Abstract:
The present invention relates to plants genetically modified to increase the level of stored carbohydrates in the plant, particularly during periods of high sink activity and low source activity through production of a glycosyl-transferase which catalyzes the formation of soluble glucans. The invention also relates to the genetic constructs used to produce the engineered plants and the method of producing the engineered plants.

Description:
This application is a 371 of PCT/AU95/00527 filed Aug. 24, 1995. 
    
    
     TECHNICAL FIELD 
     The present invention relates to plants genetically modified to increase the level of stored carbohydrates in the plant, particularly during periods of high sink activity and low source activity. The invention also relates to the genetic constructs used to produce the engineered plants and the method of producing the engineered plants. 
     BACKGROUND ART 
     The soluble storage carbohydrate found in plants, including sucrose, glucans, starch and fructans, are an important source of feed for animals, particularly grazing ruminants. These carbohydrates are stored non-structurally which makes them readily available for digestion by animals and therefore an important source of digestible energy. 
     During periods of high sink activity and low source activity, such as during a drought, the level of stored carbohydrates falls as the non-structural storage carbohydrates are mobilised for use in seed filling. The result of this mobilisation, particularly in relation to pasture grasses, is a significant loss of feed value to grazing ruminants due to the reduction in the levels of the stored carbohydrates. This reduction is caused by the enzymatic degradation of the stored carbohydrates. This enzymatic degradation is assisted by the fact that the stored carbohydrates generally have a low degree of polymerization. For example, as noted by Radojevic et al 1994, during the period from late spring to early autumn in southern Australia, the declining feed quality of the grasses causes a corresponding reduction in the lactation by dairy herds and necessitates the use of supplementary feeds. This decline in digestibility is associated with a decline in the level of soluble carbohydrates. 
     Perennial rye grass lines which accumulate high concentrations of soluble carbohydrates from late spring to early autumn do not suffer as large a decline in digestibility (Radojevic et al 1994). The result of this increased digestibility is a corresponding increase in milk production by dairy herds. 
     In addition to this, there are many pasture plants, such as white clever which do not possess any significant levels of stored carbohydrate. 
     There has, therefore, been a desire to develop methods for preventing the degradation of the stored carbohydrates during plant senescence and to increase the level of stored carbohydrates in pasture plants with low levels. 
     Glucosyltransferases of Streptococcus salivarius 
     It is known that many strains of Streptococcus salivarius and Streptococcus mutans, produce extracellular α-D-glucosyltransferase (Gtfs), an enzyme which catalyses the formation of glucan from sucrose. These Gtfs are also found in many other species of oral streptococci. 
     The Gtfs utilise the high free energy of the glycosidic bond of sucrose to synthesise glucans (Jacques N A, Giffard P M, 1991). Gtfs produce either soluble or insoluble products by transferring a glucose residue from sucrose to a growing glucan chain. 
     Gtfs which produce an insoluble product are generally considered to be primer-dependent (Walker G J, Jacques N A, 1987). These primer-dependent Gtfs require a dextran (α-(1→6)-linked glucan) as a receptor for polymerisation to proceed at an appreciable rate. In contrast, Gtfs that produce soluble products may be either primer-dependent or primer-independent. The genetic sequences for 10 gtf genes from a number of Streptococcus species have been ascertained (Gilmore K S, Russell R R B, Ferretti J J). All the Gtfs coded by these genes possess highly conserved putative signal sequences that lead to the secretion of these enzymes. The remainder of each protein is arbitrarily divided into two domains--the N-terminal two-thirds &#34;catalytic domain&#34; and the C-terminal one-third &#34;glucan-binding domain&#34;. 
     S. salivarius ATCC 25975 has been shown to possess at least four different gtf genes (Giffard et al (1991); Giffard et al (1993)). Each of these genes codes for a highly hydrophilic monomeric glucosyltransferase that possesses unique enzymic properties. These Gtfs synthesize structurally different glucans from sucrose. For example, the genes coding for GtfJ and GtfL produce enzymes which synthesize insoluble glucans. GtfJ is a primer-dependent enzyme producing essentially a linear a(1→3)-glucan while GtfL is a primer-independent enzyme that synthesizes a glucan containing 50% α-(1→3)--and 50% α-(1→6)-linked glucosyl residues. In contrast, the gtfK and gtfM genes code for enzymes which produce a soluble glucan which possess α-(1→6)-linked glucosyl residues. GtfK is primer stimulated while GtfM is primer independent. 
     SUMMARY OF THE INVENTION 
     Up until now, a gtf gene in S. salivarius or any other Streptococcus species which produces a glucosyltransfererase that synthesises a glucan which is both soluble and primer independent has not been described. 
     The significance of a glucosyltransferase produced by S. salivarius, or any other streptococci, which is both primer independent and which synthesises a soluble glucan product is twofold. First, the primer independence of the Gtf means that the enzyme should be functional when expressed in plants while the glucan that is formed from sucrose in the plant should be readily stored without detriment to the plant, due to its solubility. 
     An important characteristic of soluble glucans produced by Gtf synthesis is that they are poorly degraded by plant enzymes and are readily digested by the diverse microflora present in the rumen of grazing livestock. 
     The inventors of the present invention have isolated and characterised a novel gtf (GtfM) gene in S. salivarius which codes for a primer independent Gtf which produces a glucan which is soluble, resistant to degradation by plant enzymes and readily digested by microflora present in the rumen of grazing livestock. 
     According to a first aspect of the present invention there is provided a plant containing bacterial DNA which codes for a glucosyltransferase which catalyses the formation of glucans from sucrose. 
     Preferably, the plant contains bacterial DNA which codes for a glucosyltransferase which is primer independent. 
     More preferably, the plant contains DNA which codes for a glucosyltransferase which catalyses the formation of soluble glucans. 
     More preferably, the bacterial DNA is obtained from Streptococcus salivarius. 
     According to a second aspect of the present invention there is provided a DNA comprising a sequence according to SEQ ID NO: 1. 
     According to a third aspect of the present invention there is provided a DNA sequence which is a variant of a DNA having a sequence according to SEQ ID NO: 1. In this respect a &#34;variant&#34; is a polynucleotide which corresponds to or comprises a portion of the DNA of the invention, or is &#34;homologous&#34; to the DNA of the invention. For the purposes of this description, &#34;homology&#34; between two polynucleotide sequences connotes a likeness short of identity, indicative of a derivation of the first sequence from the second. In particular, a polynucleotide is &#34;homologous&#34; to the DNA of the invention if there is greater than 70% identity in the DNA sequence. 
     The polynucleotides of the present invention exclude those polynucleotides in the environment in which they occur in nature. They include the polynucleotides in a form in which the are substantially free of other Streptococcus salivarius polynucleotide sequences, such as sequences in isolated form, including those in substantially purified form. 
     According to a fourth aspect of the present invention there is provided a protein comprising the amino acid sequence according to SEQ ID NO: 2. 
     According to a fifth aspect of the invention there is provided a polypeptide comprising an amino-acid sequence which is a variant of SEQ ID NO:2. A variant is a polypeptide which corresponds to or comprises a portion of the polypeptide of the invention, or is &#34;homologous&#34; to the peptide of the invention. For the purposes of this description, &#34;homology&#34; between two peptide sequences connotes a likeness short of identity, indicative of a derivation of the first sequence from the second. In particular, a polypeptide is &#34;homologous&#34; to the peptide of the invention if there is greater than 70% identity in the amino acid sequence. 
     These homologous polypeptides can be produced by conventional site-directed mutagenesis of the corresponding DNA or by chemical synthesis, and fall within the scope of the invention, particularly where they retain the biological activity of a glucosyltransferase. 
     The proteins and polypeptides of the invention exclude those proteins and polypeptides in the environment in which they occur in nature. They include the proteins and polypeptides in a form in which they are substantially free of other Streptococcus salivarius polypeptide sequences, such as sequences in isolated form, including those in substantially purified form. 
     According to a sixth aspect of the present invention there is provided the microorganism E. coli containing plasmid pGSG501. 
     According to a seventh aspect of the present invention there is provided the microorganism E. coli containing plasmid pGSG502. 
     According to a eighth aspect of the present invention there is provided a plant containing DNA comprising a sequence according to SEQ ID NO: 1. 
     According to an ninth aspect of the present invention there is provided a plant containing DNA which is a variant of DNA having a sequence according to SEQ ID NO: 1. 
     According to a tenth aspect of the present invention there is provided a plant expressing a protein comprising an amino acid sequence according to SEQ ID NO: 2 or a variant thereof. 
     DNA and variants thereof of the invention can be incorporated into a variety of plant types. These include plants, such as grasses, used as fodder for livestock. They also include cereal crops or other starchy food product types, (to provide grain or other food with increased fibre); and horticultural crops, such as tomatoes and fruits, to provide fruits with increased solids. 
     In addition plants expressing the DNA and variants thereof, of the invention may also produce dextran which can in turn be used: 
     1) as a binder for use in processed foods (e.g. so called `health bars`); 
     2) in pharmaceutical preparations again as a binder; and 
     3) in medical preparations to increase antigenic activity. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 shows a restriction map of the inserts from pGSG501 and pGSG502. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The invention-is further described with reference to the accompanying Example which is no way limiting on the scope of the present invention. 
     Example 1 
     The general strategy adopted to isolate a gene from S. salivarius encoding a Gtf which produces a primer independent and soluble glucan is as follows: 
     A λ gene bank containing S. salivarius DNA was prepared. Positive clones were detected by using an E. coli strain grown on agar containing sucrose. E. coli which contained gtf DNA from S. salivarius could convert the sucrose in the medium into a polymer which resulted in opaque colonies. These opaque colonies were then picked and the S. salivarius DNA excised and subjected to restriction mapping to ascertain whether the DNA was from a previously described S. salivarius gtf gene, or whether the DNA was novel. Three clones containing novel DNA were located. These were subjected to a radioactive assay to determine whether the DNA encoded for a primer independent or primer dependent Gtf. One clone-λC-13 was found to contain a novel gtf gene which coded for a primer independent Gtf. The DNA from this clone was then isolated and sequenced. 
     The particular details of this methodology are now described below. 
     Bacterial strains and growth conditions. 
     Escherichia coli LE392 and NM522 and S. salivarius ATCC 25975 were used. E. coli strains were grown in Luria-Bertani (LB) medium at 37° C., supplemented with ampicillin (100 μg ml -1 ), isopropylthiogalactoside (IPTG) (1 mM), or 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal) (100 μg ml -1 ) as appropriate. Cultures of S. salivarius were grown at 37° C. in semi-defined medium (SDM) containing 25 mM glucose supplemented with 0.005μl Tween 80 ml -1  where appropriate. 
     Bacteriophage and phagemids. All genetic constructs, excluding sequencing subclones, are listed in Table 1. Bacteriophage-λ derivatives were grown either as 20 ml or 1 L-liquid lysates using E. coli strain LE392 as the host and DNA purified according to the method of Silhavy et al (1984). Plasmids were propagated in E. coli strains as described previously (Giffard et al, 1991). 
     Screening of Gene Bank. A bacteriophage-λ gene bank of S. salivarius ATCC 25975 (Pitty et al, 1989) was screened by detecting plaques on a lawn of E. coli LE392 grown at 37° C. on minimal agar medium containing 0.2% glucose and 50 μg ml -1  methionine as well as it (wt/vol) sucrose with or without 0.02% (wt/vol) dextran T-10. Potential Gtf clones were detected by their opacity including λC-13 containing the gtf M gene. 
     Twenty recombinant plaques were picked from minimal media plates containing sucrose and the EcoR1 restriction patterns of these recombinants were analysed. Of these recombinants, only λC-13 exhibited a unique EcoR1 restriction pattern and Gtf activity. A restriction map of λC-13 was constructed using double restriction digests. The Gtf gene encoded by λC-13 (GtfM) was located on an 8.3 kbp insert (see FIG. 1). The 5.3 kbp XbaI fragment from λC-13 was subcloned into pIBI31 30 (pGSG501; see Table 1) and was positive for Gtf activity as was the 4.8 kbp XbaI/EcoR1 from λC-13 subcloned into pIBI31 (pGSG502; see Table 1). 
     
                                           TABLE 1__________________________________________________________________________Bacterial Strains, Phages and PhagemidsBacteria, Phage or Phagemid            Description                  Source or reference__________________________________________________________________________Bacterium:Streptococcus salivarius ATCC 25975           ATCC (Hamilton, 1967)                                          Escherichia coli LE392                                         F.sup.-  e14.sup.- (merA.sup                                         .-) hsdR514 (r.sub.K.sup.-                                         m.sub.K.sup.-) supE44 and                                         supF58 Murray et al., 1977                                           lacY1 or Δ(lacIZY)6                                         galK2 gal722 metB1 trpR55                                          Escherichia coli NM522                                         F&#39;lacI.sup.4 Δ(lacZ)                                         M15 proA.sup.+ B.sup.+                                         /supE thi Δ(lac-proAB)                                          Gough and Murray, 1983                                           ΔhsdMS-mcrB)5                                         (r.sub.k.sup.- m.sub.K.sup.-                                          McrBC.sup.-)  Bacteriophage:  47.1  Loenen and Brammar, 19808 47.1 with GtfJ encoding 8.5 kbp Sau3A partial fragment Pitty et al.,1989   of S. salivarius ATCC 2597533 47.1 with GtfK encoding 9.6 kbp Sau3A partial fragment Pitty et al.,1989   of S. salivarius ATCC 2597513 47.1 with 8.3 kbp GtfM encoding Sau3A partial fragment This study  of S. salivarius ATCC 2597510 47.1 with 11 kbp GtfL encoding Sau3A partial fragment This study  of S. salivarius ATCC 2597540 47.1 with Sau3A partial fragment of S. salivarius ATCC 25975 Thisstudy   isolated from sucrose-containing medium  Phagemid:  pIBI30 Ap.sup.r, f1 origin replication, β-galactosidase, IBICorporation   T3 and T7 polymerase promoters  pIBI31 Ap.sup.r, f1 origin replication, β-galactosidase, IBICorporation   T3 and T7 polymerase promoters  pGSG101 (pGS101) pIBI30 with GtfJ encoding 6.8 kbp SacI/BamIII fragmentof -8 Giffard et al., 1991 pGSG201 (pGS201) pIBI30 with GtfK encoding 7.3 kbp Bg/II/BamIII fragmentof -33 Giffard et al., 1991 pGSG401 pIBI30 with GtfL encoding 6.2 kbp BamIII/XbaI fragment of -10This study  pGSG402 pIBI31 with 6.2 kbp BamIII/XbaI fragment of -10 This study pGSG403 pIBI30 with 4.8 kbp EcoRI fragment of -10 This study pGSG404 pIBI30 with 4.1 kbp EcoRI fragment of -10 This study pGSG502 pIBI31 with GtfM encoding 5.3 kbp XbaI fragment of -13 Thisstudy  pGSG502 pIBI31 with GtfM encoding 4.8 kbp EcoRI/XbaI fragment of -13This study  pGSG503 pIBI31 with 3.7 kbp KpnI/XbaI fragment of -13 This study__________________________________________________________________________ 
    
     Detection of Gtf activity. Gtf activity was routinely detected using a qualitative microtitre reducing sugar test for liberated fructose, outlined in Jacques N. A. (1983). Gtf activity encoded by phagemids was released from E. coli cells by permeabalizing 1 ml of a stationary phase culture. This was achieved by vortexing the cells in the presence of 50 μl 0.1% (wt/vol) SDS and 100 μl chloroform for 20 seconds. Quantification of Gtf activity utilized [U-glucosyl- 14  C]-labelled sucrose. One unit of enzyme activity was defined as the amount of Gtf that catalyzed the incorporation of 1 μmol of the glucose moiety of sucrose in 75% (vol/vol) ethanol-insoluble polysaccharide per min. 
     The assay mix used for the quantification of Gtf activity was scaled up to 8 ml and incubated with 3.2 ml of bacteriophage λ lysates at 37° C. for 2 h. After the 2 h incubation, the assay mix was boiled for a further 1 h to inactivate the enzyme and the amount of glucan formed (cpm) determined by assaying duplicate 500μl aliquots. After cooling to 37° C., C. gracile endo-(1→6)-α-D-glucanase was added to a final concentration of 500 mU/ml and the solution incubated at 37° C. Duplicate aliquots (500μl) were removed and assayed for total remaining glucan at varying time intervals over a 5 h period. Any reduction in glucan (cpm) during this period was attributed to hydrolysis by the endo-(1→6)-α-D-glucanase. 
     DNA sequence analysis. DNA sequence determination was carried out on CsCl purified double-stranded DNA using the Pharmacia T7 sequencing kit according to the manufacturer&#39;s instructions. Custom-made oligonucleotide primers (17 mers) were used and all sequencing was confirmed in both directions. DNA sequences were assembled and open reading frames (orfs) detected using the IBI-Pustell sequence analysis software version 2.03. 
     Southern Hybridizations. Chromosomal DNA from S. salivarius ATCC 25975 was extracted and purified as previously described (Giffard et al, 1991). Southern hybridizations were done essentially as outlined by Silhavy et al (1984) and in accordance with standard techniques such as those described in Maniatis et al (1989). 
     Incorporation into plants. Incorporation of gtfM gene into plants is obtained by standard transgenic techniques. The gtfM gene is obtained from λC-13 or pGSG501 by PCR. Various constructs are made using PCR primers that either do or do not contain a coding region that adds a vacuolar targeting sequence to the N- or C-terminus of the GtfM protein. These PCR constructs are cloned into a pUC18 based vector containing a Cauliflower Mosaic Virus (CaMV) 35S promoter. By this means the streptococcal promoter is replaced by a plant promoter. 
     Other methods of incorporating foreign DNA into plants are taught in Australian Patent Application No. 46881/89 by Ciba Geigy Ag. They include the use of Agrobacterium tumefaciens and the leaf disc transformation method and the use of Tobacco Mosaic Virus (TMV). 
     
         __________________________________________________________________________#             SEQUENCE LISTING   - -  - - (1) GENERAL INFORMATION:   - -    (iii) NUMBER OF SEQUENCES: 2   - -  - - (2) INFORMATION FOR SEQ ID NO:1:   - -      (i) SEQUENCE CHARACTERISTICS:       (A) LENGTH: 4853 base - #pairs       (B) TYPE: nucleic acid       (C) STRANDEDNESS: double       (D) TOPOLOGY: linear   - -     (ii) MOLECULE TYPE: DNA (genomic)   - -    (iii) HYPOTHETICAL: NO   - -     (iv) ANTI-SENSE: NO   - -     (vi) ORIGINAL SOURCE:       (A) ORGANISM: Streptococcu - #s salivarius   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:   - - CAGAGATTTA TGAAAAGAAG ATGATTTTTT CCTATTTGTA ATTTGTCTGA AT -#ATATCATA     60   - - GAGTAGAGAT GACAACAGAA AAAAGGATGA TTGATATAGA TGGAAAATAA GG -#TACGTTTT    120   - - AAACTACACA AGGTTAAGAA AAACTGGGTA ACTATTGGGG TGACCACTCT CT -#CAATGGTT    180   - - GCCTTGGCAG GTGGAAGCCT CCTAGCTCAA GGAAAAGTAG AGGCCGATGA GA -#CGAGCGCA    240   - - CCTAACGGTG ACGGCTTGCA GCAACTGAGT GAGGATGGGA CTGCCAGTCT AG -#TGACGACA    300   - - ACAACTGTTA CTGAGCAAGC TAGTGCTCAA GCAAGTGTGT CAGCAGTAGC AA -#CAGCCAGC    360   - - GTAAGTCACG AAACAAGCTT CCAGGCGGCG ACAAGTGCAG TCAGCCAGGA GG -#CAACTGCT    420   - - CAAGCACAAA CTAGTCCAGT TGCCAGTCAA GAAGTGGCAG TATCTTCGCA AA -#CTCAATCC    480   - - AGTGGCCAAG AGACACAGAC TACTGAACAG GTGTCACAAG GTCAGACATC AA -#CTCAAGTA    540   - - GCTGGGCAAA CAAGTGCTCA GTCTACTCCA AGTGTGACAG AACAAGCAAG AC -#CTAGAGTC    600   - - TTGACCAATG CAGCGCCAGC AATTGCCACA CGCGCTGCTG ATAGCACTAT TC -#GTATCAAT    660   - - GCCAACCGCA ATACTAACAT CACGATTACG GCCAGCGGTA CGACACCAAA TG -#TAACCATT    720   - - ATCACAGGGC CAAACACGCC TAAACCAAAC GTGACGGTGA CAAGTCCAAA TG -#GCACAAGA    780   - - CCAAATGTGA CCATTGTAAC GCAGCCAAAT CAACCCAACA AACCTGTTCA AC -#CAAGTCAA    840   - - CCGTCTCAAC CTAACAAGCC GGTCCAACCA AATCAGCCAA GTCTTGACTA TA -#AACCAGTA    900   - - GCCTCTAACT TGAAGACTAT CGATGGCAAG CAGTACTATG TTGAAAATGG CG -#TCGTGAAA    960   - - AAGAACGCAG CCATTGAGCT TGATGGGCGT CTTTATTATT TCGATGAGAC TG -#GAGCTATG   1020   - - GTGGATCAAA GTAAACCTTT GTATCGTGCC GATGCCATTC CAAATAACTC TA -#TCTATGCG   1080   - - GTTTATAACC AGGCCTATGA TACGTCAAGT AAGAGTTTTG AACACTTGGA TA -#ATTTCTTG   1140   - - ACGGCTGATA GCTGGTACCG TCCAAAACAG ATTTTGAAGG ATGGGAAGAA TT -#GGACAGCT   1200   - - TCAACTGAGA AAGATTATCG TCCACTTTTG ATGACTTGGT GGCCAGACAA GG -#TGACACAG   1260   - - GTCAACTACC TCAACTATAT GAGCCAACAA GGGTTTGGTA ATAAGACCTA TA -#CGACAGAT   1320   - - ATGATGAGCT ATGACTTGGC AGCTGCTGCC GAAACGGTTC AACGAGGCAT CG -#AGGAACGT   1380   - - ATTGGTCGTG AGGGCAATAC CACTTGGCTT CGCCAGTTGA TGTCAGACTT CA -#TCAAAACA   1440   - - CAGCCTGGCT GGAACTCTGA GAGTGAGGAC AATCTCTTAG TTGGTAAAGA CC -#ACTTGCAA   1500   - - GGAGGCGCTC TGACCTTCCT AAATAATAGT GCGACAAGTC ATGCCAATTC AG -#ATTTCCGC   1560   - - CTCATGAACC GCACACCGAC TAACCAAACG GGGACACGCA AATATCATAT TG -#ACCGTTCA   1620   - - AATGGTGGTT ACGAGTTGCT CTTGGCTAAC GATATCGACA ACTCTAACCC AG -#CTGTTCAG   1680   - - GCAGAGCAAC TAAACTGGCT CCACTACATC ATGAACATTG GTTCTATCCT TG -#GCAATGAT   1740   - - CCAAGTGCCA ACTTTGACGG TGTTCGTATC GATGCGGTGG ACAATGTGGA TG -#CGGATCTC   1800   - - TTGCAGATTG CTTCTGATTA CTTCAAGGAA AAATACCGTG TCGCAGATAA TG -#AAGCAAAT   1860   - - GCCATTGCTC ATTTGTCAAT CCTTGAAGCT TGGTCATACA ACGACCACCA AT -#ACAACAAG   1920   - - GATACCAAGG GTGCTCAGTT GTCTATCGAC AATCCACTAC GTGAAACGCT TT -#TGACGACT   1980   - - TTCTTGCGTA AGAGCAATTA CCGTGGCAGC TTGGAGCGCG TGATTACTAA CT -#CTCTTAAC   2040   - - AATCGTTCAA GTGAGCAGAA ACACACGCCA CGTGATGCCA ATTATATTTT CG -#TGCGAGCC   2100   - - CATGATAGTG AAGTGCAAGC TGTTTTGGCT AATATCATCA GCAAGCAGAT TA -#ATCCAAAA   2160   - - ACAGATGGTT TCACCTTCAC TATGGATGAG CTCAAACAGG CCTTCGAAAT CT -#ACAATGCG   2220   - - GACATCGCGA AGGCTGATAA AAAGTACACC CAGTACAATA TCCCAGCTGC CT -#ATGCCACA   2280   - - ATGTTGACCA ACAAGGATAG TATCACTCGT GTTTACTACG GGGACCTCTT TA -#CCGACGAT   2340   - - GGCCAATACA TGGCTGAAAA ATCACCGTAC TATAATGCCA TCGATGCCCT GC -#TCCGTGCT   2400   - - CGCATCAAAT ATGTAGCAGG TGGTCAAGAC ATGAAGGTTA CTAAGCTTAA TG -#GCTATGAA   2460   - - ATCATGTCAT CTGTGCGTTA TGGTAAGGGG GCAGAAGAAG CCAACCAGCT TG -#GTACTGCT   2520   - - GAAACACGCA ACCAAGGAAT GCTGGTCCTT ACAGCCAACC GTCCAGATAT GA -#AGTTGGGA   2580   - - GCTAATGATC GTCTGGTAGT CAATATGGGA GCTGCCCACA AAAATCAGGC TT -#ACCGTCCA   2640   - - TTGCTTCTCA GCAAATCGAC AGGTCTTGCG ACCTACCTCA AGGATTCTGA TG -#TACCAGCT   2700   - - GGATTGGTTC GCTATACGGA CAATCAAGGG AACTTGACCT TCACGGCAGA TG -#ATATTGCT   2760   - - GGTCATTCAA CCGTTGAAGT TTCAGGTTAT TTGGCAGTTT GGGTGCCAGT GG -#GTGCCTCA   2820   - - GAAAACCAAG ATGCCCGAAC CAAGGCTTCG AGCACCAAGA AGGGTGAGCA GG -#TCTTTGAA   2880   - - TCATCAGCAG CTCTTGATTC ACAAGTCATT TACGAAGGCT TCTCAAACTT CC -#AAGATTTC   2940   - - GTTAAGACAC CAAGTCAGTA CACCAACCGT GTTATTGCTC AAAATGCCAA AC -#TCTTCAAA   3000   - - GAGTGGGGAA TCACTTCCTT TGAATTTGCG CCACAGTATG TGTCTAGCCA AG -#ACGGCACT   3060   - - TTCTTGGATT CTATCATTGA AAATGGCTAC GCCTTCGAGG ACCGCTACGA TA -#TTGCCATG   3120   - - AGCAAGAACA ACAAATACGG TTCACTCAAA GACCTCATGG ATGCCCTTCG TG -#CCCTTCAC   3180   - - GCAGAAGGTA TCTCAGCCAT TGCTGACTGG GTTCCAGATC AAATTTACAA TC -#TCCCTGGA   3240   - - AAAGAAGTGG TAACAGCTTC TCGTACCAAT AGCTACGGTA CACCACGTCC AA -#ATGCTGAA   3300   - - ATCTACAATA GCCTCTACGC AGCTAAAACA CGTACCTTTG GAAATGACTT CC -#AAGGCAAG   3360   - - TACGGTGGTG CCTTCCTTGA TGAATTGAAG GCAAAATACC CAGCAATCTT TG -#AGCGCGTG   3420   - - CAGATTTCAA ACGGCCGTAA ATTGACTACC AATGAGAAAA TCACGCAATG GT -#CAGCCAAG   3480   - - TATTTCAATG GAAGCAATAT CCAAGGTACT GGAGCTCGCT ATGTCCTACA AG -#ATAACGCT   3540   - - ACCAACCAAT ACTTCAGCGT CAAAGCAGGT CAAACCTTCC TTCCTAAACA AA -#TGACTGAA   3600   - - ATTACTGGAA GTGGTTTCCG TAGGGTTGGA GATGATGTCC AATACCTCTC AA -#TTGGTGGC   3660   - - TACCTTGCTA AGAATACCTT TATTCAAGTC GGTGCCAACC AGTGGTATTA CT -#TTGATAAG   3720   - - AATGGCAACA TGGTCACAGG TGAGCAGGTC ATTGATGGCA AGAAATACTT CT -#TCCTAGAC   3780   - - AATGGTCTCC AGCTACGTCA TGTCCTTCGC CAAGGTAGTG ATGGTCATGT GT -#ATTATTAC   3840   - - GATCCTAAAG GGGTTCAGGC CTTTAACGGA TTTTATGATT TTGCGGGTCC TC -#GCCAAGAC   3900   - - GTTCGCTACT TTGATGGCAA CGGTCAAATG TATCGTGGCC TCCACGACAT GT -#ATGGCACA   3960   - - ACCTTCTATT TTGATGAAAA GACTGGTATT CAAGCCAAAG ACAAGTTCAT CC -#GTTTTGCG   4020   - - GACGGACGCA CGCGTTACTT CATCCCAGAT ACAGGAAATC TCGCAGTCAA CC -#GATTTGCG   4080   - - CAAAATCCTG AGAACAAGGC TTGGTATTAC CTCGATAGCA ACGGTTATGC CG -#TGACAGGA   4140   - - CTACAAACCA TTAACGGTAA GCAGTATTAC TTTGACAATG AAGGACGTCA GG -#TTAAGGGA   4200   - - CACTTTGTCA CTATCAATAA CCAACGTTAC TTCCTTGATG GTGATAGTGG TG -#AAATTGCT   4260   - - CCGTCACGCT TTGTGACGGA AAACAACAAG TGGTACTATG TCGATGGCAA TG -#GTAAACTG   4320   - - GTTAAAGGTG CTCAGGTCAT CAATGGTAAT CACTACTATT TCAACAATGA TT -#ATAGCCAA   4380   - - GTCAAGGGTG CCTGGGCCAA CGGCCGTTAC TATGATGGTG ACTCAGGTCA GG -#CCGTAAGC   4440   - - AACCAATTCA TTCAAATTGC GGCTAACCAA TGGGCTTACC TTAACCAAGA TG -#GTCACAAA   4500   - - GTAACAGGTC TTCAAAATAT TAACAATAAA GTTTACTATT TTGGTAGCAA TG -#GTGCTCAA   4560   - - GTCAAAGGTA AATTGCTCAC TGTCCAAGGT AAGAAATGTT ACTTTGATGC CC -#ACACAGGT   4620   - - GAGCAAGTGG TAAACCGCTT TGTCGAAGCT GCACGTGGCT GCTGGTATTA CT -#TTAACTCA   4680   - - GCTGGCCAAG CAGTGACTGG ACAACAGGTC ATCAATGGTA AACAACTTTA CT -#TCGACGGT   4740   - - TCAGGTCGTC AAGTTAAAGG ACGTTATGTT TATGTTGGTG GTAAACGACT CT -#TCTGCGAT   4800   - - GCCAAAACTG GTGAATTGAG ACAGCGTCGC TAATTAATAT GTACTTTAAA AA - #T4853  - -  - - (2) INFORMATION FOR SEQ ID NO:2:  - -      (i) SEQUENCE CHARACTERISTICS:      (A) LENGTH: 1577 amino - #acids      (B) TYPE: amino acid      (C) STRANDEDNESS:      (D) TOPOLOGY: Not Relev - #ant  - -     (ii) MOLECULE TYPE: protein  - -     (vi) ORIGINAL SOURCE:      (A) ORGANISM: Streptococcu - #s salivarius  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:  - - Met Glu Asn Lys Val Arg Phe Lys Leu His Ly - #s Val Lys Lys Asn Trp 1               5   - #                10  - #                15  - - Val Thr Ile Gly Val Thr Thr Leu Ser Met Va - #l Ala Leu Ala Gly Gly        20      - #            25      - #            30  - - Ser Leu Leu Ala Gln Gly Lys Val Glu Ala As - #p Glu Thr Ser Ala Pro    35          - #        40          - #        45  - - Asn Gly Asp Gly Leu Gln Gln Leu Ser Glu As - #p Gly Thr Ala Ser Leu50              - #    55              - #    60  - - Val Thr Thr Thr Thr Val Thr Glu Gln Ala Se - #r Ala Gln Ala Ser Val 65                  - #70                  - #75                  - #80  - - Ser Ala Val Ala Thr Ala Ser Val Ser His Gl - #u Thr Ser Phe Gln Ala            85  - #                90  - #                95  - - Ala Thr Ser Ala Val Ser Gln Glu Ala Thr Al - #a Gln Ala Gln Thr Ser        100      - #           105      - #           110  - - Pro Val Ala Ser Gln Glu Val Ala Val Ser Se - #r Gln Thr Gln Ser Ser    115          - #       120          - #       125  - - Gly Gln Glu Thr Gln Thr Thr Glu Gln Val Se - #r Gln Gly Gln Thr Ser130              - #   135              - #   140  - - Thr Gln Val Ala Gly Gln Thr Ser Ala Gln Se - #r Thr Pro Ser Val Thr 145                 1 - #50                 1 - #55                 1 -#60   - - Glu Gln Ala Arg Pro Arg Val Leu Thr Asn Al - #a Ala Pro Ala IleAla             165  - #               170  - #               175  - - Thr Arg Ala Ala Asp Ser Thr Ile Arg Ile As - #n Ala Asn Arg Asn Thr        180      - #           185      - #           190  - - Asn Ile Thr Ile Thr Ala Ser Gly Thr Thr Pr - #o Asn Val Thr Ile Ile    195          - #       200          - #       205  - - Thr Gly Pro Asn Thr Pro Lys Pro Asn Val Th - #r Val Thr Ser Pro Asn210              - #   215              - #   220  - - Gly Thr Arg Pro Asn Val Thr Ile Val Thr Gl - #n Pro Asn Gln Pro Asn 225                 2 - #30                 2 - #35                 2 -#40   - - Lys Pro Val Gln Pro Ser Gln Pro Ser Gln Pr - #o Asn Lys Pro ValGln             245  - #               250  - #               255  - - Pro Asn Gln Pro Ser Leu Asp Tyr Lys Pro Va - #l Ala Ser Asn Leu Lys        260      - #           265      - #           270  - - Thr Ile Asp Gly Lys Gln Tyr Tyr Val Glu As - #n Gly Val Val Lys Lys    275          - #       280          - #       285  - - Asn Ala Ala Ile Glu Leu Asp Gly Arg Leu Ty - #r Tyr Phe Asp Glu Thr290              - #   295              - #   300  - - Gly Ala Met Val Asp Gln Ser Lys Pro Leu Ty - #r Arg Ala Asp Ala Ile 305                 3 - #10                 3 - #15                 3 -#20   - - Pro Asn Asn Ser Ile Tyr Ala Val Tyr Asn Gl - #n Ala Tyr Asp ThrSer             325  - #               330  - #               335  - - Ser Lys Ser Phe Glu His Leu Asp Asn Phe Le - #u Thr Ala Asp Ser Trp        340      - #           345      - #           350  - - Tyr Arg Pro Lys Gln Ile Leu Lys Asp Gly Ly - #s Asn Trp Thr Ala Ser    355          - #       360          - #       365  - - Thr Glu Lys Asp Tyr Arg Pro Leu Leu Met Th - #r Trp Trp Pro Asp Lys370              - #   375              - #   380  - - Val Thr Gln Val Asn Tyr Leu Asn Tyr Met Se - #r Gln Gln Gly Phe Gly 385                 3 - #90                 3 - #95                 4 -#00   - - Asn Lys Thr Tyr Thr Thr Asp Met Met Ser Ty - #r Asp Leu Ala AlaAla             405  - #               410  - #               415  - - Ala Glu Thr Val Gln Arg Gly Ile Glu Glu Ar - #g Ile Gly Arg Glu Gly        420      - #           425      - #           430  - - Asn Thr Thr Trp Leu Arg Gln Leu Met Ser As - #p Phe Ile Lys Thr Gln    435          - #       440          - #       445  - - Pro Gly Trp Asn Ser Glu Ser Glu Asp Asn Le - #u Leu Val Gly Lys Asp450              - #   455              - #   460  - - His Leu Gln Gly Gly Ala Leu Thr Phe Leu As - #n Asn Ser Ala Thr Ser 465                 4 - #70                 4 - #75                 4 -#80   - - His Ala Asn Ser Asp Phe Arg Leu Met Asn Ar - #g Thr Pro Thr AsnGln             485  - #               490  - #               495  - - Thr Gly Thr Arg Lys Tyr His Ile Asp Arg Se - #r Asn Gly Gly Tyr Glu        500      - #           505      - #           510  - - Leu Leu Leu Ala Asn Asp Ile Asp Asn Ser As - #n Pro Ala Val Gln Ala    515          - #       520          - #       525  - - Glu Gln Leu Asn Trp Leu His Tyr Ile Met As - #n Ile Gly Ser Ile Leu530              - #   535              - #   540  - - Gly Asn Asp Pro Ser Ala Asn Phe Asp Gly Va - #l Arg Ile Asp Ala Val 545                 5 - #50                 5 - #55                 5 -#60   - - Asp Asn Val Asp Ala Asp Leu Leu Gln Ile Al - #a Ser Asp Tyr PheLys             565  - #               570  - #               575  - - Glu Lys Tyr Arg Val Ala Asp Asn Glu Ala As - #n Ala Ile Ala His Leu        580      - #           585      - #           590  - - Ser Ile Leu Glu Ala Trp Ser Tyr Asn Asp Hi - #s Gln Tyr Asn Lys Asp    595          - #       600          - #       605  - - Thr Lys Gly Ala Gln Leu Ser Ile Asp Asn Pr - #o Leu Arg Glu Thr Leu610              - #   615              - #   620  - - Leu Thr Thr Phe Leu Arg Lys Ser Asn Tyr Ar - #g Gly Ser Leu Glu Arg 625                 6 - #30                 6 - #35                 6 -#40   - - Val Ile Thr Asn Ser Leu Asn Asn Arg Ser Se - #r Glu Gln Lys HisThr             645  - #               650  - #               655  - - Pro Arg Asp Ala Asn Tyr Ile Phe Val Arg Al - #a His Asp Ser Glu Val        660      - #           665      - #           670  - - Gln Ala Val Leu Ala Asn Ile Ile Ser Lys Gl - #n Ile Asn Pro Lys Thr    675          - #       680          - #       685  - - Asp Gly Phe Thr Phe Thr Met Asp Glu Leu Ly - #s Gln Ala Phe Glu Ile690              - #   695              - #   700  - - Tyr Asn Ala Asp Ile Ala Lys Ala Asp Lys Ly - #s Tyr Thr Gln Tyr Asn 705                 7 - #10                 7 - #15                 7 -#20   - - Ile Pro Ala Ala Tyr Ala Thr Met Leu Thr As - #n Lys Asp Ser IleThr             725  - #               730  - #               735  - - Arg Val Tyr Tyr Gly Asp Leu Phe Thr Asp As - #p Gly Gln Tyr Met Ala        740      - #           745      - #           750  - - Glu Lys Ser Pro Tyr Tyr Asn Ala Ile Asp Al - #a Leu Leu Arg Ala Arg    755          - #       760          - #       765  - - Ile Lys Tyr Val Ala Gly Gly Gln Asp Met Ly - #s Val Thr Lys Leu Asn770              - #   775              - #   780  - - Gly Tyr Glu Ile Met Ser Ser Val Arg Tyr Gl - #y Lys Gly Ala Glu Glu 785                 7 - #90                 7 - #95                 8 -#00   - - Ala Asn Gln Leu Gly Thr Ala Glu Thr Arg As - #n Gln Gly Met LeuVal             805  - #               810  - #               815  - - Leu Thr Ala Asn Arg Pro Asp Met Lys Leu Gl - #y Ala Asn Asp Arg Leu        820      - #           825      - #           830  - - Val Val Asn Met Gly Ala Ala His Lys Asn Gl - #n Ala Tyr Arg Pro Leu    835          - #       840          - #       845  - - Leu Leu Ser Lys Ser Thr Gly Leu Ala Thr Ty - #r Leu Lys Asp Ser Asp850              - #   855              - #   860  - - Val Pro Ala Gly Leu Val Arg Tyr Thr Asp As - #n Gln Gly Asn Leu Thr 865                 8 - #70                 8 - #75                 8 -#80   - - Phe Thr Ala Asp Asp Ile Ala Gly His Ser Th - #r Val Glu Val SerGly             885  - #               890  - #               895  - - Tyr Leu Ala Val Trp Val Pro Val Gly Ala Se - #r Glu Asn Gln Asp Ala        900      - #           905      - #           910  - - Arg Thr Lys Ala Ser Ser Thr Lys Lys Gly Gl - #u Gln Val Phe Glu Ser    915          - #       920          - #       925  - - Ser Ala Ala Leu Asp Ser Gln Val Ile Tyr Gl - #u Gly Phe Ser Asn Phe930              - #   935              - #   940  - - Gln Asp Phe Val Lys Thr Pro Ser Gln Tyr Th - #r Asn Arg Val Ile Ala 945                 9 - #50                 9 - #55                 9 -#60   - - Gln Asn Ala Lys Leu Phe Lys Glu Trp Gly Il - #e Thr Ser Phe GluPhe             965  - #               970  - #               975  - - Ala Pro Gln Tyr Val Ser Ser Gln Asp Gly Th - #r Phe Leu Asp Ser Ile        980      - #           985      - #           990  - - Ile Glu Asn Gly Tyr Ala Phe Glu Asp Arg Ty - #r Asp Ile Ala Met Ser    995          - #       1000          - #      1005  - - Lys Asn Asn Lys Tyr Gly Ser Leu Lys Asp Le - #u Met Asp Ala Leu Arg1010             - #   1015              - #  1020  - - Ala Leu His Ala Glu Gly Ile Ser Ala Ile Al - #a Asp Trp Val Pro Asp 1025                1030 - #                1035 - #               1040  - - Gln Ile Tyr Asn Leu Pro Gly Lys Glu Val Va - #l Thr Ala Ser Arg Thr            1045 - #               1050  - #              1055  - - Asn Ser Tyr Gly Thr Pro Arg Pro Asn Ala Gl - #u Ile Tyr Asn Ser Leu        1060     - #           1065      - #          1070  - - Tyr Ala Ala Lys Thr Arg Thr Phe Gly Asn As - #p Phe Gln Gly Lys Tyr    1075         - #       1080          - #      1085  - - Gly Gly Ala Phe Leu Asp Glu Leu Lys Ala Ly - #s Tyr Pro Ala Ile Phe1090             - #   1095              - #  1100  - - Glu Arg Val Gln Ile Ser Asn Gly Arg Lys Le - #u Thr Thr Asn Glu Lys 1105                1110 - #                1115 - #               1120  - - Ile Thr Gln Trp Ser Ala Lys Tyr Phe Asn Gl - #y Ser Asn Ile Gln Gly            1125 - #               1130  - #              1135  - - Thr Gly Ala Arg Tyr Val Leu Gln Asp Asn Al - #a Thr Asn Gln Tyr Phe        1140     - #           1145      - #          1150  - - Ser Val Lys Ala Gly Gln Thr Phe Leu Pro Ly - #s Gln Met Thr Glu Ile    1155         - #       1160          - #      1165  - - Thr Gly Ser Gly Phe Arg Arg Val Gly Asp As - #p Val Gln Tyr Leu Ser1170             - #   1175              - #  1180  - - Ile Gly Gly Tyr Leu Ala Lys Asn Thr Phe Il - #e Gln Val Gly Ala Asn 1185                1190 - #                1195 - #               1200  - - Gln Trp Tyr Tyr Phe Asp Lys Asn Gly Asn Me - #t Val Thr Gly Glu Gln            1205 - #               1210  - #              1215  - - Val Ile Asp Gly Lys Lys Tyr Phe Phe Leu As - #p Asn Gly Leu Gln Leu        1220     - #           1225      - #          1230  - - Arg His Val Leu Arg Gln Gly Ser Asp Gly Hi - #s Val Tyr Tyr Tyr Asp    1235         - #       1240          - #      1245  - - Pro Lys Gly Val Gln Ala Phe Asn Gly Phe Ty - #r Asp Phe Ala Gly Pro1250             - #   1255              - #  1260  - - Arg Gln Asp Val Arg Tyr Phe Asp Gly Asn Gl - #y Gln Met Tyr Arg Gly 1265                1270 - #                1275 - #               1280  - - Leu His Asp Met Tyr Gly Thr Thr Phe Tyr Ph - #e Asp Glu Lys Thr Gly            1285 - #               1290  - #              1295  - - Ile Gln Ala Lys Asp Lys Phe Ile Arg Phe Al - #a Asp Gly Arg Thr Arg        1300     - #           1305      - #          1310  - - Tyr Phe Ile Pro Asp Thr Gly Asn Leu Ala Va - #l Asn Arg Phe Ala Gln    1315         - #       1320          - #      1325  - - Asn Pro Glu Asn Lys Ala Trp Tyr Tyr Leu As - #p Ser Asn Gly Tyr Ala1330             - #   1335              - #  1340  - - Val Thr Gly Leu Gln Thr Ile Asn Gly Lys Gl - #n Tyr Tyr Phe Asp Asn 1345                1350 - #                1355 - #               1360  - - Glu Gly Arg Gln Val Lys Gly His Phe Val Th - #r Ile Asn Asn Gln Arg            1365 - #               1370  - #              1375  - - Tyr Phe Leu Asp Gly Asp Ser Gly Glu Ile Al - #a Pro Ser Arg Phe Val        1380     - #           1385      - #          1390  - - Thr Glu Asn Asn Lys Trp Tyr Tyr Val Asp Gl - #y Asn Gly Lys Leu Val    1395         - #       1400          - #      1405  - - Lys Gly Ala Gln Val Ile Asn Gly Asn His Ty - #r Tyr Phe Asn Asn Asp1410             - #   1415              - #  1420  - - Tyr Ser Gln Val Lys Gly Ala Trp Ala Asn Gl - #y Arg Tyr Tyr Asp Gly 1425                1430 - #                1435 - #               1440  - - Asp Ser Gly Gln Ala Val Ser Asn Gln Phe Il - #e Gln Ile Ala Ala Asn            1445 - #               1450  - #              1455  - - Gln Trp Ala Tyr Leu Asn Gln Asp Gly His Ly - #s Val Thr Gly Leu Gln        1460     - #           1465      - #          1470  - - Asn Ile Asn Asn Lys Val Tyr Tyr Phe Gly Se - #r Asn Gly Ala Gln Val    1475         - #       1480          - #      1485  - - Lys Gly Lys Leu Leu Thr Val Gln Gly Lys Ly - #s Cys Tyr Phe Asp Ala1490             - #   1495              - #  1500  - - His Thr Gly Glu Gln Val Val Asn Arg Phe Va - #l Glu Ala Ala Arg Gly 1505                1510 - #                1515 - #               1520  - - Cys Trp Tyr Tyr Phe Asn Ser Ala Gly Gln Al - #a Val Thr Gly Gln Gln            1525 - #               1530  - #              1535  - - Val Ile Asn Gly Lys Gln Leu Tyr Phe Asp Gl - #y Ser Gly Arg Gln Val        1540     - #           1545      - #          1550  - - Lys Gly Arg Tyr Val Tyr Val Gly Gly Lys Ar - #g Leu Phe Cys Asp Ala    1555         - #       1560          - #      1565  - - Lys Thr Gly Glu Leu Arg Gln Arg Arg1570             - #   1575__________________________________________________________________________ 
    
     LIST OF REFERENCES 
     1. Radojevic et al. 1994 Aust J Agric Res 45, 901-12. 
     2. Jacques N A, Giffard P M, &#34;The Glycosyltransferases of Oral Streptococci&#34; Todays Life Science 1991; 3: 40-6. 
     3. Walker G J, Jacques N A, &#34;Polysaccharides of Oral Streptococci&#34; In: Reizer J, Peterkofsky A, Eds. &#34;Sugar Transport and Metabolism in Gram-Positive Bacteria&#34;. Chichester: Ellis Horwood, 1987; 39-68. 
     4. Gilmore K S, Russell R R B, Ferretti J J, &#34;Anaylsis of the Streptococcus downei gtfS gene, which specifies a glucosyltransferase that synthesises soluble glucans&#34;. Infect Immun 1990; 58: 2452-8. 
     5. Giffard P M, Simpson C L, Milward C P, Jacques N A, &#34;Molecular characterization of a cluster of at least two glucosyltransferase genes in Streptococcus salivarius ATCC25975&#34;. J. Gen. Microbiol. 1991; 137:2577-93. 
     6. Giffard P M, Allen D M, Milward C P, Simpson C L, Jacques N A, &#34;Sequence of the GtfK of Streptococcus salivarius ATCC25975 and the evolution of the gtf genes of oral streptococci&#34;. J Gen Microbiol 1993; 
     139:1511-22. 
     7. Pitty L S, Giffard P M, Gilpin M L, Russell R R B and Jacques N A, 1989. &#34;Cloning and expression of glycosyltransferase C gene (gtfC) from Streptococcus mutans LM7. Infect Immun 55: 2176-2182. 
     8. Silliary T S, Berman M L, and Enquist L W, 1984. &#34;Experiments with Gene fusions&#34;. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 
     9. Jacques N. A. 1983. Membrane perturbation by cerulenin modulates glucosyltransferase secretion and acetate uptake by Streptococcus salivarius. J. Gen. Micriobial. 129 : 3293-3302. 
     10. Maniatis T, Fritsch E F, and Sambrook J. (1989). &#34;Molecular Cloning; a laboratory manual. Second edition. Cold Spring Harbor Laboratory Press, N.Y.