (signal transduction) pathways mediating tumour cell survival and growth. EMBO J, 1994 Jul 1, 13(13), 3136 - 48 Synthetic lethality with fibrillarin identifies NOP77p, a nucleolar protein required for pre-rRNA processing and modification; Berges T et al.; The nucleolar protein fibrillarin (encoded by the NOP1 gene in yeast), is required for many post-transcriptional steps in yeast ribosome synthesis . A screen for mutations showing synthetic lethality with a temperature sensitive nop1-5 allele led to the identification of the NOP77 gene . NOP77 is essential for viability and encodes a nucleolar protein with a predicted molecular weight of 77 kDa . Depletion of NOP77p impairs both the processing and methylation of the pre-rRNA . The processing defect is greatest for the pathway leading to 25S rRNA synthesis, and is distinctly different from that observed for mutations in other nucleolar components . NOP77p contains three canonical RNA recognition motifs (RRMs), suggesting that it is an RNA binding protein . The NOP77 allele which complements the synthetic lethal nop1 strains has an alanine at position 308, predicted to lie in helix alpha 1 of RRM3, whereas the non-complementing nop77-1 allele contains a proline at the corresponding position . We propose that NOP77p mediates specific interactions between NOP1p and the pre-rRNA. Biochem J, 1994 Jul 1, 301 ( Pt 1), 275 - 81 Substitution of asparagine residues in Aspergillus awamori glucoamylase by site-directed mutagenesis to eliminate N-glycosylation and inactivation by deamidation; Chen HM et al.; Aspergillus awamori glucoamylase is a secreted glycoprotein containing N-linked carbohydrate recognition sites at Asn-171, Asn-182 and Asn-395 . Site-directed mutagenesis was performed at Asn-182 and Asn-395 to determine whether these residues were N-glycosylated by Saccharomyces cerevisiae, to investigate the function of any glycans linked to them, and to determine the effect of their deamidation on glucoamylase thermostability . Asn-171 and Asn-395, but not Asn-182, were N-glycosylated . Deletion of the glycan N-linked to Asn-395 did not affect specific activity, but greatly decreased enzyme secretion and thermostability . The mutant lacking the N-glycan linked to Asn-395 was synthesized very slowly, and was more associated with cell membrane components and susceptible to proteinase degradation than were wild-type or other mutant glucoamylases . Its secreted form was 30-fold less thermostable than wild-type enzyme at pH 4.5 . Replacement of Asn-182 by Gln to eliminate deamidation at this site did not change glucoamylase specific activity or thermostability, while replacement by Asp decreased specific activity about 25%, but increased thermostability moderately at pH 4.5 below 70 degrees C . Both mutations of Asn-182 increased glucoamylase production. Biochem J, 1994 Jul 1, 301 ( Pt 1), 257 - 65 A Kex2-related endopeptidase activity present in rat liver specifically processes the insulin proreceptor; Alarcon C et al.; The insulin proreceptor is cleaved by limited proteolysis post-translationally at an Arg-Lys-Arg-Arg site to generate its mature alpha- and beta-subunit form . An 35S-labelled insulin proreceptor substrate preparation and a 15-mer peptide substrate that mimics the amino acid sequence around and including the insulin proreceptor processing site (IRP-peptide) has revealed an endopeptidase activity that catalyses insulin proreceptor cleavage in a rat liver subcellular fraction . Under optimal conditions, normal 35S-labelled insulin proreceptor substrate processing by this fraction was quantitative . This fraction was not able to process an 35S-labelled insulin proreceptor variant substrate (where the Arg-1 of the tetrabasic cleavage site had been replaced by Ala-1), similarly to previous in vivo observations, suggesting that this endopeptidase activity has physiological relevance . Biochemical characterization of the insulin proreceptor/IRP-peptide processing revealed this rat liver endopeptidase activity to have a broad pH range (> 70% maximal activity between pH 5.5 and 10.0) and a pH optimum of pH 8-10 . It was Ca(2+)-dependent activity, maximally active between 0.5 and 5 mM Ca2+ and half-maximally activated between 50 and 90 microM Ca2+ . Endoproteolytic activity was not inhibited by group-specific inhibitors of serine-, cysteinyl or aspartyl proteinases or by 1,10-phenanthroline; however, EDTA and 1,2-cyclohexanediaminetetraacetic acid did inhibit the activity, but this was accounted for by Ca2+ chelation . The IRP-peptide substrate assay enabled measurement of an apparent Km of 22 microM and a Vmax of 18.6 pmol/min for this endopeptidase activity . These biochemical characteristics suggest that insulin proreceptor processing endopeptidase activity to be a legitimate member of the Kex2-related proprotein convertase family . Immunoblotting detected furin and PACE4 proteins (both members of this family) to be present in the rat liver subcellular fraction containing insulin proreceptor processing activity . Since the biochemical characteristics of the insulin proreceptor processing endopeptidase activity mostly resembled those of furin activity, it is likely that insulin proreceptor proteolytic maturation can be catalysed by furin in the liver. J Cell Biol, 1994 Jul, 126(2), 423 - 32 Mapping actin surfaces required for functional interactions in vivo; Holtzman DA et al.; An in vivo strategy to identify amino acids of actin required for functional interactions with actin-binding proteins was developed . This approach is based on the assumption that an actin mutation that specifically impairs the interaction with an actin-binding protein will cause a phenotype similar to a null mutation in the gene that encodes the actin-binding protein . 21 actin mutations were analyzed in budding yeast, and specific regions of actin subdomain 1 were implicated in the interaction with fimbrin, an actin filament-bundling protein . Mutations in this actin subdomain were shown to be, like a null allele of the yeast fimbrin gene (SAC6), lethal in combination with null mutations in the ABP1 and SLA2 genes, and viable in combination with a null mutation in the SLA1 gene . Biochemical experiments with act1-120 actin (E99A, E100A) verified a defect in the fimbrin-actin interaction . Genetic interactions between mutant alleles of the yeast actin gene and null alleles of the SAC6, ABP1, SLA1, and SLA2 genes also demonstrated that the effects of the 21 actin mutations are diverse and allowed four out of seven pseudo-wild-type actin alleles to be distinguished from the wild-type gene for the first time, providing evidence for functional redundancy between different surfaces of actin. Eur J Biochem, 1994 Jul 1, 223(1), 155 - 60 Mutations of iso-1-cytochrome c at positions 13 and 90 . Separate effects on physical and functional properties; Huang Y et al.; Residues at positions 13 (lysine or arginine) and 90 (glutamate or aspartate) of eukaryotic cytochromes c have been conserved during evolution; Cys102, however, is found only in yeast cytochrome c . The positively charged residue at position 13 and the negatively charged residue at position 90 are close together in those cytochromes c for which three-dimensional structures are available . We have replaced the amino acids at these two positions by cysteine in Saccharomyces cerevisiae iso-1-cytochrome c; in an earlier study, Cys102 was replaced by threonine without negatively influencing the physical or enzymic properties of the protein . The mutated proteins {R13C, C102T}cytochrome c (iso-1-cytochrome c containing Arg13-->Cys and Cys102-->Thr mutations), {D90C, C102T}cytochrome c (iso-1-cytochrome c containing Asp90-->Cys and Cys102-->Thr mutations) and {R13C, D90C, C102T}cytochrome c (iso-1-cytochrome c containing Arg13-->Cys, Asp90-->Cys, and Cys102-->Thr mutations) are functional in vivo . Free sulfhydryl titration shows that the doubly mutated forms each contain one sulfhydryl group while the triple mutant contains two sulfhydryl groups . The stability of mutant {R13C, C102T}cytochrome c resembles that of {C102T} cytochrome c, whereas the stability of {D90C, C102T}cytochrome c resembles the stability of {R13C, D90C, C102T}cytochrome c . The activity of cytochrome-c oxidase using cytochrome c was monitored polarographically . Compared to the wild-type or {C102T}cytochrome c, which shows two kinetic phases with cytochrome-c oxidase, {D90C, C102T}cytochrome c has much the same profile; {R13C, C102T}cytochrome c and {R13C, D90C, C102T}cytochrome c exhibit one kinetic phase with decreased activity . Electron-transfer activity of the mutant cytochromes c is inhibited by Hg2+ . The inhibition is highest for the triple mutant, less for {R13C, C102T}cytochrome c, even less for {D90C, C102T}cytochrome c and insignificant for the wild type . It would appear as though the stability of the triple mutant follows the changes that result from the Asp90-->Cys mutation while the activity changes follow those of the Arg13-->Cys mutation. Arch Biochem Biophys, 1994 Jul, 312(1), 285 - 91 Generation of strand breaks and formation of 8-hydroxy-2'-deoxyguanosine in DNA by a Thiol/Fe3+/O2-catalyzed oxidation system; Park JW et al.; Strand breaks are produced in pBluescript plasmid DNA by a metal-catalyzed oxidation system composed of Fe3+, O2, and a thiol as an electron donor . Formation of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) in calf thymus DNA was also observed utilizing the same system . DNA damage was observed with the physiological thiols dihydrolipoic acid, glutathione, and cysteine as well as with the synthetic thiols dithiothreitol and 2-mercaptoethanol under a wide range of conditions . Both strand breaks and formation of 8-OH-dG in DNA were temperature dependent, occurring more rapidly at higher temperatures . Sodium azide and mannitol as well as a metal chelator, diethylenetriaminepentaactic acid, decreased strand breaks and 8-OH-dG formation in DNA . Superoxide dismutase did not block damage to DNA, whereas a thiol-dependent protector protein from Saccharomyces cerevisiae and catalase inhibited DNA damage in a concentration-dependent manner . The results of the present study indicate that H2O2 may be generated from a thiol/Fe3+/O2 system and that hydroxyl free radicals may be produced by metal-catalyzed Fenton reactions and may be the ultimate species mediating the DNA damage. J Neurosci, 1994 Jul, 14(7), 4318 - 28 Chronic electroconvulsive seizure (ECS) treatment results in expression of a long-lasting AP-1 complex in brain with altered composition and characteristics; Hope BT et al.; Gene transcription is likely to play a role in the biochemical adaptations thought to underlie the long-term behavioral changes observed following various chronic treatments . The AP-1 (activator protein-1) complex is a well-studied transcription factor capable of regulating gene transcription . We therefore examined the regulation of the AP-1 complex in rat cerebral cortex and hippocampus following electroconvulsive seizures (ECS), known to induce biochemical alterations in the brain after chronic treatment . We show that 10 d of chronic ECS treatment results in an AP-1 binding complex that persists for at least 7 d in the cortex and hippocampus . In contrast, AP-1 binding returns to control levels within 18 hr of a single acute ECS . Supershift experiments and Western blots show that the chronic AP-1 complex contains two novel Fos-related antigens (Fras) of 35 and 37 kDa that do not appear following a single acute ECS . The chronically induced 35 and 37 kDa Fras and the chronic AP-1 complex show similar time courses for induction by repeated ECS . Moreover, the 37 kDa Fra band persists for at least 7 d following chronic ECS treatment, as observed for the chronic AP-1 complex . Competition experiments indicate that the relative affinities of the acute and chronic AP-1 complexes for several AP-1-like sites are similar, although there was approximately a twofold difference in the affinity for one particular AP-1-like site . The altered composition of the chronic AP-1 complex, and differences in half-life, DNA binding affinity, and possibly transcriptional activating properties are likely to cause changes in the overall pattern of gene expression, which may underlie some of the long-term biochemical adaptations observed following chronic ECS and other chronic perturbations. J Neurosci, 1994 Jul, 14(7), 3998 - 4006 Prolonged expression of AP-1 transcription factors in the rat hippocampus after systemic kainate treatment; Pennypacker KR et al.; Systemic administration of kainate, a glutamate receptor agonist, caused neuronal death in the CA1 and CA3 fields of the rat hippocampus . In the areas of cell loss, reactive astrocytes increased their expression of an astrocyte-specific protein, glial fibrillary acidic protein (GFAP) . AP-1 DNA binding activity and the expression of a 35 kDa fos-related antigen (fra) remained elevated in the rat hippocampus for at least 2 weeks after a single systemic injection of kainate, which correlated with changes in gene expression during reactive gliosis . Immunoreactivity for fras was detected in the nuclei of neurons in the dentate gyrus, but relatively few cells in CA1 and CA3 were immunoreactive 1 week after kainate treatment . However, elevated AP-1 DNA binding activity was observed in the CA1 and CA3 regions as well as in the dentate gyrus, suggesting that proteins other than the fras were involved in the astrocytic AP-1 complex . The AP-1 DNA binding activity in hippocampus recognized an AP-1 sequence from the promoter region of the GFAP gene, suggesting that GFAP is a potential target gene . Thus, a single systemic injection of kainate causes long-term activation of AP-1 DNA binding activity in the rat hippocampus and may be important for long-term changes in gene expression in hippocampal cells. J Cell Biol, 1994 Jul, 126(1), 99 - 110 Determination of four biochemically distinct, sequential stages during vacuole inheritance in vitro; Conradt B et al.; Vacuole inheritance in Saccharomyces cerevisiae can be reconstituted in vitro using isolated organelles, cytosol, and ATP . Using the requirements of the reaction and its susceptibility to inhibitors, we have divided the in vitro reaction into four biochemically distinct, sequential subreactions . Stage I requires exposure of vacuoles to solutions of moderate ionic strength . Stage II requires "stage I" vacuoles and cytosol . In stage III, stage II vacuoles react with ATP . Finally, during stage IV, stage III vacuoles at a certain, minimal concentration complete the fusion reaction without further requirement for any soluble components . Reagents that inhibit the overall vacuole inheritance reaction block distinct stages . Stage III of the reaction is sensitive to the proton ionophore CCCP, to inhibitors of the vacuolar ATPase such as bafilomycin A1, and to the ATP-hydrolyzing enzyme apyrase, suggesting that an electrochemical potential across the vacuolar membrane is required during this stage . Inhibition studies with the amphiphilic peptide mastoparan and GTP gamma S suggest that GTP-hydrolyzing proteins might also be involved during this stage . Microcystin-LR, a specific inhibitor of protein phosphatases of type 1 and 2A, inhibits stage IV of the inheritance reaction, indicating that a protein dephosphorylation event is necessary for fusion . The definition of these four stages may allow the development of specific assays for the factors which catalyze each of the consecutive steps of the in vitro reaction. J Cell Biol, 1994 Jul, 126(1), 87 - 97 G-protein ligands inhibit in vitro reactions of vacuole inheritance; Haas A et al.; During budding in Saccharomyces cerevisiae, maternal vacuole material is delivered into the growing daughter cell via tubular or vesicular structures . One of the late steps in vacuole inheritance is the fusion in the bud of vesicles derived from the maternal vacuole . This process has been reconstituted in vitro and requires isolated vacuoles, a physiological temperature, cytosolic factors, and ATP (Conradt, B., J . Shaw, T . Vida, S . Emr, and W . Wickner . 1992 . J . Cell Biol . 119:1469-1479) . We now report a simple and reliable assay to quantify vacuole-to-vacuole fusion in vitro . This assay is based on the maturation and activation of vacuole membrane-bound pro-alkaline phosphatase by vacuolar proteinase A after vacuole-to-vacuole fusion . In vitro fusion allowed maturation of 30 to 60% of pro-alkaline phosphatase . Vacuoles prepared from a mutant defective in vacuole inheritance in vivo (vac2-1) were inactive in this assay . Vacuole fusion in vitro required a vacuole membrane potential . Inhibition by nonhydrolyzable guanosine derivatives, mastoparans, and benzalkonium chloride suggest that GTP-hydrolyzing G proteins may play a key role in the in vitro fusion events. Circ Res, 1994 Jul, 75(1), 15 - 22 Heparin decreases activator protein-1 binding to DNA in part by posttranslational modification of Jun B; Au YP et al.; Heparin is a potent inhibitor of the proliferation and migration of vascular smooth muscle cells . This agent selectively inhibits the transcription of tissue-type plasminogen activator and interstitial collagenase, probably by decreasing the binding of activator protein-1 (AP-1) to phorbol ester-responsive elements in the promoters of these genes . Decreased AP-1 binding is not due to a direct inhibition by heparin, since heparinase digestion of nuclear extracts prepared from heparin-treated smooth muscle cells does not restore AP-1 binding activity . Treatment of cells with heparin suppresses the expression of Jun B, one of the components of AP-1 . The major effect of heparin is at the level of posttranslational modification of Jun B . Results from pulse-chase labeling experiments show that the newly synthesized Jun B is rapidly converted to a higher-molecular-weight form and that conversion is suppressed by heparin . Evidence is presented suggesting that the heparin-inhibited event is phosphorylation of Jun B. Mol Cell Biol, 1994 Jul, 14(7), 4802 - 14 Induction of recombination between homologous and diverged DNAs by double-strand gaps and breaks and role of mismatch repair; Priebe SD et al.; Sequence homology is expected to influence recombination . To further understand mechanisms of recombination and the impact of reduced homology, we examined recombination during transformation between plasmid-borne DNA flanking a double-strand break (DSB) or gap and its chromosomal homolog . Previous reports have concentrated on spontaneous recombination or initiation by undefined lesions . Sequence divergence of approximately 16% reduced transformation frequencies by at least 10-fold . Gene conversion patterns associated with double-strand gap repair of episomal plasmids or with plasmid integration were analyzed by restriction endonuclease mapping and DNA sequencing . For episomal plasmids carrying homeologous DNA, at least one input end was always preserved beyond 10 bp, whereas for plasmids carrying homologous DNA, both input ends were converted beyond 80 bp in 60% of the transformants . The system allowed the recovery of transformants carrying mixtures of recombinant molecules that might arise if heteroduplex DNA--a presumed recombination intermediate--escapes mismatch repair . Gene conversion involving homologous DNAs frequently involved DNA mismatch repair, directed to a broken strand . A mutation in the PMS1 mismatch repair gene significantly increased the fraction of transformants carrying a mixture of plasmids for homologous DNAs, indicating that PMS1 can participate in DSB-initiated recombination . Since nearly all transformants involving homeologous DNAs carried a single recombinant plasmid in both Pms+ and Pms- strains, stable heteroduplex DNA appears less likely than for homologous DNAs . Regardless of homology, gene conversion does not appear to occur by nucleolytic expansion of a DSB to a gap prior to recombination . The results with homeologous DNAs are consistent with a recombinational repair model that we propose does not require the formation of stable heteroduplex DNA but instead involves other homology-dependent interactions that allow recombination-dependent DNA synthesis. Mol Cell Biol, 1994 Jul, 14(7), 4788 - 801 Cell cycle-dependent transcription of CLN2 is conferred by multiple distinct cis-acting regulatory elements; Stuart D et al.; The budding yeast Saccharomyces cerevisiae CLN1, CLN2, and CLN3 genes encode functionally redundant G1 cyclins required for cell cycle initiation . CLN1 and CLN2 mRNAs accumulate periodically throughout the cell cycle, peaking in late G1 . We show that cell cycle-dependent fluctuation in CLN2 mRNA is regulated at the level of transcriptional initiation . Mutational analysis of the CLN2 promoter revealed that the major cell cycle-dependent upstream activating sequence (UAS) resides within a 100-bp fragment . This UAS contains three putative SWI4-dependent cell cycle boxes (SCBs) and two putative MluI cell cycle boxes (MCBs) . Mutational inactivation of these elements substantially decreased CLN2 promoter activity but failed to eliminate periodic transcription . Similarly, inactivation of SWI4 decreased CLN2 transcription without affecting its periodicity . We have identified a second UAS in the CLN2 upstream region that can promote cell cycle-dependent transcription with kinetics similar to that of the intact CLN2 promoter . Unlike the major CLN2 UAS, this newly identified UAS promotes transcription in cells arrested in G1 by inactivation of cdc28 . This novel UAS is both necessary and sufficient for regulated transcription driven by a CLN2 promoter lacking functional SCBs and MCBs . Although this UAS itself contains no SCBs or MCBs, its activity is dependent upon SWI4 function . The characteristics of this novel UAS suggest that it might have a role in initiating CLN2 expression early in G1 to activate the positive feedback loop that drives maximal Cln accumulation. Mol Cell Biol, 1994 Jul, 14(7), 4779 - 87 Role of Swi4 in cell cycle regulation of CLN2 expression; Cross FR et al.; Expression of the Saccharomyces cerevisiae CLN1 and CLN2 genes is cell cycle regulated, and the genes may be controlled by positive feedback . It has been proposed that positive feedback operates via Cln/Cdc28 activation of the Swi4/Swi6 transcription factor, leading to CLN1 and CLN2 transcription due to Swi4 binding to specific sites (SCBs) in the CLN1 and CLN2 promoters . To test this proposal, we have examined the effects of deletion either of the potential SCBs in the CLN2 promoter or of the SWI4 gene on CLN2 transcriptional control . Deletion of a restriction fragment containing the identified SCBs from the promoter does not prevent cell cycle regulation of CLN2 expression, although expression is lowered at all cell cycle positions . A promoter containing a 5.5-kb plasmid insertion or an independent 2.5-kb insertion at the point of deletion of the SCB-containing restriction fragment also exhibits cell cycle regulation, so involvement of unidentified upstream SCBs is unlikely . Neither Swi4 nor the related Mbp1 transcription factor is required for cell cycle regulation of the intact CLN2 promoter . In contrast, Swi4 (but not Mbp1) is required for correct cell cycle regulation of the insertion/deletion promoter lacking SCB sites . We have extended previous genetic evidence for involvement of Swi4 in some aspect of CLN2 function: a mutant hunt for CLN2 positive regulatory factors yielded only swi4 mutations at saturation . Swi4 may bind to nonconsensus sequences in the CLN2 promoter (possibly in addition to consensus sites), or it may act indirectly to regulate CLN2 expression. Vopr Virusol, 1994 Jul-Aug, 39(4), 179 - 82 {The antitumor action of double-stranded interferon inducers (larifan, ridostin) and reaferon in an experiment}; Barinskii IF et al.; Interferon inducers larifan and rhidostin, and reaferon were shown to exert an inhibiting antitumor effect manifested in the prolongation of the incubation period, decrease of the size of tumors, and longer survival of the animals . The maximal anti-tumor and immunomodulating effect was obtained by combined use of preimmunization with tumor cells and simultaneous administration of reaferon or interferon inducers, larifan and rhidostin . Larifan was also shown to have a greater antitumor activity than rhidostin . Larifan, however, was maximally active only in combination combination with vaccination using syngeneic cells of the virus-induced tumor . In this case levels of alpha and gamma interferons were 2-4 times higher than normally. Chromosoma, 1994 Jul, 103(4), 237 - 50 TEL+CEN antagonism on plasmids involves telomere repeat sequences tracts and gene products that interact with chromosomal telomeres; Enomoto S et al.; In Saccharomyces cerevisiae, circular plasmids that include either a centromere (CEN-plasmids) or a telomere sequence (TEL-plasmids) segregate more efficiently than circular ARS-plasmids . In contrast, circular plasmids that include both telomere and centromere sequences were unstable, a property we term TEL+CEN antagonism . TEL+CEN antagonism required a telomere repeat tract longer than 49 bp although the distance and relative orientation of the centromere and telomere sequences was not critical . TEL+CEN antagonism was alleviated in strains carrying different rap1 alleles including rap1ts, rap1s, and rap1t alleles . Mutations SIR2, SIR3, SIR4, NAT1 and ARD1, genes that influence transcriptional silencing at telomeres and at the silent mating type loci, abolished TEL+CEN antagonism Mutation of SIR1 also partially alleviated TEL-CEN antagonism . In some sir mutant strains short yeast artificial chromosomes (YACs), which are normally unstable, became more stable, suggesting that the same mechanism that caused TEL+CEN antagonism on circular plasmids may contribute to the instability of short linear plasmids. J Cell Sci, 1994 Jul, 107 ( Pt 7), 1817 - 24 Evidence that the 16 kDa proteolipid (subunit c) of the vacuolar H(+)-ATPase and ductin from gap junctions are the same polypeptide in Drosophila and Manduca: molecular cloning of the Vha16k gene from Drosophila; Finbow ME et al.; The 16 kDa proteolipid (subunit c) of the eukaryotic vacuolar H(+)-ATPase (V-ATPase) is closely related to the ductin polypeptide that forms the connexon channel of gap junctions in the crustacean Nephrops norvegicus . Here we show that the major protein component of Manduca sexta gap junction preparations is a 16 kDa polypeptide whose N-terminal sequence is homologous to ductin and is identical to the deduced sequence of a previously cloned cDNA from Manduca (Dow et al., Gene, 122, 355-360, 1992) . We also show that a Drosophila melanogaster cDNA, highly homologous to the Manduca cDNA, can rescue Saccharomyces cerevisiae, defective in V-ATPase function, in which the corresponding yeast gene, VMA3, has been inactivated . Evidence is presented for a single genetic locus (Vha16) in Drosophila, which in adults at least contains a single transcriptional unit . Taken together, the data suggest that in Drosophila and Manduca, the same polypeptide is both the proteolipid subunit c component of the V-ATPase and the ductin component of gap junctions . The intron/exon structure of the Drosophila Vha16 is identical to that of a human Vha16 gene, and is consistent with an ancient duplication of an 8 kDa domain . A pilot study for gene inactivation shows that transposable P-elements can be easily inserted into the Drosophila ductin Vha16 gene . Although without phenotypic consequences, these can serve as a starting point for generation of null alleles. Protein Eng, 1994 Jul, 7(7), 911 - 6 Effects of introduced aspartic and glutamic acid residues on the P'1 substrate specificity, pH dependence and stability of carboxypeptidase Y; Stennicke HR et al.; Carboxypeptidase Y is a serine carboxypeptidase isolated from Saccharomyces cerevisiae with a preference for C-terminal hydrophobic amino acid residues . In order to alter the inherent substrate specificity of CPD-Y into one for basic amino acid residues in P'1, we have introduced Asp and/or Glu residues at a number of selected positions within the S'1 binding site . The effects of these substitutions on the substrate specificity, pH dependence and protein stability have been evaluated . The results presented here demonstrate that it is possible to obtain significant changes in the substrate preference by introducing charged amino acids into the framework provided by an enzyme with a quite different specificity . The introduced acidic amino acid residues provide a marked pH dependence of the (kcat/Km)FA-A-R-OH/(kcat/Km)FA-A-L-OH ratio . The change in stability upon introduction of Asp/Glu residues can be correlated to the difference in the mean buried surface area between the substituted and the substituting amino acid . Thus, the effects of acidic amino acid residues on the protein stability depend upon whether the introduced amino acid protrudes from the solvent accessible surface as defined by the surrounding residues in the wild type enzyme or is submerged below. Curr Biol, 1994 Jul 1, 4(7), 647 - 50 Signal transduction . Switching off MAP kinases; Clarke PR; Protein phosphatases have recently been identified that inactivate MAP kinases and turn off intracellular signalling pathways . Their regulation may determine the response of cells to stimuli that signal via MAP kinases. J Dairy Sci, 1994 Jul, 77(7), 2080 - 90 Heat stress interactions with protein, supplemental fat, and fungal cultures; Huber JT et al.; Cows that were subjected to hot environmental temperatures yielded less milk (3.1 kg/d) on a diet high in CP (18.4%) and of medium degradability (65%) than on diets high in CP of low degradability (59%) or medium in CP (16.1%) . The high CP diets were associated with decreased DMI and higher water intake, ruminal NH4, and blood urea . Negative effects on yield from the high CP, medium degradability diet were not observed at moderate temperatures . Evaporative cooling of cows in hot weather resulted in a greater milk yield response to low versus medium rumen-undegradabale protein diets than for uncooled cows . Evaporative cooling of cows also affected response to protein quality . For cooled cows, high Lys diet (soybean, fish, and blood meals) increased milk yield 14% over that with low Lys diet (corn gluten meal), but, for uncooled cows, a high Lys diet only increased yield by 9% . Percentage of CP, degradability, and protein quality had no effect on body temperatures or respiration rates of lactating cows . Some, but not other, reports showed that supplementation of 2 to 2.5% fat to diets fed under hot summer conditions resulted in less yield response than when fat was added at moderate temperatures . In several studies, fungal cultures (3 to 5 g/d) in the diet decreased body temperatures and respiration rates in hot, but not cool, weather . Increased milk yields and cellulose digestibility also resulted from dietary fungal cultures in some, but not all, trials . The mechanism of action exerted by fungal cultures on body temperature and respiration rate is unclear. Biophys J, 1994 Jul, 67(1), 64 - 70 Correlation approach to identify coding regions in DNA sequences; Ossadnik SM et al.; Recently, it was observed that noncoding regions of DNA sequences possess long-range power-law correlations, whereas coding regions typically display only short-range correlations . We develop an algorithm based on this finding that enables investigators to perform a statistical analysis on long DNA sequences to locate possible coding regions . The algorithm is particularly successful in predicting the location of lengthy coding regions . For example, for the complete genome of yeast chromosome III (315,344 nucleotides), at least 82% of the predictions correspond to putative coding regions; the algorithm correctly identified all coding regions larger than 3000 nucleotides, 92% of coding regions between 2000 and 3000 nucleotides long, and 79% of coding regions between 1000 and 2000 nucleotides . The predictive ability of this new algorithm supports the claim that there is a fundamental difference in the correlation property between coding and noncoding sequences . This algorithm, which is not species-dependent, can be implemented with other techniques for rapidly and accurately locating relatively long coding regions in genomic sequences. Biophys J, 1994 Jul, 67(1), 336 - 48 Vectorially oriented membrane protein monolayers: profile structures via x-ray interferometry/holography; Chupa JA et al.; X-ray interferometry/holography was applied to meridional x-ray diffraction data to determine uniquely the profile structures of a single monolayer of an integral membrane protein and a peripheral membrane protein, each tethered to the surface of a solid inorganic substrate . Bifunctional, organic self-assembled monolayers (SAMs) were utilized to tether the proteins to the surface of Ge/Si multilayer substrates, fabricated by molecular beam epitaxy, to facilitate the interferometric/holographic x-ray structure determination . The peripheral membrane protein yeast cytochrome c was covalently tethered to the surface of a sulfhydryl-terminated 11-siloxyundecanethiol SAM via a disulfide linkage with residue 102 . The detergent-solubilized, photosynthetic reaction center integral membrane protein was electrostatically tethered to the surface of an analogous amine-terminated SAM . Optical absorption measurements performed on these two tethered protein monolayer systems were consistent with the x-ray diffraction results indicating the reversible formation of densely packed single monolayers of each fully functional membrane protein on the surface of the respective SAM . The importance of utilizing the organic self-assembled monolayers (as opposed to Langmuir-Blodgett) lies in their ability to tether specifically both soluble peripheral membrane proteins and detergent-solubilized integral membrane proteins . The vectorial orientations of the cytochrome c and the reaction center molecules were readily distinguishable in the profile structure of each monolayer at a spatial resolution of 7 A. J Struct Biol, 1994 Jul-Aug, 113(1), 1 - 12 Imaging the asymmetrical DNA bend induced by repressor activator protein 1 with scanning tunneling microscopy; Muller T et al.; The yeast Repressor Activator Protein 1 (RAP1) binds a 13-bp consensus found in many transcriptional regulatory regions, in silencer elements, and in telomeric repeat DNA of Saccharomyces cerevisiae . Gel retardation assays suggest that RAP1 bends DNA as it binds, with the vertex of the angle located 5' of the consensus . We show that removal of 230 aa in the N-terminus of RAP1 reduces the aberrant electrophoretic mobility of the protein-DNA complex, while removal of a C-terminal domain of RAP1 causes even greater distortion . To demonstrate that the aberrant electrophoretic mobility is really due to a bend in the double helix, the RAP1-DNA complex was analyzed by Scanning Tunnelling Microscopy (STM) . The efficiency and accuracy of binding is checked in parallel by standard Transmission Electron Microscopy (TEM) . Due to the use of high-angle shadowing of freeze-dried samples at low temperatures, the STM images allow us to confirm that RAP1 binding induces a DNA bend > 50 degrees, while the binding of the minimal DNA-binding domain shows significantly less distortion of the DNA helix. Bioorg Med Chem, 1994 Jul, 2(7), 639 - 45 Phosphoenolpyruvate as a dual purpose reagent for integrated nucleotide/nicotinamide cofactor recycling; Fessner WD et al.; An efficient technique is presented which integrates cofactor dependent enzymic phosphorylation and dehydrogenation into a single, closed-loop system by employing phosphoenolpyruvate as the sacrificial reagent for sequential ATP and NAD+ recycling steps . Exemplary applications are developed for the synthesis of 6-phosphogluconate from glucose, and that of dihydroxyacetone phosphate from glycerol . The latter system is combined with exergonic diastereoselective aldol additions for the one-flask synthesis of a ketosugar (D-sorbose), thiosugar (L-threo-5-thiopentulose), or a sugar acid (L-threo-pent-4-ulosonic acid) starting from a mixture of glycerol and simple aldehydes. Bioorg Med Chem, 1994 Jul, 2(7), 627 - 9 Selective inhibition of metabolic enzymes by enzymatically synthesized D-glucal-6-phosphate; Chenault HK et al.; Yeast hexokinase (EC 2.7.1.1) catalyzes the phosphorylation of D-glucal and methyl alpha- and beta-D-glucopyranosides at 1-5% of the rates of phosphorylation of D-glucose and 2-deoxy-D-glucose . Maltose, cellobiose, D-galactal and tetrahydropyran-2-methanol are not substrates of hexokinase . Enzymatically synthesized D-glucal-6-phosphate inhibits rabbit muscle phosphoglucose isomerase competitively (KI = 1.94 mM) and phosphoglucomutase noncompetitively (KI = 0.122 mM). Comput Appl Biosci, 1994 Jul, 10(4), 455 - 6 A note about computing all local alignments; Miller W et al.; A recent paper in this journal by G . Barton proposed an efficient algorithm for locating locally optimal alignments between two sequences . Although the paper claims that all such alignments are found, the approach frequently fails to detect some of the significant matches . This note explains the deficiency. Electrophoresis, 1994 Jul, 15(7), 903 - 6 Detection of exo-beta-1,3-glucanase activity in polyacrylamide gels after electrophoresis under denaturing or nondenaturing conditions; Vargic T et al.; A method for the visualization of exo-beta-1,3-glucanase activity in polyacrylamide gels is presented . The procedure consists of the enzyme reaction in the gel with the substrate alpha-naphthylglucopyranoside, and a subsequent staining of the obtained alpha-naphthol with dyes Fast Red B, or Fast Blue BB, respectively . A mixture of exoglucanases produced by the fungus Polyporus squamosus was used for the optimization of the method . The procedure is applicable for the standard Laemmli discontinuous electrophoresis system, even in the presence of sodium dodecyl sulfate, as well as for electrophoresis in linear gradients of the polyacrylamide concentration . The staining method was used for the analysis of exoglucanases secreted by several yeast genera . All yeasts tested produced two types of exoglucanases, a high molecular mass species heterogeneous in size, and one or two smaller homogeneous enzymes. J Virol, 1994 Jul, 68(7), 4152 - 66 The Cys-His motif of Ty3 NC can be contributed by Gag3 or Gag3-Pol3 polyproteins; Orlinsky KJ et al.; The major structural proteins capsid and nucleocapsid (NC) of the Saccharomyces cerevisiae retroviruslike element Ty3 are produced as domains within the Gag3 and Gag3-Pol3 precursor polyproteins . Ty3 NC contains one copy of the conserved motif CX2CX4HX4C found in most retroviral NC proteins . We show here that NC proteins derived by processing of these different precursor species differ at their carboxyl termini . To determine whether the Cys-His motifs of these nascent NC domains contribute differently to replication, Gag3 and Gag3-Pol3 fusion proteins containing wild-type or mutant Cys-His domains were expressed from separate constructs . Although the Cys-His box was shown to be essential for polyprotein processing of a wild-type Ty3 element, this domain could be contributed from Gag3 or as part of Gag3-Pol3 . These data suggest that the functions of the retroviral NC Cys-His domain contributed from Gag and Gag-Pol are redundant. Nature, 1994 Jun 30, 369(6483), 756 - 8 A mammalian protein targeted by G1-arresting rapamycin-receptor complex; Brown EJ et al.; The structurally related natural products rapamycin and FK506 bind to the same intracellular receptor, FKBP12, yet the resulting complexes interfere with distinct signalling pathways . FKBP12-rapamycin inhibits progression through the G1 phase of the cell cycle in osteosarcoma, liver and T cells as well as in yeast, and interferes with mitogenic signalling pathways that are involved in G1 progression, namely with activation of the protein p70S6k (refs 5, 11-13) and cyclin-dependent kinases . Here we isolate a mammalian FKBP-rapamycin-associated protein (FRAP) whose binding to structural variants of rapamycin complexed to FKBP12 correlates with the ability of these ligands to inhibit cell-cycle progression . Peptide sequences from purified bovine FRAP were used to isolate a human cDNA clone that is highly related to the DRR1/TOR1 and DRR2/TOR2 gene products from Saccharomyces cerevisiae . Although it has not been previously demonstrated that either of the DRR/TOR gene products can bind the FKBP-rapamycin complex directly, these yeast genes have been genetically linked to a rapamycin-sensitive pathway and are thought to encode lipid kinases. J Biotechnol, 1994 Jun 30, 35(2-3), 257 - 72 Comparing nucleotide and protein sequences by linguistic methods; Pietrokovski S; Nucleotide and amino acid sequences can be analyzed and compared by their oligomer compositions . Such methods are fundamentally different from comparison methods based on sequence alignment . They are analogous to the linguistic analysis of human texts . The methods have a wide range of sensitivity and can identify homologous as well as functionally and taxonomically related sequences . Significant sequence dissimilarity can also be identified enabling detection of foreign DNA sequences in genomes, genetic libraries and databases . The simplicity and speed of linguistic methods make them very suitable for database searching and maintenance and as a preliminary step to more specific and time-consuming analysis methods. Biochemistry, 1994 Jun 28, 33(25), 7811 - 8 Dehydration of alkyl- and arylaldoximes as a new cytochrome P450-catalyzed reaction: mechanism and stereochemical characteristics; Boucher JL et al.; The Z isomers of benzaldoxime and 4-(hexyloxy)benzaldoxime were dehydrated into the corresponding nitriles in the presence of rat liver microsomes and NADPH or dithionite . Their E isomers remained unchanged under identical conditions . Alkylaldoximes, like phenylacetaldoxime and heptanaldoxime, are also dehydrated under these conditions, the alkylaldoximes being more rapidly transformed than the arylaldoximes . A genetically well-defined P450 expressed in yeast, P450 3A4, the major P450 isozyme in human liver, was also found to be catalytically active for dehydration of (Z)-benzaldoxime . All these reactions were found to be catalyzed by P450 Fe(II) as they required the use of intact microsomes in the presence of NADPH or dithionite and were strongly inhibited by O2 and CO as well as by classical P450 inhibitors . A P450 complex characterized by a Soret peak at 442 nm was detected during these reactions; its disappearance was found to be concomitant with the consumption of the aldoxime and the formation of the corresponding nitrile . (E)-benzaldoximes and all the studied ketoximes failed to give such complexes with P450 Fe(II) . On the basis of these results, a possible mechanism for this new P450 reaction is proposed . It involves a P450 Fe(II)<--N(OH)=CHR complex as a key intermediate and a charge transfer from P450 Fe(II) to the aldoxime C=N bond which results in a cleavage of the aldoxime N-O bond. FEBS Lett, 1994 Jun 27, 347(2-3), 185 - 9 Cloning of an Arabidopsis histidine transporting protein related to nitrate and peptide transporters; Frommer WB et al.; In plants, many of the proteins involved in transport of nitrogenous compounds have not been identified so far . The use of heterologous complementation in yeast mutants has enabled the isolation of a gene family encoding amino acid permease (AAP) . A highly sensitive selection procedure was used to identify other proteins capable of transporting amino acids . In addition to members of the AAP gene family, an integral membrane protein (NTR1) that shows significant similarities to the low affinity nitrate transporter from Arabidpsis and to peptide transporters from yeast and rabbit was identified . NTR1 seems to be involved in the supply of reproductive organs with nitrogen as it is expressed at low levels in leaves and highly in developing pods. Gene, 1994 Jun 24, 144(1), 45 - 51 Expression of the Leishmania tarentolae ubiquitin-encoding and mini-exon genes; Fleischmann J et al.; To develop models for transcription and trans-splicing in kinetoplastid protozoa, we have characterized ubiquitin (Ubi) gene organization and mRNA processing in Leishmania tarentolae (Lt) . Three ubi loci were characterized: two discrete Ubi-extension protein 52 (EP52)-encoding genes (ubiA and ubiB) and a polymorphic polyubiquitin-encoding gene (ubiC) . The three loci resided on chromosomes of 2.05 Mb, 630 kb and 2.9 Mb, respectively . On the basis of upstream flanking gene identity, ubiB appears to be the homologue of the tandemly repeated ubi-EP52/1 and 2 in Trypanosoma brucei (Tb) . Similar to Trypanosoma cruzi, Lt did not contain a homologue of the ubi-EP76 that has been found in Saccharomyces cerevisiae and multicellular organisms . All three Lt ubi loci were transcribed . The primary transcripts from the ubi loci were processed at the 5'-end by trans-splicing with the mini-exon . A Lt mini-exon gene (min) that gave rise to a 95-nt primary transcript, which is the second template in the trans-splicing reaction, was also characterized. J Biol Chem, 1994 Jun 24, 269(25), 17279 - 88 Fusion proteins containing the cytochrome b2 presequence are sorted to the mitochondrial intermembrane space independently of hsp60; Rospert S et al.; hsp60 is a chaperonin located in the mitochondrial matrix . It has been suggested that hsp60 participates in two processes: protein folding in the matrix, and the sorting of imported proteins to the intermembrane space . We analyzed hsp60 function by allowing isolated mitochondria to import two model precursor proteins and then measuring the binding of these proteins to the chaperonin . Of the methods that we tested for monitoring the association of imported proteins with hsp60, only co-immunoprecipitation with specific anti-hsp60 antibodies proved to be reliable . A chimeric matrix-targeted precursor, consisting of a mitochondrial presequence fused to a chloroplast-encoded protein, bound stably to hsp60 after import . In contrast, there was no detectable binding to hsp60 with a fusion protein that was targeted to the intermembrane space by the bipartite cytochrome b2 presequence . Analysis of a translocation intermediate demonstrated that the cytochrome b2 presequence arrests import through the inner membrane, with the result that the attached passenger protein is never exposed to hsp60. Nature, 1994 Jun 23, 369(6482), 669 - 71 Mammary hyperplasia and carcinoma in MMTV-cyclin D1 transgenic mice; Wang TC et al.; Physical associations between cyclins, viral oncogenes and tumour suppressor genes imply a central role for cyclins in growth control . Cyclin D1 was identified as a candidate oncogene (PRAD1) in tumour-specific DNA rearrangements and is suspected to be a contributor to several types of neoplasms including breast cancer . Cyclin D1 also rescues G1 cyclin-defective Saccharomyces cerevisiae, and is a growth-regulated gene . Despite evidence suggesting that cyclin D1 is an oncogene, its ability to transform cells directly in culture remains controversial . To evaluate its potential to deregulate growth in vivo in a physiologically relevant tissue we overexpressed cyclin D1 in mammary cells in transgenic mice . We report here that overexpression of cyclin D1 resulted in abnormal mammary cell proliferation including the development of mammary adenocarcinomas . We conclude that overexpression of cyclin D1 deregulates cell proliferation and can induce tumorigenic changes in mammary tissues, suggesting that cyclin D1 indeed plays an important oncogenic role in breast cancer. Proc Natl Acad Sci U S A, 1994 Jun 21, 91(13), 6156 - 60 Rch1, a protein that specifically interacts with the RAG-1 recombination-activating protein; Cuomo CA et al.; RAG1 and RAG2 are lymphoid-specific genes that together induce V(D)J recombinase activity in a variety of nonlymphoid cell types . While no other lymphoid-specific factors are required to induce recombination, other factors with more widespread expression patterns have been implicated in the reaction . However, none of these factors have been cloned, and their relationship to the RAG proteins is unclear . Using the yeast two-hybrid assay, we have identified RCH1, a gene encoding a protein of molecular weight 58,000 that interacts specifically with RAG-1 . The predicted Rch1 protein sequence is 47% identical to yeast SRP1, a protein associated with the nuclear envelope . A truncated form of Rch1, which retains the ability to interact with RAG-1, reduces V(D)J recombination activity in HeLa cells. Proc Natl Acad Sci U S A, 1994 Jun 21, 91(13), 5977 - 81 New phenotype of mutations deficient in glucosylation of the lipid-linked oligosaccharide: cloning of the ALG8 locus; Stagljar I et al.; Glc3Man9GlcNAc2 is the preferred substrate of the oligosaccharyltransferase of N-linked glycosylation of proteins, but nonglucosylated oligosaccharides can be transferred to proteins in Saccharomyces cerevisiae . Mutations affecting the addition of the three terminal glucose residues lead to accumulation of Man9GlcNAc2 or Glc1Man9GlcNAc2 in vivo but do not show any detectable growth defect . When these mutations were introduced into a strain with reduced oligosaccharyltransferase activity (due to the wbp1-1 mutation), a severe growth defect was observed: accumulation of suboptimal lipid-linked oligosaccharide and reduced oligosaccharyltransferase activity resulted in a severe underglycosylation of secreted proteins . This new synthetic phenotype made it possible to isolate the ALG8 locus, encoding a potential glucosyltransferase of the endoplasmic reticulum . The ALG8 protein is a 63.5-kDa hydrophobic protein that is not essential for the vegetative growth of yeast . However, the lack of this protein resulted in underglycosylation of secreted proteins. Science, 1994 Jun 17, 264(5166), 1768 - 71 Silencers and domains of generalized repression; Loo S et al.; Gene expression can be affected by the chromosomal position of the gene . An example of this position effect is silencing of the HML and HMR mating-type loci of Saccharomyces cerevisiae . An in vitro assay revealed that silencing induced a transcription-independent general occlusion of the DNA at HMR from sequence-specific interactions of proteins with DNA . The minimum boundaries of the silenced chromatin structure were determined, as were the contributions of the E and I silencers to the size of the silenced domain . Examination of endonuclease-sensitive sites provided evidence that neither the integrity of the chromosomal duplex nor covalent linkage of the silencers to HMR was important for maintenance of the silenced structure in vitro. J Biol Chem, 1994 Jun 17, 269(24), 16684 - 8 DNA-dependent protein kinase is activated by nicks and larger single-stranded gaps; Morozov VE et al.; DNA-PK is a DNA-activated serine/threonine protein kinase capable of phosphorylating a number of nuclear DNA-binding proteins . Purified human DNA-PK has two subunits, a 350-kDa polypeptide, Prkdc, which binds ATP and is presumed to contain the catalytic site, and the Ku autoantigen which mediates DNA binding and activation . Previous studies have shown that DNA-PK is activated in vitro by linear double-stranded DNA fragments; however, the Ku subunit binds a broader range of DNA structures . Here we show that EBP-80, a protein originally isolated as a transcription factor for a retroviral long terminal repeat element and subsequently found to be similar to if not identical with Ku, also mediates kinase activation . The EBP-80-Prkdc complex is activated by nanomolar concentrations of DNA constructs containing single-to-double strand transitions, including a closed stem-loop structure and single strand gaps of 0 (nick), 6, and 30 nucleotides . Kinase activation parallels the ability of EBP-80 to bind these and other constructs . Our results extend the range of DNA configurations known to activate DNA-PK and are consistent with the participation of this enzyme complex in several nuclear functions. J Biol Chem, 1994 Jun 17, 269(24), 16574 - 8 Cbp1p is required for message stability following 5'-processing of COB mRNA; Chen W et al.; Cbp1p is a nuclear encoded protein required for the stabilization of mitochondrial COB mRNA, which codes for apocytochrome b in the yeast Saccharomyces cerevisiae . The COB gene is cotranscribed with the upstream tRNA(Glu) gene . Release of tRNA(Glu) from the initial transcript generates a precursor mRNA with a 5'-end at position -1098 . The 5'-end of mature COB message is generated by cleavage of the pre-mRNA at nucleotide -955 or -954 . Previous work indicated that Cbp1p acts through cis-elements near these cleavage sites . Here we have tested whether Cbp1p stabilizes COB mRNA solely by stimulating the processing of COB precursor RNA at nucleotide -955/-954 . Yeast strain TG955 was constructed such that the -955 COB mRNA 5'-processing site coincides with the upstream tRNA 3'-endonuclease site at position -1098, allowing the 5'-end of COB mRNA to be formed by the tRNA 3'-processing enzyme . Respiratory growth and stability of COB mRNA in TG955 are Cbp1p-dependent . Therefore, we conclude that Cbp1p is important for the stabilization of COB mRNA after 5'-processing. J Biol Chem, 1994 Jun 17, 269(24), 16521 - 4 Role of membrane anchor domain of Bcl-2 in suppression of apoptosis caused by E1B-defective adenovirus; Nguyen M et al.; Bcl-2 is an integral membrane protein that functions as a suppressor of programmed cell death . It contains a COOH-terminal signal anchor sequence that is selective for import and insertion of Bcl-2 into the mitochondrial outer membrane and, by a different mechanism, can also direct the protein to other membrane sites . Deletion of the signal anchor sequence rendered Bcl-2 cytosolic and impaired its ability to prevent apoptotic death of human KB cells infected with a mutant form of adenovirus type 5 that does not make E1B 19-kDa protein . When the predicted transmembrane domain of the Bcl-2 signal anchor was replaced with that of the signal anchor of the yeast outer mitochondrial membrane protein, Mas70p, the Bcl-2/Mas70p hybrid was found to be very similar to Bcl-2 in its distribution within transfected KB cells, in its ability to heterodimerize with Bax, and in its ability to suppress apoptosis . These results are consistent with a model in which the transmembrane segment contributes to the function of Bcl-2 by targeting and anchoring the protein to strategic membrane locations in the cell . Concentration of Bcl-2 at these sites may contribute to its proposed role as regulator, or component, of an antioxidant pathway. Cell, 1994 Jun 17, 77(6), 895 - 907 COPII: a membrane coat formed by Sec proteins that drive vesicle budding from the endoplasmic reticulum; Barlowe C et al.; In vitro synthesis of endoplasmic reticulum-derived transport vesicles has been reconstituted with washed membranes and three soluble proteins (Sar1p, Sec13p complex, and Sec23p complex) . Vesicle formation requires GTP but can be driven by nonhydrolyzable analogs such as GMP-PNP . However, GMP-PNP vesicles fail to target and fuse with the Golgi complex whereas GTP vesicles are functional . All the cytosolic proteins required for vesicle formation are retained on GMP-PNP vesicles, while Sar1p dissociates from GTP vesicles . Thin section electron microscopy of purified preparations reveals a uniform population of 60-65 nm vesicles with a 10 nm thick electron dense coat . The subunits of this novel coat complex are molecularly distinct from the constituents of the nonclathrin coatomer involved in intra-Golgi transport . Because the overall cycle of budding driven by these two types of coats appears mechanistically similar, we propose that the coat structures be called COPI and COPII. J Biol Chem, 1994 Jun 17, 269(24), 16802 - 9 Differential expression of a novel protein kinase in human B lymphocytes . Preferential localization in the germinal center; Katz P et al.; B lymphocytes which reside in the germinal center region of lymphoid follicles are functionally and phenotypically distinct from the surrounding mantle zone B cells . We have isolated cDNA clones for several genes that are differentially expressed between these two populations of B lymphocytes . One such gene, BL44, is preferentially expressed in germinal center B cells . The nucleotide sequence of a 2,874-base pair BL44 cDNA was determined and a 2,451-bp open reading frame found that encodes for a 97-kDa serine/threonine protein kinase referred to as GC kinase . It has an NH2-terminal catalytic domain most similar to that of the Drosophila NinaC protein and the yeast STE20 protein . GC kinase mRNA transcripts are not unique to germinal center B cells and are found in several other tissues, including brain, lung, and placenta . The GC kinase protein was immunoprecipitated from transfected COS cells and from the Burkitt cell line RAMOS . GC kinase immunoprecipitated from transfected COS cells phosphorylated the substrates casein and myelin basic protein . In addition, a 97-kDa phosphoprotein likely to be GC kinase itself was detected . GC kinase may participate in an important signal transduction pathway in germinal center B cells. Mol Gen Genet, 1994 Jun 15, 243(6), 660 - 5 Isolation of Arabidopsis thaliana mutants hypersensitive to gamma radiation; Davies C et al.; A screening method for mutants of Arabidopsis thaliana hypersensitive to gamma-radiation has been devised . Plants grown from ethyl methanesulfonate (EMS)-treated seeds were irradiated at the seedling stage, which is highly radiosensitive due to extensive cell division . Severe growth inhibition of mutant plants by a gamma-ray dose which only slightly affects wild-type plants was the selective criterion . Twelve true-breeding hypersensitive lines were isolated from a total of 3394 screened plants . Genetic analysis of five of the lines revealed five new genes, designated RAD1-RAD5 . These Arabidopsis RAD mutants are phenotypically similar to mutants in the RAD52 epistasis group of Saccharomyces cerevisiae, which are highly sensitive to ionizing radiation but not hypersensitive to UV light . One possibility is that the Arabidopsis mutants are defective in a nonhomologous or illegitimate recombination mechanism used by plants for repair of chromosome breaks. EMBO J, 1994 Jun 15, 13(12), 2870 - 5 The transcription factor MTF-1 is essential for basal and heavy metal-induced metallothionein gene expression; Heuchel R et al.; We have described and cloned previously a factor (MTF-1) that binds specifically to heavy metal-responsive DNA sequence elements in the enhancer/promoter region of metallothionein genes . MTF-1 is a protein of 72.5 kDa that contains six zinc fingers and multiple domains for transcriptional activation . Here we report the disruption of both alleles of the MTF-1 gene in mouse embryonic stem cells by homologous recombination . The resulting null mutant cell line fails to produce detectable amounts of MTF-1 . Moreover, due to the loss of MTF-1, the endogenous metallothionein I and II genes are silent, indicating that MTF-1 is required for both their basal and zinc-induced transcription . In addition to zinc, other heavy metals, including cadmium, copper, nickel and lead, also fail to activate metal-responsive promoters in null mutant cells . However, cotransfection of an MTF-1 expression vector and metal-responsive reporter genes yields strong basal transcription that can be further boosted by zinc treatment of cells . These results demonstrate that MTF-1 is essential for metallothionein gene regulation . Finally, we present evidence that MTF-1 itself is a zinc sensor, which exhibits increased DNA binding activity upon zinc treatment. Genes Dev, 1994 Jun 15, 8(12), 1423 - 33 The POP1 gene encodes a protein component common to the RNase MRP and RNase P ribonucleoproteins; Lygerou Z et al.; Two forms of the yeast 5.8S rRNA are generated from a large precursor by distinct processing pathways . Cleavage at site A3 is required for synthesis of the major, short form, designated 5.8S(S), but not for synthesis of the long form, 5.8S(L) . To identify components required for A3 cleavage, a bank of temperature-sensitive lethal mutants was screened for those with a reduced ratio of 5.8S(S):5.8S(L) . The pop1-1 mutation (for processing of precursor RNAs) shows this phenotype and also inhibits A3 cleavage . The pre-rRNA processing defect of pop1-1 strains is similar to that reported for mutations in the RNA component of RNase MRP; we show that a mutation in the RNase MRP RNA also inhibits cleavage at site A3 . This is the first site shown to require RNase MRP for cleavage in vivo . The pop1-1 mutation also leads to a block in the processing of pre-tRNA that is identical to that reported for mutations in the RNA component of RNase P . The RNA components of both RNase MRP and RNase P are underaccumulated in pop1-1 strains at the nonpermissive temperature, and immunoprecipitation demonstrates that POP1p is a component of both ribonucleoproteins . The POP1 gene encodes a protein with a predicted molecular mass of 100.5 kD and is essential for viability . POP1p is the first protein component of the nuclear RNase P or RNase MRP for which the gene has been cloned. Genes Dev, 1994 Jun 15, 8(12), 1400 - 10 The global transcriptional regulators, SSN6 and TUP1, play distinct roles in the establishment of a repressive chromatin structure; Cooper JP et al.; Repression of a-cell specific gene expression in yeast alpha cells requires MAT alpha 2 and MCM1, as well as two global repressors, SSN6 and TUP1 . Previous studies demonstrated that nucleosomes positioned adjacent to the alpha 2/MCM1 operator in alpha cells directly contribute to repression . To investigate the possibility that SSN6 and TUP1 provide a link between MAT alpha 2/MCM1 and neighboring histones, nucleosome locations were examined in ssn6 and tup1 alpha cells . In both cases, nucleosome positions downstream of the operator were disrupted, and the severity of the disruption correlated with the degree of derepression . Nevertheless, the observed changes in chromatin structure were not dependent on transcription . Our data strongly indicate that SSN6 and TUP1 directly organize repressive regions of chromatin. Biochemistry, 1994 Jun 14, 33(23), 7146 - 56 Interaction of spin-labeled apocytochrome c and spin-labeled cytochrome c with negatively charged lipids studied by electron spin resonance; Snel MM et al.; Apocytochrome c has been spin-labeled with a nitroxide derivative of maleimide on a cysteine residue at either position 14 or position 17 in the N-terminus . Yeast cytochrome c was spin-labeled with the same maleimide derivative on its single free cysteine residue at position 102 in the C-terminus . The ESR spectra of spin-labeled apocytochrome c have been characterized in different environments with respect both to the conformation of the protein and to its association with lipid . In buffer, the spectrum of spin-labeled apocytochrome c indicates high mobility, characteristic of the unfolded structure of the apoprotein, and that of spin-labeled cytochrome c is only slightly less mobile, suggesting that the site labeled is situated at the surface of the folded holoprotein . Upon binding the spin-labeled protein to negatively charged lipid membranes composed of dioleoylphosphatidylglycerol (DOPG), the ESR spectra of apocytochrome c evidence a large reduction in the mobility of the spin-label group, as also do those of yeast cytochrome c . In the case of apocytochrome c, this immobilization most likely arises from both an increase in secondary structure and a partial penetration of the protein into the lipid bilayer, in addition to the electrostatic interaction with the lipid headgroups, whereas for cytochrome c the immobilization observed arises primarily from an intimate association with the membrane surface . When the spin-labeled holocytochrome c is denatured by heating and is bound to DOPG bilayer membranes, a rather mobile ESR spectrum is observed, which demonstrates that the spin-label is located at the surface of the membrane in this case . The ESR spectra of spin-labeled apocytochrome c bound to mixed bilayers of dimyristoylphosphatidylglycerol and dimyristoylphosphatidylcholine (DMPC) consist of both an immobile and a mobile component . The proportion of the mobile component is increased by increasing the mole fraction of the zwitterionic DMPC in the mixed bilayers . The mobile component represents a localization of apocytochrome c at the membrane surface, whereas the immobile component most probably represents the penetration of the precursor protein into the membrane interior . The immobile component assigned to membrane penetration of the precursor protein is still present at negatively charged lipid contents comparable to those in the native mitochondrial system . The results are discussed in relation to the conformation of apocytochrome c, its interaction with lipid, and the import of the apoprotein into mitochondria. Nucleic Acids Res, 1994 Jun 11, 22(11), 1966 - 73 The upstream activator CTF/NF1 and RNA polymerase II share a common element involved in transcriptional activation; Xiao H et al.; The carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II consists of tandem repeats of a heptapeptide with the consensus YSPTSPS . It has been shown that the heptapeptide repeat interacts directly with the general transcription factor TFIID . We report here that the CTD activates transcription when fused to the DNA-binding domain of GAL4 . More importantly, we find that the proline-rich transcriptional activation domain of the CCAAT-box-binding factor CTF/NF1 contains a sequence with striking similarity to the heptapeptide repeats of the CTD . We show that this CTD-like motif is essential for the transcriptional activator function of the proline-rich domain of CTF/NF1 . Deletion of and point mutations in this CTD-like motif abolish the transcriptional activator function of the proline-rich domain, while natural CTD repeats from RNA polymerase II are fully functional in place of the CTD-like motif . We further show that the proline-rich activation domain of CTF/NF1 interacts directly with the TATA-box-binding protein (TBP), and that a mutation in the CTD-like motif that abolishes transcriptional activation reduces the affinity of the proline-rich domain for TBP . These results demonstrate that a class of proline-rich activator proteins and RNA polymerase II possess a common structural and functional component which can interact with the same target in the general transcription machinery . We discuss the implications of these results for the mechanisms of transcriptional activation in eucaryotes. J Biol Chem, 1994 Jun 10, 269(23), 16478 - 85 Preproteins of chloroplast envelope inner membrane contain targeting information for receptor-dependent import into fungal mitochondria; Brink S et al.; The amino-terminal transit sequences of two preproteins destined for the chloroplast inner envelope membrane show similarities to mitochondrial presequences in the prevalence of positive charges and the potential formation of an amphipathic alpha-helix . We studied if these preproteins could be imported into mitochondria and found a low, yet significant import into isolated plant mitochondria . The plant mitochondria were previously shown not to import precursors of chloroplast stromal or thylakoidal proteins . To analyze the specificity of import into mitochondria we used the established import systems of fungal mitochondria . The envelope preproteins were efficiently imported into Saccharomyces cerevisiae or Neurospora crassa mitochondria . Their import showed the characteristics of specific mitochondrial protein uptake, including a requirement for the main receptor MOM19 (mitochondrial outer membrane protein of 19 kDa) and a membrane potential across the inner membrane, and depended on the presence of the chloroplast transit sequence . We conclude that some chloroplast transit sequences contain sufficient information for specific interaction with mitochondrial import receptors (at least from fungal sources). J Biol Chem, 1994 Jun 10, 269(23), 16247 - 53 Two pairs of oppositely charged amino acids from Jun and Fos confer heterodimerization to GCN4 leucine zipper; John M et al.; The preferential assembly of Jun and Fos into heterodimers has been shown to be mainly driven by 16 amino acids (8 from each protein) situated in positions e and g of the leucine zipper coiled-coil structures of the two proteins (O'Shea, E . K., Rutkowski, R., and Kim, P . S . (1992) Cell 68, 699-708) . Using a similar approach, we show that among these residues two pairs of oppositely charged amino acids account in fact for most of the additional free energy of heterodimerization in this system . These residues are 2 glutamic acid side chains in positions g1 and e2 of the Fos leucine zipper and 2 lysine residues in the equivalent positions of the Jun zipper . These amino acids were placed in the context of a GCN4 leucine zipper using peptide synthesis . These peptides contain unique cysteine residues enabling the formation of covalent dimers . The gain in heterodimer free energy has been determined both by cysteine-linked dimer formation under redox conditions and by thermal melting experiments of covalent dimers using circular dichroism experiments . The two pairs of oppositely charged residues (Glu,Glu and Lys,Lys) in positions g1 and e2 contribute at least -1.9 kcal/mol of additional free energy, accounting for a 50-fold excess of the heterodimer with respect to one of the homodimers . Thermal denaturation studies as a function of pH and ionic strength suggest that electrostatic effects should indeed be a major driving force for heterodimerization . On the contrary, peptides harboring the 12 amino acids from Jun and Fos in the other e and g positions (i.e . in e1, g2, e3, g3, e4, and g4) show only a moderate tendency to form heterodimers. J Biol Chem, 1994 Jun 10, 269(23), 15981 - 4 Absence of a Ku-like DNA end binding activity in the xrs double-strand DNA repair-deficient mutant; Getts RC et al.; Double-strand DNA break repair is important in maintaining the genetic integrity of the genome . Using a mobility shift assay, we find that a protein, or complex of proteins, that is present in mammalian and yeast cells binds to the ends of double-strand DNA and renders the ends resistant to exonuclease digestion . Additionally, a mammalian DNA double-strand repair-deficient mutant, xrs, has no observable DNA end binding activity, while a revertant cell has wild-type activity . In addition, mobility supershift assays using monoclonal antibodies to the human Ku antigen (M(r) 70,000 subunit) reveal that one of the proteins of this end binding activity may be the Ku antigen or a protein with similar antigenic determinants . These observations suggest that this DNA end-binding protein may function in DNA repair. J Biol Chem, 1994 Jun 3, 269(22), 15740 - 8 Mutagenic alteration of the distal switch II region of RAS blocks CDC25-dependent signaling functions; Mirisola MG et al.; We have explored the role of the distal switch II region of the yeast RAS2 protein in determining the response to the nucleotide exchange factor CDC25 . We first constructed yeast tester strains in which the deletion of the chromosomal CDC25, RAS1, and RAS2 genes, in combination with the chromosomal suppressor CRI4, resulted in detectable phenotypes in vivo and in vitro . Phenotypes included impaired growth at 37 degrees C, defective glucose-induced cyclic AMP signaling, and low adenylyl cyclase activity of membrane preparations . Tester strains were subsequently used for the reintroduction of various combinations of wild-type and mutated RAS2 and CDC25 genes by genetic techniques, as well as for in vitro reconstitution assays with the corresponding proteins . CDC25 restored both growth and glucose-induced cyclic AMP signaling in the presence, but not in the absence of wild-type RAS2 . A gene encoding a RAS2 protein with a mutationally altered switch II region was expressed but was ineffective in reintegrating exchange factor-dependent responses in vivo . Wild-type, but not mutagenically altered, RAS2 proteins were stimulated by exchange factors in vitro . We conclude that the conserved distal switch II region is required for CDC25-dependent activation of RAS. Toxicol Appl Pharmacol, 1994 Jun, 126(2), 233 - 9 Peroxisomal proliferators inhibit acyl CoA synthetase and stimulate protein kinase C in vivo; Bojes HK et al.; The mechanism by which hypolipidemic drugs and industrial plasticizers cause hepatic tumors in rodents remains unknown . It is known, however, that protein kinase C is elevated during hepatic cell turnover, and sustained cellular replication is correlated with an increased incidence of hepatic tumors . Therefore, several peroxisomal proliferators varying in their tumorigenic potency in chronic feeding studies were examined for their ability to increase protein kinase C activity . Intragastric administration of (4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio)acetic acid (Wy-14,643; 100 mg/kg) increased protein kinase C activity threefold in 5 hr and fivefold in 10 hr . Perfluorooctanoate also increased protein kinase C activity significantly in microsomes at 5 hr . Wy-14,643 and perfluorooctanoate also diminished acyl CoA synthetase activity significantly, with Wy-14,643 exhibiting competitive type kinetics . Other peroxisomal proliferators were examined {e.g., ciprofibrate, clofibrate, 2-ethylhexanol, valproate, and di(ethylhexyl)phthalate (DEHP)} and collectively an inverse relationship between their ability to stimulate protein kinase C activity and inhibit acyl CoA synthetase was observed (r = -0.80) . All chemicals examined had no direct effect on protein kinase C activity in vitro . Interestingly, those compounds which are more potent as hepatocarcinogens (e.g., Wy-14,643) in long-term feeding studies decreased acyl CoA synthetase and elevated protein kinase C activity to a greater extent than their weaker counterparts (e.g., DEHP) . It is proposed that inhibition of acyl CoA synthetase by peroxisomal proliferators elevates free fatty acids which stimulate protein kinase C activity and ultimately promote tumor formation. Arch Biochem Biophys, 1994 Jun, 311(2), 503 - 8 Substrate activity of Rh(III)ATP with phosphoglycerate kinase and the role of the metal ion in catalysis; Pappu KM et al.; An exchange-inert Rh(H2O)4ATP complex showed a one-turnover substrate activity with yeast phosphoglycerate kinase . Transfer of the phosphoryl group between ATP and 3-phosphoglycerate occurs with both substrates in the coordination sphere of the metal ion . Because of the slow ligand exchange rates of Rh3+, the reaction product 1,3-diphosphoglycerate (1,3-dPGA) remained coordinated to the metal ion . During the course of the reaction, the enzyme was inactivated, suggesting that the metal ion is coordinated to a protein side chain . Thus the product Rh(H2O)nADP.1,3-dPGA remained bound to the enzyme even after removal of excess substrate . These results suggested that the metal ion may not only act as an electron sink to activate the electrophile, but it may also help to optimally align both substrates for phosphoryl transfer by coordination to both substrates . It is therefore likely that entry of 3-phospho-D-glycerate into the coordination sphere of metal of a metal-ATP complex may start the proposed hinge-bending motion of yeast phosphoglycerate kinase to form a "closed" active site between the two substrate binding domains of the enzyme. Mol Cell Biol, 1994 Jun, 14(6), 4011 - 9 The CRY1 gene in Chlamydomonas reinhardtii: structure and use as a dominant selectable marker for nuclear transformation; Nelson JA et al.; We have cloned and sequenced the CRY1 gene, encoding ribosomal protein S14 in Chlamydomonas reinhardtii, and found that it is highly similar to S14/rp59 proteins from other organisms, including mammals, Drosophila melanogaster, and Saccharomyces cerevisiae . We isolated a mutant strain resistant to the eukaryotic translational inhibitors cryptopleurine and emetine in which the resistance was due to a missense mutation (CRY1-1) in the CRY1 gene; resistance was dominant in heterozygous stable diploids . Cotransformation experiments using the CRY1-1 gene and the gene for nitrate reductase (NIT1) produced a low level of resistance to cryptopleurine and emetine . Resistance levels were increased when the CRY1-1 gene was placed under the control of a constitutive promoter from the ribulose bisphosphate carboxylase/oxygenase small subunit 2 (RBCS2) gene . We also found that the 5' untranslated region of the CRY1 gene was required for expression of the CRY1-1 transgene . Direct selection of emetine-resistant transformants was possible when transformed cells were first induced to differentiate into gametes by nitrogen starvation and then allowed to dedifferentiate back to vegetative cells before emetine selection was applied . With this transformation protocol, the RBCS2/CRY1-1 dominant selectable marker gene is a powerful tool for many molecular genetic applications in C . reinhardtii. Mol Cell Biol, 1994 Jun, 14(6), 4002 - 10 Nucleosomal location of the STE6 TATA box and Mat alpha 2p-mediated repression; Patterton HG et al.; It has been proposed that yeast MATa cell-specific genes are repressed in MAT alpha cells by the Mat alpha 2p repressor-directed placement of a nucleosome in a position that incorporates the TATA box of the MATa-specific gene close to the nucleosomal pseudodyad . In this study, we address this proposal directly with a series of plasmids designed to place the MATa-specific STE6 TATA box at different locations in a nucleosome and in the internucleosomal linker . These plasmids contain different lengths of synthetic random DNA between the Mat alpha 2p operator and the TATA box of the STE6 promoter, which is located upstream of a lacZ reporter gene in a multicopy plasmid . We show that in MAT alpha cells, a nucleosome is retained in an identical translational frame relative to the Mat alpha 2p operator in all the constructs investigated, irrespective of the sequence of the DNA wrapped onto the histone octamer . This result shows that the nucleosomal organization of the STE6 promoter in MAT alpha cells is not conferred by the sequence of the promoter itself . No expression of the lacZ reporter gene was detectable in MAT alpha cells in any of the constructs, even with the TATA box located in a short internucleosomal linker . These data indicate that repression of MATa-specific genes in MAT alpha cells does not require the precise translational placement of the TATA box close to the nucleosomal pseudodyad; the gene remains repressed when the TATA box is located within the investigated 250-bp region in the organized chromatin domain abutting the Mat alpha 2p operator in MAT alpha cells and may remain repressed with the TATA box located anywhere within this organized repression domain. Mol Cell Biol, 1994 Jun, 14(6), 3927 - 37 RNA polymerase II cofactor PC2 facilitates activation of transcription by GAL4-AH in vitro; Kretzschmar M et al.; We have isolated from a crude Hela cell cofactor fraction (USA) a novel positive cofactor that cooperates with the general transcription machinery to effect efficient stimulation of transcription by GAL4-AH, a derivative of the Saccharomyces cerevisiae regulatory factor GAL4 . PC2 was shown to be a 500-kDa protein complex and to be functionally and biochemically distinct from native TFIID and previously identified cofactors . In the presence of native TFIID and other general factors, PC2 was necessary and sufficient for activation by GAL4-AH . Cofactor function was specific for transcriptional activation domains of GAL4-AH . The repressor histone H1 further potentiated but was not required for activation of transcription by GAL4-AH . On the basis of the observation that PC2 exerts entirely positive effects on transcription, we propose a model in which PC2 increases the activity of the preinitiation complex in the presence of an activator, thereby establishing a specific pathway during activation of RNA polymerase II. Mol Cell Biol, 1994 Jun, 14(6), 3842 - 52 Identification of potential target genes for Adr1p through characterization of essential nucleotides in UAS1; Cheng C et al.; Adr1p is a regulatory protein in the yeast Saccharomyces cerevisiae that binds to and activates transcription from two sites in a perfect 22-bp inverted repeat, UAS1, in the ADH2 promoter . Binding requires two C2H2 zinc fingers and a region amino terminal to the fingers . The importance for DNA binding of each position within UAS1 was deduced from two types of assays . Both methods led to an identical consensus sequence containing only four essential base pairs: GG(A/G)G . The preferred sequence, TTGG(A/G)GA, is found in both halves of the inverted repeat . The region of Adr1p amino terminal to the fingers is important for phosphate contacts in the central region of UAS1 . However, no base-specific contacts in this portion of UAS1 are important for DNA binding or for ADR1-dependent transcription in vivo . When the central 6 bp were deleted, only a single monomer of Adr1p was able to bind in vitro and activation in vivo was severely reduced . On the basis of these results and previous knowledge about the DNA binding site requirements, including constraints on the spacing and orientation of sites that affect activation in vivo, a consensus binding site for Adr1p was derived . By using this consensus site, potential Adr1p binding sites were located in the promoters of genes known to show ADR1-dependent expression . In addition, this consensus allowed the identification of new potential target genes for Adr1p. J Virol, 1994 Jun, 68(6), 3803 - 8 Genetic analysis indicates that the human foamy virus Bel-1 protein contains a transcription activation domain of the acidic class; Blair WS et al.; Human foamy virus encodes a nuclear regulatory protein, termed Bel-1, that serves as a potent activator of viral transcription . Mutational analysis has identified a small, discrete activation domain within Bel-1 that is highly active in both higher and lower eukaryotic cells . Here, we demonstrate that the activation domain of Bel-1 is highly dependent on the ADA2 transcriptional adaptor for biological activity in yeast cells, a property previously shown to be a hallmark of the VP16 class of acidic transcriptional activators (S . L . Berger, B . Pina, N . Silverman, G . A . Marcus, J . Agapite, J . L . Regier, S . J . Triezenberg, and L . Guarente, Cell 70:251-265, 1992) . Using genetic selection in yeast cells, we have derived a set of point mutants within the Bel-1 activation domain that display a qualitatively similar loss in activation potential when examined in either yeast or human cells . These data indicate that the Bel-1 activation domain functions similarly in both lower and higher eukaryotes and strongly suggest that Bel-1 belongs to the VP16 class of acidic transcription factors. J Virol, 1994 Jun, 68(6), 3536 - 43 Hepadnavirus reverse transcription initiates within the stem-loop of the RNA packaging signal and employs a novel strand transfer; Tavis JE et al.; Replication of the hepadnavirus genome occurs by reverse transcription of an RNA pregenome and is mediated by the viral polymerase; the polymerase is also required for packaging of the pregenome through interaction with the RNA packaging signal, epsilon . Previous work suggested that reverse transcription of minus-strand DNA initiates within the sequence element DR1 (direct repeat 1) and that disruption of DR1 activates a cryptic initiation site in a downstream copy of epsilon . However, using active duck hepatitis B virus polymerase expressed in a yeast Ty vector system, we demonstrate that synthesis of minus-strand DNAs with 5' ends at DR1 requires the stem-loop of epsilon, whereas the production of DNAs mapping to epsilon does not require DR1 . Mutations at epsilon that remove homology between epsilon and DR1 eliminate reverse transcripts with 5' ends in DR1, and restoring homology at DR1 to a mutant epsilon partially restores DNAs mapping to DR1 . Insertions of one nucleotide into the bulge region of the epsilon stem-loop increase the length of minus-strand DNA whose 5' ends map to DR1 by one nucleotide . Thus, very short minus-strand primers are initiated within epsilon, rather than in DR1 as previously supposed; they are then transferred to a four-nucleotide homology in DR1 . Transfer was also observed in vivo during replication of duck hepatitis B virus in avian cells; in this case, transfer is from the 5' copy of epsilon to the 3' copy of DR1 . This minus-strand transfer reaction is likely to be a general feature of all hepadnaviruses. Lipids, 1994 Jun, 29(6), 437 - 9 Lymphatic fatty acids from rats fed human milk and formula containing coconut oil; Roche ME et al.; Human milk and infant formula containing coconut/soy oil were infused into the duodenum of rats to determine the incorporation of capric, lauric, myristic and palmitic acids into lymphatic triacylglycerol (TAG) . The proportion of capric and lauric acids in the lymphatic TAG reflected the fatty acid composition of the diet . Based on positional analysis, it appears that more than 50% of the capric and lauric acids could have been absorbed from the intestine as sn-2 monoacylglycerols . In the rats fed human milk, 50% of palmitic acid in lymphatic TAG was in the sn-2 position . Because of the nonrandom distribution of palmitic acid in the lymphatic TAG, the nonspecific lipase in human milk, i.e., bile salt-stimulated lipase, did not appear to be a factor in milk lipid digestion. Curr Genet, 1994 Jun, 25(6), 531 - 7 Isolation of the ERG2 gene, encoding sterol delta 8-->delta 7 isomerase, from the rice blast fungus Magnaporthe grisea and its expression in the maize smut pathogen Ustilago maydis; Keon JP et al.; The Magnaporthe grisea ERG2 gene, encoding delta 8-->delta 7 sterol isomerase, was isolated from a genomic library by heterologous hybridization to a fragment of the Ustilago maydis ERG2 gene . The isolated gene contained a reading frame of 745 bp which encoded a protein of 221 amino acids . The coding region was interrupted by a single putative 79-bp-long intron . The deduced amino-acid sequence exhibited similarity to the ERG2 gene products of U . maydis and of Saccharomyces cerevisiae, particularly in the central region of the proteins . The NH2-terminal of all three proteins contained a long stretch of amino acids that were strongly hydrophobic, suggesting that they may function by anchoring the protein to a membrane surface . The M . grisea ERG2 gene complemented a U . maydis deletion mutant in which the ERG2 gene had been removed using a one-step gene replacement procedure . The delta 8-->delta 7 sterol isomerase produced by the M . grisea ERG2 gene exhibited a level of sensitivity to the sterol biosynthesis inhibitor, tridemorph, similar to that of the enzyme derived from the U . maydis ERG2 gene. Trends Biochem Sci, 1994 Jun, 19(6), 236 - 40 Diversity in function and regulation of MAP kinase pathways; Blumer KJ et al.; Eukaryotic cells from yeast to humans use sequential protein kinase reactions to regulate complex cellular functions . Equivalent protein kinases in different pathways have significant sequence homologies; however, little crossover in phosphorylation of substrates between pathways normally occurs . Assembly of kinase complexes and discrimination of substrates provide the selectivity of sequential protein kinase pathways to regulate such diverse cellular functions as osmoregulation, cell-wall biosynthesis, growth and differentiation. Plant Cell, 1994 Jun, 6(6), 875 - 84 Functional homologs of fungal metallothionein genes from Arabidopsis; Zhou J et al.; Metallothioneins (MTs) are cysteine-rich proteins required for heavy metal tolerance in animals and fungi . Two cDNAs encoding proteins with homology to animal and fungal MTs have been isolated from Arabidopsis . The genes represented by these cDNAs are referred to as MT1 and MT2 . When expressed in an MT-deficient (cup1 delta) mutant of yeast, both MT1 and MT2 complemented the cup1 delta mutation, providing a high level of resistance to CuSO4 and moderate resistance to CdSO4 . Although the MT-deficient yeast was not viable in the presence of either 300 microM CuSO4 or 5 microM CdSO4, cells expressing MT1 were able to grow in medium supplemented with 3 mM CuSO4 and 10 microM CdSO4, and those expressing MT2 grew in the presence of 3 mM CuSO4 and 100 microM CdSO4 . In plants, MT1 mRNA was more abundant in roots and dark-grown seedlings than in leaves . In contrast, MT2 mRNA accumulated more in leaves than in either roots or darkgrown seedlings . MT2 mRNA was strongly induced in seedlings by CuSO4, but only slightly by CdSO4 or ZnSO4 . However, MT1 mRNA was induced by CuSO4 in excised leaves that were submerged in medium . These results indicated that Arabidopsis MT genes are involved in copper tolerance . Plants also synthesized metal binding phytochelatins (poly{gamma-glutamylcysteine}glycine) when exposed to heavy metals . The results presented here argue against the hypothesis that phytochelatins are the sole molecules involved in heavy metal tolerance in plants . We conclude that Arabidopsis MT1 and MT2 are functional homologs of yeast MT. J Inorg Biochem, 1994 Jun, 54(4), 297 - 304 Aluminum inhibition of hexokinase activity in vitro: a study in biological availability; Exley C et al.; We have used the HK/G6PDH coupled enzyme assay to determine the biological availability of aluminum in mixed-ligand media of biological interest . The biological availability of aluminum was measured as the inhibition of the activity rate of the assay and was shown to be dependent upon the equilibration state of the aluminum stock solutions (prior to their addition to the assay) and the comparative reaction kinetics of competitive aluminum equilibria in the assays . Aluminum was found to inhibit the assay, however, the inhibition by aluminum was abolished when silicic acid was present in both the aluminum stock solution and the assay medium . The assay is proposed as a model system for investigating the biological availability of aluminum in heterogenous media of biochemical significance. EMBO J, 1994 Jun 1, 13(11), 2686 - 98 Ubiquitin-assisted dissection of protein transport across membranes; Johnsson N et al.; We describe a new way to analyze targeting in protein translocation . A fusion in which ubiquitin (Ub) is positioned between a signal sequence and a reporter domain is cleaved by Ub-specific proteases (UBPs) in the cytosol unless the fusion can 'escape' into a compartment such as the endoplasmic reticulum (ER) . The critical step involves rapid folding of the newly formed Ub moiety, which precludes its translocation and makes possible its cleavage by UBPs . However, if a sufficiently long spacer is present between the signal sequence and Ub, then by the time the Ub polypeptide emerges from the ribosome, the latter is already docked at the transmembrane channel, allowing the translocation of both the Ub and reporter domains of the fusion into the ER . We show that Ub fusions can be used as in vivo probes for kinetic and stochastic aspects of targeting in protein translocation, for distinguishing directly between cotranslational and posttranslational translocation, and for comparing the strengths of different signal sequences . This method should also be applicable to non-ER translocation. EMBO J, 1994 Jun 1, 13(11), 2617 - 24 A short element required for turning off heat shock transcription factor: evidence that phosphorylation enhances deactivation; Hoj A et al.; Transcriptional activation of heat shock genes is mediated by a presynthesized nuclear protein, the heat shock factor (HSF), which transiently converts from an inactive to an active form in response to hyperthermia . It has been suggested that hyperphosphorylation of HSF upon heat shock triggers activation through the induction of a conformational change unmasking transcriptional activator domains . Here we report that a short conserved element is involved in returning yeast HSF to the inactive state after heat shock and show that deactivation can be enhanced by phosphorylation of adjacent serine residues . These results suggest that phosphorylation of HSF in yeast serves as a regulatory mechanism to deactivate HSF, rather than being involved in its activation. Biochem J, 1994 Jun 1, 300 ( Pt 2), 303 - 7 The Ras-related protein R-ras interacts directly with Raf-1 in a GTP-dependent manner; Spaargaren M et al.; R-ras is a member of the ras family of small GTPases that associates with the apoptosis-suppressing proto-oncogene product Bcl-2 . Using the yeast two-hybrid system we provide evidence for an interaction between R-ras and the Raf-1 kinase . This interaction requires only the N-terminal regulatory domain (amino acids 1-256) of Raf-1, and is observed with both the wild type and a constitutively active R-ras mutant, but not with a deletion mutant that lacks the potential effector domain or a mutant of R-ras impaired for GTP binding . Moreover, using an in vitro binding assay we show a direct GTP-dependent interaction of purified R-ras with a purified Raf-1 fragment corresponding to the proposed 81-amino-acid H-Ras-binding domain of Raf-1 (amino acids 51-131) . Taken together, these data indicate that R-ras may exert its biological effect by means of modulating the activity of the Raf-1 kinase as its direct downstream effector. Yeast, 1994 Jun, 10(6), 801 - 10 Isolation and DNA sequence of the STE13 gene encoding dipeptidyl aminopeptidase; Anna-Arriola SS et al.; We have isolated a mutant which exhibits partial constitutivity for a-specific gene expression in alpha cells . The wild-type gene was cloned and demonstrated to be allelic to the STE13 gene, which encodes the dipeptidyl aminopeptidase involved in processing of the alpha-factor prepropheromone . Thus, the mating defect of the ste13 mutations in alpha cells may result both from the production of incompletely processed alpha-factor and from the increased expression of a-specific genes . The STE13 open reading frame of 931 amino acids contains a putative membrane-spanning segment near its amino terminus and is 31% identical to a second yeast dipeptidyl aminopeptidase (DAP2) . A null mutant of STE13 has been constructed: it is viable and sporulation-proficient. Protein Eng, 1994 Jun, 7(6), 743 - 8 Lysozyme requires fluctuation of the active site for the manifestation of activity; Imoto T et al.; Mutations around His15 which lie far away from the active site, stimulated glycol chitin activity of lysozyme at physiological temperature . Del-Arg14His15 lysozyme, a mutant lysozyme whose Arg14 and His15 were deleted together, and has the highest activity among these mutant lysozymes, had a similar binding ability to a trimer of N-acetyl-glucosamine, a substrate analogue, relative to native lysozyme . This suggests that the increased activity was due to an increased kcat in the catalysis reaction . The H-D exchange rate of the N-1 proton in the Trp63 which is located in the active site cleft, was enhanced in the Del-Arg14His15 lysozyme, while 2-D proton NMR analysis revealed no conformational change around Trp63 . We conclude that some sort of fluctuation at the active site might be required for the manifestation of activity . This theory is supported by the finding that the Del-Arg14His15 lysozyme showed a shift in temperature dependency of activity to lower temperatures compared with that of native lysozyme. Curr Biol, 1994 Jun 1, 4(6), 532 - 3 Metal ion metabolism . The copper-iron connection; Chang A et al.; Iron and copper, key nutrients for biological redox reactions, have been found to regulate each other's metabolism. Curr Opin Cell Biol, 1994 Jun, 6(3), 396 - 402 The SNF/SWI family of global transcriptional activators; Carlson M et al.; The yeast SNF/SWI proteins have a global role in transcriptional activation . This set of five proteins assists many gene-specific activators, most likely by altering chromatin structure to relieve repression . Recent work shows that the SNF/SWI proteins function together in a multiprotein complex and that SNF2 has DNA-dependent ATPase activity . SNF/SWI homologs have now been identified in Drosophila, mice and humans, suggesting a conserved role in transcriptional activation. Curr Opin Cell Biol, 1994 Jun, 6(3), 373 - 9 Telomere maintenance and gene repression: a common end? Palladino F, Gasser SM. In yeast, the study of the teleomere has recently provided new information on the requirements for chromosome stability, on elements influencing nuclear architecture and on position-effect variegation. Curr Opin Cell Biol, 1994 Jun, 6(3), 368 - 72 Eukaryotic DNA replication; Diffley JF; The past year has seen the genetic characterization of a human replication origin as well as the identification and characterization of some key components of replication initiation complexes in budding yeast . These results should provide important information for determining how the initial events in DNA replication are regulated during the eukaryotic cell cycle. Br J Pharmacol, 1994 Jun, 112(2), 551 - 6 Relationship between plasma lipids and palmitoyl-CoA hydrolase and synthetase activities with peroxisomal proliferation in rats treated with fibrates; Alegret M et al.; 1 . The time-course of the effect of clofibrate (CFB), bezafibrate (BFB) and gemfibrozil (GFB) on lipid plasma levels and palmitoyl-CoA hydrolase and synthetase activities, as well as the correlations with the peroxisomal proliferation phenomenon have been studied in male Sprague-Dawley rats . 2 . The administration of the three drugs caused a significant reduction in body weight gain, accompanied with a paradoxical increase in food intake in groups treated with BFB and GFB . 3 . Drug treatment produced gross hepatomegaly and increase in peroxisomal beta-oxidation, and these parameters were strongly correlated . The order of potency was BFB > CFB > or = GFB . 4 . Both plasma cholesterol (BFB approximately CFB > GFB) and triglyceride (BFB approximately GFB > CFB) levels were reduced in treated animals . There was an inverse correlation between these parameters and peroxisomal beta-oxidation, although the peroxisomal proliferation seemed to explain only a small part of the hypolipidemic effect observed . 5 . Cytosolic and microsomal (but not mitochondrial) palmitoyl-CoA hydrolase activities were increased by the three drugs (BFB > CFB > GFB), probably by inducing the hydrolase I isoform, which is insensitive to inhibition by fibrates in vitro . The increased hydrolase activities were directly and strongly correlated with peroxisomal beta-oxidation . 6 . Palmitoyl-CoA synthetase activity was also increased by the treatment with fibrates (BFB > CFB > GFB), probably as a consequence of the enhancement of hydrolase activities.(ABSTRACT TRUNCATED AT 250 WORDS) Nat Struct Biol, 1994 Jun, 1(6), 383 - 7 Solution structure of a cysteine rich domain of rat protein kinase C; Hommel U et al.; Intracellular protein phosphorylation by protein kinase C (PKC) plays a major role in the translation of extracellular signals into cellular events . Speculations on the structural basis for PKC activation are based on sequence homology between their cysteine-rich domains (CRD) and the DNA-binding 'zinc-fingers' . We produced a fragment comprising the second CRD (CRD2) of rat PKC-alpha and de