Functional expression of a sequence-specific endonuclease encoded by the retrotransposon R2Bm

A fraction of the 28S ribosomal genes in certain insect species is interrupted by the insertion elements R1 and R2. These two elements from the silkworm Bombyx mori (R1Bm and R2Bm) are retrotransposons capable of transposing in a highly sequence-specific manner. We report here the functional expression in E. coli of the entire single open reading frame of R2Bm and show that it encodes a double-stranded endo-nuclease (integrase) that can specifically cleave the 28S gene at the R2 insertion site. The resulting cleavage is a 4 bp staggered 5' overhang. Deletion analysis of the 28S gene revealed that the DNA sequence required for specific cleavage is asymmetric with respect to the actual insertion (cleavage) site, with fewer than 10 bp required at one side and at least 24 bp at the other side of the site. A model is proposed based on these and previous data to account for the sequence-specific integration of the R2 retrotransposon.     

Complementarity between the mRNA 5' untranslated region and 18S ribosomal RNA can inhibit translation

In eubacteria, base pairing between the 3' end of 16S rRNA and the ribosome-binding site of mRNA is required for efficient initiation of translation. An interaction between the 18S rRNA and the mRNA was also proposed for translation initiation in eukaryotes. Here, we used an antisense RNA approach in vivo to identify the regions of 18S rRNA that might interact with the mRNA 5' untranslated region (5' UTR). Various fragments covering the entire mouse 18S rRNA gene were cloned 5' of a cat reporter gene in a eukaryotic vector, and translation products were analyzed after transient expression in human cells. For the largest part of 18S rRNA, we show that the insertion of complementary fragments in the mRNA 5' UTR do not impair translation of the downstream open reading frame (ORF). When translation inhibition is observed, reduction of the size of the complementary sequence to less than 200 nt alleviates the inhibitory effect. A single fragment complementary to the 18S rRNA 3' domain retains its inhibitory potential when reduced to 100 nt. Deletion analyses show that two distinct sequences of approximately 25 nt separated by a spacer sequence of 50 nt are required for the inhibitory effect. Sucrose gradient fractionation of polysomes reveals that mRNAs containing the inhibitory sequences accumulate in the fractions with 40S ribosomal subunits, suggesting that translation is blocked due to stalling of initiation complexes. Our results support an mRNA-rRNA base pairing to explain the translation inhibition observed and suggest that this region of 18S rRNA is properly located for interacting with mRNA.     

Genomic characterization of the filamentous integrative bacteriophages {phi}RSS1 and {phi}RSM1, which infect Ralstonia solanacearum

The genomic DNA sequences were determined for two filamentous integrative bacteriophages, phiRSS1 and phiRSM1, of the phytopathogen Ralstonia solanacearum. The 6,662-base sequence of phiRSS1 contained 11 open reading frames (ORFs). In the databases, this sequence showed high homology (95% identity) to the circular double-stranded DNA plasmid pJTPS1 (6,633 bp) isolated from a spontaneously occurring avirulent mutant of R. solanacearum. Two major differences between the two sequences were observed within phiRSS1 ORF7, corresponding to pIII, a minor coat protein required for host adsorption, and at the phiRSS1 intergenic (IG) region. The 9,004-base sequence of phiRSM1 showed 12 ORFs located on the same strand (plus strand) and 2 ORFs on the opposite strand. Compared with Ff-type phages, two insertions are obvious in the phiRSM1 replication module. Genomic DNA fragments containing the phiRSM integration junctions were cloned and sequenced from phiRSM lysogenic strain R. solanacearum MAFF211270. The att core sequence was identified as 5'-TGGCGGAGAGGGT-3', corresponding to the 3' end of the serine tRNA (UCG) gene. Interestingly, ORF14, located next to the attP site on the phiRSM1 genome, showed high amino acid sequence homology with bacterial DNA recombinases and resolvases, different from XerCD recombinases. attP of phiRSS1 is within a sequence element of the IG region.     

Structure, expression and products of the ribosomal RNA operons of Rhodopseudomonas palustris No. 7

Rhodopseudomonas palustris strains carry one or two ribosomal rRNA operons, and those with duplicated rrn operons grow faster. The two rrn operons in R. palustris No. 7 are virtually identical over a 54,70-bp stretch containing the genes for 16S rRNA, tRNAile, tRNAala, 23S rRNA and 5S rRNA, as well as the intergenic spacers and part of the extragenic spacer. In R. palustris, unlike most bacteria with multiple rrn operons, the putative promoter sequences of the two operons are highly diverged, suggesting possible functional differentiation. By simultaneous primer-extension analysis of both pre-rRNAs, we detected a two-fold higher level of expression from rrnA under photoautotrophic conditions. Alteration of the conditions of growth leads to changes in the relative levels of expression of the two operons. Within the 5,470-bp segment, only two sequence differences are found between the 23S rRNA genes; one is at the center of the 23S rRNA molecule and affects a site of unknown function, and the other is within or immediately adjacent to sequences involved in processing of the 5' 23S rRNA IVS. In vitro processing of 5' IVS-containing 23S rRNA precursors from each operon does not reveal any detectable difference between them. The 5' ends of the mature 16S, 23S, and 5S rRNAs were determined by primer-extension analysis, and the 3' end of 23S rRNA was determined by RNA linker ligation-mediated cDNA cloning. The 5' and 3' ends of the R. palustris 23S rRNA molecule are extensively processed, suggesting that, unlike the situation in the established eubacterial model, these ends cannot basepair.