Chloroplast ribosomal RNA genes in Euglena gracilis exist as three clustered tandem repeats

Chloroplast ribosomal DNA from Euglena gracilis was partially purified, digested with restriction endonucleases BamHI or EcoRI and cloned into bacterial plasmids. Plasmids containing the ribosomal DNA were identified by their ability to hybridize to chloroplast ribosomal RNA and were physically mapped using restriction endonucleases BamHI, EcoRI, HindIII and HpaI. The nucleotide sequences coding for the 16S and the 23S chloroplast ribosomal RNAs were located on these plasmids by hybridizing the individual RNAs to denatured restriction endonuclease DNA fragments immobilized on nitrocellulose filters. Restriction endonuclease fragments from chloroplast DNA were analyzed in a similar fashion. These data permitted the localization on a BamHI map of the chloroplast DNA three tandemly arranged chloroplast ribosomal RNA genes. Each ribosomal RNA gene consisted of a 4.6 kilobase pair region coding for the 16S and 23S ribosomal RNAs and a 0.8 kilobase pair spacer region. The chloroplast ribosomal DNA represented 12% of the chloroplast DNA and is G + C rich.     

Construction and restriction endonuclease mapping of hybrid plasmids containing Saccharomyces cerevisiae ribosomal DNA.

Summary Fragments produced by partial digestion of Saccharomyces cerevisiae ribosomal DNA (rDNA) with the restriction endonuclease EcoRI were ligated in vitro to the bacterial plasmid RSF2124. The resulting hybrid plasmids were cloned in Escherichia coli. Three hybrid plasmids which contain at least one intact repetitive unit of the multiple, tandem sequences of the yeast rDNA genes have been further characterized. These plasmids have been used to construct a map of the EcoRI, SmaI, HindII and HindIII restriction sites in the individual repetitive units of yeast rDNA.     

Ribosomal RNA gene variation in diploid and tetraploid Tolmiea menziesii

Evidence from gross morphology, karyology and flavonoid chemistry suggests that Tolmiea menziesii is one of the clearest examples of autopolyploidy in natural populations. To provide additional data regarding the origin of the tetraploid cytotype of Tolmiea, both the 5S and 18S-25S ribosomal RNA genes were studied at the restriction enzyme level. Using restriction enzymes that cut once per repeat, the lengths of the 5S and 18S-25S ribosomal genes were estimated in diploids and tetraploid plants. There appear to be no consistent differences between diploids and tetraploids for the repeat length of the 18S-25S ribosomal genes. Furthermore, there is no significant repeat length heterogeneity within tetraploid plants for these genes. In addition, no differences in repeat length of the 5S genes were observed among the diploid and tetraploid plants analysed. The homogeneity observed among diploid and tetraploid plants for repeat length of the 5S and 18S-25S ribosomal genes is consistent with the hypothesis that the tetraploid cytotype is of autopolyploid origin.     

Cloning and characterization of the ribosomal genes of the sea-urchin Paracentrotus lividus. Heterogeneity of the multigene family

A Paracentrotus lividus genomic library was constructed using sperm DNA prepared from a single animal. The DNA was fragmented by partial digestion with DNase II, sized on a preparative agarose gel and inserted in the Pst I site of pBR 322 by the dG X dC tailing method. Recombinant plasmids containing ribosomal DNA were isolated, a restriction map of the gene was determined and the 18S and 26S transcribed sequences were located by S1 protection mapping. The organization of the ribosomal genes in genomic DNA of individual animals and of a pool of animals was studied by blot-hybridization of the restriction fragments, using as probes nick-translated 32P-labelled cloned ribosomal DNA fragments or 18S and 26S sea-urchin ribosomal RNA. The repeat length of the ribosomal unit was about 10.5 X 10(3) bases. A comparison of the restriction patterns of DNA from different animals showed a marked sequence heterogeneity in the spacer region of these genes. Variations of about 200 base pairs were detectable in the length of the spacer of some individuals.     

Construction in vitro of hybrid plasmids carrying all the EcoRI fragments from lambdarifd18 DNA.

The cloning of all the eleven fragments obtained by degrading the phage lambdarifd18 by the restriction enzyme EcoRI into the plasmid pSF2124 has been achieved: nine of these fragments have been cloned individually, whereas two others have been cloned jointly in the same plasmid. These fragments harbor, in addition of lambda genes, the genes for ribosomal proteins, the elongation factor Tu, the beta and beta' subunits of RNA polymerase and the ribosomal RNAs. The clones carrying the ribosomal RNA genes have been constructed to provide convenient plasmids to determine the primary structure of ribosomal RNAs. Some further genetic manipulations in vitro have been performed on two of them to remove extraneous non-ribosomal RNA gene sequences; the ribosomal genes purified this way have been subcloned into the plasmid pBR322. Other clones of interest have been obtained which carry the genes for the elongation factor Tu, a number of 50-S ribosomal proteins and the beta subunit of RNA polymerase.