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1.
BMC Genet ; 21(1): 3, 2020 01 15.
Article in English | MEDLINE | ID: mdl-31941450

ABSTRACT

BACKGROUND: DNA methylation is an epigenetic modification that mainly repress expression of genes essential during embryogenesis and development. There are key ATPase-dependent enzymes that read or write DNA methylation to remodel chromatin and regulate gene expression. Structural maintenance of chromosome hinge domain containing 1 (SMCHD1) is an architectural protein that regulates expression of numerous genes, some of which are imprinted, that are sensitive to DNA methylation. In addition, SMCHD1 germline mutations lead to developmental diseases; facioscapulohumoral muscular dystrophy (FSHD), bosma arhinia and micropthalmia (BAMS). Current evidence suggests that SMCHD1 functions through maintenance or de novo DNA methylation required for chromatin compaction. However, it is unclear if DNA methylation is also essential for genomic recruitment of SMCHD1 and its role as an architectural protein. We previously isolated SMCHD1 using a methylated DNA region from mouse pituitary growth hormone (Gh1) promoter, suggesting that methylation is required for SMCHD1 DNA binding. The goal of this study was to further understand DNA methylation directed role of SMCHD1 in regulating gene expression. Therefore, we profiled SMCHD1 genome wide occupancy in human neuroblastoma SH-SY5Y cells and evaluated if DNA methylation is required for SMCHD1 genomic binding by treating cells with the DNA demethylating reagent, 5-azacytidine (5-azaC). RESULTS: Our data suggest that the majority of SMCHD1 binding occurs in intron and intergenic regions. Gene ontology analysis of genes associated with SMCHD1 genomic occupancy that is sensitive to 5-azaC treatment suggests SMCHD1 involvement in central nervous system development. The potassium voltage-gated channel subfamily Q member1 (KCNQ1) gene that associates with central nervous system is a known SMCHD1 target. We showed SMCHD1 binding to an intronic region of KCNQ1 that is lost following 5-azaC treatment suggesting DNA methylation facilitated binding of SMCHD1. Indeed, deletion of SMCHD1 by CRISPR- Cas9 increases KCNQ1 gene expression confirming its role in regulating KCNQ1 gene expression. CONCLUSION: These findings provide novel insights on DNA methylation directed function of SMCHD1 in regulating expression of genes associated with central nervous system development that impact future drug development strategies.


Subject(s)
Azacitidine/pharmacology , Binding Sites , Chromosomal Proteins, Non-Histone/metabolism , DNA Methylation/drug effects , Epigenomics , Epigenesis, Genetic/drug effects , Epigenomics/methods , Exons , Gene Expression Regulation/drug effects , Humans , Introns , Promoter Regions, Genetic , Protein Binding , Transcription Initiation Site
2.
Curr Mol Med ; 13(7): 1047-65, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23116263

ABSTRACT

The basic Helix-Loop-Helix/PER-ARNT-SIM (bHLH-PAS) domain family of transcription factors mediates cellular responses to a variety of internal and external stimuli. As functional transcription factors, these proteins act as bHLH-PAS heterodimers and can be further sub-classified into sensory/activated subunits and regulatory or ARNT-like proteins. This class of proteins act as master regulators of the bHLH-PAS superfamily of transcription factors that mediate circadian rhythm gene programs, innate and adaptive immune responses, oxygen-sensing mechanisms and compensate for deleterious environmental exposures. Some contribute to the etiology of human pathologies including cancer because of their effects on cell growth and metabolism. We will review the canonical roles of ARNT and ARNT-like proteins with an emphasis on coactivator selectivity and recruitment. We will also discuss recent advances in our understanding of noncanonical DNA-binding independent or off-target roles of ARNT that are uncoupled from its classic heterodimeric bHLH-PAS binding partners. Understanding the DNA binding-independent functions of ARNT may identify novel therapeutic options for the treatment of a large spectrum of disease states.


Subject(s)
Aryl Hydrocarbon Receptor Nuclear Translocator/genetics , DNA-Binding Proteins/genetics , Amino Acid Sequence , Aryl Hydrocarbon Receptor Nuclear Translocator/metabolism , Circadian Rhythm/genetics , DNA-Binding Proteins/metabolism , Helix-Loop-Helix Motifs , Humans , Protein Binding
3.
Insect Mol Biol ; 16(1): 25-35, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17257206

ABSTRACT

In order to understand how lepidopteran insects react physiologically to Bacillus thuringiensis crystal toxin ingestion, transcriptional profiling of Choristoneura fumiferana larvae exposed to sublethal doses of Cry1Ab protoxin were monitored using a C. fumiferana-specific cDNA microarray derived from a protoxin-specific subtractive library. Differential gene expression occurred primarily between 2 and 5 h postingestion. Metabolic enzymes such as lipases and proteases were generally repressed, whereas genes involved in detoxification, immune system regulation or general stress response were upregulated. A similar protoxin-specific transcriptional pattern was also observed with Manduca sexta larvae, using three upregulated genes (serpin, cytochrome P450 and carboxyl/cholinesterase) and one downregulated gene (beta-glucosidase), suggesting that a susceptible larval response to Cry toxin exposure might be universal among lepidopterous insects.


Subject(s)
Bacterial Proteins/pharmacology , Bacterial Toxins/pharmacology , Endotoxins/pharmacology , Gene Expression Regulation/drug effects , Hemolysin Proteins/pharmacology , Moths/drug effects , Moths/genetics , Animals , Bacillus thuringiensis Toxins , Dose-Response Relationship, Drug , Gene Expression Profiling , Insecticides/pharmacology , Larva/drug effects , Larva/genetics
4.
Insect Mol Biol ; 15(4): 475-83, 2006 Aug.
Article in English | MEDLINE | ID: mdl-16907834

ABSTRACT

Bacillus thuringiensis is a microbial control agent active against Choristoneura fumiferana, a lepidopteran defoliator of North American forests. Although the B. thuringiensis insecticidal crystal protoxins have a relatively narrow host range, there is concern about their impact on non-target species where intoxication effects may not be overt. Larval toxicity effects can be assessed at the molecular level by determining altered transcriptional profiles in response to sublethal protoxin exposure in sensitive insects. Subtraction hybridization libraries were created using two larval populations, control and protoxin-fed and were characterized by sequencing 1091 clones. Differential mRNA expression of selected clones, as measured by quantitative polymerase chain reaction, identified a number of metabolic and stress-related genes that were either transcriptionally enhanced or repressed after protoxin exposure.


Subject(s)
Bacterial Proteins/toxicity , Bacterial Toxins/toxicity , Endotoxins/toxicity , Hemolysin Proteins/toxicity , Lepidoptera/drug effects , RNA, Messenger/metabolism , Transcription, Genetic/drug effects , Animals , Bacillus thuringiensis Toxins , Base Sequence , DNA Primers , Gene Library , Larva/drug effects , Molecular Sequence Data , Nucleic Acid Hybridization , Polymerase Chain Reaction/methods , RNA, Messenger/genetics , Sequence Analysis, DNA
5.
Appl Environ Microbiol ; 67(10): 4828-33, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11571190

ABSTRACT

Streptococcus suis is an important pathogen of swine which occasionally infects humans as well. There are 35 serotypes known for this organism, and it would be desirable to develop rapid methods methods to identify and differentiate the strains of this species. To that effect, partial chaperonin 60 gene sequences were determined for the 35 serotype reference strains of S. suis. Analysis of a pairwise distance matrix showed that the distances ranged from 0 to 0.275 when values were calculated by the maximum-likelihood method. For five of the strains the distances from serotype 1 were greater than 0.1, and for two of these strains the distances were were more than 0.25, suggesting that they belong to a different species. Most of the nucleotide differences were silent; alignment of protein sequences showed that there were only 11 distinct sequences for the 35 strains under study. The chaperonin 60 gene phylogenetic tree was similar to the previously published tree based on 16S rRNA sequences, and it was also observed that strains with identical chaperonin 60 gene sequences tended to have identical 16S rRNA sequences. The chaperonin 60 gene sequences provided a higher level of discrimination between serotypes than the 16S RNA sequences provided and could form the basis for a diagnostic protocol.


Subject(s)
Chaperonin 60/genetics , Streptococcal Infections/veterinary , Streptococcus suis/classification , Swine Diseases/microbiology , Animals , Genes, Bacterial , Molecular Sequence Data , Phylogeny , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNA , Serotyping , Streptococcal Infections/microbiology , Streptococcus suis/genetics , Swine
6.
Mol Cell Biol ; 21(3): 781-93, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11154266

ABSTRACT

Steroid hormone receptors act to regulate specific gene transcription primarily as steroid-specific dimers bound to palindromic DNA response elements. DNA-dependent dimerization contacts mediated between the receptor DNA binding domains stabilize DNA binding. Additionally, some steroid receptors dimerize prior to their arrival on DNA through interactions mediated through the receptor ligand binding domain. In this report, we describe the steroid-induced homomeric interaction of the rat glucocorticoid receptor (GR) in solution in vivo. Our results demonstrate that GR interacts in solution at least as a dimer, and we have delimited this interaction to a novel interface within the hinge region of GR that appears to be both necessary and sufficient for direct binding. Strikingly, we also demonstrate an interaction between GR and the mineralocorticoid receptor in solution in vivo that is dependent on the ligand binding domain of GR alone and is separable from homodimerization of the glucocorticoid receptor. These results indicate that functional interactions between the glucocorticoid and mineralocorticoid receptors in activating specific gene transcription are probably more complex than has been previously appreciated.


Subject(s)
Receptors, Glucocorticoid/chemistry , Receptors, Mineralocorticoid/chemistry , Animals , Binding Sites , COS Cells , Cell Line , Cell Nucleus/metabolism , Cytoplasm , Dimerization , In Vitro Techniques , Protein Structure, Quaternary , Rats , Receptors, Glucocorticoid/genetics , Receptors, Glucocorticoid/metabolism , Receptors, Mineralocorticoid/genetics , Receptors, Mineralocorticoid/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Solutions , Two-Hybrid System Techniques
7.
Mol Cell Biol ; 19(10): 7106-22, 1999 Oct.
Article in English | MEDLINE | ID: mdl-10490647

ABSTRACT

Steroid hormone receptors are distinguished from other members of the nuclear hormone receptor family through their association with heat shock proteins and immunophilins in the absence of ligands. Heat shock protein association represses steroid receptor DNA binding and protein-protein interactions with other transcription factors and facilitates hormone binding. In this study, we investigated the hormone-dependent interaction between the DNA binding domain (DBD) of the glucocorticoid receptor (GR) and the POU domains of octamer transcription factors 1 and 2 (Oct-1 and Oct-2, respectively). Our results indicate that the GR DBD binds directly, not only to the homeodomains of Oct-1 and Oct-2 but also to the homeodomains of several other homeodomain proteins. As these results suggest that the determinants for binding to the GR DBD are conserved within the homeodomain, we examined whether the ectopic expression of GR DBD peptides affected early embryonic development. The expression of GR DBD peptides in one-cell-stage zebra fish embryos severely affected their development, beginning with a delay in the epibolic movement during the blastula stage and followed by defects in convergence-extension movements during gastrulation, as revealed by the abnormal patterns of expression of several dorsal gene markers. In contrast, embryos injected with mRNA encoding a GR peptide with a point mutation that disrupted homeodomain binding or with mRNA encoding the DBD of the closely related mineralocorticoid receptor, which does not bind octamer factors, developed normally. Moreover, coinjection of mRNA encoding the homeodomain of Oct-2 completely rescued embryos from the effects of the GR DBD. These results highlight the potential of DNA-independent effects of GR in a whole-animal model and suggest that at least some of these effects may result from direct interactions with homeodomain proteins.


Subject(s)
DNA-Binding Proteins/metabolism , Homeodomain Proteins/metabolism , Receptors, Glucocorticoid/metabolism , Repressor Proteins , Animals , Binding Sites/genetics , Body Patterning , Bone Morphogenetic Protein 4 , Bone Morphogenetic Proteins/isolation & purification , DNA-Binding Proteins/genetics , Goosecoid Protein , Homeodomain Proteins/isolation & purification , Host Cell Factor C1 , Leucine/genetics , Mesoderm , Mutation , Octamer Transcription Factor-1 , Peptide Fragments/genetics , Peptide Fragments/metabolism , Proline/genetics , Protein Binding/genetics , Receptors, Glucocorticoid/genetics , Tissue Distribution , Transcription Factors/metabolism , Zebrafish , Zebrafish Proteins
8.
J Biol Chem ; 274(38): 26713-9, 1999 Sep 17.
Article in English | MEDLINE | ID: mdl-10480874

ABSTRACT

Transcriptional synergism between glucocorticoid receptor (GR) and octamer transcription factors 1 and 2 (Oct-1 and Oct-2) in the induction of mouse mammary tumor virus (MMTV) transcription has been proposed to be mediated through directed recruitment of the octamer factors to their binding sites in the viral long terminal repeat. This recruitment correlates with direct binding between the GR DNA binding domain and the POU domain of the octamer factors. In present study, in vitro experiments identified several nuclear hormone receptors to have the potential to bind to the POU domains of Oct-1 and Oct-2 through their DNA binding domains, suggesting that POU domain binding may be a property shared by many nuclear hormone receptors. However, physiologically relevant binding to the POU domain appeared to be a property restricted to only a few nuclear receptors as only GR, progesterone receptor (PR), and androgen receptor (AR), were found to interact physically and functionally with Oct-1 and Oct-2 in transfected cells. Thus GR, PR, and AR efficiently promoted the recruitment of Oct-2 to adjacent octamer motifs in the cell, whereas mineralocorticoid receptor (MR), estrogen receptor alpha, and retinoid X receptor failed to facilitate octamer factor DNA binding. For MMTV, although GR and MR both induced transcription efficiently, mutation of the promoter proximal octamer motifs strongly decreased GR-induced transcription without affecting the total level of reporter gene activity in response to MR. These results suggest that the configuration of the hormone response element within the MMTV long terminal repeat may promote a dependence for the glucocorticoid response upon the recruitment of octamer transcription factors to their response elements within the viral promoter.


Subject(s)
DNA-Binding Proteins/metabolism , Mammary Tumor Virus, Mouse/genetics , Promoter Regions, Genetic , Receptors, Glucocorticoid/metabolism , Receptors, Mineralocorticoid/metabolism , Transcription Factors/metabolism , Transcription, Genetic , Animals , Binding Sites , DNA, Viral/metabolism , Drosophila , Fibroblasts/metabolism , Host Cell Factor C1 , Mice , Octamer Transcription Factor-1 , Octamer Transcription Factor-2 , Protein Folding , Rabbits , Receptors, Retinoic Acid/metabolism , Retinoic Acid Receptor alpha , Transcriptional Activation
9.
Mol Cell Biol ; 18(6): 3416-30, 1998 Jun.
Article in English | MEDLINE | ID: mdl-9584182

ABSTRACT

Glucocorticoid receptor (GR) and octamer transcription factors 1 and 2 (Oct-1/2) interact synergistically to activate the transcription of mouse mammary tumor virus and many cellular genes. Synergism correlates with cooperative DNA binding of the two factors in vitro. To examine the molecular basis for these cooperative interactions, we have studied the consequences of protein-protein binding between GR and Oct-1/2. We have determined that GR binds in solution to the octamer factor POU domain. Binding is mediated through an interface in the GR DNA binding domain that includes amino acids C500 and L501. In transfected mammalian cells, a transcriptionally inert wild-type but not an L501P GR peptide potentiated transcriptional activation by Oct-2 100-fold above the level that could be attained in the cell by expressing Oct-2 alone. Transcriptional activation correlated closely with a striking increase in the occupancy of octamer motifs adjacent to glucocorticoid response elements (GREs) on transiently transfected DNAs. Intriguingly, GR-Oct-1/2 binding was interrupted by the binding of GR to a GRE. We propose a model for transcriptional cooperativity in which GR-Oct-1/2 binding promotes an increase in the local concentration of octamer factors over glucocorticoid-responsive regulatory regions. These results reveal transcriptional cooperativity through a direct protein interaction between two sequence-specific transcription factors that is mediated in a way that is expected to restrict transcriptional effects to regulatory regions with DNA binding sites for both factors.


Subject(s)
DNA-Binding Proteins/metabolism , DNA/metabolism , Receptors, Glucocorticoid/metabolism , Transcription Factors/metabolism , Animals , Binding Sites , Cells, Cultured , Host Cell Factor C1 , Mammary Tumor Virus, Mouse/genetics , Mice , Nucleic Acid Conformation , Octamer Transcription Factor-1 , Octamer Transcription Factor-2 , Point Mutation , Promoter Regions, Genetic , Repetitive Sequences, Nucleic Acid , Transcriptional Activation
10.
FEBS Lett ; 410(2-3): 397-402, 1997 Jun 30.
Article in English | MEDLINE | ID: mdl-9237670

ABSTRACT

Disulfide bridges were introduced into CrylAa, a Bacillus thuringiensis lepidopteran toxin, to stabilize different protein domains including domain I alpha-helical regions thought to be involved in membrane integration and permeation. Bridged mutants could not form functional ion channels in lipid bilayers in the oxidized state, but upon reduction with beta-mercaptoethanol, regained parental toxin channel activity. Our results show that unfolding of the protein around a hinge region linking domain I and II is a necessary step for pore formation. They also suggest that membrane insertion of the hydrophobic hairpin made of alpha-helices 4 and 5 in domain I plays a critical role in the formation of a functional pore.


Subject(s)
Bacillus thuringiensis/metabolism , Bacterial Proteins/metabolism , Bacterial Toxins/metabolism , Disulfides/metabolism , Endotoxins/metabolism , Bacillus thuringiensis Toxins , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Toxins/chemistry , Bacterial Toxins/genetics , Binding Sites , Disulfides/chemistry , Endotoxins/chemistry , Endotoxins/genetics , Hemolysin Proteins , Models, Molecular , Mutagenesis, Site-Directed , Protein Engineering
11.
J Biol Chem ; 272(9): 5647-58, 1997 Feb 28.
Article in English | MEDLINE | ID: mdl-9038175

ABSTRACT

NRE1 is a DNA sequence element through which Ku antigen/DNA-dependent protein kinase (DNA-PK) catalytic subunit represses the induction of mouse mammary tumor virus transcription by glucocorticoids. Although Ku is an avid binder of DNA ends and has the ability to translocate along DNA, we report that direct sequence-specific Ku binding occurs with higher affinity (Kd = 0.84 +/- 0.24 nM) than DNA end binding. Comparison of Ku binding to several sequences over which Ku can accumulate revealed two classes of sequence. Sequences with similarity to NRE1 competed efficiently for NRE1 binding. Conversely, sequences lacking similarity to NRE1 competed poorly for Ku and were not recognized in the absence of DNA ends. Phosphorylation of glucocorticoid receptor (GR) fusion proteins by DNA-PK reflected Ku DNA-binding preferences and demonstrated that co-localization of GR with DNA-PK on DNA in cis was critical for efficient phosphorylation. Phosphorylation of the GR fusion protein by DNA-PK mapped to a single site, Ser-527. This site occurs adjacent the GR nuclear localization sequence between the DNA and ligand binding domains of GR, and thus its phosphorylation, if confirmed, has the potential to affect receptor function in vivo.


Subject(s)
Antigens, Nuclear , DNA Helicases , DNA Repair , DNA-Binding Proteins/metabolism , DNA/metabolism , Nuclear Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Receptors, Glucocorticoid/metabolism , Transcription Factors/metabolism , Animals , Binding, Competitive , DNA-Activated Protein Kinase , Ku Autoantigen , Mice , Peptide Mapping , Phosphorylation , Rats , Serine , Structure-Activity Relationship
12.
Nature ; 380(6571): 265-8, 1996 Mar 21.
Article in English | MEDLINE | ID: mdl-8637578

ABSTRACT

DNA-dependent protein kinase (DNA-PK) has been implicated in several nuclear processes including transcription, DNA replication, double-stranded DNA break repair, and V(D)J recombination. Linkage of kinase and substrate on DNA in cis is required for efficient phosphorylation. Recruitment of DNA-PK to DNA is by Ku autoantigen, a DNA-end-binding protein required for DNA-PK catalytic activity. Although Ku is known to translocate along naked DNA, how DNA-end binding by Ku might lead to DNA-PK-mediated phosphorylation of sequence-specific DNA-binding proteins in vivo has not been obvious. Here we report the identification of Ku as a transcription factor that recruits DNA-PK directly to specific DNA sequences. NRE1 (negative regulatory element 1) is a DNA sequence element (-394/ -381) in the long terminal repeat of mouse mammary tumour virus (MMTV) that is important for repressing inappropriate viral expression. We show that direct binding of Ku/DNA-PK to NRE1 represses glucocorticoid-induced MMTV transcription.


Subject(s)
Antigens, Nuclear , Autoantigens/metabolism , DNA Helicases , DNA, Viral/metabolism , DNA-Binding Proteins/metabolism , Nuclear Proteins/metabolism , Transcription Factors/metabolism , Transcription, Genetic , Animals , Base Sequence , Cell Line , Cricetinae , Cricetulus , DNA-Activated Protein Kinase , Ku Autoantigen , Mammary Tumor Virus, Mouse/genetics , Mice , Mice, SCID , Molecular Sequence Data , Phosphorylation , Plasmids , Protein Binding , Protein Serine-Threonine Kinases/metabolism , Repetitive Sequences, Nucleic Acid
13.
Appl Environ Microbiol ; 59(1): 114-9, 1993 Jan.
Article in English | MEDLINE | ID: mdl-8439143

ABSTRACT

Arbitrary primer polymerase chain reaction technology has been applied to the identification of commercial strains of Bacillus thuringiensis by using total DNAs extracted from single bacterial colonies as templates. Characteristic DNA banding patterns can be readily and reproducibly obtained by agarose gel electrophoresis. This method has been used to distinguish commercial products containing B. thuringiensis serovar kurstaki (3a3b). When a single primer was used this method was capable of producing discriminating DNA fingerprints for 33 known serovars. Differentiation from the closely related species Bacillus cereus is also readily achieved. This technique should prove to be a powerful tool for identification and discrimination of individual B. thuringiensis strains.


Subject(s)
Bacillus thuringiensis/classification , Bacillus thuringiensis/isolation & purification , Polymerase Chain Reaction , Bacillus cereus/classification , Bacillus cereus/genetics , Bacillus cereus/isolation & purification , Bacillus thuringiensis/genetics , Base Sequence , DNA Fingerprinting , DNA, Bacterial/chemistry , Genes, Bacterial , Molecular Sequence Data , Polymerase Chain Reaction/methods , Sequence Deletion , Serotyping , Species Specificity
14.
Biochem J ; 269(2): 507-12, 1990 Jul 15.
Article in English | MEDLINE | ID: mdl-2167072

ABSTRACT

Two commercially important strains (NRD-12 and HD-1) of the entomopathogenic bacterium Bacillus thuringiensis subsp. kurstaki each contain three genes of partially identical sequence coding for three classes of 130-135 kDa protoxins (termed the 4.5, 5.3 and 6.6 protoxins) that display toxicity towards various lepidopteran larvae. These gene products combine to form the intracellular bipyramidal P1 crystal. Each of the genes from both strains was cloned and expressed in Escherichia coli. Analysis of the cloned genes at the restriction-endonuclease level revealed no detectable differences among genes within a particular gene class. The composition of the P1 crystal from both strains was quantitatively analysed by CNBr cleavage of the purified P1 crystal, with the purified recombinant gene products as reference proteins. Independent verification of the presence of high 6.6-protoxin gene product in the P1 crystal was provided by a rapid in vitro lawn cell toxicity assay directed against a Choristoneura fumiferana (CF-1) insect cell line. The results indicate that, although all three gene products are represented within the P1 crystal of either NRD-12 or HD-1, only the contents of the 4.5 and 5.3 protoxins vary between the two crystals, whereas the 6.6 protoxin contents are similar and represent approximately one-third of the P1 crystal in either strain.


Subject(s)
Bacillus thuringiensis/genetics , Bacterial Toxins/genetics , Protein Precursors/genetics , Animals , Bacterial Toxins/analysis , Bacterial Toxins/pharmacology , Cells, Cultured , Cloning, Molecular , Crystallization , Cyanogen Bromide , DNA Restriction Enzymes , Escherichia coli/genetics , Gene Expression , Genes, Bacterial , Insecticides , Lepidoptera/drug effects , Peptide Fragments/isolation & purification , Protein Precursors/analysis , Protein Precursors/pharmacology , Recombinant Proteins/analysis , Recombinant Proteins/pharmacology
15.
FEMS Microbiol Lett ; 51(3): 273-7, 1989 Aug.
Article in English | MEDLINE | ID: mdl-2583476

ABSTRACT

A highly efficient procedure for the transformation of Bacillus thuringiensis and Bacillus subtilis using covalently closed circular plasmid DNA was developed by using the small Staphylococcus aureus plasmid pC194 and electroporation. We have achieved transformation efficiencies in B. thuringiensis subsp. kurstaki (HD-73) greater than 5 x 10(6) transformants/micrograms plasmid DNA. The electro-transformation (or electroporation) procedure also worked with B. subtilis 168 although at a 200-fold less level of efficiency. The results indicated that the plasmid exists in double and single-stranded forms both in B. subtilis and B. thuringiensis. A second single-stranded species was also observed in both species. This technique may prove to be applicable to other members of the genus Bacillus.


Subject(s)
Bacillus thuringiensis/genetics , DNA, Bacterial/analysis , Electrochemistry , Transformation, Genetic , Blotting, Southern , DNA, Bacterial/isolation & purification , Plasmids
17.
Appl Environ Microbiol ; 53(12): 2808-14, 1987 Dec.
Article in English | MEDLINE | ID: mdl-2829720

ABSTRACT

Fifteen Bacillus thuringiensis strains representing 13 serotypes were screened with five oligodeoxyribonucleotide probes specific for certain regions of two published sequences and one unpublished sequence of B. thuringiensis delta-endotoxin genes. Of the 15 cultures, 14 hybridized with at least one probe; the B. thuringiensis subsp. thompsoni strain alone did not hybridize. Two B. thuringiensis subsp. kurstaki strains of commercial interest, HD-1 and NRD-12, were found to be so closely related as to be indistinguishable with this technique; the same situation was found with strains from B. thuringiensis subspp. dendrolimus and sotto. Five strains were identified as probably containing only one endotoxin gene. A probe specific for the gene from the B. thuringiensis subsp. kurstaki HD-73 strain hybridized to only 3 of the 15 cultures tested. The hybridization data suggest that the DNA sequences coding for the C-terminal region of the endotoxin protein are as well conserved as those coding for the N-terminal toxic portion.


Subject(s)
Bacillus thuringiensis/genetics , Bacterial Proteins , Bacterial Toxins , Endotoxins/genetics , Genes, Bacterial , Oligonucleotides/genetics , Bacillus thuringiensis Toxins , DNA Restriction Enzymes , DNA, Bacterial/genetics , Hemolysin Proteins , Nucleic Acid Hybridization , Sequence Homology, Nucleic Acid
18.
Gene ; 30(1-3): 247-50, 1984 Oct.
Article in English | MEDLINE | ID: mdl-6096218

ABSTRACT

A family of universal expression vectors based on the pUC8 and pUC9 plasmids has been constructed. These vectors cover all three possible reading frames in both directions, allowing any synthetic DNA, genomic DNA or cDNA to be expressed under control of the lac promoter. The four new vectors retain the useful features of the pUC plasmids, including the blue to white color change on X-gal plates indicating the presence of an insert. This family of expression vectors is expected to be quite useful in allowing direct immunological screening of cDNA or genomic DNA banks.


Subject(s)
Cloning, Molecular , Genetic Vectors , Plasmids , Escherichia coli/genetics , Lac Operon
19.
Can J Biochem Cell Biol ; 62(4): 209-16, 1984 Apr.
Article in English | MEDLINE | ID: mdl-6722637

ABSTRACT

A 355 base pair (bp) DNA sequence coding for human preproinsulin has been assembled by joining a synthetic DNA leader sequence coding for 24 preregion amino acids to the previously synthesized DNA duplex of 277 bp constituting the sequence of BCA chain. It was next cloned in M13 mp8 single-stranded bacteriophage and subjected to site-specific mutagenesis and phase shifting to allow its inducible expression under lac operator control. An affinity leader sequence of 25 bp has been added in an attempt to facilitate purification of the preproinsulin.


Subject(s)
Proinsulin/genetics , Protein Precursors/genetics , Bacteriophages/metabolism , Base Sequence , Chemical Phenomena , Chemistry , Cloning, Molecular , Gene Expression Regulation , Humans , Insulin , Mutation , Proinsulin/biosynthesis , Protein Precursors/biosynthesis
20.
Gene ; 27(2): 201-11, 1984 Feb.
Article in English | MEDLINE | ID: mdl-6373502

ABSTRACT

A 74-bp DNA sequence coding for the pre sequence of human preproinsulin and containing EcoRI termini was synthesized by the chemical enzymatic method, joined with previously synthesized proinsulin DNA, and cloned in the M 13mp8 vector. A clone pNB82 -121 was identified by DNA sequence which confirmed the correct orientation of the pre sequence to the proinsulin DNA. The EcoRI site at the junction of pre- and proinsulin DNA was eliminated by removing a triplet ATT using a synthetic 19-mer primer. To simplify preproinsulin isolation and to study its expression in the M 13 system, a 25-bp affinity leader sequence coding for (glu)7 was inserted at the remaining EcoRI site; this put the preproinsulin DNA in a correct reading frame with the AUG initiation codon of beta-galactosidase. Preproinsulin was expressed under lac promoter control as analyzed by a radioimmunoassay (RIA) against C-peptide.


Subject(s)
DNA/chemical synthesis , Genes, Synthetic , Proinsulin/genetics , Protein Precursors/genetics , Base Sequence , C-Peptide/analysis , Cloning, Molecular , Coliphages/genetics , Escherichia coli/genetics , Gene Expression Regulation , Insulin , Mutation , Radioimmunoassay
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