A new Arabidopsis thaliana (ecotype Columbia) genomic library has been constructed in Yeast Artificial Chromosomes: the CIC library (for CEPH, INRA and CNRS). Optimization of plant culture conditions and protoplast preparation allowed the recovery of large amounts of viable protoplasts. Mechanical shearing of DNA was minimized by isolation of DNA from protoplasts embedded in agarose. Cloning of large inserts was favored by including two successive size fractionation steps (after partial EcoRI digestion and after ligation with the vector arms), which selected DNA fragments larger than 350 kb. The library consists of 1152 clones with an average insert size of 420 kb. Clones carrying chloroplast DNA and various nuclear repeated sequences have been identified. Twenty-one per cent of the clones are found to contain chloroplast DNA. Therefore, the library represents around four nuclear genome equivalents. The clones containing 5S rDNA genes, 18S-25S rDNA sequences and the 180 bp paracentromeric repeated element account for 3.6%, 8.9% and 5.8%, respectively. Only one clone was found to carry the 160 bp paracentromeric repeated element. Given the smaller size of clones carrying Arabidopsis repeated DNA, the average size of remaining clones is around 480 kb. The library was screened by PCR amplification using pairs of primers corresponding to sequences dispersed in the genome. Seventy out of 76 pairs of primers identified from one to seven YAC clones. Thus at least 92% of the genome is represented in the CIC library. The survey of the library for clones containing unlinked DNA sequences indicates that the proportion of chimeric clones is lower than 10%.
The Drosophila ovo gene, which encodes a putative transcription factor (Ovo) with TFIIIA-like zinc fingers, is required for female germline survival and proper oogenesis. Three dominant female-sterile ovoD mutations cause ovarian abnormalities that define an allelic series, with ovoD1 displaying the stronger phenotype and ovoD3 the weaker. We report here that all three ovoD mutations are point mutations that create new in-frame methionine codons in the 5′ part of ovo. There are two types of overlapping ovo transcription units, ovo alpha and ovo beta. By using various ovo-lacZ reporter genes, we determined that the long Ovo isoforms starting at methionine M1, present in transcripts ovo alpha, are expressed at low levels only in mature oocytes. Short Ovo isoforms are translated from methionine M373, the first in-frame start codon present in transcript ovo beta, and correspond to the activity defined by recessive loss of function ovo mutations. The new AUGs created in ovoD mutations all are located upstream of the M373 initiation site. Our results support the hypothesis that they can substitute for M373 as translation starts and initiate the synthesis of Ovo proteins that have extra amino acids at their N termini. We propose that premature expression of long Ovo protein isoforms occurs in ovoD mutants and interferes with wild-type Ovo function in controlling female germline differentiation.
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