No description available.

No description available.

Cytoplasmic male sterility (CMS) is a maternally inherited trait of higher plants that can be suppressed by nuclear restorers of fertility ( Rf) genes that normally down-regulate the expression of a CMS-associated mitochondrial gene. In Brassica napus, nap CMS is associated with the expression of the orf222/nad5c/orf2101 mitochondrial gene region and is suppressed by the restorer gene Rfn. I present here an extensive analysis of the expression of the orf222/nad5c/orf2101 region in nap CMS and fertility-restored plants. Using RT-PCR methodology, I mapped transcript initiation sites, processing sites, and 3' termini. I identified two processing events, one within and one immediately downstream of orf222, that are specific to fertility restored plants and I suggest possible mechanisms by which the Rfn protein may recognize cognate RNA substrates. Unexpectedly, I also found that levels of atp8 transcripts are much lower in CMS than in restored plants. / nad5c, one of the components of the nap CMS-associated region, is the small central exon of the nad5 gene. In higher plants, nad5c transcripts must be joined to exons b and d through two group II intron trans-splicing events. I found that in the dicot Brassica and the monocot wheat, proper splicing requires exon c and d joining occur prior to the splicing of c with b. Joining of c to a/b transcripts prior to c/d splicing results exclusively in mis-spliced products in which the 5' end of c is joined to cryptic sites within exon b. It is suggested that intron sequences downstream of c base-pair with exon a, leading to mis-folding of the b/c intron and mis-splicing. In Oenothera, where the c/d intron is further fragmented into a tri-partite intron, mis-splicing does not occur. I suggest that avoidance of mis-splicing may be a factor that drives fragmentation of trans -splicing group II introns and may have contributed to the eventual evolution of spliceosomal RNAs from a group II intron precursor.

This thesis will discuss the 'Polima' cytoplasmic male sterility (CMS) system (pol) in Brassica napus (Canola) and detailed genetic mapping of the region surrounding restorer gene ( Rfp) for that system This fine mapping of the Rfp region will facilitate efforts to clone the gene that will eventually lead to its characterization. Knowledge of the structure of Rfp will provide insight in the molecular mechanisms governing mitochondrial gene statement as well as pollen production and may lead to the development of alternative methods of pollination control. In addition, it is possible that nuclear restorer genes for other CMS systems in other crops may be similar to that of the 'Polima' system. / Map based cloning requires the identification of DNA markers tightly linked to Rfp. Two PCR based markers which are located on either side of Rfp were developed. These markers allowed facile screening of a large population. / RFLP markers used in this study are based on the synteny between B. napus and the well known crucifer A. thaliana. (Abstract shortened by UMI.)

To identify region(s) of the mitochondrial genome that might be involved in specifying the "Polima" (pol) cytoplasmic male sterility (CMS) of Brassica napus, transcripts corresponding to 14 mitochondrial genes and DNA clones representing $>$90% of the mitochondrial genome of Brassica campestris were analyzed in nap (male fertile), pol (male sterile) and nuclear fertility-restored pol cytoplasm plants. CMS-correlated transcriptional differences among these plants were detected only with the ATPase subunit 6 (atp6) gene. Sequence analysis of the atp6 gene regions of pol and nap mitochondrial DNAs show that rearrangements in the pol mitochondrial genome upstream of atp6 have generated a chimeric 224-codon open reading frame, designated orfJ224, that is cotranscribed with atp6. In male sterile plants, most transcripts of this region are dicistronic, comprising both orf224 and atp6 sequences. In fertility restored-plants, genes at either of two distinct nuclear restorer loci specifically alter this transcript pattern, resulting in predominantly monocistronic atp6 transcripts. The effect of the restorer locus on orf224/atp6 transcripts does not seem to be tissue or developmental stage specific. orf224 comprises a portion of the mitochondrial gene, orfB, fused to sequence of unknown origin. The pol mitochondrial genome contains an apparently functional copy of orfB. The expression of the atp6 region is developmentally regulated in pol plants such that levels of monocistronic atp6 transcripts are increased in seedlings as compared to the floral tissue. Preliminary data indicate that the chimeric gene, orf224, is expressed at the protein level in pol plants.

No description available.

No description available.

No description available.

A two-component transposable element system consisting of a stabilized Activator (Acst) and a chimeric Dissociation (Ds) element has been introduced into the genome of Brassica napus. This Acst/ Ds system incorporates the use of several highly effective screenable and selectable markers. One of these markers is the maize Lc gene, a transcriptional regulator of flavonoid biosynthetic genes. This substrate-independent screenable marker was tested for the first time in B. napus and I show that when overexpressed, there is augmented trichome production and a light-dependent, enhanced accumulation of anthocyanins in B. napus plants. The phenotypes are expressed under a wide range of conditions, are visually distinct, and are observed throughout plant development. When used as a visual marker for the Acst element, Lc B. napus plants were rapidly identified among F2 segregating populations. As part of my goal to develop a very efficient Acst/Ds system for use in B. napus, a conditional negative selectable marker, the E. coli codA gene, was also tested for the first time in B. napus. This was done because use of a substrate-dependent negative selectable marker can facilitate the rapid and reliable identification of stable Ds transposition events when used as a marker for the Acst T-DNA. The enzyme cytosine deaminase, encoded by the codA gene, catalyzes the deamination of the non-toxic compound 5-fluorocytosine (5-FC) to the highly toxic compound 5-fluorouracil. In codA transformed B. napus seedlings, expression of cytosine deaminase results in a severe suppression of growth and this phenotype is dependent on the presence of the 5-FC substrate. Wild-type seedlings, however, lack endogenous cytosine deaminase activity and appear unaffected by the presence of 5-FC in the growth media. These results indicate that codA has the potential to be used effectively in B. napus as a substrate-dependent negative selectable marker for the Acst T-DNA. To determine if Ac transposase cou

Previous studies have shown that transcriptional differences in the orf224-atp6 mitochondrial gene region are correlated with fertility restoration of the pol CMS trait by the dominant nuclear Rfp gene in Brassica napus. Recently, the recessive rfp allele, or a tightly linked gene, was found to act as a dominant gene, designated Mmt, in controlling the production of additional, smaller transcripts of two other mitochondrial loci. The results presented in this thesis reveal that Mmt-specific transcripts lack sequences found at the $5 sp prime$ end of the full-length transcripts of these loci and contain a common sequence, UUGUGG, which maps immediately downstream of their $5 sp prime$ termini. A similar sequence, UUGUUG, is found within orf224 downstream of the major Rfp-specific $5 sp prime$ transcript terminus; these hexanucleotide sequences may serve as recognition motifs in the generation of Mmt- and Rfp-specific transcripts. These results suggest that Rfp/Mmt is a novel nuclear locus affecting the expression of multiple mitochondrial gene regions, with different alleles or haplotypes affecting different mitochondrial genes.