Identification and characterization of a silencer regulatory element in the 3'-flanking region of the murine CD46 gene.

The murine membrane cofactor protein (CD46) gene is expressed exclusively in testis, in contrast to human CD46, which is expressed ubiquitously. To elucidate the mechanism of differential CD46 gene expression among species, we cloned entire murine CD46 genomic DNA and possible regulatory regions were placed in the flanking region of the luciferase reporter gene. The reporter gene assay revealed a silencing activity not in the promoter, but in the 3'-flanking region of the gene and the silencer-like element was identified within a 0.2-kb region between 0.6 and 0.8 kb downstream of the stop codon. This silencer-like element was highly similar to that of the pig MHC class-I gene. The introduction of a mutation into this putative silencer element of murine CD46 resulted in an abrogation of the silencing effect. Electrophoretic mobility-shift assay indicated the presence of the binding molecule(s) for this silencer sequence in murine cell lines and tissues. A size difference of the protein-silencer-element complex was observed depending upon the solubilizers used for preparation of the nuclear extracts. A mutated silencer sequence failed to interact with the binding molecules. The level of the binding factor was lower in the testicular germ cells compared with other organs. Thus the silencer element and its binding factor may play a role in transcriptional regulation of murine CD46 gene expression. These results imply that the effects of the CD46 silencer element encompass the innate immune and reproductive systems, and in mice may determine the testicular germ-cell-dominant expression of CD46.

The agrobacterium tumefaciens C58-6b gene confers resistance to N(6)-benzyladenine without modifying cytokinin metabolism in tobacco seedlings

The 6b gene of Agrobacterium tumefaciens has been demonstrated to modify the activity of the plant growth regulators, auxin and cytokinin. To study the possible mode of action of the gene, tobacco (Nicotiana tabacum L. cv. Samsun) plants were transformed with the A. tumefaciens C58-6b gene. Seeds obtained from morphologically normal transgenic as well as wild-type plants were germinated on media supplemented with growth-inhibitory levels of cytokinin, N(6)-benzyladenine (BA). The transgenic seedlings showed increased resistance to cytokinins, as reflected by continuous shoot development, whereas further growth of the wild-type plants beyond the cotyledonary stage was inhibited. Concurrently, the levels of 6b gene transcripts in transgenic seedlings increased greatly upon BA treatment. Since glucosylation of BA represents the main inactivation mechanism of the hormone, we analyzed BA glucoside formation during the early stages of seedling growth. Intracellular levels of the major BA metabolite, N(6)-benzyladenine-7-glucoside (80-92%), as well as other BA-derived components were found to be comparable in transgenic and wild-type seedlings. Therefore, increased resistance of the C58-6b transgenic seedlings to cytokinins could not be directly attributed to enhanced BA glucosylation and subsequent hormone inactivation.

A novel glycine-rich/hydrophobic 16 kDa polypeptide gene from tobacco: similarity to proline-rich protein genes and its wound-inducible and developmentally regulated expression.

We have isolated a cDNA clone, NT16, encoding a novel glycine-rich/hydrophobic protein from tobacco crown gall tumor tissues, which was induced by the T-DNA genes of Agrobacterium tumefaciens. The accumulation of NT16 transcripts was high in unorganized callus as well as in shoot-forming calli. In normal tobacco plants, the transcript levels were high in roots, and low in stems, whereas virtually no transcript accumulation was found in flowers or leaves. In leaves, however, NT16 transcript accumulation was induced by mechanical wounding. These results show that NT16 expression is developmentally regulated and induced by wound-stress conditions. Sequence analysis suggests that NT16 encodes a putative 16 kDa polypeptide that is apparently composed of 3 structural domains: two hydrophobic regions separated by a glycine-rich region. The NT16 polypeptide displays similarity to a number of proteins in its hydrophobic domains, but is unique in its glycine-rich domain which, in the corresponding domains of the homologous proteins, are mostly proline-rich. Since both glycine-rich and proline-rich proteins are generally reported to be mostly cell wall proteins, the NT16 gene may be involved in shoot and root formation and in wound-healing process by modifying cell wall composition.

Exogenous phytohormone-independent growth and regeneration of tobacco plants transgenic for the 6b gene of Agrobacterium tumefaciens AKE10.

The 6b gene of Agrobacterium tumefaciens AKE10 (AK-6b) induces crown gall tumors on certain plants but so far there have been no reports of the gene being able to induce tumors on culture medium. We cloned T-DNA segments containing the 6b gene but lacking the auxin and cytokinin biosynthesis genes from A. tumefaciens AKE10. Tobacco (Nicotiana tabacum) leaf discs infected with A. tumefaciens LBA4404 carrying the clones produced shooty calli on hormone-free Murashige-Skoog medium. The relevant T-DNA segment was integrated into plant DNA as determined by Southern hybridization. Some of these immature shoots spontaneously developed into mature shoots, of which several leaves displayed morphological abnormalities. When leaf discs of these mature plants were placed onto the same medium numerous shoots developed from the wounding sites, indicating that the transgenic plants possessed a high regenerative potential. Northern blot and reverse transcriptase-polymerase chain reaction analyses showed a large accumulation of the AK-6b transcripts in the shooty calli, but only a limited degree in mature plants, demonstrating that AK-6b expression is regulated in plants and essential for the early stages of regeneration. Cytokinin levels in the shooty calli were comparable to those in normal shoots, suggesting that shoot regeneration is not mediated by the modulation of cytokinin content.

Bacillus thuringiensis protoxin: location of toxic border and requirement of non-toxic domain for high-level in vivo production of active toxin.

Insecticidal crystal proteins, or protoxins, of Bacillus thuringiensis are composed of two domains, an amino-terminal half essential for toxicity, and a carboxy-terminal half with an as yet unassigned function. To define the boundary of the two domains, sequential termination codons were introduced from the 3'-end of the DNA sequence encoding the toxic domain of the 1155-residue cry1A(b) gene product. The mutated and the intact genes were placed under the control of the Escherichia coli inducible promoter PrecA, and toxicity of the cell extracts was determined using silkworm larvae. Under non-induced conditions, in which the gene products accumulated to a limited degree, mutations encoding 606 amino acid residues or more were toxic, whereas those encoding 605 residues or less were non-toxic. Comparison of the toxicities and the levels of the toxic proteins suggested that the mutant proteins had comparable activity to that of the intact protoxin. Furthermore, the non-toxic protein seemed to be unstable in the extracts. To investigate the roles of the non-toxic domain, the mutant proteins were overproduced in both E. coli and B. thuringiensis. The intact and the mutated genes carrying natural promoters were introduced into acrystalliferous B. thuringiensis. Upon induction of PrecA in E. coli, and upon sporulation in B. thuringiensis, there was a large accumulation of gene products which formed inclusion bodies. The inclusion bodies of the intact protoxin were active, whereas those of the mutant proteins were inactive. Inclusion bodies of the intact protein could be solubilized in alkali, whereas the mutant inclusion bodies were insoluble. Since solubilization under alkaline conditions in the insect midgut is considered to be the first step of toxic action, the non-toxic domain is required to direct the synthesis of inclusion bodies as an active soluble form.

Sequence analysis of an insertion element, IS1131, isolated from the nopaline-type Ti plasmid of Agrobacterium tumefaciens.

Transferred DNA (T-DNA) from several nopaline-type Ti plasmids of Agrobacterium tumefaciens was previously shown to be variable in size due to the insertion of extra DNA segments. We found an insertion sequence (named IS1131) in the T-DNA of the strain PO22 and determined the nucleotide (nt) sequence. IS1131 is 2773 bp long, contains four open reading frames, and is flanked by 12-bp perfect inverted repeats (IR). An 8-bp direct repeat was found immediately outside the IR, and represents a target site of integration. Although the IS1131 nt sequence showed a limited degree of similarity to those of the previously reported IS elements of A. tumefaciens and to the central region of T-DNA, the terminal IR of IS1131 were highly homologous (up to 83%) to those of IS66 and IS866, suggesting that these three IS elements are related to each other. A number of IS1131-related copies were found in several pathogenic, as well as nonpathogenic, nopaline-type strains from different plant sources, and were distributed on both chromosomes and plasmids.

Polymorphism of Nopaline-type T-DNAs from Agrobacterium tumefaciens.

The structure of several T-DNAs of Agrobacterium tumefaciens was determined by molecular cloning and Southern hybridization. The T-DNAs cloned in Escherichia coli vectors from four different nopaline type strains (PyTE1, PO31, PO22, and AKE10) showed various sizes of restriction enzyme fragments. Comparative analysis of the restriction maps revealed that the T-DNAs were composed of three distinct structural domains: (1) the region proximal to the right border (Domain I) containing the portion essential for tumorigenicity, (2) the proximity to the left border (Domain II), and (3) the region between the two domains (Domain III) to both of which no functional assignments have yet been made. The restriction map indicated that the Domains I and II were conserved in the most clones, including the well-characterized T37 T-DNA. The only exception was AKK1 (obtained from AKE10) which differed in Domain I. In the Domain III, insertions of 1.5- or 1.6-kb DNA were found in four clones, whereas an additional 2.5-kb insertion was found in one clone (PO22P1). The individual T-DNAs including Domain III with insertions was demonstrated in petunia and poplar tumors induced by the referred A. tumefaciens strains. However, resulting tumors differed in morphology and growth. These results suggest that the length polymorphism of the nopaline type T-DNA can be accounted by DNA insertions, and that diverse T-DNAs reflect their different roles in tumorigenicity.

Genetic mapping and physiological consequences of metE mutations of Bacillus subtilis.

Three metE mutations of Bacillus subtilis, which cause cells to have a 25- to 200-fold decrease in L-methionine S-adenosyltransferase (EC 2.5.1.6) activity, were mapped between bioB and thr. The corresponding three metE mutants contained three- to fourfold less intracellular S-adenosylmethionine (SAM) but at least sevenfold more methionine than the metE+ strain when grown in synthetic medium. This indicates a strong feedback control of SAM on its synthesis. However, only the metE2 strain, with the lowest SAM concentration, grew at a slightly lower rate than the parent, which showed that an intracellular concentration of about 25 microM SAM was critical for growth at the normal rate. Neither DNA methylation (measured by bacteriophage luminal diameter 105 restriction) nor sporulation was affected at this low SAM concentration. Addition of methionine to the growth medium caused an increase in the pool of SAM in some but not all metE mutants. Coaddition of adenine did not change this result. However, the extent of sporulation (induced by mycophenolic acid) was decreased 50-fold in all mutants by the addition of methionine and adenine. Therefore, the combination of methionine and adenine suppresses sporulation regardless of whether it causes an increase in the level of SAM.

The biotechnology of Bacillus thuringiensis.

One of the challenges in the application of biotechnology to pest control is the identification of agents found in nature which can be used effectively. Biotechnology offers the potential of developing pesticides based on such agents which will provide environmentally sound and economically feasible insect control alternatives. Such an agent, the insect pathogen Bacillus thuringiensis, is the subject of intense investigations in several laboratories. Insecticides which use the entomocidal properties of B. thuringiensis are currently produced and sold worldwide; new products are currently in the development stage. Herein, the biology and genetics of B. thuringiensis and the problems associated with current products are critically reviewed with respect to biotechnology. Moreover, the economic and regulatory implications of technologically advanced products are evaluated.

Bacillus thuringiensis entomocidal protoxin gene sequence and gene product analysis.

A 3778-bp DNA sequence of the insecticidal protoxin gene coding sequence and flanking regions from Bacillus thuringiensis subspecies berliner 1715 has been determined. The protoxin is composed of 1155 amino acids, deduced from the nucleotide sequence, and has a calculated molecular mass of 130,615 daltons. To determine the DNA portion that encodes toxicity, sequential deletions were constructed from the 3' end of the coding region using nuclease Bal-31. Using these mutants in an insect bioassay, we found that an amino-terminal 612-amino-acid peptide is toxic, whereas, a 603-amino-acid peptide is not toxic to insects. Ninety percent of the amino acid residues were homologous to the protoxins from closely related subspecies kurstaki HD-1-Dipel and sotto. The differences occurred both in the amino-terminal half, or toxic portion, and in the carboxy-terminal half. These differences were clustered in several regions. From comparative analysis of subspecies berliner and kurstaki, we propose a model whereby the protoxin molecule is divided into distinct structural and functional domains. These domains may be responsible for the differences in specific toxicities and spectra of insect host range among these subspecies.

An ethA mutation in Bacillus subtilis 168 permits induction of sporulation by ethionine and increases DNA modification of bacteriophage phi 105.

In contrast to Escherichia coli and Salmonella typhimurium, Bacillus subtilis could convert ethionine to S-adenosylethionine (SAE), as can Saccharomyces cerevisiae. This conversion was essential for growth inhibition by ethionine because metE mutants which were deficient in S-adenosylmethionine synthetase activity, were resistant to 10 mM ethionine and converted only a small amount of ethionine to SAE. Another mutation (ethA1) produced partial resistance to ethionine (2 mM) and enabled continual sporulation in glucose medium containing 4 mM DL-ethionine. This sporulation induction probably resulted from the effect of SAE, since it was abolished by the addition of a metE1 mutation. The induction of sporulation was not simply controlled by the ratio of SAE to S-adenosylmethionine, but apparently depended on another effect of the ethA1 mutation, which could be demonstrated by comparing the restriction of clear plaque mutants of bacteriophage phi 105 grown in an ethA1 strain with the restriction of those grown in the standard strain. The phages grown in the ethA1 strain showed increased protection against BsuR restriction. We propose that SAE induces sporulation through the inhibition of a key methylation reaction.

Only part of the protoxin gene of Bacillus thuringiensis subsp. berliner 1715 is necessary for insecticidal activity.

Escherichia coli strains harboring deletion mutations of the insecticidal protoxin gene of Bacillus thuringiensis subsp. berliner 1715 were constructed. Although these strains did not produce intact protoxin, cell extracts from one of the mutants were extremely toxic to tobacco hornworm (Manduca sexta) larvae, indicating that only a part of the protoxin gene is required for insecticidal activity.

Defective complex formation between single-stranded DNA and mutant recA430 or recA1 protein of Escherichia coli.

The formation of a complex between recA protein and single-stranded DNA (ssDNA) was studied by filter-binding assays and sucrose density gradient centrifugation. In the presence of excess ATP, the wild-type recA protein formed salt-resistant complexes with ssDNA. One mutant recA430 (lexB30) protein bound to ssDNA slightly less effectively than wild-type protein, and the complexes had lost the stability to salt. Another mutant recA1 protein did not form complexes with ssDNA. On the other hand, in the absence of ATP, all proteins bound to ssDNA with the same efficiency, but all of the complexes were unstable in the presence of salt. The hybridization reaction in which homologous ssDNA is converted to double-stranded DNA (dsDNA) catalyzed by the recA430 protein was more sensitive to salt than that catalyzed by the wild-type protein. No hybridization reaction was found with the recA1 protein.