Differential expression of members of the napin storage protein gene family during embryogenesis in Brassica napus.

S1 nuclease analysis and sub-family-specific oligonucleotide probes were used to characterize the expression during embryogenesis of the napin storage protein gene family of Brassica napus (oilseed rape). The expression of one sub-class represented by the napin gene gNa peaks and declines earlier than the other members of the family. This sub-class was highly expressed representing ca. 20% of napin mRNA at 26 days after anthesis.

The expression of class I patatin gene fusions in transgenic potato varies with both gene and cultivar.

Patatin is a family of glycoproteins that contributes about 40% of the total soluble protein in tubers of potato (Solanum tuberosum L.). The protein is encoded by a multigene family of 50-70 genes which have been divided into classes I and II on the basis of sequence homology. The promoters of two class I genes, PS20 and PS3/27, were transcriptionally fused to beta-glucuronidase and transformed into the potato cultivars Désirée and Maris Bard. Examination of the expression levels in large populations of microtubers indicated that the PS20 promoter produced beta-glucuronidase activities 5-fold lower in Désirée than Maris Bard whereas the PS3/27 promoter showed similar levels in both cultivars. Furthermore, the relative expression levels from the two promoters were reversed in the two cultivars. The beta-glucuronidase enzyme activity was correlated with the mRNA level but not the copy number of the introduced gene. The implications for the use of patatin promoters in the genetic modification of tubers is discussed.

Ribosomal DNA methylation in a flax genotroph and a crown gall tumour.

The methylation patterns of two flax lines are described. One, a genotroph S1, has 800 rNA genes per haploid cell while FT37/1, a crown gall tumour incited on S1, has only 300. Using the enzymes EcoRII, BstNI and ApyI to assess CXG methylation and HpaII and MspI for CG, we show that the methylation patterns of the rDNAs of both lines are identical. Both lines contain 3 fractions; the first contains repeats that are methylated at all sites examined and the second has some unmethylated sites. The third fraction contains repeats that are fully methylated but contain a discrete hypomethylated site at the 5' end of the pre-rRNA. The number of repeats which show these hypomethylated sites is constant in both lines despite the copy number difference. These may represent the active rRNA gene repeats.

The use of pNJ5000 as an intermediate vector for the genetic manipulation of Agrobacterium Ti-plasmids.

The use of broad-host-range plasmids derived from RP4 as intermediate vectors for the transfer of narrow-host-range recombinant plasmids from Escherichia coli to Agrobacterium tumefaciens as a preliminary to marker exchange is described. Recombinant plasmids having a ColE1 type origin were linked to the RP4 derivative. Cointegrate formation appeared to take place by RecA-independent, homologous recombination within a short piece of DNA derived from the beta-lactamase gene of Tn1/Tn3 carried by both vector components, so that it never disrupted the recombinant portion of the construction. pNJ5000 provides an unstable intermediate vector for use in marker exchange experiments, while its stable relative pNJ1020 provides a carrier for use in binary vector systems.

Nopaline Ti-plasmid, pTiT37, T-DNA insertions into a flax genome.

The FT37/1 plant tumor line induced on flax epicotyls by T37, a nopaline strain of Agrobacterium tumefaciens, contains multiple copies of the pTiT37, T-DNA. There are three to four distinct full-length insertions and one tandem insertion. Allowing for the different copy numbers of the inserts, this amounts to seven to eight T-DNA copies per genome unit. The genome unit in this case is the haploid DNA value (7 X 10(8) bp) which predicts a T-DNA copy number of 14-16 per diploid cell. Three novel types of abnormal insertion are also present. FIL (one copy per basic genome) comprises 3.04-4.47 kb of T-DNA derived from the left end, with a normal left border and an abnormal right border. FIR (four copies per basic genome) comprises 5.88-6.47 kb of T-DNA derived from the right end, with a normal right border and an abnormal left border. The third abnormality is represented by fragment "X," a HindIII fragment of 4.90 kb which contains homology with several noncontiguous regions of the T-DNA and which may derive from a tandem insertion. Of the two possible left-border sites (primary and secondary) in which fusion with plant DNA sequences has been observed, only the primary is used.