J Cell Biol, 1990 Dec, 111(6 Pt 1), 2537 - 42 Analysis of the molecular basis of calmodulin defects that affect ion channel-mediated cellular responses: site-specific mutagenesis and microinjection; Hinrichsen R et al.; The ability of microinjected calmodulin to temporarily restore an ion channel-mediated behavioral phenotype of a calmodulin mutant in Paramecium tetraurelia (cam1) is dependent on the amino acid side chain that is present at residue 101, even when there is extensive variation in the rest of the amino acid sequence . Analysis of conservation of serine-101 in calmodulin suggests that the ability of calmodulin to regulate this ion channel-associated cell function may be a biological role of calmodulin that is widely distributed phylogenetically . A series of mutant calmodulins that differ only at residue-101 were produced by in vitro site-specific mutagenesis and expression in Escherichia coli, purified to chemical homogeneity, and tested for their ability to temporarily restore a wild-type behavioral phenotype to cam1 (pantophobiacA1) Paramecium . Calmodulins with glycine-101 or tyrosine-101 had minimal activity; calmodulins with phenylalanine-101 or alanine-101 had no detectable activity . However, as a standard of comparison, all of the calmodulins were able to activate a calmodulin-regulated enzyme, myosin light chain kinase, that is sensitive to point mutations elsewhere in the calmodulin molecule . Overall, these results support the hypothesis that the structural features of calmodulin required for the transduction of calcium signals varies with the particular pathway that is being regulated and provide insight into why inherited mutations of calmodulin at residue 101 are nonlethal and selective in their phenotypic effects. Jpn J Cancer Res, 1990 Dec, 81(12), 1259 - 64 Inhibition of avian myeloblastosis virus reverse transcriptase by aurochloric acid; Semba M et al.; We investigated the inhibitory effects of aurochloric acid (AuCl4H) on reverse transcriptase (RT) derived from avian myeloblastosis virus and DNA polymerase alpha (pol . alpha) purified from HeLa S3 cells . The activities of RT, pol . alpha and E . coli DNA polymerase I (pol . I) with dTTP as the substrate were inhibited 50% at AuCl4H concentrations of 18 microM, 43 microM and 230 microM, respectively . AuCl4H inhibited RT activity competitively with respect to the substrate, dTTP, and uncompetitively with the template/primer, (rA)n(dT)12-18 . In assays with dGTP as the substrate, 50% inhibitions of RT, pol . alpha and pol . I activities were observed at AuCl4H concentrations of 100 microM, 450 microM and 580 microM, respectively . AuCl4H inhibited RT activity uncompetitively with respect to the substrate, dGTP, and noncompetitively with the template/primer, (rC)n(dG)12-18 . AuCl4H at concentrations causing more than 50% inhibition of RT activity had little inhibitory effect on the colony-forming ability of HeLa cells or their syntheses of DNA, RNA and protein. Arch Biochem Biophys, 1990 Dec, 283(2), 311 - 7 Expression of mammalian DT-diaphorase in Escherichia coli: purification and characterization of the expressed protein; Ma Q et al.; A full-length cDNA clone, pKK-DTD4, complementary to rat liver cytosolic DT-diaphorase {NAD(P)H:quinone oxidoreductase (EC 1.6.99.2)} mRNA was expressed in Escherichia coli . The pKK-DTD4 cDNA was obtained by extending the 5'-end sequence of a rat liver DT-diaphorase cDNA clone, pDTD55, to include an ATG initiation codon and the NH2-terminal codons using polymerase chain reaction (PCR) . Restriction sites for EcoRI and HindIII were incorporated at the 5'- and 3'-ends of the cDNA, respectively, by the PCR reaction . The resulting full-length cDNA was inserted into an expression vector, pKK2.7, at the EcoRI and HindIII restriction sites . E . coli strain AB1899 was transformed with the constructed expression plasmid, and DT-diaphorase was expressed under the control of the tac promotor . The expressed DT-diaphorase exhibited high activity of menadione reduction and was inhibited by dicumarol at a concentration of 10(-5)M . After purification by Cibacron Blue affinity chromatography, the expressed enzyme migrated as a single band on 12.5% sodium dodecyl sulfate-polyacrylamide gel with a molecular weight equivalent to that of the purified rat liver cytosolic DT-diaphorase . The purified expressed protein was recognized by polyclonal antibodies against rat liver DT-diaphorase on immunoblot analysis . It utilized either NADPH or NADH as electron donor at equal efficiency and displayed high activities in reduction of menadione, 1,4-benzoquinone, and 2,6-dichlorophenolindophenol which are typical substrates for DT-diaphorase . The expressed DT-diaphorase exhibited a typical flavoprotein spectrum with absorption peaks at 380 and 452 nm . Flavin content determination showed that it contained 2 mol of FAD per mole of the enzyme . Edman protein sequencing of the first 20 amino acid residues at the NH2 terminus of the expressed protein indicated that the expressed DT-diaphorase is not blocked at the NH2 terminus and has an alanine as the first amino acid . The remaining 19 amino acid residues at the NH2 terminus were identical with those of the DT-diaphorase purified from rat liver cytosol. J Cell Biol, 1990 Dec, 111(6 Pt 2), 3049 - 64 Deletions in epidermal keratins leading to alterations in filament organization in vivo and in intermediate filament assembly in vitro; Coulombe PA et al.; To investigate the sequences important for assembly of keratins into 10-nm filaments, we used a combined approach of (a) transfection of mutant keratin cDNAs into epithelial cells in vivo, and (b) in vitro assembly of mutant and wild-type keratins . Keratin K14 mutants missing the nonhelical carboxy- and amino-terminal domains not only integrated without perturbation into endogenous keratin filament networks in vivo, but they also formed 10-nm filaments with K5 in vitro . Surprisingly, keratin mutants missing the highly conserved L L E G E sequence, common to all intermediate filament proteins and found at the carboxy end of the alpha-helical rod domain, also assembled into filaments with only a somewhat reduced efficiency . Even a carboxy K14 mutant missing approximately 10% of the rod assembled into filaments, although in this case filaments aggregated significantly . Despite the ability of these mutants to form filaments in vitro, they often perturbed keratin filament organization in vivo . In contrast, small truncations in the amino-terminal end of the rod domain more severely disrupted the filament assembly process in vitro as well as in vivo, and in particular restricted elongation . For both carboxy and amino rod deletions, the more extensive the deletion, the more severe the phenotype . Surprisingly, while elongation could be almost quantitatively blocked with large mutations, tetramer formation and higher ordered lateral interactions still occurred . Collectively, our in vitro data (a) provide a molecular basis for the dominance of our mutants in vivo, (b) offer new insights as to why different mutants may generate different phenotypes in vivo, and (c) delineate the limit sequences necessary for K14 to both incorporate properly into a preexisting keratin filament network in vivo and assemble efficiently into 10-nm keratin filaments in vitro. EMBO J, 1990 Dec, 9(13), 4511 - 7 The methionine-rich domain of the 54 kd protein subunit of the signal recognition particle contains an RNA binding site and can be crosslinked to a signal sequence; Zopf D et al.; The 54 kd protein subunit of the signal recognition particle (SRP54) has been shown to bind signal sequences by UV crosslinking . Primary structure analysis and phylogenetic comparisons have suggested that SRP54 is composed of two domains: an amino-terminal domain that contains a putative GTP-binding site (G-domain) and a carboxy-terminal domain that contains a high abundance of methionine residues (M-domain) . Partial proteolysis of SRP revealed that the two proposed domains of SRP54 indeed represent structurally discrete entities . Upon proteolysis the intact G-domain was released from SRP, whereas the M-domain remained attached to the core of the particle . Reconstitution experiments demonstrated that the isolated M-domain associates with 7SL RNA in the presence of SRP19 . In addition, we observed a specific binding of the M-domain directly to 4.5S RNA of Escherichia coli, which contains a structural motif also present in 7SL RNA . This shows that the M-domain contains an RNA binding site, and suggests that SRP54 may be linked to the rest of SRP through this domain by a direct interaction with 7SL RNA . Using UV crosslinking, we found that in an in vitro translation system the preprolactin signal sequence contacts SRP through the M-domain of SRP54 . These results imply that the M-domain contains the signal sequence binding site of SRP54, although we cannot exclude that the G-domain may also be in proximity to bound signal sequences.(ABSTRACT TRUNCATED AT 250 WORDS) Proc Natl Acad Sci U S A, 1990 Dec, 87(24), 9966 - 70 Another mechanism for creating diversity in gamma-aminobutyrate type A receptors: RNA splicing directs expression of two forms of gamma 2 phosphorylation site; Whiting P et al.; Diversity of gamma-aminobutyrate type A (GABAA) receptors has recently been proposed to be achieved by assembly of receptor subtypes from a multitude of subunits (alpha 1-6, beta 1-3, gamma 1-2, and delta) encoded by different genes . Here we report a further mechanism for creating GABAA receptor diversity: alternative RNA splicing . Two forms of bovine gamma 2 subunit cDNA were isolated (gamma 2S and gamma 2L) that differed by the presence or absence of a 24-base-pair (8-amino acid) insertion in the cytoplasmic domain between the third and fourth putative membrane-spanning regions . Polymerase chain reaction from RNA demonstrated that the two forms of gamma 2 subunit are expressed in bovine, human, and rat brain . Sequencing of genomic DNA clones encoding the gamma 2 subunit demonstrated that the 24-base-pair insert is organized as a separate exon . Analysis of the sequence of the 8-amino acid insert revealed that it contains a protein kinase C consensus phosphorylation site . Expression of the large cytoplasmic loop domains of gamma 2S and gamma 2L in Escherichia coli, followed by phosphorylation of the recombinant proteins by protein kinase C, demonstrated that gamma 2L, but not gamma 2S, could be phosphorylated . Thus the two forms of gamma 2 subunit differ by the presence or absence of a protein kinase C phosphorylation site . This mechanism for creating GABAA receptor diversity may allow differential regulation of the function of receptor subtypes. Proc Natl Acad Sci U S A, 1990 Dec, 87(24), 9519 - 23 Structure and expression of cytosolic cyclophilin/peptidyl-prolyl cis-trans isomerase of higher plants and production of active tomato cyclophilin in Escherichia coli; Gasser CS et al.; cDNA clones encoding proteins of approximately 18 kDa in which 83% of the amino acids are conserved relative to the published sequences of mammalian cyclophilin/rotamase (CyP) have been isolated from tomato, maize, and Brassica napus . In correspondence with the mammalian genes, but in contrast with the Neurospora gene and one yeast CyP gene, the plant CyP genes encode only mature proteins lacking transit peptides . RNA blot analyses demonstrate that CyP genes are expressed in all plant organs tested . Southern blots of genomic DNA indicate that there are small families (two to eight members) of CyP-related genes in maize and B . napus . A vector was constructed for expression of the tomato cDNA in E . coli . SDS/polyacrylamide gels show that extracts of appropriately induced cells harboring this vector contain nearly 40% of the protein as a single approximately 18-kDa band . While the majority of this protein is sequestered in insoluble inclusion bodies, the soluble extracts have higher levels of peptidyl-prolyl cis-trans isomerase (rotamase) activity than extracts of wild-type cells . This additional activity is sensitive to inhibition by the cyclic undecapeptide cyclosporin A. Dev Biol, 1990 Dec, 142(2), 346 - 59 Regulatory elements from the related spec genes of Strongylocentrotus purpuratus yield different spatial patterns with a lacZ reporter gene; Gan L et al.; The Spec1 and Spec2 genes of Strongylocentrotus purpuratus are closely associated with the differentiation of aboral ectoderm . To examine cis-regulatory elements involved in the spatial expression of the Spec genes, we fused the Escherichia coli lacZ gene containing a nuclear targeting signal to 5'flanking DNA plus 5' untranslated leader sequences from Spec1, Spec2a, and Spec2c . All three genes contain 700 bp of highly conserved DNA in their upstream regions, but in Spec1 and Spec2c large insertions interrupt the conserved regions . The Spec-lacZ reporter gene plasmids were microinjected into eggs of S . purpuratus, Lytechinus variegatus, and L . pictus, and beta-galactosidase activity was determined in situ by X-gal staining . The Spec2a-lacZ fusion gene, which contained 1516 bp of 5' flanking DNA and 18 bp of 5' untranslated leader sequence, was preferentially expressed in aboral ectoderm cells in all three species . The Spec1-lacZ fusion gene was expressed in a strikingly different fashion--preferentially in primary and secondary mesenchyme cells, occasionally in aboral ectoderm cells, and less often in oral ectoderm and endoderm cells . The staining pattern was the same in either homologous or heterologous embryos . The Spec2c-lacZ fusion gene, like Spec2a-lacZ, was preferentially expressed in aboral ectoderm, but staining of other cell types was frequently observed . To further delineate sequences required for correct spatial expression, we deleted 800 bp of 5' flanking DNA from the Spec2a-lacZ fusion gene, resulting in a delta Spec2a-lacZ fusion gene that contained only the conserved DNA region . This gene fusion showed preferential expression in aboral ectoderm cells . However, the cell type specificity was not as great as with the parental Spec2a-lacZ plasmid . These experiments implied that the conserved DNA region, associated with all Spec genes examined, was insufficient for complete aboral ectoderm specificity, and suggested that a spatial repressor element existed between -1516 and -697 bp in the 5' flanking DNA of Spec2a. J Bacteriol, 1990 Dec, 172(12), 7104 - 10 An expression vector for the archaebacterium Haloferax volcanii; Nieuwlandt DT et al.; The recent development of an efficient transformation method and shuttle vectors for Haloferax volcanii has set the stage for rapid progress in archaebacterial molecular biology . We describe a shuttle-expression vector that can be selected for and maintained in either H . volcanii or Escherichia coli and permits the expression of cloned genes in H . volcanii . The vector, pWL204, was constructed by incorporating an H . volcanii tRNA(Lys) gene promoter into a derivative of the H . volcanii-E . coli shuttle vector pWL102 . The vector has been used to express a modified, intron-containing, H . mediterranei tRNA(Trp) gene (tRNA(Trp)-O167) . Transcription from the tRNA(Lys) gene promoter in vivo was detected by Northern (RNA) analysis with an oligonucleotide probe complementary to the unique intron sequence of tRNA(Trp)-O167 . Dependence of transcription on the tRNA(Lys) promoter was demonstrated by the absence of transcription when the promoter sequence was deleted from the vector and by mapping the transcription initiation site by primer extension. Infect Immun, 1990 Dec, 58(12), 3909 - 13 Immunologic characterization of a cloned fragment containing the species-specific epitope from the major outer membrane protein of Chlamydia trachomatis; Toye B et al.; A 183-bp fragment encoding variable domain IV (VD IV) of Chlamydia trachomatis serovar B major outer membrane protein (MOMP) (amino acids 273 to 333) and containing the species-specific epitope was cloned and expressed in Escherichia coli as a fusion protein with Schistosoma japonicum glutathione S-transferase (GST-VD IV) . The fusion protein was affinity purified under nondenaturing conditions and used to immunize rabbits . Antisera were characterized by microimmunofluorescence, immunoblot, dot blot, peptide enzyme-linked immunosorbent, and in vitro neutralization assays . Antisera recognized MOMP from all 12 tested serovars of C . trachomatis but not from Chlamydia psittaci . In a dot blot assay, antisera bound to elementary bodies of serovars B, D, E, L2, and K in a strong fashion and to elementary bodies of serovars F, G, A, and H in a weak fashion but not to elementary bodies of serovars C, J, and I . High-resolution peptide mapping with synthetic overlapping serovar B MOMP peptides in a solid-phase enzyme-linked immunosorbent assay showed that immunization with GST-VD IV produced a serologic response that closely mimicked the response produced with purified serovar B elementary bodies . Antipeptide antibodies with strong binding to species- and subspecies-specific epitopes were elicited . Antisera were able to neutralize only those C . trachomatis serovars that bound antibodies in the dot blot assay . These results suggest that antigenic fragments from VD IV containing the species-specific epitope may be useful in the construction of a chlamydial vaccine for some but not all C . trachomatis serovars. Proc Natl Acad Sci U S A, 1990 Dec, 87(23), 9454 - 8 Retron for the 67-base multicopy single-stranded DNA from Escherichia coli: a potential transposable element encoding both reverse transcriptase and Dam methylase functions; Hsu MY et al.; The region (retron-Ec67) required for the biosynthesis of a branched-RNA-linked multicopy single-stranded DNA (msDNA-Ec67) from a clinical isolate of Escherichia coli was mapped at a position equivalent to 19 min on the K-12 chromosome . The element containing the retron consisted of a unique 34-kilobase sequence that was flanked by direct repeats of a 26-base-pair sequence found in the K-12 chromosomal DNA . This suggests that the 34-kilobase element was probably integrated into the E . coli genome by a mechanism related to transposition or phage integration . In the 34-kilobase sequence an open reading frame of 285 residues was found, which displays 44% sequence identity with the E . coli Dam methylase . Interestingly, there are three GATC sequences, the site of Dam methylation, in the promoter region of the gene for reverse transcriptase. J Immunol, 1990 Dec 1, 145(11), 3747 - 54 Binding of IL-1 beta to alpha-macroglobulins and release by thioredoxin; Borth W et al.; Human alpha 2-macroglobulin (H alpha 2M) is a major IL-1 beta binding plasma protein . The characteristics of the H alpha 2M IL-1 beta complex formation suggested, that cleavage of the internal thiol ester in other members of the alpha-macroglobulin family (alpha M) could enable these proteins to bind IL-1 beta . Characterization of optimal conditions for binding 125I IL-1 beta to H alpha 2M showed that H alpha 2M-IL-1 beta complex formation could be obtained over a pH range of 6.3 to 9 in the presence of some metal cations (i.e., Zn2+, Cd2+, Cu2+, Ni2+) . Other divalent metal cations (i.e., Mn2+, Mg2+, Ca2+) were without effect . Time kinetic studies showed that binding of IL-1 beta to H alpha 2M was complete within 200 min and that H alpha 2M-IL-1 beta complexes became increasingly resistant to dissociation by boiling in SDS as a function of incubation time . Human pregnancy zone protein, rat alpha 1-, alpha 2-macroglobulin (R alpha 1M, R alpha 2M), all homologous with H alpha 2M, were tested for their ability to bind IL-1 beta . In each instance, alpha M-IL-1 beta complex formation was observed only after treatment of alpha M with methylamine, a primary amine that causes cleavage of the internal thiol ester in alpha M and the appearance of free thiol groups . Similarly, for each of these proteins, complex formation was increased several fold in the presence of Zn2+ . Competition experiments using cytokines or proteins of similar molecular mass as IL-1 beta established that only unlabeled IL-1 beta was effective in inhibiting binding of 125I IL-1 beta to H"F" alpha 2M . Acylation of H"F" alpha 2M by diethylpyrocarbonate blocked the binding of IL-1 beta when analyzed by native PAGE . Deacylation of H"F" alpha 2M with hydroxylamine partially restored the binding capacity of H"F" alpha 2M further supporting the involvement of histidyl residues in the Zn2(+)-dependent binding of IL-1 beta . Reduced thioredoxin, but not its alkylated form, from Escherichia coli readily releases H"F" alpha 2M bound IL-1 beta under conditions that did not lead to reduction of disulfide bonds in H"F" alpha 2M . The action of thioredoxin also augmented IL-1-like activity in two independent bioassays suggesting that H"F" alpha 2M bound IL-1 beta is partially biologically inactive or latent . These results suggest that "activated" alpha M exert a modulating role for IL-1 beta by exposing specific binding sites, which are inaccessible in the native proteins.(ABSTRACT TRUNCATED AT 400 WORDS) J Virol, 1990 Dec, 64(12), 5757 - 63 Role of the gag polyprotein precursor in packaging and maturation of Rous sarcoma virus genomic RNA; Oertle S et al.; Rous sarcoma virus nucleocapsid protein (NC) has been shown by site-directed mutagenesis to be involved in viral RNA packaging and in the subsequent maturation of genomic RNA in the progeny viral particles . To investigate whether NC exerts these activities as a free protein or as a domain of the polyprotein precursor Pr76gag, we have constructed several mutants unable to process Pr76gag and analyzed their properties in a transient-transfection assay of chicken embryo fibroblasts, the natural host of Rous sarcoma virus . A point mutation in the protease (PR) active site completely prevents Pr76gag processing . The full-length Pr76gag polyprotein is still able to package viral RNA, but cannot mature it . A shorter gag precursor polyprotein lacking the C-terminal PR domain, but retaining that of the NC protein, is however, unable even to package viral RNA . This indicates that the NC protein can participate in packaging viral RNA only as part of a full-length Pr76gag and that the PR domain is, indirectly or directly, also involved in RNA packaging . These results also demonstrate that processing of Pr76gag is necessary for viral RNA dimerization. Agric Biol Chem, 1990 Dec, 54(12), 3241 - 50 Extracellular production of human tumor necrosis factor-alpha by Escherichia coli using a chemically-synthesized gene; Nakamura S et al.; A DNA fragment of approximately 490 base pairs encoding human TNF was chemically synthesized and expressed within Escherichia coli cells . Furthermore, extracellular production of human TNF and several N-terminal deletion mutants of TNF was attempted using the excretion vector pEAP8 . The TNF mutant with two N-terminal amino acids deleted (N delta 2-TNF) was efficiently excreted into the culture medium by E . coli carrying the plasmid pEXTNF3 . In this clone, the signal peptide was correctly processed during the excretion . The E . coli-excreted N delta 2-TNF had higher antitumor activity than wild-type TNF or N delta 2-TNF produced intracellularly by E . coli. J Steroid Biochem Mol Biol, 1990 Nov 30, 37(4), 481 - 90 The steroid-binding properties of recombinant glucocorticoid receptor: a putative role for heat shock protein hsp90; Ohara-Nemoto Y et al.; The steroid-binding domain of the human glucocorticoid receptor was expressed in Escherichia coli either as a fusion protein with protein A or under control of the T7 RNA polymerase promoter . The recombinant proteins were found to bind steroids with the normal specificity for a glucocorticoid receptor but with reduced affinity (Kd for triamcinolone acetonide approximately 70 nM) . Glycerol gradient analysis of the E . coli lystate containing the recombinant protein indicated no interaction between the glucocorticoid receptor fragment and heat shock proteins . However, synthesis of the corresponding fragments of glucocorticoid receptor in vitro using rabbit reticulocyte lystate resulted in the formation of proteins that bound triamcinolone acetonide with high affinity (Kd 2nM) . Glycerol gradient analysis of these proteins, with and without molybdate, indicated that the in vitro synthesised receptor fragments formed complexes with hsp90 as previously shown for the full-length rat glucocorticoid receptor . Radiosequence analysis of the recombinant steroid-binding domain expressed in E . coli and affinity labelled with dexamethasone mesylate identified binding of the steroid to Cys-638 predominantly . However, all cysteine residues within the steroid-binding domain were affinity labelled to a certain degree indicating that the recombinant protein has a structure similar to the native receptor but more open and accessible. Gene, 1990 Nov 30, 96(1), 29 - 36 Genomic replacement in Escherichia coli K-12 using covalently closed circular plasmid DNA; Oden KL et al.; A number of gene replacements at different loci were constructed using covalently closed circular (ccc) plasmid DNA in the recB21 recC22 sbcB15 sbcC201 mutant of Escherichia coli (JC7623) . Selected constructs representing deletions and insertion mutations formed from double-crossover events involving the ccc plasmid molecules and the genome were confirmed by Southern blots, and the frequency of double-crossover events was evaluated . It is reported that such mutants may be constructed without linearizing plasmid DNA, as described previously. Gene, 1990 Nov 30, 96(1), 23 - 8 High efficiency transformation of Escherichia coli with plasmids; Inoue H et al.; We have re-evaluated the conditions for preparing competent Escherichia coli cells and established a simple and efficient method (SEM) for plasmid transfection . Cells (DH5, JM109 and HB101) prepared by SEM are extremely competent for transformation (1-3 x 10(9) cfu/microgram of pBR322 DNA), and can be stored in liquid nitrogen for at least 40 days without loss of competence . Unlike electroporation, transformation using these competent cells is affected minimally by salts in DNA preparation . These competent cells are particularly useful for construction of high-complexity cDNA libraries with a minimum expenditure of mRNA. Gene, 1990 Nov 30, 96(1), 141 - 5 HU-1 mutants of Escherichia coli deficient in DNA binding; Goshima N et al.; We constructed four mutants of the Escherichia coli hupB gene, encoding HU-1 protein, by synthetic oligodeoxyribonucleotide-directed, site-specific mutagenesis on M13mp18 vectors . The HupBR45 protein contained alterations of Arg58----Gly and Arg61----Gly, and the HupBF3, HupBK2 and HupBA1 proteins contained Phe47----Thr, Lys37----Gln and Ala30----Asp alterations, respectively . HupBF3 and HupBR45 were unable to maintain normal cell growth in a hupA-hupB-himA triple mutant at 42 degrees C, mini-F or RSF1010 proliferation, or Mu phage development in a hupA-hupB double mutant, whereas HupBA1 and HupBK2 supported these cellular activities . DNA-affinity column chromatography showed that the HupBF3 and HupBR45 had reduced affinities to DNA . These observations indicate that two highly conserved Arg residues in the arm structure of the C-terminal half of the HU-1 molecule and a Phe residue in the short beta-sheet connecting the two halves of the molecule are important for the DNA-binding ability and biological functions of this protein. Cell, 1990 Nov 30, 63(5), 1085 - 97 Molecular cloning and expression of a hexameric Drosophila heat shock factor subject to negative regulation; Clos J et al.; We report the cloning of the transcriptional activator of heat shock genes, HSF, from Drosophila . The predicted sequence of Drosophila HSF protein is surprisingly divergent from that of yeast HSF, except in regions important for DNA binding and oligomerization . A segment of the DNA binding domain of HSF bears an intriguing similarity to the putative DNA recognition helix of bacterial sigma factors, while the oligomerization domain contains an unusual arrangement of conserved hydrophobic heptad repeats . Drosophila HSF produced in E . coli under nonshock conditions forms a hexamer that binds specifically to DNA with high affinity and activates transcription from a heat shock promoter in vitro . In contrast, when HSF is expressed in Xenopus oocytes, maximal DNA binding affinity is observed only after heat shock induction . These results suggest that Drosophila HSF has an intrinsic affinity for DNA, which is repressed under nonshock conditions in vivo. Science, 1990 Nov 30, 250(4985), 1259 - 62 Binding of the Wilms' tumor locus zinc finger protein to the EGR-1 consensus sequence; Rauscher FJ 3rd et al.; The Wilms' tumor locus (WTL) at 11p13 contains a gene that encodes a zinc finger-containing protein that has characteristics of a DNA-binding protein . However, binding of this protein to DNA in a sequence-specific manner has not been demonstrated . A synthetic gene was constructed that contained the zinc finger region, and the protein was expressed in Escherichia coli . The recombinant protein was used to identify a specific DNA binding site from a pool of degenerate oligonucleotides . The binding sites obtained were similar to the sequence recognized by the early growth response-1 (EGR-1) gene product, a zinc finger-containing protein that is induced by mitogenic stimuli . A mutation in the zinc finger region of the protein originally identified in a Wilms' tumor patient abolished its DNA-binding activity . These results suggest that the WTL protein may act at the DNA binding site of a growth factor-inducible gene and that loss of DNA-binding activity contributes to the tumorigenic process. Gene, 1990 Nov 30, 96(1), 17 - 22 A phosphate group at the cos ends of phage lambda DNA is not a prerequisite for in vitro packaging: an alternative method for constructing genomic libraries using a new phasmid vector, lambda pGY97; Vincze E et al.; It is shown here that the phosphate groups at the cos ends of phage lambda DNA are not a prerequisite for in vitro packaging . Molecules with phosphatase-treated cos ends are packaged in vitro as efficiently as native lambda DNA . This observation can be used for an alternative strategy to improve the efficiency of gene library construction, since cos-cos ligation decreases in vitro encapsidation and infectivity . Dephosphorylated cos ends and a new phasmid vector lambda pGY97 have been used to construct a representative gene bank of alfalfa in a Mcr- (5-methylcytosine restriction deficient) Escherichia coli host strain . These recombinant clones can be propagated as phages or more conveniently as plasmids in recA- E . coli, to prevent possible homologous recombination events between repetitive sequences of the insert that would otherwise interfere with clone stability . The 5-19-kb inserts can be easily recloned as plasmids from the recombinant phasmids with simple EcoRI digestion and re-ligation . This observation also implies that the construction of gene libraries in cosmid vectors can be made more efficient if cos-cos ligates were cleaved by lambda terminase just before in vitro packaging. Cell, 1990 Nov 30, 63(5), 1013 - 24 Cyclin activation of p34cdc2; Solomon MJ et al.; The gradual accumulation of cyclin in the frog egg induces an abrupt and concerted activation of p34cdc2 that initiates mitosis . Activation is delayed even after the accumulation of cyclin to a critical threshold concentration . We have reproduced these unusual kinetic properties of p34cdc2 activation in vitro using bacterially expressed cyclin proteins and extracts derived from Xenopus eggs . Abrupt activation follows a lag period, the length of which is independent of the concentration of cyclin . The threshold concentration of cyclin and the length of the lag period are regulated by INH, an inhibitor of MPF activation in oocytes recently identified as a type 2A protein phosphatase . Binding to cyclin induces both tyrosine and threonine phosphorylation of the previously unphosphorylated p34cdc2, rendering it inactivated . The concerted transition into mitosis involves both a reduction in the rate of p34cdc2 phosphorylation on tyrosine and an increase in its rate of dephosphorylation. Biochem Biophys Res Commun, 1990 Nov 30, 173(1), 141 - 8 Molecular cloning of mouse acid beta-galactosidase cDNA: sequence, expression of catalytic activity and comparison with the human enzyme; Nanba E et al.; A full-length cDNA coding for mouse lysosomal acid beta-galactosidase has been isolated on the basis of homology with the human gene . Catalytic activity toward 4-methylumbelliferyl beta-D-galactoside in the COS-1 cell expression system provided positive proof for its authenticity . The sequence analysis showed that the degree of similarity between the human and mouse enzymes was approximately 70% in the nucleotide sequence and nearly 80% in the amino acid sequence . The deduced primary amino acid sequences of the enzymes from the two species indicated that, of the seven possible N-glycosylation sites in the human enzyme, five are conserved in the mouse enzyme . Three additional possible N-glycosylation sites, not present in the human enzyme, are found in the primary amino acid sequence of the mouse enzyme . All seven cysteine residues in the mouse enzyme are conserved in the human enzyme . Although the nucleotide sequence could be aligned to 60% identity with the E . coli beta-galactosidase, similarity in the amino acid sequence was minimal. Biochim Biophys Acta, 1990 Nov 30, 1087(3), 336 - 8 Nucleotide sequence of mouse HSP60 (chaperonin, GroEL homolog) cDNA; Venner TJ et al.; The cDNA sequence of the 60 kDa heat-shock protein from mouse 3T3 cells has been determined . The deduced amino acid sequence of mouse hsp60 protein differs from the corresponding proteins from Chinese hamster and human cells in 7 and 13 residues, respectively, most of which are conservative replacements. Cell, 1990 Nov 30, 63(5), 941 - 8 Guanylyl cyclase is a heat-stable enterotoxin receptor; Schulz S et al.; Plasma membrane forms of guanylyl cyclase have been shown to function as natriuretic peptide receptors . We describe a new clone (GC-C) encoding a guanylyl cyclase receptor for heat-stable enterotoxin . GC-C encodes a protein containing an extracellular amino acid sequence divergent from that of previously cloned guanylyl cyclases; however, the protein retains the intracellular protein kinase-like and cyclase catalytic domains . Expression of GC-C in COS-7 cells results in high guanylyl cyclase activity . In addition, heat-stable enterotoxin from E . coli, but not natriuretic peptides, causes marked elevations of cyclic GMP and is specifically bound by cells transfected with GC-C . The enterotoxin fails to elevate cyclic GMP in nontransfected cells or in cells transfected with the natriuretic peptide/guanylyl cyclase receptors . These results show that a heat-stable enterotoxin receptor responsible for acute diarrhea is a plasma membrane form of guanylyl cyclase. Biochem Biophys Res Commun, 1990 Nov 30, 173(1), 231 - 9 Expression of DNA sequences containing neuron specific enolase gene in Escherichia coli; Dutta SK et al.; There is evidence that the gene for gamma-gamma enolase (neuron specific enolase, NSE) is regulated during cell differentiation and development, conserved in a variety of organisms and contains mRNA destabilizing sequences . In order to investigate further the mechanisms of these processes and to obtain large quantity of this protein, the NSE gene was isolated from neuroblastoma cells and cloned in E . coli using standard molecular biology techniques . The NSE gene expression was studied and the expressed protein (recombinant NSE) was characterized extensively . The recombinant NSE behaves like parental NSE in antisera specificity, resistance for chaotropic agents like urea, thermal stability at higher temperatures etc . The physical parameters like secondary structure, hydrophilicity, antigenic index and flexibility of the expressed protein were studied . The results of the present investigation collectively form the basis for initial investigations of how the expression of NSE gene is regulated . This is the first report where the recombinant NSE gene has been characterized so extensively. J Chromatogr, 1990 Nov 28, 522, 171 - 7 Use of metal chelate affinity chromatography for removal of zinc ions from alkaline phosphatase from Escherichia coli; Lubinska VK et al.; Alkaline phosphatase from Escherichia coli (APEC) is not retained at 4 degrees C on a metal-free tris(carboxymethyl)ethylenediamine (TED) column, but at 15 degrees C the metalloenzyme becomes bound to the gel . Chromatography of phosphatase on metal-free TED gel indicates a decline in its enzymic activity and zinc content to about 26% and 40%, respectively . The activity of chromatographed APEC can be partially restored by addition of zinc ions, indicating that metal-free TED gel is capable of removing zinc ions from alkaline phosphatase. J Chromatogr, 1990 Nov 28, 522, 163 - 70 Reversed-phase chromatography of Escherichia coli ribosomal proteins . Correlation of retention time with chain length and hydrophobicity; Champney WS; The elution times of 52 bacterial ribosomal proteins from a C4 reversed-phase column have been predicted . The prediction is based upon the use of the hydrophobicity coefficients for the protein amino acid content as defined by Guo et al . {J . Chromatogr., 359 (1986) 499-518} . A strong positive correlation was observed when the difference between predicted and observed protein retention time was plotted against the product of net hydrophobicity and natural log of protein chain length . Observations with this class of related proteins strengthens and extends the observations of Mant et al . {J . Chromatogr., 476 (1989) 363-375} . Observed deviations from predicted chromatographic behavior can be explained for several proteins which elute as dimers or which have modified amino acid residues. Biochemistry, 1990 Nov 27, 29(47), 10590 - 3 Remodeling hexose-1-phosphate uridylyltransferase: mechanism-inspired mutation into a new enzyme, UDP-hexose synthase; Kim J et al.; Hexose-1-phosphate uridylyltransferase catalyzes the interconversion of UDP-galactose and glucose-1-P with UDP-glucose and galactose-1-P by a double-displacement mechanism through a covalent intermediate (E-UMP), in which UMP is bonded to one of two histidine residues at the active site, H164 or H166 . To identify which histidine is the nucleophilic catalyst, we prepared two specific mutants of the enzyme from Escherichia coli, H164G and H166G, in each of which the imidazole ring and methylene carbon of one histidine are deleted . To determine whether the function of the deleted imidazole in these mutants could be carried out by the imidazole ring in uridine 5'-(phosphoimidazolate) (UMP-Im), we examined the mutant proteins for catalytic activity in the reaction of UMP-Im with glucose-1-P to form UDP-glucose and imidazole . The mutant H166G catalyzes this reaction, as well as the reverse reaction, by a sequential kinetic mechanism involving ternary complexes as intermediates . The mutant enzyme also accepts galactose-1-P as a substrate to form UDP-galactose . Hexose-1-P uridylyltransferase does not catalyze these reactions, and H166G does not catalyze the wild-type reaction . The substrate Km values for the mutant enzyme are similar to those for hexose-1-P uridylyltransferase . The value of kcat in the direction of UDP-glucose formation is 1.31 +/- 0.01 s-1, compared with 350 s-1 for hexose-1-P uridylyltransferase, and in the reverse direction kcat is 4.8 +/- 0.4 s-1, compared with 960 s-1 for the wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1990 Nov 27, 29(47), 10659 - 63 The membrane-bound domain of the phosphotransferase enzyme IImtl of Escherichia coli constitutes a mannitol translocating unit; Lolkema JS et al.; The orientation of the mannitol binding site on the Escherichia coli phosphotransferase enzyme IImtl in the unphosphorylated state has been investigated by measuring mannitol binding to cytoplasmic membrane vesicles with a right-side-out and inside-out orientation . Enzyme IImtl is shown to catalyze facilitated diffusion of mannitol at a low rate . At equilibrium, bound mannitol is situated at the periplasmic side of the membrane . The apparent binding constant is 40 nM for the intact membranes . Solubilization of the membranes in detergent decreases the affinity by about a factor of 2 . Inside-out membrane vesicles, treated with trypsin to remove the C-terminal cytoplasmic domain of enzyme IImtl, showed identical activities . These experiments indicate that the translocation of mannitol is catalyzed by the membrane-bound N-terminal half of enzyme IImtl which is a structurally stable domain. Biochemistry, 1990 Nov 27, 29(47), 10695 - 702 Sequence requirements of the hammerhead RNA self-cleavage reaction; Ruffner DE et al.; A previously well-characterized hammerhead catalytic RNA consisting of a 24-nucleotide substrate and a 19-nucleotide ribozyme was used to perform an extensive mutagenesis study . The cleavage rates of 21 different substrate mutations and 24 different ribozyme mutations were determined . Only one of the three phylogenetically conserved base pairs but all nine of the conserved single-stranded residues in the central core are needed for self cleavage . In most cases the mutations did not alter the ability of the hammerhead to assemble into a bimolecular complex . In the few cases where mutant hammerheads did not assemble, it appeared to be the result of the mutation stabilizing an alternate substrate or ribozyme secondary structure . All combinations of mutant substrate and mutant ribozyme were less active than the corresponding single mutations, suggesting that the hammerhead contains few, if any, replaceable tertiary interactions as are found in tRNA . The refined consensus hammerhead resulting from this work was used to identify potential hammerheads present in a variety of Escherichia coli gene sequences. FEBS Lett, 1990 Nov 26, 275(1-2), 91 - 4 Construction and characterization of a recombinant tripartite enzyme, galactose dehydrogenase/beta-galactosidase/galactokinase; Ljungcrantz P et al.; The in-frame gene fusion between 3 enzymes, galactose dehydrogenase, beta-galactosidase and galactokinase, is described . The purified artificial tripartite enzyme displayed all three enzymic activities . Two major forms of the hybrid protein were found, consisting of 4 and 8 subunits respectively, but other forms could also be identified . Each subunit was made up of one monomer each of galactose dehydrogenase, beta-galactosidase and galactokinase . Proximity effects exhibited by the hybrid enzyme could be demonstrated using {14C}galactose as a reporter molecule. Eur J Biochem, 1990 Nov 26, 194(1), 103 - 8 Independent folding of individual components in hybrid proteins . Evidence that the carboxy-terminal 135 residues of the LexA repressor constitute a single autonomous domain; Slilaty SN et al.; Inactivation of the Escherichia coli repressor protein, LexA, takes place through a cleavage reaction which hydrolyzes the Ala84-Gly85 peptide bond near the center of the molecule . The mechanism of cleavage has previously been shown to be an intramolecular reaction stimulated in vitro by elevated pH or by the addition of activated RecA protein . The entire self-cleavage activity of LexA has been found to lie within a 135-residue tryptic fragment extending from Leu68 to the end of the protein at Leu202 . Since the activity of self-cleavage is dependent on the proper three-dimensional structure of the protein, we have used it as a probe to investigate the extend of folding autonomy and functional independence of this 135-residue carboxy-terminal domain of LexA by applying a protein fusion approach . A series of twelve different hybrid proteins, containing LexA sequences in a variety of predefined primary structural arrangements, were constructed and evaluated for whether or not self-cleavage activity has been retained . The results revealed that retention or loss of activity is independent of the nature or size of the foreign protein used . Loss of self-cleavage was found to be a function of amino- or carboxy-terminal deletions in the self-cleaving LexA component of the fusion proteins . The present findings, together with the observations of other artificial fusions proteins and the naturally occurring bifunctional and multifunctional proteins, along with the data on helix packing, provide further support for the notion of modular architecture of proteins and suggest that when these autonomous units are fused, they retain their tendency to fold independently of the remainder of the polypeptide to generate physically linked active domains, rather than to fold dependently and yield scrambled structures. Nucleic Acids Res, 1990 Nov 25, 18(22), 6641 - 7 Statistical analysis of nucleotide sequences; Stuckle EE et al.; In order to scan nucleic acid databases for potentially relevant but as yet unknown signals, we have developed an improved statistical model for pattern analysis of nucleic acid sequences by modifying previous methods based on Markov chains . We demonstrate the importance of selecting the appropriate parameters in order for the method to function at all . The model allows the simultaneous analysis of several short sequences with unequal base frequencies and Markov order k not equal to 0 as is usually the case in databases . As a test of these modifications, we show that in E . coli sequences there is a bias against palindromic hexamers which correspond to known restriction enzyme recognition sites. Nucleic Acids Res, 1990 Nov 25, 18(22), 6537 - 44 Codon recognition in polypeptide chain termination: site directed crosslinking of termination codon to Escherichia coli release factor 2; Tate W et al.; An RNA synthesized in vitro was positioned on the Escherichia coli ribosome at the P site with tRNAala, and with a termination codon, UAA, as the next codon in the A site . Such a complex bound stoichiometric amounts of release factor 2 (RF-2); a corresponding RNA with UAC in place of UAA was not a template for the factor . An RNA containing 4-thio-UAA in place of the UAA supported binding of RF-2, and this has allowed site-directed crosslinking from the first position of the termination codon to answer two long standing questions about the termination of protein biosynthesis, the position of the termination codon and its proximity to the release factor during codon recognition . An RF-2.mRNA crosslinked product was detected, indicating the release factor and the termination codon are in close physical contact during the codon recognition event of termination . The 4-thio-U crosslinked also to the ribosome but only to the 30S subunit, and the proteins and the rRNA site concerned were identified . RF-2 decreased significantly the crosslinking to the ribosomal components, but no new crosslink sites were found . If the stop codon was deliberately displaced from the decoding site by one codon's length then a different pattern of crosslinking in particular to the rRNA resulted . These observations are consistent with a model of codon recognition by RF-2 at the decoding site, without a major shift in position of the codon. Nucleic Acids Res, 1990 Nov 25, 18(22), 6517 - 22 Frameshift autoregulation in the gene for Escherichia coli release factor 2: partly functional mutants result in frameshift enhancement; Donly BC et al.; The regulation of release factor 2 (RF-2) synthesis in Escherichia coli occurs, at least in part, through autoregulatory feedback exerted at a unique frameshifting step required during RF-2 translation . We have constructed fusions between the genes for RF-2 and E . coli trpE which make direct measurement of frameshifting efficiency possible since both products of regulation, the termination product and the frameshift product, are stable . The addition of purified RF-2 to in vitro expressions of these fusion genes was found to result in decreased frameshifting and increased termination at the regulation site . The frame-shifted trpE-RF-2 products synthesized from these fusions are unique with respect to their functional release factor activities; when tested in assays of two intermediate steps of translational termination, they were found to be partially active for the function of ribosome binding, but inactive for peptidyl-tRNA hydrolysis (release) . These are the first examples of release factor mutants selectively active for only one of these function . In vivo these chimeric proteins promote large increases in frameshifting at the RF-2 frameshift region, thereby reversing normal negative autoregulatory feedback and instead supporting fully efficient frameshifting in their own synthesis . This activity provides new evidence for the importance of ribosomal pausing in directing efficient frameshifting at the RF-2 frameshift region. J Biol Chem, 1990 Nov 25, 265(33), 20673 - 7 Cellular processing of the interleukin-2 fusion toxin DAB486-IL-2 and efficient delivery of diphtheria fragment A to the cytosol of target cells requires Arg194; Williams DP et al.; We have used site-directed mutagenesis to examine the role played by Arg191, Arg193, and Arg194 of the fusion toxin DAB486-IL-2 in the intoxication of high affinity interleukin-2 receptor-bearing T-lymphocytes . These arginine residues are positioned in the proteolytically sensitive 14-amino acid loop subtended by the disulfide bond between Cys187 and Cys202 in this fusion toxin . DAB486-IL-2 was formed by the genetic substitution of the native diphtheria toxin receptor binding domain with human interleukin-2 (Williams, D.P., Parker, K., Bacha, P., Bishai, W., Borowski, M., Genbauffe, F., Strom, T.B., and Murphy, J.R . (1987) Protein Eng . 1, 493-498) . We demonstrate that substitution of Arg194 with Gly results in a 1000-fold loss of DAB486-IL-2 potency . Since trypsin "nicking" of the Gly194 mutant restores biologic activity, we conclude that Arg194 is required for the cellular processing of the fusion toxin which results in the release of fragment A into the cytosol. J Biol Chem, 1990 Nov 25, 265(33), 20609 - 15 Expression of wild-type and mutated forms of the catalytic (alpha) subunit of Caenorhabditis elegans casein kinase II in Escherichia coli; Hu E et al.; A full-length Caenorhabditis elegans cDNA that encodes the alpha subunit of casein kinase II was inserted into the inducible bacterial expression vector pET3a to generate the plasmid pCK alpha . Escherichia coli DE21 lysozyme S that was transformed with pCK alpha expressed soluble, catalytically active casein kinase II alpha upon induction with isopropyl beta-D-thiogalactopyranoside . The expressed alpha subunit was purified to homogeneity with a 60% yield by chromatography on CM-Sephadex, P-11 phosphocellulose, and heparin-agarose . The Mr values estimated from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr = 42,000) or calculated from hydrodynamic measurements (s20,w = 3.3 S, Stokes radius = 2.8 nm, Mr = 37,000) were similar, thereby indicating that the expressed enzyme is monomeric . The native holoenzyme and the expressed alpha subunit exhibited several similar properties including the utilization of both ATP and GTP as substrates and the susceptibility to inhibition of phosphotransferase activity by low concentrations of heparin . However, the kcat for E . coli-derived alpha was only 9% of the kcat for the native holoenzyme, and catalytic activity was not stimulated by polyamines . Recombinant casein kinase II alpha aggregates at low ionic strength, and the aggregation is partially reversible . A mutant alpha subunit in which Lys74 and Lys75 were substituted by glutamic acid residues was constructed by site-directed mutagenesis . The mutant enzyme was not inhibited by typically effective concentrations of heparin (e.g . IC50 = 0.3 micrograms/ml) because the affinity of modified recombinant casein kinase II Glu-74Glu-75 for heparin decreased approximately 70-fold . Thus, Lys74 and Lys75 are implicated in the heparin binding, inhibitory domain . The successful expression of casein kinase II alpha in E . coli will facilitate the analysis of the structural basis for functional domains in this enzyme. J Biol Chem, 1990 Nov 25, 265(33), 20421 - 9 Disruption of active site interactions with pyridoxal 5'-phosphate and substrates by conservative replacements in the glycine-rich loop of Escherichia coli D-serine dehydratase; Marceau M et al.; We have used site-directed mutagenesis to examine the function of three putative active site residues (C278, G279, and G281) of the vitamin B6 enzyme D-serine dehydratase . These residues lie in or adjacent to a conserved glycine-rich loop that is known to interact with the pyridoxal 5'-phosphate cofactor in several B6 enzymes and that resembles the GXGXXG loop of nucleotide-binding sites . The cofactor affinity, catalytic properties, and spectral properties (UV, CD, fluorescence, and 31P NMR) of alanine variants C278A, G279A, and G281A were measured as well as the susceptibility of each variant to thiol modification by 5,5'-dithiobis(2-nitrobenzoic acid) . The specific thiols modified in each variant and wild type D-serine dehydratase were identified by amino acid sequencing of labeled tryptic peptides . C278A, G279A, and G281A displayed 10-, 33-, and 22-fold lower affinities for pyridoxal 5'-phosphate than did wild type D-serine dehydratase and turnover numbers with D-serine that were 50, 6, and 60% of normal, respectively . The introduction of a methyl side chain into G281 enhanced catalytic efficiency with the substrates D-threonine, D-allo-threonine, and L-serine, whereas the methyl side chain at position 279 impaired catalysis of all substrates as well as cofactor affinity . The 31P NMR spectrum of D-serine dehydratase was minimally perturbed by the alanine substitutions, consistent with the view that neither G279 nor G281 interacts with the phosphate group of the cofactor (in contrast to the arrangement found in several other B6 enzymes) . C311 was the single thiol modified by 5,5'-dithiobis(2-nitrobenzoic acid) in wild type D-serine dehydratase . Two normally inaccessible thiol groups, C233 and C278, were rendered susceptible to modification as a consequence of either G----A substitution, and modification of C278 was associated with inactivation of G279A and G281A . These observations suggest that small perturbations in the glycine-rich loop induce conformational changes spanning a considerable area around the active site. J Biol Chem, 1990 Nov 25, 265(33), 20085 - 6 Isolation of subtilisin pro-sequence mutations that affect formation of active protease by localized random polymerase chain reaction mutagenesis; Lerner CG et al.; In order to analyze the role of the pro-sequence in folding of the alkaline serine protease subtilisin, localized random mutagenesis using the polymerase chain reaction with Taq DNA polymerase was employed to obtain mutations in the pro-sequence which prevent production of active protease . The unique aspect of this procedure is that random mutations can be easily generated in vitro over large but defined regions of a specific gene . The method was applied to a 458-base pair fragment encompassing the coding region of the pro-sequence of subtilisin, a region of the protein which has been shown to be required for proper folding . Protease-deficient mutants containing a variety of amino acid substitutions were isolated with a frequency of 4.3% . From analysis of these mutants, four independent amino acid substitution mutations in the pro-sequence were identified . The present results demonstrate that polymerase chain reaction is an efficient and simple method for obtaining random mutations within a localized region of a given gene. J Biol Chem, 1990 Nov 25, 265(33), 20069 - 72 Minimum substrate sequence for signal peptidase I of Escherichia coli; Dev IK et al.; The minimum substrate sequence recognized by signal peptidase I (SPase I or leader peptidase) was defined by measuring the kinetic parameters for a set of chemically synthesized peptides corresponding to the cleavage site of the precursor maltose binding protein (pro-MBP) . The minimum sequence of a substrate hydrolyzed by SPase I at a measurable rate was the pentapeptide Ala-Leu-Ala decreases Lys-Ile . The rates of hydrolysis of this substrate, however, were several hundred-fold lower than those observed for the maturation of MBP in Escherichia coli, suggesting that in addition to these minimal sites involved in recognition, other features of pro-MBP are also needed for the optimal rate of signal peptide cleavage by SPase I . One parameter may be the length of the polypeptide chain . Studies of the synthetic peptides showed that decreasing the length of the polypeptide chain of substrates decreased the substrate efficiency measured as kcat/Km . However, in one case a decrease in the length of a peptide corresponding to -7 to +3 positions of pro-MBP to a nonapeptide (-7 to +2) increased the substrate efficiency by about 900-fold . The nonapeptide is the most efficient substrate for the enzyme in vitro so far reported . It is speculated that better peptide substrates are the ones which are able to adopt folded structures. Nucleic Acids Res, 1990 Nov 25, 18(22), 6659 - 63 Purification and characterization of a DNA polymerase from the cyanobacterium Anacystis nidulans R2; Lin HJ et al.; A DNA polymerase has been highly purified from Anacystis nidulans R2 . Electrophoretic analysis in sodium dodecyl sulfate-polyacrylamide gels revealed that the final fraction contains three bands of Mr 107,000, 93,000, and 51,000, respectively . Analysis of purified DNA polymerase activity in situ indicates that of the three polypeptides the Mr 107,000 species has the catalytic activities . The native molecular weight of the enzyme was estimated by glycerol gradient sedimentation to be 100,000 . The enzyme has an absolute requirement for a divalent cation . Mg2+ can be replaced with Mn2+, but the DNA polymerase is less active . Potassium chloride stimulates the enzyme, while potassium phosphate has no apparent effect . The enzyme is active over a pH range from 7.5 to 9.5 in 50mM Tris-HCl buffer . The ability of the cyanobacterial DNA polymerase to use activated DNA as a template, its associated 3'----5' and 5'----3' exonuclease activities, as well as its resistance to N-ethylmaleimide, dideoxynucleotides, arabinosyl-CTP and aphidicolin suggest a similarity between this enzyme and E . coli DNA polymerase I . This is the first characterization of a DNA polymerase from a cyanobacterium. J Biol Chem, 1990 Nov 25, 265(33), 20384 - 9 Active-site residues of 2-keto-4-hydroxyglutarate aldolase from Escherichia coli . Bromopyruvate inactivation and labeling of glutamate 45; Vlahos CJ et al.; Treatment of pure 2-keto-4-hydroxyglutarate aldolase from Escherichia coli, a "lysine-type," Schiff-base mechanism enzyme, with the substrate analog bromopyruvate results in a time- and concentration-dependent loss of enzymatic activity . Whereas the substrates pyruvate and 2-keto-4-hydroxyglutarate provide greater than 90% protection against inactivation by bromopyruvate, no protective effect is seen with glycolaldehyde, an analog of glyoxylate . Inactivation studies with {14C} bromopyruvate show the incorporation of 1.1 mol of 14C-labeled compound/enzyme subunit; isolation of a radioactive peptide and determination of its amino acid sequence indicate that the radioactivity is associated with glutamate 45 . Incubation of the enzyme with excess {14C}bromopyruvate followed by denaturation with guanidine.HCl allow for the incorporation of carbon-14 at cysteines 159 and 180 as well . Whereas the presence of pyruvate protects Glu-45 from being esterified, it does not prevent the alkylation of these 2 cysteine residues . The results indicate that Glu-45 of E . coli 2-keto-4-hydroxyglutarate aldolase is essential for catalytic activity, most likely acting as the amphoteric proton donor/acceptor that is required as a participant in the overall mechanism of the reaction catalyzed. J Biol Chem, 1990 Nov 25, 265(33), 20293 - 301 Structure-function studies of the SERPIN plasminogen activator inhibitor type 1 . Analysis of chimeric strained loop mutants; Lawrence DA et al.; Three chimeric mutants of plasminogen activator inhibitor 1 (PAI-1) have been constructed where the strained loop of wild type PAI-1 (wtPAI-1) has been replaced with a 19-amino acid region from either plasminogen activator inhibitor 2 (PAI-2), antithrombin III, or with an artificial serine protease inhibitor superfamily consensus strained loop . The inhibitors were expressed in Escherichia coli, and the purified proteins had specific activities toward urokinase-type plasminogen activator (uPA) or the single- and two-chain forms of tissue type plasminogen activator (tPA) that were similar to wtPAI-1 . Experiments suggest that the strained loop of PAI-1 is not responsible for the transition between the latent and the active conformations or for binding to vitronectin . Second-order rate constants for the interactions with uPA and single- or two-chain tPA were similar to those of wtPAI-1 . Values range from a low of 1.8 x 10(5) M-1 s-1 for the interaction of the PAI-2 chimera with single-chain tPA to a high value of 1.6 x 10(7) M-1 s-1 for the consensus mutant with two-chain tPA . This former value is 200 times higher than the reported rate constant for the interaction between PAI-2 and single-chain tPA, suggesting that structures outside of the strained loop are responsible for the major differences in specificity between PAI-1 and PAI-2. J Chromatogr, 1990 Nov 23, 521(2), 267 - 77 Purification of a recombinantly produced transmembrane protein (gp41) of HIV I; Soutschek E et al.; The transmembrane protein gp41, a component of the viral envelope of HIV I, and its analogue gp36 of HIV II are important antigens for the sensitive and specific detection of anti-HIV antibodies . The immunodominant region of the protein gp41, which reacts with 100% of sera of infected persons, was produced by gene technological means in Escherichia coli . The protein accumulates in the form of insoluble inclusion bodies in the bacterial cell . Purification strategies for this aggregated material depend mainly on the isolation of these "inclusion bodies" and subsequent washing procedures . Growth conditions of the recombinant E . coli cells and the method of the cell disruption are important for the efficiency of purification and the recovery of the antigen . Owing to the insolubility of the expressed antigen, a significant concentration of recombinant gp41 was possible by extracting the soluble cell components . For this purpose, mild detergent solutions and low-molarity chaotropic buffer solutions were used . After final solubilization in 8 M urea buffer at pH 12.5, further chromatographic purification steps followed . The reduction of disulphide bridges with beta-mercaptoethanol or dithiothreitol was important . Gel filtration on a Sephacryl S-200 or Superose 12 column and/or ion-exchange chromatography on a DEAE-Sepharose Fast Flow or Mono Q HR (5/5) column finally resulted in the desired purity of the antigen. Nature, 1990 Nov 22, 348(6299), 339 - 42 (Mg-ATP)-dependent self-assembly of molecular chaperone GroEL; Lissin NM et al.; The important Escherichia coli heat-shock protein GroEL of relative molecular mass 57,259 is a typical molecular chaperone . It possesses ATPase activity and interacts in ATP-driven reactions with non-folded proteins to stimulate their correct folding and/or assembly by preventing the formation of improper protein structures or aggregates . As GroEL is isolated and functions as a 20-25S tetradecameric particle (GroELp), the question arises--what is the mechanism of its own assembly? Here we show the (Mg-ATP)-dependent self-stimulation ('self-chaperoning') in vitro of GroELp reassembly from its monomeric state. Biochemistry, 1990 Nov 20, 29(46), 10455 - 60 Formation and stability of repairable pyrimidine photohydrates in DNA; Boorstein RJ et al.; Ultraviolet irradiation of poly(dG-dC) and poly(dA-dU) in solution produces pyrimidine hydrates that are repaired by bacterial and mammalian DNA glycosylases {Boorstein et al . (1989) Biochemistry 28, 6164-6170} . Escherichia coli endonuclease III was used to quantitate the formation and stability of these hydrates in the double-stranded alternating copolymers poly(dG-dC) and poly(dA-dU) . When poly(dG-dC) was irradiated with 100 kJ/m2 of 254-nm light at pH 8.0, 2.2% of the cytosine residues were converted to cytosine hydrate (6-hydroxy-5,6-dihydrocytosine) while 0.09% were converted to uracil hydrate (6-hydroxy-5,6-dihydrouracil) . To measure the stability of these products, poly(dG-dC) was incubated in solution for up to 24 h after UV irradiation . Cytosine hydrate was stable at 4 degrees C and decayed at 25, 37, and 55 degrees C with half-lives of 75, 25, and 6 h . Uracil hydrate produced in irradiated poly(dA-dU) was stable at 4 degrees C and at 25 degrees C and decayed with a half-life of 6 h at 37 degrees C and less than 0.5 h at 55 degrees C . Uracil hydrate and uracil were also formed in irradiated poly(dG-dC) . These experiments demonstrate that UV-induced cytosine hydrate may persist in DNA for prolonged time periods and also undergo deamination to uracil hydrate, which in turn undergoes dehydration to yield uracil . The formation and stability of these photoproducts in DNA may have promoted the evolutionary development of the repair enzyme endonuclease III and analogous DNA glycosylase/endonuclease activities of higher organisms, as well as the development of uracil-DNA glycosylase. J Mol Biol, 1990 Nov 20, 216(2), 411 - 24 Crystallographic study at 2.5 A resolution of the interaction of methionyl-tRNA synthetase from Escherichia coli with ATP; Brunie S et al.; The crystal structure of the tryptic fragment of the methionyl-tRNA synthetase from Escherichia coli, complexed with ATP, has been refined to a crystallographic R-factor of 0.220, at 2.5 A resolution (for 4433 protein atoms) . In the last stages of the refinement, the simulated annealing refinement method was fully applied, contributing to a drastic improvement of the model and the identification of the missing atoms . In the final model, the root-mean-square deviation from ideality for bond distances is 0.021 A and for angle distances is 0.054 A . The position of the zinc ion has been confirmed and is located near the active site . The tryptic fragment is composed of two globular domains . The first domain, from the N terminus to Thr360, contains a nucleotide-binding fold into which two long polypeptides of 101 and 70 residues are inserted . The nucleotide-binding fold is strengthened by the presence of the zinc ion in the vicinity of the active site . The second domain, up to Pro526, is mainly alpha-helical . The C-terminal polypeptide, Phe527 to Lys551, folds back towards the first domain, making a link between the two domains . The heptapeptide 528-534 partly shapes a deep cavity that plunges into the central core of the nucleotide-binding fold, where the ATP molecule is located . The adenine ring, deeply buried in the bottom of the cleft, is blocked between the first helix HA, and the strands A and D of the beta-sheet and makes no polar interaction with the enzyme . The 2' and 3' hydroxyl groups of the ribose, whose conformation is C2' endo, interact with the main-chain carbonyl oxygen atoms of Ile231 and Glu241, respectively . The side-chain nitrogen atom of Lys142 is at hydrogen-bonding distance from the ring oxygen O-4' of the ribose . One of the alpha-phosphate oxygen atoms and one of the gamma-phosphate oxygen atoms interact with the imidazole ring of His21, which is well conserved in many of the known synthetases; this indicates a possible crucial role for this residue in binding ATP . The beta-phosphate group is linked to the main-chain carbonyl oxygen atom of Tyr15 through an intermediate water molecule . The gamma-phosphate group interacts with the carbonyl oxygen atom and the side-chain of Asn17.(ABSTRACT TRUNCATED AT 400 WORDS) J Mol Biol, 1990 Nov 20, 216(2), 375 - 84 Co-operative interactions between the catalytic sites in Escherichia coli aspartate transcarbamylase . Role of the C-terminal region of the regulatory chains; Xi XG et al.; In aspartate transcarbamylase (ATCase) each regulatory chain interacts with two catalytic chains each one belonging to a different trimeric catalytic subunit (R1-C1 and R1-C4 types of interactions as defined in Fig . 1) . In order to investigate the interchain contacts that are involved in the co-operative interactions between the catalytic sites, a series of modified forms of the enzyme was prepared by site-directed mutagenesis . The amino acid replacements were devised on the basis of the previously described properties of an altered form of ATCase (pAR5-ATCase) which lacks the homotropic co-operative interactions between the catalytic sites . The results obtained (enzyme kinetics, bisubstrate analog influence and pH studies) show that the R1-C4 interaction is essential for the establishment of the enzyme conformation that has a low affinity for aspartate (T state), and consequently for the existence of co-operativity between the catalytic sites . This interaction involves the 236-250 region of the aspartate binding domain of the catalytic chain (240s loop) and the 143-149 region of the regulatory chain which comprises helix H3'. J Mol Biol, 1990 Nov 20, 216(2), 353 - 62 Structural study of the yeast RNA polymerase A . Electron microscopy of lipid-bound molecules and two-dimensional crystals; Schultz P et al.; Two-dimensional crystals of yeast RNA polymerase A (I) were obtained by interaction with positively charged lipid layers . The analysis of single molecular images of lipid-bound RNA polymerases showed that the enzyme was preferentially oriented by the lipid phase, which probably facilitated crystallization . Electron micrographs of the crystals revealed a rectangular unit cell 25.8 nm by 45.6 nm in size containing four RNA polymerase dimers related by P22(1)2(1) symmetry . The projection map showed, at about 2.5 nm resolution, two different views of the enzyme characterized by two bent arms, which appeared to cross at one end . These arms are likely to contain the A190 and A135 subunits and delimit a 3 to 4 nm wide groove . Additional structural features were observed and compared to the Escherichia coli enzyme. J Mol Biol, 1990 Nov 20, 216(2), 299 - 310 Escherichia coli threonyl-tRNA synthetase and tRNA(Thr) modulate the binding of the ribosome to the translational initiation site of the thrS mRNA; Moine H et al.; Escherichia coli threonyl-tRNA synthetase binds to the leader region of its own mRNA at two major sites: the first shares some analogy with the anticodon arm of several tRNA(Thr) isoacceptors and the second corresponds to a stable stem-loop structure upstream from the first one . The binding of the enzyme to its mRNA target site represses its translation by preventing the ribosome from binding to its attachment site . The enzyme is still able to bind to derepressed mRNA mutants resulting from single substitutions in the anticodon-like arm . This binding is restricted to the stem-loop structure of the second site . However, the interaction of the enzyme with this site fails to occlude ribosome binding . tRNA(Thr) is able to displace the wild-type mRNA from the enzyme at both sites and suppresses the inhibitory effect of the synthetase on the formation of the translational initiation complex . Our results show that tRNA(Thr) acts as an antirepressor on the synthesis of its cognate aminoacyl-tRNA synthetase . This repression/derepression double control allows precise adjustment of the rate of synthesis of threonyl-tRNA synthetase to the tRNA level in the cell. J Mol Biol, 1990 Nov 20, 216(2), 289 - 98 FLP protein of 2 mu circle plasmid of yeast induces multiple bends in the FLP recognition target site; Schwartz CJ et al.; The FLP recombinase of the 2 mu plasmid of Saccharomyces cerevisiae binds to a target containing three 13 base-pair symmetry elements called a, b and c . The symmetry elements b and c are in direct orientation while the a element is in inverted orientation with respect to b and c on the opposite side of an eight base-pair core region . Each symmetry element acts as a binding site for the FLP protein . The FLP protein can form three different complexes with the FLP recognition target (FRT site) according to the number of elements within the site that are occupied by the FLP protein . Binding of FLP to the FRT site induces DNA bending . We have measured the angles of bends caused by the binding of the FLP protein to full and partial FRT sites . We find that FLP induces three types of bend in the FRT-containing DNA . The type I bend is approximately 60 degrees and results from a molecule of FLP bound to one symmetry element . The type II bend is greater than 144 degrees and results from FLP molecules bound to symmetry elements a and b . The type III bend is approximately 65 degrees and results from FLP proteins bound to symmetry elements b and c . Certain FLP proteins that are defective in recombination can generate the type I and type III bends but are impaired in their ability to induce the type II bend . We discuss the role of bending in FLP-mediated recombination. J Mol Biol, 1990 Nov 20, 216(2), 275 - 87 Specific sequences downstream from -6 are not essential for proper and efficient in vitro utilization of the Escherichia coli lactose promoter; Lorimer DD et al.; A series of deletion mutants of the wild-type Escherichia coli lactose promoter, with endpoints at +25, +19, +14, +1 and -6 (relative to the start of transcription at +1), was constructed and the deleted DNA replaced with non-lac DNA . These mutants were used to show that no specific DNA sequences downstream from -6 are required for efficient promoter utilization in vitro . In all cases transcription is dependent on the presence of the catabolite activator protein (CAP) and cAMP, and begins at +1 at a level indistinguishable from that at the wild-type promoter . A set of lac DNA fragments deleted to -6 was constructed, having an A, C, G or T residue at +1 and heterologous DNA downstream . These synthetic promoters allow systematic testing of the effect of the initiating nucleotide on the transcription process . Again, transcription occurs mainly from +1, at a level similar to the normal wild-type level . No substantial differences between these promoters are observed in the rates of formation of stable complexes, in the degree of complex formation, in the rate at which polymerase "escapes" from the complex or in abortive transcription products . Equivalent results are seen with a related set of constructs based on the CAP-insensitive lac UV5 promoter . Thus, lac promoter sequences including consensus hexamers at -10 and -35, plus the spacer region between them, provide specificity and efficiency both in initiation of transcription by RNA polymerase and in CAP-polymerase interactions . A question as to whether there is a third RNA polymerase binding site at lac, in addition to the known overlapping P1 and P2 regions, was not unambiguously answered . However, if a "P3" site does exist, it must lie between P1 and P2 . Alternatively, the variety of polymerase interactions at wild-type lac may reflect different structural states of the enzyme . The results presented here indicate that DNA downstream from -6 plays little part in determining the conformation of the enzyme at the lactose promoter. J Mol Biol, 1990 Nov 20, 216(2), 261 - 73 Positive and negative regulation of transcription by a cleavage product of Ada protein; Akimaru H et al.; The 39,000 Mr Ada protein of Escherichia coli that carries two distinct methyltransferase activities and activity to promote transcription of the ada and the alkA genes is cleaved by a cellular proteinase . As a result, the 20,000 and the 19,000 Mr proteins are formed, which are derived from the N-terminal and the C-terminal halves of the protein, respectively . To elucidate the molecular mechanism of transcriptional control by Ada protein, the N-terminal 20,000 Mr protein was overproduced by manipulating the cloned ada gene . The protein possessed an activity to transfer a methyl group from the methylphosphotriester of the alkylated DNA to its own molecule and retained the potential to promote transcription of the alkA gene . The methylated form of the 20,000 Mr proteins binds to the proper alkA regulatory sequence, as does the intact Ada protein, and facilitates further binding of RNA polymerase to the promoter, thus forming an active transcription initiation complex . The non-methylated 20,000 Mr protein was incapable of binding itself or supporting RNA polymerase binding to the alkA promoter . When the 20,000 Mr protein was produced under the control of the lac promoter in E . coli and then exposed to a methylating agent, a considerable amount of 3-methyladenine-DNA glycosylase II, the product of the alkA gene, was formed . Thus, the results obtained in in vitro experiments were confirmed by the events observed in vivo . The methylated 20,000 Mr protein also binds to the ada promoter; however, it does not facilitate further binding of RNA polymerase to the promoter nor does it promote ada transcription in vitro . These findings indicate that the N-terminal half of Ada protein is mainly responsible for recognition of and binding to alkA and the ada regulatory sequence . The methylated 20,000 Mr protein occupies the same region of the ada promoter to which the intact Ada protein would bind, thereby suggesting that it acts as a repressor for expression of the ada gene . The ada transcription promoted by the Ada protein was greatly inhibited by the methylated, but not the non-methylated, form of the 20,000 Mr protein . In an in vivo system, formation of the 20,000 Mr protein leads to inhibition of transcription from the ada promoter . We suggest that termination of the adaptive response may come about by proteolytic cleavage of the Ada protein, the result being a loss of the activator as well as formation of the repressor for ada transcription. J Mol Biol, 1990 Nov 20, 216(2), 207 - 11 Purification and secondary structure determination of simian immunodeficiency virus p27; Burns NR et al.; We have developed a novel method for the expression and purification of p27, the major core protein of simian immunodeficiency virus . Circular dichroism measurements of purified p27 were used to determine the relative amounts of alpha-helix, beta-sheet and unordered secondary structural elements . These empirically determined values appear to be inconsistent with previously published theoretical models based on homology comparisons. J Mol Biol, 1990 Nov 20, 216(2), 195 - 9 Supercondensed structure of plasmid pBR322 DNA in an Escherichia coli DNA topoisomerase II mutant; Huang XT et al.; An unusual structural component, supercondensed pBR322 DNA, has been found in plasmid pBR322 DNA samples isolated from a DNA topoisomerase II mutant of Escherichia coli, SD108 (topA+, gyrB225) . The supercondensed pBR322 DNA moved faster than supercoiled pBR322 DNA as a homogeneous band in agrose gels when the DNA samples were analysed by electrophoresis . The mobility of the supercondensed DNA was not substantially affected by chloroquine intercalation . The supercondensed pBR322 DNA migrated as a high density "third DNA band" when the samples were subjected to caesium chloride/ethidium bromide gradient equilibrium centrifugation . The unusual pBR322 DNA visualized by electron microscopy was a globoid-shaped particle . These observations suggest that the pBR322 plasmid can assume a tertiary structure other than a supercoiled or relaxed structure . DNA topoisomerases may be involved in the supercondensation of plasmid DNA and chromosomal DNA. J Mol Biol, 1990 Nov 20, 216(2), 335 - 52 Energetics of RecA-mediated recombination reactions . Without ATP hydrolysis RecA can mediate polar strand exchange but is unable to recycle; Rosselli W et al.; We demonstrate that the step of DNA strand exchange during RecA-mediated recombination reaction can occur equally efficiently in the presence or absence of ATP hydrolysis . The polarity of strand exchange is the same when instead of ATP its non-hydrolyzable analog adenosine-5'-O-(3-thiotriphosphate) is used . We show that the ATP dependence of recombination reaction is limited to the post-exchange stages of the reactions . The low DNA affinity state of RecA protomers, induced after ATP hydrolysis, is necessary for the dissociation of RecA-DNA complexes at the end of the reaction . This dissociation of RecA from DNA is necessary for the release of recombinant DNA molecules from the complexes formed with RecA and for the recycling of RecA protomers for another round of the recombination reaction. J Mol Biol, 1990 Nov 20, 216(2), 189 - 94 Expression of a ciliate gene in Escherichia coli using a suppressor tRNA to read the UAA and UAG glutamine codons; Cohen J et al.; Most ciliates use a particular genetic code where the standard stop codons UAA and UAG encode glutamine . Ciliate genes cannot therefore be expressed in heterologous systems such as Escherichia coli . To overcome this problem, we worked out a system of inducible suppression to permit efficient readthrough of UAAs and UAGs: a strong UAA tRNA suppressor that inserts glutamic acid was cloned downstream from a tac promoter whose efficiency was reduced by a transcription terminator . This system proved to be operational (1) to suppress UAG mutations by wobble pairing in an E . coli lacI-lacZ gene fusion and (2) to read through at least eight UAA glutamine codons in a Paramecium alpha-tubulin gene, as detected by Western blotting and colony hybridization . This work opens the way for cloning Ciliate genes from expression libraries and for expressing particular sequences without extended in vitro mutagenesis . A similar approach can be envisaged for expression of genes from Mycoplasma, mitochondria or other genomes that use non-standard genetic codes. Cell, 1990 Nov 16, 63(4), 687 - 95 The hepatitis B virus-encoded transcriptional trans-activator hbx appears to be a novel protein serine/threonine kinase; Wu JY et al.; To study the functional mechanism of the hepatitis B virus (HBV) X (hbx) gene product, we have expressed the hbx protein in E . coli and purified it by HPLC . The purified hbx protein was shown to be active in transactivating transcription directed by the LTR sequence of HIV-1 . The hbx protein was found to have an intrinsic serine/threonine protein kinase activity . The hbx protein was detected in hepatitis B virions, and tryptic phosphopeptide maps of the hbx protein phosphorylated in the virion and of the in vitro phosphorylated bacterially expressed hbx protein were similar . Inactivation of the hbx protein by heat, protein-denaturing agents, or an ATP affinity analog, p-fluorosulfonylbenzoyl 5'-adenosine, resulted in loss of both protein kinase activity and trans-activation activity . These results suggest that the HBV-encoded trans-activator hbx is a novel protein kinase. Cell, 1990 Nov 16, 63(4), 773 - 81 Mapping of a higher order protein-DNA complex: two kinds of long-range interactions in lambda attL; Kim S et al.; To map the protein-protein and protein-DNA interactions involved in lambda site-specific recombination, Int cleavage assays with suicide substrates, nuclease protection patterns, gel retardation experiments, and quantitative Western blotting were applied to wild-type attL and attL mutants . The results lead to a model in which one IHF molecule bends the attL DNA and forms a higher order complex with the three bivalent Int molecules required for excisive recombination . It is proposed that each of the Int molecules binds in a unique manner: one bridges two DNA binding sites in cis, one is held via its high affinity amino-terminal DNA binding domain, and the third depends upon protein-protein interactions in addition to its low affinity carboxy-terminal DNA binding domain . This protein-DNA complex contains two unsatisfied DNA binding domains, each with a different sequence specificity, and is well suited to specific interactions with an appropriate recombination partner. Cell, 1990 Nov 16, 63(4), 815 - 25 Control of c-Jun activity by interaction of a cell-specific inhibitor with regulatory domain delta: differences between v- and c-Jun; Baichwal VR et al.; Analysis of transcriptional activation properties of c-Jun chimeras in different cell lines suggests that it contains an activator domain (A1) that is negatively regulated by a cell type-specific inhibitor . A regulatory domain of c-Jun, delta, previously identified by in vitro experiments, also regulates transcriptional activation by c-Jun in vivo . The delta domain facilitates or stabilizes the interaction of the cellular inhibitor with A1 . v-Jun, which lacks delta, is a stronger transcriptional activator than c-Jun, since its activity is not efficiently repressed by the cellular inhibitor . In vitro transcription with chimeric Jun proteins and extracts from different cell types confirms that the A1 and delta domains are repressed in a cell type-specific manner . These findings implicate a specific cellular factor in the negative regulation of c-Jun activity and suggest a molecular basis for the observed difference in transcriptional properties between v-Jun and c-Jun. J Biol Chem, 1990 Nov 5, 265(31), 19091 - 9 Characterization of transcriptional initiation from promoters P1 and P2 of the pyrBI operon of Escherichia coli K12; Donahue JP et al.; Expression of the pyrBI operon of Escherichia coli K12, which encodes the pyrimidine biosynthetic enzyme aspartate transcarbamylase, is negatively regulated by pyrimidine availability, primarily through an attenuation control mechanism, and also appears to be subject to stringent control . Previous in vitro transcription studies indicated that the pyrBI operon is transcribed from tandem promoters designated P1 and P2, which appeared to be of similar strength . In this study, we characterized these promoters in detail and examined their role in pyrBI expression . Our results show that although transcription is initiated at both promoters in vivo, greater than 99% of the pyrBI transcripts are initiated at promoter P2, indicating that this is the only physiologically significant promoter . The level of transcripts initiated at promoter P2 was found to be higher in cells grown under pyrimidine-limiting conditions compared to that in cells grown under conditions of pyrimidine excess, indicating pyrimidine-mediated regulation at the level of transcriptional initiation . In vitro characterization of transcription from the pyrBI promoter region showed that non-physiological reaction conditions used in the original identification of the two promoters greatly overestimated the strength of promoter P1 . Further in vitro characterization of the two promoters showed that transcription from promoter P2, but not from promoter P1, is inhibited by guanosine tetraphosphate and exhibits salt and heparin sensitivity typical of a stringently controlled promoter . In addition, heparin-challenge experiments revealed a UTP-induced instability of transcriptional initiation complexes at promoter P2 which may be of regulatory significance. J Biol Chem, 1990 Nov 15, 265(32), 19990 - 5 The tryptophan cluster: a hypothetical structure of the DNA-binding domain of the myb protooncogene product; Kanei-Ishii C et al.; In the DNA-binding domain of the c-myb protooncogene product (c-Myb) which consists of three repeats of 51-52 amino acids, there are 3 perfectly conserved tryptophans in each repeat . Site-directed mutagenesis of these tryptophans showed that any single or multiple mutations of tryptophan to hydrophilic residues or alanine abolished or greatly reduced the sequence-specific DNA-binding activity, but mutations to hydrophobic amino acids retained considerable activity . Raman spectroscopic study showed that these tryptophans were buried in the protein core . These 3 tryptophans are proposed to form a cluster in the hydrophobic core in each repeat . This hypothetical structure is referred to as the "tryptophan cluster," and it may represent a characteristic property of a group of DNA-binding proteins including the myb- and ets-related proteins. J Biol Chem, 1990 Nov 15, 265(32), 19582 - 7 The absence of a m7G cap on beta-globin mRNA and alfalfa mosaic virus RNA 4 increases the amounts of initiation factor 4F required for translation; Fletcher L et al.; beta-Globin mRNA and alfalfa mosaic virus (AMV) RNA 4, two naturally capped mRNAs, and satellite tobacco necrosis virus (STNV) RNA, a naturally uncapped mRNA, were prepared by in vitro transcription with and without a 5' m7G cap structure (m7G(5')ppp(5')N) . The translation of the capped and uncapped forms of these mRNAs was measured in a crude S30 system and a partially purified system from wheat germ . In the S30 system the uncapped forms of beta-globin mRNA and AMV RNA 4 are much less active (greater than or equal to 10%) than their capped forms, whereas the uncapped and capped forms of STNV RNA are equally active . The low activity of uncapped beta-globin mRNA and AMV RNA 4 in the S30 system is due, in part, to inactivation of the uncapped mRNAs in this system . Additional studies, carried out in the partially purified system in which very little inactivation of the mRNAs occurs, show that the uncapped and capped forms of beta-globin mRNA or AMV RNA 4 differ markedly with respect to the amount of eukaryotic initiation factor (eIF)-4F required for translation . For beta-globin mRNA the absence of the 5' cap structure increases the concentration of eIF-4F required for half-maximal translation about 6-fold (from 10 to 60 nM) and for AMV RNA 4 it increases the concentration of eIF-4F about 12-fold (from 5 to 60 nM) . The concentrations of eIF-3, eIF-4A, and eIF-4B required for half-maximal translation of the uncapped forms of beta-globin mRNA and AMV RNA 4 are either the same or only slightly higher (1.5- to 2-fold) than the concentrations required for the capped forms . With STNV RNA the concentration of eIF-4F required for half-maximal translation of either uncapped or capped STNV RNA is 3 nM, and the concentrations of eIF-3, eIF-4A, and eIF-4B required for the two forms are also the same . The translation of the capped and uncapped forms of beta-globin mRNA and AMV RNA 4 is inhibited strongly by low concentrations of m7GTP in the partially purified system containing low concentrations of eIF-4F . Under the same conditions, the translation of capped or uncapped STNV RNA is inhibited only slightly by m7GTP . These findings suggest the possibility that the mechanism by which eIF-4F interacts and initiates translation with naturally uncapped mRNAs may not be identical to the mechanism by which eIF-4F interacts and initiates translation of naturally capped mRNAs. J Biol Chem, 1990 Nov 15, 265(32), 19560 - 7 Electron transfer from menaquinol to fumarate . Fumarate reductase anchor polypeptide mutants of Escherichia coli; Westenberg DJ et al.; Fumarate reductase (FRD) of Escherichia coli is a four-subunit membrane-bound complex that is synthesized during anaerobic growth when fumarate is available as a terminal oxidant . The two subunits that comprise the catalytic domain, FrdA and FrdB, are anchored to the cytoplasmic membrane surface by two small hydrophobic polypeptides, FrdC and FrdD, which are also required for the enzyme to interact with quinone . To better define the individual roles of the FrdC and FrdD polypeptides in FRD complex formation and quinone binding, we selectively mutagenized the frdCD genes . Frd- strains were identified by their inability to grow on restrictive media, and the resulting mutant FRD complexes were isolated and biochemically characterized . The majority of the frdC and frdD mutations were identified as single base deletions that caused premature termination in either FrdC or FrdD and resulted in the loss of one or more of the predicted transmembrane helices . Two additional frdC mutants were characterized that contained single base changes resulting in single amino acid substitutions . All mutant enzyme complexes were incapable of oxidizing the physiological electron donor, menaquinol-6, in the presence of fumarate . Additionally, the ability of the mutant complexes to oxidize reduced benzyl viologen or reduce the ubiquinone analogue 2,3-dimethoxy-5-methyl-6-pentyl-1,4-benzoquinone and phenazine methosulfate with succinate as electron donor were also affected but to varying degrees . The separation of oxidative and reductive activities with quinones suggests there are two quinone binding sites in the fumarate reductase complex and that electron transfer occurs in two le- steps carried out at these separate sites. J Biol Chem, 1990 Nov 15, 265(32), 19551 - 9 Peptide and protein carboxyl-terminal labeling through carboxypeptidase Y-catalyzed transpeptidation; Berne PF et al.; A survey of carboxypeptidase Y-catalyzed carboxyl-terminal modification of short peptides in the presence of various amino acids revealed that transpeptidation occurred in significant yield only with peptides containing a proline at the penultimate or antepenultimate position . For these peptides, transpeptidation was shown to occur specifically at the carboxyl side of the proline, thus suggesting a determining role of this residue for transpeptidation . Two model peptides, YPFP-GPI and YPFVEPI, were studied in detail . Initial yields of transpeptidation in the presence of various nucleophiles were compared . Among natural amino acids, the highest yield was obtained with methionine, followed by other amino acids bearing hydrophobic side chains . In order to transpose the method of transpeptidation to a protein, a variant of Escherichia coli methionyl-tRNA synthetase bearing the carboxyl-terminal Glu-Pro-Met sequence was genetically created . Under the conditions optimized for the transpeptidation of YPF-VEPI with methionine, this protein could be labeled specifically at its carboxyl-terminal end . Moreover, the parameters of the labeling reaction were in agreement with those observed in the transpeptidation of the model peptide. J Biol Chem, 1990 Nov 15, 265(32), 19397 - 400 Nucleotide sequence and 40 S subunit assembly of Xenopus laevis ribosomal protein S22; Keiper BD et al.; We have isolated and determined the nucleotide sequence of a cDNA encoding Xenopus laevis ribosomal protein S22 . A synthetic S22 mRNA derived from this cDNA directs the synthesis of an in vitro translation product that is indistinguishable from S22 purified from Xenopus ovarian ribosomes . In vitro translated S22 is assembled into 40 S subunits when microinjected into the cytoplasm of oocytes . Analysis of the derived amino acid sequence indicates that Xenopus S22 is homologous to Escherichia coli ribosomal protein S10. J Biol Chem, 1990 Nov 15, 265(32), 19377 - 80 Molecular cloning, sequencing, and expression of mouse ferrochelatase; Taketani S et al.; The cDNA encoding mouse ferrochelatase (protoheme ferrolyase, EC 4.99.1.1) was isolated from a mouse erythroleukemia (MEL) cell cDNA library in lambda gt11 expression vector, by immunoscreening with a polyclonal antibody . Two full-length clones containing cDNA inserts of 2.2 and 2.90 kilobases were obtained . These clones have the same entire enzyme coding region, but alternative putative polyadenylation sites in the 3'-noncoding regions . From the deduced primary structure, a putative leader sequence of 53 amino acid residues resulted in a precursor protein of 420 amino acid residues (Mr 47,130) and a mature protein of 367 residues (Mr 41,692) . The cDNA allows for the expression of active ferrochelatase by transfected culture cells . RNA blot analysis showed two species of ferrochelatase mRNA consistent with findings of two polyadenylation sites . Both the mRNAs increased by treatment of the MEL cells with dimethyl sulfoxide . The band pattern of the RNA of the mouse liver was the same as that of the MEL cells . Based on these results, we deduce that ferrochelatase in erythroid and hepatic cells can be only of one type. Biochem Biophys Res Commun, 1990 Nov 15, 172(3), 1230 - 5 Molecular cloning and expression of human arachidonate 12-lipoxygenase; Yoshimoto T et al.; The cDNA for a 12-lipoxygenase was isolated from cDNA library of human erythroleukemia cells . The cDNA had an open reading frame encoding 663 amino acids with a calculated molecular weight of 75,513 . The deduced amino acid sequence of human 12-lipoxygenase exhibited 41.5%, 65.3% and 65.4% identity with human 5-lipoxygenase, human 15-lipoxygenase and porcine 12-lipoxygenase, respectively . Blot hybridization analysis of RNA from human erythroleukemia cells demonstrated a single species (3.1 kb) of mRNA with the cDNA probe for 12-lipoxygenase of these cells, but not with the cDNA for porcine leukocyte enzyme . The cytosol of Escherichia coli transformed with a recombinant pUC19 plasmid oxygenated the position 12 of arachidonic acid. Arch Biochem Biophys, 1990 Nov 15, 283(1), 96 - 101 The kinetic mechanisms of the bifunctional enzyme aspartokinase-homoserine dehydrogenase I from Escherichia coli; Angeles TS et al.; The kinetic mechanisms of the reactions catalyzed by the two catalytic domains of aspartokinase-homoserine dehydrogenase I from Escherichia coli have been determined . Initial velocity, product inhibition, and dead-end inhibition studies of homoserine dehydrogenase are consistent with an ordered addition of NADPH and aspartate beta-semialdehyde followed by an ordered release of homoserine and NADP+ . Aspartokinase I catalyzes the phosphorylation of a number of L-aspartic acid analogues and, moreover, can utilize MgdATP as a phosphoryl donor . Because of this broad substrate specificity, alternative substrate diagnostics was used to probe the kinetic mechanism of this enzyme . The kinetic patterns showed two sets of intersecting lines that are indicative of a random mechanism . Incorporation of these results with the data obtained from initial velocity, product inhibition, and dead-end inhibition studies at pH 8.0 are consistent with a random addition of L-aspartic acid and MgATP and an ordered release of MgADP and beta-aspartyl phosphate. Arch Biochem Biophys, 1990 Nov 15, 283(1), 141 - 9 Human immunodeficiency viral protease is catalytically active as a fusion protein: characterization of the fusion and native enzymes produced in Escherichia coli; Boutelje J et al.; Processing of the gag and pol gene precursor proteins of retroviruses is essential for the production of mature infectious virions . The processing is directed by a viral protease that itself is part of these precursors and is presumed to cleave itself autocatalytically . To facilitate study of this process, the protease was produced as a fusion protein in Escherichia coli . In this construct, the 10,793-Da protease was preceeded by two copies of a modified IgG binding domain derived from protein A . The IgG binding domain was linked to the protease by an Asp-Pro peptide bond which could not be cleaved by the viral protease . A dimer of the 25,400-Da fusion protein was catalytically active, specifically cleaving a substrate peptide at the correct Tyr-Pro bond . Thus, the fusion protein could serve as a model of the viral gag-pol polyprotein . The finding that the fusion protein was catalytically active supports the suggestion that a gag-pol dimer can initiate a proteolytic cascade after budding of the immature virus . The fusion protein also provided a source of authentic protease . The protease was released from the fusion construct by incubation with formic acid, cleaving the Asp-Pro linkage which had been inserted between the IgG binding domain and the protease. Biochem J, 1990 Nov 15, 272(1), 107 - 12 Cloning and expression in Escherichia coli of a rat brain cDNA encoding a Ca2+/calmodulin-sensitive inositol 1,4,5-trisphosphate 3-kinase; Takazawa K et al.; Inositol 1,4,5-trisphosphate (InsP3) 3-kinase catalyses the phosphorylation of InsP3 to inositol 1,3,4,5-tetrakisphosphate (InsP4) . InsP3 3-kinase activity was stimulated by Ca2+ in the presence of calmodulin (CaM) and the protein was associated with two silver-stained bands which migrated with an apparent Mr of approx . 50,000 on SDS/polyacrylamide gels . Upon limited proteolysis with trypsin, the native InsP3 3-kinase was converted into polypeptides of Mr 44,000 and 36,000 . Both tryptic fragments displayed InsP3 3-kinase activity that was Ca2+/CaM-sensitive . A cDNA clone, C5, that encodes the C-terminal part of the InsP3 3-kinase, was isolated by immunoscreening of a rat brain cDNA library . The 5' end of this clone was used in turn to probe the same library, yielding a clone (CP16) containing the entire coding sequence of InsP3 3-kinase . The encoding protein of 459 amino acids (calculated Mr 50,868) has several putative phosphorylation sites for cyclic AMP-dependent protein kinase, protein kinase C and CaM-dependent protein kinase II . When clone C5 was expressed in Escherichia coli, the truncated fusion protein showed Ca2+/CaM-sensitive InsP3 3-kinase activity . Our data demonstrate that the N-terminal part of the protein is not essential for either enzymic or CaM-regulatory properties. J Biol Chem, 1990 Nov 15, 265(32), 19679 - 84 Molecular structure of microtubule-associated protein 2b and 2c from rat brain; Kindler S et al.; Full length cDNA clones encoding microtubule-associated proteins (MAP) 2b and 2c from rat brain have been isolated and sequenced . The cDNA fragments spanning the coding regions for both MAP2b and MAP2c were assembled and expressed in Escherichia coli . The mobility of these bacterial expressed proteins in sodium dodecyl sulfate gels is identical to that of MAP2b and MAP2c from rat brain . The protein sequence of rat MAP2b has been compared to the full length sequence from mouse and the partial sequence from human high molecular weight MAP2 . This comparison has revealed that MAP2b is composed of several highly conserved domains flanked by domains with extensive sequence divergence . Two of the conserved domains, found either at the NH2 or COOH terminus, overlap with the binding domain for the regulatory subunit of the cAMP-dependent protein kinase II and the microtubule-binding domain, respectively . A third homologous domain of unknown function lies in a central region of MAP2b . Secondary structure prediction suggests that the portion of MAP2b which extends from the microtubule surface is composed of an extensive number of alpha-helices separated by small turns which may account for the extended yet flexible structure of MAP2 . Interestingly, the 4000-base pair deletion from the middle of MAP2b which generates MAP2c not only removes these helices, but also this third highly conserved MAP2b domain. J Biol Chem, 1990 Nov 15, 265(32), 19502 - 6 Holoenzymes of cAMP-dependent protein kinase containing the neural form of type I regulatory subunit have an increased sensitivity to cyclic nucleotides; Cadd GG et al.; Specific isoforms of the cAMP-dependent protein kinase are preferentially expressed within discrete neuronal regions in mouse brain (Cadd and McKnight (1989) Neuron 3, 71-79) suggesting that these subunits might have different functional properties . We have used recombinant techniques to express and purify the type I regulatory subunits, RI alpha and RI beta, the catalytic subunits C alpha and C beta, and then reconstituted holoenzymes with the various combinations of R and C subunits . The ability of the subunits to form inactive holoenzymes and then to be activated in the presence of cyclic nucleotides was examined . Holoenzymes containing C beta had essentially the same activation properties exhibited by C alpha holoenzymes . However, the presence of the neural form of RI, RI beta, led to formation of a holoenzyme which was activated at a 3-7-fold lower concentration of cyclic nucleotides compared to holoenzymes containing RI alpha . Expression of the RI beta protein in discrete regions of the central nervous system may provide a mechanism for increasing the sensitivity of the kinase to what would otherwise be subthreshold levels of stimulation . Two mutant forms of RI beta were constructed that converted the RI beta sequence to that of RI alpha at position 98 (RI beta Ala) or positions 98 and 99 (RI beta Ala/Ile) . These sequences form part of a pseudosubstrate site thought to interact with the C subunit . Wild type and mutant R subunits were combined in vitro with purified bovine C subunits and half maximal activation constants (Ka) were determined with cyclic nucleotides . Holoenzymes containing RI beta Ala and RI beta Ala/Ile gave Ka values which were higher than wild type RI beta, with the double mutant shifting toward the Ka value of RI alpha holoenzymes by about 30% . These results suggest that amino acid differences in the pseudosubstrate site may account for some, but not all, of the increased sensitivity to cyclic nucleotides exhibited by RI beta. Biochem Biophys Res Commun, 1990 Nov 15, 172(3), 1157 - 62 Protein conformational "annealing": binding to cytochrome c refolds the active site region of recombinant cytochrome c peroxidase; Hake R et al.; When initially isolated with heme reconstitution, recombinant cytochrome c peroxidase molecules exhibit a conformation, revealed by visible spectra which observably differ from the corresponding holo proteins isolated from yeast . Binding yeast iso-1 cytochrome c to these recombinant cytochrome c peroxidases (either in solution or via an affinity column) catalyses a local refolding of the recombinant proteins to a form that is indistinguishable from the native (yeast) protein. Biochem J, 1990 Nov 15, 272(1), 223 - 9 Annexin proteins PP4 and PP4-X . Comparative characterization of biological activities of placental and recombinant proteins; Romisch J et al.; The human placental proteins PP4 and PP4-X, belonging to the annexin protein family, were expressed in Escherichia coli at high yield . The proteins were purified to homogeneity . The physicochemical parameters of the recombinant proteins were determined and compared with those of their natural placental counterparts . Except for a minor change in the pI, the proteins appeared to be indistinguishable by several criteria . Both recombinant PP4 and recombinant PP4-X were biologically active in a thromboplastin inhibition test and in a phospholipase A2 inhibition test. Biochem Biophys Res Commun, 1990 Nov 15, 172(3), 1217 - 23 Quantitative renaturation from a guanidine-denatured state of the SecA dimer, a 200 KDa protein involved in protein secretion in Escherichia coli; Shinkai A et al.; SecA is an essential component of the protein secretory machinery of Escherichia coli . SecA denatured in 6 M guanidine hydrochloride was quantitatively renatured through dilution and dialysis . The renatured SecA was the same as native SecA as to the CD spectrum, fluorescence spectrum for tryptophan residues and dimeric structures . It was as functionally active as native SecA as to interactions with ATP and presecretory proteins, and in vitro translocation . SecA-N95, which lacks the carboxyl-terminal 70 amino acid residues including three of four cysteine residues and yet is as active as intact SecA as to in vitro translocation, was also renatured to an active form from the guanidine solution . Furthermore, the renaturation of SecA took place in the presence of 1 mM dithiothreitol . It is concluded that disulfide bridges, both intra- and intermolecular ones, do not play a role in the folding and functioning of the SecA molecule. J Biol Chem, 1990 Nov 15, 265(32), 19848 - 52 Nucleosome linking number change controlled by acetylation of histones H3 and H4; Norton VG et al.; High levels of acetylation of lysines in the amino-terminal domains of all four core histones, H2A, H2B, H3, and H4, have been shown to reduce the linking number change per nucleosome core particle in reconstituted minichromosomes (Norton, V . G., Imai, B . S., Yau, P., and Bradbury, E . M . (1989) Cell 57, 449-457) . Because there is evidence to suggest that the acetylations of H3 and H4 have functions that are distinct from those of H2A and H2B, we have determined the nucleosome core particle linking number change in minichromosomes containing fully acetylated H3 and H4 and very low levels of acetylation in H2A and H2B . This linking number change was -0.81 +/- 0.05, in close agreement with the linking number change for hyperacetylated nucleosome core particles which contain high levels of acetylation in all four core histones (approximately 70% of full acetylation in H3 and H4) . Therefore, high levels of acetylation of H3 and H4 alone are responsible for the reduction in the linking number change per nucleosome core particle. Gene, 1990 Nov 15, 95(2), 223 - 30 Stable, high-level expression of a carcinoembryonic antigen-encoding cDNA after transfection and amplification with the dominant and selectable asparagine synthetase marker; Cartier M et al.; The introduction and expression of cloned genes in a wide variety of animal cells requires the convenient use of dominant selectable markers . Very few of these markers can be amplified in copy number, a necessary feature if variable and high-level expression of the gene of interest is required . We describe the successful dominant transfection and amplification of a vector containing the Escherichia coli asparagine synthetase (AS)-encoding gene, asnA, transfected into a variety of human and rodent cell lines . An unlinked co-transfected expression vector containing the CEA cDNA, encoding human carcinoembryonic antigen, can be co-amplified with the asnA marker leading to extremely high levels of CEA synthesis . In addition, we show that the expression of both the asnA marker and the co-transfected CEA construct are stable in normal and amplified transfectants after prolonged culture in the absence of selective pressure. Anal Biochem, 1990 Nov 15, 191(1), 65 - 9 Use of biotinylated inorganic pyrophosphatase for detection of biotin bound to solid support; Vener AV et al.; A colorimetric procedure to detect biotin bound to microtiter plates with a sensitivity down to 10(-16) mol was developed using biotinylated inorganic pyrophosphatase of Escherichia coli . Reaction of pyrophosphatase with 1 mM N-biotinyl-6-aminocaproic acid N-hydroxy-sulfonosuccinimide ester yielded a stable 87% active enzyme containing 5.6 mol biotin/mol . In the measurements of human immunoglobulin G, a biotinylated pyrophosphatase.streptavidin complex provided a sensitivity superior to that of conventional enzyme immunoassay due to low nonspecific binding . The new procedure was also more sensitive compared with that using biotinylated alkaline phosphatase . Together with high thermostability of pyrophosphatase and its substrate, low background staining allowed measurement of enzymatic activity to be performed at 60 degrees C for 4 h resulting in a marked increase in assay sensitivity. Anal Biochem, 1990 Nov 15, 191(1), 35 - 40 The use of ubiquitin-peptide extensions as protein kinase substrates; Yoo Y et al.; Four ubiquitin-peptide extensions prepared as cloned products in E . coli were tested as casein kinase II substrates . Two extensions containing the sequence Ser-Glu-Glu-Glu-Glu-Glu were readily phosphorylated by partially purified rabbit reticulocyte casein kinase II . The other two fusion proteins, which lack a consensus phosphorylation site for casein kinase II, did not serve as substrates under identical reaction conditions . Native ubiquitin was not phosphorylated by reticulocyte casein kinase II, nor have we observed its phosphorylation in crude extracts from HeLa cells, mouse liver, or Xenopus eggs . Ubiquitin's apparent lack of phosphorylatable residues coupled with its remarkable heat stability and rapid migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels make the protein an attractive carrier for carboxyl-terminal peptides containing specific phosphorylation sites . Such ubiquitin extension proteins should prove valuable as protein kinase substrates. Eur J Biochem, 1990 Nov 13, 193(3), 801 - 6 The collagen-binding site of type-II units of bovine seminal fluid protein PDC-109 and fibronectin; Banyai L et al.; A single type-II domain has been isolated by limited proteolysis of the collagen-binding bovine seminal fluid protein, PDC-109 . The 45-residue fragment corresponding to the second type-II domain of the parent molecule was found to have retained affinity for immobilized colla