Additional evidence implicating Triticum searsii as the B-genome donor to wheat

In vitro DNA:DNA hybridizations and hydroxyapatite thermal-elution chromatography were employed to identify the diploid wheat species ancestral to the B genome of Triticum turgidum. ³H-T. turgidum DNA was hybridized to the unlabeled DNAs of T. urartu, T. speltoides, T. sharonensis, T. bicorne, T. longissimum, and T. searsii. ³H-Labeled DNAs of T. monococcum and a synthetic tetraploid AADD were hybridized with unlabeled DNAs of T. urartu and T. searsii to determine the relationship of the A genome of polyploid wheat and T. urartu. The heteroduplex thermal stabilities indicated that T. searsii was most closely related to the B genome of T. turgidum (AB) and that the genome of T. urartu and the A genome have a great deal of base-sequence homology. Thus, it appears that T. searsii is the B-genome donor to polyploid wheat or a major chromosome donor if the B genome is polyphyletic in origin.Published with the approval of the Director of The West Virginia Agricultural Experiment Station as Scientific Paper No. 1837.     

The ribosomal RNA genes from chloroplasts of mustard (Sinapis alba L.): mapping and sequencing of the leader region

Summary The genes coding for rRNAs from mustard chloroplasts were mapped within the inverted repeat regions of intact ctDNA and on ctDNA fragments cloned in pBR322. R-loop analysis and restriction endonuclease mapping show that the genes for 16S rRNA map at distances of 17 kb from the junctions of the repeat regions with the large unique region. The genes for 23S rRNA are located at distances of 2.8 kb from the junctions with the small unique region. Genes for 4.5S and 5S rRNA are located in close proximity to the 23S rRNA genes towards the small unique region. DNA sequencing of portions of the 5 terminal third from the mustard 16S rRNA gene shows 96–99% homology with the corresponding regions of the maize, tobacco and spinach chloroplast genes. Sequencing of the region proximal to the 16S rRNA gene reveals the presence of a tRNA^Val gene in nearly the same position and with identical sequence as in maize, tobacco and spinach. Somewhat less but still strong homology is also observed for the tDNA ^Val/16S rDNA intercistronic regions and for the regions upstream of the tRNA^Val gene. However, due to many small and also a few larger deletions and insertions in the leader region, common reading frames coding for homologous peptides larger than 44 amino acids can not be detected; it is therefore unlikely that this region contains a protein coding gene.     

Use of lambda phasmids for deletion mapping of non-selectable markers cloned in plasmids

A nonselectable gene carried on a poorly selectable recombinant plasmid has been physically mapped by deletion analysis. Our method involved cloning the plasmid into a coliphage lambda vector and treating the recombinant phage with a chelator. Virtually all particles surviving this treatment carried large deletions within the plasmid insert. Further deletion analysis was done by inserting a selectable lambda sequence into one such deletion derivative and repeating the chelator selection. Chelator selection was also used to isolate deletions constructed in vitro. The deleted phage are readily characterized by restriction mapping, and the gene in question scored after infection of a mutant host strain. These techniques have enabled us to physically assign the cyclopropane fatty acid synthase gene of Escherichia coli to 0.8 kb of a 16-kb segment after characterizing only a small number of isolates. This approach should be generally useful in the mapping of plasmids for which no convenient method exists for selecting or scoring the gene in question.     

Nucleotide sequence of the genome of the filamentous bacteriophage I2-2: Module evolution of the filamentous phage genome

Summary The nucleotide sequence of the circular single-stranded genome of the filamentous Escherichia coli phage I2-2 has been determined and compared with those of the filamentous E. coli phages Ff(M13, fl, or fd) and IKe. The I2-2 DNA sequence comprises 6744 nucleotides; 139 nucleotides less than that of the N- and I2-plasmid-specific phage IKe, and 337 (336) nucleotides more than that of the F-plasmid-specific phage Ff. Nucleotide sequence comparisons have indicated that I2-2, IKe, and Ff have a similar genetic organization, and that the genomes of I2-2 and IKe are evolutionarily more closely related than those of I2-2 and Ff. The studies have further demonstrated that the I2-2 genome is a composite replicon, composed of only two-thirds of the ancestral genome of IKe. Only a contiguous I2-2 DNA sequence of 4615 nucleotides encompassing not only the coat protein and phage assembly genes, but also the signal required for efficient phage morphogenesis, was found to be significantly homologous to sequences in the genomes of IKe and Ff. No homology was observed between the consecutive DNA sequence that contains the origins for viral and complementary strand replication and the replication genes. Although other explanations cannot be ruled out, our data strongly suggest that the ancestor filamentous phage genome of phages I2-2 and IKe has exchanged its replication module during evolution with that of another replicon, e.g., a plasmid that also replicates via the so-called rolling circle mechanism. Offprint requests to: R.N.H. Konings     

Liverwort genomes display extensive structural variations

PIKE, L. M., HU, A., RENZAGLIA, K. S. & MUSICH, P. R., 1992. Liverwort genomes display extensive structural variations. Analyses of the total genomic DNA of eight species of liverworts and two species of green algae by thermal denaturation and CsCl buoyant density gradient centrifugation reveal a high degree of structural complexity and interspecific heterogeneity. The hepatic taxa exhibit two or more DNA components of varying base composition. Average G4-C contents of total cellular DNA calculated from melting profiles are similarly variable, ranging from 38% to 53% G + C. The green alga Chara , a member of the ancestral line to land plants, shows similarities with liverworts in possessing multiple DNA components of comparable complexity, whereas Hydrodiciyon DNA displays a single component. Detailed hybridization analyses of individual density gradient fractions using α-tubulin, rRNA and ribulose 1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene probes were performed to locate the low-copy number and moderately repetitive nuclear genes, and the chloroplast chromosome, respectively. The location of each gene within the density gradient is highly variable among the organisms examined; a-tubulin occurs in fractions ranging from 44–64% G + C, rDNA in 50–64% G + C fractions, and the RbcL gene is located in fractions from 30–59% G + C. For a given species, the two nuclear genes normally overlap in their distributions within the gradient. In most instances, neither gene occurs in the major DNA components, indicating that these components may contain repetitive DNAs. The observed variation in the density of the rbcL gene implies substantial reorganization of the chloroplast genome. The overall differences in the genomic components within and between taxa provide insight into the dynamics of DNA structure that have occurred during the extended evolutionary history of these organisms.     

Construction of a yeast artificial chromosome library of tomato and identification of cloned segments linked to two disease resistance loci

Summary We have constructed a yeast artificial chromosome (YAC) library of tomato for chromosome walking that contains the equivalent of three haploid genomes (22 000 clones). The source of high molecular weight DNA was leaf protoplasts from the tomato cultivars VFNT cherry and Rio Grande-PtoR, which together contain loci encoding resistance to six pathogens of tomato. Approximately 11 000 YACs have been screened with RFLP markers that cosegregate withTm-2a andPto — loci conferring resistance to tobacco mosaic virus andPseudomonas syringae pv.tomato, respectively. Five YACs were identified that hybridized to the markers and are therefore starting points for chromosome walks to these genes. A subset of the library was characterized for the presence of various repetitive sequences and YACs were identified that carried TGRI, a repeat clustered near the telomeres of most tomato chromosomes, TGRII, an interspersed repeat, and TGRIIl, a repeat that occurs primarily at centromeric sites. Evaluation of the library for organellar sequences revealed that approximately 10% of the clones contain chloroplast sequences. Many of these YAC clones appear to contain the entire 155 kb tomato chloroplast genome. The tomato cultivars used in the library construction, in addition to carrying various disease resistance genes, also contain the wild-type alleles corresponding to most recessive mutations that have been mapped by classical linkage analysis. Thus, in addition to its utility for physical mapping and genome studies, this library should be useful for chromosome walking to genes corresponding to virtually any phenotype that can be scored in a segregating population.     

Alteration of mitochondrial genomes containing atpA genes in the sexual progeny of cybrids between Raphanus sativus cms line and Brassica napus cv. Westar

Summary We have investigated the fate of the mitochondrial genomes of cybrids derived from donor-recipient protoplast fusion between X-irradiated Raphanus sativus (cms line) and iodoacetamide-treated Brassica napus cv. Westar. Two out of ten fusion products were male-sterile with the diploid chromosome number of B. napus. The mitochondrial (mt) genomes of the cybrids and their progeny were further analyzed by DNA-DNA hybridizaion using the pea mitochondrial ATPase subunit gene (atpA) as a probe. One cybrid, 18-3, had a 3.0 kb fragment characteristic of B. napus and a 2.0 kb non-parental fragment when the BamHI-digested DNA was hybridized with the probe. In the first-backcrossed progeny of this cybrid, the hybridization pattern was not stably inherited. A 4.0 kb radish fragment, not detectable in the cybrid, appeared in one of the BC₁ generation siblings, and the 2.0 kb non-parental fragment was lost in another. The hybridization patterns in BC₁ progeny siblings of cybrid 12-9 were also varied. The alteration of mtDNA in the cybrid progeny continued to the BC₂ generation. There was no clear evidence of a heteroplasmic state or of sub-stoichiometric molecules in the mt genome of cybrid 18-3. A possible cause of the observed alteration in the mt genome is discussed.     

An aberrant plastid ribosomal RNA gene cluster in the root parasite Conopholis americana

The plastid ribisomal RNA (rRNA) operon of the achlorophyllous root parasite Conopholis americana was completely sequenced. Full-length rRNA genes are retained in the gene cluster, but significant divergence has occurred in the 16S, 23S and 5S genes. Both the 16S–23S intergenic spacer and the 4.5S–5S intergenic spacer have suffered substantial deletions, including the two tRNA genes typically found in prokaryotic and plastid 16S–23S spacers.     

Nucleotide sequence and phylogenetic implication of the ATPase subunits β and ε encoded in the chloroplast genome of the brown alga Dictyota dichotoma

We have cloned and sequenced the genes atpB and atpE, coding for CF₁ subunits and , respectively, of the chloroplast genome of the brown alga Dictyota dichotoma. Although the coding site of atpE cannot be demonstrated by heterologous Southern hybridizations, a 417 bp reading frame 3 to atpB was identified as the gene atpE by sequence similarities with atpE genes from other sources. A maximum sequence identity of 30% is found between the predicted amino acid sequence of the Dictyota subunit and the corresponding cyanobacterial subunits. Including conserved amino acid replacements, the Dictyota subunit exhibits about 70% sequence similarity with the cyanobacterial and land plant subunits. As in cyanobacteria, the atpE gene does not overlap the preceding gene atpB. The deduced amino acid sequence of atpB is 74–79% identical to the corresponding cyanobacterial and chloroplast subunits. Entirely conserved are regions referred to as the catalytic and/or regulatory sites of ATP formation, including interacting regions between subunits and . A phylogram predicted from F₁/CF₁- subunits of eleven different organisms suggests a common evolutionary origin of plastids from chlorophytes and brown algae.