Mapping of genes on the chloroplast DNA of Spirodela oligorhiza

With the use of spinach chloroplast RNAs as probes, we have mapped the rRNA genes and a number of protein genes on the chloroplast DNA (cpDNA) of the duckweed Spirodela oligorhiz. For a more precise mapping of these genes we had to extend the previously determined [14] restriction endonuclease map of the duckweed cpDNA with the cleavage sites for the restriction endonucleases Sma I and Bgl I. The physical map indicates that duckweed cpDNA contains two inverted repeat regions (18 Md) separated by two single copy regions with a size of 19 Md and 67 Md, respectively.By hybridization with spinach chloroplast rRNAs it could be shown that each of the two repeat units contains one set of rRNA genes in the order: 16S rRNA gene — spacer — 23S rRNA gene — 5S rRNA gene.A spinach chloroplast mRNA preparation (14S RNA), which is predominantly translated into a 32 Kilodalton (Kd) protein [9], hybridized strongly to a DNA fragment in the large single copy region, immediately outside one of the inverted repeats. With another mRNA preparation (18S), which mainly directs the in vitro synthesis of a 55 Kd protein [9], hybridization was observed with two DNA regions, located between 211° and 233° and between 137° and 170°, respectively. Finally, with a spinach chloroplast genomic probe for the large subunit of ribulose 1,5-bisphosphate carboxylase [17], hybridization was found with a DNA fragment located between 137° and 158° on the map.     

The nucleotide sequence of the 4.5S and 5S rRNA genes and flanking regions from Spirodela oligorhiza chloroplasts

The base sequence of Spirodela oligorhiza chloroplast DNA coding for 4.5S and 5S ribosomal RNA, the flanking regions and the spacer between these two genes has been determined. We have compared these sequences with the corresponding ones in other higher plants. Besides a high degree of homology, some interesting differences are found.     

Repeated sequences on mitochondrial DNA ofSpirodela oligorhiza

Summary Mitochondrial DNA ofSpirodela oligorhiza (duck weed) was analyzed with restriction enzymes. The genome size appears to be at least 250 kbp. Four different PstI fragments were cloned. These four clones contain a sequence which is reiterated about 100-fold on theSpirodela mitochondrial DNA. Hybridization analysis showed that a similar sequence is present onZea mays mitochondrial DNA, although much less reiterated here. The presence of these reiterated sequences might contribute to the physical heterogeneity of plant mitochondrial DNA.     

Analysis of nucleotide sequences polymorphism of chloroplast trnL-trnF spacer of tRNA genes in giant duckweed Spirodela polyrrhiza (L.) Schleiden

Chloroplast DNA trnL-trnF spacer sequences of tRNA genes of 14 specimens of the fam. Lemnaceae have been characterized. Nucleotide polymorphism analysis of the spacer trnL-trnF of geographically isolated and morphologically differing accessions of S. polyrrhiza that is the most widespread species of Spirodela genus showed the low level of intraspecific variability. Five trnL-trnF haplotypes of S. polyrrhiza are identified. Both mono-, and polynucleotide repeats, and also extensive indels, specific to representatives Spirodela polyrrhiza, Landoltia punctata and Lemna sp. are revealed. Competency of Landoltia genus allocation as separate entity was confirmed.     

Variations in chloroplast proteins and nucleotide sequences of three chloroplast genes in Triticum and Aegilops.

Two alloplasmic wheat lines having the same common wheat nucleus but the cytoplasms of Aegilops crassa and Ae. columnaris together with the corresponding normal line (control) were used in the two-dimensional gel electrophoresis of soluble and thylakoid membrane proteins of the chloroplast. Three chloroplast polypeptides: the Rubisco large subunit, the beta subunit of ATP synthase, and an unidentified 31 kDa protein, differed in the common wheat and two Aegilops cytoplasms. Three chloroplast genes, atpB, atpE and trnM, that respectively encode the beta and epsilon subunits of ATP synthase and tRNA(met), were sequenced. The atpB gene differed by two synonymous base substitutions, whereas the other two genes were identical in the two Aegilops cytoplasms. From the predicted amino acid sequences, the beta subunits of the ATP synthase in the Aegilops cytoplasms were assumed to have three amino acid substitutions: Ala by Val, Asp- by Ala, and Gln by Lys+, in contrast to the cytoplasm of common wheat. This accounts for the difference in pI values found for the common wheat and Aegilops cytoplasms. The two base substitutions for the atpE genes of common wheat and the Aegilops cytoplasms were synonymous. The differences detected in the genes encoding the two subunits of ATP synthase do not appear to be ascribable to the differences in phenotypic effects for the common wheat and Aegilops cytoplasms. The base substitution rate of the atpB-atpE-trnM gene cluster was similar to that of the rbcL gene. From the rate for the atpB gene alone, evolutionary divergence of the wheat-Aegilops complex is assumed to have begun ca. 3.0 x 10(6) years ago, as compared to ca. 8.0 x 10(6) years ago for the divergence of the wheat-Aegilops complex and barley.     

The Effect of Ultraviolet Radiation and Sucrose on IAA Levels in Spirodela oligorhiza

U.v. radiation significantly lowers the IAA level in frondsof Spirodela oligorhiza. Fronds grown in the light on mediumsupplemented with sucrose had much higher IAA levels than frondsgrown on medium lacking sucrose. The results suggest that u.v.-inducedabscission in Spirodela and the reduction of abscission by sucroseare both related to their effect on endogenous IAA levels.     

Statistical analysis of nucleotide runs in coding and noncoding DNA sequences

A statistical analysis of the occurrence of particular nucleotide runs in DNA sequences of different species has been carried out. There are considerable differences of run distributions in DNA sequences of procaryotes, invertebrates and vertebrates. There is an abundance of short runs (1-2 nucleotides long) in the coding sequences and there is a deficiency of such runs in the noncoding regions. However, some interesting exceptions from this rule exist for the run distribution of adenine in procaryotes and for the arrangement of purine-pyrimidine runs in eucaryotes. The similarity in the distributions of such runs in the coding and noncoding regions may be due to some structural features of the DNA molecule as a whole. Runs of guanine (or cytosine) of three to six nucleotides occur predominantly in noncoding DNA regions in eucaryotes, especially in vertebrates.     

Nucleotide sequences of transfer RNA genes in the Pisum sativum chloroplast DNA

Summary Eight transfer RNA (tRNA) genes which were previously mapped to five regions of the Pisum sativum (pea) chloroplast DNA (ctDNA) have been sequenced. They have been identified as tRNA^Val(GAC), tRNA^Asn(GUU), tRNA^Arg(ACG), tRNA^Leu(CAA), tRNA^Tyr(GUA), tRNA^Glu(UUC), tRNA^His(GUG), and tRNA^Arg(UCU) by their anticodons and by their similarity to other previously identified tRNA genes from the chloroplast DNAs of higher plants or from E. gracilis. In addition,two other tRNA genes, tRNA^Gly (UCC) and tRNA^Ile(GAU), have been partially sequenced. The tRNA genes are compared to other known chloroplast tRNA genes from higher plants and are found to be 90–100% homologous. In addition there are similarities in the overall arrangement of the individual genes between different plants. The 5 flanking regions and the internal sequences of tRNA genes have been studied for conserved regions and consensus sequences. Two unusual features have been found: there is an apparent intron in the D-loop of the tRNA^Gly(UCC), and the tRNA^Glu(UUC) contains GATTC in its T-loop.     

Characteristics of nucleotide blocks in coding and non-coding DNA sequences from different organisms

A statistical analysis of occurrence of particular nucleotide runs (1 divided by 10 nucleotides long) in DNA sequences of different species has been carried out. There are considerable differences in run distributions in DNA sequences of prokaryotes, invertebrates and vertebrates. Distribution of various types of runs has been found to be different in coding and non-coding sequences. There is an abundance of short runs 1 divided by 2 nucleotides long in coding sequences, and there is a deficiency of such runs in the non-coding regions. However, some interesting exceptions from this rule exist: for run distribution of adenine in prokaryotes and for distribution of purine-pyrimidine runs in eukaryotes. This may be stipulated by the fact that the distribution of runs are predetermined by structural peculiarities of the entire DNA molecule. Runs of guanine or cytosine of three to six nucleotides long occur predominantly in the non-coding DNA regions in eukaryotes, especially in vertebrates.     

Complete nucleotide sequences of seven eubacterial genes coding for the elongation factor Tu: functional,structural and phylogenetic evaluations

The nucleotide sequences of cloned genes coding for the elongation factor Tu of seven eubacteria have been determined. These genes were fiom Anacystis nidulans, Bacillus subtilis, Bacteroides fragilis, Deinonema spec., Pseudomonas cepacia, Shewanella putrefaciens and Streptococcus oralis. The primary structures of the genes were compared to the available sequences of prokaryotic elongation factors Tu and eukaryotic elongation factors 1 alpha. A conservation profile was determined for homologous amino acid residues. Sites of known or putative functions are usually located at highly conserved positions or within highly conserved sequence stretches. The aligned 24 amino acid sequences were used as basis for a phylogenetic analysis. The phylogenetic tree corroborates the kingdom as well as phylum concept deduced from 16S rRNA data.Abbreviations EF-Tu elongation factor Tu - GDP guanosine 5-diphosphate - GTP guanosine 5-triphosphate; tuf gene, gene coding for elongation factor Tu     

Characterization of the trnL-trnF spacer sequence of the chloroplast tRNA genes in Spirodela species

The trnL-trnF spacer region of the chloroplast tRNA genes was sequenced and characterized in 14 accessions of the genus Spirodela (Lemnaceae). Only a low intraspecific variation of the spacer was observed in geographically isolated and morphologically different accessions of S. polyrrhiza, the most widespread Spirodela species. Five haplotypes of the spacer were identified, differing in mono-and oligonucleotide repeats and extended indels, specific to S. polyrrhiza, Landoltia punctata, and Lemna sp. The result supported the isolation of Landoltia from Spirodela.     

Localization and nucleotide sequences of the tRNA GCC Gly,tRNA GUC Asp and tRNA GCA Cys genes from wheat chloroplast

The location on the wheat chloroplast DNA map and the nucleotide sequences of the genes coding for tRNA _GCC ^Gly (trnG-GCC), tRNA _GUC ^Asp (trnD-GUC) and tRNA _GCA ^Cys (trnC-GCA) have been determined. These three genes are located in the large single copy region of the chloroplast genome, about half-way between one of the inverted repeats and the gene for the α subunit of ATP synthase. They are located on two Bam H1 fragments, called B6 and B18 by Bowmanet al. (1), which are separated by about 450 bp and which were cloned in our laboratory to allow sequencing. ThetrnD-GUC andtrnC-GCA sequences show 98.6 and 89% homology, respectively, with the corresponding spinach chloroplast tRNA genes sequences (2), which are the only other higher plant chloroplasttrnD-GUC andtrnC-GCA sequenced so far, while no othertrnG-GCC sequence has been published. ThetrnG-GCC sequence shows only 58% homology with the corresponding gene sequence inEuglena chloroplasts (3).     

Molecular evolution of chloroplast DNA sequences

Comparative data on the evolution of chloroplast genes are reviewed. The chloroplast genome has maintained a similar structural organization over most plant taxa so far examined. Comparisons of nucleotide sequence divergence among chloroplast genes reveals marked similarity across the plant kingdom and beyond to the cyanobacteria (blue-green algae). Estimates of rates of nucleotide substitution indicate a synonymous rate of 1.1 x 10(-9) substitutions per site per year. Noncoding regions also appear to be constrained in their evolution, although addition/deletion events are common. There have also been evolutionary changes in the distribution of introns in chloroplast encoded genes. Relative to mammalian mitochondrial DNA, the chloroplast genome evolves at a conservative rate.      

Suicidal nucleotide sequences for DNA polymerization.

Studying the activity of T7 DNA polymerase (Sequenase) on open circular DNAs, we observed virtually complete termination within potential triplex-forming sequences. Mutations destroying the triplex potential of the sequences prevented termination, while compensatory mutations restoring triplex potential restored it. We hypothesize that strand displacement during DNA polymerization of double-helical templates brings three DNA strands (duplex DNA downstream of the polymerase plus a displaced overhang) into close proximity, provoking triplex formation, which in turn prevents further DNA synthesis. Supporting this idea, we found that Sequenase is unable to propagate through short triple-helical stretches within single-stranded DNA templates. Thus, DNA polymerase, by inducing triplex formation at specific sequences in front of the replication fork, causes self-termination. Possible biological implications of such 'conformational suicide' are discussed. Our data also provide a novel way to target DNA polymerases at specific sequences using triplex-forming oligonucleotides.     

The 3'-terminal nucleotide sequences of lambda DNA

The base sequences of the 3′-termini of coliphage λ DNA have been analyzed by a new technique. Escherichia coli DNA polymerase I was used to add a single radioactive nucleotide to the 3′-OH terminus of one of the DNA strands. The DNA was then digested with pancreatic DNase I, and the resulting oligonucleotides were separated by two dimensional ionophoresis. Terminal oligonucleotides were identified by the presence of the radioactive label, and the base sequence of the labelled terminus was deduced from the base compositions of the terminal di-, tri-, tetra-, etc., oligonucleotides. It is found that the left 3′-terminus of λ DNA ends with the sequence d(pCpGpCpG) and the right 3′-terminus ends with the sequence d(pCpG).