Signal peptidase I overproduction results in increased efficiencies of export and maturation of hybrid secretory proteins in Escherichia coli

Summary The effects of 25-fold overproduction ofEscherichia coli signal peptidase I (SPase I) on the processing kinetics of various (hybrid) secretory proteins, comprising fusions between signal sequence functions selected from theBacillus subtilis chromosome and the mature part of TEM-β-lactamase, were studied inE. coli. One precursor (pre[A2d]-β-lactamase) showed an enhanced processing rate, and consequently, a highly improved release of the mature enzyme into the periplasm. A minor fraction of a second hybrid precursor (pre[Al3i]-β-lactamase), which was not processed under standard conditions of SPase I synthesis, was shown to be processed under conditions of SPase I overproduction. However, this did not result in efficient release of the mature β-lactamase into the periplasm. In contrast, the processing rates of wild-type pre-β-lactamase and pre(A2)-β-lactamase, already high under standard conditions, were not detectably altered by SPase I overproduction. These results demonstrate that the availability of SPase I can be a limiting factor in protein export inE. coli, in particular with respect to (hybrid) precursor proteins showing low (SPase I) processing efficiencies.     

A 2.6 kb intron separates the signal peptide coding sequence of an anther-specific protein from the rest of the gene in sunflower

Summary Metabolism of sulfonylurea herbicides by Streptomyces griseolus ATCC 11796 is carried out via two cytochromes P-450, P-450_SU1 and P-450_SU2. Mutants of S. griseolus, selected by their reduced ability to metabolize a fluorescent sulfonylurea, do not synthesize cytochrome P-450_SU1 when grown in the presence of sulfonylureas. Genetic evidence indicated that this phenotype was the result of a deletion of > 15 kb of DNA, including the structural genes for cytochrome P-450_SU1 and an associated ferredoxin Fd-1 (suaC and suaB, respectively). In the absence of this monooxygenase system, the mutants described here respond to the presence of sulfonylureas or phenobarbital in the growth medium with the expression of only the suhC,B gene products (cytochrome P-450_SU2 and Fd-2), previously observed only as minor components in wild-type cells treated with sulfonylurea. These strains have enabled an analysis of sulfonylurea metabolism mediated by cytochrome P-450_SU2 in the absence of P-450_SU1, yielding an in vivo delineation of the roles of the two different cytochrome P-450 systems in herbicide metabolism by S. griseolus.     

2-Amino-N6-hydroxyadenine induces gene/point mutations and multiple-locus mutations, but not multilocus deletion mutations, in the ad-3 region of a two-component heterokaryon of Neurospora crassa.

The mutagenicity of 2-amino-N6-hydroxyadenine (AHA) has been studied in Neurospora crassa by treating a two-component heterokaryon (H-12) and recovering specific-locus mutations induced in the ad-3 region. This assay system permits the identification of ad-3A and/or ad-3B mutants resulting from gene/point mutations, multilocus deletion mutations, and multiple-locus mutations of various genotypes, involving one or both loci. Genetic characterization of the ad-3 mutants recovered from experiments with AHA in H-12 shows that 98.9% (270/273) of the ad-3 mutants are gene/point mutations (ad-3R), 1.1% (3/270) are unknowns, and none is a multilocus deletion mutation (ad-3IR). Among the gene/point mutations, 3.3% (9/273) are multiple-locus mutations (gene/point mutations with a closely-linked recessive lethal mutation [ad-3R + RLCL]). Another 25.3% (69/273) are multiple-locus mutations with a recessive lethal mutation located elsewhere in the genome [ad-3R + RL]. Heterokaryon tests for allelic complementation among the ad-3BR mutants showed that 90.8% (139/153) of the mutants were complementing, and 20.3% (31/153) were leaky. In addition, 32.5% (38/117) of the ad-3AR mutants were leaky. These data are consistent with the hypothesis that AHA produces specific-locus mutations in the ad-3 region of N. crassa by base-pair substitution. The data from the present experiments are compared with the data for 2-aminopurine (2AP)-induced ad-3 mutants in H-12 (de Serres and Brockman, 1991). Whereas, 2AP is a weak mutagen in H-12, AHA is extremely potent (Brockman et al., 1987). In contrast with 2AP, AHA induces ad-3 mutants exclusively by gene/point mutation in H-12. We conclude that whereas AHA induces ad-3 mutants predominantly by AT to GC base-pair transitions, 2AP induces ad-3 mutants by a wide variety of mechanisms including: (1) AT to GC and GC to AT base-pair transitions, (2) frameshift mutations, (3) other, as yet unidentified, intragenic alterations, (4) small multilocus deletion mutations, and (5) multiple-locus ad-3R mutations with closely linked recessive lethal mutations.     

Qualitative differences in the spectra of genetic damage in 2-aminopurine-induced ad-3 mutants between nucleotide excision-repair-proficient and -deficient strains of Neurospora crassa.

The mutagenic effects of 2-aminopurine (2AP) have been compared in the adenine-3 (ad-3) region of two-component heterokaryons of Neurospora crassa: nucleotide excision repair-proficient (uvs-2+/uvs-2+) heterokaryon 12 (H-12) and nucleotide excision repair-deficient (uvs-2/uvs-2) heterokaryon 59 (H-59). This forward-mutation, morphological and biochemical, specific-locus assay system permits the recovery of ad-3A and/or ad-3B mutants in 3 major classes: gene/point mutations, multilocus deletion mutations, and unknowns, and 3 different subclasses of multiple-locus mutations. Previous studies (Brockman et al., Mutation Res., 218 (1989) 1-11) showed that 2AP treatment of growing cultures of H-12 and H-59 gave no difference between ad-3 forward-mutation frequencies over a wide range of 2AP concentrations in each strain. In the present experiments, genetic analyses of ad-3 mutants recovered from these experiments has demonstrated qualitative differences between the spectra of the 3 main classes of ad-3 mutations. In H-12, 84.2% (203/241) resulted from gene/point mutation, 11.6% (28/241) from multilocus deletion mutation, and 4.1% (10/241) were unknowns. In contrast, in H-59, 43.0% (99/230) resulted from gene/point mutation, 55.7% (128/230) from multilocus deletion mutation, and 1.3% (3/230) were unknowns. In addition, quantitative differences were also found between the spectra of ad-3 mutations in 1 subclass of multiple-locus mutations, but not 2 additional subclasses. The first subclass consisted of 1.7% (4/241) and 9.6% (22/230) gene/point mutations with a closely linked recessive lethal mutation, in H-12 and H-59, respectively. The second two subclasses consisted of (a) 0.4% (1/241) and 0.4% (1/230) multilocus deletion mutations with a closely linked recessive lethal mutation, and (b) 13.3% (32/241) and 15.2% (35/230) gene/point mutations with a separate recessive lethal mutation elsewhere in the genome, in H-12 and H-59, respectively. Data from studies by others have shown that 2AP inhibits adenosine deaminase, resulting in nucleotide precursor pool inbalance, and that 2AP can saturate the mismatch repair system. As a consequence of either effect of 2AP, the spectrum of 2AP-induced mutation could include frameshift mutations and chromosome aberrations such as multilocus deletions in addition to base-pair substitutions. The defect in DNA repair due to the uvs-2 allele, which has been shown to be a deficiency in pyrimidine dimer excision (Worthy and Epler, 1974), most probably has some other excision-repair deficiency (Macleod and Stadler, 1986; Baker et al., 1991).(ABSTRACT TRUNCATED AT 400 WORDS)     

The genetic toxicology of 2-amino-N6-hydroxyadenine in eukaryotic organisms: significance for genetic risk assessment

A collaborative study was designed to assess the mutagenicity of 2-amino-N6-hydroxylaminopurine (AHA) in a wide variety of eukaryotic assays systems in terms of potency and specificity. Earlier studies in Salmonella and Neurospora had shown that AHA was an extremely potent mutagen which appeared to cause predominantly AT to GC base-pair transitions. This discovery was viewed as an unusual opportunity to explore the general utility of different eukaryotic assay systems for genetic risk assessment. The objective was to determine whether AHA would show comparable potency and specificity in those eukaryotic organisms used to evaluate mutagenic potential of environmental chemicals for the human population. The data presented in this report show that AHA was mutagenic in all the eukaryotic assays utilized; however, the level of effect was found to be assay system-dependent. In addition, in assays where other base analogs were used as positive controls, differences in relative potency were observed from those obtained in the earlier studies with Salmonella and Neurospora. When alkylating agents were used as positive controls in the higher eukaryotic assays, AHA was found to have a mutagenic potency comparable to ethylnitrosourea (ENU), ethyl methanesulfonate (EMS) or methyl methanesulfonate (MMS) for many of the assays. With regard to mutagenic specificity, AHA appears to induce gene/point mutations in eukaryotic organisms, resulting predominantly from base-pair substitutions, predominantly AT to GC base-pair transitions; however, there was some unexplained variation in the ratio of these base-pair transitions and other transitions and transversions as a function of assay system. In addition, studies on the induction of micronuclei have shown that AHA induces chromosomal damage at high concentrations and low levels of survival.     

Glyoxylate Cycle Enzymes in Peroxisomes Isolated from Petals of Pumpkin (Cucurbita sp.) during Senescence

The activities of the two unique enzymes of the glyoxylate cycle,isocitrate lyase (EC 4.1.3.1 [EC] ) and malate synthase (EC 4.1.3.2 [EC] ),were undetectable in petals of pumpkin (Cucurbita sp. AmakuriNankin) until the end of blooming, but they appeared duringsenescence. The activity of catalase (EC 1.11.1.6 [EC] ) increased,glycolate oxidase (EC 1.1.3.1 [EC] ) activity did not change, whilehydroxypyruvate reductase (EC 1.1.1.81 [EC] ) activity peaked at fullblooming stage and declined thereafter. After fractionationof cellular organelles on a sucrose density gradient, we detectedisocitrate lyase and malate synthase activities in peroxisomalfractions only from petals at the senescing stage. Northernblot analysis revealed that malate synthase mRNA increased duringpetal senescence. Citrate synthase (EC 4.1.3.7 [EC] ) and malate dehydrogenase(EC 1.1.1.37 [EC] ) activities were also present, while aconitase(EC 4.2.1.3 [EC] ) was not detectable in peroxisomal fractions. Moreoverthe presence of 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35 [EC] )and urate oxidase (EC 1.7.3.3 [EC] ) in the peroxisomal fractionsfrom senescing petals indicates that peroxisomes could be involvedboth in the ß-oxidation pathway and in the purinecatabolism during petal senescence. (Received May 25, 1991; Accepted September 25, 1991)     

Cardenolides from Asclepias asperula subsp. Capricornu and A. Viridis

6′-O-(E-4-hydroxycinnamoyl) Desglucouzarin, the first cardenolide containing a cinnamoyl ester moiety, has been isolated from the ethanolic extract of the milkweed, Asclepias asperula. In addition, five known cardenolides were isolated and identified from A. asperula and A. viridis.     

Quinovic acid glycosides from Uncaria guianensis.

From the bark of Uncaria guianensis, two new quinovic acid glycosides, quinovic acid 3 beta-O-beta-D-quinovopyranoside and quinovic acid 3 beta-O-beta-D-fucopyranosyl-(27----1)-beta-D-glucopyranosylester, have been isolated, in addition to known quinovic acid 3 beta-O-[beta-D-glucopyranosyl-(1----3)-beta-D-fucopyranosyl]-(27----1)- beta-D-glucopyranosylester and quinovic acid 3 beta-O-beta-D-fucopyranoside. Their structures were elucidated by spectral and chemical studies.     

Stromal low temperature compartment derived from the inner membrane of the chloroplast envelope

Leaf discs of four dicotyledonous species, when incubated at temperatures of 4 to 18°C (optimum at 12°C) for 30 or 60 minutes, responded by accumulations of membranes in the chloroplast stroma in the space between the inner membrane of the envelope and the thylakoids. The accumulated membranes, here referred to as the low temperature compartment, were frequently continuous with the envelope membrane and exhibited kinetics of formation consistent with a derivation from the envelope. Results were similar for expanding leaves of garden pea (Pisum sativum), soybean (Glycine max), spinach (Spinacia oleracea), and tobacco (Nicotiana tabacum). We suggest that the stromal low temperature compartment may be analogous to the compartment induced to form between the transitional endoplasmic reticulum and the Golgi apparatus at low temperatures. The findings provide evidence for the possibility of a vesicular transfer of membrane constituents between the inner membrane of the chloroplast envelope and the thylakoids of mature chloroplasts in expanding leaves.