PRODUCTION OF PHENYLALANINE AMMONIA LYASE

Abstract: For use in genetic engineering a gene is provided, derived from a PAL-producing strain of Rhodosporidium toruloides, from which non coding introns have been excised. The gene may be inserted into plasmid vectors which may be introduced into heterologous organisms so that PAL is expressed. A method of preparing the gene is provided, and its polynucleotide sequence is listed.

Description: PRODUCTION OF PHENYLALANINE AMMONIA LYASE This invention relates to genetic material which encodes the protein phenylalanine ammonia lyase (herein abbreviated to 'PAL') and in particular to such genetic material which lacks the intervening non- coding DNA (introns) normally found in the PAL - encoding gene in its natural state. Phenylalanine ammonialyase (PAL; EC 4.3.1.5) which occurs in plants, yeasts, fungi, and streptomycetes catalyzes the nonoxidative deamination of L-phenylalanine to trans-cinnamic acid (see Gilbert et al., 1985). The enzyme has a potential role in the treatment and diagnosis of phenylketonuria (Ambrus et al. , 1978) and has industrial applications in the synthesis of L-phenylalanine from trans-cinnamic acid (Yamada et al.. 1981). In plants the enzyme, involved in flavanoid biosynthesis, is induced by illumination while in gherkin and mustard seedlings induction is the result of activation of a constitutive pool of inactive enzyme (Attridge et al. , 1974). Illumination elicits de novo synthesis of the enzyme in other botanical species (Schroder et al., 1979). Gherkin, apple, sweet potatoe, and sunflower PAL is also regulated by a specific inactivating system (Tan, 1980). In some basidiomycete yeast phenylalanine can act as sole source of carbon, nitrogen, and energy. As PAL catalyzes the initial reaction in the catabolism of the amino acid, the enzyme plays a key role in regulating phenylalanine metabolism. In Rhodosporidium toruloides PAL is induced by the presence of L-phenylalanine or L-tyrosine (Marusich et al. , 1981). Glucose, and ammonia in the presence of glucose, repress PAL synthesis (Marusich et al., 1981), while induction of PAL activity is the result of de novo synthesis of the enzyme rather than activation of an inactive precursor or a decrease in the rate of PAL degradation (Gilbert and Tully, 1982). Glucose represses PAL synthesis but has no effect upon stability of the enzyme, whereas ammonia prevents uptake of phenylalanine and so may repress enzyme synthesis through inducer exclusion (Gilbert and Tully, 1982). In vitro translation data of mRNA, isolated from R. toruloides grown under different physiological conditions, showed that phenylalanine, ammonia and glucose regulate PAL synthesis by adjusting the level of functional PAL mRNA (Gilbert et al., 1983). In recent years genetic engineering methods have been developed whereby microorganisms which are common or which can easily be grown - on an industrial scale, in particular certain bacteria or yeasts, have their genetic material (DNA sequences) modified so that they produce a desired compound eg a protein. Broadly this is achieved by inserting into the host microorganism a plasmid consisting of a gene which is a polynucleotide sequence which eπcodesthe compound, 0 together with other genetic material which instructs the host's genetic apparatus to synthesise the compound. The gene encoding PAL has recently been cloned as a 8.5 kb genomic PstI fragment (Gilbert et al. , 1985). These studies indicated that PAL is synthesised from a monocistronic mRNA of 2.5 kb, and that the gene is present as a single copy in the the R. toruloides genome. The introduction of the cloned PAL gene into both E. coli (Gilbert et al., 1985) and Saccharomyces cerevisae (Tully and Gilbert, 1985) did not result in the production of PAL protein_ 0 Although attempts have been made along these lines to introduce the cloned PAL - encoding gene from R-toruloides into the microorganism E-coli (Gilbert et al; 1985) and into the yeast Saccharomyces Cerevisae (Tully and Gilbert, 1985), these heterologous hosts did not then produce PAL protein. 25 It is an object of the invention to provide genetic material which may be introduced into host organisms other than R-toruloides, which hosts will then produce PAL protein. Other objects and advantages of the invention will be apparent from the following - Q description. According to a first aspect of the invention there is provided an intron-free structural gene, derived from a corresponding intron- containing structural gene from a eu aryotic microorganism, both genes coding for the same gene product provided that the intron- free gene is capable of expressing the product within a prokaryotic or eukaryotic microorganism. The gene product may be a chemical compound the production of which is desired, for example a protein. According to a second, preferred aspect of the invention there is provided an intron-free structural gene which encodes PAL or a polypeptide which displays PAL activity. The gene is preferably derived from a PAL - producing strain of a eukaryotic organism, most preferably a strain of R toruloides. A portion of the genetic DNA polynucleotide sequence of R.toruloides is shown in Fig 3. The methods used by the inventors to determine this sequence are described later. The PAL encoding sequence extends from the location marked "start codon" to the location marked "stop codon", and the introns, six in number are marked IVS 1 to IVS 6. The amino acids encoded by these codons are shown, as also are various restriction sites. The gene of the second aspect of the invention therefore preferably consists of a DNA sequence identical to, related to, derived from or complementary to the sequence of codons from the start codon to the stop codon in Fig 3, from which the six introns IVS 1 to IVS 6 have been deleted, having the following polynucleotide sequence: ATG GGG GCT GGA CCA AGC TGG GAG TGG GAG GCT GGG AGC TGG GGC ACA AGG GAG GTG AGG GAG GTC GAG ATG GTC GAG AAG ATG CTG GGC GGG GCG AGC GAG TCG AGG GTG GAA GTG GAG GGG TAG TGG CTG AAG GTG GGA GAG GTC GTC TGG GCC GGG AGG AAG GGG AGG GCT GTG CGC GTC AAG GAG AGC GAG GAG ATC CGC TGA AAG ATT GAG AAA TGG GTG GAG TTG TTG GGG TCG CAA GTG TCC ATG AGG GTG TAG GGG GTG AGG ACT GGA TTT GGG GGA TGG GGA GAG ACC GGG ACC GAG GAG GGC ATC TCG CTC CAG AAG GCT GTG GTC GAG GAC CAG GTC TGG GGT GTT CTC CCT TCG TGG TTG GAG TGG TTG GGC CTC GGG GGG GGT GTC GAG AAG TGG GTT GGG GTC GAG GTT GTT GGC GGC GCG ATG ACA ATG GGC GTG AAG AGC TTG ACC CGC GGC CAC TCG GCT GTC CGC GTC GTC GTC GTC GAG GGG GTC ACC AAC TTG CTG AAG GAC GGC ATG ACC GCC ATC GTC CGC GTC CGC GGC ACC ATG TGT GCG TCG GGC GAG CTC TCT GCT CTC TCC TAC ATT GGA GGG GCC ATG AGC GGT CAC CCG GAG AGC AAG GTG CAG GTG GTC GAG GAG GGG AAG GAG AAG ATC CTG TAG GCC CGC GAG GGG ATG GCG GTC TTG AAG GTC GAG GCC GTG GTC CTC GGC CCG AAG GAA GGT CTC GGT GTG GTG AAG GGG AGC GGG GTC TGA GGA TCG ATG GCC ACC CTG GGT GTG GAC GAG GCA GAC ATG GTC TCG GTC GTC TCG GAG TCG GTG AGG GGC ATG AGG GTG GAA GCG ATG GTC GGG CAG GGC GGC TGG TTG GAG GCG TTG GTT CAC GAC GTG ACG CGC CCT CAG CCG AGG CAG ATC GAA GTC GCG GGA AAC ATC CGC AAG GTC CTC GAG GGA AGC CGC TTT GCT GTG CAC CAT GAG GAG GAG GTG AAG GTG AAG GAG GAG GAG GGC ATT GTC CGC CAG GAG GGC TAC CCC TTG CGC ACG TGT CCT CAG TGG GTC GGC CGG CTC GTG AGC GAC CTC ATT CAG GCC CAG GCC GTC GTC ACC ATG GAG GGC GGC CAG TCG AGG ACC GAG AAG CCT CTC ATC GAC GTC GAG AAG AAG ACT TCG GAC CAC GGC GGC AAT TTC CAG GGT GCC GCT GTG GCC AAC ACC ATG GAG AAG ACT CGC CTC GGG CTC GCG CAG ATC GGC AAG CTG AAC TTC AGG CAG GTC ACC GAG ATG CTC AAC GCC GGC ATG AAC CGC GGC CTC CCC TCC TGC CTC GCG GCC GAA GAC CCC TCG CTC TCC TAC CAC TGG AAG GGC CTC GAC ATC GGC GCT GCG GCG TAC ACC TCG GAG TTG GGA CAC CTG GCC AAC CCT GTG ACG ACG CAT GTG CAG CCG GCT GAG ATG GCG AAG GAG GCG GTC AAG TGG GTT GCG CTC ATC TCG GCT CGT GGC AGG ACC GAG TCC AAC GAC GTC GTT TCT GTC CTC CTC GGC ACC CAC GTG- TAC TGG GTT GTC GAA GCC ATC GAG TTG GGC GGG ATC GAG TTG GAG TTG AAG AAG CAG TTG GGC CCA GCC ATG GTC TCG CTG ATG GAC CAG CAC TTT GGC TCC GCC ATG AGC GGC TCG AAC CTG CGC GAG GAG CTC GTG GAG AAG GTG AAG AAG AGG GTC GCC AAG GGC CTC GAG GAG AGC AAG TCG TAG GAC CTG GTG CCG GGC TGG GAC GAC GCC TTC TCG TTC GGC GCC GGC ACC GTC GTG GAG GTG CTC TCG TCG ACG TGG GTC TCG CTG GGC GGC GTG AAC GCC TGG AAG GTC GCC GCC GCC GAG TGG GGC ATG TCG CTC ACC CGG GAA GTG GGC GAG ACC TTC TGG TCC GCC GGG TGG ACC TCG TGG CCC GCG CTC TCG TAC CTC TGG GCG CGC ACT CAG ATC GTC TAC GCC TTG GTG CGG GAG GAG CTT GGG GTC AAG GGC GGC CGC GGA GAC GTC TTC GTC GGC AAG CAA GAG GTG ACG ATC GGG TGG AAC GTC TCC AAG ATC TAC GAG GGC ATG AAG TGG GGG AGG ATG AAG AAG GTC CTC GTC AAG ATG CTC GCT TAG . It is well known in the field of genetics that DNA sequences which are related to or derived ficom a defined sequence may encode the same protein or a polypeptide having similar activity to that expressed by the defined sequence. For example the related or derived sequence may lack some bases or may include some additional bases. Also it is known that the genetic code is degenerate, in that several codons may encode the same amino acid. The related or derived sequence may therefore contain some codons which are different to those listed in Fig 3 but which prefera ' bly encode the same amino acid. Genes which are related to or derived from this sequence of codons in one or more of these ways are included in the invention. Genes related to or derived from this sequence may also be defined in terms of the degree of conformity to this sequence. This is preferably as high as possible, ideally 100%, but 70% or higher, eg 85% or higher conformity to that sequence is generally satisfactory. To enable a gene according to the first or second aspects of the invention to be introduced into a host organism, it is common to include the gene into a recombinant DNA molecule. According to a third aspect of the invention there is therefore provided a recombinant DNA molecule, especially a plasmid, which contains a gene according to the first or second aspects of the invention. The plasmid according to this aspect of the invention may be used as a vector to introduce the gene into a host and may therefore also contain additional genetic material appropriate to a host into which it is intended to introduce the plasmid. Such genetic material may preferably contain an expression control sequence operatively linked to said gene, and/or transcription/translation signals from other genes appropriate to the organism into which the plasmid is to be introduced and from which expression of the product, eg PAL, is hoped. The structure of the plasmid according to this aspect of the invention will vary according to the host organism for which it is to be used as a vector, but by positioning the gene of the first or second aspect of the invention downstream of the appropriate regulatory signals, vectors may be prepared using which expression of R. toruloides PAL may be obtained in any of the currently used production organisms. These include E. coli K12, Bacillus subtilis, Saccharomyces cerevis ae, Pseudomonas putida, Erwinia chrysanthe i and mammalian cell lines. Similarly the nature of the regulating DNA sequences immediately upstream of the PAL cDNA coding region in the plasmid will be composed of appropriate, characterised transcription/translation signals. For example for use in S. cerevisae a ribosome binding site (conforming to the sequence CCACCTT) may be positioned at the appropriate position upstream of the translational start of the PAL gene, and powerful transcriptional signals, such as those derived from the S. cerevisae phosphoglycerate kinase and mating facter genes, placed 5' to the ribosome binding site. The plasmid itself may use standard replicons (eg 2p) and selectable markers (e.g. Leu2, Trp etc)_ Similarly, for use in E. coli use will be made of the PL, tac trp, rac or lac promoters, with appropriate bacterial ribosome binding sites, and plasmids based on ColEl (e.g. pBR322 and pϋC plasmids), RSF1010, and runaway replicons of RI. As the introns present, in the natural PAL gene act as a barrier to the expression of PAL in organisms other than R. toruloides, the invention may be used to produce PAL in a wide range of procaryotic and eukaryotic hosts which are unable to express the natural PAL gene due to the presence of the 6 introns. In accordance with a fourth aspect of the invention there is provided a host organism, especially a strain of E. coli, Erwinia sp., Clostridia sp. , Streptomyces sp., B. subtilis, B. s earothermophilus, Pseudomonas, other microorganisms such as bacilli, yeasts, other fungi, animal or plant hosts, and preferably a prokaryotic host, transformed with at least one recombinant DNA molecule according to the third aspect. The invention also provides a process for the preparation of a gene from which introns have been deleted which includes the steps of. (i) isolating PAL mRNA from a strain of R. toruloides, (ii) synthesising two intron-free complementary DNA ('cDNA' ) sequences from the mRNA, the two cDNA sequences each - containing a portion of a gene which encodes PAL or a polypeptide which displays PAL activity, the two portions together containing the 3' and the 5' ends of the gene. (iii) joining the two cDNA sequences proposed in (ii) to form an intron-free structural gene which encodes PAL or a polypeptide which displans PAL activity. The method used in step (ii) may use a cloning method which forms the cDNA sequences contained in plasmids. In such a case the sequences may be isolated from the plasmids which contain them by cleavage of the plasmids at a suitable restriction site, followed by ligation of the two sequences to form the gene. The gene may then be combined with other genetic material to form a plasmid containing it for example following cleavage of a suitable known plasmid such as pϋC9 at appropriate sites. If desired the gene may then in turn be excised from this plasmid and combined with yet other genetic material to form other plasmids which may be used as vectors. Standard recombinant DNA techniques, familiar to those skilled in the art may be used for the process of the invention. . The gene and/or plasmid produced in step (iii) of this process is preferably one of the genes or plasmids encompassed by the second and/or third aspects of the invention, and the cDNA sequences produced in step (ii) are consequently preferably portions of these. The cDNA sequences produced in step (ii) and plasmids containing them are further aspects of the invention. The invention therefore also includes DNA polynucleotide sequences, eg plasmids, the same as or substantially the same as or derived from or related to those produced by the process of the invention. The invention will now be described by way of examole only with reference to the accompanying figures: Fig 1 is a schematic diagram illustrating how the genetic DNA carrying the PAL gene was sequenced. Fig 2 illustrates the production of the two plasmids carrying the PAL gene which lack the intron sequences of the natural gene. Fig 3 shows the complete nucleotide sequence of the genomic clone, the intron sequences removed in the invention being labelled IVS1 to IVS6, and the corresponding amino acid sequence of PAL. Fig 4 shows the formation of the recombinant plasmid pPAL 3 containing the intron-free gene, by combination of the two cDNA plasmids pPALl and pPAL2. Figs 5 illustrates the DNA nucleotide sequences of the over- & 6 lapping cDNA clones pPALl and pPAL 2 respectively. Fig 7 shows the expression of PAL protein from the plasmid pPAL4. In this description and the figures the following abbreviations are used: Amino acid symbol Nucleotide bases symbol Alanine Ala Uracil ϋ Arginine Arg Thymine T Asparagine Asn Cytosine C Aspartic acid Asp Adenine A Asn + Asp Asx Guanine G Cysteine Cys Glutamine Gin Glutamic Acid Glu Gin + Glu Glx Glycine Gly Histidine His Isoleucine He Leucine Leu L sine Lys Methionine Met Phenylalanine Phe Prσline Pro Serine Ser Threonine Thr Tryptophan Trp Amino acid Symbol Tyrosine Tyr Valine Val Referring to Figs 1 to 6 in more detail: In Fig 1. Region 2 was isolated from the appropriate clone (pHG3), circularised by treatment with T4 DNA ligase, fragmented by sonication, and fragments of between 500 and 1000 bp inserted into Ml3mp8. Region (1) was inserted into Ml3mp8 and M13mp9 as various specific fragments utilising the restriction sites BamHI, Bell and Sail. The sequence of the DNA spanning the BamHI site was obtained by cloning the indicated fragment (3) into M13mp8. In Fig 2. Clone 1 (pPALl) was obtained by the method of Heidecker and Messing (1983). Total mRNA from pal-induced R. toruloides cells was annealed to oligo(dT)-tailed pϋC9, and the first strand cDNA copy synthesised using reverse transcriptase in the presence of all four dNTP's. The newly synthesised strands were tailed with oligo(dC) using terminal deoxynucleotidyl tranferase. Following fractionation by an alkaline sucrose gradient, single-stranded plasmid DNA carrying cDNA sequences were annealed to denatured oligo(dG)-tailed pUC9 and the second strand synthesised using DNA polymerase (Klenow) and the addition of all four dNTP's. Clone 2 (pPAL2) was constructed using the procedure of Gubler and Hoffman (1982) . The first strand cDNA copy was synthesised using reverse transcriptase and a 19-mer oligodeoxynucleotide primer (GATCAGAGGGTTGTCGGTC) complementary to pal mRNA. The RNa within the RNA-DNa hybrid was then nicked with RNase H and the RNA strand replaced with DNA by E. coli DNA polymerase, utilsing the nicked RNA as a primer. The double stranded DNA was then blunt ended by the action of T4 DNA polymerase, tailed with oligo(dC), and annealed to oligo(dG) tailed pBR322. cDNA clones produced using both methods were transformed into E. coli JM83, and colonies screened for Pal cDNa sequences using [ ^.--^^p] dATP-labelled pHG3 restriction fragments. In Fig 3. The determined amino acid sequences of the 5 randomly derived peptide fragments are indicated by overlining of the relevant residues. The introns are labelled IVS 1-6, and the sequence common to all 6 is indicated by underlining of the relevant region. A dashed overline in the 5" non-coding region represents the TC rich > region of the sequence, while the under- and overlining immediately downstream marks a repetitive region. The sequence extends from the most leftward Bell site of Figure 1, to the 3' end of cDNA clone PPALl (Figure 2). 10 In Fig 4. The single line of the pPALl and pPAL3 circular maps represent pUC9 and pUC8 derived DNA, respectively, while the single line of pPAL2 represents pBR322 DNA (see Fig. 2 for construction of pPALl and pPAL2) . The double line of pPAL2 represents the "intron-free" 5* end of the PAL gene, while the thick line of ,e pPALl represents the 3" end of the gene. The 5* end of the PAL gene was isolated as a l.Okb PstI - Fspl fragment from pPAL2 and ligated to a 1.25kb Fspl - BamHI fragment, isolated from pPALl, which carried the 3'end of the gene. The ligated fragment was inserted between the BamHI and PstI sites of pUC8 to yield pPAL3. The positions 20 of the PAL gene translational start (ATG) and stop (TAG) codon (see Fig. 3) are marked by arrows. The orientation of insertion of the PAL gene is such that transcriptional read through from the vector borne lac promoter (lac po) will not occur. in Fig 5 & 6. The Fsp 1 site..used to join these two clones to form 25 pPAL 3 is indicated by underlining of the relevant nucleotides. In Fig ~'. The plasmid pPAL4 contains the complete PAL gene from pPAL 3 (Fig 4) cloned into pϋC9 as an EcoRI — Hind III fragment such that transcriptional read through from the adjacent lac promoter 30 can occur. Gene product formation was assessed using a plasmid- directed in vitro translation kit obtained from Amersham International PLC. Samples in the numbered tracks are as follows; 1, no DNA added; 2, plasmid pUC9; 3, plasmid pPAL4. Molecular weights of the protein markers are given as M_. In the following description reference will be made to the following general procedures: MICROBIAL STRAINS AND PLASMIDS 5 Microbial strains and plasmids used in accordance with the invention are listed in Table 1. MEDIA 10 coli strains were cultured in L-broth (1% tryptone, 0.5% yeast extract, 0.5% NaCl). Media were solidified with the addition of 2% (w/v) Bacto-agar (Difco). Ampicillin (100 υg ml was used for the selection and growth of transformants. Functional β -galactosidase was detected by ■t e the addition of 5-bromo-_-4-chloro-indoyl- fi -D-galactoside (X-Gal) to a -1 final concentration of 2 υg ml CHEMICALS 32 20 [ O P] dATP and the cDNA synthesis kit were obtained from Amersham International. Agarose, restriction enzymes, T4 DNA ligase, terminal deoxynucleotidyl transferase and 17mer universal sequence primer were purchased from Bethesda Research Laboratories. Klenow DNA polymerase was from Boehringer Mannheim, while dT tailed pUC9 was from PL-Biochemicals. Reverse transcriptase was purchased from Anglicon Biotechnology Ltd. 25 while all other reagents were obtained from Sigma Chemical Co. or BDH. ~0 35 TABLE 1 Microbial Strains and Vectors Used Strain E. coli JM83 ara, (lac-pro) Vieria and Messing (1982) rpsL, thi, O80d lad ZM15 E. coli JM101 (lac-pro) , supE, thi/ Messing and Vieria (1982) Flacl ZM15 traD pro Plasmids pUC9 Amp Vieria and Messing (1982) pBR322 Amp Tet Bolivar et al. (1977) pGH3 Amp ('PAL genomic clone) Gilbert et al. (1985) ^ pPALl Amp (3* end PAL cDNA clone) Novel plasmids pPA 2 Amp (5' end PAL cDNA clone) Novel plasmids pPAL3 Amp (entire PAL cDNA gene) Novel plasmids pPAL4 Amp (entire PAL cDNA gene) Novel plasmids Bacteriophaqe M13mp8 Messing and Vieria (1982) M13mp9 Messing and Vieria (1982) DNA MANIPULATIONS All restriction enzymes and DNA/RNA modifying enzymes were used in th buffers and under the conditions recommended by the suppliers. Plasmid transformation techniques and all manipulation of DNA have previously been described (Minton et al. , 1984). PLASMID DNA ISOLATION E. coli plasmids were purified from 1 litre of L-broth cultures containing a picillin by "Brij lysis" and subsequent CsCl density gradient centrifugation (Clewell and Helinski, 1969). The rapid boiling method of Holmes and Quigley (1981) was employed for small scale plasmid isolation screening purposes. TEMPLATE GENERATION BY SONICATION The DNA to be sequenced was fragmented into random blunt-ended fragments by the procedure of Deininger (1983). The fragments obtained were cloned into the Smal site of M13mp8 and template DNA prepared as described by Sanger et al (1980). NUCLEOTIDE SEQUENCING Nucleotide sequencing was undertaking by the dideoxy method of Sanger e_t al (1980). The data obtained was compiled into a complete sequence using the computer programmes of Staden ( I98O ). ISOLATION OF PAL mRNA PAL mRNA was isolated as has previously been described (Gilbert et al. , 1985) employing publicly available R. toruloides strain IFO 0559 (equivalent to NCYC 1589 deposited at the National Collection of Yeast Cultures, Norwich (GB) under the terms of the Budapest Treaty on 8 September 1986) . CDNA CLONING (SYNTHESIS) i) Heidecker-Messing Method c The method utilised was essentiallyas described by Heidecker and Messing (1983). Total mRNA from PAL induced R. toruloides cells was annealed to oligo dT tailed pUC9 and the first strand cDNA copy synthesised using reverse transc iptase in the presence of all 4 dNTP's. The newly synthesised strands were tailed with oligo dC using terminal - j o deoxynucleotidyl transferase. Following fractionation by an alkaline sucrose gradient, single stranded plasmid DNA carrying cDNA sequences were annealed to denatured oligo dG tailed pUC9 and the second strand synthesised using Klenow DNA polymerase and the addition of all 4 dNTP's. ii) Gubler-Hoffman Method 15 The second method employed in the synthesis of cDNA was that of Gubler and Hoffman (1983). The first strand cDNA copy was synthesised using reverse transcriptase and a 19-mer primer (GATGAGAGGGTTGTCGGTC) complementary to PAL mRNA. The RNA within the RNA-DNA hybrid was then 20 "nicked" with RNaseH and the RNA strand replaced with DNA by E. coli DNA polymerase, utilising the nicked RNA as a primer. The double stranded DNA was then blunt-ended by the action of T4 DNA polymerase, tailed with oligo dC and annealed to oligo-dG tailed pBR322. 25 DETECTION OF PAL cDNA CLONES Plasmid DNA carrying cDNA inserts were transformed into E. coli JM83 and the Amp transformants screened for PAL specific DNA. This was undertaken by in situ colony hyridisation (Grunstein and Hogness, 1975), 30 utilising radio labelled pHG3 DNA subfragments carrying portsion of the PAL gene. AMINO ACID SEQUENCING 35 Peptide- fragments of purified (according to Gilbert et al., (1985) PAL protein were isolated as previously described (Minton et al. , 1984). Amino acid sequencing was undertaken using automated Edman degradation using an Applied Biosystems gas phase sequencer, model 470A. IN VITRO TRANSLATION The bacterial ion-free coupled transcrip ion-translation system used was a modification of that first described by De Vries and Zubay (1967). The E. coli S-30 extract and the supplement solutions required for in vitro expression of genes contained on a bacterial plasmid were purchased as a kit from Amersham International PLC. Proteins produced were labelled 35 with S-methionine (Amersham), and analysed by SDS-PAGE on 12% acrylamide gels (Laemmli, 1970). Gels were dried prior to autoradiography for 16 hours. 1. NUCLEOTIDE SEQUENCING OF THE P L GEHOtUC CLONE The PAL gene was previously shown (Gilbert et al. , 1985) to occupy a 2.5 kb region of DNA within a 6.7 kb Bell fragment cloned into pUC8 to yield the recombinant plasmid pHG3 (see Fig. 1). The majority of the gene resided on a 3 kb BamHI fragment, while the remaining 5' end of the gene lay on a 0.7 kb BamHI-BclI fragment. Accordingly, the 3 kb fragment was isolate from an appropriate clone (fragment 2, Fig. 1) and random subfragments, generated by sonication (Deininger, 1983), cloned into M13mp8. A total of some 250 templates were prepared and sequenced, the data obtained being compiled into a complete sequence using the computer programmes of Staden (1980). The sequence of the 5" end of the gene was obtained by the site directed cloning of the relevant Bell-BamHI, Bcll-Sall and BamHI-Sall fragments (region 1, Fig. 1.) into the appropriate sites of M13mp8 and M13mp9. Sequence determination of the DNA spanning the BamHI site was achieved by cloning the Sall-Xhol fragment (3) indicated in Fig. 1. The translation of the appropriate DNA strand of the sequenced region indicated that an open reading frame (ORF) capable of coding for PAL was not present. Confirmation that this region does encode PAL, however, was obtained by comparing the translated amino acid sequences with the determined sequence of 5 randomly derived peptide fragments. All 5 peptide sequences were located within the translated sequence but occurred in various translational reading frames (Fig. 2.). The absence of a contiguous ORF suggested that, in common with other fungal genes, the PAL gene contains introns. 2. ISOLATION OF cDNA CLONES CARRYING THE PAL GENE To enable the identification of the PAL intervening sequences we elected to reclone the gene from cDNA. In the initial experiments the procedure of Heidecker-Messing (1983) was adopted, utilising the vector pUC9 and purified PAL mRNA. Clones carrying PAL DNA sequences were identified utilising the 3 kb BamHI-BclI fragment of pHG3 as a DNA probe. The largest clone obtained, pPALl, contained some 1.3 kb from the 3' end of the PAL gene (Fig. 3). The 5' end of the gene was obtained by cloning C-tailed cDNA, prepared by the method Gubler and Hoffman (1983), into G-tailed pBR322, to yield pPAL2. In this case the primer utilised durin first strand synthesis was a synthesised 19-mer oligonucleotid complementary to the PAL coding strand 150 bp downstream from the 5' en of the previously obtained cDNA (see Fig. 3.). The nucleotide sequenc of the two cDNA clones was determined by site directed cloning o appropriate restriction fragments into M13mp8 and M13mp9. 3. INDENTIFICATION OF THE PAL INTRONS 10 Sequence determination of the 2 clones confirmed the presence of 6 introns within the PAL coding sequence. Thus the 6 regions of DNA labelled IVS1 to IVS6 were completely absent from the appropriate regions of pPALl and pPAL2. Examination of the 6 missing regions revealed that they all contained the nucleotides CAG at their 3' ends, exhibiting l_r perfect agreement to the consensus intron accepter sequence generally observed in eukaryotic genes (Mount, 1982). A number of the sequences at the 5' end of some of these introns demonstrated less conformity to the eukaryotic consensus donor sequence (GTA/GAGT). Thus the donor sequences of IVS 2, 4 and- 5 were GTGCGT, GTGCGC and GTGCGC respectively. The 20 introns of eukaryotic genes have been generally shown to contain sequences necessary for the accurate splicing of the intervening non-coding regions. Seqeunces conforming to consensus sequences observed in the introns of other eukaryotics (e.g. TACTTAACA in S. cerevisae; see Orbach et al., 1986) are not present in the R. toruloides introns. In their place a sequence is present conforming to the consensus G/ANG/CTGAC (the relevant sequence within each intron has been underlined in Fig. 3). Such a sequence may be specific to R. toruloides and closely related organisms. The PAL gene has been shown not to express in either E. coli (Gilbert et al. , 1985) or S. cerevisae (Tully and Gilbert, 1985). The reason for lack of expression in the former is undoubtedly due to the presence of introns in the PAL gene. Furthermore, although S. cerevisae is capable of splicing introns, the differences in the nucleotide sequences of the PAL introns and those found in S. cerevisae intron probably explains the 5 inability of this yeast to express the R. toruloides PAL gene. 4. DERIVATION OF A CONTIGUOUS CDRA PAL GENE The procedure utilised in the cloning of the PAL gene from cDNA had resulted in two clones, pPALl, which carried the 3' end of the gene, and pPAL2, carrying the 5' end of the gene. A third plasmid was constructed, carrying the entire PAL structural gene by amalgamating the inserts of the above two plasmids. This was achieved by isolating a l.Okb Fspl - PstI fragment from pPAL2, carrying the 5' end of PAL, and ligating it to a 1.25kb Fspl - BamHI fragment carrying the 3' end of the gene isolated from pPALl. The ligated DNA was then inserted into pUC9 cleaved with PstI and BamHI (Fig. 4). The plasmid pPAL3 therefore carries the entire PAL structural gene, but lacks all 6 introns found in the natural R. toruloides chromosomal gene. 5. SYNTHESIS OF PAL PROTEIH The fragment containing the complete intron-free PAL gene from pPAL3 has been cloned into the pUC plasmids in both orientations relative to the lac promoter, to give pPAL3 (pϋC8) and pPAL4 (pUC9). In pPAL4, the PAL gene is in phase with the lacZ promoter from pUC9, and synthesis of PAL protein has been demonstrated in a plasmid-directed in vitro translation system. This is shown in Fig. . With the PAL gene in the opposite orientation (pPAL3) no PAL protein is produced. We are currently developing vector systems to enable us to express the PAL gene in ~ Saccharomyces cerevisiae. 0 5 Ambrus, C ., J.L. Ambrus, C. Hoarvath, H. Pedersen, S. Sharma, C. Kent, E. Mirand, R. Guthrie, and T. Paul. 1978. Phenylalanine depletion for the management of phenylketonuria: use of enzyme reactors with immobilized enzymes. Science 201: 837-839. 5 Attridge, T.H., C.B. Jonhson, and H. Smith. 1974. Density-labelling evidence for the phytochrome-mediated activation of phenylalanine ammonia-lyase in mustard cotyledons. Biochim. Biophys. Acta. 343: 440-451. 10 Boulivar, F., R.L. Rodriquez, P.J. Green, M.C. Betlach, H.L. Heyneker and H.W. Boyer. 1977. Construction and characterisation of new cloning vehicles II. A multipurpose cloning system. Gene 2: 95-113. TcClewell, D.B., and D.R. Helinski. 1969. Supercoiled circular DNA-protein complex in E. coli. Purification and induced conversion to an open circular form. Proc. Natl. Acad. Sci. USA 6_2: 1159-1166. DeVries, J.K., and G. Zubay. 1967. DNA-directed peptide synthesis II. 20 The synthesis of the -fragment of the enzyme b -galactosidase. Proc. Natl. Acad. Sci. USA 57.: 1010-1012. Gilbert, H.J., J.R. Stephenson, and M. Tully. 1983. Control of synthesis of functional mRNA coding for phenylalanine ammonia-lyase for 2_. Rhodospori ium toruloides.- J. Bacteriol. 153: 1147-1154. Gilbert, H.J., and M. Tully. 1982. Synthesis and degradation of phenylalanine ammonia-lyase of Rhodosporidium toruloides. J. Bacteriol. 150: 498-505. 3 J 0 Gilbert, H.J., I.N. Clarke, R.K. Gibson, J.R. Stephenson, and M. Tully. 1985. Molecular cloning of the phenylalanine ammonia lyase gene from Rhodosporidium toruloides in Escherichia coli K-12. J. Bacteriol. 161: 314-320. -^ Grunstein, M. , and D.S. Hogness. 1975. Colony hybridisation: a method for the isolation of cloned DNA's that contain a specific gene. Proc. Natl. Acad. Sci. USA 72: 3961-3965. Gubler, U., and'B.J. Hoffman. 1983. A simple and very efficient method of ^ " 0 generating cDNA libraries. Gene 2_5: 263-269. Heidecker, G., and J. Messing. 1983. Sequence analysis of zein cDNA; obtained by an efficient mRNA cloning method. nacl. Acids. Res. 11: 4891-4906. 5 Holmes, D.S., and M. Quigley. 1981. A rapid boiling method for the preparation of bacterial plasmids. Anal. Biochem. 114: 193-197. Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly 50 of the lead of bacteriophage T4. Nature 227: 680-685. Marusich, W.C., R.A. Jensen, and L.O. Zamir. 1981. Induction of 5 L-phenylalanine ammonia-lyase during utilization of phenylalanine as carbon or nitrogen source in Rhodotorula glutinis. J. Bacteriol. 146: 1013-1019. Messing, J. , and J. Vieria. 1982. A new pair of M13 vectors for selecting either DNA strand of double-digest restriction fragments. 19 ^ : 269-276. Minton, N.P., T. Atkinson, C.J. Bruton and R.F. Sherwood. 1984. The complete nucleotide sequence of the Pseudomonas gene coding for carboxypeptidase G . Gene 3_1: 31-38. Mount, S.M. 1982. A catalogue of splice junction sequences. Nacl. Acids. Res. 10.: 459-472. Orbach, M.J., E.B. Porro, and C. Yanotsky. 1986. Cloning and characterisation of the gene for -tublin from a benomyl-resistant mutant of Neurospora crassa and its use as a dominant selectable marker. Mol. Cell. Biol. 6: 2452-2461. Sanger, F., A.R. Coulson, B.G. Barrell, A.J.H. Smith and B.A. Roe. 1980. Cloning in single-stranded bacteriophage as an aid to rapid DNA sequencing. J. Mol. Biol. 143: 161-178. Schroder, J., F. Kreuzaler, E. Schafer, and K. Hahlbrock. 1979. Concomitant induction of phenylalanine ammonia-lyase and flavanone synthase in irradiated plant cells. J. Biol. Che . 254: 57-65. Tan, S.C. 1980. Phenylalanine ammonia-lyase and the phenylalanine ammonia-lyase inactivating system: effects of light, temperature, and mineral deficiencies. Aust. J. Plant. Physiol. 1.: 159-167. Tully, M. , and H.J. Gilbert. 1985. Transformation of Rhodosporidium toruloides. Gene 36: 235-240. Vieria, J., an J. Messing. 1982. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19_: 259-260. Yamada, S-, K. Kabe, N. Izuo, K. Nakamichi, and I. Chibata. 1981. Production of L-phenylalanine from trans-cinnamic acid with Rhosdotorula glutinis containing L-phenylalanine ammonia-lyase activity. Appl. Environ. Microbiol. 42: 773-778.

Claims: CLAIMS 1. A gene, characterised in that it is an intron-free structural gene, derived from a corresponding intron-containing structural gene from a eukaryotic microorganism, both genes encoding the same product provided that the intron-free gene is capable of expressing the product within a prokaryotic or eukaryotic microorganism. 2. A gene as claimed in claim 1, characterised in that the gene encodes phenylalanine ammonia lyase ('PAL') or a polypeptide which displays PAL activity. 3. A gene as claimed in claim 2, characterised in that it is derived from an intron-containing gene of a PAL-producing strain of a eukaryotic microorganism. 4. A gene as claimed in claim 3, characterised in that the micro¬ organism is R toruloides. 5. A gene characterised in that it has a structure identical to, related to, derived from or complementary to the following polynucleotide sequence: ATG GCG CCT CGA CCA ACC TCG CAG TCG CAG GCT CGC ACC TGC CCC ACA ACC CAG GTC ACG CAG GTC GAC ATC GTC GAG AAG ATG CTC GCC GCG CCG ACC GAC TCG ACG CTC GAA CTC GAC GGC TAC TCG CTC AAC CTC GGA GAC GTC GTC TCG GCC GCG AGG AAG GGC AGG CCT GTC CGC GTC AAG GAC AGC GAC GAG ATC CGC TCA AAG ATT GAC AAA TCG GTC GAG TTC TTG CGC TCG CAA CTC TCC ATG AGC GTC TAC GGC GTC ACG ACT GGA TTT GGC GGA TCC GCA GAC ACC CGC ACC GAG GAC GCC ATC TCG CTC CAG AAG GCT CTC CTC GAG CAC CAG CTC TGC GGT GTT CTC CCT TCG TCG TTC GAC TCG TTC CGC CTC GGC CGC GGT CTC GAG AAC TCG CTT CCC CTC GAG GTT GTT CGC GGC GCC ATG ' ACA.ATC CGC GTC AAC AGC TTG ACC CGC GGC CAC TCG GCT GTC CGC CTC GTC GTC CTC GAG GCG CTC ACC AAC TTC CTC AAC CAC GGC ATC ACC CCC ATC GTC CCC CTC CGC GGC ACC ATC TCT GCG TCG GGC GAC CTC TCT CCT CTC TCC TAC ATT GCA GCG GCC ATC AGC GGT CAC CCG GAC AGC AAG GTG CAC GTC GTC CAC GAG GGC AAG GAG AAG ATC CTG TAC GCC CGC GAG GCG ATG GCG CTC TTC AAC CTC GAG CCC GTC GTC CTC GGC CCG AAG GAA GGT CTC GGT CTC GTC AAC GGC ACC GCC GTC TCA GCA TCG ATG GCC ACC CTC GCT CTG CAC GAC GCA CAC ATG CTC TCG CTC CTC TCG CAG TCG 5- (contd. ) CTC ACG GCC ATG ACG GTG GAA GGG ATG GTG GGC CAC GCC GGC TCG TTG CAC CCC TTG CTT CAC GAC GTC ACG GGG CCT CAG GCG ACG GAG ATC GAA GTC GCG GGA AAC ATC CGC AAG CTC CTC GAG GGA AGG CGC TTT GGT GTG CAC CAT GAG GAG GAG GTC AAG GTG AAG GAC GAC GAG GGG ATT GTC GGC CAG GAC CGC TAC CCC TTG CGG ACG TCT CCT CAG TGG CTC GGG CCG CTC GTC AGG GAC CTC ATT CAC GCC CAC GCC GTC CTC ACC ATG GAG GCG GGC CAG TCG ACG ACC GAC AAC CCT CTC ATC GAG GTC GAG AAC AAG ACT TCG GAC CAC GGC GGC AAT TTC CAG GGT GCC GCT GTG GCC AAC ACC ATG GAG AAG ACT CGC CTC GGG CTG GCC CAG ATC GGC AAG CTC AAG TTC ACG GAG CTC ACC GAG ATG CTC AAG GCC GGG ATG AAG CGC GGG CTG CCC TCC TGC CTC GCG GCC GAA GAG CCC TCG CTC TCC TAC CAG TGC AAG GGG GTC GAC ATC GCC GCT GCG GCG TAG ACC TCG GAG TTG GGA CAC CTG GCC AAG CCT GTG ACG ACG CAT GTC CAG CCG GGT GAG ATG GGG AAC CAG GGG GTG AAC TCG CTT GCG CTC ATC TCG GCT CGT CGC ACG ACC GAG TCC AAC GAC GTC CTT TCT CTC CTC CTC GCC ACC CAC CTC TAC TGG GTT GTC CAA GGC ATC GAC TTG CGC GCG ATC GAG TTC GAG TTC AAG AAG CAG TTC GGG GCA GCC ATC GTC TCG CTC ATC GAG CAG CAG TTT GGG TCC GCC ATG ACC GGG TGG AAC CTG CGC GAC GAG CTG GTC GAG AAG GTG AAC AAG ACG CTC GCC AAG GGC CTC GAG CAG ACC AAC TCG TAC GAG CTC GTC CCG CGG TGG CAG GAC GCC TTC TCC TTC GCC GCC GGC ACC GTC GTC GAG GTC CTC TCG TCG ACG TCG GTC TCG CTC GCC GCC GTG AAC GCC TGG AAG GTC GCC GCC GCC GAG TCG GCC ATC TCG GTC ACC CGG CAA GTG CGC GAG AGC TTC TGG TCC GCC GCG TCG ACC TCG TGG GCC GGG CTC TCG TAC CTC TCG GCG CGG ACT CAG ATC CTC TAC GCG TTC GTC CGC GAG GAG CTT GGG GTG AAG GCC CGC CGG GGA GAC GTG TTC CTC GGC AAG CAA GAG GTG ACG ATC GGC TCG AAG GTG TGC AAG ATC TAG GAG GCC ATG AAG TGG GGG AGG ATC AAG AAG GTC CTG CTC AAG ATG CTC GGT TAG which encodes PAL or a polypeptide which displays PAL activity. 6. A gene as claimed in claim 5 characterised in that it^lacks some bases or includes some additional bases or has some of the listed codons replaced by other codons, provided that the gene encodes PAL or a polypeptide displaying PAL activity. 7. A recombinant DNA molecule characterised in that it contains a gene as claimed in any one of claims 1 to 4. 8. A recombinant DNA molecule characterised in that it contains a gene as claimed in claim 5 or claim 6. 9. A molecule as claimed in claim 8 characterised in that it is a plasmid. 10. A plasmid as claimed in claim 9 characterised in that it is a vector and also contains an expression control sequence operatively linked to the gene, and/or transcription/translation signals appropriate of PAL, or a polypeptide which displays PAL activity, from E. coli K12, Bacillus subtilis, Saccharomyces cerevisae, Preudomonas putida, Erwinia chrysanthemi or mammalian cell lines. 11. A molecule as claimed in claim 10 characterised in that is contains a ribosome binding site upstream of the translational start of the gene and transcriptional signals derived from the S cerevisae phosphoglycerate kinase and mating facter genes placed 5' to the ribosome binding site. 12. A recombinant DNA molecule characterised in that it consists of a gene as claimed in claim 5 inserted into the plasmid pUC9 with the gene in phase with the lac Z promoter of pUC 9. 13. A host microorganism characterised in that it is transformed with a recombinant DNA molecule as claimed in claim 8. 14. A process for the preparation of a gene from which introns have been deleted characterised in that it includes the steps of: (i) isolating PAL mRNA from a strain of R. toruloides. (ii) synthesising two intron-free cDNA sequences from the mRNA, the two cDNA sequences each containing a portion of a gene which encodes pAL or a polypeptide which displays PAL activity, the two portions together containing the 3' and the 5' ends of the gene, ( iii ) joining the two cDNA sequences to form an intron-free structural gene which encodes PAL or a polypeptide which displays PAL activity. 15- A polynucleotide sequence characterised in that it is a portion of an intron-free gene which encodes PAL or a polypeptide which displays PAL activity and contains the y or the 5' end of the gene. 16. A sequence as claimed in claim 15 characterised in that it contains a polynucleotide sequence identical to, related to or derived from the following polynucleotide sequence: ATG GCG CCT CGA CCA ACC TCG CAG TCG CAG GCT CGC AGC TGG CCC ACA ACC CAG GTC ACG CAG GTC GAC ATC GTC GAG AAG ATG CTG GCC GCG CCG ACC GAC TCG ACG GTC GAA CTG GAC GGG TAC TCG CTC AAC GTC GGA GAC GTG GTC TGG GCC GCG AGG AAG GGC AGG GCT GTC CGC GTC AAG GAC AGC GAC GAG ATC CGC TCA AAG ATT GAC AAA TGG GTC GAG TTC TTG CGC TCG CAA CTG TCC ATG AGC GTC TAC GGC GTG ACG ACT GGA TTT GGC GGA TCC GCA GAC ACC CGC ACC GAG GAC GCC ATC TCG GTC CAG AAG GGT CTC CTG GAG CAC CAG CTG TGG GGT GTT CTC CCT TCG TGG TTC GAG TCG TTC CGC CTC GGC CGG GGT GTC GAG AAC TCG CTT CCC CTC GAG GTT GTT CGC GGC GCC ATG ACA ATC CGC GTC AAC AGC TTG ACC CGC GGC CAC TCG GCT GTC GGG CTC GTC GTC CTC GAG GGG GTC ACC AAC TTC CTC AAC CAC GGC ATC ACC CCC ATC GTC CCC CTC CGC GGC ACC ATC TCT GCG TGG GGC GAC CTC TCT CCT CTC TCC TAC ATT GCA GCG GGC ATG AGG GGT CAC CCG GAG AGC AAG GTG CAC GTC GTC CAC GAG GGC AAG GAG AAG ATG CTG TAG GCC CGC GAG GCG ATG GCG CTG TTC AAC CTG GAG CCC GTG GTC CTC GGC CCG AAG GAA GGT GTC GGT CTC GTC AAC GGC AGC GCC GTC TCA GCA TCG ATG GCC ACC CTC GCT CTG CAC GAC GCA CAC ATG CTC TCG CTC CTC TCG CAG TCG CTC ACG GCC ATG ACG GTC GAA GCG ATG GTC GGG CAC GCC GGC TCG TTC • CAC CCC TTC CTT CAC GAG GTC ACG CGC CCT CAC CCG ACG CAG ATC GAA GTC GCG GGA AAC ATC CGC AAG CTC CTG GAG GGA AGC CGC TTT GGT GTC CAC CAT GAG GAG GAG GTC AAG GTG AAG GAC GAC GAG GGG ATT CTC CGC CAG GAG CGC TAC CCC TTG CGC ACG ' . 17. A sequence as claimed in claim 15 characterised in that it contains a polynucleotide sequence identical to, related to or derived from the following polynucleotide sequence: 17. ( contd. ) TCT CCT CAG TGG CTC GGC CCG GTC GTC AGC GAC CTC ATT CAC GCC CAC GCC GTG CTC ACC ATC GAG GCC GGC CAG TCG ACG ACC GAC AAC CCT CTC ATC GAC GTC GAG AAG AAG ACT TCG CAC CAC GGG GGG AAT TTC CAG GCT GCG GCT GTG GCC AAC ACC ATG GAG AAG ACT CGC CTC GGG CTC GCC CAG ATC GGC AAG CTC AAG TTC ACG CAG GTC ACC GAG ATG GTC AAC GGC GGG ATG AAG CGC GGC CTC CCC TCC TGC CTC GGG GCC GAA GAC CCC TCG CTC TCC TAC CAC TGC AAG GGC CTC GAC ATC GCC GCT GCG GCG TAC ACC TCG GAG TTG GGA CAC CTC GGC AAC CCT GTG ACG ACG CAT GTC CAG GCG GCT GAG ATG GCG AAC CAG GGG GTC AAC TCG CTT GCG CTC ATC TCG GCT CGT CGC ACG ACC GAG TCC AAC GAG GTG CTT TCT CTC CTC CTC GCC ACC CAG CTC TAC TGC GTT GTC CAA GCC ATG GAC TTG CGC GCG ATC GAG TTC GAG TTG AAG AAG CAG TTC GGG GCA GCC ATC GTC TCG CTG ATC GAC CAG CAC TTT GGG TCC GCC ATG ACC GGC TCG AAG CTG CGC GAC GAG CTC GTC GAG AAG GTG AAC AAG AGG CTC GCC AAG CGC CTC GAG CAG AGC AAC TCG TAC GAC CTC GTC CCG CGG TGG CAC GAC GCC TTC TCC TTC GCC GCC GGC ACC GTC GTG GAG GTC CTC TCG TCG ACG TCG CTC TCG CTC GCC GGC GTG AAC GCC TGG AAG GTG GCG GCC GCC GAG TCG GCC ATC TCG CTC ACC CGC CAA GTC CGC GAG ACC TTC TGG TCC GGC ! GCG TCG ACC TCG TCG GCC GCG CTC TCG TAG CTC TCG CCG CGC ACT CAG ATC CTC TAC GCC TTC GTC CGC GAG GAG CTT GGG GTC AAG GGC CGG CGC GGA GAC GTC TTC CTC GGG AAG CAA GAG GTG ACG ATC GGG TCG AAC GTC TCC AAG ATC TAC GAG GCC ATC AAG TCG GGC AGG ATG AAG AAC GTG CTC CTC AAG ATG CTC GCT TAG . 18. A polynucleotide sequence as claimed in claim 16 or 17 characterised in that it lacks some bases or includes other bases or has some of the listed codons replaced by other codons. 19. A recombinant DNA molecule characterised in that it contains a polynucleotide sequence as claimed in claim 15. 20. A recombinant DNA molecule characterised in that it contains a polynucleotide sequence as claimed in claim 16 or 17. 21. A recombinant DNA molecule as claimed in claim 20 characterised in that the polynucleotide sequence is combined with pBR322 or pUC9.

Inventors: ANSON JOHN, GILBERT HAROLD, MINTON NIGEL PETER, ORAM JON, ANSON, JOHN, GILBERT, HAROLD, MINTON, NIGEL, PETER, ORAM, JON
Applicants: HEALTH LAB SERVICE BOARD, THE PUBLIC HEALTH LABORATORY SERVICE BOARD
Application year: 1987
Main classification: C12N15
IPC codes: C12N1, C12N9, C12N15, C12R1