AU519528B2 - Purification of nucleotide sequences - Google Patents

Description

i Pi~cAoN A PATENT lot 3648 8- aI) ffere insert (in K1 THE REGENTS OF THE UNIVERSITY OF;CAIFONI full) Name or Names of A1pplicant or (eana educational institution chartered by. lkpplicants, followed by Address (es). the State of California) of 2.200 Universit~y

California 94720,UNITED STATES OF AMERICA

Here hereby apply for the grant of a Patent for tun invention entitled, insert Title of Invention. "PURIFICATION OF NUCLEOTIDE SEQUENCES .SUITTABLE

EXPRESSION IN BACTERIA" ATwC)w ACCED~L AND "EN~t"

(n)mHere(ar which is described in the accompanying complete specification. This application is a of basic appicaions) Convention application and is based on the application numbered 836,218 (4eoinsert

basi UNIED SATESOF AMERICA Csurtry or(4 CountrIes, an for a pa e t or sim ilar d n ST A TESm basMi date or aen rtcio aei orto Z i n 23rd Septem ber 9 7_7

2 1 AY K4 i

mx address for service is Messrs. EdIwd. Waters Sons, Patent Attorneys,

Ou-r

Queen Street, Melbourne, Victoria, Austialia.

DATED this 24th day Y

THE REGENTS OF THE UNIVERSITY OF' CALIFORNIA

tureSigna.- of Applicant (s) or Seal of Company and Signatures of by: 7 its Oficers as prescribed by its Articles of Association. F. Dancer)

Reg'd. Patent Attorney THE COMMISSIONER OF PATFNTS.

Edwd. Wsters Sons. Melbourne,

t

PURIFICATION OF NUCILEOTIDE SEQUENCES SUITABLE FOR EXPRESSION IN BACTERIA

The U.S. Government has rights in this invention pursuant

DECLARATION IN SUPPORT OF CONYENTIQN APPL'CATION FOR A PATENT OR PATENT OF ADDITION

(The dcclaration shall be made by the applicant, or, i/ the applicant is a body corpor- ate, by a person azahorized by the body corporate to make the declaration on iW beh~1f.)

Ina support of the Zoavention Application made for Ai patent an invention entitled "PURIFICATION OF NUCLEOTlDE SEQUENCES SUITABLE FOR EXPRESSION IN BACTERIA" Insert full name(s) Elizabeth 0. Hansen and addre'ss(es) of I declarant(s). of220O Universiity Avenue, Berkeley, California 94720, United States of Americal

3648 9/78 do solemnly and sincerely declare as follows:- Delete the words which are not applicable. Iriert full name of or I am authorized by THE REGENTS -OF THE UNIVERSITY OF CALIFORNIA, applicant Company. 2200 University Avenue, City of Berkely, St-ate of California, United States of America the appiaL.. for the patent, RR". 4A~~mlomake this declaration on its behalf. 2. The basic application.K.... as defined by Section 141 of the Act li was Insert country and i~.h~.X~xit.d the..2 .3.Se date of filing of each ap- plication on which priority by Howard Michael Goodman, Peter Horst See urg John Shine is based.

Insert full name of appli. cant in each basic application.

leta &e.,words~.which,_

Ciyof San Francisco, State of California, John Shine, '7 ,gilies St., Curtin,-A.C.T. 2605, Australia are are i actual inventor-s.-. of the invention and -the facts upon which the applicarL....

'~entitled to make the application are as follows:- State mnanner in which applicant(s) derive title The applicant company is assignee of the inventir(S) from actual inientor(s).

4, The basic application referred to in paragraph 2 of this Declaration gmjwas

*~Jw rst apiicti~rL.....: maa VCuineLjin COUnTIry in respecit of i1heiiifiiiiif the subject of the application.

Siznature(s) of Declared atBerkeleX Calif this 23rd day of June, 1978 declarant(a). Il (inaftresto r e THE REGENTS OF THE UNIVERSITY OF CALIFORNIA

lo Injitial &UI AltcicI06.I Q~r AS8S T' NTr00.. 8 LL0'"i T ASSOCIATE SECRETA TO: OF THE UNIVERSITY OF CAL FORIA T't~ C.mnrnLsior'er of Patents

only in mi~aiscule amounts. Often, the source organism cannuot be obtained in quantities sufficient to provide an adeauate amount of the desired -or,-ein. Cn qety Consequently, it

(12) PATENT ABRIDGMENT (19) AU (11) AU-B1-36 489/78

(54) PURIFICATION OF NUCLEOTIDE SEQUENCES (71) THE REGENTS OF THE UNIVERSITY OF CALIFORNIA (21) 36 489/78 519 528 (22) 25.5.78 (23) 25.5.78 (24) 23.9.77 (31) 836218 (32) 23.9.77 (33) US (43) 29.11.79 (44) 10.12.81 (51) 2 C12K 1/00 C07G 17/00 C07H 21/04 (72) Goodman, Shine, and Seeburg, P.H. (74) WM (56) 36488/78 C12N (57) Claim 1. A method for purifying a fragment of a specific deoxyribonucleotide sequence for recombination with a DNA transfer vector, and transfer to a microorganism, from a population of polyribonucleotides heterogeneous in length and sequence, comprising the steps of:

providing a population of cDNA transcripts of the polyribonucleotides, at least a portion of said cDNA transcripts having at least two restriction sites, subjecting the cDNA transcripts to the action of a restriction endonuclease preparation capable of catalyzing the hydrolysis of the cDNA transcripts at each of the two restriction sites in order to produce a fragment of a specific deoxyribonucleotide sequence which is homogeneous in length, fractionating the fragments produced by restriction endonuclease action according to their length, thereby separating the homogeneous length fragments of the specific deoxyribonucleotide sequence from cDNA transcripts of different length, whereb. a fragment of the specific deoxyribonucleotide sequence is pu ified for recombination with a DNA transfer vector and transfer to a microorganism.

Di 46*3

*113/11.,

:1

Form COMMONWEALTH OF AUSTRALIA

PATENTS ACT 1952-69

COMPLETE SPECIFICATION (ORIGINAL)

3648 9/78 Application Number: 36489/78 Lodged: 25th May 1978

Complete Specification Lodged: Accepted: Published: Priority

Related Art: i.!

4;

Name of Applicant: THE REGENTS OF THE UNIVERSITY OF CALIFORNIA

Address of Applicant: 2200 University Avenue, Berkeley, California 94720, United States of America

Actual Inventor: HOWARD MICHAEL GOODMAN, JOHN SHINE and PETER HORST SEEBURG

EDWD. WATERS SONS, Address for Service: 50 QUEEN STREET, MELBOURNE, AUSTRALIA, 3000.

Complete Specification for the invention entitled:

"PURIFICATION OF NUCLEOTIDE SEQUENCES SUITABLE FOR EXPRESSION IN BACTERIA"

The following statement is a full description of this invention, including the b(est method of performing it known to US

j i u4 uncT6 as prescribed by siArtiole. o F. Dancer)

Reg'd. Patent Attorny To: THE COMMISSIONER OF PATENTS.

Edwd. Waters Sons, Melbourne,

PURIFICATION OF NUCLEOTIDE SEQUENCES SUITABLE FOR EXPRESSION IN BACTERIA

The U.S. Government has rights in this invention pursuant

to Grants No. AM-18878 and CA 14026 awarded by the

Department of Health, Education and Welfare.

BACKGROUND OF THE INVENTION

C0 Proteins and peptides Ere synthesized in almost endless

variety by living organisms. Many have proven to have

C medical, agricultural or industrial utility. Some proteins

are enzymes, useful as specific catalysts for complex

chemical reactions. Others function as hormones, which

act to affect the growth or development of an organism or

to affect the function or specific tissues in medically

significant ways. Specific binding proteins may have

commercial significance for the isolation and purification

of trace substances and for the removal of contaminating

substances. Both proteins and peptides are composed of

linear chains of amino acids, the latter term being applied

to short, single-chain sequences, the former referring to

long-chain and multi-chain substances. The principles of

the present invention apply equally to both proteins and

ieptides.

Proteins and peptides are generally high molecular weight

substances, each having a specific sequence of amino acids.

Except for the smaller peptides, chemical synthesis of

peptides and proteins is frequently impractical, costly

and time consuming, if not impossible. In the majority

of instances, in order to make practical use of a desired

protein, it must first be isolated from the organism which

makes it. Frequently, the desired protein is present

-1A-

I

Zuojec i tne m appLLca.In.

Signature(.) of Declared atBerkeley, Calif this 23rd day of June, 1978 declara t(s) U. S C i S(o attetationNorA TO F^ M signature i THE REGENTS OF THE UNIVERSIrTY OF CALIFORNIA

Tit6 allJ 7 B B'oU: SSO9T:ANT~ CCOFGt. f T!r Ns ASSOCIATE SECRETARY TO: OF THE UNIVERSITY OF CALIFORNIA The Cnmmrnioper of Patents

only in miniscule amounts. Often, the source organism

cannot be obtained i quantities sufficient to provide

an adequate amount of the desired protein. Consequently,

many potential agricultural, industrial and medical

applications ior specific proteins are known, but remain co Ct undeveloped simply because an adequate supply of the desired.

Co protein or peptide does not exist. Recently developed techniques have made it possible to

c employ micro-organisms, capable of rapid and abundant

growth, for the synthesis of commercially useful proteins

and peptides, regardless of their source in nature. These

techniques make it possible to genetically erdow a suitable

microorganism with the ability to synthesize a protein or

peptide normally made by another organism. The technique

makes use of a fundamental relationship which exists in

all living organisms between the genetic material, usually

DNA, and the proteins rynthesized by the organism. This

relationship is such that the amino acid sequence of the

protein is reflected in the nucleotide sequence of the

DNA. Thereare jne or more trinucleotide sequence groups

specifically related to each of the twenty amino acids most

commonly occuring in proteins. The specific relationship

between each given trinucleotide sequence 'and its corres-

ponding amino acid constitutes the genetic code, The

genetic code is believed to be the same or similar for all

living organisms. As a consequence, the amino acid

sequence of every protein or peptide is reflected by a

corresponding nucleotide sequence, according to a well

understood relationship. Furthermore, this sequence of

nucleotides can, in principle, be translated by any living

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I: 1j1 organisms.

TABLE I

Genetic Code

Phenyl al aine (The) TTK istidine(His') CAK Ieucine(Leu) XTY Glut amine(Gin) CAJ Isoleucine(Ilie) ATM Asparagine Asn) AAK tZ CO~ Methionine(Met) ATG Lysine(Lys) AAJ Valine(Val) GTL Aspartic acid(Asp GAK Serine(Ser) QRS Glutamic acid(Gu) GAJ C. Proline(Pro) CCL Cysteine(Cys) TGK Threonine(Thr) ACL Tryptophan(Try) TGG Alanine(Ala) GCL Arginine(Arg) WGZ Tyrosine(Tyr) TAK Glycine (Gly) C-GL Termination signal TAJ Termination signal TGA

Key: Each 3-letter triplet represents a trinucleotide of

DNA, having a 5' end on the left and a 3' end on the right.

The letters stand for the purine or pyrimidine bases forming

the nucleotide sequence.

A adenine J A or G G guasnine K T or C C cytosine L A, T, C or G T thymine M A, C or T X T or C if Y is A or G X C if Y is C or T Y A, G, C or T if X is C Y A or G if X is T W C or A if Z is A or G Z A, G, C or T if W is C Z A or G if W is A W C if Z is C or T

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A

QR TO if S is A, G, C or T QR AG if S is T or C S A, G, C or T if QR is TC S T or C if QR is AG

The trinucleotides of Table I, termed codons, are

presented as DNA trinucleotides, as they exist in the

genetic material of a living organism. Expression of Co

these codons in protein synthesis requires the intermediate

formation of messenger RNA (mRNA), as described more fully, CYZ infra. The mRNA codons have the same sequences as the DNA

codons of Table I, except that uracil is found in place of

thymine. Complementary trinucleotide DNA sequences having

opposite strand polarity are functionally equivalent to the

codons of Table I, as is understood in the art. An

important and well known feature of the genetic code is its

redundancy, whereby, for most of the amino acids used to make

proteins, more than one coding nucleotide triplet may be

employed. Therefore, a number of different nucleotide

sequences may code for a given amino acid sequence. Such

nucleotide sequences are considered functionally equivalent

since they canl result in the production of the same amino

acid sequence in all organisms, although certain strains

may translate some sequences more efficiently than they

do others. Occasionally, a methylated variant of a

purine or pyrimidine may be found in a given nucleotide

sequence. Such methylations do not affect the coding

relationship in any way.

In its basic outline, a method of endowing a microorganism

with the ability to synthesize a new protein involves three

general steps: isolation and purification of the specific gene cr nucleotide sequence containing the genetically coded information for the amino acid sequence of the desired protein, recombination of the isolated nucleotide sequence with an appropriate transfer vector,

ZZ typically the DNA of a bacteriophage or plasmid, and (3) transfer of the vector to the appropriate microorganism. __and selection of a strain of the recipient microorganism i

Scontaining the desired genetic information. A fundamental difficulty encountered in attempts to exploit commercially the above-described general process lies in the first step, the isolation and purification of the desired specific genetic information. DNA exists irL a~l living cells in the f orm of extremely high molecular weighat chains of nucleotides. A cell may contain more than 10,000 stru~ctural genes, coding for the amino acid sequences of over 10,000 specific proteins, each gene havinLg a sequence many hundreds of nucleotides in length. For the most part, four different nucleotide bases make up all the existing sequences. These are adenine guanine cytosine and thymine The long sequences comprising the structural genes of specific proteins are consequently very similar in overall chemical- composition and physical properties. The separation of one such sequence from the plethora of other sequences present in isolated DNA cannot ordinarily be accomplished by conventional physical and chemical preparative methods. M~essenger RlNA fun'3tions in the process of' converting the nucleotide sequence information of DNA into the amino acid sequence structure of a.protein. In the first step of ti- process, termed transcription, a local segment of

DNA having a nucleotide sequence which specifies a protein to be made, is first copied into RNA. RN~A is a polynucleotide similar to DNA except that ribose is substituted for deoxyribose and uracil is used in place of thymine. The nucleotide bases in RN~'A are capable of Sentering into the same-kind of base pairing relationships Sthat are known to exist between the complementary strands of DNA. A and U are complementary, and G and C arG complementary. The RNIA transcript of a DNA nucleotide sequence wiLll be complementary to the copied sequence. Such

RN~A is termed messenger RlNA (miNA) because of its status as intermediary between the genetic apparatus of the cell and its protein synthesizing apparatus. Generally, the only mRNA sequences present in the cell at any given time are those which correspond to proteins beiJ-g actively

syi aiesized at that time. Therefore, a -ifferentiated cell whose function is devoted primarily to the synthesis of a single protein will contain primarily the RNA species corresponding to that protein. In those instances where it is feasible, the isolation and purification cf the

appropriate nucieotide sequence cerding for a given protein can be accomplished by isolating mPRNA coding for the specializedL synthesis of such protein in differentiated cells. A major disadvantage of the foregoing procedure is that it is applicable only in the relatively rare instances where

cells can be found engaged in synthesizing primarily a single protein. The majority of proteins of commercial interest are not synthesized in such a specialized way. The desired proteins may be one of a hundred or so different

proteins being produced by the cells of a tissue or organism at a given time. Nevertheless, 'the mRNA isolation technique is potentially -useful since the set of RNA species present in the cell usually represents only a fraction of the total sequences existing in the DNA, and thus provides an initial purification. In order -to take advantage of such purification, however, a method is needed whereby sequences present in low frequencies, such as a few percent, can be isolated in high purity. The present invention provides a process whereby nucleotide sequences can be isolated and purified even when present at a frequency as low as 2% of a heterogeneous population of m:RNA sequences. Furthermore, the method may be combined with known methods of fractionating mRNA to isolate and puri~y sequences present in even lower frequ6.acy in the total RNA population as initially isolated. The method is generally applicable to mRNA species extracted from virtually any organism and is therefore expected to provide a powerful basic tool for th. ultimate production of proteins of comnercial and research interest, in useful quantities. Human growth hormone has medical utility in the treatment of defective pituitary function. Animal growth hormones have commercial utility in vetberinary medicine and in agriculture, particularly in the case of animals used as food sources, where large size and rapid maturation are desirable attributes. Human chorionic somatomainmotropin is of medical significance because of its role in the fetal maturation process. The process of the present invention takes advantage of certain structural features of mRNBYA and DNA, and makes use of certain enzyme catalyzed reactions. The nature of

C. I these reactions and structural details as they are understood in the prior art are described herewith. The symbols ani. abbreviations used herein are set forth in the following table:

Co TABLE 2 SDNA deoxyribonucleic acid A Adenine C0 RNA ribonucleic acid T Thymine cDNA complementary DNA G Guanine (enzymatically synthesized) C Cytosine from an mRNA sequence) U Uracil mRNA messenger RNA Tris 2-Amino-2-hydroxyethyl- dATP deoxyadenosine 1,3-propanediol triphosphate EDTA ethylenediamine dGTP deoxyguanosine tetraacetic acid triphosphate ATP adenosine triphosphate dCTP deoxycytidine dTTP thymidine triphosphate triphosphate HCS Human Chorionic Somato- mammotropin TCA Trichloroacetic acid HGH Human Growth Hormone

As a consequence of the known base-pairing relationships

that govern DNA replication and transcription, isolating

the mRNA which contains the nucleotide secuence coding for

the amino acid sequence of a particular protein is equivalent

to the isolation of the same sequence, or gene, from the

DNA itself. If thp mRNA is retranscribed to form DNA

complementary thereto (cDNA), the exact DNA sequence is

thereby reconstituted and can, by appropriate techniques,

be inserted into ,he genetic material of another organism.

The two complementary versions of a given sequence are

-8- .i i: therefore interconvertible, and functionally equivalent to each other. The nucleotide subunits of DNA and RNA are linked together by ihosphodiester bonds between the 5' position of on Co nucleotide sugar and the 3' position of its next neighbor. 00 Reiteration of such linkages produces a linear polynucleotide cM which has polarity in the sense that one end can be distinguished from the other. The 3' end may have a free 3'-hydroxyl, or the hydroxyl may be substituted with a phosphate or a more complex structure. The same is true of the 5' end. In eucaryotic organisms/ thcee having a nuclcua and mit.ti..aaratu.s d.fi the synthesis of functional mRNA usually includes the addition of polyadenylic acid to the 3' end of the mRNA. Messenger RNA can therefore be separated from other classes of RNA isolated from an eucaryotic organism by column chromatography on cellulose to which is attached polythymidylic acid. See Aviv, and Leder, Proc. Nat. Acad. Sci. U.S.A, 69. 1408 (1972). Other chromatographic methods, exploiting the base-pairing affinity of poly A for chromatographic packing materials containing oligo dT, poly U, or combinations of poly T and poly U, for example, poly U-Sepharose, are likewise suitable. Reverse transcriptase catalyzes the synthesis of DNA complementary to an RNA template strand in the presence of the RITA template, a primer which may be any complementary oligo or polynucleotide having a 3'-hydroxyl, and the four deoxynucleoside triphosphates, dATP, dGTP, dCTP, and dTTP. The reaction is inititated by the non-covalent association of the oligo-deoxynucleotide primer near the 3' end of mRNA followed by stepwise addition of the appropriate

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N, S ' fe deoxynucleotides, as determined by base-pairing relationships with the mRNA nucleotide sequence, to the 3' end of the growing chain. The product molecule may be described as a hairpin structure in which the original RNA is paired by hydrogen bonding with a complementary strand of DNA partly folded back upon itself at one end. The DNA and RNA strands are not covalently joined to each other. Reverse transcriptase is also capable of catalyzing a similar reaction using a single-stranded DNA hairpin having a loop of single- stranded DNA joining one set of ends. See Avid, H. and Leder, Proc. Nat'l. Acad. Sci. U.S.A. 69, 1408 (1972) and Efstratiadis, Kafatos, F.C. Maxam, and

Maniatis, Cell 7, 279 (1976). Restriction endonucleases are enzymes capable of hydrolyzing phosphodiester bonds in DNA, thereby creating a break in the continuity of the DNA strand. If the DNA is in the form of a closed loop, the loop is converted to a linear structure. The principal feature of a restriction enzyme is that its hydrolytic action is exerted only at a point where a specific nucleotide sequence occurs. Such a sequence is termed the restriction site for the restriction endonuclease. Restrict- ion endonucleases from a variety of sources have been isolated and characterized in terms of the nucleotide sequences of their restriction sites. When acting on double-stranded DNA, some restriction endonucleases hydrolyze the phospho- diester bonds on both strands at the same point, producing blun, ends. Others catalyze hydrolysis of bonds separated by a few nucleotides from each other, producing free single- stranded regions at each end of the cleaved molecule. Such single-stranded ends are self-complementary, hence cohesive,

0<

U I- and may be used to rejoin the hydrolyzed DNA. Since any DNA susceptible to cleavage by such an enzyme must contain the same recognition site, the same cohesive ends will be produced, so that it is possible to joint heterogen- eous sequences of DNA which have been treated with restrict- ion endonuclease to other sequences similarly treated. See

Roberts, Crit.. Rev. Biochem 4, 123 (1976). I0 It has been observed that restriction sites for a given

enzyme are relatively rare and are nonuniform]r distributed.

Whether a specific restriction site exists within a given

segment is E. matter which must be empirically determined.

However, there is a large and growing number of restriction

endonucleases, isolated from a variety of sources with

varied site specificity, so that there is a reasonable

probability that a given segment of a thousand nucleotides

will contain one or more restriction sites.

SUMMARY OF INVENTION

A novel purification procedure for cDNA of desired nucleotide

scquence complementary to an individual mRNA species is,

disclosed. The method employs restriction endonuclease

cleavage of cDNA transcribed from a complex mixture of mRNA.

The method does not requi5.e any extensive purification of

RNA but instead makes use 'of traiscription of RNA into cDNA,

the sequence specific fragmentation of this cDNA with one

or two restriction endonucleases, and the fractionation of

the cDNA restriction fragments on the basis of their length,

The use of restriction endonucleases eliminates size

heterogeneity and produces homogeneous length DNA fragments

from any cDNA, species which contains at least two restriction

sites. From the initially heterogeneous population of

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t lit cEPA transcripts, uniform size fragments of desired sequence are produced. The fragments may be several hundred nucleotides in length and may in some instances include the entire structural gene for the desired protein. oa The length of the fragments depends on the number of nucleo- 0- tides separating the restriction sites and will usually be

^y different for different regions of DNA. Frectionation by length enables purification nf a homogeneous population of fragments having the desired sequence. The fragments will be homogeneous in size and highly pure in terms of nucleo- tide sequence. Current separation and analysis methods enable the isolation of such fragments from a corresponding mRNA species representing at least 2% of the mass of the RNA transcribed. The use of prior art RNA fractionation methods to prepurify the mRNA before transcription will result in

lowering the actual lower limit of detection to less than 2% of the total mRNA isolated from the organism. Specific sequences purified by the procedure outlined above may be further purified by a second specific cleavage with a restriction endonuclease capable of cleaving the desired sequence at an internal site. This cleavage results in formation of two sub-fragments of the desired sequence, separable on the basis of the-L lengths. The sub-fragment. are separated from uncleaved and specifically cleaved contaminating sequences having substantially the same original size. The method is founded upon the rarity and rc :domness of placement of restriction endonuclease recogni- tion sites, which results in an extremely low probability that a contaminant having the same original length will be cleaved by the same enzyme to yield fragments having the same length as those yielded by the desired sequence. After separation from the contaminants, the sub-fragments of the desired sequence may be rejoined using techniques knowvn.in the art, to reconstitute the original sequence. The two sub-fragments must be prevented from joining together in the reverse ord.er of their original sequence. A method is

Sa D disclosed whereby the sub-fragments can only join to each

other in the proper order.

Variations of the above-recited methods may be used in

combination with the appropriate labelling techniques to

obtain accurate, quantitative measurements of the purity of

the isolated sequences. The combined techniques have been

applied to produce a known nucleotide sequence with greater

than 99% purity.

The cDNA isolated and purified by the described methods may

be recombined with a suitable transfer vector and transferred

to a itable host microorganism by known methods. Novel

plasmids have been produced, containing the nu.-eotide

sequences coding for the major portions of human chorionic

somatomammotropin and human growth hormone, respectively.

Novel microorganisms have been produced having as part of

their genetic makeup the major portion of HCS and the major

portion of HGH, respectively. T.e disclosed techniques

may be used for the isolation and purification of growth

hormones from other animal species and for the construction

of novel transfer vectors and microorganisms containing

these genes.

DETAILED DESCRIPTION OF THE INVENTION

The prep -t invention employs as starting material polyadeny-

lated, .e or partially purified messenger RNA, which may

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electrophoresis runs of P-labeled cDNA, as described

:n detail in example 1.

Figure 2 is a schematic representation of the be heterogeneous in sequence and in molecular size. The selectivity of the P A isolation procedure is enhanced by any method which results in an enrichment of the de%~i:iL a mRNA in the heterodisperse population of mRNA isolated. Any such prepurification method may be employed in conjunuti.- with the method of the present invention, provided the method does not introduce endonucleolytic cleavage of the mRNA. An important initial consideration is the selectio:a of an appropriate source tissue for the desired mRNA. Often, this choice will be dictated by the fact that the protein ulti- mately to be produced is only made by a certain specialized tissue of a differentiated organism. Such is the case, for example, with the peptide hormones, such as growth hormone or HCS. In other cases, it will be found that a variety of cell types or microbial species can serve as a source of the desired mRNA. In those cases, some preliminary experiment- a't ion will be necessary in order to determine the optimal source. Frequently, it will be found that the proportion of desired mRNA can be increased by taking advantage of cellular respones to enviroiimental stimuli. For example, treatment with a hormone may cause increased production of the desired mRNA. Other techniques include growth at a particular temperature and exposure to a specific nutrient or other chemical substance.

Prepurification to enrich for desired mRNA sequences may

also be carried out using conventional methods for fraction-

ating RNA, after its isolation from the cell. Any techn-

ique which does not result in degradation of the RNA may

be employed. The techniques of preparative sedimentation

in a sucrose gradient and gel electrophoresis are especially

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California) at 27,000 rpm for 16 hr. at 15 0 C. Fullerton,

Crkvenjakov, and Ryus, Biochem, 13, (G-isin, or C (Qai7A After centrifuaation, the tube contents suitable.

The mRNA must be isolated from the Sourse cells under

conditions which preclude degradation of the mRNA. The

action of RNase enzymes is particularly to be avoided

because these enzymes are capable of hydrolytic cleavage

of the RNA nucleotide sequence. The hydrolysis of one

bond in the sequence results in disruption of that sequence

and loss of the RNA fragment containing the original 5' end

of the sequence. A suitable method for inhibiting RNase

duing extraction from cells is disclosed in/- Sy application

/8 ,23-r incorporated herein by reference, assigned to the

same assignee as the instant application. The method

involves the use of 4N guanidiaium thiocyanate and 1 M

mercapt o etaanol during the cell disruption step. In addition,

a low tempe:.-atire and a pH near 5.0 are helpful in further

reducing RNase degradation of the isolated RNA.

Prior to application of the method of the present invention

mRNA must be prepared essentially free of contaminating

protein, DNA, polysaccarides and lipids. Standard methods

are well known in the art for accomplishing such purification.

RNA thus isolated contains non-messenger as well as messenger

RNA. A convenient method for separating the mRNA of eucar-

yotes is chromatography on columns of oligo-dT cellulose, or

other oligonucleotide-substituted column material such as

poly U-Sepharose, taking advantage of the hydrogen bonding

specificity conferred by the presence of polyadenylic acid

on the 3' end of eucaryotLic mRNA.

The initial step in theprocess of the present invention

is the formation of DNA complementary to the isolated

heterogeneous sequences of mRnRA. The enzyme of choice

N.

Academic Press, N.Y. (1974), p. 150. Reactions were started by the addition of enzyme at 0°0C and synthesis was for 6 min at 420C. Under these conditions approximately for this reaction is reverse transcriptase, although in principle any enzymj capable of forming a faithful comple- mentary DNA copy of the mRNA template could be used. The reaction may be carried out under conditions described in the

Co prior art, using mRNA as a template and a mixture of the four

Co deoxynucleoside triphosphates dATP, dGTP, dCTP and dTTP, as O 4 precursors for the DNA strand. It is convenient to provide

that one of the deoxynucleoside triphosphates be labeled CYD with a radioisotope, for example 32p in the alpha position,

in order to monitor the course of the reaction, to provide

a tag for recovering the product after separation procedures

such as chromatography and electrophoresis, and for the pur-

pose of making quantitative estimates for recovery. See

Efstratiadis, et al. supra.

The cDNA transcripts produced by the reverse transcriptase

reaction are somewhat heterogeneous with respect to sequences

at the 5' end and the 3' end due to variations in the initia-

tion and termination points of individual transcripts, relative

to the mRNA template. The variability at the 5' end is

thought to be due to the fact that the oligo-dT primer used

to initiate synthesis is capable of binding at a variety of

loci along the polyadenylated region of the mRNA. Synthesis

of the cDNA transcript begins at an indeterminate point in

the poly-A region, and a variable length of poly-A region is

trascribed depending on the initial binding site of the oligo-

dT primer. It is possible to avoid this indeterminacy by the

use of a primer containing, in addition to an oligo-dT tract,

one or two nucleotides of the RNA sequence itself, thereby

producing a primer which will have a preferred and defined

binding site for initiating the transcription reaction.

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ized by autoradiography of the dry gel.

Figure 1 shows the results of gel electrophoresis and

autoradiography of 3 2 P-labeled cDNA, prepared as described

The indeterminacy at the 3'-end of the cDNA transcript

is due to a variety of factors affecting the reverse

transcriptase reaction, and to the possibility of partial

degradation of the RNA template. The isolation of specific

Ir cDNA transcripts of maximal length is greatly facilitated if

conditions for the reverse transcriptase reaction are chosen CO which not only favor full length synthesis but also repress

Ce the synthesis of small DNA chains. Preferred reaction

conditions for avian myeloblastosis virus reverse transcript-

ase are given in the examples section. The specific para-

meters which may be varied to provide maximal production of

long-chain DNA transcripts of high fidelity are reaction

temperature, salt concentration, amount of enzyme, concen-

tration of primer relative to template, and reaction time.

The conditions of temperature and salt concentration are

chosen so as to optimize specific base-pairing between the

oligo-dT primer and the polyadenylated portion of the RNA

template. Under properly chosen conditions, the primer

will be able to bind at the polyadenylated region of the

RNA template, but non-specific initiation due to primer

binding at other locations on the template, such as short,

A-rich sequences, will be substantially prevented. The

effects of temperature and salt are interdependent. Higher

temperatures and lower salt concentrations decrease the stability ofspecific base-pairing interactions. The reaction

time is kept as short as possible, in order to prevent non-

specific initiations and tominimize the opportunity for

degradation. Reaction times are interrelated with tempera-

ture, lower temperatures requiring longer reaction times.

At 420C., reactions ranging from 1 min. to 10 minutes are

-17-

HhaI produces a fragment of about 470 nucleotides, and HaeIII digestion produces a fragment of a-roximatelv 5r n l on+ suitable. The primer should be present in 50 to 500- fold molar excess over the RNA template and the enzyme should be present in similar molar excess over the RNA template.

The use of excess enzyme and primer enhances initiation

and cDNA chain growth so that long-chain cDNA transcripts CO are produced efficiently within the confines of the short

incubation times.

e In many cases, it will be possible to carry out the remainder of the purification process of the present invention using

single-stranded cDNA sequences transcribed from mRNA. However,

as discussed below, there may be instances in which the

desired restriction enzyme is one which acts only on double-

stranded DNA. In these cases, the cDNA prepared as described above may be used as a template for the synthesis of double-

stranded, DNA, using a DNA polymerase such as reverse transcript-

ase and a nuclease capable of hydrolyzing single-stranded

DNA. Methods for preparing double-stranded DNA in this

manner have been described in the prior art. See, for example, Ullrich, Shine, Chirgwin, Pictet, R.,

Tischer, Rutter, and Goodman, Science 196

1313 (1977).

Heterogeneous cDNA, prepared by transcription of heterogen-

eous mRNA sequences, is then treated with one or two restrict-

ion endonucleases. The choice of endonuclease to be used depends in the first instance upon a prior determination

that recognition sites for the enzyme exist in the sequence

of the cDNA to be isolated. The method depends upon the

existance of two such sites. If the sites are identical, a single eilzyme will be sufficient. The desired sequence will be cleaved at both sites, eliminating size heterogeneity

-18-

the difference in the electrophoretic mobility of the band

appearing in Lane B and Lane D of figure 1. The size of DNA fragment A is known exactly from a determination of its

'i T. C I

as far as the desired cDNA sequence is concerned, and

creating a population of molecules, termed fragments,

containing the desired sequence and homogeneous in length.

If the restriction sites are different, two enzymes will be

co required in order to produce the desired homogeneous length rZ fragments. 0O The choice of restriction enzyme(s) capable of producing

an optimal length nucleotide sequence fragment coding for all

or part of the desired protein must be made empirically. If the amino acid sequence of the desired protein is known, it

is possible to compare the nucleotide sequer.e of uniform

length nucleotide fragments produced by restricti.on endonuc-

lease cleavage with the amino acid sequence for which it

codes, using the known relationship of the genetic code common to all forms of life. A complete amino acid sequence

for the desired protein is not necessary, however, since a

reasonably accurate identification may be made on the basis

of a partial sequence. Where the amino acid sequence of

the desired protein is not known, the uniform length polynucleotides produced by restriction endonuclease cleavage

may be used as probes capable of identifying the synthesis

of the desired protein in an appropriate in vitro protein

synthesizing system. Alternatively, the mRNA may be

purified by affinity chromatography. Other techniques

which may be suggested to those skilled in the art will be

appropriate for this purpose.

The number of restriction enzymes suitable for use depends

upon whether single-stranded or double-stranded cDNA is used.

The preferred enzymes are those capable of acting on single- stranded DNA, which is the immediate reaction product of

-19-

Figure 3 shows the results of the experiments. The gels

were run essentially as described in example 1. Size

markers prepared by endonuclease HaeIII cleavage of phage mRNA reverse transcription. The number of restriction enzymes now known to be capable of acting on single-stranded DNA is limited. The enzymes HaeIII, and HhaI and Hin(f)I are presently known to be suitable. In addition, the enzyme MboII may act on single-stranded DNA. Where further study reveals that other restriction enzymes can act on CO single-stranded DNA, such other enzymes may appropriately be Ct included in the list of preferred enzymes. Additional suitable enzymes include those specified for double-stranded cDNA. Such enzymes are not preferred since additional reactions are required in order to produce double-stranded cDNA, providing increased opportunities for the loss of longer sequences and for other losses due to incomplete recovery. The use of double-stranded cDNA presents the additional technical disadvantage that subsequent sequence analysis is more complex and laborious. For these reasons, single-stranded cDNA is preferred, but the use of double- stranded DNA is feasible. The cDNA prepared for restriction endonuclease treat ment Im be raiioactively labeled so that it may be detected after subsequent separation steps. A preferred techn:.que is to incorporate a radioactive label such as 32p in the alpha position of one of the four deoxynucleoside triphos- phate precursors. Highent activity is obtained when the concentration of radioactive precursor is high relative to the concentration of the non-radioactive form. However, the total concentration of any deoxynucleoside triphosphate shouldbe greater than 30j/M, in order to maximize the length of cDNA obtained in the reverse transcriptase reaction. See Efstratiadis, Maniatis, Kafatos., Jeffrey, method of measuring the purity of the isolated sequence.

The purified fragment is demonstrated to be greater than

99% pure. Phiri-Pinar inn n-P W Tf rlTVA

ILI I i II I 1;

and Vournakis, Ce1? 367 (1975). For the

purpose of determining the nucieoaide sequence of cDNA,

the 5' ends may be conveniently labeled with 32p in a reaction catalyzed by the enzyme polynucleotide kinase.

'O See Maxam, and Gilbert, Proc. Nat'l. Acad. Sci. cr USA 74, 560 (1977). 00 .a Fragments which have been produced by the action of a

C_ restriction enzyme or combination of two restriction

enzymes may be separated from each other and from hetero-

disperse sequences lacking recognition sites by any

appropriate technique capable of separating polynucleotides

on the basis of differences in length. Such methods include

a variety of electrophoretic techniques and sedimentation

techniques using an ultracentrifuge. Gel electrophoresis is preferred because it provides the best resolution on the

basis of polynucleotide length. In addition, the method

readily permits quantitative recovery of separated materials.

Convenient gel electrophoresis methods have been described

by Dingman, and Peacock, Biochemistry, 7, 659 (1968), and by Maniastis, Jeffrey, A. and van de Sande, Biochemistry 14, 3787 (1975). Prior to restriction endonuclease treatment, cDNA transcripts

obtained from most sources will be found to be heterodisperse

in length. By the action of a properly chosen restriction endonuclease, or pair of endonucleases, polynucleotide

chains containing the desired sequence will be cleaved at

the respective restriction sites to yield polynucleotide

fragments of uniform length. Upon gel electrophoresis,

these will be observed to form a distinct band. Depending

on the presence or absence of restriction sites on other

-21-

The remaining material corresponding to the 550 base-pair

fragment shown in figure 4(E) was digested with 4 units of

HhaI endonculease in 50,a1 of the same buffer used for dige- iL sequences, other discrete bands may be formed as well, which will most likely be of different length than that of the desired sequence. Therefore, as a consequence of restriction endonuclease action, the gel electrophoresis

CO pattern will reveal the appearance of one or more discrete

CO bards, while the remainder of the cDNA will continue to be

heterodisperse. In the case where the desired cDNA

CO sequence comprises the major polynucleotide species present,

the electrophoresis pattern will reveal that most of the

cDNA is present in the discrete band.

Although it is unlikely that two different sequences will

be cleaved by restriction enzymes to yield fragments of

essentially similar length, a method for determining the

purity of the defined length fragments is desirable.

Sequence analysis of the electrophoresis band may be used

to detect impurities representing 10% or more of the material

in the band. A method for detecting lower levels of

impurities has been developed, as part of the present

invention, founded upon the same general principles applied

in the initial isolation method. The method requires that

the desired nucleotide sequence fragment contain a recogni-

tion site for a restriction endonuclease not employed in

the initial isolation. Treatment of polynucleotide material,

eluted from a gel electrophoresis band, with a restriction

endonuclease capable of acting internally upon the desired

sequence will result in cleavage of the desired sequence

into two sub-fragments, most probably of unequal length.

These sub-fragments upon electrophoresis will form two

discrete bands at positions corresponding to their respective

lengths, the sum of which will equal the length of the

22-

the pattern of the eluted material is shown in figure 4(B).

Figure 4(A) represents the electrophoresis Dattern of 32p_ p !I II U polynucleotide prior to cleavage. Contaminants in the original band that are not susceptible to the restriction enzymes may be expected to migrate to the original position.

Contaminants containing one or more recognition sites for

the enzyme may be expected to yield two or more sub-fragments.

Since the distribution of recognition sites is believed to

be essentially random, the probability that a contaminant

will also yield sub-fragments of the same size as those of

the fragment of desired sequence is extremely The amount of material present in any band of radioactively

labeled polynucleotide can be determined by quantitative

measurement of the amount of radioactivity present in each

band, or by any other appropriate method. A quantitative

measure of the purity of the fragments of desired sequence can be obtained by comparing the relative amounts of material

present in those bands representing sub-fragments of the

desired sequence with the total amount of material.

Following the foregoing separation, the desired sequence

may be reconstituted. The enzyme DNA ligase, which catalyzes the end-to-end joining of DNA fragments, may be

employed for this purpose. The gel electrophoresis bands

representing the sub-fragments of the desired sequence may

be separately eluted and combined in the presence of DNA

ligase, under the appropriate conditions. See Sgaramalle, Van de Sande, and Khorana, Proc. Natl. Acad.

Sci. USA 67, 1468 (1970). Where the sequences to be joined

are not blunt-ended, the ligase contained from E. Coli may

be used, Modrich, and Lehman, J. Biol. Chem.

245, 3626 (1970). The efficiency of reconstituting the original sequence from

-22- A

cleavage products and by quantitation of the distribution

of radioactivity in each of the two restriction endonuclease

digests. Such measurements reveal that the purified human

TI-

sub-fragments produced by restriction endonuclease treat-

ment will be greatly enhanced by the use of a method for

preventing reconstitution in improper sequence. This unwanted result is prevented by treatment of the homogenous

EZ length cDNA fragment of desired sequence with an agent

CO capable of removing the 5'-terminal phosphate groups on

the cDNA prior to cleavage of the homogenous cDNA with a cv restriction endonuclease. The enzyme, alkaline phosphatase, is preferred. The 5'-terminal phosphate groups are a

structural prerequisite for the subsequent joining action of DNA ligase used to reconstitute the cleaved sub-fragments.

Therefore, ends which lack a 5'-terminal phosphate cannot

be covalently joined. The DNA sub-fragments can only be joined at the ends containing a 5'-phosphate generated by the restriction endonuclease cleavage performed on the

isolated DNA fragments. The method is essentially that described in detail in U.S. application 805,023.

The majority of cDNA transcripts, under the conditions employed, are derived from the mRNA region containing the 5'-end of the mRNA template by specifically priming on the

same template with a fragment obtained by restriction

endonuclease cleavage. In this way, the above-described

method may be used to obtain not only fragments of specific

nucleotide sequence related to a desired protein, but also the entire nucleotide sequence coding for the protein of

interest.

The purification process is of a special significance in the cloning of human genes, which, under Federal regulations, can only be put into recombinant DNA and then into bacteria after the genes have been very carefully purified, or if

-23-

hours. Digestion with the endonuclease resulted in

cleavage at the EcoRI site of the decamers giving rise to

HCS cDNA with EcoRI cohesive ends as well as cleaved un- the experiments are carried out in special high-risk facilities. See Federal Register, Vol. 41, No. 131, July 7,

1967, pp. 27902-27943. The present method has enabled the

production of sufficiently pure human genes, comprising the

majority of the structure of HCS and HGH. Human genetic

CO material, isolated and purified as described above, may be

ko incorporated into recombinant plasmids or other transfer

vectors. Double stranded chemically synthesized oligon-

ucleotide linkers, containing the recognition sequence for

a restriction endonuclease, may be attached to the ends of

the isolated cDNA, to facilitate subsequent enzymatic temoval

of the human gene portion from the transfer vector DNA. See

Scheller, et cl., Science 196, 177 (1977). The trans-

fer vector DNA is converted from a continuous loop to a

linear form by treatment with an appropriate restriction

endonuclease. The ends thereby formed are treated with

alkaline phosphate to remove 5'-phosphate end groups so

that the transfer vector DNA may not reform a continuous

loop in a DNA ligase reaction without first incorporating a segment of the human DNA. The cDNA, with attached

linker oligonucleotides, and the treated transfer vector

DNA are mixed together with a DNA ligase enzyme, to join

the cDNA to the vector DNA, forming a continuous loop

of recombinant vector DNA having the cDNA incorporated therein. Where a plasmid transfer vector is used, usually

the closed loop will be the only form able to transform

a bacterium. Transformation, as is underatood in the art

and used herein, is the term used to denote the process

whereby a microorganism incorporates extracellular DNA

into its own genetic constitution. Plasmid DNA in the

of the plasmid and prevents self-ligation of the plasmid

DNA, insuring that circle formation and hence transformation

is dependent on the insertion of a DNA fragment containing c *n'hn a *nr m'r1 o~ t- av i" ni -1 M1I1- ^.Tn form a closed loop may be so incorporated under appropriate environmental conditions. The incorporated closed loop plasmid undergoes replication in the transformed cell, and the replicated copies are distributed tc progen/ co cells when cell division occurs. As a reLlt, a new cell line is established, containing the plasmid and carrying the genetic determinants thereof. Transformation by a plasmid in this manner, where the plasmid genes are main- tained in the cell line by plasmid replication, occurs at high frequency when the transforming plasmid DNA is in closed loop form, and does not or rarely occurs if linear plasmid

DNA is used. Once a recombinant transfer vector has been

made, transformation of a suitable microorganism is a

straightforward process, and novel microorganisms strains

containing the human gene may readily be isolated, using

appropriate selection techniques, as understood in the art.

Using the above-described methods of purification and

analysis, a desired nucleotide sequence containing most of

the structural gene for HCS has been isolated and shown to

be greater than 99% pure. The structural ger for HGH has

been isolated to a comparable degree of purity. Novel

plasmids containing the isolated HCS or HGH sequences have

been synthesized. Novel microorganisms containing the

isolated HCS or HGH sequences as part of their genetic

material have beaaproduced.

BRIEF DESCRIPTION OF THE FIGURES

The accompanying figures and drawing are provided to

demonstrate the results obtained in the specific examples

illustrating the invention.

Figure 1 is an autoradiogram of a series of gel

of the same transformation buffer. The cell suspension,

ml, was added to 0.25 ml of diluted ligation reaction

mixture and incubated on ice for 15 minutes, then transferred lated, e or partially purified messenger RNA, which may

-13-

electrophoresis runs of P-labeled cDNA, a described

detail in example 1.

Figure 2 is a schematic representation of the

nucleotide sequence coding for HCS, showing the relative co locati of various restriction sites, as described in Qo detail example 1. s> Figure 3 is an autoradiogram of gel electrophoresis CO 32 results using 3P-labeled cDNA, as described in detail in

example 2.

Figures 4 and 5 are autoradiograms of gel electro- 3 2 P-labeled cDNA, as described in phoresis results using

detail in example 3.

EXAMPLE 1

The general procedure for isolating a sp-i.fic cDNA sequence

has been demonstrated by isolating F sequence comprising a

portion of the coding region for nOS, extracted from placental

tissue.

mRNA Extraction From Placenta

Human term placentas obtained from Caesarean section were

quick-frozen ir liquid nitrogen and stored at -600C. For

extraction of total RNA, 40g of the frozen placental tissue

was broken into small pieces and dissolved with the aid of

a blender in 140 ml of freshly prepared 7 M guanidinium-HC1

(Cox. Methods in Enzymology 12, 120 (1968), 20 mM

Tris-HC1, pH 7.5, 1 mM EDTA, 1% sarcosyl* at 000. After

adding 0.5g CsC1 to each ml, the dark brown solution was

heated at 650 C, for 5 min., quick-cooled in ice, layered

on top of a 5 ml cushion of 5.7 M CsC1, 10 mM Tris-HCl, pH

1 mM EDTA in 1 in. x 3-1/2in. nitrocellulose tubes and

centrifuged in an SW27 rotor (Beckman Instruments Corp.,

-26-

*Trade Mark, Ciba-Geigy, Corp., Greensboro, N/C.

ii the amino acid sequence predicted therefrom on the basis

of the known genetic code. The amino acid sequence'deter- in a sucrose gradient and gel electrophoresis are especially

-14-

I Fullerton, California) at 27,000 rpm for 16 hr. at 150C.

(Glisin, Crkvenjakov, and Ryus, Biochem, 13,

2633 (1974). After centrifugation, the tube contents

owere decanted, the tubes were drained, and the bottom 1/2 cm

3 containing the clear RNA pellet was cit off with a razor OO blade. Pellets were transferred into a sterile erlenmeyer

flask and dissolved in 20 ml 10 mM Tris-H1l, pH 7.5, 1 mM

EDTA, 5% sarcosyl and 5% phenol. The solution was then

made 0.1 M in NaC1 and vigorously shaken with 40 ml of a phenol-50% chloroform mixture. RNA was precipitated from

the aqueous phase with ethanol in the presence of 0.2 M Na-

acetate pH 5.5. RNA pellets were washed with 95% ethanol,

dried, and dissolved in sterile wauer. Usually 40g of

placental tissue yielded about 30mg of RNA from which appro-

ximately 300 ug of polyadenylated RNA was obtained after

twice chromatographing on oligo-dT cellulose. See Aviv,

and Leder, supra.

Synthesis of cDNA.

Analytical reactions were performed in 5,ul containing mM Tris-HC1, pH 8.3; 0.1 mM EDTA; 7 mM MgC1 2 20 mM KC1; 3 2p mM mercaptoethanol; 40 iM dCTP (50,000 cpm per

pmole); 500 uM each dCTP, dATP, and dTTP; 100 ,g/ml of

polyadenylated RNA; 20 ug/ml oligo-dT1 2 -18 obtained from

Collaborative Research, Waltham, Mass.; and 100 units/ml

reverse transcriptase from avian myeloblastosis virus. The

enzyme is available from Dr. D.J. Beard, Life Science

Incorporated, St. Petersburg, Florida, who produces the

enzyme under contract with the National Institutes of Health,

by the procedure of Kacian, and Spiegelman, in

Methods in Enzymology 29, L. Grossman, and K. Moldave, eds.,

-27-

TABLE 3

WE-i E- -4 11 4 fti 01. bG=< o heterogeneous sequences of miNA. The enzyme of choice

Academic Press, N.Y. (1974), p. 150. Reactions were

started by the audition of enzyme at 000 and synthesis was

for 6 min at 4200. Under these conditions approximately 6 32 Co 10 cpm P were incorporated into TCA-pre-ipitable material

cn and each ug of RNA yielded about 50 ng of cDNA. To obtain

sp enough cDNA for sequence analysis, the reaction volumes

were increased to 100 ul and the dCTP concentration was

raised to 250 )A (specific activity of 500 cpm 3 2P per pmole).

Under these conditions about 200,000 cpm of 3 2 P-labeled

dCMP were incorporated into cDNA. Restriction Endonuclease Treatment.

For restriction endonuclease digestions the analytical

reactions were stopped by the addition of 20 jl of ice-cold

water, boiled for 2 min, quick-cooled on ice, and made 7 mM

in MgCl 2 Aliquots (5 ul, about 2 x 10 5 cpm) were digested

using an excess amount of restriction endonuclease(s) Hae III

or Hhal or both, for 1 hr at 37 0C HaeIII was prepared

according to the method of Middleton, Edgell, M.H.,

and Hutchinson, III J. V -ol 10, 42 (1972). HhaI and

HpaII were obtained from New England Bio-Labs, Beverly, Mass.

HaeIII is also available from the latter source. The amount

of enzyme used was empirically determined to be in excess

of the amount needed to completely digest an equivalent

amount of restriction-sensitive DNA under identical reaction

conditions. Reactions were stopped with 5 pl of 20 mM EDTA, sucrose, 0.05% bromophenoiblue, heated to 1000°C for 1

min and then analyzed by polyacrylamide gel electrophoresis.

The products were separated on a composite 4.5% 10% poly-

acrylamide slab gel for 2.5 hr at 150V in Tris-Borate-EDTA

buffer (Dingman, and Peacock, supra) and visual- binding site for initiating the transcription reaction.

-16-

ized by autoradiography of the dry gel.

Figure 1 shows the results of gel electrophoresis and

autoradiography of 3 2 P-labeled cDNA, prepared as described

above. The samples were initially spotted at the origin co E and migrated electrophoretically through 4.5% acrylamide

O and then through 10% acrylamide. A bar is placed on the

"r4 left-hand side of the figure to indicate the position of the

CO boundary between the two gel regions. Lane A represents the

electrophoretic migration of the total cDNA transcript. Lane

B shows the migration of HhaI treated cDNA. Lane C shows

the migration of HaeIII treated cDNA. Lane D shows the

electrophoretic migration of total cDNA treated with both

HhaI and HaeIII. Lane E demonstrates the electrophoretic

migration of the material isolated from the prominent band

in Lane C. Lane F shows electrophoretic migration of isolated material from the prominent band of Lane C after treatment

with Hhal. Lane G shows the electrophoretic migration of

HaeIII cleaved 5 "-32P end-labelled single-straaded phage

M13 DNA used as a size standard, according to Horiuchi,K.,

and Zinder, Proc. Nat. Acad. Sci. USA 72, 2555 (1975).

The approximate lengths in nucleotides of these DNA frag-

ments are indicated by the numbers on the right.

The result in Lane A demonstrates that the cDNA transcript

from term placental mnNA is heterodisperse. Treatment with

HhaI, Lane B or HaeIII, Lane C results in the accumulation

of polynucleotides of discrete length. The production of

such discrete bands indicates the presence, in a heterogen-

eous population of cDNA transcripts, of at least one

sequence present in multiple copies and having two restriction

sites for Hhal and HaeIII, respectively. Cleavage with

At 42°C., reactions ranging from 1 mi. to 10 minutes are

-17-

haI produces a fragment of about 470 nucleotides, and HaeIII

digestion produces a fragment of approximately 550 nucleotides in length. Digestion by both enzymes yields three fragments designated A, 90 nucleotides long, B 460 nucleotides long, c and C, approximately 10 nucleotides long. Due to itssmall

size, fragment C migrated off the gel under the conditions

.qiused in figure 1. The band of material appearing at the interface between 10% and 4.5% gel represents heterogeneous

materialvhich was too large to enter the 10% gel and there- fore accummulated at the interface. As judged from the

simple band pattern of Lane D, fragments A and B seem to originate from the same cDNA molecule. This conclusion

was confirmed by elution of the larger HaeIII fragment from the gel, migrating as shown in Lane E, followed by re- digestion with HhaI. Such treatment produced two fragments

comigrating with the bands released by combined HaeIII and HhaI digestion of the total cDNA, as seen by comparing Lanes D and F. In the total cDNA digest, Lane D, the auto- radiographic density, which is a measure of the total radioactivity present in the band, is greater for fragment A

than fragment B, although the reverse may be expected on the basis of size differences, This observation suggests that fragment A is -;ranscribed from a region closer to the 3'-end

of the mRNA than is fragment B. Figure 2 is a schematic representation of the cDNA molecule showing the relative locations of the HaeIII and Hha.I

restriction sites. DNA fragments A and B, derived from the same cDNA molecule, were ordered on the basis of their

relative intensity on the autoradiogram shown in figure 1, Lane D. The existence of DNA fragment C was inferred from

EXAMPLE E ThePurn1Ificain of--PDNTAwhose-nucleoIdeseunc cmrie

11 V a U L'J. v JI 5 K xLu.Uy

-18-

the difference in the electrophoretic mobility of the band

appearing in Lane B and Lane D of figure 1. The size of

DNA fragment A is known exactly from a determination of its

nucleotide sequence by the method of Maxam, A. and Gilbert.

supra. The size of DNA fragment B was determined by c0 comparison with the M13 DNA size markers shown in figure 1,

Lane G.

The nucleotide sequences of DNA fragment A and a portion of

the 5'-end of fragment B were determined by the procedure of Maxam, A. and Gilbert supra. Since the amino acid

sequence of HCS is known, the nucleotide sequence of the

two fragments could be compared with the amino acid sequence,

using the known relationships of the genetic code. On the

basis of these relationships it was demonstrated that the specific sequences did in fact code for portions of the HCS

molecule, and further confirmed the ordering of these frag-

men's shown in figure 2.

EXAMPLE 2

The ability of the process of the present invention to purify

a desired nucleotide sequence that is a minority proportion

of the total population of nucleotide sequences is demon-

strated in the following reconstruction experiment. Defined

RNA mixtures containing purified rabbit globin RNA and human

polyadenylated placental RNA were used as template for reverse transcriptase in the presence of alpha- 3 P dCTP, final speci-

fic activity, 105 cpm per pmole. The cDNA products were

cleaved with endonuclease HaeIII and the cleavage products

were separated on 4.5% 10% composite polyacrylamide slab

gel. The cDNA fragments were visualized by autoradiography

of the dried gel. -31-

further purification.

For further purification, the previously described

technique of dividing the DNA into sub-fragments and separately strande I NA, whicl is $he immediate reaction product of

-19-

Figure 3 shows the results of the experiments. The gels

were run essentially as described in example 1. Size

markers prepared by endonuclease HaeIII cleavage of phage

M1 3 DNA and 32P end labelling of the fragments thereby t" produced, were run in lanes A and H. The approximate

CO lengths in nucleotides of these DNA fragments are indicated

CO by the numbers on the left. Lanes B-G show the electrophoresis

patterns produced by initiating the foregoing sequence of

reactions with mixtures of globin RNA and placental RNA in vprying proportions, as shown in the following table.

Globin RNA Placental RNA Lane nanograms nanograms

B 300 0 C 60 240 D 30 270 E 15 285 F 7.5 292.5 G 0 300

It can be seen that a 320 nucleotide long HaeITI fragment

is derived from globin cDNA. The globin cDNA transcript

can still be detected if globin RNA represents as little

as 2-5% of the total RNA. If an RNA species is present

as isolated in too low a copy number to be amenable to this

mode of analysis, it can be first partially purified by

any one of the known RNA purification schemes until it

represents about 2-5% of the remaining species mixture.

EXAMPLE 3

The purification of a nucleotide sequence fragment

approxiJately 550 base-pairs in length comprising a portion

of the coding region for HCS is described, together with a bee Etstratiadis, Maniatis, Kafatos., Jeffrey, method of measuring the purity of the isolated sequence.

The purified fragment is demonstrated to be greater than

99% pure.

Purification of HCs cDNA

Polyadenylated placental RNA isolated as described in

z example 1 was enriched for HCS mRNA by sedimentation in a

to 20% sucrose gradient at 400 in the SW 27 rotor of a Beckman Instruments ultra-centrifuge at 25,000 rpm for

16 hrs. The 11S-1$S region of the gradient was pooled and 100 g of this RNA used for the synthesis of double-stranded

cDNA as described by Ullrich, et al., supra. Synthesis

of the second strand was stopped by extraction of the

reaction mixture with one volume of ethanol at -700C.

Digestion of the cDNA with HaeIII endonuclease was carried

out in 50,Al of 6 mM Tris-HcL, pH 7.5, 6 mM MgC1 2 6mM

p -mercaptoethanol with 2 units of HaeIII enzyme at 3700

for two hours, following which 0.1 units of bacterial alkaline

phosphatase (type BAPF, Worthington Biochemical Corp.,

Freehold. units as defined by manufacture) we. e added

and digestion continued at 6000 for ten minutes. Following

extraction with one volume of phenol-chloroform, the DNA

was precipitated with two volumes of ethanol -700C, dissolved

in 20 zl of 10 mM Tris-HC1, pH 8, 1 mM EDTA, and subjected

to electrophoresis on a 6% poly-acrylamide gel.

Figure 4(F) shows the electrophoresis pattern of the fore-

going reaction mixture, which reveals a prominent band

corresponding to a nucleotide sequence approximately 550

base-pairs in length. The 550 base-pair fragment was

excised from the gel, and eluted electrophoretically, with

the result shown in figure 4(E).

-33-

TABLE 4

OJH :3 uE4 PI C) E-4 Q) on the presence or absence of restriction sites on other

-21-

The remaining material corresponding to the 550 base-pair

fragment shown in figure 4(E) was digested with 4 units of

HhaI endonculease in 5041 of the same buffer used for dige-

stion with HaeIII endonuclease, at 3700 for 2 hours.

L Following phenol-chloroform extraction and ethanol precipi-

CO tation, the digestion products were separated by electro-

C phoresis on a 6% polyacrylamide gel. The result is CO shown in figure 4(D).

The two fragments were eulted electrophoretically, combined

and rejoined by incubation in 20yl of 66 mM Tris-HC1, pH

7.6, 6 mM MgC12, 15 mM dithiotheitol, 1 mM ATP containing

of T4 DNA ligase at 1500 for two hours. The

reaction mixture was then diluted to 200 1 with 0.1 M NaC1,

extracted with 1 volume of phenol-chloroform and the DNA

precipitated with 2 volumes of ethanol. After resuspension

in 201al of 10 mM Tris-HC1, pH 8, 1 mM EDTA, the ligation

products-were separated by electrophoresis in the 6% (w/v)

polyacrylenide gel. The result is shown in figure 1(C).

It can be seen from the elec-rophoresis pattern of figure

4 that the 55.0 nucleotide fragment was reconstituted by the ligation treatment. The prior treatment with

alkaline phosphatase insured thatthe two Hhal fragments

were rejoined in the original sequence relative to each

other to reconstitute the 550 nucleotide segment. The

additional bands seen in figure 4(C) were the result of

dimer formation between the HhaI fragments, since dimer

formation is not prevented by the alkaline phosphatase

treatment.

The reconstituted 550 nucleotide fragment was excised from

the gel and eluted electrophoretically. The electrophoresis

-33-A- lengths, the sum of which will equal tlne engtn o1ne

22-

the pattern of the eluted material is shown in figure 4(B). Figure 4(A) represents the electrophoresis pattern of 32p_

labeled HaeIII digest of double-stranded M13 DNA used as

Ssize marker. The electrophoretic analyses were con- o ducted in a 6% polyacrylamide gel in 50 mM Tris-

borate, pH 8, 1 nm EDTA at 100 volts for two hours. Follow- 4 ing electrophoresis, the gel was dried and exposed to Kodak NS2T x-ray film to produce the autoradiograms. CvD Purity of Reconstituted 550 Nucleotide Fragment of HCS cDNA. The isolated reconstituted HCS cDNA HaeIII fragments was

labeled with P at its 5' ends using the enzyme poly- nucleotide kinase obtained from bacteriophage T4-infected

E. Coli by the method of Panet, et al. Biochemistry 12,

5045 (1973). Polynucleotide kinase is also commercially available from P-L Biochemical, Milwaukee, Wisconsin. The fragment was then digested with either HhaI or HpaII in

1p of 6 mM Tris-Hcl, pH 7.6, 6 mM MgC12, 6 mMI f -mercapto-

ethanol at 37 C for two hours. Following extraction with an

equal volume of pnenol-chloroform, the DNA was precipitated with two volumes of ethanol at -700C, resuspended in 20 41l, mM Tris-HC1, pH 8, 1 mM EDTA and subjected to electro-

phoresis, the gel was exposed to x-ray film to visualize

the labeled fragments, as described previously.

Results are shown in figure 5. Figures 5(B) and 5(E) re- present duplicate runs of the 550 nucleotide fragment prior

to restriction enzyme digestion. Figure 5(0) represents the pattern resulting from Hhal cleavage and figure

represents the pattern resulting from HpaII cleavage.

The purity of the 550 nucleotide fragment was measured

by scanning the autoradiograms of the restriction enzymes

-22- A

cleavage products and by quantitation of the distribution

of radioactivity in each of the two restriction endonuclease

digests. Such measurements reveal that the purified human

HOCS cDNA reconstituted HaeIII fragment was greater than 99%

homogeneous. rz EXAMPLE 4 Synthesis of a plasmid containing a nucleotide sequence of st' 550 base-pairs comprising the majority of the coding region

for HOS is described. A 550 nucleotide fragment of HCS cDNA of greater than 99%

purity was prepared as described in example 3. Terminal

phosphate end groups were restored in a reaction mixture

containing 50 mM Tris-HC1, pH 8.5, 10 mM MgOl 2 0.1 mM

spermidine, 5 mM P -mercaptoethanol, 5% glycerol, 333pmole ATP, 5 units of T4 polynucleotide kinase incubated

in a final volume of 4001 at 37 for two hours. DNA was

separated from the reaction mixture by phenol extraction

followed by ethanol precipitation. Synthetic decanucleotide

linkers having restriction site specificity for EcoRI and

having the sequence, 5'-CCGAATTCGG-3', prepared according

to Scheller, et al. supra, were then ligated to the HCS DNA

in a molar ratio of approximately 50:1 in 50x4l of 66 mM

Tris-HC1, pH 7.6, 9 mM MglC 2 15 mM dithiothreitol, 1 mM

ATI and 20 p/ml T4 DNA ligase. Linkers are commercially

available from Collaborative Research, Waltham, Massachusetts,

After incubation a 400°C for 18 hours, the reaction was

stopped by extraction with phenol-chloroform. The ligation

products were precipitated with ethanol, redissolved in

A 1 100 mM NaCl, 50 mM Tris-HC1, pH 7.6, 7mM MgC12, and digested with 50 units EcoRI endonuclease at 3700 for 2

GENERAL CONOLUDING REMARKS

The process of the present invention provides for the first tD j Y -l L U Y VL .L

-23-

hours. Digestion with the endonuclease resulted in

cleavage at the EcoRI site of the decamers giving rise to

HCS cJNA with EcoRI cohesive ends as well as cleaved un-

reacted decanucleotides and selif-ligated decanucleotides.

As the cleaved decamers also contained EcoRI termini and

would compete with the HOS cDNA for recombination with the col similarly cleaved plasmid, the HCS cDNA-was isolated by gel

CID, electrophoresis before reaction with the transfer vector.

The use of the foregoing decanucleotide linker has the

advantage that the HCS cDNA fragment may be reisoltted from

the plasmid in a form identical to that of the original

fragment.

The transfer vector employed was the bacterial plasmid

p1V-9, a 3.5x 10 6 molecular weight molecule containing a

single EcoRI site, prepared as described by Rodriguez, R.L.,

Bolivar, Goodman, Boyer, H.W. and Betlach, M.

in ION-UCLA Symposi-Lum On Molecular and Genetic Bioloy, D.P.

Wierlich, W. J. Rutter, and C. F. Fox, Eds. (Academic Press,

New York, 1976), pp 471-477. The plasmids pMB-9 and pBR-

322 (Example 5) are commercially available from Bethesda

Research Labs, Rockville, Maryland. Infection of B. Coli

with. pMB-9 confers resistance to tetracycline. Incorporation

of-DNA into the EcoRI site of pMB-9 does not affect the tetra-

cyoline resistance or any other known property of the plasmid. 0 Consequently, there are no phenotypic differences between

recombinant 9.ird normal plasmids. Therefore the EcoRI cut

pMB-.9 was first treated with alkaline phosphatase, according

to a method described in detail in application 805,023. See

also, Ullrich, et al., supra. Alkaline phosphatase treat-

ment removes the 5' phosphates from the EcoRI generated ends

-36-

I: :i same amino acid sequence, during the course.of in vivo

transcription, and translation. Consequently, all such

U n

of the plasmid and prevents self-ligation of the plasmid

DNA, insuring that circle formation and hence transformation

is dependent on the insertion of a DNA fragment containing

phosphorylated termini. The alkaline phosphatase treat-

ment was carried out in a reaction mixture at the level of cr5 1.0 enzyme units/mg of plasmid DNA in 25 mM Tris-HC1, pH 8,

for 30 minutes at 650C, followed by phenol extraction to

C remove the phosp.atase, and ethanol precipitation of the DNA. Ligation of HCS cDNA to plMB-9 treated as described was carr-

ied out in 50 pl reactions containing 60 mM Tris-HC1, pH 8,

mMO-mercaptoethanol, 8 mM MgCl 2 between 10 and 50 ng of the purified HCS cDNA and approximately 500 ng of EcoRI

cleaved 5' dephosphorylated plasmid DNA. Reactions were

begun by addition of T4 DNA ligase to 5pg/ml, allowed to proceed at 150C for 1 hour and mixture diluted to 0.25 ml

with 120 mM NaC1, 1 mM EDTA. The diluted reaction mixture was used directly for transformation of E. Coli X-1776. E. Coli X-1776 is a host strain especially developed for

recombinant DNA work, certified by NIH as an EK-2 host under the Federal guidelines. The strain is available from Dr.

Roy Curtiss III, University of Alabama, Department of Micro-

biology, Birmingham, Alacama. The bacteria were grown in

150 ml of nutrient broth supplemented with 100 ug/ml dia-

minopimelic (DAP) and 40 ug/ml thymine to a cell of approx- imately 2 x 10 cells/ml. The cells were harvested by

centrifugation and washed in 60 ml of 10 mM NaCI, recent- rifuged and resuspended in 60 ml of transformation buffer

containing 10 mM Tris-HC1, pH 8, 140 mM NaC1, 75 mM CaC1 2

The cell suspension was kept on ice for 15 minutes, the cells collected by centrifugation and resuspended in 1.5 ml

I iure an au-oraaogram oi a series of gel

of the same transformation buffer. The cell suspension,

ml, was added to 0.25 ml of diluted ligation reaction

mixture and incubated on ice for 15 minutes, then transferred

to 250C for 4 minute then on ice again for 30 minutes. The co C cell suspension, 0.2 ml, was plated directly onto nutrient

cO agar plates supplemented with 100 ug/ml DAP and 40 ug/ml

:4 thymine and 20 ug/ml tetracycline, Four transformants were S obtained, all of which contained a 550 base-pair insertion

which was released from the plasmid DNA by either EcoRI or

HaeIII endonuclease digestion.

A transformant clone designated pHCS-1 was selected for

sequence a-aalysis. E. Coli X-1776--pHCS-1 was grown in

suitable nutrient medium, plasmid DNA was isolated therefrom

and cleaved with EcoRI endonuclease. The 550 base-pair

insertion was isolated from linear pMB-9 by electrophoresis

in a 6% polyacrylamide gel and subjected to a DNA sequence

analysis using the procedure of Maxam and Gilbert, supra.

Sub-fragments of the HCS DN&A ere prepared by incubation

with HpaII restriction endonuclease and the 5' termin' were

labelled using _/32P-ATF and polynucleotide kinase. Follow-

ing the sequence analysis procedure of Maxam and Gilbert, the

nucleotide sequence of cloned HCS-DNA was determined. By

comparison with the known amino acid sequence of HCS, the

5'7 nucleotide sequence represented that portion of the coding

region of HCS mRNA from amino acids 24 to 191, plus

nucleotides of the 3'-untraslated region. See Niall, H.D., Hogan, Sauer, Rosenblum, and Greenwood, F.C.

Proc. Nat. Acad. Sci. USA 68, 866 (1971). The primary structure of HCS mRNA as determined from the DNA squence

of cloned fragment pHCS-1 is shown in Table 3, together with

-38-

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A method for purifying a fragment of a specific deo-

xyribonucleotide sequence for recombination with a DNA

*Trade Mark, Ciba-Geigy, Corp., G-reensboro, N/C.

the amino acid sequence predicted therefrom on the basis ofEthe known genetic code. The amino acid sequence* eter- mined from the nucleotide sequence is identical with the previously published amino acid sequence determined by chemical means,. This demonistrates that the initially isolated HCS mRNA has been copied in vitro with high fidelity and that the cloned HCS DNA fragment was replicated with high fidelity in the transformed bacteria. TABLE 3 Nucleotide sequence of one strand of HOS DNA from cloned pHCS-1. e number, -refer to the amino acid sequence beginning at the amino terminus. The DNA sequence shown corresponds to the inRNA sequence for HCS, except that U replaces T in the mRnNA. The amino acid sequence from positi-ons 1 through 2.3 is also shown.

-39-

~2

3 6489n78 Vai-Gin-Thr-Val-Pro-Leu-SerAgLuPeAp sAaMtLu-l-l-i-r-l-i-l-e

24 Ala Ile Asp Thr Tyr Gin Glu Phe Glu Giu Thr Tyr Ile Pro Lys Asp Gin Lys Tyr Ser Phe Leu His Asp Ser Gin GCC ATT GAC ACC TAG GAG GAG TTT GAA GAA ACC TAT ATC CCA AAG GAC GAG AAG TAT TCG TIC CTG CAT GAG TCC GAG

Thr 5cr Phe Cys Phe Ser Asp Ser Ie Pro Thr Pro 5cr Asn Met Giu Giu Thr Gin Gin Lys Ser Asn Leu Giu Leu Leu AGC TCC TTG TGC TTG TGA GAG TCT ATT CCG ACA CCC TGC AAC ATG, GAG GAA ACG GAA GAG -AAATCG AAT GTA GAG GTG CTC

100 Arg Ie Ser Lou Leu Lou Ie Glu Ser Trp Leu Glu Pro Vai Arg Phe Lou Arg Ser Met Phe Ala Asn Asn Leu Vai Tyr GGG MGC TGG CGG GIG CTG ATC GAG TGG TGG GIG GAG GGG GIG GGG TTC GTG AGO ACT AIG TTG GGG AAG AAG GIG GIG JAT

120 Asp Thr Scr Asp 5cr Asp Asp Tyr His Leu Lou Lys AsD Leu Glu Giu G].y Ile Gin Thr Leu Met Gly Arg Levu Giu Asp CAG AGG TGG GAG AGCGCAT GAG TAT GAG GTG CTA AAG GAGC CTA GACGCAA GGG AIC GAA AGG GIG ATC CCC AGO GIfG GAA GAG

140 Cly Ser Arg Arg Thr Gly Gin Ile Leu Lys Gin Thr Tyr Ser Lys Phe Asp Thr Asn Ser Hlis Asa His Asp Ala Lcu Lou CCC AGG CCC CGG AGT GGG GAG AIG CUG AAG GAG AGG TAG AGG AAG TTT GAG AGA AAC TGG GAG AAG GAT CAG GGA GIG GIG

160 180 Lys Asn Tyr Gly Lou Lou Tyr Gys Phe Arg Lys Asp Met Asp Lys Val Giu Thr Phe Lou Arg,Met Val Gin Cys Arg Ser

I. MGC AG TAG GGG GTG "1JG TM' TGG TTG AGG MAG GAG AIGC A G GTC GAG AGA TTG GTG GGC ATG GTG GAG TGG CGG IGII

Val Giu Giy Ser Gys Gly Phe GIG GAG GGG AGC IGI GGG TTG TAG GTCGGGAGTAGGATGCGTGCGGGTGGGGAGTCGTGTCGTGGG------ 3

3 648978 110 Vai-Gin-Thr-Vai-P ro-Leu-S er-Ar g-Leu.-Phe-Asp-His-Aia-Met-Leu-Giu-Aia-Hi.,-Arg-Ala-His-Giln-Leu

24 Ala Ile Asp Thr Tyr Gin Giu The Giu Giu Thr Tyr le Pro Lys Asp Gin Lys Tyr Ser The Leu His Asp SE GOG ATT GAG ACC TAO CAG GAG TTT GAA GAA AGO TAT ATC CCA AAG GAG GAG AAG TAT TCG, TTG CIG CAT GAC TC

Thr Ser Phe Gys Phe Ser Asp Ser Ile Pro Thr Pro Ser Asn Met Glu Giu Thr Gin Gin Lys Ser Asn Leu Glu AGC TOG TTG TGG TTO TGA GAG TOT ATT CCG ACA GOG TOG AAG ATG GAG CAA AGG CAA GAG AAA TOG AAT OTA GAG

Arg Ile Ser Leu Leu Leu Ie Gin Ser Trp Len Giu Pro Val Arg Phe Len Arg 5cr Met The Ala Asn Asn Leu CGO ATG TOG CTC :TG GTO ATC GAG TOG TGG GTG GAG GOG GTG CGG TTO OTG AGO ACT ATG TTG CG MGC MC GTG

120 Asp Thr Scr Asp Scr Asp Asp Tyr His Leu Leu Lys Asp Leu Glu Gin Gly Ile Gin Thr Leu Meat Gly Arg Leu GAG AGO TGG GAG AGO CAT GAG TAT CAC GTG OTA A-AG GAG OTA CAC GMA000 ATG GM. AGG 070 ATO COG AGO CTC-

140 Oly Ser Arg Arg Thr Gly Gin Ile Leu Ly s Gin Thr Tyr Ser Lys Phe Asp Thr Asn 5cr His Asn Hlis Asp Al, CG AGC CCC CGG ACT COG GAG ATG GUC AAG GAG AGO TAO AGO AAG TTT GAG ACA MOC TOG GAG MO GAT GAO Ci

160 IS0 Lys Asn Tyr Giy Leu Leu Tyr Cys Pbs Arg Lys Asp Met Asp Lys Val Giu Thr Phe Le- Arg,Met Vai Gin Cy MAG AMO TAG 000 OTO 0130 TAG TG TTG AGO MAG GAO ATG GAG MG OTO GAG ACA TTO C1, OC ATG GTG GAG TC,

Val Oiu Gly Ser Cys Gly Phe CTG; GAG GC AGC TOT 000 TTC TAG GTGCOGGAGTAGCATGGTGTCACGCOTCCOOAGTCOGTOTGGTGGGCC------3'

3 648978 Y-His-Ala-Met-Leu-Glu-Ala-Hif;-Arg-Ala-H-is-Gln-Leu

rTyr Ie Pro Lys Asp Gin Lys Tyr Ser Phe Leu His Asp Ser Gin ZTAT ATO CCA MAG GAG CAC AAG TAT TCG TTC CTG CAT GAG TCC GAG

Pro Ser Asn Met Giu Giu Thr Gin Gin Lys Ser Asn Leu Ciu Leu Leu CCC TGG AAC ATG GAG GAA ACG CAA CAG AAA TGC AAT CTA GAG CTG CTC

100 Giu Pro Val Arg Phe Leu Arg Ser Met Phe Ala Asn Asn Leu Val Tyr GAG CCC OTO CGG TTC CTG AGO ACT ATG TTC CCC MGC AAG CTC CTG TAT

Lys Asp Leu Glu Clii Ile Gin Thr Leu Net Gly Arg Leu Giu Asp AAG CAC CTA GAG GMA ATC CAA AG ATO COG AGO CTG CAA GAC

Thr Tyr Ser Lys Phe Asp Thr Asn Ser His Asn Hlis Asp Ala Lcu Lcu AGC TAC AGC AAC TTT GAG ACA MAC TGG GAG MAC CAT GAG OCA CTG CTC

Asp Met Asp Lys Val Giu Thr Phe Le- Arg,Met Gin Cys Arg Ser GAG ATG GAG MAG GTC GAG ACA TTG C1, OCC ATG GAG TGC CGC TCT

'CATCGTGTGACCCCTCCCCACTGCCTGTCTCCGCC 3

EXAMPLE

The purification of DNA whose nucleotide sequence comprises

most of the coding region for HGH is described, together

with the synthesis of a plasmid transfer vector containing

the purified DNA and the construction of a microorganism P"* strain having the DNA as part of its genetic makeup. HGH C0 was purified essentially as described for HCS in Example 3, c except as noted below. CD Five benign human pituitary tumors, quick-frozen in liquid

nitrogen after surgical removal, weighing 0.4 g to 1.5 g

eacn were thawed and homgenized in 4 M guanidinium thiocyanate

containing 1 M mercaptoethanol buffered to pH 5.0 at

The homogenate was layered over 1.2 ml 5.7 M CsC1 containing

100 mM EDTA and centrifuged for 18 hours at 37,0Cj rpm in the

SW 50.1 rotor of a Beckman ultra-centrifuge at 1500 (beckman

instrument Company, Pullerton, California). RNA travelled

to the bottom of the tube. Further purification, using an

oligo-dT column and sucrose gradient sedimentation was as

described previously in Examples 1 and 3. About 10% of the

RNA thus isolated coded for growth hormone, as judged by

incorporation of a radioactive amino acid precursorinto anti-

growth hormone precipitable material in a cell-free trans-

lation system derived from wheat germ. See Roberts, B.E.

and Patterson, B,iV. Proc. Nat. Acad. Sci. USA 70 2330 (1973).

Single-stranded cDNA and double-stranded cDNA were synthesized

as described in Example 3. IIGH cDNA was then treated with

restriction endonuclease HaeIII and alkaline phosphatase as

described in Example 3, then fractionated by gel electropho-

resis. A distinct band in a position corresponding to about

550 nucleotides in length was observed, and isolated for

-41-

A is deoxyadenyl,

G is deoxyguanylA

C is deoxycytosyl,

T is thymidyl, T A 0 further purification.

For further purification, the previously described

technique of dividing the DNA into sub-fragments and separately .purifying and recombining the sub-fragments was carried out

as previously described, except that for HGH, the restriction 0 1 endonuclease PvuII was used to produce two sub-fragments of 00O approximately 490 and approximately 60 nucleotides length,

1 respectively. All restriction en ymes used herein are

commercially available from New England, Biolabs, Beverly, Massachusetts. The religated product, about 550 base-pairs

in length, was greater than 99% pure as judged by sub-

fractionation in four separate restriction endonuclease

systems.

Synthesis of a recombinant transfer vector containing HGH

DNA was carried out essentially as described in Example 4

except that the decanucleotide linkers and plasmid employed

were different. A decanucleotide linker having Hind III

specificity was employed, sequence 5'-CCAAGCTTGG-3'. Treat-

ment with HsuI, yielded HGH cDNA with cohesive ends. HsuI and Hind III have the same site specificity and may be used

interchangeably. The plasmid pBR-322 was used as the

transfer vector. This plasmid confers host resistance to

the antibiotics ampicillin and tetracycline. DNA insertions

into the Hind III site have been found to reduce or abolish tetracycline resistance. Recombinants were therefore

selected by growth on nutrient plates containing ampicillin,

and by their inability to grow on 20 ug/ml of tetracycline.

HGH-cDNA was recombined with HnuI-cleaved alkaline phosphatase

treated pBR-322, under conditions essentially as described

in Example 4.

-42-

12. A recombinant DNA transfer vector comprising

codons for human growth hormone comprising the nucleotide

sequence:

Products of the ligase reaction were used to transform E.

Coli X-1776 under conditions as describe1 in Example 4.

Seven colonies were isolated based upon the ability to grow

in the presence of ampicillin and their inability to grow Co in the presence of tetracycline. Five of the seven colonies OC carried the recombinant plasmid containing the approximately

C.D 550 base-pair portion of HGH DNA. One of the bacterial

strains, pHGH-1, carrying HGH DNA as part of its genetic

makeup, was grown in quantity to provide a source of plasmid

DNA from which the HGH DNA could be reisolated by treatment with Hind III or HsuI. This isolated HGH DNA, having under-

gone many replications, was subjected to sequences analysis

as described in Example 4. The results are shown in Table

4.

TABLE 4

Nucleotide sequence of one strand of HGH-DNA of cloned

pHGH-1. The numbers refer to the amino acid sequence of HGH

beginning at the amino terminus. The DNA sequence shown

corresponds to the mRNA sequence for HGH, except that U

replaces T in the mRNA.

-43-

A is deoxyadenyl,

G is deoxyguanyl, rF i f l4Pr~r r4-flr xT

3 64 89J78

24 34 40 43 Ala Phe Asp Thr Tyr Gin Gin Phe Gin Gin Ala Tyr Ie Pro Lys Glu Gin Lys Tyr Ser Phe Leu Gin Asn Pro Gin GCG TTT GAG ACC TAG GAG GAG TTT GAA GAA GCC TAT ATG CCA AAG CAA GAG MAG TAT TGA TIOC GG AG AAG GGG GAG

Thr Ser Leu Gys Phe Ser Gin Ser Ie Pro Thr Pro Ser Asn Asg Gin Gin Thr Gin Gin Lys Ser Asn Leu Giu Leu Leu AGG TGG GIG TGT TTG TGA GAG TGT ATGGG AGA GGG TGG MGC AGG GAG GAAGMCAAGAG AAA TGG MG GITA GAG CGG GI

Arg Ie Ser Leu Leu Leu Ie Gin Ser Trp Len Gin Pro Val Gin Phe Leu Arg Ser Val Phe Ala Asn Len Val Tyr GGG ATG TOG GI G IGGIGAIG GAG TGG TGG GIG GAG GGG GTG GAG TTG GIG AGG AGT GIG ITCGCGG AAG GIG GIG TAG

GIly Ala Ster Asp Ser Asu Val Tyr Asp Leu Lou Lys Asp Leu Giu Giu Gly ile Gin Thr Lou Met Giy Arg Lou Glu Asp GC CC GTCT GAC ACG AACGTGIGTAT GAG GIG GTA MGAG ACCTA GAG GAA CCCOGAGGMACGIG ATGC CC AGGO GIGCGA GAG

Gly Ser Pro Arg Ihr Giy Gin ile Phe Gin Thr Tyr Ser Lys Phe Asp Thr Asn Ser Hius Asn His Asp Ala Leu Leu GCG AGG GGG CGG AGT GGG GAG ATG TTG GAG AGG TAG AGG MG TTG GAG CAGACGTGA GAG MG GCAT CACGCGA GTA GIG

Lys Asn Tyr Gly Leu Lou Tyr Gys Phe Arg Ly.. Asp Met Asp Lys Val Gin Thr Phe Leu Arg Ie Val Gin Gys Arg Ser MAG AAGTAG1GGG GIGG TG TAG GITCG AGGGAG AIG GACG G GIGGAG AGA TG GGGGATG GIG GAG TGG GGG IGI

191 Val Gin Gly Ser Gys Gly Phe GIG GAG GGG AGO IGI GGG TTG TAG CTGGGCGGGGGGGATGGGTGTGAGGGGTGGGGAGTGGTGTGGTGG-----

-33*A-

K;~

3 648978

24 34 43 Ala Phe Asp Thr Tyr Gin Giu Phe Glu Glu Ala Tyr Ile Pro Lys Glu Lys Tyr Ser Phe Leu Gin Asn P, G CC TTT CAC ACC TAG GAG GAG TTT GMA GAA GCC TAT ATC CCA AAG GMA MAG TAT TCA TTC CTG CAG MAC C:

Thr Ser Leu Cys Phe Ser Giu Ser Ile Pro Thr Pro Ser Asn Asg Giu Giu Thr Gin Gin Lys Ser Asn Leu Giti ACC TCG CTC TGT TTC TCA GAG TCT ATT CCC ACA CCC TCC MAC AGG GAG GMA ACA CMA GAG AAA TCC MAC CTA GAC

Arg Ile Ser Leu Leu Leu Ie Gin Ser Trp Leu Giu Pro Val Gin Phe Leu Arg Ser Val Phe Ala Asn Asn Lei CCC ATC TGC CTG CTG. CTC ATC GAG TCG TOG CTG AG CCC GTG CAG TTC CTC AGG AGT OTC TTC CCC MAC MGC CT(

Gly Al-a Sur Asp Ser Asti Val Tyr Asp Leu Lou Lys Asp Lemi 0Th Glu Ile Gin Thr Lc~u Met: Gly Arg L~e GGCc tCC TCT GAC AGG AAC CTG TAT GAC G'rC GIA MAG GAG CTA GAG GAA ATC CAA AG CTG ATC CCC AGG CT

140 Oly Ser Pro Arg Thr Oly Gin Ile Phe Lys Gin Thr Tyr Scr Lys Phei Asp Thr Asti Ser His Asn His Asp Al GCC ACC CCC COO ACT COG GAG ATG TTC MOG GAG ACC TAG ACC MAG TTC GAG AGA MAC TGA GAG MGC CAT CACGC

160 1 180 Lys Asni Tyr Oly Leu Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Giu Thr Phe Leu Arg Ile Val Gin C, AAC MG TAG 000 CTO CTG TAG TOG TTC AGO MOG CAC ATO GA AG C TG GAO ACA TTG CG CGC ATG GTO CG D~

Val 0Th Gly Ser Cys Oly Phe OTO GAO 000 AG TOT GCG TTC TAG GTOGCGGGTGGCATCGCTGTGAGCGCTGGGCAGTGGCTCTCCTGCCC------3'

4

3 648 9 n8

43 Tyr Ile Pro Lys Glu Lys Tyr Ser Phe Leu Gin Asn Pro Gin TAT ATC CCA AAG GMA MAG TAT TCA TTC CTG GAG AAG CCC CAG

Pro Ser As- Asg Glu Giu Thr Gin Gin Lys Ser Asn Leu Clu Leu Leu CCC TCC MGC AGG GAG GMA ACA GMA GAG AMA TOG MGC CTA GAG GTC OTC

100 Glii Pro Val Gin Phe Leu Arg Ser Vai Phe Ala Asn Asn Leu Val Tyr 6AG CCC GTGCGAG TTC CTC ACC ACT CTC TTG CCC MGC MC CTC GTG TAG

Lys Asp Leu Glu Gin Ile Gin Thr Leu Met Gly Arg Lou Glu Asp AAG GAC CTA GAG GAA ATC CAA AGC CT(; ATG CCC AGGO CTG GAA GAC

Thr Tyr Ser Lys Phe Asp Thr Asn Ser His Asn His Asp Ala Lou Leu ACC TAG AGO AAG TTG GAG ACA MGC TCA GAC MGC OAT CAC GCA G'IA CTC

1 180 Asp Met Asp Lys Val Gin Thr Phe Leu Arg Ile Val Gin Cys Arg Ser GAO ATC GAG AAO C GAG ACA TTG CTC CCC ATO CGC GAG TCC CCC TGT

'JCATCCCTGTGACGGCTCCCCAGTGCCTCTCCTGGCC------- 31

GENERAL CONGLUDING REMARKS

The process of the present invention provides for the first

time a method of general applicability for purifying desired

specific nucleotide sequences. These sequences may be

correlated with the production of a specific protein of

commercial or medical significance. The disclosed process

CO results in the purification of nucleotide sequences which may

e- be fragments of a larger sequence coding for the desired

protein. The present method may be used in combination with

known ancillary procedures to produce the entire nucleotide

sequence coding for a specific protein.

In addition, a method has been disclosed whereby a nucleotide

sequence of specific length, however derived, may be highly

purified. A method for measuring the degree of purity of

such fragments is also disclosedo By these means, a nucleo-

tide sequence coding for a portion of human HOS has been

isolated, purified and shown to be at least 99% pure.

Transfer vectors containing most of the nucleotide sequence i- coding for HCS and most of the sequence coding for HGH have

been synthesized. Novel microorganism strains containing

the foregoing genes and portions of genes have been produced.

The foregoing nucleotide sequences have been reisolated after

many cycles of replication in the host microorganism and

found to contain essentially the identical nucleotide sequence

to that existing in the source organism.

On the basis of the genetic code, there exists a finite

set of nucleotide sequences which can genetically code for

a given amino acid sequence. All such equivalent nucleotide

sequences are operable variants o$ the disclosed sequences,

since all give rise to the same protein hormone, having the

K:

same amino acid sequence, during the course of in vivo

transcription, and translation. Consequently, all such

variants areincluded in the. scope of the present invention.

While the invention has been described in connection with

specific embodiments thereof, it will be understood that it I:00 is capable of further modifications and this application

is intended to cover any variations, uses, or adaptations

of the invention following, in general, the principles of

the invention and including such departures from the present

disclosure as come within known or customary practice within

the art to which the invention pertains and as may be applied

to the essential features hereinbefore set forth, and as

follows in the scope of the appended claims.

I

Claims (1)

1. THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

   1. A method for purifying a fragment of a specific deo-

   xyribonucleotide sequence for recombination with a DNA

   transfer vector, as.d transfer to a microorganism, from a

   S population of polyri. onucleotides heterogeneous in length

   0C and sequence, comprising the steps of:

   providing a population of cDNA transcripts of

   the polyribonucleotides, at least a portion of said cDNA

   transcripts having at least two restriction sites,

   subjecting the cDNA transcripts to the action

   of a restriction endonuclease preparation capable of catal-

   yzing the hydrolysis of the cDNA transcripts at each of the

   two restriction sites in order to produce a fragment of a

   specific deoxyribonucleotide sequence which is homogeneous

   in length,

   fractionating the fragments producced by restriction

   endonuclease action according to their length, thereby

   separating the homogeneous length fragments of the specific

   deoxyribonucleotide sequence from cDNA transcripts of

   different length, whereby a fragment of the specific deo-

   xyribonucleotide sequence is purified for recombination with a DNA transfer vector and transfer to a microorganism.

   2. A method according to claim 1 of further purifying

   linear DNA of specific nucleotide sequence containing a

   restriction site, from an essentially homogeneous length

   population of DNA molecules prepared according to the method

   of claim 1 comprising the additional steps of:

   pretreating the DNA to remove any 5' phosphate end groups,

   incubating the DNA with a restriction endonuclease capable of acting on the specific desired nucleotide sequence to produce two linear sub-fragments thereof, fractionating the sub-fragments according to their length, isolating the two sub-fragments, rejoining the sub-fragments covalently, in a &r DNA ligase catalyzed reaction, to reconstitute the original oO 1 specific sequence, and fractionating the rejoined DNA molecules according to their length, whereby the specific sequence is

   ,urified from a population of DNA molecules of essentially homogeneous length, for recombination with a DNA transfer vector and transfer to a microorganism.

   3. A method according to Claim 2 wherein alkaline

   phosphatase is used to remove the 5'-phosphate groups.

   4. A method according to Claim 2 wherein fractionation by length is accomplished by gel e ect.e chioresis.

   A recombinant DNA transfer vector comprising a

   fragment of a specific deoxyribontdleotide sequence prepared by the method of Claim 1. 6. A recombinant DNA transfer vector comprising a

   fragment of a specific deoxyribonucleotide sequence prepared by the method of Claim 2.

   7. A microorganism strain modified to contain a fragment of a specific deoxyribonucleotide sequence prepared by the method of Claim 1.

   8. A microorganism strain modified to contain a fragment

   of a specific deoxyribonucleotide sequence prepared by the

   method of Claim 2.

   9. A microorganism strain modified to contain the DNA

   *3648 9i c+ H+

   GCL 24 ATM25 J30TTF31G3 GAJ ACL 34TAX 35 25 GAK 26ACL27 TAX A 28CAJ29 2 8 A 2 GA 3 ~3 3 2 GA 3 3 A 3 5 A1M33 co~ AV'~W~C~.T(D (D OCL 3 7AJ 3 A 8 3 9 uu 4A0 J 4 1 TAK 4 2R 4 3S 43 44 4545 46 47 48 48449Z (D j 1 5 0 QB ACL S TTK TGK TTK QP GAK R S A TM 5 9 ACL C0L 1 0 C C H CD 5051 51 52 53 5 5 5 AK56 QR57 57 58 S AA 61R6 YGJ 62 X YHo ooc 09 00 jC-

   77 7 7 ATM 7 8 QR7 9 S7 9 X8oTy 8 0X 1lTy 8 lX8 2 2 YAT 83 A 8 4 QR8 5S 8 5 78 8r jr o p 4fCCL GTLrW91GZ91TTK 89 92X9393W94G'944&Y7 TT R 95S95 ATGTTK 97 GOL 98 AAr 99 AAK 1 R~gAA100 X 101 TY1 01p >i P

   GTL 1 2 TAK 1 3GAK1 0 4ACL 10 5 QR 1 6 S 10 6 GAK 07 QR 0 8 S 1 O-GAK 1 9GAK 1 GAX 1 1 1 CAK 1 2 C i

   x113TY113XK 114 T 114 I115 Y TY A A .TY G-AJ GAJ GGL ATM CAJ ACL c 116 117 117 118 119 120 121 122 123 0 H- C+I CD 0 1 2 4 TY 2 4 ATGGGL1 2 6 W GZT1 2 7 8 8 GAJ GAK GGL QR1 3 GWP) 1 i

   W GZ AOL GGL AT 1 T AJ CJTAL A SAJ CD (1ATy ci 0 IC AAJ TTK GA ACL AAK GL 0 145 146 A47148 149 150 1:0 151 152 153 154 X T155 16 056 c-f Fj 0 x YA-K AJ TK GL Y x T TAXL T-K TTK ~W GCZ [J 9 0 X 1 5 7TY AAJ1 5 8 A 1 57 1 5 Ta 9 160''1 61"i 621 621 63 163C 1 641 65 166l167 1 7 W' P. F C-

   16 8169 ATGGAK 171AA172 SG-AK AA GTL 173 G-AJ 174ACL 175 TTK1677x TY GZ 177W 1 78 G ATGGTLY 17 8 A 180 to

   CJ TGK W GZ s T A G R s TKTTK GGL 19 CD 0J 18 1 182 1 8 3 81Q 3 8 14 8 GTL 1 4 1 8GAJ 5 18 6 L 18 7 Q 18 8 18 8 G G 189 L909 191 0C

   TAGGTGCCCGAGTACATCCTGTGACOCCTCCCAGTGCTCTCCTGGCC 3t wherein o 0 H (D m ~ii-0 ii

   36489n1 RJ 0

   51-G GCL, M 4 25 A( 2 6 ACL2 7 TAK 2 8 AJ 0 3 1 GJ 3 IJ2 id3 29 3 3 4 3 5ALIV 3 6 CCL AM GAK CAJAAJ TAK QR S TTK X TY CAKCAKQRSCAJ (D 37 394041 4243434445 45 46 47484849 p TGK~~( ~cDC SATM mms Q CD AC 50 QR 1 s 1 TTK5 2 TGK 5 TK 54555 5GAK C o 5 AK5 6 QR 57 7 58 ccL ACLC 59 L6 1QR6 2 S6 2 C0 0

   AAA ATGGAJ 63 6 5G66. 67 68 fAJ 69 A7Q 7 AA72"73T Y73 GA J74"75 T 7576 T0 76O 771 '-4

   W77GZ77 WQR AM X YX T XYT T78Q79S79X888181X82T GAJQB. TGGX87TY8GAJ8 C+ 82A83 84 R8585 8 CCL8 9 QTL89 W GZ TTK X TYWG QR ATG-TTGOGL A ATY 09 0 91 01 91 92 93 93 94GZ94R95S, T97 098AA99AA100X101 101p te GTL 102 TAX GK ACL QB. S GAK QR G CeS ae 103 104 105 106 106 107R1 0l0 so8 1GAK09 GAI1 1 0GAK1C11 A K 1 12

   X 1 13Y 1 13X 1 1 4 Y 11 4AAJ 1 1 5GAK 1 16X 1 17T 1 1GAJ 7 11 GAJ1 1GGL 9 1 2 0 P 1 21 CAJ 12 2 A.C1 2 3 0 X1 2 4 TY 1 2 4 ATGGGL 1 2 6 W GZ X 28 TY T 1 28 AJ GAK GL 3 Q 127 12712 18 129 130 G131 Q'132'132W133 1330 '-d4 0 W134 213JC 1 35L jCAY1 3 7AT1 381X 1 3 9TY 13 9 AAJ1 4 OCAJ 0 1 41 ACL 1 4 TAK 2 14 3 QR1 l445144 0' 0 0F AAJ TVGAT( AACI QR S1 CAKAPJK C(XGA T. TY J1 4T 5 14 6 1 47A 14 8 K 14 150 1) 151 152 1 5 3CK 1 5 4 1 55 1 56 156 0Fd 0

   X1 5 7 Y 1 57Cl 58 K1 9TAK 1 6 0 GL 1 X 6 1 6 2TY 1 62X 1 6 3 TY1 6 3 AK 1 64T K 1 6 5TTK 1 6 6 W1 GZ 7 167 H' H y

   AAJ GAKATGGA AAJ GTL GAJ AOL TTK X TY W GZATGTL180 168169 171 172 173 174 175 176 177 177 178 178 H

   CAJ 181 TGKWGZ 182W183 SGTLGT8 183QR1843184 AJ 186GGL 1 87 18(8 8~ 188 19GGL 190 18 TGK 19 818 TTK 91 19 TK D0 9 S0 TAGxGTGCCCGAGTAGCATCCTGTGACCCCTCCCCAGTGCCTCTCCTGGCC 3' wherein CD M' 01

   i c. rlPII. _II i '1

   64889 74 P. 0

   C 3 2 GAJ 3 3 AOL 3 AK ATMI36 (D

   CAJ co 4 5AK 4 6AK 4 7 QR4 8 4 8C 49 CD TM CCL AC CL 8 0 5 9 AL CCL 60 6 1 QR6 2 62 2 0 0

   K7 2 73 Y 7 3 CAJ 7 4i5 TY 7 5X 7 TY 76

   8 4 Q 8 5 s8 5 TGGX 87 TY 87 GAJ 8 8 0 t TK 9GGL 7 9 8 AA 99AAK 1 00X 1 0 1TY 10 1 C+ C 8GAK 1 0 GAK 1 1 0 GAK 1 1 1 OAK K0 1 12

   J GGL ATM OAJ AOL 119 120 121 122 123 0 H

   GG0L13 1 132 1 2W 1 3 3 3 3 1 0 'dd H CD J0AJ1 4 1 TA sS 0R 14A L1 42 A 1 4 3 QR1 44 144 0+ 0 CAK GAI QGI T 0 152 153 154 1 5 5 X 1 5 6TY1 5 6 H~ 0 f TAY, TGK TTK W Zp 163 16 4 16 5 166 1670 167 m C H' X. TY W GZ ATGGTII 176XI77 177 178 178 180 H

   QR s TGK GGL CTKW QT 18 8 18 8 1 89 19 0 191 CD 0

   '-CC 3' wherein 0 H, CD m ca

   550 nucleotides in length was observed, and isolated for

   -41-

   ii A is deoxyadeyl,

   G is deoxyguanyl,

   C is deoxycytosyl,

   T is thymidyl,

   J is A or G;

   K is.T or C;

   L is A, TO or G;

   M is A, C or T;

   X is T or C, if Y is A or G, and C if Yn is C or T;

   Y is A, G, C or T, if Xn is C, and A or G if Xn is T; W is C or A, if Zn is G or A, and C if Z is C or T; Z is A, G, C or T, if Wn is C, and A or G if Wn is A;

   QR is TO, if Sn is A, G, C or T, and AG is Sn is T or C;

   Sn is A, G, C or T, if oRn is TO, and T or C if QRn is AG and subscript numerals, n, refer to the amino acid position

   in human chorionic somatomammotropin, for which the

   nucleotide sequence corresponds, according to the genetic

   code, the amino acid positions being numbered from the amiotr%enudeCc -cud/.kad bir- Ate W Sl vC 0r amino en

   iL -A L +IULV L VGI- UU1 CL: U UV ;V4

   comprising a plasmid.

   13. The DNA transfer vector of Claim 11 wher n the

   plasmid is pBR-322.

   14. A microorganism modified to conta' the DNA transfer vector of Claim 11

   A microorganism according o Claim 14, comprising a bacterium. i

   16. A microorganism acc ding to Claim 15, comprising Escherichia coli X-177 containing plasmid pBR-322. tJ DAT> THIS 16TH DAY OF NOVEMBER, 1978. i UN; THE REGENTS THEIUNIVERSITY OF CALIFORNIA.

   EDWD. WATERS SONS, Patent Attorneys, Queen Street, i

   u1fythot this and the prece g pages are a tw MELBOURNE, VIC. 3000. te 1 and ex act copy if AUSTRALIA. -50- specaion io,w. -6;tt i. 50 7: ;i-E~

   12. A recombinant DNA transfer vector comprising

   codons for human growth hormone comprising the nucleotide

   sequence:

   51-GGCL TTK GAK ACL TAX CAJ GAJ TTK GAJ GAJ GCL 5'-GGCL 24 25 26 27 28 29 30 31 32 33 34

   TAK 35ATM36 CCL AAJ GAJ CAJ AAJ TAK QRS TTK X4 TY4 3 T 36 C 37 38 39 40 41 42 434, 44 45

   CAJ AAK47 CCL 48CAJ ACL QR S 51X52 TY TGK TTK 5 4 QR 5 5S 55GAJ56 C 4 6 AA C7 4 8 CA 4 9 AC 5 QQR 5 1S 5 1X 5 2 52 53 5 5555 56 R S ATM 58CCL59ACL CCL QR S AAK W GZ CAJ GAJ 66ACL6 7 S 5 7 ATN 5 8 59 60 61 62 62 63 64 64 65 66 67

   CAJ 68 CAJ AAJ QOR S AAK X TY GAJ X TY X TY W GZ 68 69 70 71 71 72 73 73 74 75 75 76 76 77 77

   AT R 4 QR8 S 79 X 80 TY 80 X 81TY81X 82 TY 82 ATM 83 CAJ884 87GG 87 ATM 7 8 QR 79 85 TGGX 87 TY87

   GAJ CCL GTL CAJ TTK92 X TY W 94GZ94 QR9SGTL TTK97GCL 88~ 89 90 91 92 93 93 94 QR 95 GTL TT6 GCL9 AAK AAK X TY GTL TAK GGL GCL QR 1 0 6 S 16 AK 99 100 101 101 102 103 104 105 106 106 AK107

   QR 0 8S 1 0 8 AAK 1 0 9 GTLlTAK1 1 GAK 11 2 X 1 1 3 TY 1 1 3 X 1 1 4 TY 114 AAJ 1

   f GAK 116 X 117 TY 117 GAJ 118 GAJ 119 GGL 120 ATM 121 CAJ 122 ACL 123 124 X TY 124

   ATGGGL 1 2 6 W GZ X TY GAJ GAK GGL QR S CCL 126 127Z127 128 128 129 130 13 1 132 132 133

   W GZ ACL GGL CAJ ATM TTK AAJ CAJ ACL 134GZ134 L135 136 137 139 139 140 141 142

   TAK QR S AAJ TTK GAK ACL AAK QR S CAK 143 144 144 145 146 147 148 149 150 150 151

   AAK CAK GAK GCL X TY X TY AAJ AAK TAK AAK152AK153AK154 L155 156 156 157 157 158 159 160

   GGL 161 X 162 TY 162 X 163 TY 163 TAK 164 TGK 165 TTK 166 W 167 GZ 167 AAJ 168

   GAK169 ATGGAK GAK AAJ ATGGAK171 AAJ172 GTL AJ174 ACL 175 TTK 176 X 177 TYWGZ GTL173 GAJ TY177 178 178

   ATM GTL CAJ TGK W GZ GLT CCL 187 G.AJ 186GG ATM179 GTL180CAJ 181 TGK182 183GZ 183 QR 1 8 4 S 1 8 4 GLT185G-

   R 188S 188TGK 189 GGL 190 TTK 191 TAGCTGCCCGGGTGGCATCCCTGTGACCCC 18188

   TCCCCAGTGCCTCTCCTGGCC-3', wherein

   A is deoxyadenyl,

   G is deoxyguanyl,

   C is deoxycytosyl,

   T is thymidyl,

   J is A or G,

   K is T or C,

   L is A, T, C or G,

   M is A, C or T,

   X is T or C if Y is A or G or X is C if Yn is C or T, n n n Y is A, G or C if X is C or Y is A or G if X is T, n n n n W is C or A if Z is G or A or W is C if Z is C or T, n n n n Z is A, G, C or T if W is n C or Z n is A or G if W is A, n n QR is TC if S is A, G, C or T or QR is AG if S is T or C,

   Sn is A, G, C or T if QR is TC or S is T or C if QR is AG, n n n

   and subscript numerals, n, refer to the amino acid position

   in human growth hormone, for which the nucleotide sequence

   corresponds, according to the genetic code, the amino acid

   positions being numbered from the amino end, wherein

   said nucleotide sequence is purified by the method of

   claim 1 or 2.

   4, mnnn*~l~ui~;;ih~ i-~i~n~z ewF

   13. A DNA transfer vector according to Claim

   11 or 12 comprising a plasmid.

   14. The DNA transfer vector of Claim 11 or 12

   wherein the plasmid is pBR-322.

   A microorganism modified to contain the DNA

   transfer vector of Claim 11 or 12.

   16. A microorganism according to Claim 15, compris-

   ing a bacterium.

   17. A microorganism according to Claim 16, compris-

   ing Escherichia coli X-1776 containing plasmid pBR-322.

   DATED this 13th day of October, 1981.

   THE REGENTS OF THE UNIVERSITY OF CALIFORNIA.

   EDWD. WATERS SONS, Patent Attorneys, Queen Street, MELBOURNE VICTORIA. AUSTRALIA.

   -53-

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