Volume 38, Issue No. 3

The in vivo Expression of Streptomyces rimosus tRNA Genes

Sonja Durajlija Žinić*, Miroslav Plohl and Vera Gamulin

Department of Molecular Genetics, »Ruđer Bošković« Institute, Bijenička c. 54, HR-10000 Zagreb, Croatia

Article history:
Received February 17, 2000
Accepted April 7, 2000

Key words:
Streptomyces rimosus, expression, promoter, tRNA genes

Summary:
The expression of seven tRNA genes from Streptomyces rimosus, cloned on bifunctional plasmids, has been studied in a homologous (S. rimosus) and a heterologous (Escherichia coli) system. Analyzed genes included cluster of genes containing two tRNA^Gln and three tRNA^Glu genes and two independent tRNA^fMet genes. Northern hybridization analysis showed that all tRNA genes on plasmids are transcribed and processed in the homologous system. In the E. coli system only the cluster of Gln-Glu tRNA genes is properly expressed. From the deletion experiments it can be concluded that in both species all five genes in the cluster are cotranscribed from the same promoter, located 140–65 bp upstream from the first gene. A sequence TTGGAC-17-TAATGT resembling to Streptomyces-E. coli (SEP) promoter is located in this region. Similar sequence TTGCGC-18-TAGACT was also found 13 bp upstream from the tRNA^fMet1 gene. However, this gene is not properly expressed in E. coli. Putative promoter of the tRNA^fMet2 gene could not be easily identified by sequence homology in relation to two other presumptive promoters of tRNA genes. Streptomycetes promoters show huge sequence heterogeneity and two tRNAfMet genes obviously have very different promoters. 

*Corresponding author:           duras@rudjer.irb.hr
                                               ++385 (0)1 4561 115
                                               ++385 (0)1 4561 177

The Relevance of Solid-state Substrate Moisturing on Ganoderma lucidum Biomass Cultivation

Jožica Habijanič¹ and Marin Berovič^1,2*

¹National Institute of Chemistry, Hajdrihova 19, SI-1115 Ljubljana, Slovenia
²Department of Chemical, Biochemical & Ecology Engineering, University of Ljubljana Hajdrihova 19, SI-1115 Ljubljana, Slovenia

Article history:
Received December 10, 1999 
Accepted May 17, 2000

Key words:
solid-state cultivation, moisture content, Ganoderma lucidum 

Summary:
Solid-state cultivation of Ganoderma lucidum biomass based on originally isolated Slovenian strain and production of its polysaccharides in horizontal stirred tank reactor (HSTR) were developed. For solid state G. lucidum biomass cultivation and polysaccharide production, moisture fraction of solid substrate 70 % was found as a critical value. Moisture fractions higher than 70 % promote the growth and polysaccharide production, while below 70 % the growth of mycelium is slower and polysaccharide production is reduced. As the moisture of the substrate drops to 55 %, growth of mycelium stops, therefore, after 7 days of cultivation continuous moisturing of the substrate is necessary for high biomass and polysaccharide production. 

*Corresponding author:      marin.berovic@uni-lj.si

Agrobacterium-mediated Transformation of Broad Bean Vicia faba L.

Srećko Jelenić¹*, Petar T. Mitrikeski², Dražena Papeš¹ and Sibila Jelaska<sup>1

1</sup>Department of Molecular Biology, Faculty of Science, University of Zagreb, Rooseveltov trg 6, HR-10000 Zagreb, Croatia
²Laboratory for Biology and Microbial Genetics, Faculty of Food Technology and Biotechnology, University of Zagreb, Pierottijeva 6, HR-10000 Zagreb, Croatia

Article history:
Received May 22, 2000
Accepted July 5, 2000

Key words:
Vicia faba L., Agrobacterium tumefaciens, Agrobacterium rhizogenes, peroxidase

Summary:
Three broad bean cultivars, which varied in morphology and geographical origin, were inoculated with nine Agrobacterium strains to determine the best combination for potential use in transformation and to investigate the possibility of regenerating genetically transformed plants. The stems of seedlings were inoculated with A. tumefaciens wild type strains (A281 and B6S3), transconjugant strains (C58C1(pArA4abc) and C58C1(pArA4b)), the B6S3 rooty and shooty mutants (GV3101(pGV2255), GV3101(pGV2215) and GV3101(pGV2235)), and A. rhizogenes wild type strains (8196 and 15834). With all the tested strains only unorganized tumour tissue was obtained. Cultivars differed in their susceptibility to bacterium strains, and plant genotype vs. strain interaction was detected. The strain most virulent to cv. Lobab Lippoi was C58C1(pArA4b), while with the less susceptible cvs. Topolo and Ošlje the best results were obtained with strains A281 and B6S3, respectively. However, the size and phenotype of tumours depended on the bacterial strain exclusively, indicating that the difference in transformation efficiency of each bacterial strain might be the result of a different efficiency in one or more of the steps prior to expression of T-DNA genes within each particular cultivar. Established tumorous calli grown on hormone-free MS medium showed enhanced peroxidase activity. The in vitro transformation of cotyledon, leaf and internodal stem segments with Ri plasmids containing bacterium strains, was not successful. 

*Corresponding author:          sjelen@zg.biol.pmf.hr 
                                              ++385 (0)1 4826 260
                                              ++385 (0)1 4826 260