Shigeyuki Tanifuji

Nucleotide sequences of the 5.8S rRNA gene and internal transcribed spacer regions in carrot and broad bean ribosomal DNA.

SummaryNucleotide sequences of the first and second internal transcribed spacers (ITS1 and ITS2, respectively) of ribosomal DNA (rDNA) from two dicot plants, carrot and broad bean, were determined. These sequences were compared with those of rice, a monocot plant, and other eukaryotic organisms. Both types of ITS region in some species of Angiospermae were the shortest among all eukaryotes so far examined and showed a wide range of variation in their G+C content, in contrast to a general trend toward very high G+C content in animals. Phylogenetic relationships of plants with animals and lower eukaryotes were considered using the nucleotide sequences of carrot and broad bean 5.8S rDNA that were determined in the present study, together with that of wheat 5.8S rRNA, which has been reported previously.

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.

Molecular and physiological evaluation of transgenic tobacco plants expressing a maize phosphoenolpyruvate carboxylase gene under the control of the cauliflower mosaic virus 35S promoter

The expression of maize (Zea mays) phosphoenolpyruvate carboxylase (PEPC) gene constructs in transgenic tobacco plants (Nicotiana tabacum) was studied. Where transcription was under the control of a CaMV 35S promoter, maize PEPC transcripts of the correct size were detected. Western blot analysis indicated that the transgenic plants contained about twice as much PEPC as non-transformed plants. Furthermore, the enzymatic activity of PEPC in the leaves of these transgenic plants was up to twice as high as that in non-transformed plants. Two forms of PEPC with different kinetic properties were identified in leaf extracts of the transgenic plants: one form (the maize isoform) gave a high apparentKm value for phosphoenolpyruvate (PEP) and a high maximum activity, and the other (the tobacco isoform) exhibited a low apparentKm value for PEP and a low maximum activity. These biochemical differences resulted in several significant physiological changes in the transgenic plants: (1) the growth rate of the transgenic plants was lower than that of non-transgenic plants: (2) chlorophyll content per leaf area was relatively lower in the transgenic plants; and (3) the quantum yield of photosynthesis in the transgenic plants was not affected by changes in leaf temperature.

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.

High molecular weight RNA synthesized in nucleoli of higher plants

Abstract When decotyledonized pea embryos, at the early stage of germination, were labeled for 20 or 30 min with radioactive uridine, a large-size RNA component sedimenting far faster than 25 S was most rapidly synthesized in a nucleoli-rich fraction. At this time, the radioactivity was not significantly detected in the cytoplasmic fraction. Even after chasing in excess amounts of uridine, the radioactivity of the nucleolar fractions continued to increase, but the extent of accumulation of the isotopes was far greater in a nucleolar component sedimenting near the region of 25 S. The appearance of this second radioactive component in nucleoli, near the region of 25 S, was accompanied by the appearance of labeled 16 S in cytoplasm. The radioactive 25-S RNA emerged into cytoplasm after some delay of time. These two nucleolar RNA components of high molecular weight were labeled with 3 H, when embryos were incubated in the media containing [ Me - 3 H]methionine. The results in this report indicate that these nucleolar RNA components of high molecular weight are ribosomal precursor molecules and the mode of processing of rRNA in higher plant cells is similar to that described in animal cells.

Difference between two major size classes of carrot rDNA repeating units is due to reiteration of sequences of about 460 bp in the large spacer

SummaryBased on the previous observation that the different regions between two major size classes of carrot rRNA genes are located in their large spacer, detailed physical maps of the spacer region were constructed by cloning, followed by restriction analysis. As a results, the different regions were restricted to BamHI segments of 1.3 kb and 1.8 kb. Sequence analysis of these segments revealed that the shorter one carried one truncated and two complete copies of about 460 bp of repetitious sequences, while the longer one contained one truncated and three full copies of the repetitious sequences. S1 nuclease mapping data suggest that either transcription initiation sites or processing sites of precursor rRNA are located in the repetitious sequence closest to the 18 S rRNA coding region.

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.

Sequence analysis of three tRNAPhe nuclear genes and a mutated gene, and one gene for tRNAAla from Arabidopsis thaliana.

Three genes and one mutant gene for tRNAPhe (GAA) and one gene for tRNAAla (UGC) were isolated from a whole-cell DNA library of Arabidopsis thaliana. All three tRNAPhe genes are identical in their nucleotide sequence, but differ in their 5′ and 3′ flanking regions. The mutant tRNAPhe (GAA) gene differs from the other three genes by one nucleotide change from highly conserved G to C at the 57th nucleotide position. The primary structure of the first tRNAAla gene was also determined in this experiment.

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.