From a potato genomic library a phage lambda clone was isolated that carried nucleotide sequences of two patatin genes, thus demonstrating a close physical linkage between these two members of the patatin gene family. Sequence and restriction analysis showed the genes to be oriented in tandem. The more upstream gene was a pseudogene truncated at the 3' end, whereas the downstream gene was a class II patatin gene. In addition to a 208 bp fragment also present in patatin class I promoters, the region in between both genes contained various direct repeats also found in other patatin genes. To study the promoter activity of this intergenic region, a 2.78 kb fragment was transcriptionally fused to the beta-glucuronidase gene and reintroduced into potato cultivar Bintje. Histochemical analysis revealed expression in the outermost layer of cells of the cortex, in the tuber phellogen, in or around the root vascular system, and also in the abaxial phloem layer of the vascular bundle in leaves.

The class-specific expression of patatin genes was investigated by analysing four new patatin genes. A class I patatin gene from cv. Berolina as well as a class I and two class II patatin genes from the monohaploid cultivar AM 80/5793 were isolated and partially sequenced. Sequence comparison indicates rearrangements as the major source for the generation of diversity between the different members of the classes. The expression of single genes was studied in potato plants transformed with chimaeric genes where the putative patatin promoters were fused to the GUS reporter gene. A detailed histochemical analysis reveals that both class I genes are expressed as the previously described class I patatin gene B33 from cv. Berolina [1], i.e. in the starch-containing cells of potato tubers and in sucrose-induced leaves. The class II gene pgT12 shows the same pattern as the previously described class II gene pgT2 [2], i.e. expression in root tips and in the vascular tissue of tubers, whereas no activity was detectable for pgT4. Thus the expression pattern of both classes of genes seems to be stable at least within or even between different cultivars.

The promoter of the PGT3 patatin gene belonging to the class II subfamily is highly homologous to other class II patatin genes except for a 736 bp insertion in front of the putative transcription start site. The insertion is characterized by structural features resembling a transposable element such as an 11 bp inverted repeat at the termini and an 8 bp duplication flanking the insertion site. Despite the high homology to active patatin genes, fusion of its promoter to the beta-glucuronidase reporter gene does not lead to detectable beta-glucuronidase (GUS) activity in transgenic potato or tobacco plants, suggesting that the inactivation of this gene might be caused by the insertion of the transposon like element.

A new member of the patatin gene family belonging to the class II subfamily was isolated and characterized by DNA sequencing. In order to study the expression profile of this gene, the promoter was fused to the beta-glucuronidase gene and transferred to potato and tobacco. Histochemical analysis revealed high expression in a few defined cells in potato tubers and in a specific layer of both potato and tobacco root tips. In contrast to the developmentally and metabolically regulated class I patatin gene B33 this gene was not inducible by elevated levels of sucrose. Expression of this chimaeric gene was also found in callus and suspension cultures of potato.

We have used a combined genetical and molecular approach to study the structural diversity of the patatin gene family in tetraploid Solanum tuberosum L. cv. Desiree (2n = 4x = 48). Nine dihaploid derivatives (2n = 2x = 24) of cv. Desiree were isolated by gynogenesis through prickle pollination with S. phureja Juz et Buk. Patatin DNA sequences in Desiree and in the dihaploids were examined by probing Southern blots of restriction endonucleases HindIII and XbaI digested DNA with patatin cDNA region-specific gene probes and by more detailed examination (restriction endonuclease mapping and partial DNA sequencing) of 10 patatin genomic clones in bacteriophage lambda replacement vector EMBL4. This provided positive identification for most individual patatin gene family members and some estimate of their organisation and diversity. Most of the 64-72 patatin DNA copies showed little allelic variation based on HindIII and XbaI restriction fragment length polymorphism (RFLP) mapping and did not appear to be very tightly clustered. Four of the 6-8 class I patatin genes (without a characteristic 22 bp insert in their untranslated leader DNA), showed apparent allelic homogeneity, whilst the remaining class I genes comigrated with a single class II patatin gene RFLP subclass. Of the isolated clones, 4 contained apparent pseudogenes lacking 5' control sequences and exon-1 DNA while another clone contained a patatin gene truncated at the 5' region due to the cloning event. The remaining 5 all contained class II genes (with the 22 bp insert) and these showed varying degrees of sequence homology for 400 bp of conserved 5' coding and non-coding DNA (from 77%-95%). In one case the extent of homology differed, with complete sequence divergence upstream of position -80 from the start of transcription. The structural diversity of the patatin gene family is discussed in relation to expression of individual patatin genes and the use of cv. Desiree as a host for potato transformation experiments.

Patatin is a family of glycoproteins that accounts for up to 40% of the total soluble protein in potato tubers. We isolated and characterized 25 patatin genomic clones. All of these exhibit different restriction patterns, but can be divided into two classes based on the presence (class II) or absence (class I) of a 22-bp sequence within the 5'-untranslated region. We determined the complete nucleotide sequence of the class-I clone PS20 and the 5'-flanking sequence of seven additional clones. The transcribed region of PS20 spans 3197 bp and is divided by six introns. The seven exons encode a transcript which is identical to the cDNA, cloned in pGM203 [Mignery et al., Nucl. Acids Res. 12 (1984) 7987-8000]. The 5'-flanking sequences of both class-I and class-II patatin genes are highly homologous up to bp position -87 and then diverge. This conserved region contains the CAAT and TATA homologies as well as a homology to the core enhancer sequence. Within a class there are additional large regions of homology, but differences exist that allow the patatin genes to be further divided into several discrete subclasses. S1 nuclease protection experiments with both class-I and class-II probes showed that class-I transcripts are the predominant species present in tubers. Class-II transcripts are present in tubers, but are 50-100-fold less abundant. In potato roots, class-II transcripts are the predominant species and few, if any, class-I transcripts are present.