Kimitaka Yakura

Length heterogeneity of the large spacer of Vicia faba rDNA is due to the differing number of a 325 bp repetitive sequence elements

SummaryThe DNA fragments including the whole large spacer region of Vicia faba rDNA were cloned in plasmid pBR325. Sixteen clones were classed into five groups which differed from each other in the lengths of the rDNA inserts. Physical maps of these length variants cloned were constructed using EcoRI, SalI, HpaI, MluI and AccI and evidence was obtained that the length heterogeneity was due mainly to the differing number of 325 base pairs (bp) subrepeating elements in the large spacer. Sequence analysis of this subrepeating element revealed that it consisted of a duplet of an approximately 155 bp sequence and a 14 bp unrelated sequence. This structure of the repetitive element is novel.

The structure of the large spacer region of the rDNA in Vicia faba and Pisum sativum

The complete nucleotide sequence of the rDNA large spacer region was determined for two related legume plants,Vicia faba andPisum sativum. In the large spacers of both plants, there were one or several tandem arrays of repeated sequences which accunt well for the length heterogeneity of the rDNA repeating units. When the entire lengths ofVicia andPisum rDNA large spacers were compared, significant sequence similarity ranging from approximately 50 to 80% was observed, thus a close phylogenetical relationship was revealed. However, organization patterns of these rDNA spacers were clearly different from each other. ThePisum rDNA large spacer contained only one type of repeated sequence of about 175 bp, whereas theVicia contained five kinds of repeated sequence. In addition, the initiation site for transcription was tentatively determined by S1 protection experiment.

Repeated DNA sequences found in the large spacer of Vicia faba rDNA

Abstract In the large spacer of the rDNA of Vicia faba , multiples of a 0.32 kilobasepair (kb) sequence reiterate to various degrees. We sequenced the repetitious region consisting of the repeating sequences and its flanking regions using two cloned plasmids, which contain V. faba rDNA segments encompassing the whole region of the large spacer. The repetitious region was found to consist of multiple complete copies and one truncated copy of a 325 bp repeat unit and to be flanked by direct repeat sequences of about 150 bp. The set of direct repeats located at either side of the repetitious region differed from each other with about 10% sequence heterogeneity. However, nucleotide sequences of the direct repeats were well conserved between the two clones examined. Southern blot hybridization indicated a widespread distribution within the whole V. faba genome of some related sequences with high homologies to the 325 bp repeat unit and to the direct repeats.

Sequence analysis of Vicia faba highly repeated DNA: the BamHI repeated sequence families

Cleavage of Vicia faba nuclear DNA with the restriction endonuclease BamHI yielded discrete size classes of 250, 850, 900, 990, 1 150, 1 500 and 1 750 bp of highly repetitive DNA. Each of these sequence families comprised about 3% of the total genomic DNA. Some sequence members from each sequence family were cloned in pBR322 and their primary structures determined. Computer analyses of nucleotide sequences suggested the existence of about 60 bp sequence periodicity within the repeating unit of the 990 bp sequence family, though the extent of homology among the surmised shorter subrepeat units was very low. With other BamHI sequence families, however, the data did not show any clear internal sequence periodicity. The repeat units of the 850 bp and 1 750 bp sequence families contained nucleotide sequences homologous to the 250 bp family sequence. No sequence relationship between or among other sequence families was observed. There was 13–25% sequence variation among 6 cloned members of the 250 bp family and probably also among those of other BamHI repeat families. DNA sequences homologous to these V. faba BamHI repeat families were detected in Pisum sativum DNA by Southern blot hybridization. Furthermore, very weak cross-hybridization was observed with plant DNAs from Phaseolus vulgaris, Triticum aestivum, Cucumis sativus and Trillium kamtschaticum.

Non-coordinated histone synthesis with DNA replication and close temporal association of ribosomal proteins and rRNA syntheses in germinating Vicia faba embryos

Syntheses of DNA, RNA and basic proteins during germination of Vicia faba seeds were examined. Incorporation of [3H]thymidine into DNA reached a maximum peak at approximately 34 h after beginning imbibition and the phase of DNA replication was preceded by active RNA synthesis. When Vicia embryos were labeled with [3H]lysine or E13H]arginine, these isotopes were incorporated into ribosomal proteins and histones. The incorporation studies indicated that the synthesis of ribosomal structural proteins is closely linked with rRNA synthesis and histone synthesis occurs during and also before the phase of DNA replication. Polyacrylamide gel electrophoresis of Vica histones indicated that some histones of Vicia embryos differ from corresponding calf thymus histones in their mobilities.

Chromatin replication in a higher plant, Vicia faba.

Abstract Embryo axes of Vicia faba seeds were collected and pulse-labeled with [3H]thymidine for 5–6 min at the first DNA synthesis period (34 h stage of germination) and chromatin was isolated. About 67% of the pulse-labeled DNA in the chromatin was converted to acid-soluble material after extensive digestion with micrococcal nuclease. On the other hand, bulk chromatin uniformly labeled with [14C]thymidine for a 12-h period showed acid-solubility of about 56% under identical conditions of digestion. Sedimentation analyses of the nuclease digests of chromatin and extracted DNA samples showed that the nucleosome structure was already organized in the chromatin containing 10–11-S single-strand DNA fragments of an intermediate molecule during DNA replication. These fragments were detected as a major component after labeling for 10 to 15 min with [3H]thymidine. Further-more, the result of the pulse-labeling for 5–6 min indicated the presence of the nucleosome structure in the chromatin containing 5–6-S and 8–9-S nascent DNA fragments as the main labeled components, thus suggesting the involvement of at least the 8–9-S intermediate fragment in the reorganization of the nucleosome structure. Electrophoresis of DNA fragments obtained after micrococcal nuclease digestion of chromatin showed that after a pulse of 5–6 min with [3H]thymidine, labeled dimer and trimer DNA fragments were significantly shorter than corresponding [14C]thymidine-labeled bulk dimer and trimer DNA fragments, though the peaks of pulse-labeled and uniformly labeled monomer DNA fragments were coincident with each other in 3.5% gels. The difference in size of dimer and trimer DNA fragments between the newly formed and non-replicating bulk chromatin disappeared after a prolonged labeling for 15 min, irrespective of the fact that the major part of labeled DNA was still the 10–11-S intermediate fragment. The mode of chromatin assembly in the replicating chromatin in higher plant cells is discussed.