Spectinomycin resistance mutations in the rrn16 gene are new plastid markers in Medicago sativa.

We report here the isolation of spectinomycin-resistant mutants in cultured cells of Medicago sativa line RegenSY-T2. Spectinomycin induces bleaching of cultured alfalfa cells due to inhibition of protein synthesis on the prokaryotic type 70S plastid ribosomes. Spontaneous mutants resistant to spectinomycin bleaching were identified by their ability to form green shoots on plant regeneration medium containing selective spectinomycin concentrations in the range of 25-50 mg/l. Sequencing of the plastid rrn16 gene revealed that spectinomycin resistance is due to mutations in a conserved stem structure of the 16S rRNA. Resistant plants transferred to the greenhouse developed normally and produced spectinomycin-resistant seed progeny. In light of their absence in soybean, a related leguminous plant, the isolation of spectinomycin-resistant mutants in M. sativa was unexpected. The new mutations are useful for the study of plastid inheritance, as demonstrated by detection of predominantly paternal plastid inheritance in the RegenSY-T2 × Szapko57 cross, and can be used as selective markers in plastid transformation vectors to obtain cisgenic plants.

Dwarf plants of diploid Medicago sativa carry a mutation in the gibberellin 3-beta-hydroxylase gene.

In this paper we describe the identification of a gene, MsDWF1 coding for a putative gibberellin 3-beta-hydroxylase (GA3ox), whose natural mutation is conditioning a dwarf growth phenotype in Medicago sativa. The dwarf phenotype could not be complemented with grafting, which indicates that the bioactive gibberellin compound necessary for shoot elongation is immobile. On the contrary, exogenously added gibberellic acid restored normal growth. The genetic position of the Msdwf1 gene was mapped to linkage group 2 (LG2) and the physical location was delimited by map-based cloning using Medicago truncatula genomic resources. Based on the similar appearance and behavior of the dwarf Medicago sativa plants to the pea stem length mutant (le) as well as the synthenic map position of the two genes it was postulated that MsDWF1 and pea Le are orthologs. The comparison of wild type and mutant allele sequences of MsGA3ox revealed an amino acid change in a conserved position in the mutant allele, which most probably impaired the function of the enzyme. Our results indicate that the dwarf phenotype was the consequence of this mutation.

Transposition and target specificity of the typical IS30 family element IS1655 from Neisseria meningitidis.

We have analysed the transposition and target selection strategy of IS1655, a typical IS30 family member resident in Neisseria meningitidis. We have redefined IS1655 as a 1080 bp long element with 25 bp imperfect inverted repeats (IRs), which generates a 3 bp target duplication and have shown that it transposes using an intermediate with abutted IRs separated by 2 bp. IS1655 exhibits bipartite target specificity inserting preferentially either next to sequences similar to its IRs or into an unrelated but well defined sequence. IR-targeting leads to the formation of a new junction in which the targeted IR and one of the donor IRs are separated by 2 bp. The non-IR targets were characterized as an imperfect 19 bp palindrome in which the central five positions show slight GC excess and the distal region is AT-rich. Artificial targets designed according to the consensus were recognized by the element as hot spots for insertion. The organization of IS1655 is similar to that of other IS30 family members. Moreover, it shows striking similarity to IS30 in transposition strategy even though their transposases differ in their N-terminal regions, which, for IS30, appears to determine target specificity. Comparative analysis of the transposases and the evolutionary aspects of sequence variants are also briefly discussed.