Differential expression of the msp130 gene among skeletal lineage cells in the sea urchin embryo: a three dimensional in situ hybridization analysis.

In order to examine the ontogeny of tissue-specific expression of the msp130 gene during early embryogenesis of the sea urchin, we have developed a whole-mount, non-radioactive in situ hybridization protocol suitable for these embryos. This protocol is adapted from the existing technology of immunohistochemical localization of digoxygenin-labelled hybridization probes in tissue sections. Transcript distribution patterns in the whole embryo are seen in three dimensions, and at much higher resolution and sensitivity than can be achieved using radioactive probes and sectioned material. We have traced the ontogeny of expression of the skeleton-specific gene, msp130, during the development of Strongylocentrotus purpuratus. Transcripts are first detected at the blastula stage, in micromere-lineage cells just prior to ingression. Appearance of msp130 transcripts remains strictly limited to this lineage through the pluteus stage. Estimated from the relative intensity of staining of the PMCs of an embryo, the relative abundance of msp130 transcripts is uniform among the 32 cells of this lineage in secondary mesenchyme blastulae and in gastrulae, indicating that expression is homogeneous among these cells up to the early prism stage. However, the relative intensity of stain, and therefore abundance of transcripts, changes dramatically and in a consistent pattern among the PMCs of an embryo during prism and pluteus stages, suggesting that these cells switch from an autonomous mode of regulation of the msp130 gene, to an inductive mode. In the pluteus larva, the highest levels of expression occur in those cells associated with the rapidly growing tips of the spicular skeleton.

Three classes of homologous Bacillus thuringiensis crystal-protein genes.

Four homologous genes encoding insecticidal crystal proteins from Bacillus thuringiensis have been cloned in Escherichia coli. Differences in lengths of HindIII restriction fragments containing the 5' ends of the genes allowed the identification of three classes termed the '4.5- and 5.3- and 6.6-kb-class genes'. A survey of 24 strains from subspecies kurstaki and thuringiensis revealed strains containing one, two, or three of these classes of crystal protein genes. The 4.5- and 6.6-kb-class genes encode polypeptides of Mr 133,500 and 133,330, respectively, while the two 5.3-kb-class genes encode a ca. 130-kDa polypeptide. The polypeptide composition of crystals from the B. thuringiensis strains agreed with the composition predicted from the content of the three classes of genes. The representative genes from each class were isolated from B. thuringiensis plasmids of different sizes. An analysis of plasmid DNA flanking three of these genes revealed a complex pattern of two different inverted repeat (IR) sequences, IR2150 and IR1750. Four variant forms of IR1750 were found. The IR elements were located near deletions and rearrangements adjacent to crystal protein genes and may account for the diversity of plasmids carrying crystal protein genes in other subspecies of B. thuringiensis.

Isolation of a Bacillus thuringiensis RNA polymerase capable of transcribing crystal protein genes.

We report the isolation of an RNA polymerase from sporulating cells of Bacillus thuringiensis subsp. kurstaki HD-1-Dipel that directs transcription from the promoter region of an insecticidal crystal protein gene. The core components of this RNA polymerase are associated with a polypeptide that has an apparent mass of 35 kDa. Neither RNA polymerase holoenzyme isolated from vegetative B. thuringiensis, nor the core derived from this enzyme, is capable of transcribing from the crystal protein gene promoter region; the addition of gel-purified 35-kDa polypeptide to the core reconstitutes the specific transcribing capability. The reconstituted enzyme does not direct transcription from the promoters for the ctc or spoVG genes of Bacillus subtilis; however, this form of RNA polymerase does direct transcription from a promoter for the 27-kDa crystal protein of B. thuringiensis subsp. israelensis and from a promoter for a 29-kDa polypeptide present in cuboidal crystals of B. thuringiensis subsp. kurstaki HD-1. We propose a tentative consensus sequence based on the alignment of the three B. thuringiensis promoters. This consensus sequence is different from consensus sequences reported for promoters recognized by enzymes containing other sigma subunits, suggesting that the 35-kDa polypeptide is an unusual sigma subunit.

Insecticidal crystal proteins of Bacillus thuringiensis.

A classification for crystal protein genes of Bacillus thuringiensis is presented. Criteria used are the insecticidal spectra and the amino acid sequences of the encoded proteins. Fourteen genes are distinguished, encoding proteins active against either Lepidoptera (cryI), Lepidoptera and Diptera (cryII), Coleoptera (cryIII), or Diptera (cryIV). One gene, cytA, encodes a general cytolytic protein and shows no structural similarities with the other genes. Toxicity studies with single purified proteins demonstrated that every described crystal protein is characterized by a highly specific, and sometimes very restricted, insect host spectrum. Comparison of the deduced amino acid sequences reveals sequence elements which are conserved for Cry proteins. The expression of crystal protein genes is affected by a number of factors. Recently, two distinct sigma subunits regulating transcription during different stages of sporulation have been identified, as well as a protein regulating the expression of a crystal protein at a posttranslational level. Studies on the biochemical mechanisms of toxicity suggest that B. thuringiensis crystal proteins induce the formation of pores in membranes of susceptible cells. In vitro binding studies with radiolabeled toxins demonstrated a strong correlation between the specificity of B. thuringiensis toxins and the interaction with specific binding sites on the insect midgut epithelium. The expression of B. thuringiensis crystal proteins in plant-associated microorganisms and in transgenic plants has been reported. These approaches are potentially powerful strategies for the protection of agriculturally important crops against insect damage.

Two highly related insecticidal crystal proteins of Bacillus thuringiensis subsp. kurstaki possess different host range specificities.

Two genes encoding insecticidal crystal proteins from Bacillus thuringiensis subsp. kurstaki HD-1 were cloned and sequenced. Both genes, designated cryB1 and cryB2, encode polypeptides of 633 amino acids having a molecular mass of ca. 71 kilodaltons (kDa). Despite the fact that these two proteins display 87% identity in amino acid sequence, they exhibit different toxin specificities. The cryB1 gene product is toxic to both dipteran (Aedes aegypti) and lepidopteran (Manduca sexta) larvae, whereas the cryB2 gene product is toxic only to the latter. DNA sequence analysis indicates that cryB1 is the distal gene of an operon which is comprised of three open reading frames (designated orf1, orf2, and cryB1). The proteins encoded by cryB1 and orf2 are components of small cuboidal crystals found in several subspecies and strains of B. thuringiensis; it is not known whether the orf1 or cryB2 gene products are present in cuboidal crystals. The protein encoded by orf2 has an electrophoretic mobility corresponding to a molecular mass of ca. 50 kDa, although the gene has a coding capacity for a polypeptide of ca. 29 kDa. Examination of the deduced amino acid sequence for this protein reveals an unusual structure which may account for its aberrant electrophoretic mobility: it contains a 15-amino-acid motif repeated 11 times in tandem. Escherichia coli extracts prepared from cells expressing only orf1 and orf2 are not toxic to either test insect.

Activity of uridine diphosphate N-acetylglucosamine-4-epimerase during spherulation of Physarum polycephalum.

The specific activity of uridine diphosphate N-acetylglucosamine-4-epimerase increases during spherulation of Physarum polycephalum, a process that involves the synthesis of galactosamine walls. This increase is prevented by the addition of cycloheximide.

Deoxyribonucleic acid-dependent ribonucleic acid polymerases in the dimorphic fungus Mucor rouxii.

Three forms of DNA-dependent RNA polymerase have been separated by chromatography of extracts of yeast-like cells and mycelium of the dimorphic fungus Mucor rouxii. Each of the three eznymes has been purified by means of protamine sulfate precipitation, ion exchange chromatography, affinity chromatography, and velocity sedimentation. Electrophoresis under denaturing conditions showed differences in the subunit compositions of all three purified enzymes. The properties of the enzymes from M. rouxii were similar to those of polymerases from other eukaryotic organisms. Denatured DNA was a better template than native DNA for all three enzymes but each enzyme had a distinct pattern of activities with different templates. Enzymes I and III displayed optimal activity with Mn-2gs the divalent cation and were stimulated significantly by Kcl and (NH4)2S04. Enzyme II had a greater activity with Mg-2gnd was only slightly stimulated by KCl and (NH4)2SO4. None of the enzymes were inhibited by cycloheximide or by rifampicin: all were inhibited by actinomycin C and rifampin AF/018: only enzyme II was inhibited by alpha-amanitin. No differences could be found in the properties of the same enzymes isolated from yeast-like cells or mycelium.

Molecular characterization of two novel crystal protein genes from Bacillus thuringiensis subsp. thompsoni.

Two genes encoding the predominant polypeptides of Bacillus thuringiensis subsp. thompsoni cuboidal crystals were cloned in Escherichia coli and sequenced. The polypeptides have electrophoretic mobilities of 40 and 34 kDa, with the deduced amino acid sequences predicting molecular masses of 35,384 and 37,505 Da, respectively. No statistically significant similarities were detected between the 40- or 34-kDa crystal protein and any other characterized B. thuringiensis crystal protein, nor were they detected between the 40- and 34-kDa crystal proteins. A 100-MDa plasmid carries both crystal protein genes, which appear to be part of an operon, with the 40-kDa gene 64 nucleotides upstream of the 34-kDa gene. Both crystal proteins are synthesized in approximately the same amounts. Even though small compared with other crystal proteins, the 34-kDa crystal protein has insecticidal activity against lepidopteran larvae (Manduca sexta). The 40-kDa polypeptide appears to have no insecticidal activity, but it could have a role in crystal structure.

The role of the delta peptide of the Bacillus subtilis RNA polymerase in promoter selection.

We have examined the effect of the delta subunit of the Bacillus subtilis RNA polymerase on the formation of closed, open, and stably initiated complexes with Hha I restriction fragments of phage SP82 DNA; the effect of delta on the transcription of these DNA fragments has also been investigated. In vitro, the holoenzyme (core-sigma-delta) bound to and transcribed the same regions of the phage genome that are transcribed in vivo early in infection. In the absence of the delta subunit, the polymerase (core-sigma) bound nonspecifically and transcribed regions of the genome other than those containing early phage genes. Addition of delta to preparations of core-sigma restored the pattern of binding and transcription observed with the holoenzyme. Similarly, delta-less preparations of two SP82-modified forms of polymerase (the enzyme isolated at 8 min after infection and the enzyme isolated 20 min after infection) bound nonspecifically and transcribed regions of the genome other than those containing "middle" and "late" genes. Addition of delta to these preparations resulted in patterns of binding and transcription expected for enzymes functioning a middle and late times of infection, respectively. Quantitation of polymerase-DNA complexes at various temperatures, NaCl concentrations, and polymerase-DNA ratios supported the conclusion that delta enhanced promoter selection.

Early RNAs in SP82- and SP01-infected Bacillus subtilis may be processed.

Transcription of SP82 and SP01 DNAs in vitro by Bacillus subtilis RNA polymerase yielded mostly large RNA species, with many in excess of 1,500 bases in length, whereas most of the RNAs synthesized in vivo early in infection were much smaller. Addition of an extract from uninfected B. subtilis to reaction mixtures containing RNAs synthesized in vitro generated additional discrete RNAs whose mobilities on polyacrylamide gels matched the mobilities of some of the smaller RNAs synthesized in vivo.

Cloning and expression of the Bacillus thuringiensis crystal protein gene in Escherichia coli.

Sau 3A1 partial digestion fragments from Bacillus thuringiensis var. kurstaki HD-1 plasmid DNA were ligated into the BamHI site of the cloning vector pBR322 and transformed into Escherichia coli strain HB101. Colonies presumed to contain recombinant plasmids were screened for production of an antigen that would react with antibody made against B. thuringiensis crystals. One strain, ES12, was isolated by using this procedure. ES12 contains a plasmid of Mr 11 X 10(6) that has DNA sequence homology with pBR322 as well as with Mr 30 X 10(6) and Mr 47 X 10(6) plasmids of B. thuringiensis. It makes a protein antigen, detected by antibodies to crystal, which has the same electrophoretic mobility as the B. thuringiensis crystal protein. Protein extracts of ES12 are toxic to larvae of the tobacco hornworm Manduca sexta.

Analysis of bacteriophage SP82 major "early" in vitro transcripts.

A restriction map was constructed for the 17.5-kilobase SalI C fragment of SP82 DNA. Unfractionated SP82 DNA, the SalI C fragment, and restriction fragments derived from SalI-C were used as templates for in vitro synthesis by the Bacillus subtilis RNA polymerase, and the resulting transcripts were analyzed by gel electrophoresis. Comparison of the RNA species obtained from SalI-C with those produced from unfractionated DNA indicated that most of the RNAs and all of the major transcripts originate from the SalI C fragment; this fragment contains one copy of the terminally redundant portion of the genome. Seven major transcripts, a bidirectional terminator site, and 5 of the 13 minor transcripts were located within the 13-kilobase redundant region. The binding of polymerase to fragments of DNA produced by digestion of SalI-C with HpaII and HhaI was used to identify promoter-bearing regions within SalI-C.

Effect of a 20-kilodalton protein from Bacillus thuringiensis subsp. israelensis on production of the CytA protein by Escherichia coli.

CytA, a 27-kDa cytolytic crystal protein of Bacillus thuringiensis subsp. israelensis, is produced only at very low levels by recombinant Escherichia coli cells unless a 20-kDa B. thuringiensis subsp. israelensis protein is also present (K. M. McLean and H. R. Whiteley, J. Bacteriol. 169:1017-1023, 1987; L. F. Adams, J. E. Visick, and H. R. Whiteley, J. Bacteriol. 171:521-530, 1989). However, the data reported here demonstrate that the 20-kDa protein is not required for high-level CytA production in E. coli strains carrying mutations in rpoH, groEL, or dnaK, all of which affect the proteolytic ability of the cells. The 20-kDa protein also increases the amount of CryIVD (another B. thuringiensis subsp. israelensis crystal protein) and LacZX90 (a mutant of beta-galactosidase) made by E. coli. The latter phenomenon is attributable to an increase in the half-life of LacZX90, suggesting that the 20-kDa protein may stabilize this protein. The effect of the 20-kDa protein was also examined in vitro and in a T7 RNA polymerase expression system, and the possible significance of these results for the timing of proteolysis and of 20-kDa protein activity is discussed. Finally, the ability of a single antibody to coimmunoprecipitate CytA and the 20-kDa protein from E. coli extracts provides evidence for a protein-protein interaction that may be related to the mechanism of action of the 20-kDa protein.

Translation of RNAs synthesized in vivo and in vitro from bacteriophage SP82 DNA.

The synthesis of 69 phage-specific polypeptides during the infection of Bacillus subtilis with bacteriophage SP82 was detected by pulse-labeling, one-dimensional electrophoresis, and autoradiography. SP82 virions were found to contain approximately 22 polypeptides, most of which were synthesized late in infection; evidence was obtained for the processing of the major virion protein. RNAs extracted at different times during infection were translated by using an Escherichia coli cell-free extract. Only smaller-molecular-weight peptides were produced efficiently in vitro; in the 9,000- to 60,000-molecular-weight range, 50 to 60% of the peptides synthesized in vivo were produced by translation of RNAs extracted from infected cells. Eight of the virion peptides were produced by in vitro translation of RNAs extracted from infected cells. RNAs were synthesized under defined conditions by RNA polymerase extracted from uninfected B. subtilis and by polymerases isolated from cells 8 and 20 min after infection with SP82. Translation of these RNAs yielded characteristic and different patterns of polypeptides. Nine of the 12 polypeptides produced by translation of RNAs synthesized by the host polymerase corresponded in mobility to peptides appearing in vivo in the 0 to 3 and 3 to 6 min intervals of pulse-labeling after infection; 12 of the 25 peptides synthesized from RNAs produced by polymerase extracted 8 min after infection corresponded in mobility to peptides detected in vivo 8 min after infection, and 15 of the 22 peptides directed by RNAs made by the polymerase isolated 20 min after infection corresponded to peptides present in vivo late in infection. Five of the peptides produced in vitro from the latter RNA corresponded to virion peptides.

Inverted repeat sequences flank a Bacillus thuringiensis crystal protein gene.

Two sets of inverted repeat DNA sequences, IR2150 and IR1750, were discovered flanking the crystal protein gene on the 75-kilobase plasmid of Bacillus thuringiensis subsp. kurstaki HD73. A restriction map of ca. 40 kilobases around the crystal protein gene was constructed, and the positions of the copies of IR2150 and IR1750 were determined. Three copies of IR2150 were found flanking the crystal protein gene in an inverted orientation, and one partial and three intact copies of IR1750 were found in both inverted and direct orientations around the gene. Hybridization experiments with fragments from within IR2150 and IR1750 demonstrated the presence of multiple copies of these sequences on the chromosome of B. thuringiensis subsp. kurstaki HD73 and also revealed a strong correlation between the presence of these sequences and the presence of the crystal protein gene on plasmids from 14 strains of B. thuringiensis.

UV cross-linking of the Bacillus subtilis RNA polymerase to DNA in promoter and non-promoter complexes.

Complexes between Bacillus subtilus RNA polymerase and 32P-labeled DNA were irradiated with UV light and digested with nuclease; electrophoresis and autoradiography were used to identify the polymerase subunits cross-linked to DNA. These experiments showed: 1) that cross-linkage of promoter complexes yielded predominantly the beta and sigma subunits; 2) that beta, beta', and sigma were detected in non-promoter complexes; 3) that addition of the delta subunit or high concentrations of NaCl decreased cross-linkage of all subunits, especially the cross-linkage of the sigma subunit in non-promoter complexes and the binding of polymerase at DNA ends; 4) that different patterns of cross-linkage were obtained at 0 degrees C (conditions favoring the formation of closed complexes) and 37 degrees C (conditions favoring the formation of open complexes); and 5) predominantly beta and possibly alpha were cross-linked by irradiation of core-DNA complexes whereas similar experiments with core-delta complexed to DNA showed the efficient cross-linkage of beta' and beta.

Delineation of a toxin-encoding segment of a Bacillus thuringiensis crystal protein gene.

Crystals of Bacillus thuringiensis subsp. kurstaki HD-1-Dipel contain a Mr 134,000 protoxin which can be cleaved by proteolysis to a peptide of Mr approximately 70,000; this peptide is lethal to lepidopteran larvae. We have analyzed the peptides produced by recombinant Escherichia coli strains bearing deletions and fusions of the protoxin gene in order to delineate the portion of the gene which encodes the toxic peptide. The recombinant strains produced the toxic peptide as well as larger peptides whose size was related to the length of the deleted gene. The results indicate that the amino-terminal 55% of the protoxin protein is sufficient for toxicity. While two different gene fusions to the 10th codon allowed the synthesis of toxic polypeptides, fusions to the 50th codon did not. 3' end deletions up to the 645th codon allowed synthesis of the toxic peptide whereas a deletion to the 603rd codon yielded a non-toxic peptide. Some of the 5' and 3' end alterations to the gene caused changes in the proteolytic cleavage patterns of the polypeptides synthesized by E. coli, suggesting that the alterations led to conformational changes in the proteins. The presence of different 3' end segments affected the levels of synthesis of the altered crystal proteins.

Location of the dipteran specificity region in a lepidopteran-dipteran crystal protein from Bacillus thuringiensis.

Two highly related crystal protein genes from Bacillus thuringiensis subsp. kurstaki HD-1, designated cryIIA and cryIIB (previously named cryB1 and cryB2, respectively), were used to study host range specificity. Their respective gene products are 87% identical but exhibit different toxicity spectra; CryIIA is toxic to both mosquito and tobacco hornworm larvae, whereas CryIIB is toxic only to the latter. Hybrids of the cryIIA and cryIIB genes were generated, and their resultant gene products were assayed for toxicity. A short segment of CryIIA corresponding to residues 307 through 382 was shown to be sufficient for altering host range specificity-i.e., when this region replaced the corresponding segment of CryIIB, the resulting hybrid protein acquired toxicity against mosquitoes. The CryIIA and CryIIB polypeptides differ by only 18 amino acids in this region, indicating that very few amino acid changes can have a substantial effect on the toxicity spectra of these proteins.

Isolation of the second Bacillus thuringiensis RNA polymerase that transcribes from a crystal protein gene promoter.

A crystal protein gene of Bacillus thuringiensis subsp. kurstaki HD-1-Dipel is transcribed in vivo from two overlapping promoters that are activated at different times during sporulation. We reported earlier (K. L. Brown and H. R. Whiteley, Proc. Natl. Acad. Sci. USA 85:4166-4170, 1988) that an RNA polymerase containing a sigma subunit with an apparent Mr of 35,000 can transcribe in vitro from the promoter utilized from early to midsporulation. We now report the isolation of an RNA polymerase containing a sigma subunit with an Mr of ca. 28,000; this polymerase activates transcription in vitro from the promoter used from mid- to late sporulation. This form of RNA polymerase also directs transcription in vitro from promoters preceding two other crystal protein genes and a gene coding for a spore coat protein. On the basis of a comparison of the four promoters, we propose the following consensus sequence for the -10 region recognized by RNA polymerase containing the Mr-28,000 sigma subunit: 5'-TNATANNaTGag-3'. No consensus sequence could be derived for the -35 region. When the N-terminal amino acid sequence of the sigma 28 polypeptide was aligned with the amino acid sequences of known sigma subunits, significant homology was found with the N terminus of the mature form of the sigma K subunit of RNA polymerase isolated from sporulating cells of Bacillus subtilis.

Expression in Escherichia coli of a cloned crystal protein gene of Bacillus thuringiensis subsp. israelensis.

A ca. 10-kilobase (kb) HindIII fragment of plasmid DNA from Bacillus thuringiensis subsp. israelensis was cloned into plasmid pUC9 and transformed into Escherichia coli. Extracts of the recombinant strain contained a 27-kilodalton (kDa) peptide that reacted with antibodies to a 27-kDa peptide isolated from crystals produced by B. thuringiensis subsp. israelensis. Extracts of the recombinant strain were hemolytic and toxic to Aedes aegypti larvae. Full expression of the 27-kDa peptide required the presence of a ca. 0.8-kb region of DNA located 4 kb upstream from the structural gene; the 0.8-kb region could be present in cis or trans relative to the gene and apparently acted post-transcriptionally. Analysis of maxicells showed that the 10-kb insert also coded for peptides of 67, 20, and 16 kDa; data obtained with different subclones suggest that the 20-kDa peptide is encoded in the 0.8-kb DNA region.