IE851225L - Peptides - Google Patents

Abstract

1. Claims for the contracting states : BE. CH. LI. DE. FR. GB. IT. LU. NL. SE DNA sequence B, obtainable from Streptomyces tendae strains, which produce tendamistat and have preferably been treated with sublethal doses of acriflavine, by isolation of the complete DNA, digestion with Pst l, Southern hybridization with the DNA sequence A, 5'-(**32 P-)CCT TCA GTG TCG TCT TCG TA-3' (A) isolation of the 2.3 kb Pst l fragment, cutting with BamHl, Southern hybridization with the sequence A, isolation of the 0.94 kb Pst l-BamHl subfragment, cutting with Sau 3a, Southern hybridization with the sequence A, isolation of the 0.525 kb BamHi-Sau 3a subfragment and sequencing of the DNA, and which has the following features : a) it is located immediately upstream of the tendamistat structural gene, b) it codes at the amino terminal end for Met-Arg-Val-Arg-Ala-Leu-Arg, c) it codes at the carboxyl terminal end for Ala-Ser-Ala and d) it codes in the middle for a hydrophobic region X which comprises 10 to 25, preferably 17 to 20, amino acids. 1. Claims for contracting state AT A process for the preparation of polypeptides composed of genetically codable amino acids, characterized by the expression, in a host cell of the genus Streptomyces, of a gene structure which contains in the reading frame with the structural gene for the desired polypeptide the DNA sequence B of a prepeptide, which sequence is obtainable from Streptomyces tendae strains, which produce tendamistat and have preferably been treated with sublethal doses of acriflavine, by isolation of the complete DNA, digestion with Pst l, Southern hybridization with the DNA sequence A, 5'-(**32 P-)CCT TCA GTG TCG TCT TCG TA-3' (A) isolation of the 2.3 kb Pst l fragment, cutting with BamHI, Southern hybridization with the sequence A, isolation of the 0.94 kb Pst l-BamHl subfragment, cutting with Sau 3a, Southern hybriridization with the sequence A, isolation of the 0.525 kb BamHl-Sau 3a subfragment and sequencing of the DNA, and a) is located immediately upstream of the structural gene, b) codes at the amino terminal end for Met-Arg-Val-Arg-Ala-Leu-Arg, c) codes at the carboxyl terminal end for Ala-Ser-Ala and d) codes in the middle for a hydrophobic region X which comprises 10 to 25 amino acids.

Description

58385 - 2 - The invention relates to a signal peptide which is a constituent of a propeptide which,, in a Streptornyces cell which contains a signal peptidase, is cleaved into the signal peptide and a polypeptide,, the latter being 5 removed from the cell and excreted into the culture medium. The invention further relates to DMA sequences which code for this signal peptide, gene structures which contain this DMA sequence in the reading frame with a structural gene, plasmids which contain a gene structure of this type,, and 10 host organisms containing plasmids of this type* Further aspects of the invention and its preferred embodiments are illustrated in detail below.

A process for the preparation of tendamistat by fermentation of Streptornyces tendae has already been pro-15 posed^ in German Patent Application P 33 31 860.3which process comprises using S„ tendae strains which produce tendamistat and have been treated with sublethal doses of acriflavine. A DMA fragment containing the gene for tendamistat was isolated from strains thus obtained,, namely a 20 2.3 kb Pst I fragment. It was possible^. by incorporation of this fragment in pBR 322 which had been cut with Pst I to amplify this DMA in E, coli and to re-isolate this DNA in pure form.

It has now been found that the signal peptide 25 (prepeptide) of the formula I Met-Ars-V?.l-Arg- Ala-Leu- Arg-X-Ala-Ser-Ala CI ) in which X represents a hydrophobic region comprising 10 to 25, preferably 17 to 20 amino acids, is coded on this 2.3 kb fragment immedi-30 ate ly upstream of the structural gene for tendaroi stat- It has also been found that by use of this signal peptide other* peptides ©re excreted from host cells which contain an appropriate signal peptidase. Thus the inven tion also relates to propeptides of the formula II Sig-R (II) in which Sig denotes the amino acid sequence of the formula I? and R represents the residue of a genetically codable peptide linked by its amino terminal end,* in which an acidic amino acid,* in particular aspartic acid,* is preferably located at the amino terminal end.

Thusone aspect of the invention relates to peptides of the formula II in which R denotes hydrogen or a peptide residue,, for example the tendamistat residue. Another aspect of the invention relates to the corresponding DMA sequence which can be obtained from Streptornyces tendae strains which produce tendamistat and which have preferably been treated with sublethal doses of acri-flavine,,. by isolation of the total digestion with Pst I /. Southern hybridization with the D N A sequence A., 5'~(32P-)CCT TCA GTG TCG TCT TCG TA-3' (A) isolation of the 2.3 kb Pst I fragment, cutting with BamHI,. Southern hybridisation with the sequence A, isolation of the 0-94 kb PstI BamHI subf ragment,, cutting with Sau 3 a r Southern hybridization with the sequence A, isolation of the 0»525 kb BamHI Sau 3a subf ragment and sequencing of the and which has the following features: a) it is located immediately upstream of the tendamistat structural gene, b) it codes at the amino terminal end for Met-Arg-Val-Arg-Ala-Leu-Arg, c) it codes at the carboxy terminal end for Ala-Ser-Ala and d) it codes in the1 middle for a hydrophobic region which comprises 10 to 25preferably 17 to 20 amino acids* This DMA sequence is called sequence B or signal peptide in the following text.

The kb figures which Mere determined by comparison with standard markers have the customary accuracy.

In place of the sequence it is possible to select for the Southern hybridization any desired sequence which is complementa ry to the tendamistat gene or the — 4 *■* counterpart strand.

For the various steps for the characterization of the DMA sequence B , in each case in practice the DNA is introduced into a suitable vector, the latter is trans-5 formed into a host cell,, amplified there,, the transfor- mants determined by colony hybridization with the sequence A, and the DNA reisolated. These steps are known per se.

The DNA sequence C , whose coding strand is represented in the attachment,, has the nucleotide sequence of 10 the tendamistat structural gene from S » tendae.

Thus,, the gene structures mentioned contain the DMA sequence B in the reading frame with a structural gene which codes, for example,, for tendamistat, preferably the DNA sequence C , 15 The invention also relates to plasmids which com prise the DMA sequence B in the reading frame with a structural gene which codes,, for example, for tendamistat, in particular for the DNA sequence C „ These plasmids can contain a replicon which is effective in E» coli, and are 20 then able to amplify and, possibly,, also to express the DNA in E . coli.

Preferred plasmids additionally contain a replicon which is effective in Streptomycetes. If a Streptornyces is transformed with a plasmid of this type, it becomes 2 5 able to express the peptide, which is determined by the structural gene, in the form of the propeptide of the formula II, which is then cleaved by a signal peptidase during the processing, and the desired peptide is excreted into the culture medium, 30 So-called shuttle plasmids which contain both a replicon which is effective in E. coli and one which is effective in Streptomycetes are also advantageous. These shuttle vectors can be amplified in E„ coli and, after re-isolation, transformed into St reptomycetes, where 35 production of the desired polypeptide then takes place™ The invention also relates to host organisms which have been transformed with the plasmids ment i oned, in particular host organisms of the genus Streptornyces, especially of the species S„ tendae or, in particular, - 5 - S „ lividans- Furthermore, the invention relates to a process for the preparation of a polypeptide of the general formula III 5 H2N-R (III) in which R has the meaning indicated for formula II,. in which is used one of the transformed host organisms mentioned, which contains a signal peptidase uhich splits off the propeptide of the formula II and secretes the desired 10 polypeptide- While a plethora of vectors are available for Gram-negative bacteria, only a few vectors have been described for Gram-positive bacteria,, in particular for St reptomycetes. Vectors for bacteria of the species S. tendae 15 have not hitherto been disclosed* Thus,, an approach for the utilization of S - tendae as host organisms is made possible by the i nvent i on.

A particular advantage of the invention is that transformed Streptornyces strains, in particular S - lividans 20 strains, sporulate optimally, that is to say the content of recombinant plasmid does not adversely affect these strains in their generative phase- Thus, the transformed organisms are also suitable for further strain improvements, for example for the production and selection of 25 metabolic mutants involving the use of spores- Compared with untransformed strains of S. tendae, the transformed strains, in particular S» lividans, do not form melanin. Thus, there is no necessity to remove it, and this makes the isolation of the desired peptide, for 30 example tendamistat, considerably easier and it prevents losses in yield- Another advantage of the invention is that foreign genes are also expressed in S „ lividans, and the corresponding polypeptides are excreted, which likewise offers 35 a variety of possibilities for strain improvement and for modification of the polypeptide thus produced™ However,, it is also possible according to the invention to transform other species of Streptomyces, for example S „ ghanaensis or aureofaciens- When strains which contain no plasmid and are able to synthesize a specific signal peptidase are transformed with the hybrid plasmids according to the invention, then stable trans-formants which express and secrete the coded peptide are 5 obtained.

Particularly preferred embodiments of the invention are illustrated in detail in the examples which follow. In these examples, percentage data relate to weight unless otherwise specified. The figures repre-10 senting the hybrid plasmids show the restriction sites true to scale„ The following vectors, known from the literature^ were used in the examples; single-strand phages M 13 mp 8 and M 13 mp 9" Messing et a I.Gene 19 (1982) 269; pUC 8: 15 Vierra et a I.Gene 19 (1982) 259; pAC 177 and 184* Chang et a I., J. Bacteriology 134 (1978) 1141; pIJ 102 and 350: Kieser et aL, Hoi. Gen. Genet„ 185 (1982) 223.

Maintenance of the S . tendae strain is described in U.S. Patent 4^,226^764. In principle,. the tendamistat 20 gene can be isolated from any strain which produces tendamistat. However, the procedure of German Patent Application P 33 31 850.3 is particularly advantageous, the isolation of the DNA being described in Example 3 therein.

This isolated complete DNA is the starting material for 25 Example 1 below.

Example 1 5 pg of DMA is completely digested with the restriction enzyme Pst I and, after fractionation in a 0.8% agarose gel,, transferred to nitrocellulose filters 30 (Southern transfer). The filter with the bound, denatured DNA is prehybridized for 6 hours in 5 ml of prehybridiza-t i on medium (0.6 M NaCl^ 0.06 ft Ma EDTA, 0.1% sodium dodecyl sulfate solution,, 100 ug/ir, 1 sonicated calf thymus DNA and 4-fold concentrated Oenhardt's solution)., It is 35 then again treated with 5 ml of the prehybridization medium to which,, however,* 500,?000 cpm/ml of radiolabeled DNA have been added. This radiolabeled probe is obtained as follows* The DNA sequence A is synthesised chemically by - 7 - the phosphite process. It contains 20 nucleotides (molecular weight about 13,000) and is complementary to the putative DNA sequence for tendamistat^. derived from the amino acid sequence of tendamistat beginning from amino 5 acid 37 of tendamistatusing the triplets preferred by E„ coli™ This DNA sequence A is radiolabeled at the 5* end using y ^P-ATP and nucleotide kinase.

For the hybridization of this radioactive probe to the complementary DNA sequence in the complete DMA,, the 10 mixture is allowed to stand at 37°C for 24 hours. Then the non-bound radioactive DNA is removed,* and the filter is washed at 37°C with 5 x 200 ml of hybridization medium for 30 minutes each time,, and then subjected to autoradiography™ After exposure for 24 hours,* the hybridiza-15 tion signals show that the gene is located on the 2.3 kb Pst I fragment. This fragment is obtained by electro-elution of a section corresponding to this fragment size,, cut out of a preparative agarose gel on which the Pst I digested total DNA had been fractionated. The eluted DNA 20 is cloned in the Pst I restriction site of the plasmid pUC 8» These hybrid plasmids are transformed into E„ coli JW 103 and are amplified. The; clones which carry the insertion with the desired tendamistat gene are detected by colony hybridization using the radioactive DNA probe A. The 25 hybrid plasmids pKAI la and 1 b thus obtained are represented in Figures 1 a and b„ The localization of the gene can be determined exactly by further Southern hybridization steps against the isolated 2»3 kb Pst I fragment and its subfragments 30 (Figures 2a to 2 c ) „ Example 2 The 2.3 kb Pst I fragment from S. tendae is cloned in the unique Pst I restriction site in the plasmid pIJ 102. The hybrid plasmid pAX 1 a and 1 b thus obtained,, which 35 differ in their orientation of the insertion,* confer the ability to produce tendamistat on S. lividans strains after having been transformed into them. Figure 3 shows the plasmid pAX 1 a. - 8 - Example 3 The commercially available strain S. lividans TK 24 (John Innes Institute, Norwich, England) is converted Q into protoplasts in known manner,, and 10° protoplasts 5 are added to 1 pg of hybrid plasmid p A X 1 a in the presence of 20% polyethylene glycol 6000. The transformed protoplasts are incubated on regeneration medium R2YE (Thompson et al«, Nature 286 (1980) 525) at 30°C for 5 days. 10 The formation of an extracellular amylase inacti- vator can be demonstrated by a plate test: 5 ml of an aqueous solution containing 0 „ 4 to 1„0 mg/rol pancreatin is poured over the regenerated colonies, and the mixture is incubated at 37°C for 1 hour. 15 The solution is then removed and replaced by 5 ml of a 2% starch agar. After incubation of the plates at 37°C for 2 hours, 5 ml of an iodine/potassium iodide solution is poured over them to develop them. Colonies with a blue halo indicate that the clones synthesize and excrete ten-20 damistat.

As a check, the plasmid DNA of strains which produce tendamistat and sporulate well can be isolated and mapped. All strains which produce tendamistat carry p A X 1 plasmid D M A „ 25 Example 4 The process is carried out in accordance with Example 2, but the plasmid pIJ 350 which carries a thio-strepton-resistance gene as a selectable marker in Strepto-mycetes is used. The hybrid plasmids pAX 350 a and b are 30 thus obtained (which differ in the orientation of the insertion). Figure 4 shows the plasmid pAX 350 a™ After transformation in accordance with Example 3 , resistant clones are selected on minimum medium (Hopwood, Bacteriological Reviews 31 (1967) 373 - 403) in the pre-35 series of 50 jjg/m I ? h i ost repton^ and are tested for production of tendamistat either directly on minimum medium or after being transferred to non-selective R2YE agar.

Example 5 Hybrid plasmids which contain the 2.3 kb Pst I fragment and,, in addition to the Streptornyces replicon, contain an E„ coli replicon have a number of advantages as shuttle vectors; because of the E. coli replicon and resistance markers effective in E» coli^, they are able to be well amplified in these organisms. After isolation and transformation into Streptomycetes, in particular into S„ lividans^, they have high stability™ As a consequence of their selection markers which are effective in Strepto-mycetes, and of the Streptornyces replicon, they can also be well amplified in these organisms and can express and secrete tendamistat.

The plasmid pAC 184 is completely digested with the restriction enzyme Sal I, and the enzyme is removed by extraction with phenol/chloroform. The protruding 5" ends are filled using the enzyme DMA polymerase (KI enow fragment) in the presence of ATP, OTP, GTP and TTP. A Pst I linker of the structure 5'TCG AGC TGC AGC TCG A 3' 3'AGC TCG ACG TCG AGC T 5' is ligated to the blunt ends in a ligase reaction at 22°C (2 jjg of linker to 0.4 jjg of DNA). The DHA is extracted with phenol/chloroform and, after precipitation, is digested with the enzyme PSt I to obtain Pst I ends which can be ligated. The Pst I ends of the vector are then dephos-phorylated, again extracted with phenol/chloroform, and ligated with plasmid pAX 1 a which has been partially digested with Pst I. The ligation mixture js transformed into E . coli (HB 101 or M C 1061). Clones resistant to chloramphenicol are rinsed off the plate,, and the plasmid DNA is isolated. S. lividans TK 24 is transformed with 1 to 2 jj g of plasmid DMA and tested for production of tendamistat.

Clones with a positive reaction in the tendamistat test are isolated, the plasmid DNA is isolated by rapid alkaline lysis and introduced into E ■ coli HB 101 or P3C 1061 by ba ck-t ransf o r ma t i on. Plasmids re-isolated after amplification do not differ from the plasmids isolated from S „ lividans strains,. The recombinant plasmids - 10 - are designated pSA 2 a or b (Figures 5a and b) depending on the orientation of the insertion which carries the tendamistat gene.

Example 6 The process is carried out in accordance with Example 5r but starting from plasmid pAX 350 a^, selecting in E „ coli for chloramphenicol resistance and in S. I i v i -dans for thiostrepton resistance and production of tendamistat^ and the plasmids pSA 351 a and b (Figures 6a/b) a re obtai ned.

Example 7 The process is carried out in accordance with Example 5, but starting from the plasmid pAC 177 in place of pAC 184, and the plasmids pSA 3 a and b (Figures 7 a/b) are obtained.

For this purpose,, the plasmid pAX 1 a is partially cut with Pst 1^. and the enzyme is heat-inactivated by heating at 68°C for 15 minutes. The DNA is ligated into the plasmid pAC 177 which has been cut with Pst 1, dephos-phorylated and deproteinized. After transformation of E» coli HB 101 or HC 1061^ clones resistant to kanamycin are rinsed off the plate^ the plasmid DNA is isolated, and S » lividans TK 24 is transformed with 1 to 2 jjg of this D N A „ Clones which produce tendamistat are selected and the plasmids are characterized.

Examp le 8 The process is carried out in accordance with Example 7, but the plasmid pAX 350 a is used in place of the plasmid pAX 1, selection in S» lividans for thiostrepton resistance is carried out, and the plasmids pSA 352 a and b (Figures 8 a/b) are obtained.

E xamp le 9 As is evident from Figure 2f the gene which codes for tendamistat and the signal sequence is 0»3 kb long.

The 2.3 kb fragment used in the examples detailed above can thus be used in a shortened form for the construction of hybrid plasmids which bring about the production of tendami stats The plasmid pKhl 1 a is digested with Sal I and - 11 - religated. In this manner, the plasmid pKAI 2 which has been shortened by about 750 base-pairs is obtained,. It is cloned, isolated and cut with Pst I. The DNA is dephos-phorylated using alkaline phosphatase from calf intestines, 5 and is deproteinized with phenol/chloroform.

The plasmid pSJ 102 is completely cut with Pst I and, after heat-inactivation of the enzyme, the fragments are ligated in the Pst I restriction site of pKAI 2„ The ligation mixture is transformed into E _ coli HB 101 or MC 10 1061* The plasmid DNA from clones which are resistant to ampicillin is isolated by the rapid alkaline lysis process, and S„ lividans TK 24 is transformed with 1 to 2 ug of this DNA. Clones which produce tendamistat are selected and the plasmid DNA from them is isolated by rapid alkaline lysis. 15 After re-transformation into E„ coli HB 101 or MC 1061 and after isolation of the plasmid DNA from the transformed E * coli strains, the plasmid pSA 1 is obtained and characterized by restriction analysis (Figure 9) „ The plasmid shows no difference from the plasmids isolated from the S. 20 lividans. strains, but the DNA work-up from E„ coli is more productive and possible in a shorter time-E xamp le 10 The process is carried out in accordance with Example 9, but the plasmid pIJ 350 is used in place of 25 the plasmid pIJ 102, it then being possible additionally to select for thiost repton resistance in S. lividans, an d the plasmids pSA 350 a and b are obtained* Figure 10 shows the plasmid pSA 350 a. In shake cultures, this plasmid leads to higher yields of tendamistat than does 30 the plasmid pSA 350 b which contains the insertion with the tendamistat gene in the reverse orientation,. In contrast, the reduction in size of the insertion to 1»5 kb has no significant effect on the formation of product. Example 11 35 To determine the structure and nucleotide sequence of the tendamistat gene, the 0,-94 kb Pst I/Bam HI sub-fragment (Figures 2a and b) and 295 bp Sau 3a/Bam HI sub-fragment (Figure 2c) were cloned in the single-strand phages H 13 mp 8 and M 13 mp 9. The primer used for the - 12 - dideoxy sequencing reaction was the 20 nucleotide f>Un sequence A and a commercially available 15 bp primer (Bethesda Research Laboratories GmbH,,, IMeu-Isenburg) . The DNA sequence C was found. 5 Example 12 Upstream of the structural gene of tendamistat,, there is on the DNA an open reading frame up to the start codon ATG (Het) for a protein which is located immediately upstream of the amino terminal end of tendamistat. This 10 signal peptide corresponds to DNA sequence B.

Appendix: DNA sequence C (coding strand) 5 10 5'-GAC ACG ACC GTC TCC GAG CCC GCA CCC TCC TGC GTG NHg-Asp Thr Thr Val Ser Glu Pro Ala Pre Ser Cys Val 15 20 ACG CTC TAC CAG AGC TGG CGG TAC TCA CAG GCC GAC Thr Leu Tyr Gin Ser Trp Arg Tyr Ser Gin Ala Asp 25 30 35 AAC GGC TGT GCC GAG ACG GTG ACC GTG AAG GTC GTC Asn Gly Cys Ala Glu Thr Val Thr Val Lys Val Val HO H 5 TAG GAG GAC GAC ACC GAA GGC CTG TGC TAC GCC GTC Tyr Glu Asp Asp Thr Glu Gly Leu Cys Tyr Ala Val 5 0 55 60 GCA CCG GGC CAG ATC ACC ACC GTC GGC GAC GGC TAC Ala Pro Gly Gin lie Thr Thr Val Gly Asp Gly Tyr 6 5 7 0 ATC GGC TCG CAC GGC CAC GCG CGC TAC CTG GCT CGC He Gly Ser His Gly His Ala Arc Tyr Leu Ala Arg TGC CTT TAG-3' Cys Leu Stp

Claims (9)

1. 1. -13- CLA IMS: 1. DS^A sequence B obtainable frotn Streptornyces tendae strains,, which produce tendatni stat and have preferably been treated with sublethal doses of acriflavine, by isolation of the b complete DNA,. digestion with Pst I, Southern hybridisation with the DNA sequence A, 5'~(32P-)CCT TCA GTG TCG TCT TCG TA-3' CA) isolation of the 2-3 kb Pst I fragment^, cutting with BamHI,. Southern hybridisation with the sequence isolation of 10 the 0.94 kb PstX BamHI subf ragment, cutting with Sau 3a, Southern hybridisation with the sequence A, isolation of the 0,525 kb BamHI Sau 3a subf ragment and sequencing of the DNA, and which has the following features: a) it is located immediately upstream of the tendami stat 15 structural gene, b) it codes at the amino terminal end for Met-Arg-Val-Arg-Ala-Leu-Arg, c) it codes at the ca rboxy terminal end for Ala-Ser-Ala end 20 d) it codes in the middle for a hydrophobic region which comprises 10 to 25, preferably 17 to 20., amino acids.

2. A peptide of the formula Me t-A'rg-Val-Arg-Ala-Leu-Arg-X-Ala-Ser-Ala-R in which R denotes hydroxy or the residue of a genetically 25 codable peptide, and X represents the hydrophobic region defined in Claim 1.

3. a gene structure containing the DNA sequence B defined in Claim 1.

4. a gene structure as claimed in claim 3, containing the DNA sequence B in the reading frame with a struc-tural gene.

5. A gene structure as claimed in claim 3 or 4, wherein the structural gene codes for tendani stat - 6_ a gene structure as claimed in claim 5, havi ng DNA sequence C - 35 7_ a plason d having e DNA sequence as claimed in any one of claims 1 or 3 to

6. 8„ a plasencl a© c laiaed i n c I a "3 tn having a replicon effective in St reptomycetes. 9„ A plasmid as claimed in claim 7 or 8^ hawing a replicon effective in E. coli, 10„ A host organism belonging to the genus Streptornyces containing a plasmid as claimed in any one of claims 7 to 9. 11. A host organism as claimed in claim 10^, belonging to the species S„ tendae or S. lividans. 12. A process for the preparation of a poly peptide which comprises using a host organism as claimed in claim 10 or 11 , which contains a signal peptidase which detaches the prepeptide corresponding to the DUh sequence B. 13. A process as claimed in Claim 12, wherein the polypeptide is, after detachment of "the prepeptide, released from the cell. 14- A process as claimed in claim 12 or 13, wherein the host organism used has a plasmid which contains a gene structure as claimed in any one of claims 3 to 6. 15. DMA sequence B as claimed in Claim 1, substantially as hereinbefore described and exempli£led. 16. A peptide of the formula given and defined in Claim 2, substantially as hereinbefore described and exemplified. 1

7. A gene structure as claimed in Claim 3, substantially as hereinbefore described and exemplified. 1

8. A plasmid as claimed in Claim 7, substantially as hereinbefore described. 1

9. A host organism as claimed in Claim 10, substantially as hereinbefore described. 20. A process as claimed in Claim 12 for the preparation of a polypeptide, substantially as hereinbefore described and exemplified. 21. A polypeptide whenever prepared by a process claimed in a preceding claim. F. R. KELLY a CO., AGENTS FOR THE APPLICANTS.