Treatment of sensitive Escherichia coli cells with colicin E3 leads to inactivation of 30S ribosomal subunits. In vitro reconstitution of 30S subunits indicates that the E3-induced defect lies solely in the 16S RNA. 16S RNA from E3-treated cells lacks several T1 RNase oligonucleotides of normal 16S RNA, including the one from the 3'-end of the 16S RNA, as analyzed by the fingerprint technique of Sanger. An RNA fragment about 50 nucleotides long has been isolated from E3-treated cells. This RNA contains the original 3'-terminal oligonucleotide and other oligonucleotides missing in the E3-16S RNA. The results show that colicin E3 treatment causes the cleavage of 16S RNA at a specific position near the 3'-terminus. Preparation of 16S RNA and total 30S proteins, as well as the method of reconstitution of 30S subunits, was described previously (5,8). Ribosomal RNA was analyzed on 3% acrylamide-0.5% agarose column gels or 10% acrylamide slab gels in Tris-EDTA-borate buffer, pH 8.3 (9).The purified RNAs were studied by the fingerprint technique of Sanger et al. (10, 11) after they were digested with T,RNase and alkaline phosphatase. The fingerprints obtained were compared with those obtained by Fellner and his coworkers (12), in order to assign numbers to the various oligonucleotides. The molar yield of each product was determined by counting, in a scintillation counter, the area of paper containing it. Particular oligonucleotides were characterized after elution from the paper by digestion with 5 ul of pancreatic RNase (0.1 mg/ml in 10 mM Tris (pH 7.6)-i mM EDTA plus 2 mg/ml of carrier RNA) for 30 min at 37°C and electrophoresis at pH 3.5 on DEAE-cellulose paper (13). RESULTS Reconstitution of 30S particles with components from E3-inactivated 30S subunits ("E3-30S")We have used the ribosome reconstitution technique (5) to determine which component is responsible for the inactivity of E3-30S. As shown in Table 1
We have determined the DNA sequences of regions involved in two of the three inversions known to have occurred during the evolution of wheat chloroplast DNA. This establishes the extent of the second largest of the three inversions. Examination of these sequences suggests that although short repeated sequences are present, the endpoints of the second and third inversions are not associated with repeated sequences as long as those associated with the first inversion. However the endpoints of all three inversions are all adjacent to at least one tRNA gene, and there is evidence that three of the tRNA genes have been subjected to partial duplication, possibly at the time of inversion. This suggests that tRNA genes might be involved with rearrangements of chloroplast DNA, as has also been postulated for mitochondrial DNA.
To perform a detailed study of genome evolution in the natural Brassica amphidiploid B. juncea, we have constructed two linkage maps based on RFLP (restriction fragment length polymorphism) markers; one generated from a cross between a resynthesized B. juncea (a chromosome doubled interspecific B. rapa x B. nigra hybrid) and a natural B. juncea cultivar, the other from a cross between two B. juncea cultivars. By using a common cultivar in both crosses, the two maps could be unambiguously integrated. All loci exhibited disomic inheritance of parental alleles in the natural x resynthesized cross, showing that B. rapa chromosomes paired exclusively with their A-genome homologues in B. juncea and that B. nigra chromosomes likewise paired with their B-genome homologues. The maps derived from the two crosses were also perfectly collinear. Furthermore, these maps were collinear with maps of the diploid progenitor species (B. nigra and B. rapa) produced using the same set of RFLP probes. These data indicate that the genome of B. juncea has remained essentially unchanged since polyploid formation. Our observations appear to refute the suggestion that the formation of polyploid genomes is accompanied by rapid change in genome structure.
Chloroplast DNA variation has been used to examine some of the maternal lineages involved in the evolution of the intraspecific polyploid complex, Dactylis glomerata L. Diploid (2x) and tetraploid (4x) individuals were collected from natural populations of the subspecies glomerata (4x), marina (4x) and lusitanica (2x), as well as from sympatric 2x/4x populations of the Galician type. Digestion of their ctDNA with 11 restriction endonucleases revealed enough variation to characterise three ctDNA variants, designated MBMK, MBmK and mBMK. The distribution of these ctDNA variants reflects different stages in their spread among the populations. The MBMK ctDNA variant predominated at both ploidy levels in subspecies glomerata, lusitanica and marina, and in recent tetraploid Galician/glomerata hybrids. The MBmK variant was detected in a single tetraploid individual and probably results from a relatively recent mutation. Fixation of the mBMK minority variant in the diploid and tetraploid Galician populations adds to the evidence concerning the possible origin of the Galician tetraploids. It means that the Galician diploids were maternal ancestors of the tetraploids. This result complements evidence from earlier studies based on morphology or biochemical markers, and reduces the likelihood that the tetraploids arose by hybridisation between an ancient Galician diploid and an alien tetraploid. It is, however, consistent with a true autopolyploid origin of the tetraploids.
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