PLANT U14 NUCLEIC ACID SEQUENCES AND DERIVATIVES THEREOF

Abstract: The invention concerns a nucleic acid sequence comprising: [Fa-Ub-Fc]n wherein a + b + c >/= 1 and n >/= 1; and U represents a sequence, "U14", present in a plant genome as a non-translated sequence, said non-translated sequence comprising: i) at least one of the following phylogenetically conserved sequences, wherein N represents any one of the nucleotides A, C, G or T : I : TGATG(A/T) or II : CATTCGCAGTNNCCNCCTAAGA, or III : CCTTCCTNGGATGTCTGA or a sequence having at least 90 % homology with one of sequences I, II or III, and ii) at least one plant-specific region and, iii) a pair of inverted repeats, and F represents a non-translated flanking sequence which, in a plant genome, is immediately adjacent to the said "U14" sequence.

Description: PLANT U14 NUCLEIC ACID SEQUENCES AND DERIVATIVES THEREOF The invention concerns U14 sequences and fragments and derivatives thereof, as well as the use of the sequences in eukaryotic cells. The two major classes of eukaryotic small nuclear RNAs are the spliceosomal sn NAs, involved in pre- messenger RNA splicing and small nucleolar RNAs (snoRNAs) involved in processing of pre-ribosomal RNA (pre-rRNA) transcripts and ribosome formation. At least 12 snoRNAs have been identified in yeast (reviewed by Fournier & Maxwell, 1993) and may be involved in pre-rRNA processing or ribosome formation due to their nucleolar localisation, propensity for base pairing with rRNA transcripts and their association with nucleolar proteins. The best characterised animal snoRNAs are U3 , U8, U13 and U14. The properties of these snoRNAs and the role in pre-rRNA processing of U3 in particular have recently been reviewed (Filipowicz & Kiss, 1993) . U14snoRNA is required for a normal growth phenotype and for processing of pre-rRNA transcripts in yeast (Jarmolowski et al., 1990). U14 contains the conserved Box C and D sequences, of which Box C is required for binding of the nucleolar protein, fibrillarin (Baserga et al., 1991), and are found in a number of nucleolar snoRNAs, and two conserved sequence elements which are complementary to the 18S rRNA (Trinh-Rolik and Maxwell, 1986 ; Li and Fournier, 1992) . The genomic organisation of animal U14s is of particular significance in that in mouse, rat, Xenopus and human, three copies of U14 are located in introns 5, 6 and 8 of the constitutively expressed cognate hsc70 heat shock gene (Liu & Maxwell, 1990) . The orientation of the U14 sequences in the coding strand of the hsc70 introns, the lack of typical UsnRNA promoter sequences, and the lack of a cap structure at the 5' end suggest that these U14 sequences are transcribed as part of the hsc70 pre-mRNA and processed from the intron sequences containing them. Recently, a number of other snoRNAs have been shown to be encoded within introns of, for the most part, nucleolar proteins. U15, a single copy gene, was found in the first intron of the S3 ribosomal protein gene in human (Tycowski et al., 1993) and U16 and U18 in introns of the LI ribosomal protein gene of Xenopus species and human (Fragapane et al., 1993 ; Prislei et al., 1993). Two U17 genes lie in introns 1 and 2 of a human cell cycle regulatory protein gene, RCCI. Examples of other snoRNAs, U19-U21, Y, E2 and E3 which appear to be or are likely to be encoded in introns of ribosomal or nucleolar protein genes have been recently reviewed (Filipowicz & Kiss, supra) The evolutionary conservation of the U14 genomic organisation among different animal species and the now widespread occurrence of intron-encoded snoRNA genes suggests that this kind of gene arrangement may function to ensure co-ordination of expression of nucleolar or ribosomal components. In yeast U14snoRNA is not intron-encoded but is likely to be processed from a precursor containing both U14 and a second nucleolar snRNA, snR190 (Zagorski et al., 1988 ; Balakin et al., 1994). The common features of the termini of the yeast and mammalian U14 and many of the other intron-encoded snoRNAs and the successful processing of U14 from transcripts of the mouse hsc70 intron 5 in Xenopus nuclei suggests conservation of the processing machinery. Although this mechanism remains to be elucidated, processing of U15 and U17 in human in vitro extracts suggested that 5' and 3• end processing were independent of one another and the discrete products detected were indicative of initial endonucleolytic cleavage steps with maturation particularly at the 3' end due to exonucleolytic digestion of bordering sequences (Kiss & Filipowicz, 1993 ; Tyckowski et al., 1993). The difference in U14 genomic organisation between yeast and metazoans and the lack of U17s in introns of Drosophila and Saccharomyces pombe RCCI analogues raises the question of whether or not such gene arrangements are only found in higher eukaryotes and vertebrates. To date, the only information available on plant snoRNA genes concerns U3 (Kiss & Solymosy, 1990 ; Marshallsay et al., 1990, 1992 ; Kiss et al., 1991) and MRP/7-2 snRNA (Kiss et al., 1992). No data on plant U14 genes is available. The technical problem underlying the present invention was to identify whether or not U14 genes are present in plants, and if so, to elucidate their structure and function. The inventors have succeeded in characterising plant U14's using a PCR approach. They have isolated genomic clones of maize and potato and analysed their genomic organisation. Two genomic clones, one from maize and one from potato contained single U14 genes while the second maize genomic contained a cluster of four closely linked U14 sequences. Clustering of plant U14 genes was confirmed by intergenic PCR with maize, barley and potato DNA and intergenic RT-PCR on maize and potato RNA demonstrated expression of polycistronic U14 transcripts. No eukaryotic RNA polymerase II or III promoter elements were recognisable and the immediate flanking sequences were AU-rich, a property of plant introns (Goodall & Filipowicz, 1989) suggesting that the plant U14s may be intron encoded. The lack of ho ology of the flanking sequences with sequences in the databases has not allowed identification of the host gene(s). Furthermore, this novel genomic organisation, the absence of known promoter elements and the detection of polycistronic transcripts by RT-PCR suggests cotranscription of the genes and processing to release individual U14s. The invention concerns a nucleic acid sequence or molecule comprising or consisting of : [F a -U b -F c ] n wherein a + b + c > 1 and n > 1; and U represents a sequence, "U14", present in a plant genome as a non-translated sequence, said non- translated sequence comprising : i) at least one of the following phylogenetically conserved sequences wherein N represents any one of the nucleotides A, C, G or T : I : TGATG(A/T) or II : CATTCGCAGTNNCCNCCTAAGA, or III : CCTTCCTNGGATGTCTGA or a sequence having at least 90% homology with one of sequences I, II or III, and ii) at least one plant-specific region and, iii) a pair of inverted repeats, and F represents a non-translated "flanking" sequence which, in a plant genome, is immediately adjacent to the said "U14" sequence. In the conserved sequence I above, the indication of A and T in the sixth position signifies the presence of either A or T. In the context of the present invention, a "phylogenetically conserved region" signifies a nucleic acid sequence having a length of at least 6 nucleotides and being present either identically or with at least 90 % homology, and preferably at least 95 % homology, in U14 sequences of eukaryotes, including yeast. In the context of the invention "non-translated" signifies a sequence which is present in the genome and in transcribed RNA, but which is not translated into a protein sequence. The U14 gene coding sequence is processed from an RNA precursor and is addressed as a stable molecule to the nucleolus. In the context of the present invention, percentage homologies are calculated as the number of identical bases in the alignment of 2 sequences, divided by the length, between indicated boundaries. If, between the indicated boundaries, the two sequences are not of the same length, the percentage homology is the number of identical bases in the alignment divided by the length of the longest sequence. Thus, the invention firstly concerns isolated nucleic acid sequences having the same sequence and structure as the U14 genes (small nucleolar RNA's) occurring naturally in the plant nuclear genome and in transcribed RNA, and/or the flanking intergenic sequences of the U14 genes. Secondly, the invention relates to derivatives and fragments of these genomic sequences. According to the first embodiment, sequences of the invention, including those illustrated in the figures, can be isolated, using the above phylogenetically conserved sequences I, II or III, preferably II and III, or part thereof, or a sequence having about 90%, for example 95% homology with these sequences, as primers in a nucleic acid amplification reaction e.g. Polymerase Chain Reaction (PCR), preferably reverse-transcriptase PCR as described by Simpson et al., 1992, from plant total RNA preparations (see examples below) . For example, RT-PCR is carried out, using conserved sequences II and III as primers, on RNA from various plant tissues. This normally gives products of around 70 to 100 base pairs, corresponding to an internal fragment of the U14 coding sequence. Next, primers can be designed from the products of the RT-PCR, to allow amplification between closely linked U14 genes (intergenic PCR or IgPCR) . This normally gives products in the 150 to 300 bp size range. Alternatively, hybridisation probes corresponding to the conserved regions or regions amplified between closely - linked genes can be used to screen genomic plant DNA libraries. This process of isolation of U14 genes, with or without their flanking sequences, also forms part of the invention. The U14 sequences of the invention (also referred to as "U14 coding sequences") are easily identifiable in the amplification products, since they contain at least one, and usually all three, of the above conserved sequences and are bordered at each extremity by a pair of inverted repeats. They are also characterised by the absence of known eukaryotic promoters, and by their potential to bind the nucleolar protein fibrillarin. The total length of the U14 sequence of the invention including inverted repeats, varies according to the plant species from which it originates, and according to the particular U14 gene, but is generally between 50 and 150 nucleotides long. Typical lengths are between 100 and 150 nucleotides. The inverted repeats are usually 4 to 20 nucleotides long. The conserved sequence I defined above is also known as Box C, and is most commonly TGATGA but can also be TGATGT. It occurs at the 5 1 end of the U14 sequence, separated from the 5' inverted repeat sequence by 2 or 3 nucleotides. The conserved sequence II is roughly central in the U14 sequence and contains the universal 18-SA element CATTCGCATNNC (Li and Fournier, 1992) complementary to the 18-S rRNA. The conserved sequence III is at the 3' end of the U14 sequence, containing the "Box D" element, GTCTGA an element universal in all U14 sequences identified to date. The conserved sequence III also contains part of the putative 18-SB element CCTTCCT (Li and Fournier, 1992) . The U14 sequences of the invention contain, in addition to the phylogenetically conserved sequences defined above, one or more plant specific regions. In the context of the present invention "plant-specific" signifies that the region has less than 40% and preferably less than 30 % or 20% homology with U14 sequences from other eukaryotes, particularly metazoans and yeast. The plant specific regions identified so far have no homologous counterparts in any known U14 sequences. They can vary in length from about 5 or 6 nucleotides to over 200, depending on the species. In angiosperms, typical lengths are between 20 to 60 nucleotides, for example 30 to 35, or 40. Lower plants such as fern have been shown to have much longer plant-specific regions, for example 100-160 nucleotides. The U14 sequences typically contain two plant- specific regions, one major one having a length of about 35 to 200 nucleotides (around 35 for angiosperms) positioned between the 3' and central conserved sequences, and a minor one, having 7 or 8 nucleotides, immediately 3 • to the Box C conserved sequence. Within the plant kingdom, these sequences are generally conserved with a homology of at least 70%. The major and minor plant specific regions often have the following sequences, respectively : GCCNNGCCAGGCTNGAGAGNTNNTGCTGNNNNAT and YRNARNN wherein N represents any one of the nucleotides A, C, G, T, Y is a pyrimidine (T, C) and R is a purine (G, A) . In both these sequences, insertions may occur between any two consecutive nucleotides shown, particularly following a position represented by "N" in the consensus. The insertion may comprise one or more bases, and can be as long as 50 to 150 bases or more (see figure 11) . Shorter insertions are more likely in the minor plant-specific sequence, whereas longer ones occur in the major sequence. Examples of the major plant specific sequences are GCCNYGCCAGGCTYGAGAGNTNRTGCTGYNNNAT. Examples of the minor plant specific regions are TANANYT wherein Y, R and N have the previously given meanings. Accordingly, the major and minor plant-specific regions may have the following sequences, respectively : GCCNTGCCAGGCTTGAGAGNTNNTGCTGNNNAAT and TGAAGTT or alternatively they have a sequence having at least 70%, and preferably 80 or 90% homology with one of these sequences. Within a species, homology of the plant-specific region can be as high as 80%, 90% or 95%. The minor plant specific sequence has no counterpart in yeast or vertebrate U14 genes. The major conserved sequence has a counterpart in yeast although the degree of homology between these two regions in plants and yeast is very low. In vertebrates, this region is missing completely. The major plant-specific region can also be considered as comprising 2 smaller plant specific sub-regions separated by a relatively conserved region having about 10 nucleotides with some similarity to corresponding sequences in both vertebrate and yeast U14 genes (see figure 15 - plant-specific sub-regions P2 and P3) . P2 often has the sequence GCCNNGCCAGGC with or without an insertion of the type indicated above. P3 often has the sequence TGCTGNNNNAT. The sequence of the invention may therefore comprise or consist of one of the two plant-specific sub-regions P2 and P3, or a fragment thereof having at least 6 nucleotides and preferably at least 8 nucleotides. In the same way as for the major plant-specific region, insertions may occur between any 2 consecutive bases shown in the above sequences for P2 and P3. The insertions can be from one to several tens of bases long, for example 1 to 150, or 1 to 50 or 60, and may occur in the following positions : P2 : GCCNN(X)GCCAGGC P3 : TGCTG(X)NNNNAT wherein X represents an insertion of at least 1 nucleotide. The U14 sequences of the invention further contain, at the extremities of the coding sequence, a pair of inverted repeats, which usually have a length of between 4 and 20, for example 6 to 12 nucleotides. and which are complementary to each other to allow the formation of a stem by base-pairing. The stem structure is thought to protect the U14 gene from digestion by RNase. Examples of such inverted repeats are : WTGCCN wherein W = T or G, with its complement N = T, G, C or A TATGGC and GCCATA, TXYZTTGC wherein X = G or C, with its complement, Y = C or T, Z = A or T, TTGGGGGATT and AATCCCCCAA GCCZTGTT, Z being A or T, with its complement. In the above sequences, XYZ is often GCA (as in barley and maize) or CTT (as in potato) , and N is often C, A or T. The structure of a typical plant U14 sequence is shown in figures 1, 2 and 15. The sequences of the invention may comprise, in addition to the U14 sequence, or instead of the U14 sequence, a sequence "F" which corresponds in the plant genome to the non-translated flanking sequence which is immediately adjacent to the U14 sequence. The sequence F is also referred to as an intergenic region, in the case of polycistronic (clustered) U14 genes, because it separates one U14 gene from the next. If plant U14 genes are intron encoded, the sequence F will separate the U14 sequence from the adjacent exons. Normally, therefore, each U14 gene is immediately flanked, on the 5' and 3' sides, by a flanking sequence. The 5' and 3 1 "F" sequences may be identical to each other of different. Where the sequences of the invention are obtained by amplification of genomic DNA, or by RT-PCR of total plant RNA, the sequences "F" can easily be identified among the amplification products by their position immediately 5* and 3 1 to the U14 sequence, more particularly, immediately 5 1 and 3 1 to the inverted repeats which delimit the U14 sequences. The "F" sequence extends from the inverted repeats upto either the adjacent U14 sequence in the case of polycistronic organisation, and potentially upto the adjacent exon, which can be identified either by comparison with the mRNA or cDNA of the same genomic sequence, the U14 and flanking sequences not being present in the mRNA, or by inverse PCR. The flanking sequences "F" vary in length, for example, from about 20 to 140 nucleotides, typically 20 to 50 in the case of polycistronic U14 genes, and from 20 to 500 or more in the case of monocistronic U14 genes. Since the plant U14 genes are most probably in introns, the intergenic sequences F are commonly AU rich. Although the sequences of the flanking regions vary greatly between species, some zones of conservation have been identified. For example the following sequence can be considered to be an inter- species consensus sequence occuring in the flanking regions of the U14 genes of all species tested so far : TTCT(T)GYYY wherein (T) may be present or absent, Y = pyrimidine (T or C) . Y is most often T. This sequence occurs, in the case of polycistronic genes, in the intergenic region between about 5 to 30 nucleotides downstream from the 3' inverted repeat, and in the case of monocistronic genes, about 50 to 60 bases downstream from the 3' inverted repeat. It has also been shown, by the inventors, that within a species, particularly for polycistronic U14 genes, a high degree of homology, for example 70% or more, is exhibited between flanking sequences. For example, in maize, the following two sequences occur in a plurality of U14 flanking sequences : TTCTTGTCCAGCTC and CCTATGTTTGATACTTGT In potato, particularly in the intergenic regions of polycistronic genes, the following sequence occurs : GTTCTTG(C/T)TTGATGATGAATA. In barley, the intergenic region often contains the following sequence : GTTTCTG(C/T)TTTAT. These interspecies and species-specific consensus flanking sequences also form part of the invention. It is thought that these conserved flanking sequences may be important for the recognition or cleavage of the intergenic regions of polycistronic U14's, enabling the release of individual U14s's. The sequences of the invention may be composed of a U14 sequence alone, an intergenic or flanking sequence "F" alone, or a combination of U14 and F sequences. Particularly preferred sequences have the structure [F a -U b -F c ] n , in which a, b and c each have the value 1, and n is an integer from 1 to 200, preferably 1 to 10, for example 3, 4 or 5. This type of structure corresponds to the polycistronic genomic organisation naturally occurring in plant genomes. Also preferred are variants wherein only the U sequence is present, i.e. b > 1, and a and c are both 0, or also variants in which only one F sequence is present, i.e, a or c in the above general formula, is 0. Thus, according to a preferred embodiment, the sequence of the invention comprises a U14 sequence comprising : i) at least one of the said phylogenetically conserved regions I, II or III as defined above, or a sequence having at least 90 % homology with one of sequences I, II or III, and ii) at least one plant specific region chosen from PI, P2 and P3 wherein : PI = YRNARNN P2 = GCCNN(X)GCCAGGC P3 = TGCTG(X)NNNNAT Y, R and N being as previously defined, (X) being present or absent and representing an insertion of 1 to 150 bases, and iii) a pair of inverted repeats having from 4 to 20 nucleotides each and being capable of hybridising to each other to form a stem structure, and said sequence F contains TTCT(T)GYYY. This variant also englobes derivatives or fragments of the above sequence comprising at least sequences (ii) and/or F, optionally in association with sequence (i) and/or with sequence (iii) . A typical example of a U14 sequence of the invention is the maize U14 sequence : TATGGCAATGATGTTGAAGTTAAAGGCTTGTTTCTCAACAT TCGCAGTAGCCGCCTAAGAGCTTTCGCCCTGCCAGGCTTGA GAGCTTGTGCTGTTTAATCCTTCCTTGGATGTCTGAGCCATA, or a sequence exhibiting at least 70 % homology, preferably at least 80 %, with this sequence. Other examples are shown in the figures, particularly potato and fern. U14 sequences of monocotyledonous and dicotyledonous plants, for example the cereals, e.g. maize, wheat, barley, oats, rye, etc... and of the Solanacea (e.g. potato) , the Cruciferae and the Cucurbitacea are particularly preferred. In addition to the U14 and "F" sequences defined above, the sequences of the invention may also include sequences other than F or U14 sequences, for example, vector sequences, linkers, adaptors, restriction enzyme sites, etc.. , or any engineered sequence appended to the 5 1 or 3* ends of U14 (e.g. promoter/terminator sequences) . In a preferred embodiment, the invention also relates to derivatives of the genomic-type sequences defined above, said derivatives comprising fragments of either or both of the F and U14 sequences, or of sequences showing at least 70% and preferably at least 80% or 85% homology with the F or U14 sequences. The fragments may be combined with other types of nucleic acid sequence. Advantageously the fragments have a length of between 6 nucleotides to upto 5 kb preferably 6 to 1000, such as 10 to 500, or 10 to 200, for example 10 to 40. According to a preferred variant, the U14- derivative comprises or consists of the plant-specific region of the U14 sequence as defined above, or a fragment comprising at least 6, and in the case of fragments of the major plant-specific region, particularly at least 10 or 20, consecutive nucleotides of this region. Preferably, the plant- specific fragment has a length of 6 to 25, for example 6 to 10 nucleotides. For example, according to this variant, the plant-specific fragments may comprise at least 6 consecutive nucleotides of one of the following sequences, and preferably, when the fragment has a length of 6 nucleotides, it does not contain more than two consecutive "N"'s : YRNARNN or GCCNNGCCAGGCTNGAGAGNTNNTGCTGNNNNAT wherein N represents any nucleotide, Y represents a pyrimidine and R represents a purine, or of a sequence exhibiting at least 70% and preferably at least 80% homology with one of these sequences, or a sequence corresponding to one of these two sequences with insertions between two consecutive bases as defined above preferably having a length of about 1 to 150 nucleotides. The plant-specific fragment may also comprise or consist of the sub- regions P2 and P3, with or without insertions as defined above. The U14 sequence derivative may also be a sequence comprising or consisting of one or both of the inverted repeats found at the 5• and 3* extremities of the U14 sequence in the plant genome, as described above. In particular, the derivative may comprise or consist of one of the following sequences : WTGCCN wherein W = T or G, with its complement, N = T, G, C or A, TATGGC and GCCATA, TXYZTTGC wherein X = G or C, with its complement, Y = C or T, Z = A or T, TTGGGGGATT and AATCCCCCAA, GCCZTGTT with its complement wherein Z = A or T. According to this variant, the length of the inverted repeat fragment may correspond to that naturally occurring in the plant genome, for example between 4 and 20 nucleotides, or may be lengthened or shortened by addition or deletion of nucleotides. If the inverted repeats are lengthened by addition of nucleotides, base-pairing between the two halves of the repeat must be conserved. The U14 sequence derivative may also be a fragment of the flanking or intergenic sequence "F" comprising or consisting of at least 6, and preferably at least 10 or 20, consecutive nucleotides of the sequence F, or of a sequence having at least 70%, and preferably at least 80% homology with the F sequence occurring in the plant genome. For example, the F sequence fragment may comprise or consist of at least 6 and preferably at least 8 consecutive nucleotides of one of the following sequences : TTCT(T)GYYY wherein (T) may be present or absent, and Y = pyrimidine (T or C) . Y is most often T. Examples of this tye of sequence are : TTCTTGTCCAGCTC GTTCTTG(C/T)TTGATGATGAATA, and GTTTCTG(C/T)TTTAT Another example of a flanking sequence fragment is : CCTATGTTTGATACTTGT According to a most preferred embodiment, the sequence of the invention comprises a nucleic acid sequence containing or consisting of an association of fragments as defined above, i.e. containing or consisting of at least two of the sequences derived from the F-U14 sequences, (plant-specific region, inverted repeats, flanking sequences and derivatives) as defined above. An example of this embodiment of the invention is a plant specific sequence or fragment thereof in association with an inverted repeat or a fragment of the flanking or intergenic sequence "F" with an inverted repeat. In this case, the inverted repeat may correspond to an inverted repeat naturally occurring in the plant U14 gene, or alternatively, may be any inverted repeat capable of forming a base- paired stem. The sequence comprising or consisting of an association of fragments of the invention may also contain one or more of the sequence derivatives as defined above, in association with all or part of one of the phylogenetically conserved regions. By "part of the phylogenetically conserved regions" is meant a fragment of at least 4 and preferably at least 6, for example at least 8 or 10 consecutive nucleotides of these regions. According to this latter variant, particularly preferred associations are the Box C and Box D regions in association with an inverted repeat, or with a flanking sequence fragment. Another preferred chimeric construction is an inverted repeat, together with box C and box D elements and parts of the flanking sequences, for example the consensus regions shown above. Another example is the 18-SA or 18-SB regions in association with an inverted repeat, optionally also with parts of the flanking sequence. According to another variant of the invention, the chimeric construction may comprise a promoter to drive expression of U14 genes or sequences, an intron with splice sites, and, embedded in the intron, the U14 sequence or fragments or derivatives thereof. It is possible according to this embodiment of the invention, to associate in one sequence, fragments originating from different plants, for example inverted repeats from maize with a plant specific sequence from barley, or another cereal etc... giving rise to hybrid U14 genes and fragments, or even to create hybrids originating from different kingdoms or classes of organisms, e.g. plant/human or plant/yeast. According to this latter variant, the hybrids preferably contain at least part of the plant specific region defined above, or its complementary sequence, and/or a plant U14 inverted repeat, in association with a U14 sequence, fragment or derivative from a eukaryotic organism other than a plant. The invention also concerns sequences which are complementary to any of the above sequences, derivatives and fragments. By "complementary" is to be undestood a degree of complementarity sufficiently high, for example at least 80 or 90%, to allow hybridisation of the sequence to its complement in stringent conditions by standard techniques (Sambrook et al., 1989). For example : - hybridisation on nylon membrane filters in 20 ml of hybridisation solution (5xSSPE, 0.5 % SDS, 5xDenhardt's, 20 μg/ml sonicated salmon sperm DNA) , - a pre-hybridisation for 1 hour at 65°C is followed by a hybridisation of 12-16 hours (solution containing labelled probe) at 65°C. The filters are washed as follows : * 1 wash for 30 min in 2xSSC, 0.5%SDS at 65°C ; * 1 wash for 20 min in 2xSSC, 0.1%SDS at 65°C ; * 1 wash for 10 min in 0.5xSSC, 0.1%SDS at 45"C Most preferably, the sequences are 100% complementary, possibly with 3 or 4 mismatches, at most. This embodiment of the invention includes antisense sequences to plant whole U14 genes and flanking or intergenic sequences, as well as antisense to all of the derivatives and fragments defined above. Also included in this embodiment of the invention are ribozymes, for example of the hammerhead or hairpin type, whose hybridising arms are complementary to the U14 sequences and derivatives as defined above. and of the RNaseP type, using External Guide Sequence technology. For details of hammerhead and hairpin ribozymes see European and International patent applications EP-A-321201 and WO-A-9119789 respectively. The ribozymes are preferably capable of cleaving the U14 coding sequences or intergenic regions. The sequences of the invention may be either RNA or DNA, or a mixture of the two. The sequences of the invention have a number of applications and uses. The identification, by the inventors, of the existence of U14 sequences showing regions of homology with vertebrate and yeast U14 sequences signifies that U14 sequence of any eukaryotic origin and fragments and derivatives thereof, can be used in plants, e.g. in transformation. Furthermore, plant U14 sequences and fragments and derivatives thereof as defined above, can be used in other eukaryotes, e.g. yeast or mammals, particularly in transformation. Any of the above sequences, derivatives and fragments can be used, optionally labelled with a detectable marker, as a probe to detect plant U14 sequences, or as a primer in a nucleic acid amplification reaction for amplication of sequences within and adjacent to plant U14 genomes. As labels, all the conventional systems such as radio-labelling, enzyme-labelling, fluorescein and biotin etc.. can be used. Use can be made of the particular functions of certain parts of the U14 genes and flanking sequences, for example the inverted repeats can be used as stabilisors to protect heterologous RNA from digestion by RNase. The anti-sense and ribozyme variants of the invention, since they are capable of inactivating or cleaving, respectively, the U14 genes and flanking sequences, are useful in the regulation of the processing of pre-rRNA transcripts and ribosome formation. Indeed, it has been shown in yeast (reviewed by Filipowicz and Kiss, 1993) that impairment of U14 results in impairment of 18SrRNA production, and accumulation of 23SrRNA. Inactivation or cleavage of U14 genes by ribozymes therefore results in cell death due to impairment of these essential functions. According to this variant of the invention, the antisense and ribozyme constructs may be coupled with a tissue-specific promoter to direct tissue specific ablation, e.g. to create male- sterility. The invention further relates to transformation vectors containing any of the sequences of the invention, suitable for stably transforming plant cells, and to the transformed cells thus obtained. Clearly all necessary signals for transcription initiation and termination etc.. , must be present. Conventional transformation techniques are applied. Transgenic plants, obtained by regeneration of the transformed cells, also fall within the scope of the invention, particularly plants capable of producing the U14 anti-sense and ribozyme sequences discussed above. As examples of plants capable of being transformed by the sequences of the invention, mention can be made of monocotyledons and dicotyledons such as the cereals e.g. wheat, maize, barley, rice, etc... ; Brassicae e.g. oilseed rape, cabbage, broccoli, etc... ; Cucurbitacea e.g. melon, cucumber and also sunflower, soya, etc... The invention also concerns eukaryotic cells other than plant cells, particularly mammalian and yeast cells, containing the plant U14 sequences and derivatives particularly the chimeric fragment . associations defined above. The cells may be stably transformed by the U14 and derived sequences. Different aspects of the invention are illustrated in the figures : Figure 1 is a schematic representation of the different regions of mouse, yeast and plant U14 snoRNAs. Thick boxes represent conserved regions, as do the Boxes C and D. Figure 2 shows the aligned U14 coding sequences of Yeast, Mouse, Maize (MU14.1a → MU14.4) and Potato (PU14.1), the conserved and specific regions of the sequences being illustrated. The consensus sequences I, II and III are shown. Conserved region II can be extended both in the 5' and 3' direction as shown. Putative 18S-A and 18-SB regions are also marked, PL.sp. = Plant specific. X = region highly conserved between plants and yeast but absent in vertebrates. The minor plant specific region immediately 3' to Box C is absent in all other classes of organisms so far tested and shows around 60 % homology between plant species. The major plant specific region is conserved with at least 70 % homology between species and about 90 to 95 % within a species. Phylogenetically conserved regions II and III have at least 70 % homology, (often at least 90 % homology in the case of region III) between classes of organisms. Capital letters indicate the presence of the base in all positions ; small case letter indicate the presence of the base in all but one position. Figure 3 shows a strategy for cloning U14 snRNA genes from plants. In the first stage, primers 1 and 2, specific to conserved sequences in animal and yeast U14 were used in RT-PCR reactions with plant RNA. The sequences of the generated products (approximately 90bp) were used to design primers for amplifying between closely linked genes using total plant DNA in stage 2. Figure 4 shows the alignment of Plant (PL) , Yeast (YST) and vertebrate (VERT) U14 snoRNA Consensus sequences. Figure 5 shows the alignment of plant (MU = Maize, PU = potato) , yeast and vertebrate U14 snoRNA sequences, with the consensus sequence. Figure 6 shows the polycistronic Maize U14.1 sequences, including flanking intergenic sequences and inverted repeats, as obtained by sequencing of fragments of isolated genomic clones. This transcript contains four full U14 sequences MU14.1a, MU14.1b, MU14.1c and MU14.1d. Inverted repeats and Box C and D elements are marked. Figure 7 shows the maize single U14 sequence U14.4, with flanking intergenic sequences, as obtained by sequencing of fragments of isolated genomic clones. Inverted repeats and Box C and D elements are marked. Figure 8 shows the potato single U14 sequence PU14.1 with flanking intergenic sequences, as obtained by sequencing of fragments of isolated genomic clones. Inverted repeats and Box C and D elements are marked. Figure 9 shows approximately 80 nucleotides of the 3' end of a U14 gene from Arabidopsis thaliana, in cDNA. Box D element is marked. Figure 10 shows alignment of sequenes of U14 IgPCR products from maize (Zm) and potato (St) . Conserved boxes C and D at the 3' end of the upstream gene and 5' end of the downstream gene respectively are indicated. The positions of maize primers III and IV (and the corresponding potato primers V and VI) are shown by arrows. Partial yeast and mouse U14 coding sequences are presented. Figure 11 shows alignment of fern (A. nidans) U14 sequences with those of maize U14.4. The fern sequences were obtained by RT-PCR, using primers I and II on total fern RNA. Figures 12 and 13 show barley U14 intergenic sequences, obtained by intergenic PCR using maize primers III and IV on barley DNA. Boxes C and D and putative inverted repeats are underlined. Figure 14 shows regions of potato and maize U14 genomic clones showing positions of coding regions (black boxes) and the positions of three blocks of sequence homology between the 5' flanking regions of the two maize genomic clones (shaded boxes) . Figure 15 shows alignment of plant U14 genomic sequences with mouse and yeast U14 coding regions. Inverted repeats at the 5' and 3' ends of the genes are shown, boxes C and D are in bold, 100 bp of 5* and 3 1 flankings sequences of the single genes MU14.4 and PU14.1 and flanking the U14 gene cluster of MU14.1, and the sequences downstream of genes la, lb and lc are presented, adjoining downstream U14 coding regions in the MU14.1 gene cluster are indicated by brackets. The gene fragment downstream of mU14.1b is shaded. Regions of complementarity to 18S rRNA (18S-A and 18S-B) are indicated by asterisks and conserved region 2 (Reference 22) is overlined and possible extensions of this region of homology are indicated by dotted lines. The three plant specific sequences (P1-P3) are shown by brackets and the putative extended inverted repeats in PU14.1 are underlined in bold. EXAMPLES Details of method : Total RNA preparations of potato "Cara" and Maize "Kelvedon Glory" were prepared by standard procedures. For reverse transcriptase-polymerase chain reaction (RT-PCR) the RNA preparations were extensively DNased with RQI RNase-free, DNase prior to first strand cDNA synthesis with primer II, 5'-TCAGACATCCAAGGAAGG-3• , complementary to a conserved region at the 3' end of mouse (positions 65 to 82) and yeast U14 (positions 107 to 124) . PCR reactions were carried out following addition of a second oligonucleotide primer I, 5 1 - CCATTCGNNGTTTCCAC-3' homologous to mouse (positions 24 to 41) and yeast U14 (positions 31 to 46) . RT-PCR reactions were carried out by standard procedures (Simpson et al., 1992). Amplified products were isolated following agarose gel electrophoresis, subcloned into pGEM 3zf(+) (Promega) and sequenced. Primer III 5'-CCCGGGAGCTCAGGCTTGAGAGCTAGTGC-3' , and Primer IV 5•-GAATTCTGCAGCANGGCGAAAGCTCTTAG-3 ' (or primers corresponding to III and IV but lacking the first 10 5' nucleotides) were designed on the basis of the maize RT-PCR amplified sequences and were used to amplify intergenic sequences between adjacent, closely linked U14 genes in both PCR and RT-PCR reactions (figure 3) . For potato, primers V 5 1 - CCCGGGAGCTCAGGCTTGAGAGCATATGC-3• , and VI 5•- GAATTCTGCAGCGAAGGCGAAAGCACTTAG-3' , also designed to amplify intergenic sequences between adjacent, closely linked U14 genes, were used in PCR and RT-PCR reactions with potato DNA and RNA. For fern, primer II and a primer designed to plant U14 sequences, primer VII, 5'- CGGAAGCTTNNAAGGCTTGTTTCTC-3 ' were used in RT-PCR on fern RNA. Materials : Restriction enzymes and T4 DNA ligase were purchased from Boehringer (Mannheim) , Pharmacia and Promega. SP6 RNA polymerase, T7 RNA polymerase and RNase inhibitor were from Pharmacia. Radionucleotides, T4 polynucleotide kinase and Hybond N* filters were obtained from Amersham. RNase A, RNase T-, and tag DNA polymerase were purchased from Boehringer-Mannheim. MMLV reverse transcriptase and RNase-free, DNase RQI were obtained from Gibco-BRL and Sequenase from United States Biochemicals. RNA and DNA extraction : RNA was isolated from leaf, stem and tuber tissue of the potato (Solanum tuberosum L.) cultivars Cara and Desiree, and from leaf, ovule and silk material of the maize (Zea mays L.) variety, Kelvedon Glory. DNA was extracted from leaf tissue of Cara and Kelvedon Glory. For Northern analysis, total RNA was isolated from leaf tissue of maize, wheat, barley, asparagus, potato, bean, cucumber and bird's nest fern (Asplenium nidans) . All techniques were standard (Sambrook et al., 1989). Primer extension : Mapping of the 5• end of U14 transcripts by primer extension was carried out on DNase-treated total RNA preparations using [ 32 P]-end labelled oligonucleotide primers : PotPex 1 : 5•-TTAGGCGGCCACTGCGAATG-3• ; MzPex 1 : 5 -TTAGGCGGCAACTGCGAATG-3' ; MzPex 2 : 5'-TTAGGCGGCTACTGCGAATG-3• . The two maize primers differed in a single nucleotide to reflect sequence variation seen in the cloned maize U14 genes. Primers were end-labelled with polynucleotide kinase and gel purified. Primer extension was carried out by standard procedures (Sambrook et al., 1989) and products were separated on DNA sequencing gels. RNase /Ti protection analysis : Preparation of [ 32 P]-labelled RNA transcripts complementary to maize and potato U14 genes were prepared from linearised plasmids as described previously (Waugh et al., 1991). RNase A/T- | protection analysis were performed on 5 μg of total RNA as described by Goodall and Filipowicz, 1989 and products separated on DNA sequencing gels. Nucleolar localisation of U14snRNAs in plant tissue : Anti-sense U14 probes corresponding to maize and potato U14 coding/flanking sequences incorporating digoxygenin-UTP was generated by in vitro transcription of linearised plasmids containing the maize U14.la and potato U14.1 genomic sequences and used as an in situ probe on pea root cells as described by Highett et al., 1993. This showed a clear localization to the nucleoli with little or no labelling of the rest of the nucleoplasm or the cytoplasm, thus confirming that this sequence is a nucleolar snRNA. Control in situ hybridisations were carried out using oligonucleotides complementary to plant UlsnRNA and U3snoRNA sequences and showed specific labelling to the nucleoplasm and nucleoli respectively. RESULTS : Isolation of U14 sequences from maize, potato, barley and fern by PCR : Expressed plant U14 sequences were amplified by RT-PCR from total RNA preparations of maize and potato using primers complementary to highly conserved regions of mouse and yeast U14s (primers I and II described above) . Amplified products of the expected size of approximately 80 bp were subcloned and sequenced. Four maize and two potato sequences showed homology to mouse and yeast U14 sequences suggesting that putative plant U14 sequences had been generated. The amplification was also carried out using primer II and primer VII on total RNA preparations from fern (A. nidans) (figure 11) . To amplify more of the U14 sequence in the 5 1 and 3' direction from U14 coding sequences two further primers (primers III and IV for maize, and primers V and VI for potato) were designed using the above plant RT-PCR sequences. PCR reactions with both potato and maize DNA generated amplified DNA products of only 50-350 bp (particularly 150-350) which represented the amplification of sequences between adjacent, closely linked genes. The PCR products were subcloned and the sequences of four maize and four potato clones were obtained (figure 10) . Each of the clones contained approximately 45-50 bp of the 3' end of the upstream gene (including the primer III or V sequence) and approximately 70 bp of the 5' end of the downstream U14 gene (including the primer IV or VI sequence) . The PCR reaction amplified across the regions containing the initial pair of primers confirming their presence and similarity to mouse and yeast U14s. The intergenic regions of the four maize sequences varied in size from 53 bp to 140 bp (distances were measured between the box D of the upstream gene and box C of the downstream gene) while those of the potato clones ranged from 124 to 156 bp (figure 10) . Some homology was apparent among intergenic sequences from the same species. For example ZmIgPCR3 and 4 differed by a 16 bp insertion in the latter. In addition, all four potato clones contained a sequence GTTCTTG(C/T)TTGATGATGAATA (interestingly containing a box C sequence) 41-44 bp downstream of the box D sequence and approximately 75-100 bp upstream of box C in the adjacent downstream gene, however this sequence was not present in its entirely in the potato genomic clone. Primers III and IV from maize were also used in intergenic PCR reactions on barley DNA. Products of 60 to 200 bp were generated (figures 12 and 13) . Isolation of maize and potato U14 genomic clones : Screening of maize and potato genomic libraries with a maize U14 and potato U14 probe generated by RT-PCR identified a number of positive clones. Two maize and one potato genomic clone were purified and hybridising fragments subcloned into pUC19. Maize U14.1 gave an extremely strong hybridising signal and was subcloned on four consecutive Pstl fragments of 1.1, 1.0, 0.7 and 0.065 kb which were sequenced. Four complete U14 coding sequences and a fragment of a fifth coding sequence were identified in a 760 bp region. The four apparently intact genes were called MzU14.1a-d. In order to isolate gene specific clones, sub-fragments were subcloned into pGEM3zf+. i) Gene (a) was cloned as a 432 bp Rsal fragment (pgMU14.1a) and contained 276 bp of upstream flanking sequence ; ii) Gene (b) was isolated as a 266 bp Rsal fragment (pgMU14.1b) and contained the U14.1b coding sequence fragment and 35 bp of upstream and 101 bp of downstream flanking sequence, iii) The whole of gene (d) , the 3'-most two thirds of gene (c) and 327 bp of downstream flanking sequence were isolated on a 581 bp Tag I/Bcfl II fragment (pgMU14.Id) . The second maize genomic clone, MzU14.4, was subcloned as a 2.2 kb EcoRI fragment. This clone contained a single U14 coding sequence which was subcloned as a 750 bp Pstl/EcoRI fragment and a 254 bp Nsil/Bfrl fragment. Similarly, the potato genomic clone, StU14.1 contained a single U14 coding sequence which was subcloned as a 873 bp EcoRI/Pstl fragment and a 438 bp EcoRI/Hindlll fragment. The coding sequences and extensive flanking sequences of all of the genomic U14 genes were generated by standard procedures. Putative plant U14 genomic sequences show regions of homology with vertebrate and yeast U14s : Screening maize and potato genomic libraries with maize U14 and potato U14 probes generated by intergenic PCR (IgPCR) identified a number of positive clones. Two maize and one potato genomic clone were purified and U14-containing fragments subcloned and sequenced (figure 15). Maize U14.1 gave an extremely strong hybridising signal and contained four apparently intact U14 coding sequences (MU14.1a-d) and a fragment of a fifth U14 sequence, all contained within a 761 bp region (figure 14 and 15). MU14.4 and StU14.1 each contained a single U14 coding sequence. The sequences of the four apparently full length U14 coding regions of maize U14.1, and the single genes of maize U14.4 and potato U14.1 can be aligned with yeast and mouse U14 sequences (figures 2 and 15) . The putative coding regions have been defined by the inverted repeat sequences adjacent to the Box C and Box D sequences. All six plant sequences show high homology with extensive regions of identity to one another. When compared to yeast and mouse U14's, sequence homologies ranged from 53 to 63% and 68 to 76% (expressed as the percentage of the yeast and mouse U14 sequences respectively) . While sequence variation exists among the plant, yeast and animal U14 sequences, there are regions of high homology : the Box C sequence and an 19 nt region, including Box D, and lying directly upstream of the 3' inverted repeat. These two regions were called regions 1 and 3 in the comparison of yeast and mouse U14 (Li and Fournier, 1992) and the latter contains a sequence (18S-B) , complementary to 18S rRNA which is also highly conserved in plants. In addition, a region of extensive similarity was identified in the 5' halves of the molecules and includes region 2 of Li and Fournier, 1992 which again has a sequence, 18S-A, with complementarity to 18S rRNA. However, the plant U14 sequence information allows a realignment of the mouse U14 central region which extends region 2 in the 3' direction for all of the sequences. Region 2 is also extended in the 5* direction in the comparison of the plant and sequences. The new alignment gives the functionally essential yeast-specific sequence 1 (Li and Fournier, 1992) counterparts in both mouse and plant U14s, while the essential yeast-specific region 2 is still absent in mouse but overlaps one of the three plant specific sequences. In addition, the plant sequences contain an additional 7 or 8 nucleotides between the 5' extended region 2 sequence and Box C, which are found only in plant U14s. The much longer central yeast-specific region, which is absent in mouse U14 has a counterpart in plant U14s but the plant sequences are shorter and show little homology to the yeast sequence. The sequences and lengths of the inverted repeats in the plant genes vary and there are 2-3 nt between the 5 1 IR and Box C sequence and the 3" IR is usually directly adjacent to the Box D sequence. The predicted sizes of the plant U14s are intermediate between those of mouse and yeast with maize and potato U14s being approximately 117-120 nt long. Of the genomic clones MU14.1, MU14.4 and StU14.1, 2872 bp, 1280 bp and 873 bp were sequenced respectively. Notably, the sequence across the junction of the MU14.1c and Id genes was identical to one of the sequences generated by IgPCR (Zm U14 IgPCRl - figure 10) and the sequence of Zm U14 IgPCR2 corresponded to the junctions between either MU14.1a and lb or the gene fragment downstream of lb and gene lc. These latter sequences are highly homologous to one another as they are contained within a large direct repeat in the gene cluster. The U14 flanking regions were analysed for open reading frames (ORFs) and AU/GC content in an attempt to identify potential protein-encoding exon sequences. Although three regions of nucleic acid homology were observed in the 5' flanking regions of the two maize genomic clones (figure 14) , these regions did not encode similar ORFs and therefore did not appear to correspond to exon sequences. Database searches with ORFs (> 50 amino acids) and flanking DNA sequences failed to identify significant homologies with known genes. In addition, no typical plant gene promoter elements were identified in the flanking or coding regions of the U14 genes. A search with the maize U14 coding region identified > 95% homology to an 80 bp region of an Arabidopsis expressed sequence tag. Northern analysis and primer extension mapping of 5 « end of U14 transcripts : From the sequence alignment, plant U14 snoRNAs were expected to be intermediate in size between yeast and mouse U14s. The sizes of maize, potato and a number of other plant U14s were estimated from Northern analysis of total RNA hybridised with the 316 bp EcoRI/Hindlll fragment of pgMU14.1b containing the maize U14.1b gene. All of the plant species showed a hybridising RNA band of approximately 120 nt consistent with the sequence data, with the exception of the fern, Asplenium nidans which showed considerably larger U14 hybridising bands of approximately 240 to 320 nt. There was no evidence from Northern analyses of larger hybridising species representing unprocessed or partially processed U14 precursor RNA's. Primer extension analysis of total maize ovule RNA with primers complementary to the maize and potato sequences generated a number of major products. Two maize primers (differing by a single nucleotide) were used to reflect a single nucleotide difference between U14.1b, c and d (primer MzPex 1) and U14.1a and U14.4 (primer MzPex 2) . The MzPex 1 primer produced a number of primer extension products in the size range of 54 to 59 nt of which those of 56 nt were the most intense. All of the products mapped within the 5' inverted repeat. The MzPex 2 primer gave a narrower range of products (54 to 57 nt) , of which the 56 nt band was again most intense and all mapped to the 5' IR. Primer extension with total RNA from potato leaf using primer PotPex 1 gave a range of products from 56 to 60 nt which again mapped to the 5' IR. In addition to the major primer extension products described above, other products in the size range of approximately 80-350 nt were observed and were indicative of 5' extensions to, at least, some U14 transcripts. In contrast, a control primer extension reaction with total maize ovule RNA and a primer complementary to maize UδsnRNA gave a single primer extension product of expected size. By comparison to the intensities of the major products, the extended products were low in abundance in the total RNA preparations. They were not derived from residual DNA as the same RNA preparations were used in RT-PCR reaction analyses where PCR controls gave no DNA- derived products (see below) . Detection of polycistronic U14 transcripts by RT-PCR : Intergenic PCR and the gene arrangement of MzU14.1 suggested that some U14 genes are clustered in both potato and maize raising the possibility that these U14 genes are transcribed together as a polycistronic transcript and processed to individual U14snoRNAs. RT-PCR on maize RNA using primer III for initial cDNA production and primers III and IV in the PCR reaction produced a number of labelled products of 135, 152, 180, 261, 266 and 272 nt. Similar reactions with potato RNA preparation from three different potato organs using primers V and VI gave products of 82, 118, 258, 277, 278 and approximately 360 nt. Control reactions without reverse transcriptas ' e gave no products showing no residual DNA in the RNA preparations and confirming that the RT-PCR products in lanes 1-4 were derived from RNA. Cloning and sequencing of some of the products from these lg RT- PCR reactions confirmed the presence of 3' and 5' fragments of adjacent genes divided by an intergenic sequence. From the positions of the primers, the RT- PCR products would contain approximately 120 nt of coding region from the two adjacent genes leaving intergenic regions in the range of 15-150 nt for maize and 140-240 nt for potato. The identity of the smaller RT-PCR products is unknown but could arise from rearranged U14 genes or gene fragments transcribed as part of a polycistronic U14 transcript. Cloning and sequencing of some IgRT-PCR products confirmed the presence of 3' and 5* fragments of adjacent genes separated by an intergenic sequence as already obtained by IgPCR (figure 10) . Four sequenced clones were virtually identical to the sequence of St IgPCR4 (figure 10) , two were identical to St lgPCR2 and one was identical to Zm IgPCRl (figure 10) . Two further maize-derived clones contained the 3' and 5' fragments of adjacent genes separated by only 17 and 18 bp which did not have a counterpart in the IgPCR clones nor in the MU14.1 gene cluster. Thus, RT-PCR has detected a number of transcripts containing adjacent U14 genes suggesting that U14 gene clusters as seen in the genomic MU14.1 clone are likely to be transcribed as a single polycistronic transcript and inferring that individual U14s would be released by processing. RNase / i protection mapping : RNase A/T, protection mapping using antisense RNA probes specific for MU14.1a, MU14.1b, MU14.1d, MU14.4 and PU14.1 detected full length products of 118-126 nt indicating the presence of mature U14 transcripts from these genes (or genes virtually identical to them within the limitations of the RNase A/T, technique) in total RNA. The production of full length products from those genes in the MU14.1 gene cluster taken together with the detection of polycistronic U14 transcripts would suggest that the individual U14s are processed from these larger RNA precursors. Consistent with the sequence analysis, the potato U14 products were slightly larger than those of maize. The range of sizes of the products may reflect sequence variation in the U14s and/or different 5' termini as already observed with primer extension. Much less intense, larger products were observed higher in the RNase A/T, gels and could represent precursor U14 transcripts, but their identity has not been investigated further. Mapping with the StU14.1 probe showed a protected fragment of 134-135 nt which may reflect a particularly stable precursor possibly due to formation of a terminal stem involving 10 bp GC-rich inverted repeats flanking this potato U14 gene. All probes showed smaller protected fragments arising from hybridisation of the probes to transcripts from other U14 gene variants. Control reactions without added plant RNA showed no protected fragments. Taken together, the positions of the 5 1 ends mapped by primer extension and the lengths of RNase A/T, full length products suggest that the 3' ends of the U14 transcripts would map to or immediately adjacent to the 3' inverted repeat. Summarizing, close linkage of some of the plant U14 genes has been demonstrated by IgPCR and the organisation of the cluster of maize U14 genes. In some cases, intergenic distances were too short to accommodate known plant promoter elements. For example, plant snRNA promoters require two elements, the Upstream Sequence Element (USE) and TATA-box, which normally lie within 80 base pairs from the start of transcription. As shown in figures 6, 10, 12 and 13, the intergenic sequences of the invention often have lengths of less than 80 bases. The absence of these signals and other plant gene promoter elements suggested that the U14 genes were either transcribed from novel promoter sequences or as part of larger precursor RNA molecules. The detection of transcripts containing at least two U14 coding sequences by RT-PCR and the identification of an RNA-derived RT-PCR product with the sequence of part of the MU14.1 cluster, further suggest that clusters of U14 genes are transcribed as polycistronic transcripts from which individual U14s are processed. On the basis of flanking sequences, it is not possible, at present, to confirm that these plant U14 gene sequences are intron-encoded. In animal systems only a single snoRNA gene is contained in any one intron. In the maize U14 cluster, obvious 3' and 5' splice sites are not observed in the intergenic regions, suggesting that if these genes are intron-encoded, they would all be contained within the same intron. It is, therefore, possible that the gene clusters are transcribed as a specialised U14 transcript from a single upstream promoter, from novel promoter elements contained within short intergenic or coding sequences, or as part of a pre-mRNA transcript. The location of snoRNAs in introns of genes encoding proteins involved in pre-rRNA processing or ribosome assembly suggest that their expression is both achieved and regulated by exploiting the active or constitutive expression of the host genes (References 3, 24). In addition, it has been postulated that the relative abundance of intron- encoded snoRNAs is reflected in the number of copies of the genes, such that multiple copies (but only one per intron) , as observed with vertebrate U14 and Xenopus U17 and U18 sequences may represent a means of achieving higher levels of snoRNA transcripts (Reference 24) . The novel polycistronic U14 gene arrangements in plants may also ensure abundant levels of U14 transcripts regardless of whether the precursor is intronic or transcribed from a novel promoter. Analyses of splicing and processing of U16 and U18 snoRNA-containing intron transcripts have suggested a two step processing pathway where endonucleolytic cleavages generate snoRNAs with terminal extensions which are then rapidly removed by either endo- or exonucleolytic activity (References 9, 18, 25-27). However, recent in vivo and in vitro analysis of processing of human U17 and U19 snoRNAs have demonstrated that processing requires splicing of the intron and proceeds by exonucleolytic digestion of flanking regions from the debranched intron lariat. The tight clustering of some plant U14 genes suggest that, in plants, initial endonucleolytic cleavages would be involved in generating individual U14s. The low abundance of potential U14 precursor transcripts in primer extension and RNase A/T, mapping would point to rapid processing of plant U14 precursors in vivo. The stability of mature, processed vertebrate snoRNAs and certain intermediates of the processing reaction suggest that these molecules are protected from the exonucleolytic activity required to generate the mature snoRNAs (References 28, 8, 10). While this may be due to stable stem formation between the 5• and 3' termini of, for example, U14-U16snoRNAs and binding of proteins such as fibrillarin (References 9, 21, 28, 8, 18) some processed snoRNAs (U17 and U18) do not contain inverted repeats or box C or D sequences and protection may therefore involve binding of other protein factors (Reference 27) . References 1. Fournier M. J. and Maxwell E. S., 1993, Trends Biochem. Sci., 18, 131-135 ; 2. Zagorski J. , Tollervey D. and Fournier M. J., 1988, Mol. Cell Biol., 8, 3282-3290 ; 3. Filipowicz F. and Kiss T., 1993, Mol. Biol. Rep., 149-156 4. Jarmolowski A., Zagorski J. , Li H. V. and Fournier M. J. , 1990, Embo J. , 9, 4503-4509 ; 5. Baserga S. J. , Yang W. and Steitz J. A., 1991, Embo J., 10, 2645-2651 ; 6. Trinh-Rohlik Q. and Maxwell E. S., 1986, Nucl. Acids Res., 16, 6041-6056 ; 7. Liu J. and Maxwell E. S., 1990, Nucl. Acids Res., 18, 6565-6571 ; 8. Tycowski K. T., Shu M. D. and Steitz J. A., 1993, Gene Dev. , 7, 1176-1190 ; 9. Fragapane P., Prislei S., Michienzy A., Corffourelli E. and Bozzoni I., 1993, Embo J., 12, 2921-2928 ; 10. Kiss T. and Filipowicz W. , 1993, Embo J., 12, 2913-2920 ; 11. Kiss T. and Solymosy F. , 1990, Nucl. Acids Res., 18, 1941-1949 ; 12. Marshallsay C. , Kiss T. and Filipowicz W. , 1990, Nucl. Acids Res., 18, 3459-3466 ; 13. Kiss T., Marshallsay C. and Filipowicz W. , 1991, Cell., 65, 517-526 ; 14. Goodall G. A. and Filipowicz W. , 1989, Cell, 58, 473-483 ; . 15. Simpson C. G. , Sinibaldi R. and Brown J.W.S., 1992, Plant J., 2, 835-836 ; 16. Highett M. I., Beven A. F. and Shaw P. J., 1993, J. Cell Sci., 105, 1151-1158 ; 17. Waugh R. , Clark, G. , Vaux, P. and Brown, J.W.S. (1991) Nucl. Acids Res. 19, 249-256 ; 18. Prislei S., Michienzy A., Presutti C. , Fragapane P. and Bozzoni I., 1993, Nucl. Acids Res., 21, 5824-5830 ; 19. Kiss T. , Marshallsay C. and Filipowicz W. , 1992, Embo J., 11, 3737-3746 ; 20. Marshallsay C. , Connelly S. and Filipowicz W. , 1992, Plant Mol. Biol., 19, 973-983 ; 21. Balakin A. G. , Lempicki R. A., Huang G. M. and Fournier M. J., 1994, J. Biol. Chem., 269, 739-746 ; 22. Li H. V. and Fournier M. J. , 1992, Embo J., 11, 683-689 ; 23. Sambrook J., Fritsch E. F. and Maniatis T. , 1989, Molecular Cloning-A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cols Spring Harbor, New York ; 24. Sollner-Webb B. , 1993, Cell, 75, 403-405 ; 25. Fragapane P., Caffarelli E., Lener M. , Prislei S., Santoro B. and Bozzoni I., 1992, Mol. Cell Biol., 12, 1117-1125 ; 26. Prislei S., Sperando S., Fragapane P., Caffarelli E., Presutti C. and Bozzoni I., 1992, Nucl. Acid. Res., 20, 4473-4479 ; 27. Caffarelli E. , Arese M. , Santoro B., Fragapane P. and Bozzoni I., 1994, Mol. Cell Biol., 14, 1966-1974 7 28. Leverette R.D., Andrews M.T. and Maxwell E.S., 1992, Cell, 71, 1215-1221.

Claims: CLAIMS 1. A nucleic acid sequence comprising : [F a -U b -F c ] n wherein a + b + c > 1 and n > 1 ; and U represents a sequence, "U14", present in a plant genome as a non-translated sequence, said non- translated sequence comprising : i) at least one of the following phylogenetically conserved sequences, wherein N represents any one of the nucleotides A, C, G or T : I : TGATG(A/T) or II : CATTCGCAGTNNCCNCCTAAGA, or III : CCTTCCTNGGATGTCTGA or a sequence having at least 90% homology with one of sequences I, II or III, and ii) at least one plant-specific region and, iii) a pair of inverted repeats, and F represents a non-translated flanking sequence which, in a plant genome, is immediately adjacent to the said "U14" sequence. 2. Nucleic acid sequence derived from the sequence of claim 1 comprising the plant-specific region of the said U14 sequence or a fragment comprising at least 6 consecutive nucleotides of this region. 3. Nucleic acid sequence according to claims 1 or 2 wherein the plant-specific region comprises : - one of the following sequences : YRNARNN or GCCNNGCCAGGCTNGAGAGNTNNTGCTGNNNNAT wherein N represents any nucleotide, Y represents a pyrimidine and R represents a purine. - or a sequence exhibiting at least 70 % homology with one of these sequences, - or a fragment of any of these sequences having at least 6 nucleotides, said fragment containing not more than 2 consecutive "N"s when the fragment has a length of 6 nucleotides, - or one of these sequences in which an insertion of one or more nucleotides occurs between two given consecutive nucleotides. 4. Nucleic acid sequence derived from the sequence of claim 1, comprising the flanking sequence "F", or a sequence having at least 70%, and preferably at least 80% homology with the flanking sequence "F", or a fragment comprising at least 6 consecutive nucleotides of either of these sequences. 5. Nucleic acid sequence according to claims 1 or 4 wherein the flanking sequence "F" comprises : TTCT(T)GYYY wherein (T) may be present or absent, Y = pyrimide (T or C) , - or a sequence having at least 70% homology with this sequence, - or a sequence comprising at least 6 consecutive nucleotides of this sequence. 6. Nucleic acid sequence according to claim 5 , , wherein the flanking sequence F comprises : TTCTTGTCCAGCTC or GTTCTTG(C/T)TTGATGATGAATA or GTTTCTG(C/T)TTTAT - or a sequence having at least 70% homology with one of these sequences, - or a sequence comprising at least 6 consecutive nucleotides of one of these sequences. 7. Nucleic acid sequence according to claim 1 or 4 wherein the flanking sequence F comprises : CCTATGTTTGATACTTGT - or a sequence exhibiting at least 70% homology with this sequence, - or a sequence comprising at least 6 consecutive nucleotides of this sequence. 8. Nucleic acid sequence derived from the sequence of claim 1 comprising one or both of the inverted repeats of the U14 sequence. 9. Nucleic acid sequence according to claims 1 or 8, wherein the inverted repeats are chosen from : WTGCCN wherein W = T or G, with its complement, N = T, G, C or A, TATGGC and GCCATA, TXYZTTGC wherein X = G or C, with its complement, Y = C or T, Z = A or T, TTGGGGGATT and AATCCCCCAA, GCCZTGTT wherein Z = A or T, with its complement. 10. Nucleic acid sequence comprising, in association, at least one of the sequences according to claims 2 to 9 with a second sequence according to one of claims 2 to 9, or one of the sequences according to claims 2 to 9 with a phylogenetically conserved region according to claim 1, or a fragment of the phylogenetically conserved region comprising at least 4 and preferably at least 6 consecutive nucleotides. 11. Nucleic acid sequence according to claim 10 wherein the fragment of the conserved region comprises at least one of the regions chosen from Box C, Box D, 18S-A and 18S-B regions. 12. Nucleic acid sequence according to claim 11 comprising an inverted repeat and a Box C and Box D region, and optionally all or part of a flanking sequence F. 13. Nucleic acid sequence according to claim 1, wherein said U14 sequence comprises : i) at least one of the said phylogenetically conserved regions I, II or III or a sequence having at least 90 % homology with one of sequences I, II or III, and ii) at least one plant specific region chosen from PI, P2 and P3 wherein : PI = YRNARNN P2 = GCCNN(X)GCCAGGC P3 = TGCTG(X)NNNNAT Y, R and N being as previously defined, (X) being present or absent and representing an insertion of 1 to 150 bases, and iii) a pair of inverted repeats having from 4 to 20 nucleotides each and being capable of hybridising to each other to form a stem structure, and said sequence F contains TTCT(T)GYYY, or a derivative of this sequence comprising at least sequence(s) (ii) and/or F, optionally in association with sequence (i) and/or with sequence (iii) . 14. Nucleic acid sequence according to claim 1, comprising the maize U14 sequence : TATGGCAATGATGTTGAAGTTAAAGGCTTGTTTCTCAACAT TCGCAGTAGCCGCCTAAGAGCTTTCGCCCTGCCAGGCTTGA GAGCTTGTGCTGTTTAATCCTTCCTTGGATGTCTGAGCCATA or a sequence having at least 70% homology with this sequence. 15. Nucleic acid sequence comprising the complementary sequence of any of the sequences according to claims 1 to 14. 16. Sequence according to any one of the preceding claims, comprising RNA or DNA, or a mixture of RNA and DNA. 17. Sequence according to any one of the preceding claims, optionally labelled with a detectable marker, for use as a probe for plant U14 sequences. 18. Sequence according to any one of the preceding claims for use as a primer in a nucleic acid amplification reaction. 19. Transformation vector containing a nucleotide sequence according to any one of claims 1 to 15. 20. Plant cells stably transformed by at least one of the sequences according to claims 1 to 15. 21. Transgenic plants stably transformed by one of the sequences according to claims 1 to 15. 22. Method for the regulation of rRNA production and accumulation in plants by transforming the plant with a sequence according to any one of claims 1 to 15. 23. Use of U14 sequences of eukaryotic origin in plant cells. 24. Use according to claim 23 wherein the plant cell is stably transformed by a eukaryotic U14 sequence or fragment or complementary sequence thereof. 25. Use of a plant U14 sequence according to claims 1 to 16 in eukaryotic cells, particularly mammalian or yeast cells. 26. Use according to claim 25 wherein the eukaryotic cell is stably transformed by a sequence according to claims 1 to 16. 27. Use according to any one of claims 23 to 26 wherein the U14 sequence, fragment or derivative is a hybrid U14 comprising parts of a plant U14 sequence and parts of a U14 sequence of eukaryotic origin other than plant. 28. Nucleic acid sequence according to any one of claims 4 to 16, further comprising a U14 sequence of eukaryotic origin other than plant, or fragments or derivatives thereof.

Inventors: BROWN JOHN WILLIAM SLESSOR, LEADER DAVID JOHN, WAUGH ROBBIE, BROWN, JOHN, WILLIAM, SLESSOR, LEADER, DAVID, JOHN, WAUGH, ROBBIE
Applicants: BROWN JOHN WILLIAM SLESSOR, GENE SHEARS PTY LTD, LEADER DAVID JOHN, WAUGH ROBBIE, BROWN, JOHN, WILLIAM, SLESSOR, GENE SHEARS PTY. LTD., LEADER, DAVID, JOHN, WAUGH, ROBBIE
Application year: 1995
Main classification: C12N15
IPC codes: A01H5, C12N1, C12N5, C12N15, C12Q1