SVIBOR - Project code: 1-08-197

MINISTRY OF SCIENCE AND TECHNOLOGY

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tel.: +385 1 459 44 44, fax: +385 1 459 44 69
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Project code: 1-08-197


Structure, organization and expression of genes in Streptomyces and some higher organisms


Main researcher: GAMULIN, VERA (13030)


Assistants
Type of research: basic
Duration from: 01/01/91. to 12/31/93.
Papers on project (total): 43
Papers on project quoted in Current Contents: 16
Institution name: Institut "Ruđer Bošković", Zagreb (98)
Department/Institute: Department of Molecular Genetics
Address: Bijenička cesta 54, P.O.Box 1016
City: 10000 - Zagreb, Croatia
Communication
Fax: 385 (0)41-425-647
E-mail: gamulin@olimp.irb.hr
Phone: 385 (0)4561-115
Summary: This project deals with the investigation of primary structures, genomic organization and mode of expression of genes in Streptomyces (especially S. rimosus) and in sponge from Adriatic sea, Geodia cydonium. Streptomycetes are most important industrial microorganisms and sponges, oldest multicellular animals, are very interesting for phylogenetic studies. We recently determined primary structure of seven transfer RNA genes (two individual genes, one cluster of five genes) and a sequence of one complete ribosomal RNA operon (rrnF) from S. rimosus. Promoter sequences, responsible for the expression of these genes, and terminators of the transcription (rho independent) were also defined as well as the organization of rRNA and tRNA genes on the chromosome of S. rimosus. rRNA genes in S. rimosus are clustered in six very similar gene sets, with genes organized in the order 16S-23S-5S rRNA. In other studied streptomycetes (about ten species) the number of rRNA operons varied from five to seven. tRNA genes were found individually on the chromosome, or organized in small clusters. None of seven analyzed tRNA genes encode the CCA end of mature tRNAs, what is the characteristics of tRNA genes in eukaryotes. Expression of tRNA genes was studied in homologous system, but also in Escherichia coli heterologous system. For that purpose bifunctional plasmid vectors pZG5 and pZG6, suitable for gene transfer between Streptomyces and E. coli, were constructed. All genes in the cluster of five tRNA genes are transcribed from the same promoter. This promoter belongs to SEP group of promoters and is also active in E. coli. Promoters of two other analyzed tRNA genes are not functional in E. coli. rRNA genes in S. rimosus code for 1529 nucleotides (nt) long 16S rRNA, 3121 nt long 23S rRNA and 120 nt long 5S rRNA. 16S rRNA contains highly variable species-specific region (nt 175-200) suitable for a quick determination of the species. 3121 nt long 23S rRNA belongs to the largest prokaryotic 23S rRNA analyzed so far. rrnF operon contains only one consensus promoter (P4). Other operons (rrnA-rrnE) possess at least two promoters (P3 and P4), or three promoters (duplicated P3 and P4). At the moment molecular genetic investigations of streptomycetes are focused on the cloning of genes coding for enzymes involved in degradation of nucleic acids (DNases, RNases) or protein biosynthesis (aminoacyl-tRNA synthetases). Investigation of genes from Geodia cydonium is a collaborative project with University of Mainz, Germany. Several cDNA clones were analyzes and a structure of few phylogenetically conserved genes was determined (i.e. genes coding for sponge lectins, polyubiquitin, tyrosine kinase). Several more cDNA clones are under investigation. During 1994-1995. we cloned and sequenced recA gene from S. rimosus and the results are prepared for publication. Phosphorylation of proteins in streptomycetes was also studied, as well as the evolution of phylogenetically conserved genes. In the period

Keywords: molecular genetics, gene cloning, gene structure, gene expresion, recombinant DNA, Streptomyces, sponges, RNA genes, RecA protein, phylogeny

Research goals: Molecular genetic investigations of bacteria from the genusStreptomyces and sponge from Adriatic sea (Geodia cydonium)performed on this project, were initiated with the idea toinvestigate structure, organization and mode of expression ofgenes in these very interesting prokaryotic and eukaryoticorganisms. The main goal was (better) understanding of fundamental biological rules and processes in nature as well asdefinition of specificities in the organization of thehereditary material in different species. However, many results from these investigations may havecommercial application. Bifunctional plasmid vectors pZG5 andpZG6 (for the system Streptomyces- E. coli), constructed for theneeds of this project, are stable in both hosts, very usable forgene cloning, and can therefore be used for aplicable purposes.Promoters of tRNA and rRNA genes, cloned and analyzed in detailsduring the work on the project, can serve as (aditional orbetter) promoters for genes of commercial interest. We alsodefined species-specific region in 16S rRNA from S. rimosus.These results can be used for quick determination of the smallamount of S. rimosus in any sample, with no need for classicalanalyses. During last year we defined regulatory sequences involved in the expression of the recA gene from S. rimosus and these results can have commercial application.

COOPERATION - PROJECTS


  1. Name of project: 1-08-058 Ekspresija gena u micelijskim mikroorganizmima
    Name of institution: Istraživački institut "Plive"
    City: 10000 - Zagreb, Croatia

  2. Name of project: KRO-BIO 1 Cloning of sponge genes; recombinant lectins
    Name of institution: Johannes Gutenberg University, Mainz, Germany, Laboratory of Prof. W.E.G. Muller and H.C. Schroder, Institut for Applied Molecular biology
    City: Mainz, Njemačka

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