Rapid selection and characterization of Cry1F resistance in a Brazilian strain of fall armyworm

Transgenic maize (Zea mays L., Poaceae) event TC1507, producing the Cry1F protein of Bacillus thuringiensis Berliner, has been used for management of the fall armyworm, Spodoptera frugiperda (JE Smith) (Lepidoptera: Noctuidae), in Brazil since 2009. A strain of S. frugiperda, obtained from field collections of larvae in TC1507 maize in Minas Gerais state in 2010, was selected in the laboratory for resistance to Cry1F using leaves of TC1507 maize in two selection regimes. Continuous exposure of larvae to Cry1F was more effective than exposure for 6, 8, and 10 days in the selection of resistant S. frugiperda individuals. With only four generations of laboratory selection, a strain with high levels of resistance to Cry1F was obtained, as indicated by the survival of insects reared on leaves of TC1507 maize plants and by the more than 300‐fold resistance level measured in bioassays with the purified Cry1F protein. Importantly, reciprocal crosses between control and the Cry1F‐selected strains revealed that the resistance is autosomal and incompletely recessive, and the response obtained in the backcross of the F₁ generation with the resistant strain was consistent with simple monogenic inheritance. Additionally, there were no apparent fitness costs associated with resistance either for survival or larval growth on non‐Bt maize leaves. Our findings provide experimental evidence for rapid evolution of Cry1F resistance in S. frugiperda in the laboratory and further reinforce the potential of this species to evolve field resistance to the TC1507 maize as previously reported. The resistant strain isolated in this study provides an opportunity to estimate the resistance allele frequency in the field and to determine the biochemical and molecular basis of the resistance, which should provide further information to assist in the resistance management of S. frugiperda on transgenic maize producing B. thuringiensis proteins.     

A Comparative Analysis of the Molecular Features of MANF and CDNF

Cerebral dopamine neurotrophic factor (CDNF) is a paralogous protein of mesencephalic astrocyte-derived neurotrophic factor (MANF). Both proteins have been reported to show a common cytoprotective effect on dopaminergic neurons as a secretory protein containing the KDEL-like motif of the ER retrieval signal at the C-terminus, RTDL in MANF and [Q/K]TEL in CDNF among many species, although functions of paralogous proteins tend to differ from each other. In this study, we focused on post-translational regulations of their retention in the endoplasmic reticulum (ER) and secretion and performed comparative experiments on characterization of mouse MANF and mouse CDNF according to our previous report about biosynthesis and secretion of mouse MANF using a NanoLuc system. In this study, co-expression of glucose-regulated protein 78 kDa (GRP78), KDEL receptor 1 or mutant Sar1 into HEK293 cells similarly decreased MANF and CDNF secretion with some degree of variation. Next, we investigated whether CDNF affects the secretion of mouse cysteine-rich with EGF-like domains 2 (CRELD2) because mouse wild-type (wt) MANF but not its KDEL-like motif deleted mutant (ΔC_MANF) was found to promote the CRELD2 release from the transfected cells. Co-expressing CRELD2 with wt or ΔC CDNF, we found that CDNF and ΔC_MANF hardly elevated the CRELD2 secretion. We then investigated effects of the four or six C-terminal amino acids of MANF and CDNF on the CRELD2 secretion. As a result, co-transfection of mouse CDNF having the mouse MANF-type C-terminal amino acids (CDNF_RTDL and CDNF_SARTDL) increased the CRELD2 secretion to a small extent, but mouse CDNF having human CDNF-type ones (CDNF_KTEL and CDNF_HPKTEL) well increased the CRELD2 secretion. On the other hand, the replacement of C-terminal motifs of mouse MANF with those of mouse CDNF (MANF_QTEL and MANF_YPQTEL) enhanced the CRELD2 secretion, and the mouse MANF having human CDNF-type ones (MANF_KTEL and MANF_HPKTEL) dramatically potentiated the CRELD2 secretion. These results indicate that the secretion of mouse MANF and mouse CDNF is fundamentally regulated in the same manner and that the variation of four C-terminal amino acids in the MANF and CDNF among species might influence their intracellular functions. This finding could be a hint to identify physiological functions of MANF and CDNF.     

Molecular cloning of cDNA for argininosuccinate lyase of rat liver

A cDNA expression library constructed from poly(A)+ RNA of rat liver was screened immunologically using an antibody against argininosuccinate lyase (EC 4.3.2.1), a urea cycle enzyme, of rat liver. A cDNA clone was isolated and identified by hybrid-selected translation. The clone contained an insert approximately 1.5 kilobase pairs in length. In the bacterial clone, a specific protein of Mr = about 25,000 was expressed. The argininosuccinate lyase mRNA of about 2.1 kilobases long was detected in the liver and in a lesser amount in the kidney and spleen, but not in the small intestine and heart of the rats.     

Molecular cloning of cDNA for rat mitochondrial 3-hydroxyacyl-CoA dehydrogenase

Messenger RNA for 3-hydroxyacyl-CoA dehydrogenase, a mitochondrial matrix enzyme of fatty acid beta-oxidation, was purified from livers of di(2-ethylhexyl)phthalate-treated rats by immunoadsorption of hepatic free polysomes to fixed cells of Staphylococcus aureus and enrichment for poly(A)-rich RNA by oligo(dT)-cellulose chromatography. Plasmid cDNA was constructed from this poly(A)-rich RNA by a modification of the method of Okayama and Berg and was transformed into the Escherichia coli DH1 strain. Plasmids containing cDNA sequences coding for 3-hydroxyacyl-CoA dehydrogenase were screened by differential colony hybridization, and were identified by hybrid-arrested translation and hybrid-selected translation. Plasmid pHADH-1, which contains a 1400-base-pair insert, hybridized to rat 3-hydroxyacyl-CoA dehydrogenase mRNA with a length of 1700 bases. Determination of the dehydrogenase mRNA by in vitro translation and dot-blot analysis with the cDNA probe showed that the induction of the enzyme in rat liver by di(2-ethylhexyl)phthalate could be attributed to an increase in the mRNA concentration.     

cDNA-derived amino acid sequence of rat mitochondrial 3-oxoacyl-CoA thiolase with no transient presequence: structural relationship with peroxisomal isozyme.

The sorting of homologous proteins between two separate intracellular organelles is a major unsolved problem. 3-Oxoacyl-CoA thiolase is localized in mitochondria and peroxisomes, and provides a good system for the study on the problem. Unlike most mitochondrial matrix proteins, mitochondrial 3-oxoacyl-CoA thiolase in rats is synthesized with no transient presequence and possess information for mitochondrial targeting and import in the mature protein. Two overlapping cDNA clones contained an open reading frame encoding a polypeptide of 397 amino acid residues (predicted Mr = 41,868), a 5' untranslated sequence of 164 bp, a 3' untranslated sequence of 264 bp and a poly(A) tract. The amino acid sequence of the mitochondrial thiolase is 37% identical with that of the mature portion of rat peroxisomal 3-oxoacyl-CoA thiolase precursor. These results suggest that the two thiolases have a common origin and obtained information for targeting to respective organelles during evolution. Two portions in the mitochondrial thiolase that may serve as a mitochondrial targeting signal are presented.     

Reconstitution of mitochondrial protein transport with purified ornithine carbamoyltransferase precursor expressed in Escherichia coli

Ornithine carbamoyltransferase (OTC; subunit, 36,000 Da) is initially synthesized as a precursor (pOTC) with a transient NH2-terminal presequence of 32 amino acid residues and imported posttranslationally into the mitochondrial matrix. The rat pOTC was synthesized in Escherichia coli using an expression vector containing a thermoinducible lambda pL promoter. The recombinant pOTC represented 5-10% of the total bacterial protein and was present in the precipitate of the disrupted bacteria. The precipitate was washed and pOTC was extracted with 8 M urea or 0.1% cetyltrimethylammonium bromide. The extracted pOTC was essentially homogeneous, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified pOTC was cleaved to the intermediate-sized product of 37,000 Da by a processing protease partially purified from the matrix fraction of rat liver mitochondria. The purified recombinant pOTC, but not the mature form of OTC synthesized in E. coli and purified, competed with the in vitro-synthesized, radiolabeled pOTC for uptake and processing by the isolated rat liver mitochondria. The radiolabeled and purified recombinant pOTC could be imported into the isolated mitochondria and processed to the mature form in an energy- and rabbit reticulocyte lysate-dependent manner. When the purified pOTC was subjected to sucrose gradient centrifugation, it sedimented as a large aggregate of greater than 60 S in the absence of reticulocyte lysate, whereas it sedimented as a complex of about 5 S in the presence of the lysate. These observations together with our previous results indicate that a protein factor(s) present in the lysate interacts with pOTC and holds it in an import-competent form.     

Isolation of a yeast gene, SRH1, that encodes a homologue of the 54K subunit of mammalian signal recognition particle

A 1.7 kilobase HindIII fragment of Saccharomyces cerevisiae DNA was cloned by cross-hybridization with the Escherichia coli secY gene. The complete nucleotide sequence of the 2.6 kb fragment of the yeast genomic DNA containing the cross-hybridizing HindIII fragment was determined. The sequence showed no apparent similarity with that of the E. coli secY gene with the exception of a completely matched sequence of 21 bp, but it contained a 1,623 nucleotide open reading frame coding for a protein of 541 amino acids with a calculated Mr of 59,600. The N-terminal portion of 303 residues of the predicted sequence was homologous to the cytosolic domain of the alpha-subunit of the signal recognition particle receptor (SR alpha), including consensus sequence elements for a GTP binding site, whereas the C-terminal portion of 238 residues had an unusual methionine-rich domain containing several repetitive sequences. An mRNA of 2.0 kb was detected on Northern blotting analysis. The predicted sequence was 48% identical with the reported sequences of the 54K subunit of the mammalian signal recognition particle (SRP54) (Romisch K. et al. (1989) Nature 340, 478-483; Bernstein, H.D. et al. (1989) Nature 340, 482-486). We designated this gene as SRH1 (SRP54 homologue). Gene disruption experiments showed that the SRH1 gene product is essential for cell growth.     

Comparative study of behavioural tests in the SOD1G93A mouse model of amyotrophic lateral sclerosis

In preclinical trials, a sensitive functional test is required to detect changes in the motor behaviour of the SOD1G93A mouse model of amyotrophic lateral sclerosis (ALS). We evaluated changes in body weight and motor impairment in behavioural tests, such as the rotarod, the hanging-wire test and the treadmill, of transgenic and wild type mice. We found differences in detection of the onset of symptoms and progression of the disease between the different tests assessed. Moreover, the data showed significant gender differences in the motor behaviour of this mouse model. The rotarod and the hanging-wire test were more sensitive to detect early motor impairment. Moreover, the results suggested that the rotarod and hanging-wire became the most accurate tests rather than treadmill to characterise the ALS disease phenotype.