Note: Descriptions are shown in the official language in which they were submitted.
!L2~
STABL~ DNA CONSTRUCTS FOR EXPRESSION
OF ALPHA-1-ANTITRYPSIN
Background of the Invention
The use of microorganisms for the production of useful
polypeptide products through recombinant DNA technology is
becoming established as an industry. Foreign genetic
material may be introduced into a culture of microorgan-
isms, and, given the proper intracellular and extracellular
conditions, the desired protein product(s) may be synthe-
10 sized from the foreign gene(s). Such genetic material iscommonly introduced into microorganisms in the form of
plasmids, which are autonomously replicating extrachromo-
somal elements. In order to ensure the maintenance of
plasmids within a culture of transformed cells, it has been
15 necessary to grow those cells under special conditions. In
the absence of such conditions, the plasmids, which may be
inherently unstable; will not be maintained, and the cell
population will revert to the untransformed state.
Increased plasmid stability and copy number are important
20 to the biotechnology industry as a means of maintaining the
production of plasmid-encoded proteins at a consistently
high level. Previously reported attempts to increase
plasmid stability do not appear to be optimal for commer~
cial application. The introduction of yeast centromeres
25 into _ -bearing plasmids, while enhancing stability, has
been shown to markedly decrease plasmid copy number (Clarke
and Carbon, Nature 287: 504-509, 1980 and Stinchcomb, et
al., J. Molec. Biol. 158; 157~179, 1982). Linear centro-
meric yeast plasmids similarly show an inverse relationship
--2--
between stability and copy number ~Murray and Szostak,
Nature 305 189-193 1983~
_ , .
Plasmids typically contain gene sequences, known as select-
able markers, which encode antibiotic resistance or comple-
ment nutritional requirements of the host cell. To selectfor the presence of such plasmids, transformed cells must
thus be grown in special media which contain a selective
drug or which are depleted for specific nutrients. These
media requirements may be both expensive and prohibitive of
10 optimal cell growth rates during the large-scale fermenta-
tion process. ~any such plasmids have been reported in the
literature. Those comprising antibiotic drug resistance
genes include pBR322 (Bolivar, et al., Gene 2: 95-113,
1977) and its derivatives, such as the pUC vectors IVieira
15 and Messing, Gene lg: 259-268, 1982) which carry a gene for
ampicillin resistance; and pBR325 (Prentki, et al., Gene
14: 289, 1981) which carries resistance genes for ampicil-
lin, tetracycline, and chloramphenicol. Plasmids which
complement host nutrient requirements include the yeast
20 vectors YEpl3 (Broach, et al., Gene 8: 121-133, 1979),
which carries the LEU2 gene; and YRp7' (Stinchcomb, et al.,
Nature 2 : 39, 1979), which carries the TRP1 gene.
Alpha-l-antitrypsin is a protease inhibitor, the principal
function of which is to inhibit elastase, a broad spectrum
25 protease. Lung tissue in mammals is particularly vulner-
able to attack by elastase, therefore alpha-l-antitrypsin
deficiency or inactivation may lead to loss of lung tissue
elasticity and subsequently to emphysema. Loss or reduc-
tion of alpha-l-antitrypsin activity may be a result of
30 oxidation of alpha-l-antitrypsin due to environmental
pollutants, including tobacco smoke. Deficiency of alpha-
l-antitrypsin may result from one of several genetic
disorders. See Gadek, James E., and R. D. Crystal, "Alpha-
l-Antitrypsin Deficiency", The Metabolic Basis of Inherited
35 Disease, Stanbury, J. B., et al., Ed. McGraw-Hill, New York
~g~120
(1982) pp. 1~50-1467; and Carroll, et al., Nature 2988,
329-334 (1982).
It is therefore an object of the present invention to
provide DNA constructs containing a DNA sequence encoding
5 alpha-1-antitrypsin and, as selectable markers, gene
sequences whose products are essential for the viability or
normal growth of the host cell on complex media.
It is another object of the present invention to provide
transformant strains of microorganisms containing plasmids
10 which are selectable by growth on complex media and which
are capable of expressing alpha-l-antitrypsin.
It is a further object of the present invention to provide
strains of microorganisms that are deficient in essential
functions which may act as hosts for DNA constructs
15 carrying gene sequences which complement these defective
essential functions and are capable of expressing
alpha-antitrypsin.
It is yet another object of the present invention to
provide methods for producing alpha-l-antitrypsin in trans-
20 formed microorganisms, wherein the alpha-l-antitrypsin is a
product of a gene carried on a DNA construct which con-
tains, as a selectable marker, a gene sequence which
complements a deficiency in an essential gene in the host
microorganism.
25 Other objects of the invention will become apparent to
those skilled in the art.
Summary of the Invention
According to the present invention, there are provided DNA
constructs and appropriate host cells such that the con-
30 structs are capable of expressing alpha-l-antitrypsin and
are maintained at high copy number without the need for
special selective media. Growth in such conditions may
~zg9~20
- 4 - 69140-31
result in faster growth, greater cell density, and reduced
production costs.
According to another aspect of the present inventlon
there is provided a method for producing alpha-1-antitrypsin in
a microorganism host cell having a de~iciency in a function
necessary for normal cell growth on complex media comprising the
steps of:
(a) transforming said microorganism host cell with a
DNA molecule comprlsing a gene which complements said deficiency
and a sequence coding for said alpha-1-antitrypsin;
(b) culturing the transformants from s~ep (a) in a
growth medium which need not contain antibiotics or heavy metals,
and need not be depleted of specific nutrlents, under conditions
whereby said gene functions as a selectable marker for
transformant cells
The present invention provides a method for producing
alpha-1-antitrypsin in a host cell having a deficiency in a
function necessary for normal cell growth in complex media, with
the step of transformlng the host cell with a DNA molecule com-
prising a gene which complements the deficiency and a sequencecoding for alpha-1-antitrypsin.
In some preferred embodiments: said gene is a gene
required for host cell division, cell wall biosynthesis, membrane
biosynthesis, organelle biosynthesis, protein synthesis, carbon
source utilization, RNA transcription, or DNA replication; said
gene is a gene required for host cell division, cell wall bio-
synthesis, membrane biosynthesis, organelle biosynthesis, protein
synthesis, carbon source utilization, RNA transcription, or DNA
~;Z9~2~
- 4a - 69140--31
replication and sai.d gene i5 selected from the group consisting of
genes of the yeast cell division cycle and genes of the yeast
glycolytic pathway; and said gene is selected from the group con-
sisting of genes of the yeast cell division cycle and genes of the
yeast glycolytic pathway.
As used herein the term "DNA construct" means any DNA
molecule which has been modified by man in a manner such that the
nucleotide sequences in the molecule are not identical to a
sequence which is produced naturally. The term "DNA construct"
also includes clones of DNA molecules which have been so modified.
The term "expression vector" is defined as a DNA construct which
includes an autonomous site of replication, a site of transcript-
ion initiation and at least one structural gene coding for a
protein which is to be expressed in the host organism. The
expression vector will usually also contain appropriate control
regions such as a promoter and terminator which control the
expression of the protein in the host organism. Expression
vectors according to the present invention will also contain a
selection marker comprising an essential gene as described herein.
The term "plasmid" wlll have its commonly accepted
meaning, i.e., autonomously replicating, usually closed-looped,
DNA.
In the accompanying drawings:
Figure 1 illustrates the construction of plasmid pB4.
Figure 2 illustrates the construction of plasmid pB5.
Figure 3 illustrates the construction of plasmid pB15L.
120
--5--
Figure 4 shows a Southern blot of DNA from S. cerevisiae
strain A2.7.c co-transformed with plasmids pB5 and pB15L.
The blot was probed with a 2.5 kb BamHI-HindIII fragment
from the 5' flanking region of CDC4 in order to test for
disruption of the genomic CDC4 locus. Lane a contains DNA
from cells transformed with pB5 alone; Lane b, untrans-
formed cells; Lanes c-h, co-transformants. Arrows indicate
the genomic fragments hybridizing to the probe.
Figure 5 shows the sequences of the S. pombe POTl and S.
10 cerevisiae TPIl genes together with the respective inferred
protein sequences. The entire S. ~ombe TPI protein
sequence is given. The sequence of the S. cerevisiae
protein is given only where it differs from the S. pombe
sequence. The methionine at position 1 in the S. cerevis-
15 lae protein sequence is not present in the mature protein.
Figure 6 illustrates the construction of the plas~id pCPOT.
Figure 7 illustrates the construction of the plasmidpFATPOT.
Figure 8 illustrates the construction of the plasmid
20 pTPI-LEU2.
Detailed Description
The present invention is based in part upon the discovery
that essential genes may be used as selectable markers on
DN~ constructs such as plasmids which are capable of
25 expressing alpha-l-antitrypsin. An "essential gene" is
defined as any gene that codes for a function necessary for
cell viability or normal growth on complex media. Complex
media are those media in which the nutrients are derived
from products whose composition is not well defined, such
30 as crude cell extracts, meat extracts, fruit juice, serum,
protein hydrolysates, etc. Hence, to select for a desired
transformant according to the present invention, the
selection growth medium will be merely a conventional
~2~
--6--
complex growth medium, not a special medium containing a
relatively expensive antibiotic, metal antagonist, or other
agent lethal to the untransformed host cell, or lacking one
or more specific nutrients required by the untransformed
host. Essential genes include, but are not limited t~,
genes required for cell division, membrane biosynthesis,
cell wall biosynthesis, organelle biosynthesis, protein
synthesis, carbon source utilization, RNA transcription,
and DNA replication.
10 In order to use an essential gene as a selectable marker on
a DNA construct, such as a plasmid, it is necessary to
provide an appropriate mutant host cell strain. Using the
one-step gene disruption method of Rothstein (Meth. ln
Enzymology 101: 202-210, 19~3) or the co-transformation
15 procedure described herein, suitable host strains may be
constructed which carry deletions in an appropriate essen-
tial gene in the genome. Such deletion mutants grow when
the mutation is complemented by a function coded by plas-
mid-borne genetic material. It is preferred that the
20 deletions in the essential gene or genes of the genome of
the host comprise substantial segments of the coding region
and/or flanking regions. If the mutation or mutations in
the essential gene are accomplished in a manner to achieve
only point mutations, then there is a likelihood that the
25 mutant host cell will revert to wild-type by mutation or a
recombination repair mechanism, thereby reducing or
eliminating the selectivity achievable by use of the
plasmid-borne gene.
Essential genes often exist in multiple copies (such as
30 histone or ribosomal RNA genes) and/or in multiple, related
forms called gene families (such as different hexokinase
genes, or different DNA polymerase genes). In such cases,
these redundant functions may be sequentially mutated to
make a host cell which is multiply deficient for a given
35 essential function. ~owever, by using a high copy number
plasmid to increase the activity of the gene, a single
lZ9912~
essential gene on a plasmid may complement multiple host
cell deficiencies. A high copy number plasmid is desirable
because an increase in copy number of a cloned foreign gene
may result in an increase in the production of the protein
product encoded by said gene.
The selection for transformants containing high copy
numbers of plasmids with essential genes may be accom-
plished by reducing the expression levels of each plas-
mid-borne essential gene and/or by reducing the activities
10 of the gene products encoded by the plasmid-borne select-
able marker. One approach is to mutate the essential genes
such that the transcription and/or translation rates of the
genes are reduced or the gene products are altered to have
lower specific activities. Another method for decreasing
15 the expression levels of essential genes used as selectable
markers is to use a gene from another organism to comple-
ment defects in the host cell. Such foreign genes may be
naturally defective for expression in a host cell because
the signals for transcription and/or translation may be
20 suboptimal in a different species or the gene product may
have decreased activity or stability because it is in a
foreign cellular milieu.
A broad range of functions necessary for cell viability or
normal growth on complex media exists. A defect or dele-
25 tion in an essential gene may result in lethality, adecrease in the rate of cell division, cessation of cell
division, termination of DNA, RNA, or protein synthesis,
termination of membrane synthesis, termination of cell wall
synthesis, termination of organelle synthesis, defects in
3~ sugar metabolism, etc. Examples of essential genes include
the CDC ~cell division cycle) genes of the yeast Sac-
charomyces cerevisiae (for review see Pringle and Hartwell,
"The Saccharomyces cerevisiae Cell Cycle", in Strathern, et
al., eds., The Molecular Biology of the Yeast SaccharomYces
35 Life C~ycle and Inheritance, 97-142, Cold Spring ~larbor,
1981), the genes coding for functions of the S. cerevisiae
~Z99~120
--8--
and E. coli glycolytic pathways, and the SEC (Novick and
Schekman, Proc. Nat. Acad. Sci. USA 76: 1856-1862, 1975 and
Novick, et al., Cell 21: 205-215, 1980) and INO (Culbertson
and Henry, Genetics 80: 23-40, 1975) genes of S. cerevis-
lae.
One preferred class of essential yene-deficient host cells
contains defects in CDC genes known as cdc mutations, which
lead to stage-specific arrests of the cell division cycle.
~ost cdc mutations produce complete blockage of events
10 essential to the cell cycle by affecting either the synthe-
sis or function of the particular CDC gene products. Such
mutations may be identified by their effects on events
which can be monitored biochemically or morphologically.
Most known cdc mutations are conditionally lethal (l.e.,
15 temperature sensitive) mutations, which result in the
cessation of normal development of mutant cells grown under
restrictive conditions. However, the primary defect
resulting from a cdc mutation need not be a defect in a
stage-specific function per se. For example, continu-
20 ously-synthesized gene products may have stage specific
functions; a defect in the yeast glycolytic gene PYK1 (for
the enzyme pyruvate kinase) is allelic to the cell division
cycle mutation cdcl9 (Kawasaki, Ph.D. Thesis, University of
Washington, 1979). This mutation results in cell cycle
25 arrest at the Gl phase of cells incubated in the typical
yeast complex medium YEPD (1% yeast extract, 2% bactopep-
tone, and 2~ dextrose). Thus, whether the cdc mutation
results in a defect in a stage-specific function, or
whether it causes an inhibition or disabling mutation of a
30 gene product having a stage-specific function, the effect
of the defect may be monitored.
Pringle and Hartwell (ibid.) describe the function of some
51 CDC genes. For use in carrying out the present
invention, such genes may be isolated irom gene libraries
by complementation in a strain carrying the desir~d
mutation. Gene libraries may be constructed by commonly
lZ99~
g
known procedures (for example, Nasmyth and Reed, Proc.
Natl. Acad. Sci. ~SA 77: 2119-2123, 1980; and Nasmyth and
Tatchell, Cell _: 753-764, 198G). Strains carrying the
desired cdc mutation may be prepared as described herein,
or may be obtained from depositories accessible to the
public, such as, the American Type Culture Collection and
the Berkeley Yeast Stock Center.
A second preferred class of essential genes are those
encoding products involved in the glycolytic pathway,
10 including genes coding for metabolic enzymes and for
regulatory functions. Examples of glycolytic pathway genes
in S. cerevisiae which have been identified are the
glycolysis regulation gene GCRl and the genes coding for
the enzymes triose phosphate isomerase, hexokinase 1,
15 hexokinase 2, phosphoglucose isomerase, phosphoglycerate
klnase, phosphofructokinase, enolase, fructose 1, 6-bis-
phosphate dehydrogenase, and glyceraldehyde 3-phosphate
dehydrogenase. As noted above, the pyruvate kinase gene
has been identified and described by Kawasaki. A plasmid
20 containing a yeast phosphoglycerate kinase gene and
accompanying regulatory signals has been described by
Hitzeman, et al. (J. Biol. Chem. 225: 12073-12080, 1980).
Isolation and sequencing of the yeast triose phosphate
isomerase gene TPIl has been described by Alber and
25 Kawasaki (J. Mol. ~ . Genet. 1: 419-434, 1982) and by
Kawasaki and Fraenkel (Biochem. Biophys. Res. Comm. 108:
1107-1112, 1982).
A particularly preferred glycolytic gene is TPIl, which
codes for the yeast triose phosphate isomerase, an enzyme
30 which catalyzes the interconversion of glyceraldehyde-3-
phosphate and dihydroxyacetone-3-phosphate and is therefore
essential for glycolysis and gluconeogenesis. In S.
cerevisiae the single genetic locus, TPIl, codes for this
function. Cells carrying mutations in TPIl do not grow on
35 glucose and grow poorly on other carbon sources.
~Z~91~0
--10--
The S. cerevisiae TPI1 gene was isolated by complementation
of the tpil mutation (Alber and Kawasaki, i_ ., and
Kawasaki and Fraenkel, ibid.). The triose phosphate
isomerase gene from the fission yeast Schizosaccharomyces
~ (POT1) has been isolated by complementation of the
same S. cerevisiae mutation, and has been sequenced as
shown in FIG. 5. Sequencing of the S. ~ombe gene,
designated POT1, has demonstrated that the S. ~ombe TPI
protein is homologous to the TPI protein of S. cerevisiae.
10 While in the usual case the essential gene which is
utilized in the DNA construct (plasmid) will be a wild-type
gene from the host species, in some cases it will be
preferable to use an essential gene which is foreign to the
host cell because the foreign gene may be naturally
15 defective, and thereby selectable to high plasmid copy
number. As an example of such a foreign essential gene
being used, one of the examples herein shows that the S.
~ POT1 gene may be effectively used as a selectable
marker in an S. cerevisiae host.
20 The DNA constructs according to the present invention
containing essential genes as selectable markers will be
transformed into mutant host cells which are defective in
the function of the essential gene. Properly mutated host
cells must either be prepared or, may be readily available
25 from a public depository. Mutation of the wild-type cell
to obtain a proper mutant may be accomplished according to
conventional procedures. For example, wild-type cells may
be treated with conventional mutagenizing agents such as
ethane methyl sulfonate and transformed with a plasmid
30 containing an essential gene to identify the colonies where
complementation occurs. Alternatively, the genome ma~ be
disrupted to create a specific mutation (Rothstein, ibid).
The stability of the plasmid containing the essential gene
in the host cell may be dependent on the absence of
35 homologous essential gene sequences in the host cell. The
.
~25'9120
genetic defects in the host ensure that the plasmid will be
maintained since growth of the host cell will not occur or
will be severely limited by the lack of the essential gene
function. Additionally, the integrity of the plasmid
itself may be dependent upon the absence of homology
between the plasmid-borne essential gene and the
corresponding locus in the host genome, because recom-
bination between respective plasmid and genomic loci may
cure the cell of both the mutation and the plasmid. Thus,
10 it is preferred that mutation in the host cell genome which
inactivates the genomic essential gene be of a substantial
nature, i.e., deletions be made from the DNA sequences of
the coding section and/or flanking regions of the
chromosomal gene. Once this is accomplished, curing of the
15 genomic mutation by recombination is less likely to occur.
The plasmids of the present invention are unexpectedly
stable when maintained in the appropriate mutant host
cells. A preferred host cell is yeast; however, other
eukaryotic cells may be utilized, as well as prokaryotic
20 cells. In the case of yeast cells, the stability of the
plasmids according to the present invention appears to
exceed even that of yeast plasmids containing centromeres.
Circular centromere plasmids are among the most stable
plasmids previously reported for yeast, but suffer from an
25 extremely low copy number (Clarke and Carbon, ibid. and
Stinchcomb, et al., 1982, ibid.). Linear centromeric yeast
plasmids are either unstable or present at low copy number,
depending on plasmid length (Murray and Szostak, ibid.).
It is therefore an unexpected advantage that improved
30 stability of plasmids bearing an essential gene is
achieved.
The POTl and CDC4 genes are two examples of the utility of
essential genes as selectable markers on expression
vectors. These two genes belong to a broad class of genes
35 that are required for cell proliferation on complex media.
The use of other essential genes may allow for plasmid
~Z~9~
-12-
selection in plant Dr animal tissue culture which involves
complex growth conditions and at the extreme may allow for
the maintenance of plasmids in cells receiving nutrition
from blood, serum, or sap of living animals or plants.
Data obtained from experiments using plasmids described
herein show that human alpha-1-antitrypsin (AT) productiGn
is doubled by the use of the S. pombe POTl gene as the
selectable marker, when compared to AT production obtained
with similar plasmids bearing a traditional auxotrophic
10 selectable marker, LEU2. These results indicate that POTl
containing plasmids are functionally greater in copy number
than the non-POTl plasmids from which they are derived.
The techniques used to produce the DNA constructs, l.e., in
particular the plasmids, according to the present inven-
15 tion, involve conventional methods. The essential gene tobe utilized in the DNA construct may be isolated from a
library by using a labeled DNA probe if the structure of
the gene is known, or identified by ligating segments of
the DNA library to conventional vectors, transforming the
20 vectors into a mutant cell deficient in the particular
essential gene and searching for colonies which are comple-
mented. Once an appropriate DNA fragment containing the
essential gene is identified it will be ligated to a vector
which contains a DNA sequence coding for the structural
25 protein which will be expressed. The essential gene may be
utilized together with its own promoter and other controls
necessary for expression within the host organism. Alter-
natively, a heterologous promoter may be utilized to
increase or decrease expression of the essential gene.
30 Methods of ligation of DNA fragments are amply described
and are well within the skill of those of ordinary skill in
the art to perform.
After preparation of the DNA construct it will be trans-
formed into the host organism under transforming condi-
35 tions. Techniques for transforming prokaryotes and
~Z99~20
-13-
eukaryotes (including tissue culture cells) are known in
the literature.
As described above the host organism must be deficient in
the essential function for selection of the essential gene
on a plasmid. Mutant host strains are available from
conventional depositoriec or may be made by conventional
means from wild-types by mutagenesis and screening for the
mutant carrying the proper mutation.
The transformed host may then be selected by growth on
lO conventional complex medium. In the case of yeast, a
conventional medium such as YEPD (1% yeast extract, 2
bactopeptone, and 2~ dextrose) may be used. The selectable
markers comprising essential genes according to the present
invention may be used as markers wherever appropriate in
15 any DNA construction and thus it will be recognized that
constructs containing the essential gene selection markers
according to the present invention have many uses. The
following examples are offered by way of illustration of
such use, not by way of limitation.
20 Unless otherwise indicated, standard molecular biology
methods were used. Enzymes were obtained from Bethesda
Research Laboratories, New England BioLabs, and Boehringer
Mannheim Biochemicals, and were used as directed by the
manufacturer or as described by Maniatis, et al. (Molecu-
25 lar Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, 1982). E. coli cultures were transformed by
the calcium chloride method, also disclosed in Maniatis, et
al. (ibid.). Yeast cultures were transformed by the method
of Beggs (Nature 275: 104-108, 1978), with modifications as
30 described herein.
1;~99~ZO
-14-
~xample 1
The S. cerevisiae CDC4 qene as
selectable marker
A. Construction of a stable CDC4-containing plasmid
A yeast genomic library was constructed by partial diges-
tion of yeast DNA with Sau3A, size selection on sucrose
gradients, and insertion of the selected fragments into the
yeast vector YRp7 which had been digested with BamHI
(Nasmyth and Reed, Proc. Natl. Acad. Sci. USA. 77: 2119-
10 2123, 1980). A recombinant plasmid containing the CDC4gene was isolated by transformation of yeast strains GEB5
(MATa cdc4-4 leu2 trpl lysl ural) and GEB7 (MATa cdc4-3
leu2 trpl lysl) with the library. These strains were
derived from strains A364A cdc4-3 and A364A cdc4-4 (Hart-
15 well, et al., Genetics 74: 267-286, 1973) by crossing with
a strain known to transform at high frequency (K79 [MAT~
leu2 trpl] (Nasmyth, et aI., Nature 289: 244-250, 1981;
Tatchell, et al., Cell 27:25-35, 1981) followed by back-
crossing to high transformlng strains (K79 and K80 [MATa
20 leu2 trpl lysl]) to obtain the cdc4-3 and cdc4-4 mutations
in the desired genetic background (leu2 ~ ). Selection
of transformants for tryptophan prototrophy and the ability
to grow at the restrictive temperature (37) identified one
such plasmid (designated pJY35) which was shown to inte-
25 grate into the genome and map to the CDC4 locus. Spontan-
eous plasmid integrants were identified on the basis of
their selective growth advantage. This growth advantage is
due to the presence, on the original plasmid, of a CDC4-
linked gene which is deleterious to cell growth when
30 present at high copy number (l.e., when the plasmid is not
integrated into the host genome). In the integrants, the
TRPl plasmid marker was shown to be genetically linked to
SUPll, which is linked to CDC4 on chromosome VI (Mortimer
and Schild, "Cenetic Map of Saccharomyces cerevisiae" in
35 Strathern, et al., eds., The Molecular Biology of the Yeast
Saccharomyces cerevisiae Life Cycle and Inheritance,
641-651, Cold Spring Harbor, 1981). The cdc4-3
~LZ991~:0
-15-
complementing region was purified from pJY35 as a 6.4kb
BamHI fragment and was joined, using T4 DNA ligase, to the
vector YRp7 (Struhl, et al., Proc. Natl. Acad. Sci. USA 76:
1035-1039, 1979) which had been cleaved with BamHI. This
construct is known as pJY51, and is illustrated in FIG. 1.
Referring to FIG. 1 the CDC4 coding region was purified
away from flanking genomic DNA sequences in the following
manner. Plasmid pJY51 was cleaved with HindIII and the
3.6kb fragment comprising the CDC4 region was subcloned in
lO the bacterial plasmid pBR322. This construct was digested
to completion with BamHI, partially digested with HincII,
and the ca. 2.3 kb CDC4-containing fragment was purified.
The HincII fragment end was converted to a BamHI end by the
addition of linker sequences (sequence:5'CCGGATCCGG3')
15 (obtained from Collaborative Research) and subsequent
digestion with BamHI to remove excess linkers. The result-
ing fragment, comprising approximately 1.9 kb of the CDC4
gene, was inserted into the BamHI site of YRp7 to produce
plasmid pJY70. This plasmid was shown to complement the
20 cdc4-3 mutation as described above. Although the 1.9 kb
fragment lacks small portions of both the 5'- and 3'-coding
regions of the CDC4 gene, it surprisingly complements the
temperature-sensitive defect. Presumably, transcription
and translation of the CDC4 sequence is controlled by se-
25 quences located in the pBR322 regions of the plasmid,allowing for production of a functional gene product.
Plasmid pJY70 was cleaved with EcoRI to remove the yeast
TRPl and ARS1 sequences and was re-ligated, yielding a
hybrid plasmid comprising pBR322 and CDC4 sequences. This
30 plasmid is known as pJY71, and is illustrated in Figure 1.
The l.9 kb yeast sequence was purified from pJY71 as a
BamHI-HindIII fragment. This fragment was joined to pBR322
which had been linearized by digestion with BamHI and
HindIII, to produce the plasmid pB4, and is illustrated in
35 Figure 1.
~29S~ZO
-16-
The CDC~ region was re-isolated from pB4 for insertion intG
a high copy number yeast vector. Such a vector will
contain an origin of replication of the yeast 2~ plasmid,
and one or more restrlction enzyme cleavage sites which
will serve as cloning sites for foreign genes of interest.
Preferably such sites will be unique sltes on the plasmid.
A preferred vector is MW5, which comprises the yeast 2~
plasmid replication origin and unique EcoRI and BamHI
cloning sites. Referring to FIG. 2 plasmid MW5 was derived
10 from plasmid YRp7' (Stinchcomb, et al., Nature 282: 39-43,
1979) by partial digestion with EcoRI to cleave, on aver-
age, one of the two EcoRI sites per molecule. The result-
ing unpaired ends of the linear molecules were filled in
using DNA polymerase I (Klenow fragment) and the resulting
15 blunt ends were re-joined using T4 DNA ligase. The result-
ing plasmid which retained the EcoRI site adjacent to the
ARS1 sequence was then selected. The ARS1 sequence was
removed by digestion with PstI and EcoRI, and replaced with
the PstI-EcoRI fragment of plasmid YEpl3 (Broach, et al.,
20 Gene 8: 121-133, 1979) which comprises the replication
origin of yeast 2~ DNA. The resulting plasmid, designated
MW5, is illustrated in FIG. 2.
To construct the final CDC4-containing stable plasmid, MW5
was cleaved with EcoRI and BamHI. The CDC4 fragment was
25 purified from plasmid pB4 by digesting the plasmid with
BamHI and EcoRI. The two fragments were joined, using T4
DNA ligase, and the chimeric molecules so produced were
transformed into E. coli strain RRI (Nasmyth and Reed,
ibid.) with selection for ampicillin-resistant, tetracy-
30 cline-sensitive colonies. Plasmid pB5 (shown in FIG. 2),
isolated from one such colony, comprises the yeast 2~
replication origin, pBR322 plasmid sequences, the select-
able marker TRP1, 1.9 kb of the yeast CDC4 coding sequence,
and a unique EcoRI cloning site.
~Z99120
-17-
B. Construction of a plasmid for disruption of host CDC4
gene
The stability, in a transformed host, of the CDC4-contain-
ing plasmid according to the present invention is dependent
on the lack of a functional CDC4 gene in the host. It is
further desirable that no homology exists between the host
genome and the CDC4-containing stable plasmid in order to
prevent recombination between plasmid and chromosomal
D~A's. To obtain a yeast strain having a suitably deleted
lO CDC4 locus, a yeast host containing the wild-type CDC4 gene
mav be transformed with a linearized plasmid fragment
having a "disrupted" CDC4 gene (Rothstein, ibid.). The
linearized plasmid fragment is a preferred transforming
agent because the free ends of the fragment may enhance
15 recombination within the CDC4 region. Such a plasmid
fragment will have intact CDC4 flanking regions at its ends
to facilitate recombination with the intact genomic CDC4
locus. The genetic material inserted between the CDC4
flanking regions of the plasmid fragment will code for a
20 phenotypic characteristic which can be selected in the
transformed host (a selectable marker such as TRPl or
LEU2). The disrupting plasmid will preferably also lack a
yeast origin of replication in order to select for the
integration of the disrupted CDC4-selectable marker
25 sequence into the host genome. Following transformation
with the linearized plasmid, genetic recombination results
in the substitution of the disrupted sequence for the
genomic sequence of the host. Cells in which the _DC4 gene
has now been deleted are then selectable according to the
30 marker used in the disruption.
A method for a one-step disruption of a host genome is
described by Rothstein (ibid.). As described above,
disruption is performed with the added improvement of
co-transforming a host strain with an intact stable plasmid
35 and a linearized plasmid such that in a-ddition to achieving
~299120
-18-
disruption of the host genome, transformation of the host
with the stable plasmid is also effected.
A preferred plasmid for disruption of the host CDC4 locus
is pBl5L, shown in Figure 3. It comprises the yeast LEU2
gene inserted between the flanking regions of CDC4, and the
vector pUC13 (Vieira and Messing, Gene 19: 259-268, 1982
and Messing, Meth. in Enzymology 101: 20-77, 1983). When
linearized at the junctions of yeast and vector sequences
and transformed into a suitable yeast host strain, the
lO plasmid produces a deletion of CDC4 in the host genome
resulting from the substitution of the LEU2 sequence for
the C _ region. In a host strain auxotrophic for leucine,
disrupted transformants may then be selected on the basis
of leucine prototrophy.
15 To construct plasmid pB15L, a 6.4 kb fragment comprising
the CDC4 gene and its 5'- and 3'-flanking regions was
purifled from a BamHI digest of pJY51. This fragment was
inserted into BamHI-digested pUC13 to produce the plasmid
pB14. Most of the CDC4 coding region was removed by
20 digesting pB14 with ClaI and purifying the larger fragment
which comprises the pUC13 and CDC4 flanking sequences. The
fragment ends were modified by the addition of XhoI (BglII)
"smart" linkers (Worthington Diagnostic), and the 2.8 kb
BglII LEU2 fragment of YEpl3 (Broach, et al., Gene 8:121-
25 133, 1979) was joined to the resultant cohesive termini~DNA so prepared was used to transform E. coli strain RRI.
Transformants were selected on the basis of leucine proto-
trophy, since the yeast LEU2 sequence complements the leuB
defect in the E. coli host. Plasmid pB15L was purified
30 from one such transformed colony.
Plasmid pB15L comprises only about 50 base pairs of the 5'
end of the CDC4 coding sequence in addition to the 5' and
3' flanking sequences. A comparision of the maps of
plasmids pB5 and p~15L shows a lack of homology between
35 their respective CDC4 sequences as the junction points of
lZ~20
--19--
the CDC4-LEU2 gene fusion of pB15L are located outside the
region of the CDC4 fragment present in pB5. This lack of
homology prevents recombination between pB5 and the disrup-
ted CDC4 locus in the host cell.
C. Co-transformation of S. cerevisiae
To simultane~usly delete the genomic CDC4 gene and intro-
duce plasmid pB5, yeast cells were co-transformed wi~h
BamHI-cleaved pB15L and intact plasmid pB5. The host
strain to be used in the transformation should be auxotro-
10 phic for tryptophan and leucine in order to select simul-
taneously for plasmid pB5 and the genomic CDC4 disruption.
Strain A2.7.c (MAT~ cdc4-3 trpl leu2-2,112 lysl his3-11,15
canl obtained from a cross of strain A2 (MAT~ leu2-2,112
_s3-11,15 canl; see Szostak, Meth. ln Enzymology 101:
15 245-252, 1983) with strain GEB7 (see Example lA) was used.
In a typical co-transformation experiment, lOml of a
culture of S. cerevisiae A2.7.c in log phase growth were
transformed with approximately 6~g of BamHI-digested pB15L,
l~g pB5, and lO~g calf thymus DNA as carrier. Transforma-
20 tion conditions were as described by Beggs (ibid.). Cellswere plated on a medium lacking leucine and tryptophan.
They were grown overnight at 22 and shifted to 37.
Approximately 30 colonies were obtained. The control
transformation with pB5 alone and selection for tryptophan
25 prototrophy produced approximately 1,000 transformants.
Six co-transformed colonies were analy~ed to verify the
disruption of the CDC4 locus and to test the stability of
the pB5 plasmid. Genomic DNA was isolated from co-trans-
formants by the method of Abraham, et al. (Cold Spring
30 Harbor Symposium Quant. Biol. 47: 989-998, 1983) and was
digested with EcoRI and BamHI, electrophoresed on an
agarose gel, and transferred to nitrocellulose (Southern,
J. Mol. Biol. 98: 503-517, 1975). The blot was probed with
the 2.5 kb BamHI-HindIII fragment from the 5' flanking
9~ZO
-20-
region of CDC4 present in pB15L but absent from pB5.
Figure 4 shows that the probe hybridized to a 6.4 kb
fragment of DNA from untransformed cells (lane b); there is
no EcoRI site within this 6.4 kb BamHI fragment. As the
LE~2 sequence contalns an EcoRI site, disruption of the
CDC4 locus will result in a reduction in size of the
hybridizing band (indicated by arrows in Figure 4). This
is the case for the transformants represented in lanes c,
d, f, g, and h. Lane e shows a somewhat different pattern
10 and retains the genomic-size hand~ indicating that deletion
of the genomic CDC4 did not occur. (The smaller bands seen
in lanes c through h are due to contamination of the
gel-purified probe, as shown by the patterns of the con-
trols in lanes a and b.)
15 The six co-transformants were tested for plasmid stability
by growing on complex medium (YEPD). Cells were grown for
30 generations in liquid YEPD at 25, then plated on YEPD
at 25, and replica plated onto YEPD at 37, tryptophanless
medium, and leucineless medium. Results summarized in
20 Table 1 indicate that all co-transformants except #3 were
100% stable for the plasmid markers on complex media.
(Isolate number 3 is the same co-transformant represented
in lane e of Figure 4).
Further stabllity tests were performed Oll two co-trans-
25 formants, numbers 1 and 2. Testing was performed on 663
and 681 colonies respectively. After growth for 30 gener-
ations on YEPD at 30, all colonies were prototrophic for
tryptophan and leucine.
Co-transformant #1 was tested for growth rate at 22 and
30 was found to grow at ~he same rate as an untransformed
A2.7.c control.
Co-transformant #l has been designated BELLl. It has been
deposited with ATCC under accession number 20698.
lZ9gl~0
-21-
Example 2
Schizosaccharomyces pombe POT1 gene
A. S. pombe POT1 gene as a selectable marker
The Saccharomyces _revisiae TPI1 gene codes for the triose
phosphate isomerase protein and has been obtained by
complementing the ~1 deLiciency (Kawasaki and Fraenkel,
ibid.; Alber and Kawasaki, ibid.). Surprisingly, the
homologous gene from S. pombe has been isolated by comple-
menting the same _. cerevisiae tpil mutation. The S. pombe
10 TPI gene, designated as POTl (for pombe triose phosphate
isomerase), has been cloned from a library described by
Russell and Hall (J. Biol. Chem. 25~: 143-149, 1983) which
contains genomic S. pombe DNA that has been partially
digested with Sau3A and inserted into the vector YEpl3. A
15 preliminary DNA sequence (by the method of Maxam and
Gilbert, Meth. in Enzymology 65: 497-559, 1980) has demon-
strated that the POTl gene codes for the TPI protein and
said protein is homologous with TPI proteins from other
organisms (see Alber and Kawasaki, ibid.). This POTl DNA
20 sequence is given in Figure 5, together with the S. cere-
visiae TPI1 DNA sequence and the respective protein
sequences.
The S. pombe POTl gene is preferred in this example over
the S. cerevisiae TPIl gene as a selectable marker in S.
25 cerevisiae. Foreign genes, such as POTl in S. cerevisiae,
may not function well in an alien host cell and therefore
may necessitate a higher copy number to complement a host
cell defect. Also the selectable POT1 gene on a yeast
plasmid allows for the use of the endogenous TPIl promoter
30 and TPIl terminator (control regions that show no homology
with POTl) for expression of commercially important genes
on the same vector. Because POTl and the flanking regions
of TPIl show no homology, intramolecular recombination and
subsequent plasmid instability are reduced. Finally, the
35 POTl gene is not likely to recombine with the S. cerevisiae
chromosomal DNA because it shares little homology at the
lZ99120
-22-
DNA level with the TPIl sequence and much of the TPI1 gene
has been deleted in the host strains. Thus, POTl contain-
ing plasmids may remain at high copy numbers which are
desirable for the elevated expression of foreign genes of
commercial interest in yeast.
A plasmid comprlsing the POTl gene was identified from the
S. ~ library of Russell and Hall (ibid.) by complemen-
tation of the ~1 mutation in S. cerevisiae strain N587-2D
(~awasaki and Fraenkel, ibid.).
10 A restriction map of this plasmid, pPOT, is depicted in
Figure 6. Because pPOT contains the vector YEpl3, it is
inherently unstable, since it lacks replication functions
necessary for the maintenance of 2-micron plasmids in
yeast. Therefore, the POTl gene may be moved into more
15 competent vectors, such as Cl/l and related vectors that
contain the entire 2-micron plasmid sequences. Plasmid
Cl/l was derived from pJDB248 (Beggs, Nature 275: 104-109,
1978) and pBR322 as described in Example 3 herein. It
contains all of the yeast 2-micron plasmid DNA, a select-
20 able LEU2 gene, and pBR322 sequences.
The POTl gene was isolated from pPOT as a BamHI-XbaI
restriction fragment of nearly 3,400 base pairs and was
inserted into the corresponding polylinker sites of pUC13.
The resulting plasmid is pUCPOT, a partial restriction map
25 of which is shown in Figure 6.
The pUCPOT plasmid was cut with SalI and religated to
delete about 1,800 base pairs of S. pombe and S. cerevisiae
DNA. This resulting pUCPOT-Sal plasmid is illustrated in
Figure 6.
30 The POTl gene was put into Cl/l in the following manner.
As both Cl/l and pUCPOT-Sal have a BglI site in the ampi-
cillin resistance gene and a unique BamHI site at some
other location, the POTl fragment of pycpoT-sal may be
, .
~299~20
-23-
substituted for a portion of the pBR322 region of Cl/l.
Cl/l was cut with BglI and BamHI to liberate a large
fragment of nearly 7,700 base pairs that contains part of
the ampr gene, all 2-micron DNA, and the LEU2 gene.
Likewise, pUCPOT-Sal was cut with BglI and BamHI to liber-
ate a fragment of nearly 3,400 base pairs that contains the
other portion of the ampr gene and the POTl gene. These
two fragments were ligated to form pCPOT, which contains a
"restored" selectable ampr gene, the POTl gene, the LEU2
10 gene, all 2-micron DNA, and the bacterial origin of repli-
cation region from pUC13 (the bacterial origin region from
pUC13 allows for a higher copy number of plasmids in E.
coli than does the origin region of pBR322).
E. coli strain HB101 transformed with pCPOT has been
15 deposited with ATCC under accession number 39685.
The POTl gene may also be inserted into Cl/l-derived
vectors by a similar construction. For example, the
plasmid pFAT5 (FIG. 7) contains an expression unit for the
production of human alpha-l-antitrypsin (AT) inserted into
20 Cl/l. This expression unit, prepared as described in
Example 4 consists of the TPIl promoter, the AT cDNA
sequence, and the TPIl transcription terminator. A re-
striction map of pFAT5 is given in Figure 7.
pFAT5 was cut with BglI and BamHI to liberate a fragment
25 (2,200 base pairs) that contains the AT gene and the TPIl
terminator. Also liberated is a BglI-BamHI fragment which
is identical to the Cl/l BglI-BamHI fragment described
above, except that the fragment from pFAT5 contains an
additional 900 base pairs that comprise the TPIl promoter.
30 This latter pFAT5 piece and the pUCPOT-Sal 3400 bp
Bgll-BamHI fragment (described above) are ligated to form
the plasmid pFPOT, which has the restriction map shown in
Figure 7.
~Z~9~20
-24-
The vector pFPOT was cut at the unique BamHI site to allow
for the insertion of the 2,200 base pair AT gene and TPIl
terminator fragment from pFAT5. The cloning of the 2,200
base pair fragment in the proper orientation into pFPOT
allows for the expression of human AT in this yeast vector.
The properly ligated product is designated pFATPOT, whose
restriction map is given in Figure 7.
B. Disruption of host TPI gene
The Saccharomyces cerevisiae TPIl gene has been cloned and
10 sequenced (Kawasaki and Fraenkel, ibid. and Alber and
Kawasaki, ibid.). The plasmid pTPIC10, comprising the
structural gene for the TPI protein, has been described in
Alber and Kawasaki (ibid.). A BglII site exists at DNA
position 295 in the coding region of TPIl, and another
15 BglII site is located approximately 1,200 base pairs away
in the 5' flanking resion. These BglII sites are conven-
ient cloning sites for deleting part of the TPIl gene and
for inserting another gene, such as the yeast LEU2 gene.
Such a construct can be used to produce a disruption of the
20 genomic TPIl locus in a transformed host.
At approximately -1800 in the 5' flanking region of TPIl is
a Pstl site. In pTPICl0, therefore, the TPIl gene is
flanked by a PstI site on the 5' side and by a SalI site
(in the tetr gene) on the 3' side. This PstI-SalI fragment
25 which contains TPIl was inserted into pUC13 at the PstI and
SalI sites to produce pUCTPI. A restriction map of the
PstI-SalI insert (into pUC13) is given in Figure 8.
The plasmid pUCTPI was then cut with BglII and the two DNA
fragments were separated by electrophoresis. The larger
fragment was purified and phosphatased to prevent self-li-
gation. Into the BglII sites of this DNA was ligated the
yeast LEU2 gene, which was removed from the plasmid YEpl3
(Broach, et al., Gene 8: 121-133, 1979~ as a BglII frag-
ment. The resulting plasmid was pUCTPI-LEU2, which carries
,.
~Z9~120
- 25 - 69140-31
a partial deletion of TPIl and an insertion of LEU2. pUCTPI-LEU2
is depicted in Figure 8.
The plasmid pUCTPI-LEU2 was cut with PstI and BamHI to
linearize the DNA. The yeast sequences were then isolated from
the pUC13 sequences by electrophoresis and gel purification. The
yeast DNA portion depicted in Figure 8 was used to transform S.
cerevisiae strain E2-7B (ATCC No. 20689), which is deficient for
LEU2, in order to "disrupt" the TPIl chromosomal gene (Rothstein,
ibid.). Leu+ transformants were selected on a synthetic (modi-
fied Wickerham's) medium (Mortimer and Hawthorne, in Rose andHarrison, eds., The Yeasts vol. 1, 385-460, Academic Press, 1969)
which contained 3% glycerol and 1% lactate (neutralized to pH 7),
lM Sorbitol, and no leucine. The transformants were screened for
a TPI deficiency by their inability to grow on YEP-Dextrose. One
tpi- transformant was found among the first 99 transformants
screened. This strain was designated as E2-7B~tpi#29 (hereinafter
~tpi#29). ~tpi#29 grew on YEP-3% Glycerol-l~ Lactate but not on
YEP-Dextrose. Enzyme assays (Clifton, et al., Genetics 88: 1-11,
1980) were run on crude cellular extracts and confirmed that
~tpi#29 was lacking detectable levels of triose phosphate iso-
merase activity.
~tpi#29 may be crossed to other yeast strains to form
diploids that are heterozygous for the tpi- deletion. Such
diploids may be sporulated so that other strains deficient for
triose phosphate isomerase can be generated. For example, ~tpi#29
has been crossed to E8-lOA (MAT~ eu2) (a spore segregant of the
cross E2-7BxGK100[ATCC 20669]) to form the diploid, Ell. This
diploid has been sporulated to generate the haploid descendant,
E11-3C, which has the following genotype: MAT~ pep4-3 ~ .
E11-3C has been crossed back to ~tpi#29 to form a diploid, E18,
that is homozygous for the ~ deletion. E18 may be preferred
over ~tpi#29 as a host strain for a plasmid because it has no
amino acid requirements, has larger cells, and grows
~;,z99~z~
-26-
faster. These tpi strains are deleted for the genetic
material which codes for the glycolytic functlon and are,
therefore, es~pected to be nonreverting (i.e., stable)
mutants.
C. Transformation of the POTl gene into S. cerevisiae
tpi deletion strains.
The plasmids pFPOT and pFATPOT were trans~ormed into
~tpi#29 and related tpi deletion strains. The yeast
mutants were grown aerobically overnight to late log phase
10 in YEP-2~ Galactose at 30. Transformation conditions were
as described by Beggs (ibid.), except that the cells were
allowed to recover at 30 for 1-2 hours in lM Sorbitol
containing YEP-3% Glycerol-1% Lactate or YEP-2% Galactose,
instead of YEP-Dextrose, before plating the cells in top
15 agar. The top agar and plates contained synthetic, modi-
fied Wickerham's medium with lM Sorbitol and 2% Dextrose.
After three days at 30, transformants were visible and
were picked out of the agar for replating onto YEPD.
Thereafter, the transformants were maintained on YEPD or
20 other complex media containing dextrose.
Strain E18 transformed with pFATPOT was designated ZYM-3.
It has been deposited with ATCC under accession number
20699.
Stability of pFPOT and pFATPOT on complex media. To study
25 plasmid stability, colonies from a single celi were inocu-
lated into tubes containing YEPD and allowed to grow to a
total population of 109 cells (approximately 30 divisions).
The yeast cells were sonicated to break up clumps, diluted
to appropriate numbers, and plated onto YEP-2% Galactose or
30 YEP-2% Glycerol-1% Lactate, which allows the growth of tpi
cells (with or without the plasmids carrying the POT1
gene). The colonies which arose on YEP-Galactose were then
replica plated onto YEPD to screen for the loss of the
plasmid (l.e., tpi cells which have lost the
. ~
lZ9~1~0
-27-
POT1-containing plasmid will not grow on dextrose). The
xesults, summarized in Table 2, indicate that the pFPOT and
pFATPOT plasrnids are stable in the yeast tpi deletiGn
strains. They are surprisingly much more stable than yeast
plasmids containing centromeres. Centromere-bearing
plasmids (which are low in copy number) are among the mGst
stable plasmids reported for yeast and are generally lost
at a frequency of around 1~ of cells per division on
complex media (see Murray and Szostak, ibid., for a review
10 of centromere plasmid stability). As Table 2 indicates,
the POTl plasmids described herein are lost at a frequency
of less than 1% after 30 divisions on complex media in tpi
deletion strains.
D. Expression of human alpha-1-antitrypsin in S. cerevis-
15 lae using POTl plasmids
To test the use of the POT1 plasmids for enhancing expres-
sion of foreign proteins in a transformed yeast, plasmids
pFATPOT and pFAT5 were used to transform S. cerevisiae
strains ~tpi#29 and E2-7B respectively. Transformed cells
20 were selected in leucineless media containing dextrose.
Cultures were grown at 30 to an O.D.600 of 3-4. Celi
extracts were prepared and assayed for AT as described in
Example 5.
AT produced by pFATPOT/~tpi#29 represented 4-6% of total
25 soluble protein. AT produced by pFAT5/E2-7B represented
2-3% of total soluble protein.
Although plasmid copy numbers are difficult to accurately
measure and represent a population average, empirical
observations of gene product quantities provide an indica-
30 tion of relative plasmid levels, given that the expressionunit (promoter, gene of interest, terminator) remains the
same. pFATPOT therefore appears to be functionally greater
in number than pFAT5, from which it was derived. Because
the two transformed strains are nearly identical
lZ9~0
-28-
genetically (~tpi#29 being derived from E2-7B by
plasmid-directed mutagenesis) and were grown under the same
conditions, these results are indicative of the value of
the herein~ described stable plasmid expression system over
previously described vectors.
TABLE 1: STABILITY OF CDC4 PLASMIDS
Isolate Cv onies CDC4 Trp Leu
l(BELL 1) 123 123 123 123
2 80 80 80 80
3 ~3 80 80 83
4 96 96 96 96
~8 88 88 88
6 115 115 115 115
Cells were grown in liquld complex medium (YEPD) at 25
for 30 generations, then plated on YEPD at 25.
bCells were replica plated to YEPD at 37. Cells lacking
an intact CDC4 gene failed to grow at this (restrictive)
temperature.
20 CCells were replica plated to medium lacking tryptophan.
dCells were replica plated to medium lacking leucine.
~299~2o
-29-
TABLE 2: STABILITY OF POTl PLASMIDS VS. pTPIC10
Total
Experiment Plasmid/Strain Coloniesa TPI ~ Loss
1 pTPIC10/Qtpi#29 234163 30.3
2 pFPOT/Qtpi#29 308 308 0
3 pFATPOT/Qtpi#29 471471 0
4 pFATPOT/E18(ZYM-3) 1104 1104 0
pFATPOT/E18(~Y~l-3l 634 632 0.32
6 pFATPOT/Qtpi#29 426426 0
2-6 pooled data 2943 2941 0.07
aThe plasmid/strain combinations were grown on YEPD plates
until easily visible colonies of approximately lQ4 to 105
cells were seen. These colonies were used to inoculate 6ml
15 Of YEPD liquid medium. The cultures were grow~ aerobically
overnight to a cell density of 1-3x10~ cells/ml and were
plated onto YEP-2%Glycerol-1%Lactate or YEP-2~Galactose.
Each of these media would allow tpi strains to grow,
although the resulting tpi colonies arose more slowly than
20 tpi colonies. Only 100-300 cells were distributed on each
plate so that each colony (whether tpi or tpi+) would be
countable.
bThe colonies were replica plated onto synthetic media
containing dextrose at a 2% final concentration. Cells
25 which had lost the triose phosphate isomerase gene on the
plasmids were unable to grow.
CThe "% Loss" represents the frequency of ce]ls that had
lost the plasmid after nearly 30 divisions in YEPD. The
pooled data for experiments 2 to 6 indicate that the POTl
30 plasmids are extremely stable over these many divisions and
are lost at a combined frequency well below 1% in 30 cell
doublings.
1;~99~20
-30-
Example 3
Preparation of Plasmid C1/_
C1/1 was constructed from plasmid pJDB248 (Beggs, J.,
Nature 275, 104-109 (1978)). The pMB9 sequences were
removed from pJDB248 by partial digestion with Eco RI and
were replaced by pBR322 DNA which was cut with Eco RI. The
restriction map of Cl/1 is given in FIG. 6. The C1/1
plasmid contains the entire 2-micron DNA from yeast (S.
cerevisiae), with a pBR322 insertion at an EcoRI site. It
10 also contains the LFU2 gene.
Example 4
PreDaration of Plasmid pFAT5
_
The gene coding for the predominant form of human alpha-1-
antitrypsin (AT) was isolated from a human liver cDNA
15 library by conventional procedures using the baboon
sequence (Kurachi et al., Proc. Natl. Acad. Sci. USA 78:
6826-6830, 1980; and Chandra et al., Biochem. Biophys. Res.
Comm. 103: 751-758, 1981) as a DNA hybridization probe.
The library was constructed by inserting human liver cDNA
20 into the PstI site of the plasmid pBR322 (Bolivar et al.,
Gene 2: 95-113, 1977). The AT gene was isolated from the
library as a 1500 base pair (bp) PstI fragment. This
fragment was inserted into the PstI site of pUC13 to
produce the plasmid pUC~1. In pUC1, the AT sequence is
25 flanked on the 3' end by XbaI and EcoRI sites in the
polylinker.
The TPI terminator was purified from plasmid pFG1 (Alber
and Kawasaki, ibid) as a XbaI-EcoRI fragment of approxi-
mately 700 bp and inserted into pUCl which had been
30 cleaved with XbaI and EcoRI. This construct was then cut
with EcoRI, and oligonucleotide linkers (sequence:
AATTCATGGAG
5TACCTCCTAG) were added, in multiple linked copies, to
provide a BamHI site to the 3' end of the TPI terminator.
35 The resultant plasmid is known as BAT5.
,.
'1299~20
-31-
The TPI promoter fragment was obtained from plasmid pTPIC10
(Alber and Kawasaki, ibid). This plasmid was cut at the
unique KpnI site, the I'PI coding region was removed with
Bal31 exonuclease, and an EcoRI linker (sequence: GGAATTCC)
was added to the 3' end of the promoter. DigestiGn with
BglII and EcoRI yielded a TPI promoter fragment having
BglII and EcoRI sticky ends. This fragment was then joined
to plasmid YRp7' (Stinchcomb, et al. Nature 282: 39-43,
1979) which had been cut with BglII and EcoRI. The result-
lO ing plasmid, TE32, was cleaved with EcoRI and BamHI toremove a portion of the tetracycline resistance gene. The
linearized plasmid was then recircularized by the addition
of the above described EcoRI-BamHI linker to produce
plasmid TEA32. TEA32 was then cleaved with ~glTI and
15 BamHI, and the TPI promoter was purified as a fragment of
approximately 900 bp.
To construct plasmid pFAT5, plasmid Cl/l was linearized
with BamHI, and was joined to the 900 bp TPI promoter
fragment from TEA32. The resulting construct, known as
20 plasmid F, has a unique BamHI site located at the 3' end of
the TPI promoter. This plasmid was cut with BamHI and a
2200 bp BamHI fragment, comprising the AT coding sequence
and TPI terminator, was purified from BAT5 and inserted
into the BamHI site. The resulting plasmid, known as
25 pFAT5, is illustrated in Figure 7.
Example 5
Assay for Alpha-l-Antitrypsin
As a control, 10 microliters (1 microgram) of a solution of
100 microgram/ml trypsin, 100 microgram (lOO microliters)
of bovine serum albumin and 100 microliters of 0.05 molar
TRIS, pH 8.0 buffer containing lmM benzoylargininoyl-p-ni-
troanilide were mixed, and the increase in absorbance at
405 nm was measured over time in a spectrophotometer. The
absorbance value of this solution was used as a standard
for 100~ trypsin activity. All assayed samples contain
equal concentrations of substrate and bovine serum albumin.
