Characterization of the Ly49I promoter

 Fourteen potential Ly49 genes have been identified in the C57Bl/6 mouse strain, and cDNAs containing a complete coding region have been isolated for 10 members of this gene family. Ly49 proteins are primarily expressed in natural killer (NK) cells. Although the sequence of the Ly49a promoter region has been published, no study of the cell-specific activity of the promoter has been reported. A 12-kb genomic fragment of the Ly49I gene was isolated and characterized by DNA sequencing. Approximately 5 kb of DNA sequence upstream of the first Ly49I exon was determined and this region was used to perform promoter analysis using luciferase reporter plasmid constructs. A core promoter was identified that was preferentially transcribed in a Ly49-expressing cell line, EL-4. Electrophoretic mobility shift assays using oligonucleotide probes from the core Ly49i promoter and comparable regions from the Ly49a promoter demonstrated the importance of TATA-related elements in generating EL-4 and NK cell-specific DNA/protein complexes. Received: 15 October 1999 / Revised: 26 November 1999     

Species-specific, symbiotic plasmid-located repeated DNA sequences in Rhizobium trifolii

Summary A repeated DNA sequence has been characterized in the clover symbiont, Rhizobium trifolii. Analysis of three copies of this repeated sequence revealed that it constitutes a reiteration of the nifHDK promoter region and, in some copies, an additional reiteration of the N-terminal end of the nifH gene. This sequence, as exemplified by the nifHDK promoter region, is highly conserved within all the geographically-distinct isolates of R. trifolii examined, and is located exclusively on the Sym (symbiotic) plasmid. The R. trifolii repeated sequences (designated RtRS) were shown by DNA hybridization analysis to be specific for R. trifolii and not to hybridize to DNA of any other fastgrowing Rhizobium species examined. Based on the observed species-specificity and Sym-plasmid location of these sequences, as well as the available genetic evidence, we propose a model in which the expression of symbiotic genes is host-specifically activated via these species-specific repeated (promoter) sequences. The results presented indicate that the RtRS sequences can be used as a molecular probe for both species and strain identification and should facilitate the molecular taxonomy of Rhizobium.Dedicated to Professor Georg Melchers to celebrate his 50-year association with the journal     

Production of riboflavin by metabolically engineered Corynebacterium ammoniagenes

Improved strains for the production of riboflavin (vitamin B₂) were constructed through metabolic engineering using recombinant DNA techniques in Corynebacterium ammoniagenes. A C. ammoniagenes strain harboring a plasmid containing its riboflavin biosynthetic genes accumulated 17-fold as much riboflavin as the host strain. In order to increase the expression of the biosynthetic genes, we isolated DNA fragments that had promoter activities in C. ammoniagenes. When the DNA fragment (P54-6) showing the strongest promoter activity in minimum medium was introduced into the upstream region of the riboflavin biosynthetic genes, the accumulation of riboflavin was 3-fold elevated. In that strain, the activity of guanosine 5′-triphosphate (GTP) cyclohydrolase II, the first enzyme in riboflavin biosynthesis, was 2.4-fold elevated whereas that of riboflavin synthase, the last enzyme in the biosynthesis, was 44.1-fold elevated. Changing the sequence containing the putative ribosome-binding sequence of 3,4-dihydroxy-2-butanone 4-phosphate synthase/GTP cyclohydrolase II gene led to higher GTP cyclohydrolase II activity and strong enhancement of riboflavin production. Throughout the strain improvement, the activity of GTP cyclohydrolase II correlated with the productivity of riboflavin. In the highest producer strain, riboflavin was produced at the level of 15.3 g l⁻¹ for 72 h in a 5-l jar fermentor without any end product inhibition. Received: 23 August 1999 / Received revision: 13 October 1999 / Accepted: 5 November 1999     

Agrobacterium-mediated transformation of monocot and dicot plants using the NCR promoter derived from soybean chlorotic mottle virus

The NCR promoter (PNCR) from soybean chlorotic mottle virus (SoyCMV) was used to express the selectable marker, neomycin phosphotransferase (nptII) gene, in Agrobacterium-mediated transformation of both monocot (rice) and dicot (tobacco) plants. A multi-cloning site for insertion of a gene of interest into the binary vector pTN is located proximal to the right border region of T-DNA. When chimeric genes under the control of other strong promoters were located in a head-to-head orientation to the PNCR-nptII gene, kanamycin-resistant tobacco shoots were generated more efficiently than when using the original pTN vectors. This suggests that the enhancer-like sequences in the promoters adjacent to PNCR may promote expression of the PNCR-nptII gene. Received: 20 August 1999 / Revision received: 16 November 1999 / Accepted: 19 November 1999     

Expression of orf1 from the Bacillus thuringiensis NRD-12 cry2Aa1 Operon

The 5′ untranslated region and the orf1 sequence from the cry2Aa1 operon from Bacillus thuringiensis subsp. kurstaki NRD-12 were sequenced and compared to that from strain HD-1. The start codon described in HD-1 does not yield in NRD-12 a protein of the expected size of 20 kDa, but a 10-amino acid peptide. A second, highly conserved start codon is located 25 bp downstream from the first one and corresponds to an open reading frame of the same size in all known orf1-related sequences. Expression of lacZ gene fusions created at the level of the first ATG, second ATG, and stop codon of the NRD-12 orf1 sequence showed that orf1 is translated from the second ATG. The expected protein is 19 kDa in size. The expression starts at t2, which is in agreement with the presence of a BtI promoter in the cry2Aa1 operon. Received: 8 January 1999 / Accepted: 9 February 1999