Yeast expression vectors

There are described a number of plasmid vectors suitable for the expression of genetic material, at various levels in yeasts. The plasmids each comprise a yeast selective marker, a yeast replication origin and a yeast promoter positioned relative to a unique restriction site in such a way that expression may be obtained of a polypeptide coding sequence inserted at the restriction site. The promoters used are derived from the 5' region of a gene coding for a yeast glycolytic enzyme e.g. phosphoglycerate kinase (PGK), or from the 5' region of the yeast TRP1 gene. In one Example a plasmid contains a promoter derived from both the 3' and 5' regions of the PGK gene. The replication systems used involve the yeast 2.mu. replication origin or an autonomous replicating sequence (ARS) stabilized with an ARS stabilizing sequence (ASS). The replication systems allow for a choice of high or low copy number per cell. The promoter sequences allow for a choice of high or low expression level. A kit including vectors having a combination of these alternative features is described. Yeast expression vectors including a gene for coding for human interferon-.alpha. are described.

This invention relates to the field of molecular biology and in particular 
to plasmid vectors suitable for the expression, at various levels, of 
genetic material in yeasts. 
Recently plasmids have been developed that can be used as replication 
vectors in yeast (Struhl et al (1979) PNAS 76 1035 and Kingsman et al 
(1979) Gene 7 141). 
Yeast replication vectors are capable of autonomous replication within a 
yeast host organism and are therefore suitable for introducing foreign DNA 
into yeasts. 
The vectors have also been used to isolate a portion of yeast DNA for 
further analysis. Whilst such known systems are capable of reliable 
replication within a yeast host organism they are not, to a significant 
extent, themselves capable of expression of inserted DNA. 
The production of useful and interesting polypeptides by the exploitation 
of recombinant DNA techniques has hitherto been centred around E. coli as 
a host/vector system (Martial et al (1979) Science 205 602 and Nagata et 
al (1980) Nature 284 316). In general these expression systems have 
depended on a plasmid vector containing an E. coli promoter sequence, a 
ribosome binding site (Shine-Delgarno sequence) and often the first few 
codons of an E. coli coding sequence to which the "foreign" coding 
sequence is joined (Hallewell and Emtage (1980) Gene 9 27). In many cases, 
therefore, fusion proteins are synthesised, although more recently 
procedures have been developed to allow synthesis of "foreign" proteins 
without attached E. coli amino acid sequences (Guarente et al (1980) Cell 
20 543). 
In some situations E. coli may prove to be unsuitable as a host/vector 
system. For example E. coli contains a number of toxic pyrogenic factors 
that must be eliminated from any potentially useful pharmaceutical 
product. The efficiency with which purification can be achieved will, of 
course, vary with the product. Also the proteolytic activities in E. coli 
may seriously limit yields of some useful products (e.g. Itakura et al 
(1977) Science 198 1056). These and other considerations have led to 
increased interest in alternative host/vector systems, in particular the 
use of eukaryotic systems for the production of eukaryotic products is 
appealing. Amongst the eukaryotic organisms suitable for exploitation 
perhaps the easiest to manage is the yeast Saccharomyces cerevisiae. Yeast 
is cheap, easy to grow in large quantities and it has a highly developed 
genetic system. 
It is an object of this invention to provide a yeast vector system capable 
of expressing an inserted polypeptide coding sequence. 
According to the present invention we provide a yeast expression vector 
comprising a yeast selective marker, a yeast replication origin and a 
yeast promoter positioned relative to a unique restriction site in such a 
way that expression may be obtained of a polypeptide coding sequence 
inserted at the restriction site. Preferably the expression vector should 
include at least a portion of a bacterial plasmid. This enables the yeast 
expression vector to be manipulated in a bacterial host system (e.g. E. 
coli). 
We have used two types of yeast replication origin and selective marker 
which are known to the art of yeast replication vector construction. The 
first is based on the replication region of the natural yeast plasmid 
2.mu. (2 micron). This plasmid is cryptic, that is it confers no readily 
detectable phenotype and it is present in about 100 copies per cell. In a 
particular example a 3.25kb fragment from a 2.mu. plasmid derivative 
pJDB219 (Beggs (1978) Nature 275 104) has been used. The fragment 
concerned comprises two EcoRI fragments (2.5kb and 0.75kb) as follows: 
##STR1## 
The LEU2 selective marker surrounds the internal EcorRI site and may be 
disrupted by cleavage at this site. The 2.mu. sequences have been 
described in detail (Hartley and Donelson (1980) Nature 286 560) and the 
LEU2 region has also been the subject of study (Dobson et al (1981) Gene 
16 133). The 3.25kb EcoRI fragment shown above has been used in the 
expression vectors of the present invention as a selection/replication 
module. Expression vectors of the present invention including the fragment 
may be stably maintained in yeast with a copy number of about 50-100 
plasmids per cell. 
The second type of yeast replication origin and marker sequence depends 
upon autonomous replicating sequences (ARS) derived from yeast chromosomal 
DNA. The best characterised of these sequences is 1.45kbp EcoRI fragment 
which contains both the yeast TRP1 gene and an ARS (ARS1) (Kingsman et al 
(1979) Gene 7 141 and Struhl et al (1979) P.N.A.S. 76 1035). This fragment 
has been inserted into pBR322 (a bacterial vector) to give the plasmid 
known as YRp7, which is capable of replication in both E. coli and yeast 
host systems. The ARS-based plasmids are extremely unstable, being lost 
almost entirely in the absence of selection and being maintained at only 
about 50% in the presence of selection, unless a second sequence, an ARS 
stabilising sequence (or ASS) is covalently joined to the ARS sequence. It 
now seems likely that an ASS is a centromeric DNA sequence (L. Clarke and 
J. Carbon (1980) Nature 287 504). A useful fragment is the 1.45kb TRP1: 
ARS EcoRI fragment modified to contain a 627 Sau3a fragment which contains 
an ASS: 
##STR2## 
The EcoRI fragment shown immediately above has been used in the expression 
vectors of the present invention as a selection/replication module. 
Expression vectors of the present invention containing this fragment may 
be stably maintained in yeast with a copy number of about 1 plasmid per 
cell. They segregate in an ordered fashion at mitosis and meiosis. 
According to the present invention there is further provided a yeast 
expression vector wherein the yeast promoter comprises at least a portion 
of the 5' region of a gene coding for a yeast glycolytic enzyme. The yeast 
glycolytic enzyme may be; phosphoglucose isomerase, phosphofructo kinase, 
aldolase, triose phosphate isomerase, glyceraldehyde 3 phosphate 
dehydrogenase, enolase pyruvate kinase, phosphoglycerate kinase. 
Especially preferred is a yeast expression vector wherein the yeast 
promoter comprises at least a portion of the 5' region of the yeast 
phosphoglycerate kinase (PGK) gene. Yeast expression vectors which include 
at least a portion of the 5' region of a yeast glycolytic enzyme are 
susceptible to expression control by varying the level of a fermentable 
carbon source in the nutrient medium of a yeast transformed with such a 
vector. A preferred fermentable carbon source is glucose. In a further 
preferred aspect of the present invention we provide a yeast expression 
vector wherein at least a portion of the 5' region of the PGK gene is 
located upstream of the unique restriction site and at least a portion of 
the 3' region of the PGK gene is located downstream of the unique 
restriction site. The terms "upstream" and "downstream" relate to the 
direction of transcription and translation. 
In an alternative aspect of the invention we provide a yeast expression 
vector wherein the yeast promoter comprises at least a portion of the 5' 
region of the TRP1 gene. 
The expression vectors of the present invention include a yeast replication 
origin and a yeast selective marker. In a preferred embodiment these may 
comprise a fragment containing at least a portion of the yeast plasmid 
2.mu. replication origin and at least a portion of the LEU2 yeast 
selective marker. In an alternative preferred embodiment these may 
comprise a fragment containing at least a portion of an autonomous 
replicating sequence and at least a portion of an autonomous replicating 
sequence stabilising sequence. 
A gene inserted into a yeast expression vector of the present invention may 
be expressed as a fusion protein in the correct reading frame depending 
upon the vector chosen. 
In a preferred embodiment of the present invention we provide a yeast 
expression vector containing at least a portion of a gene coding for a 
polypeptide, preferably human interferon-.alpha.. 
According to another aspect of the present invention we provide a process 
for the production of a polypeptide comprising expressing the said 
polypeptide in a yeast host organism transformed by a yeast expression 
vector containing a gene coding for the said polypeptide. 
According to another aspect of the invention we provide a kit of yeast 
expression vectors. The kit may comprise two or more yeast expression 
vectors of the present invention. The object of providing such a kit is to 
facilitate the molecular biologist's routine expression work by affording 
him a variety of vectors having either high or low copy number per cell 
and either high or low levels of expression. The reading frame of inserted 
DNA may also be selectable by choice of an appropriate vector from the 
kit. In a preferred embodiment we provide a kit comprising four or more 
yeast expression vectors wherein each vector has either of the 
TRP1:ARS1:ASS or LEU2:2.mu. replication origin selective marker and 
replication systems and at least a portion of either of the TRP1 or PGK 5' 
region yeast promoters.

In the drawings restriction endonuclease maps are not drawn to scale. The 
restriction sites are in some cases abbreviated as follows: 
RI=EcoRI 
Pst or P=PstI 
Bam or 
Ba=Bam HI 
Bg=Bgl II 
Pv=Pvu II 
Sal or S=Sal I 
Ha 3=Hae III 
H3=Hind III 
The yeast expression vectors to be described are based on the bacterial 
plasmid pBR322 and use one or other of the yeast replication 
origin/selective marker modules described above. Both modules are EcoRI 
fragments and are therefore readily manipulated. 
We have constructed, using standard techniques, a vector designated pMA3 
which is composed of the E. coli vector pBR322 and the EcoRI fragment 
containing part of the 2.mu. yeast plasmid as described above. This 
plasmid in contrast to many known chimaeric yeast plasmids appears to be 
relatively stable and is maintained in yeast at a high copy number of 
about 50-100 plasmids per cell. 
We have constructed, again using standard techniques, a second vector 
designated pMA91 which is composed of the E. coli vector pBR322 and the 
ARS:ASS EcoRI fragment described above. This plasmid is again stable in 
yeast but is present at a copy number of 1. 
The two vectors pMA3 and pMA91 are described by partial maps in FIG. 1. 
They are not vectors falling within the ambit of the present invention but 
rather important precursors in the production of vectors of this 
invention. In each case in FIG. 1 the thick line indicates the sequence 
derived from yeast DNA. 
pMA3 and pMA91 DNAs were prepared by standard procedures (Chinault and 
Carbon (1979) Gene 5 111). pMA3 was partially digested with EcoRI and the 
products separated on a 1% agarose gel. The 3.25kb double EcoRI fragment 
containing the 2.mu. origin of replication and the LEU2 gene was purified 
by the method of Tabak and Flavell (1978) Nucleic Acids Res. 5 2321). 
Similarly pMA91 was digested to completion with EcoRI and the 1.0kb 
fragment containing the TRP1 gene, ARS1 and an ASS was purified. Two DNA 
fragments were therefore available as replication/selection system 
modules. These are referred to hereinafter as the 2.mu.:LEU2 module and 
the TRP1:ARS1:ASS module respectively. 
In the specific embodiment of the invention to be described in the 
expression vectors contain one of two types of useful functional promoter 
sequence. The first comes from the 5' region of the yeast TRP1 gene and 
the second from the 5' region of the yeast PGK gene. In some of the 
vectors the 3' region of the yeast PGK gene has been included. 
The 1.45kb EcoRI fragment containing the yeast TRP1 gene and the ARS1 has 
been completely sequenced (Tschumper and Carbon (1980) Gene 10 157). The 
organisation of the fragment is shown in FIG. 2 in which the shaded area 
to the left of the TRP1 coding sequence is the 5' region of the gene. The 
5' control region has considerable homology with the analogous regions of 
the iso-1-cytochrome C and GPD genes from yeast (Smith et al (1979) Cell 
16 759) Holland & Holland (1979) JBC 254 5466). In each case there is a 
region containing a purine rich strand of about 30 nucleotides which 
terminates 48-76 nucleotides up-stream from the initiation codon. There is 
also a CACACA sequence 10-15 nucleotides up-stream from the initiation 
codon. This hexanucleotide has been seen only in yeast and its proximity 
to the initiation codon may implicate it in translation, possibly ribosome 
binding, although the existence of ribosome binding sites other than the 
5' CAP-structure in eukaryotes seems in doubt (Naksishima et al (1980) 
Nature 156 226; Stiles et al (1981) Cell 25 277). That signals necessary 
for TRP1 expression are within the 5' flanking region on the 1.45kb 
fragment in plasmid YRp7 (FIG. 2) is certain since the gene is expressed 
with the fragment in both orientations in pBR322. However, it is likely 
that all the signals for maximal TRP1 expression are not present since 
there are only 103 nucleotides 5' to the initiating ATG and most 
eukaryotic genes possess 5' control regions considerably longer than this. 
A 95bp EcoRI-AluI fragment at the very left end of the 1.45kb EcoRI 
fragment (as shown in FIG. 2) should contain signals sufficient for TRP1 
expression since the AluI site is only 8 nucleotides away from the 
initiating ATG. This fragment therefore provides a potentially useful 
"mobile promoter" although additional sequences up-stream from this 
fragment may be necessary for maximal expression. The level of expression 
from the promoter is expected to be relatively low since TRP1 mRNA is 
present in about 0.1-0.01% of total mRNA. 
The second available yeast promoter sequence is that of the 
phosphoglycerate kinase (PGK) gene isolated originally by Hitzeman et al 
(1979), ICN-UCLA SYMP. 14 57). The cloned PGK gene is less well 
characterised than TRP1 but is potentially more useful for higher levels 
of expression in yeast as the single structural PGK gene produces 1-5% of 
total polyA-mRNA and protein. The glycolytic enzyme genes of yeast are 
regulated by carbon source (Maitra and Lobo (1981) JBC 246 475) giving the 
potential of developing a simple control system for the production of 
heterologous proteins in yeast. Analysis of protein and nucleic acid 
sequences have enabled us to define the co-ordinates of the PGK coding 
sequence. 
In summary two plasmids, high and low copy number, and two promoter 
sequences, high and low expression, are available for use in yeast 
expression system. It is one aim of the invention to provide a set of 
vectors suitable for the expression, at various levels, of "useful" genes 
in yeast so that expression characteristics for a given heterologous 
protein can be determined quite simply by selecting the appropriate 
plasmid. 
This set comprises all four pairwise combinations of the two promoters, 
TRP1 and PGK and the TRP1:ARS1:ASS and LEU2:2.mu. replication origin, 
selective marker and replication systems. In addition the kit contains 
molecules based on the PGK expression system which will permit fusion of 
useful polypeptides to the amino-terminal amino acids of yeast 
phosphoglycerate kinase in all three codon reading frames. In PGK based 
expression systems expression can be regulated by the availability of 
glucose. The kit will, therefore, cover all possible expression, selection 
and replication requirements so that any polypeptide coding sequence, 
complete or partial, can be expressed under almost any control condition. 
Table 1 lists the designations of the plasmids in the kit and lists their 
basic properties. 
TABLE 1 
______________________________________ 
Saccharomyces Cerevisiae Expression Kit 
E. coli Selection 
Plasmid & Replication 
Yeast Selection 
Expression 
class/number 
System & Replication 
System 
______________________________________ 
pMA 103 Ampicillin.sup.R 
LEU2:2.mu. TRP1 
PBR322 
pMA 113 Ampicillin.sup.R 
TRP1:ARS1:ASS 
TRP1 
PBR322 
pMA 36 Ampicillin.sup.R 
LEU2:2.mu. TRP1 
PBR322 (extended) 
pMA 200 p 
Ampicillin.sup.R 
" PGK 
PBR322 
pMA 200 f1 
Ampicillin.sup.R 
" PGK 
PBR322 
pMA 200 f2 
Ampicillin.sup.R 
" PGK 
PBR322 
pMA 200 f3 
Ampicillin.sup.R 
" PGK 
PBR322 
pMA 250 p 
Ampicillin.sup.R 
TRP1:ARS1:ASS 
PGK 
PBR322 
pMA 250 f1 
Ampicillin.sup.R 
" PGK 
PBR322 
pMA 250 f2 
Ampicillin.sup.R 
" PGK 
PBR322 
pMA 250 f3 
Ampicillin.sup.R 
" PGK 
PBR322 
______________________________________ 
p = vector expresses by transcription promotion 
f1 = vector produces fusion protein with junction between codons 
f2 = vector produces fusion protein with junction at PGK reading frame +1 
f3 = vector produces fusion protein with junction at PGK reading frame + 
 
EXAMPLE 1 
A number of yeast expression vectors based on the 5' region of the yeast 
TRP1 gene were constructed. The scheme for the construction of yeast 
expression plasmid designated pMA103 is shown in FIGS. 3(a), 3(b). Partial 
restriction endonuclease site maps and sequence information are shown; 
detailed information is in Tschumper and Carbon (1980) Gene 10 157 Hartley 
and Donelson (1980) Nature 286 860 and Sutcliffe (1979) C.S.H.S.Q.B.43 
79). The use of T4 ligase and Bam HI linkers is according to Maniatis et 
al (1978) Cell 15 687 and restriction fragment purification from 
polyacrylamide gels was by the method of Maxam and Gilbert (1980) Methods 
in Enz. 65 499. E. coli transformation was as described in Cameron et al 
P.N.A.S. (1975) 72 3416. The AluI site which defines one terminus of the 
EcoRI-AluI fragment at the 5' end of the TRP1 gene is located only 8 
nucleotides up-stream from the ATG initiation codon. Therefore any 
sequence inserted at this AluI site should be efficiently transcribed from 
the TRP1 promoter. If the sequence also contains an ATG initiation codon 
close to the 5' end we would also expect efficient translation. Therefore 
the EcoRI-AluI fragment (93bp) was purified from other restriction 
fragments produced by an EcoRI and AluI digest of YRp7 after fractionation 
on a 7% acrylamide gel. This fragment was then ligated to pBR322 cleaved 
with EcoRI and BamHI linkers, more ligase and spermidine was then added to 
the reaction. After incubation for 6 h at 20.degree. C. the DNA was phenol 
extracted ethanol precipitated and then digested with BamHI to cleave the 
linker. The BamHI was then removed by phenol extraction and the mixture of 
molecules ligated and used to transform E. coli AKEC28. (AKEC 28=K.12 
trpC1117 leuB6 Thy hsdr.sup.- hsdm.sup.-). Transformant colonies 
containing plasmid which had the small EcoRI-BamHI fragment of pBR322 
replaced by the 93bp EcoRI-AluI fragment from YRp7 with a BamHI linker 
attached to the AluI terminus were identified on the basis of their 
tetracycline sensitivity, their positive signal in a "Grunstein and 
Hogness" hybridisation ((1975) P.N.A.S. 72 3961) with the 1.45kb TRP1:ARS1 
fragment as probe and subsequently by a detailed restriction analysis of 
their plasmid DNA. The plasmid thus formed is pMA101 FIG. 3b). pMA101 was 
then cleaved at its unique EcoRI site, mixed with the 2.mu.:LEU2 
replication/selection module, ligated and used to transform E. coli AKEC 
28 selecting for ampicillin resistance and leucine prototrophy. All 
transformants of this phenotype contained molecules with the same map as 
that shown as pMA103 FIG. 4 or with the 2.mu.:LEU2 module in the other 
orientation. The expression site in pMA103 is BamHI and it transforms 
yeast at a frequency of 10.sup.5 /.mu.g. 
Similarly the TRP1:ARS1:ASS module was inserted into the EcoRI site of 
pMA101 to construct pMA113 but in this case selection was for ampicillin 
resistance and tryptophan prototrophy. A partial map of pMA113 is shown in 
FIG. 5. The yeast transformation frequency is 10.sup.4 /.mu.g with pMA113. 
EXAMPLE 2 
A region of the yeast genome beyond the bounds of the 1.45kb EcoRI TRP1 
fragment was cloned in order to make use of the entire TRP1 5' control 
region. 
DNA sequences beyond the limits of the 1.45kb EcoRI:TRP1 fragment are 
required for maximal expression from the TRP1 promoter. We isolated the 
Hind III fragment that overlaps the 1.45kb EcoRI fragment and which 
contains the entire TRP1 5' control region (shown as the shaded area FIG. 
2). In order to find the size of that Hind III fragment we used the 
smaller of the EcoRI-Hind III fragments from the 1.45kb EcoRI fragment 
(FIG. 2) as a probe in a Southern hybridisation to total yeast DNA cleaved 
with Hind III. A single, approximately 2.0kb band was visible after 
autoradiography. Hind III digested total yeast DNA was then distributed in 
a 1% agarose gel and all the DNA in the size range 1.5-2.5kb was purified 
by the method of Tabak & Flavell (1978) NAR 5 2321) and ligated with Hind 
III digested pTR262 (Roberts et al (1980) Gene 12 123). 700 Tetracycline 
resistant colonies were then screened by the "Grunstein-Hogness" procedure 
using the purified 1.45kb EcoRI:TRP1 fragment as a probe. A single colony 
showed hybridisation with this probe and plasmid DNA was prepared from 
this clone. The plasmid contained a 2.2kb Hind III fragment which 
hydridised specifically to the smaller of the EcoRI-Hind III fragments 
from the 1.45kb EcoRI TRP1 fragment. The nucleotide sequence of the region 
up-stream from the EcoRI site at position-103 (A in ATG is +1) was 
determined by standard M13/dideoxy sequencing procedures (Sanger et al 
(1977) P.N.A.S. 74 6463) and is shown in FIG. 6. In this Figure the 
nucleotide sequence from 169 to 275 was after Ischumper and Carbon (1980) 
Gene 10 157. Potentially important features are underlined. New sequence 
data includes all sequences not overlined. In order to construct a 
derivative of pMA103 that contains the entire TRP1 5' control region a set 
of constructions were performed as outlined in FIG. 7. (In this Figure the 
thick lines indicate DNA derived from yeast). The 2.2kb Hind III fragment 
was purified by the method of Tabak & Flavell (1978) NAR 5 2321) and 
inserted into the Hind III site of pBR322 to form plasmid pMA33. The small 
EcoRI fragment from pMA33 was purified and then inserted into the unique 
EcoRI site of pMA101 (see FIG. 3(b)). The orientation of the fragment was 
checked to ensure reconstitution of the TRP1 5' region. The resulting 
plasmid is designated pMA35. pMA35 was then cleaved partially with EcoRI 
and the 2.mu.:LEU2 module inserted. Recombinant molecules were screened 
for the presence of the 2.mu.:LEU2 fragment at the pBR322 EcoRI site 
rather than the EcoRI site at - 103. Such a molecule is pMA36 (FIG. 7). 
EXAMPLE 3 
A number of yeast expression vectors based on the 5' region of the yeast 
PGK gene were constructed. 
The yeast PGK gene exists on a 2.95kb Hind III fragment in the yeast-E.coli 
vector, pMA3, (FIG. 1). A partial restriction map of this molecule is 
shown in FIG. (8a). The PGK Hind III fragment was isolated from a Hind III 
fragment collection inserted into .lambda.762 (Murray et al (1977) 
Molec.gen.Genet 150 53) using a .sup.32 P labelled cDNA prepared from 
yeast poly-A RNA. The fragment is identical to the "3.1kb" fragment 
described in Hitzeman et al (1980) JBC. 255, 12073 in plasmid pB1 and in 
hybrid selection translation experiments (Ricciardi et al (1979) P.N.A.S. 
76 4927) the fragment was shown to encode a protein of identical mobility 
to pure PGK in SDS-PAGE. A restriction map of the 1.95kb fragment is shown 
in FIG. 8(b). 
The amino acid sequence of residues 270-400 of yeast PGK is shown in FIG. 
9(a). The sequence was determined by manual and automated Edman 
degradation. The amino acid sequence data allowed us to match restriction 
sites on the 2.95kb Hind III fragment with groups of two or three amino 
acids in the protein sequence. FIG. 9(b) shows the relevant restriction 
sites and those sites are marked on the amino acid sequence in FIG. 9(a). 
The positions of the four sites on the restriction map and the protein 
sequence are congruent allowing us to orientate the gene with respect to 
the sites on the 1.95kb Hind III fragment. Given that the molecular weight 
of PGK is 40Kd (415 amino acid residues) and assuming that there are no 
large introns we can also predict the positions of the 5' and 3' ends of 
the coding sequence. The extent of the coding sequence, assuming 
colinearity, is shown in FIG. 8(b), the initiation codon is about 900 
nucleotides to the left of the EcoRI site and the termination codon about 
300 nucleotides to the right. 
The position of the 5' end of the PGK transcript was located by the S1 
protection method (Berk and Sharp (1978) P.N.A.S. 75 1274). The 1.2kb Hae 
III fragment spanning the 5' end of the coding sequence (FIG. 8(b) was 
purified from an agarose gel and hybridised to total yeast RNA. The 
hybrids were treated with various concentrations of S1 nuclease and the 
products were analysed on a 1.5% agarose gel by Southern hybridisation 
using the 1.95kb Hind III fragment as probe. FIG. 10 shows that the size 
of the single protected fragment was 680bp. In this Figure the 
concentrations of S1 in each lane are as follows (a) 25 units (b) 50 units 
(c) 100 units. Lane (d) has the 1.2kb Hae III fragment untreated. On the 
basis of our previous mapping data this would place the 5' end of the PGK 
transcript about 960bp to the left of the EcoRI site on the 2.95kb Hind 
III fragment. This agrees well with our estimate of the position of the 
initiation codon and suggests that if there are any introns between the 5' 
end of the transcript and the Bgl II site than they are very small. 
The 5' "control" region of the PGK gene is in a region that contains very 
few convenient restriction sites, making the design of a sequencing 
strategy relatively difficult. We adopted a procedure to solve this 
problem that may be of general use. Plasmid pMA3-PGK was digested with Sal 
I (FIG. 8) and then with exonuclease BAL 31 to remove about 500bp from 
each end. This resulted in the loss of the two small Sal I fragments and 
the creation of a series of deletions starting at the leftmost Sal I site 
in the PGK sequence and the Sal I site in pBR322 and ending around the 
initiation codon in PGK and nucleotide 1150 in pBR322 respectively. These 
deleted molecules were then ligated in the presence of a 50-fold molar 
excess of Bam HI linkers and then used to transform AKEC28 to LEU.sup.+, 
Amp.sup.R. The general structure of these molecules, designated the pMA22a 
deletion series is shown in FIG. 11. Seventy of these deleted molecules 
have been analysed by measuring the length of the EcoRI-BAM HI fragment 
containing the 5' region of the PGK gene. While they show a mean length of 
1.5kb they have a spread of 500 nucleotides. This collection therefore 
provides a number of molecules that are useful for the sequence analysis 
of the 5' region of the PGK gene. Two such deletions, C and W are shown in 
FIG. 8(b). The small EcoRI-Bam HI fragments from these molecules were 
purified and cloned in M13mp701 and sequenced by the dideoxy-chain 
termination method Sanger et al (1977) P.N.A.S. 74 5463, starting in each 
case at the Bam HI site and elongating towards the EcoRI site. The 
nucleotide sequence of 226 nucleotides up-stream from the inition codon 
and the first seven codons are shown in FIG. 12. (In this Figure the box 
marks the approximate position of the 5' end of the transcript). The 
sequence was confirmed by sequencing four other deletions with overlapping 
end-points (data not shown). 
The pMA22a deletion series constitutes a collection of molecules amongst 
which are many potential PGK based expression vectors. Each with a 
different sized small EcoRI-BAM HI fragment and therefore each with a 
different "amount" of the PGK 5' region. They all have unique Bam HI sites 
at which genes may be inserted and expressed. FIG. 13 shows the sequence 
of the PGK gene from -226 to +624 with the positions of various deletion 
end-points marked. The deletion end point numbers (FIG. 13) are carried 
through to the name of the plasmid that bears that deletion e.g. plasmid 
pMA279 is a pMA22a deletion with the deletion end-points between the 
codons for amino acids 32 and 33. At that position the Bam HI linker of 
sequence CCGGATCCGG has been inserted. At each of the deletion end-points 
there is the same BAM HI linker with the exception of pMA301 which has the 
Bgl II linker CAAAAGATCTTTTG inserted at position -1. This Bgl II linker 
was used in order to increase the A content of the region around the 
initiating ATG. 
Clearly plasmids pMA278 and pMA301 will produce transcriptional fusions 
with any coding sequence inserted at their expression sites and are 
therefore of the pMA200p type in Table 1, whereas all the others will 
produce both transcriptional and translational fusions (i.e. fusion 
proteins will be made). pMA230 is a +1 (reading frame) fusion vector, 
pMA283 is an in frame (+3) fusion vector. The molecules are of the 
pMA200f1, f2 and f3 type in Table 1. 
EXAMPLE 4 
We have constructed a PGK based expression vector designated pMA3013 which 
comprises both 5' and 3' regions from the yeast PGK gene. 
We have determined the nucleotide sequence of the 3' region of PGK by 
standard procedures and this is shown in FIG. 14. FIG. 15 shows the scheme 
for constructing pMA3013. Plasmid pMA3-PGK was cut with Bgl II and Pst I 
and the fragment containing the 3' end of the PGK gene (shown as a wavy 
line in FIG. 15) was purified by the method of Tabak and Flavell (1978) 
NAR 5 2321). This fragment was then ligated with Bgl II and Pst I cleaved 
pMA301 and the mix was used to transform E.coli strain AKEC28 to 
ampicillin resistance and leucine prototropy. Resulting clones were 
screened for a plasmid with three Hind III sites. Such a plasmid is 
pMA3013. pMA3013 has a unique Bgl II expression site flanked by the PGK 5' 
and 3' regions. 
EXAMPLE 5 
The various yeast expression vectors described have been tested using a 
human interferon-.alpha. as a heterologous, potentially useful coding 
sequence. The sequence is contained on a Bam HI fragment that is a 
derivative of plasmid N5H8 originally constructed by Prof. D. C. Burke, 
University of Warwick. Our modification places a Bam HI site followed by 
an ATG at a position corresponding to amino acid S15 in the interferon 
signal sequence. The nucleotide sequence of this Bam HI fragment is given 
in FIG. 16. The Bam HI fragment can be used in transcription fusion 
constructions because it has its own translation initiation codon and it 
can also be used in vectors designated to produce fusion proteins. This 
fragment was inserted into the expression sites of a variety of molecules 
the general structure of which is shown in FIG. 17. The resulting 
molecules were then introducted into yeast strain MD40-4C 
(MD40-4C=.alpha.ura2 trp1leu2-3 leu2-112 his3-11 his3-15) by standard 
transformation procedures (Hinnen et al (1978) P.N.A.S. 75 1919) and the 
levels of interferon produced in yeast were measured using bovine EBTr) 
cells in a viral RNA reduction assay with Semliki Forest virus (SFV) as 
the challenge (Atherton & Burke, (1975) J. Gen.Virol 29 197). Table 2 
shows levels of interferon produced in yeast cells containing various 
recombinant molecules. 
TABLE 2 
______________________________________ 
Interferon Expression from Various Vectors 
Molecules of a 
Expression Interferon per 
Vector 5' Region 3' Region 
cell* 
______________________________________ 
pMA 103 TRP1 -- 600 
pMA 36 TRP1 (extended) 
-- 1.7 .times. 10.sup.4 
pMA 278 PGK (.DELTA.278) 
-- 2.0 .times. 10.sup.4 
pMA 301 PGK (.DELTA.301) 
-- 1.5 .times. 10.sup.7 
pMA 3013 PGK (.DELTA.301) 
PGK 1.0 .times. 10.sup.7 
pMA 230 PGK (.DELTA.230) 
-- 1.5 .times. 10.sup.7 
pMA 3 (control) 
-- -- &lt;50 (not 
detectable) 
______________________________________ 
*These figures assume 2 .times. 10.sup.8 units of interferon/mg. 
It can be seen that there is a considerable range of expression 
capabilities in the system depending on which expression vector is used. 
The highest levels are obtained with the fusion protein vector pMA230 and 
the transcription vectors pMA301 and pMA3013 in which as much as 2% of the 
total cell protein is present as interferon protein (FIG. 18). This Figure 
shows Coomassie stained SDS-PAGE protein profiles in which the lanes 
contain 
(a) Total protein from MD40-4c containing pMA230 
(b) Total protein from MD40-4c containing pMA230/interferon 
(c) Protein from MD40-4c containing pMA230/interferon after partial 
purification on an NK2 column. The position of molecular weight markers 
are shown. An arrow marks the position of the PGK-interferon fusion 
protein. 
All interferon producing plasmids are maintained stably for at least 40 
generations as measured by the proportion of cells in the population with 
the phenotype conferred by the expressing plasmid. 
EXAMPLE 6 
PGK in yeast is "induced" by glucose, therefore it was of interest to 
determine whether the structures necessary for the recognition of this 
regulatory system are present on the 1500 nucleotide PGK fragment in for 
example pMA230 and if so whether human interferon-.alpha. expression could 
be regulated by glucose. 
Yeast strain MD40-4c containing pMA230 with the interferon-.alpha. sequence 
inserted at the Bam HI site was grown in rich medium with acetate as 
carbon source for twelve generations to a density of 2.times.10.sup.6 
cells/ml. These cells were used as inocula for two flasks of fresh medium. 
One containing glucose as carbon source and the other acetate. A second 
batch of cells grown on glucose was used to inoculate a fresh glucose 
culture. Therefore there were three inoculum/culture conditions: 
acetate/acetate; acetate/glucose; glucose/glucose. Aliquots of these 
cultures were taken at various intervals, extracts were prepared and 
interferon levels were assayed. The results of these assays are given in 
FIG. 19 in which =glucose/glucose; =acetate/acetate and 
.DELTA.=acetate/glucose. The data in FIG. 19 show that the glucose/glucose 
culture contains relatively high interferon levels while the 
acetate/acetate culture has low levels over the course of the experiment. 
The acetate/glucose culture exhibits increasing levels of interferon after 
the cells are transferred to glucose medium (time 0, FIG. 19). This 
induction of interferon occurs over a period of about 8 hrs. and the 
levels of interferon produced by cells grown on glucose are 20-30 fold 
higher than in cells grown on acetate. 
While these results strongly suggest that carbon source control of 
interferon levels is being mediated by the 5' control region of the PGK 
gene it is important to establish that there is no difference in plasmid 
stability in cells grown on acetate or glucose. Therefore total DNA was 
prepared from aliquots of yeast cells taken at various points during the 
experiment described in FIG. 19. The DNA was digested with EcoRI and 
fragments were separated on a 1% agarose gel. The fractionated bands were 
then blotted onto nitrocellulose and hybridised with .sup.32 P-YRp7. The 
pBR322 component of this probe served to measure levels of plasmid in the 
yeast DNA preparations while the sequence of the 1.45kb fragment were used 
to establish a control for amounts of DNA, transfer efficiencies and 
hybridisation efficiencies. In addition to this Southern blot analysis the 
proportion of Leu.sup.+ cells in the aliquots was measured by comparing 
colony counts on media with and without leucine. In all cases Southern 
hybridisation profiles were identical and &gt;99% of cells were Leu.sup.+ 
(data not shown) showing that growth on acetate or glucose has no effect 
on plasmid copy number or stability.