Showing papers in "Molecular Genetics and Genomics in 1989"

The complete sequence of the rice (Oryza sativa) chloroplast genome: Intermolecular recombination between distinct tRNA genes accounts for a major plastid DNA inversion during the evolution of the cereals

Abstract: The entire chloroplast genome of the monocot rice (Oryza sativa) has been sequenced and comprises 134525 bp. Predicted genes have been identified along with open reading frames (ORFs) conserved between rice and the previously sequenced chloroplast genomes, a dicot, tobacco (Nicotiana tabacum), and a liverwort (Marchantia polymorpha). The same complement of 30 tRNA and 4 rRNA genes has been conserved between rice and tobacco. Most ORFs extensively conserved betweenN. tabacum andM. polymorpha are also conserved intact in rice. However, several such ORFs are entirely absent in rice, or present only in severely truncated form. Structural changes are also apparent in the genome relative to tobacco. The inverted repeats, characteristic of chloroplast genome structure, have expanded outward to include several genes present only once per genome in tobacco and liverwort and the large single copy region has undergone a series of inversions which predate the divergence of the cereals. A chimeric tRNA pseudogene overlaps an apparent endpoint of the largest inversion, and a model invoking illegitimate recombination between tRNA genes is proposed which accounts simultaneously for the origin of this pseudogene, the large inversion and the creation of repeated sequences near the inversion endpoints.

Genetic and structural characterization of the avirulence gene avrBs3 from Xanthomonas campestris pv. vesicatoria

Abstract: The avirulence gene avrBs3 from Xanthomonas campestris pv. vesicatoria was cloned and found to be localized on a self-transmissable plasmid. Genetic analysis of an avrBs3 insertion mutation revealed that avrBs3 constitutes a single locus, specifying the resistant phenotype on pepper plants. Southern blot experiments showed that no DNA sequences homologous to avrBs3 were present in other races of X. c. pv. vesicatoria, which are unable to induce a hypersensitive reaction on ECW-30R. However, the DNA of several different pathovars of X. campestris hybridized to the avrBs3 probe. A deletion analysis defined a region of 3.6–3.7 kb essential for avrBs3 activity. The nucleotide sequence of this region was determined. A 3561 nucleotide open reading frame (ORF1), encoding a 125000 dalton protein, was found in the 3.7 kb region that was sufficient for avrBs3 activity. A second long ORF (2351 nucleotides) was identified on the other strand. A remarkable feature of both ORFs is the presence of 17 direct repeats of 102 bp which share 91%–100% homology with each other.

Nucleotide sequence, organization, and nature of the protein products of the carotenoid biosynthesis gene cluster of Rhodobacter capsulatus

Abstract: Carotenoid pigments are essential for the protection of both photosynthetic and non-photosynthetic tissues from photooxidative damage. Although carotenoid biosynthesis has been studied in many organisms from bacteria to higher plants, little is known about carotenoid biosynthetic enzymes, or the nature and regulation of the genes encoding them. We report here the first DNA sequence of carotenoid genes from any organism. We have determined the complete nucleotide sequence (11 039 bp) of a gene cluster encoding seven of the eight previously known carotenoid genes (crt A, B, C, D, E, F, I) and a new gene, designated crtK, from Rhodobacter capsulatus, a purple non-sulfur photosynthetic bacterium. The 5′ flanking regions of crtA, I, D and E contain a highly conserved palindromic sequence homologous to the consensus binding site for a variety of prokaryotic DNA-binding regulatory proteins. This putative regulatory palindrome is also found 5′ to the puc operon, encoding the light-harvesting II antenna polypeptides. Escherichia coli-like σ70 promoter sequences are located 5′ to crtI and crtD, suggesting for the first time that such promoters may exist in purple photosynthetic bacteria. The crt genes form a minimum of four distinct operons, crtA, crtIBK, crtDC and crtEF, based on inversions of transcriptional orientation within the gene cluster. Possible rho-independent transcription terminators are located 3′ to crtI, B, K, C and F. The 3′ end of crtA may overlap transcription initiation signals for a downstream gene required for bacteriochlorophyll biosynthesis. We have also observed two regions of exceptional amino acid homology between CrtI and CrtD, both of which are dehydrogenases.

Isolation and expression of an anther-specific gene from tomato.

Abstract: We have isolated and sequenced an anther-specific cDNA clone and a corresponding genomic clone from tomato. The gene (LAT52) encodes an 800-nucleotide-long transcript that is detectable in pollen, anthers and at 20- to 50-fold lower levels in petals. LAT52 mRNA is not detectable in pistils, sepals or non-reproductive tissues. Steady-state levels of LAT52 mRNA are detectable in immature anthers containing pollen at the tetrad stage and increase progressively throughout microsporogenesis until anthesis (pollen shed). The LAT52 gene contains 5' and 3' untranslated regions of 110 and approximately 150 nucleotides, respectively, and a single intron with a highly repetitive sequence. A TATA box motif is located 28 nucleotides upstream of the transcription start site. The gene encodes a putative protein of 18 kDa that is cysteine rich and has an N-terminal hydrophobic region with characteristics similar to eucaryotic secretory signal sequences. LAT52 is a single or low copy gene in tomato and shares homology with sequences in tobacco.

Biochemical and genetic analysis of the nifUSVWZM cluster from Azotobacter vinelandii.

Abstract: Azotobacter vinelandii genes contained within the major nif-cluster and designated orf6, nifU, nifS, nifV, orf7, orf8, nifW nifZ, nifM, and orf9 are organized into at least two overlapping transcriptional units. Nitrogenase derepressed crude extracts of Azotobacter vinelandii mutant strains having individual deletions located within nifU, nifS, nifV, nif, nifZ, or nifM were examined for nitrogenase component protein activities. The results of these experiments indicated that, in A. vinelandii, the nifU, nifS and nifM gene products are required for the full activation or the catalytic stability of the nitrogenase Fe protein. Deletion of the nifV gene resulted in lower MoFe protein activity, probably resulting from the accumulation of an altered FeMo-cofactor. The nifW and nifZ gene products were required for the full activation or catalytic stability of the MoFe protein. Deletion of nijZ alone or nifM alone did not appear to affect FeMo-cofactor biosynthesis. However, deletion of both niJZ and nifM eleminated either FeMo-cofactor biosynthesis or the insertion of FeMo-cofactor into the apo-MoFe protein. Other genes contained within the nifUSVWZM gene cluster (orf6, orf7, orf8, and orf9) were not required for Mo-dependent diazotrophic growth.

A vector system with temperature-sensitive replication for gene disruption and mutational cloning in streptomycetes

Abstract: Replication of the Streptomyces ghanaensis plasmid pSG5 was shown to be temperature sensitive. The pSG5 replicon is stably inherited at temperatures below 34° C, but is lost at incubation temperatures above this. A family of cloning vectors was constructed using the pSG5 minimal replicon and different marker genes. The vectors obtained are small in size, have an intermediate copy number, possess a broad host range and are compatible with some other streptomycete vector systems. By increasing the incubation temperature, these vectors can be eliminated from their host cells very efficiently. The suitability of the pSG5 vector family for mutating chromosomal genes by gene disruption was demonstrated: pBN10, a pSG5 derivative containing an internal fragment of the phosphinothricyl-alanyl-alanine (PTT) resistance gene pat, was integrated into the chromosomal pat gene of the PTT-producer Streptomyces viridochromogenes thus inactivating PTT resistance. The integrated pBN10 plasmid was rescued from the chromosome, together with an adjacent fragment carrying DNA of the PTT biosynthetic cluster.

FemA, a host-mediated factor essential for methicillin resistance in Staphylococcus aureus: molecular cloning and characterization.

Abstract: The methicillin resistance determinant (mec) in Staphylococcus aureus resides on additional DNA not present in isogenic sensitive cells. However, besides mec, other chromosomally determined factors are essential for expression of methicillin resistance. We cloned and characterized a chromosomally determined gene which encodes a factor essential for the expression of methicillin resistance (femA) in S. aureus. femA mapped in chromosomal segment number 18, genetically very distant from the methicillin resistance determinant (mec). The product of femA was a protein of an apparent size of 48 kDa. FemA restored methicillin resistance in S. aureus that had become sensitive to methicillin by insertion of Ω22003 (femA::Tn551). Although FemA was needed for cell growth in the presence of β-lactam antibiotics, it had no influence on the synthesis of the low affinity, additional penicillin-binding protein (PBP2′) encoded by mec and known to be essential for cell wall synthesis in the presence of inhibitory concentrations of methicillin. Nucleotide sequence analysis, Northern RNA blotting and S1 nuclease RNA mapping suggested that femA was transcribed on a polycistronic mRNA. This mRNA contained the coding region for FemA (ORF433) and a second coding region (ORF419) producing a protein of 47 kDa. The nucleotide and amino acid sequence of FemA showed homologies with ORF419, suggesting that these genes arose by gene duplication. In addition we present evidence for a second chromosomal factor, femB, involved in expression of methicillin resistance which maps close to femA.

Sequences responsible for the tissue specific promoter activity of a pea legumin gene in tobacco.

Abstract: Maturing pea cotyledons accumulate large quantities of storage proteins at a specific time in seed development. To examine the sequences responsible for this regulated expression, a series of deletion mutants of the legA major seed storage protein gene were made and transferred to tobacco using the Bin19 disarmed Agrobacterium vector system. A promoter sequence of 97 bp including the CAAT and TATA boxes was insufficient for expression. Expression was first detected in a construct with 549 bp of upstream flanking sequence which contained the the leg box element, a 28 bp conserved sequence found in the legumin-type genes of several legume species. Constructs containing -833 and -1203 bp of promoter sequence significantly increased levels of expression. All expressing constructs preserved seed specificity and temporal regulation. The results indicate that promoter sequences between positions -97 and -549 bp are responsible for promoter activity, seed specificity, and temporal regulation of the pea legA gene. Sequences between positions -549 and -1203 bp appear to function as enhancer-like elements, to increase expression.

Identification of a gene conferring resistance to zinc and cadmium ions in the yeast Saccharomyces cerevisiae

Abstract: A DNA fragment conferring resistance to zinc and cadmium ions in the yeast Saccharomyces cerevisiae was isolated from a library of yeast genomic DNA. Its nucleotide sequence revealed the presence of a single open reading frame (ORF; 1326 bp) having the potential to encode a protein of 442 amino acid residues (molecular mass of 48.3 kDa). A frameshift mutation introduced within the ORF abolished resistance to heavy metal ions, indicating the ORF is required for resistance. Therefore, we termed it the ZRC1 (zinc resistance conferring) gene. The deduced amino acid sequence of the gene product predicts a rather hydrophobic protein with six possible membrane-spanning regions. While multiple copies of the ZRC1 gene enable yeast cells to grow in the presence of 40 mM Zn2+, a level at which wild-type cells cannot survive, the disruption of the chromosomal ZRC1 locus, though not a lethal event, makes cells more sensitive to zinc ions than are wild-type cells.

Identification and nucleotide sequence of the delta-lysin gene, hld, adjacent to the accessory gene regulator (agr) of Staphylococcus aureus.

Abstract: A Tn551 insertional mutation in the accessory gene regulator (agr) locus of the Staphylococcus aureus chromosome resulted in the decreased production of at least seven extracellular toxins and enzymes and a simultaneous increase in the production of protein A and coagulase (Recsei et al. 1986). Adjacent to this locus we have now identified another gene, hld, transcribed into a 0.5 kb RNA which codes for the staphylococcal delta-lysin. The expression of hld was totally repressed in a strain carrying the agr insertional mutation. Hybridization with strand-specific probes and primer extension analysis revealed that hld and agr are transcribed in opposite directions, starting 188 nucleotides apart. The hld gene is mainly expressed during the post-exponential growth phase and is totally repressed during early exponential growth. Determination of hld mRNA half-life in different growth phases indicated that this regulation is at the level of transcription.

Analysis of the gene encoding the largest subunit of RNA polymerase II in Drosophila.

Abstract: We have characterized RpII215, the gene encoding the largest subunit of RNA polymerase II in Drosophila melanogaster. DNA sequencing and nuclease S1 analyses provided the primary structure of this gene, its 7 kb RNA and 215 kDa protein products. The amino-terminal 80% of the subunit harbors regions with strong homology to the β′ subunit of Escherichia coli RNA polymerase and to the largest subunits of other eukaryotic RNA polymerases. The carboxyl-terminal 20% of the subunit is composed of multiple repeats of a seven amino acid consensus sequence, Tyr-Ser-Pro-Thr-Ser-Pro-Ser. The homology domains, as well as the unique carboxyl-terminal structure, are considered in the light of current knowledge of RNA polymerase II and the properties of its largest subunit. Additionally, germline transformation demonstrated that a 9.4 kb genomic DNA segment containing the α-amanitinresistant allele, RpII215 C4 , includes all sequences required to produce amanitin-resistant transformants.

The genetics of position-effect variegation modifying loci in Drosophila melanogaster

Abstract: The dose dependent effects of position-effect variegation (PEV) modifying genes were studied in chromosome arms2L, 2R and3R. Four groups of PEV modifying genes can be distinguished: haplo-abnormal suppressor and enhancer loci with or without a triplo-effect. using duplications four triplo-abnormal suppressor and four triplo-abnormal enhancer functions were localized. In two cases we proved that these functions correspond to a converse haplo-abnormal one. Altogether 43 modifier loci were identified. Most of these loci proved not to display significant triplo-effects (35). The group of haplo-abnormal loci with a triplo-effect may represent genes which play an important role in heterochromatin packaging.

Differential expression within a family of novel wound-induced genes in potato

Abstract: Wounding in higher plants leads to an increased synthesis of specific messenger RNAs. A cDNA clone complementary to a wound-induced message from potato tubers was used to isolate a lambda clone from a genomic library of Salanum tuberosum var. Maris Piper. DNA sequence analysis has shown that this single genomic clone contains two novel wound-induced genes, called win1 and win2, organised in close tandem array. The coding sequences of these two genes are highly homologous and are interrupted by a single intron. However, the sequences of the introns and flanking regions have diverged widely. Win1 and win2 encode cysteine-rich proteins of 200 and 211 amino-acids, respectively, which show striking homologies to several chitin-binding proteins. Southern analysis of genomic DNA has shown that win1 and win2 are members of a small multi-gene family which is estimated to have a minimum of five members per haploid genome of Maris Piper and appears to be conserved within the Solanaceae. We have shown by Northern analysis and S1 mapping that the two genes exhibit differential organ-specific expression after the wounding of a potato plant.

Alfalfa nodulation in the absence of Rhizobium

Abstract: Certain alfalfa plants can develop non-nitrogen fixing structures on their root systems when grown in testtubes under strictly axenic conditions. We demonstrate that these structures possess all the histological features characteristic of indeterminate nodules and that their formation is inhibited by combined nitrogen. The nodule morphogenesis-related gene ENOD2 is expressed in these nodules whereas leghemoglobin transcripts cannot be detected. The capacity to Nodulate in the Absence of Rhizobium (NAR) is maintained during clonal propagation of these alfalfa plants. Our results show that Rhizobium is not absolutely required for nodule morphogenesis and suggest that plant genetic determinants are involved in the NAR phenomenon.

Transport across the outer membrane of Escherichia coli K12 via the FhuA receptor is regulated by the TonB protein of the cytoplasmic membrane.

Abstract: Point mutations in the “TonB box” offhuA were suppressed by point mutations in thetonB gene, suggesting both a functional and physical interaction between the FhuA receptor protein in the outer membrane and the TonB protein in the cytoplasmic membrane ofEscherichia coli K12. Mutations influA were classified into four types according to the extent by which they impaired mutant cells in their growth on ferrichrome as sole iron source and in their sensitivity to the antibiotic albomycin and to colicin M. ThetonB mutation with a glutamine to leucine replacement at position 165 was less efficient in restoring the FhuA functions than the glutamine to lysine exchange at the same position. The better the coupling between FhuA and TonB the poorer was the inhibition of phage T1 binding to FhuA by ferrichrome. A working model is proposed in which the TonB protein assumes different conformations in response to the energized state of the cytoplasmic membrane and thereby allosterically regulates the activity of the FhuA receptor. This model implies an intermembrane coupling between two proteins in adjacent membranes.

The gene family encoding the Arabidopsis thaliana translation elongation factor EF-1α: molecular cloning, characterization and expression

Abstract: The gene family encoding the Arabidopsis thaliana translation elongation factor (EF-1 alpha) was analysed. This family contains four genes (A1-A4) organized in a similar manner in different varieties of Arabidopsis. Based upon both their physical separation and a comparison of their sequences, it is suggested that the A4 gene and the A1, A2, and A3 genes constitute two distinct subfamilies within the genome. By introducing chimaeric gene constructs into Arabidopsis cells, we showed that the A1 gene promoter mediates a transient expression about twofold higher than that obtained using the CaMV 35 S promoter. This expression depends on a 348 bp DNA fragment extending from -982 to -634 bp upstream of the initiation codon. This element contains a characteristic telomeric sequence (AACCCTAA) which is also found in the promoters of the A2 and A4 genes as well as in the promoters of the Drosophila EF-1 alpha F1 gene and of several highly expressed plant genes.

Gentic evidence for superoperonal organization of genes for photosynthesis pigments and pigment-binding proteins in Rhodobacter capsulatus

Abstract: Three adjacent operons, each concerned with photosynthesis in Rhodobacter capsulatus, have been shown by genetic means to be cotranscribable. In the course of describing the characteristics of the bchCA operon, which encodes two enzymes essential for bacteriochlorophyll synthesis, we found that the expression of the bchCA genes is influenced by readthrough from the upstream crtE and crtF genes. The crtE and crtF genes encode enzymes required for carotenoid biosynthesis and function as an operon. Furthermore, the distal structural gene of the bchCA operon, bchA, contains within it both the major oxygen-regulated promotor (Ppuf1) and the constitutive (Ppuf2) promotor for the puf operon. Since these three operons, crtEF, bchCA, and puf, are all transcribed in the same direction, it appears that polymerases traversing the down-stream regions may start at any of several promoters. This pattern of transcription, which is unusual among bacteria, demonstrates that the activities of individual operons in a superoperonal cluster may be affected by their positions within the cluster.

Nitrate reductase of Escherichia coli: completion of the nucleotide sequence of the nar operon and reassessment of the role of the alpha and beta subunits in iron binding and electron transfer.

Abstract: The nucleotide sequence of the narGHJI operon that encodes the nitrate reductase of Escherichia coli was completed. It encodes four polypeptides NarG, NarH, NarJ and NarI of molecular weight 138.7, 57.7, 26.5 and 25.5 kDa, respectively. The analysis of deduced amino acid sequence failed to reveal any structure capable of binding iron within the NarG polypeptide. In contrast, cysteine arrangements typical of iron-sulfur centers were found in the NarH polypeptide. This suggested that the latter is an electron transfer unit of the nitrate reductase complex. Such a view is opposite to the current description of the nitrate reductase. The findings allowed us to propose a model for the electron transfer steps that occur during nitrate reduction. The NarG polypeptide was found to display a high degree of homology with numerous E. coli molybdoproteins. Moreover, the same genetic and functional organizations as well as the presence of highly conserved stretches of amino acids were noted between both NarG/NarH and DmsA/DmsB (encoding the dimethyl sulfoxide reductase) pairs.

Efficient Transformation of Arabidopsis-Thaliana Using Direct Gene-Transfer to Protoplasts

Abstract: Direct gene transfer has proved to be an efficient transformation method for Arabidopsis thaliana, a member of the Brassicaceae. Transgenic Arabidopsis plants resistant to hygromycin B have been regenerated from mesophyll protoplasts treated with polyethylene glycol and plasmid DNA carrying the hygromycin phosphotransferase (HPT) gene under the control of the 35 S promoter of cauliflower mosaic virus. The transformation procedure reproducibly yields transformants at frequencies of approximately 1 x 10(-4) (based on the number of protoplasts treated) or 5% (based on the number of regenerating calli). DNA from plants regenerated from hygromycin resistant colonies was analysed by Southern blot hybridization demonstrating that the foreign gene is stably integrated into the plant chromosome. Genetic analysis of several hygromycin resistant plants showed that the HPT gene is transmitted to the progeny. Transformation experiments performed with a selectable and a non-selectable gene on separate plasmids resulted in a co-transformation rate of functionally active copies in about 25% of the transformants analysed. Hence this approach can be used to introduce non-selectable genes into the Arabidopsis genome.

Pulsed field gel electrophoresis and physical mapping of large DNA fragments in the Tm-2a region of chromosome 9 in tomato

Abstract: A method has been developed which allows the isolation of very high molecular weight DNA (>2 million bp) from leaf protoplasts of tomato (Lycopersicon esculentum). The DNA isolated in this manner was digested in agarose with rare-cutting restriction enzymes and separated by pulsed field gel electrophoresis. The size range of the reslting fragments was determined by hybridization to a number of single copy clones and the suitability of these enzymes for the mapping of large DNA fragments was evaluated. Furthermore, five genetically tightly linked single copy clones have been used to begin the construction of a physical map in a region of the genome containing the Tm-2a gene which confers resistance to tobacco mosaic virus. Two of the five clones were found to be on the same 560 kb SalI fragment and therefore are no further apart than that distance. The remaining three markers are distributed over at least 3 million bp, so that the total minimum physical distance of that cluster is at least 4 million bp. The results are discussed with respect to correlations between recombination frequencies and physical distance as well as physical mapping large regions of a complex plant genome like tomato.

Transgenic tobacco resistant to a bacterial disease by the detoxification of a pathogenic toxin

Abstract: Some plant pathogens produce toxins which cause disease in infected plants. One of the pathogenic toxins, tabtoxin, is produced by Pseudomonas syringae pv. tabaci, which causes wildfire of tobacco. A tabtoxin resistance gene (ttr) coding for an acetyltransferase isolated from Pseudomonas syringae pv. tabaci was fused to the 35S promoter of the cauliflower mosaic virus (CaMV) to construct a chimeric gene for introduction into tobacco cells by Agrobacterium-mediated transformation. The transgenic tobacco plants showed high specific-expression of the ttr gene and no chlorotic symptoms caused by tabtoxin treatment or with infection by Pseudomonas syringae pv. tabaci. These results demonstrate a successful approach to obtain disease-resistant plants by detoxification of the pathogenic toxins which play an important role in pathogenesis.

Molecular cloning, nucleotide sequence, and abscisic acid induction of a suberization-associated highly anionic peroxidase.

Abstract: A highly anionic peroxidase induced in suberizing cells was suggested to be the key enzyme involved in polymerization of phenolic monomers to generate the aromatic matrix of suberin. The enzyme encoded by a potato cDNA was found to be highly homologous to the anionic peroxidase induced in suberizing tomato fruit. A tomato genomic library was screened using the potato anionic peroxidase cDNA and one genomic clone was isolated that contained two tandemly oriented anionic peroxidase genes. These genes were sequenced and were 96% and 87% identical to the mRNA for potato anionic peroxidase. Both genes consist of three exons with the relative positions of their two introns being conserved between the two genes. Primer extension analysis showed that only one of the genes is expressed in the periderm of 3 day wound-healed tomato fruits. Southern blot analyses suggested that there are two copies each of the two highly homologous genes per haploid genome in both potato and tomato. Abscisic acid (ABA) induced the accumulation of the anionic peroxidase transcripts in potato and tomato callus tissues. Northern blots showed that peroxidase mRNA was detectable at 2 days and was maximal at 8 days after transfer of potato callus to solid agar media containing 10−4 M ABA. The transcripts induced by ABA in both potato and tomato callus were identical in size to those induced in wound-healing potato tuber and tomato fruit. The anionic peroxidase peptide was detected in extracts of potato callus grown on the ABA-containing media by western blot analysis. The results support the suggestion that stimulation of suberization by ABA involves the induction of the highly anionic peroxidase.

Molecular characterization of an active wheat LMW glutenin gene and its relation to other wheat and barley prolamin genes

Abstract: The isolation and characterisation by DNA sequencing of a low molecular weight (LMW) glutenin gene from wheat is described. The deduced protein contains a signal peptide, a central repetitive region rich in proline and glutamine and N and C terminal non-repetitive domains, similar to other prolamins. A detailed comparison of the C terminal domain of 20 prolamin genes enabled us to divide them into 4 families. The LMW glutenin family is distinct from the α, β-and γ-gliadin families of wheat and is closest to the B hordein genes of barley. This and other comparisons were also used to assess the pattern of genetic variation among prolamin sequences and to provide a molecular basis for the interpretation of prolamin size polymorphism. The 5′ flanking fragment of the isolated gene was previously shown to direct endosperm-specific expression of a reporter gene in transgenic tobacco. Evidence is provided that the isolated gene is also active in wheat and its transcription initiation site was determined. Features of the gene which may be relevant to its activity are discussed.

On the evolution of Tn21-like multiresistance transposons: sequence analysis of the gene (aacC1) for gentamicin acetyltransferase-3-I(AAC(3)-I), another member of the Tn21-based expression cassette.

Abstract: The aminoglycoside-3-O-acetyltransferase-I gene (aacC1) from R plasmids of two incompatibility groups (R1033 [Tn1696], and R135) was cloned and sequenced. In the case of R1033, it was shown that theaacC gene is coded by a precise insertion of 833 bp between theaadA promoter and its structural gene in a Tn21 related transposon (Tn1696). This insertion occurs at the same target sequence as that of the OXA-1 β-lactamase gene insertion in Tn2603. Upstream of theaacC gene, we found an open reading frame (ORF) which is probably implicated in the site-specific recombinational events involved in the evolution of this family of genetic elements. These results provide additional confirmation of the role of Tn21 elements as naturally occurring interspecific transposition and expression casssettes.

The phosphoenolpyruvate carboxylase gene of Corynebacterium glutamicum: molecular cloning, nucleotide sequence, and expression.

Abstract: The ppc gene of Corynebacterium glutamicum encoding phosphoenolpyruvate (PEP) carboxylase was isolated by complementation of a ppc mutant of Escherichia coli using a cosmid gene bank of chromosomal C. glutamicum DNA. By subsequent subcloning into the plasmid pUC8 and deletion analysis, the ppc gene could be located on a 3.3 kb SalI fragment. This fragment was able to complement the E. coli ppc mutant and conferred PEP carboxylase activity to the mutant. The complete nucleotide sequence of the ppc gene including 5' and 3' flanking regions has been determined and the primary structure of PEP carboxylase was deduced. The sequence predicts a 919 residue protein product (molecular weight of 103 154) which shows 34% similarity with the respective E. coli enzyme.

Overproduction of alfalfa glutamine synthetase in transgenic tobacco plants.

Abstract: We have obtained transgenic tobacco plants overexpressing the enzyme glutamine synthetase (GS) by fusing an alfalfa GS gene to the cauliflower mosaic virus 35S promotor and integrating it intoNicotiana tabacum var. W38 plants byAgrobacterium tumefaciens mediated gene transfer. The amount of RNA specific to alfalfa GS was about 10 times higher in transgenic tobacco plants than in alfalfa. The alfalfa GS produced by these transgenic plants was identified by Western blotting and represented 5% of total soluble protein in the transformed plants, amounting to a 5-fold increase in specific GS activity and in a 20-fold increase in resistance to the GS inhibitorl-phosphinothricin in vitro. Tissue from GS overproducing plants showed a sevenfold lower amount of free NH3. The amino acid composition of the plant tissue was not altered significantly by GS overproduction. GS overproducing plants were fertile and grew normally. These data show that a high level of expression of a key metabolic enzyme such as glutamine synthetase does not interfere with growth and fertility of plants.

Transformation of various species of gram-negative bacteria belonging to 11 different genera by electroporation.

Abstract: We have undertaken a systematic study to test the transformation of various species of gram-negative bacteria using the electroporation method. The data obtained show very clearly that a great variety of gram-negative bacteria--15 different species belonging to 11 different genera--including freshly isolated wild-type strains can be transformed efficiently by use of the electric-field mediated transformation technique. These include species of the families Enterobacteriaceae, Pseudomonadaceae, Rhizobiaceae, photosynthetic bacteria and strains for which transformation could not be achieved, up to now, by other methods.

The development of a homologous transformation system for Aspergillus oryzae based on the nitrate assimilation pathway: A convenient and general selection system for filamentous fungal transformation

Abstract: The development of an efficient and homologous transformation system for Aspergillus oryzae is described. This is based on nitrate reductase (niaD) of the nitrate assimilation pathway. The niaD system offers a number of inherent advantages over many other systems and may be of general use for nitrate-utilising filamentous fungi. Transformation frequencies of up to 800 transformants per microgram DNA are observed with A. oryzae. The preponderance of integration events take place at the resident niaD locus either by gene conversion (41%), single integration (23%) or multiple tandem integration (36%). Heterologous expression of the A. oryzae niaD gene in the filamentous fungi A. nidulans, A. niger and Penicillum chrysogenum is observed. That heterologous putative niaD hybridisation signals are seen with other fungal DNAs affords the oppotunity to isolate the corresponding niaD from various fungi in order to develop homolgous transformation. Co-transformation with the introduction of the non-selected markers pyrG, tub-2, and uidA has been achieved.

DNA damage activates transcription and transposition of yeast Ty retrotransposons.

Abstract: A set of genes isolated from Saccharomyces cerevisiae showed increased transcript levels after yeast had been exposed to ultraviolet (UV) light or 4-nitroquinoline-1-oxide (4NQO). Included among these DNA damage responsive (DDR) genes were members of the Ty retrotransposon family of yeast. Northern hybridization analysis indicated that maximal levels of a 5.6 kb transcript encoded by the Ty elements accumulated in cells after 4 to 6 h of exposure to 4NQO. The induced levels of transcripts varied from two- to tenfold for different Ty probes although similar kinetics and dose responses were observed for transcripts hybridizing to the different Ty family members. Pulse labeling experiments suggested that the accumulation of Ty transcripts was due, in part, to an increased rate of Ty message synthesis. Transposition of Ty elements to two target loci encoding distinct alcohol dehydrogenase enzymes, ADH2 and ADH4, was examined in cells exposed to increasing doses of UV light or 4NQO. The frequency of Ty insertion into these genetic regions following DNA damaging treatments increased by as much as 17-fold compared with untreated cells. These results provide direct evidence that transposable elements can be activated by physical and chemical mutagens/carcinogens and that transpositional mutagenesis is induced by these agents in S. cerevisiae.

Control of formation of active soluble or inactive insoluble baker's yeast α-glucosidase PI in Escherichia coli by induction and growth conditions

Abstract: Using standard growth conditions (LB medium, 37°C, induction with 5 mM IPTG) yeast α-glucosidase PI expressed under the control of the regulated tac-hybrid promoter results in the synthesis of insoluble aggregated α-glucosidase granules in Escherichia coli. Under these conditions active soluble α-glucosidase amounts to less than 1% of the heterologously produced protein. However, the amount of soluble active α-glucosidase was dramatically increased when the strong tac-hybrid promoter was to a limited extent induced. This was achieved at concentrations of 0.01 mM IPTG or of 1% lactose or lower in a lactosepermease deficient host strain containing the lacI qrepressor gene on an R-plasmid. The formation of active soluble α-glucosidase was almost 100% when E. coli cells induced in this manner were cultivated under conditions that reduced growth rate, i.e. at decreased temperature, extreme pH values or in minimal and complete media supplemented with different carbon sources.