Methods of regulating protein glycosylation

Methods for producing a heterologous protein or polypeptide are disclosed. A preferred method utilizes a fungal cell carrying a defect in a gene whose product is required for the addition of outer chain oligosaccharide moieties to glycoproteins, the cell transformed with a first DNA construct comprising a regulated promoter followed downstream by a DNA sequence which complements the defect, and a second DNA construct comprising a second promoter followed downstream by a DNA sequence encoding a secretion signal and a DNA sequence encoding a heterologous protein or polypeptide. A yeast cell having a Mnn9.sup.- phenotype and capable of producing colonies of normal morphologies in the absence of osmotic stabilization is also disclosed.

BEST MODE FOR CARRYING OUT THE INVENTION 
Prior to setting forth the invention, it may be helpful to an understanding 
thereof to set forth definitions of certain terms to be used hereinafter. 
DNA construct: A DNA molecule, or a clone of such a molecule, either 
single- or double-stranded, which has been modified through human 
intervention to contain segments of DNA combined and juxtaposed in a 
manner which as a whole would not otherwise exist in nature. 
Mating-type regulatory element: A DNA sequence to which yeast MAT gene 
products will bind, resulting in the repression of expression of genes 
linked to the sequence. The terms "operator" and "operator sequence" are 
also used to describe these elements. 
Mnn9.sup.- phenotype: A yeast cell phenotype characterized by the 
production of exported or secreted glycoproteins which run on 
SDS-polyacrylamide gels as discrete, homogeneous species. These 
glycoproteins lack the hyperglycosylation characteristic of glycoproteins 
produced by wild-type yeast cells. 
Modified core oligosaccharide: An N-linked carbohydrate side chain of a 
glycoprotein which contains two N-acetylglucosamine (GlcNAc) residues 
coupled to from 9 to 13 mannose residues. Representative modified core 
oligosaccharide structures are illustrated in FIGS. 1A and 1B. 
Regulated promoter: A DNA sequence which directs transcription of a linked 
DNA sequence at levels which vary in response to external stimuli. 
Regulated promoters are, in general, either "on" (maximum transcription 
level) or "off" (little or no transcription), depending on a cell's 
environment, although in some cases intermediate levels may be obtained. 
Secretion signal: A DNA sequence encoding a secretory peptide. A secretory 
peptide is an amino acid sequence characterized by a core of hydrophobic 
amino acids which acts to direct the secretion of a mature polypeptide or 
protein from a cell. Secretory peptides are typically found at the amino 
termini of newly synthesized proteins and are cleaved from the mature 
protein during secretion. 
As noted above, the present invention describes methods by which 
heterologous glycoproteins may be secreted from fungal cells with modified 
core glycosylation. The methods described herein are particularly 
advantageous in that they allow the production of glycoproteins containing 
modified core oligosaccharide moieties through the use of a host cell 
having a defect in a gene whose product is required for the addition of 
outer chain oligosaccharide moieties to glycoproteins. These methods do 
not rely on the more expensive methods of post-production modification of 
the glycoproteins nor do the methods rely on the addition of 
glycosylation-inhibiting factors to the cells or cell products. 
Fungal cells, including species of yeast (e.g., Saccharomyces spp., 
Schizosaccharomyces pombe), or filamentous fungi (e.g., Aspergillus spp., 
Neurospora spp.) may be used as host cells for the present invention. The 
yeast Saccaromyces cerevisiae, for example, carries genes (MNN7-MNN10) 
which enable yeast cells to add outer chain oligosaccharide moieties to 
the oligosaccharide core structure of secretion-directed proteins. Mutants 
with defects in these genes (mnn7-mnn10) do not add outer mannose moieties 
to glycoproteins, resulting in glycoproteins with a homogeneous amount of 
glycosylation. 
A gene required for the addition of outer chain oligosaccharide moieties 
may be identified in a number of ways. One method, for example, has been 
described by Ballou et al. (J. Biol. Chem. 255:5986-5991, 1980). In this 
method, antibodies are raised against the mannose moieties present on the 
surface of yeast cells, preferably against those mannose moieties present 
on the surface of yeast mnn2 mutant cells. Yeast cells, preferably haploid 
mnn2 cells, are mutagenized. The antibodies, preferably labeled 
antibodies, are then used to identify populations amongst the mutagenized 
cells which fail to bind antibody. These mutants are then crossed with 
each other to establish genetic complementation groups. Complementation 
between two mutations results in a diploid with the pre-mutagenized parent 
phenotype. A preferred method for screening for the pre-mutagenized parent 
phenotype is to use antibodies directed against the mannose moieties of 
the parent strain. By this method, four complementation groups (designated 
mnn7-mnn10) are established. Glycosylation mutants of other fungi may be 
isolated using this method of mutant identification. 
An alternative method is to use the properties of concanavalin A, a lectin 
which has a high specificity for oligosaccharides containing three or more 
mannose residues. Mutagenized cells are passed over a concanavalin A 
column. Cells which exhibit cell surface glycoproteins with less than 
three mannose residues will elute off such a column and may be isolated 
from the effluent. Similarly, a third method consists of identifying 
glycosylation mutants using labeled concanavalin A which will bind to 
cells which exhibit cell surface glycoproteins with more than three 
mannose residues and not to glycosylation mutants exhibiting glycoproteins 
with three or fewer mannose moieties. 
A fourth method of isolating glycosylation mutants is to introduce a DNA 
construct comprising a sequence encoding a secretion signal followed by a 
heterologous gene or cDNA encoding a glycoprotein, preferably a 
glycoprotein known to be highly glycosylated, into a host strain. The 
transformed host strain is mutagenized and the mutagenized population is 
screened for the production of heterologous protein with reduced 
glycosylation. 
A preferred method of screening for mnn9 mutants involves the unexpected 
response of mnn9 cells when assayed for proteinase B. Briefly, cells which 
are prB.sup.+ (cells which have active proteinase B) are grown on a solid 
complex-rich (chemically undefined) medium, comprising a nitrogen source, 
inorganic salts, vitamins, a carbon source, and an osmotic stabilizer, or 
under selective conditions, on solid synthetic medium supplemented with an 
osmotic stabilizer. Solid media include those which contain agar, agarose, 
gelatin or similar agents. A particularly preferred complex-rich medium is 
YEPDS (2% yeast extract, 1% peptone, 2% glucose, 1 M sorbitol). Colonies 
grown on complex-rich media are permeabilized by spheroplasting or 
exposure to fumes of a solvent which affects membranes without causing 
widespread lysis. Suitable solvents include toluene, chloroform or other 
similar solvents generally known in the art. Colonies grown on synthetic 
medium are either grown on or transferred to filters (to compensate for 
the low pH of synthetic medium), such as nitrocellulose filters or paper 
filters, and lysed by exposure of the filters to zymolyase or preferably 
by immersion in liquid nitrogen. Colonies grown on or transferred to paper 
filters may be permeabilized by exposure to solvent fumes. The filters are 
then laid on solid rich medium. The permeabilized colonies are then 
overlayed with top agar, comprising azocoll, preferably approximately 10 
mg/ml of top agar and having pH greater than 4.0 and less than 7.4, 
preferably about 7.0. The plates are incubated at a temperature between 
20.degree. C. and 40.degree. C., preferably at 37.degree. C., for between 
3 hours and 24 hours, preferably within the range of 5 to 8 hours. 
Colonies which exhibit a Mnn9.sup.- phenotype form a clear halo around the 
colonies. 
The mnn7-mnn9 mutants are then used to clone the corresponding genes. By 
way of example, the MNN9 gene was cloned from a pool of yeast DNA 
fragments, more specifically, a pool of genomic yeast DNA fragments. 
Within the present invention, a library of DNA fragments cloned into a 
yeast/E. coli vector is made, for example, by the method described by 
Nasmyth and Reed (Proc. Natl. Acad. Sci. USA 77:2119-2123, 1980). Briefly, 
genomic yeast DNA is made and partially digested with a suitable 
restriction enzyme to generate fragments that are between about 5 kb and 
about 20 kb. Preferred enzymes are four-base cutters such as Sau 3A. The 
generated fragments are then ligated into a suitable yeast/E. coli shuttle 
vector which has been linearized by digestion with the appropriate 
restriction enzyme. It is preferable to dephosphorylate the linearized 
vector to prevent recircularization. Suitable vectors include YEp13 
(Broach et al., Gene 8:121-133, 1979), YRp7 (Stinchcomb et al., Nature 
275:39-45, 1979), pJDB219 and pJDB248 (Beggs, Nature 275:104-108, 1978), 
YCp50 (Kuo and Campbell, Mol. Cell. Biol. 3:1730-1737, 1983) and 
derivatives thereof. Such vectors will generally include a selectable 
marker. Selectable markers may include any dominant marker for which a 
method of selection exists. Such selectable markers may include a 
nutritional marker, for example, LEU2, which allows selection in a host 
strain carrying a leu2 mutation, or a gene which encodes antibiotic 
resistance, for example, chloramphenicol transacetylase (CAT), which 
enables cells to grow in the presence of chloramphenicol. Alternatively, 
they may include an "essential gene" as a selectable marker (Kawasaki and 
Bell, EP 171,142), for example, the POT1 gene of Schizosaccharomyces 
pombe, which complements a tpi1 mutation in the host cell, allowing cells 
to grow in the presence of glucose. It is preferable to transform the 
ligation mixture into an E. coli strain, for example, RR1 (Bolivar et al., 
Gene 2:95-113, 1977), to amplify the library of yeast DNA fragments. To 
facilitate selection of transformants in yeast, plasmid DNA is made from 
the E. coli transformant library and is introduced into yeast cells which 
are genotypically mnn9 and may contain a genetic defect which is 
complemented by a suitable marker present on the yeast/E. coli shuttle 
vector. Transformant colonies are selected by an appropriate selection 
method for the presence of the plasmid in the host cell. The transformants 
are then screened for the complementation of the mnn9 deficiency. 
Screening methods may include using antibodies directed against the 
mannose moieties of wild-type yeast cells, and determining the 
carbohydrate content of the mutants. A preferred method of screening for 
the complementation of the mnn9 mutation utilizes the proteinase B assay 
described above. Colonies containing a cloned MNN9 gene are identified by 
the absence of a clear halo. 
MNN9 gene clones may be confirmed to be plasmid borne, as opposed to 
revertants, by testing for the loss of plasmid. Plasmid loss is achieved 
by growing the yeast cells under nonselective conditions to determine if 
the Mnn.sup.+ phenotype is lost with the loss of the plasmid. DNA from the 
positive clones is made using methods known in the art (for example, 
Hartig et al., Mol. Cell. Biol. 6:1206-1224, 1986). Restriction mapping 
may be carried out to determine the smallest fragment of the genomic 
insert needed to complement the mnn9 deficiency. The DNA sequence may also 
be determined for the cloned gene. 
The genes or cDNAs encoding MNN7, MNN8 and MNN10 may be cloned using a 
library of yeast DNA fragments as described above to complement a genetic 
deficiency in MNN7. MNN8, or MNN10, respectively, in a host strain. 
However, the preferred screening method used for identifying MNN9 gene 
clones may not be as well suited for identifying MNN7, MNN8, or MNN10 gene 
clones. In this case, preferred screening methods include using antibodies 
directed against wild-type oligosaccharide moieties and the determination 
of oligosaccharide content (as described in Ballou, ibid., 1970, and 
Ballou, ibid., 1980). Positive clones may be further characterized as 
described for the MNN9 gene clone. 
According to the present invention, the addition of outer chain 
glycosylation may be controlled through the use of a regulated promoter to 
drive the expression of a cloned MNN7, MNN8, MNN9 or MNN10 gene. Cells 
which exhibit the mnn7-mnn10 phenotype are slow to grow and are 
exceptionally sensitive to cell lysis in the absence of osmotic support. 
The regulated expression of these genes during active cell growth will 
allow the cells to grow in a wild-type manner with wild-type glycosylation 
of glycoproteins and cell wall components. The regulated promoter is then 
turned off to permit production of a heterologous protein or polypeptide 
with only core glycosylation. The expression unit, comprising a regulated 
promoter fused to the cloned MNN gene, may be plasmid borne, in which case 
the expression unit will complement a corresponding mnn mutation in the 
host strain. Alternatively, the expression unit may be integrated into the 
host genome. 
The use of regulated promoters to drive the expression of both heterologous 
and homologous DNA sequences in yeast is well known in the art. The 
regulation of such sequences is realized through the use of any one of a 
number of regulated promoters. Preferred regulated promoters for use in 
the present invention include the ADH2 promoter (Young et al., in Genetic 
Engineering of Microorganisms for Chemicas, Hollaender et al., eds., New 
York:Plenum, p. 335, 1982), and the ADH2-4C promoter (Russell et al., 
Nature 304:652-654, 1983). 
A particularly preferred promoter is the MF.alpha.1 promoter (Kurjan and 
Herskowitz, Cell 30:933-943, 1982). Other particularly preferred promoters 
are the SXR promoters (described in co-pending, commonly assigned U.S. 
patent application Ser. No. 889,100, which is incorporated by reference 
herein in its entirety), which combine one or more mating-type regulatory 
elements and a constitutive promoter (e.g., the TpI1 promoter). 
Mating-type regulatory elements may be isolated from the upstream regions 
of yeast genes which are expressed in a mating-type specific manner or may 
be constructed de novo and are generally from about 20 base pairs to about 
32 base pairs in length. Promoters of this type are used in a yeast strain 
that contains a conditional mutation in a gene required for the expression 
of the silent mating-type loci. The term "conditional mutation" is 
understood to mean a mutation in a gene which results in the reduction or 
lack of the active gene product under one set of environmental conditions 
and a normal (wild-type) level of the active gene product under a 
different set of environmental conditions. The most common conditional 
mutations are temperature-sensitive mutations. Temperature-sensitive 
mutations in genes required for the expression of the silent mating-type 
loci including the sir1, sir2, sir3 and sir4 mutations. The 
temperature-sensitive mutation sir3-8 is particularly preferred. 
The sir3-8 mutation (also known as ste8, Hartwell, J. Cell Biol. 85:811, 
1980) is a temperature-sensitive mutation which blocks the expression of 
information at the HML and 11MR 1loci at 25.degree. C. while at 35.degree. 
C. the expression of these loci is not blocked and the information at the 
HML and HMR loci is expressed. The mating-type regulatory elements used in 
the SXR promoters are derived from the STE2 gene. These elements, placed 
within a promoter, will regulate the expression of the gene of interest 
dependent upon the presence or absence of an active SIR3 gene product. 
Yeast host strains for use in the present invention will contain a genetic 
defect within the MNN7, MNN8, MNN9 or MNN10 genes, resulting in the 
inability of the cell to add outer chain oligosaccharide moieties. This 
defect may be, for example, a mnn9 mutation as described by Ballou et al. 
(ibid., 1980) or, preferably, a gene disruption, such as a disruption of 
the MNN9 gene. A gene disruption may be a naturally occurring event or an 
in vitro manipulation in which the coding sequence of a gene is 
interrupted, resulting in either the production of an inactive gene 
product or no gene product. The interruption may take the form of an 
insertion of a DNA sequence into the coding sequence and/or the deletion 
of some or all of the coding sequence resulting in no protein product or 
premature translation termination. A gene disruption, comprising insertion 
of a DNA sequence and deletion of native MNN coding sequence, will not 
revert to wild-type as has been found with the mnn point mutations. 
Gene disruptions may be generated essentially as described by Rothstein (in 
Methods in Enzymology, Wu et al., eds., 101:202-211, 1983). A plasmid is 
constructed which comprises DNA sequences which are homologous to the 
region in the genome containing, for example, the MNN9 gene, preferably 
including the coding sequence and both 5' and 3' flanking sequences of the 
cloned MNN9 gene. The sequence encoding the MNN9 gene is disrupted, 
preferably by the introduction of a selectable marker. This selectable 
marker may interrupt the coding sequence of the gene, or it may replace 
some or a1 of the coding sequence of the gene. The selectable marker may 
be one of any number of genes which exhibit a dominant phenotype for which 
a phenotypic assay exists, to enable deletion mutants to be selected. 
Preferred selectable markers are those which may complement host cell 
auxotrophy, provide antibiotic resistance or enable a cell to utilize 
specific carbon sources, including URA3 (Botstein et al., Gene 8:17, 
1979), LEU2 (Broach et al., ibid.) and HIS3 (Struhl et al., ibid.). The 
URA3 marker is particularly preferred. Other suitable selectable markers 
include the CAT gene, which confers chloramphenicol resistance on yeast 
cells, or the lacZ gene, which results in blue colonies due to the 
expression of active .beta.-galactosidase. Linear DNA fragments comprising 
the disrupted MNN gene, preferably isolated from the vector fragments, are 
introduced into the host cell using methods well known in the literature 
(e.g., Beggs, ibid.). The yeast host cell may be any one of a number of 
host cells generally available, for example, from the American Type 
Culture Collection, Rockville, Md. or the Yeast Genetic Stock Center, 
Berkeley, Calif. The host cell may carry a genetic defect which is 
complemented by the selectable marker used to disrupt the MNN coding 
sequence. Suitable yeast strains include SEY2101 (MATa ade2-101 leu2-3,112 
ura3-52 suc2-.DELTA.9 ga12) or ZY100 (MATa ade2-101 leu2-3,112 ura3-52 
suc2-.DELTA.9ga12 .DELTA.pep4::CAT). Integration of the linear fragments 
comprising selectable markers is detected by selection or screening using 
the dominant marker and proven by, for example, Southern analysis 
(Southern, J. Mol. Biol. 98:503-517, 1975) and phenotype testing. 
It is preferable that the host cell contain a deficiency in the MNN1 gene 
as well as a deficiency in the MNN7, MNN8, MNN9, or MNN10 gene. A 
deficiency in the MNN1 gene eliminates the terminal 
.alpha.1.fwdarw.3-linked mannose in all of the N-linked glycoproteins of 
the cell (for review, see Ballou, ibid., 1982). This mutation in 
combination with, for example, a mnn9 mutation, will allow the host cells 
to produce glycoproteins containing modified core oligosaccharide 
structures with 9 or 10 mannose moieties. A mnn1 mutation may be 
introduced into a mnn9 strain, preferably a strain carrying a mnn9 gene 
disruption, by crossing it into the desired strain or preferably by 
disrupting the MNN1 gene in a strain carrying a mnn9 disruption. To 
disrupt the MNN1 gene, it must first be cloned. The MNN1 gene may be 
cloned as described previously, using a library of yeast DNA fragments in 
a suitable yeast shuttle vector. It is preferable to amplify the library 
by first transforming it into an E. coli host, preferably strain RR1. DNA 
is made from the transformed E. coli, and it is transformed into a yeast 
host which is mnn1 and may contain a genetic deficiency which is 
complemented by a selectable marker present on the yeast/E. coli shuttle 
vector. To facilitate identification of MNN1 gene clones, transformants 
are first selected for the presence of the plasmid in the host cell. The 
transformants are then screened for the complementation of the mnn1 
deficiency. Screening methods for the complementation of mnn1 include 
using antibodies directed against either wild-type oligosaccharide 
moieties or oligosaccharide moieties present on mnn1 cells to identify 
transformants carrying DNA sequences which confer a Mnn1+ phenotype on the 
host cell (described by Ballou, ibid., 1970 and Ballou, ibid., 1982). A 
preferred method for screening transformants for MNN1 complementation is 
to use antibodies directed against the terminal .alpha.1.fwdarw.3-linked 
mannose units of wild-type cells to identify positive clones. MNN1 gene 
clones may be confirmed to be plasmid borne by testing for the loss of 
plasmid coupled with loss of the Mnn1+ phenotype as described previously. 
Restriction mapping may be carried out to determine the smallest fragment 
of the genomic insert needed to complement the mnn1 deficiency. The DNA 
sequence may also be determined for the cloned gene. 
In a preferred embodiment, a yeast host cell which contains a genetic 
deficiency in MNN7, MNN8, MNN9 or MNN10 also contains a conditional 
mutation in a gene which is required for the expression of the silent 
mating-type loci. Mutations in these genes permit the use of promoters 
containing mating-type regulatory elements as described above. A 
particularly preferred conditional mutant is sir3-8. Yeast strains having 
defects such as sir3-8 are widely available, such as from the Yeast 
Genetic Stock Center, Berkeley, Calif., or may be prepared using standard 
techniques of mutation and selection. The sir3-8 mutation may be 
introduced into a strain containing a genetic deficiency in MNN7, MNN8, 
MNN9, OR MNN10 by crossing or by using standard techniques of mutation and 
selection. To optimize production of heterologous proteins, it is 
preferred that the host strain carries a mutation, such as the pep4 
mutation (Jones, Genetics 85:23-33, 1977), which results in reduced 
proteolytic activity. 
As noted above, the regulated MNN7, MNN8, MNN9 or MNN10 expression unit, 
whether plasmid borne or integrated, is used in conjunction with a second 
DNA construct comprising a second promoter and a sequence encoding a 
secretion signal fused to a heterologous gene or cDNA of interest. A 
preferred embodiment of the invention is the use of a regulated promoter 
different from that directing expression of the cloned MNN gene as the 
second promoter, the use of which provides the ability to vary the 
expression of the heterologous gene or cDNA to prevent the production of a 
protein product containing outer chain oligosaccharide moieties. Preferred 
secretion signals include those derived from the yeast MF.alpha.1 (Kurjan 
et al., U.S. Pat. No. 4,546,082; Singh, EP 123,544), PHO5 (Beck et al., WO 
86/00637), SUC2 (Carlson et al., Mol. Cell. Biol. 3:439-447, 1983) and 
BAR1 (MacKay et a-., U.S. Pat. No. 4,613,572; MacKay, WO 87/02670) genes. 
The expression unit comprising the heterologous gene or cDNA of interest 
may be carried on the same plasmid as a plasmid-borne regulated MNN 
expression unit and subsequently transformed into a host cell. 
Alternatively, the expression unit comprising the heterologous gene or 
cDNA may be on a separate plasmid, or integrated into the host genome. 
Integration is a recombination event which occurs at a homologous site and 
results in the insertion of a DNA sequence at that site. These expression 
units may be used in any combination with a plasmid-borne or integrated, 
regulated MNN gene: These combinations allow normal expression of the MNN 
gene with unimpaired cell growth during the exponential phase of cell 
growth, with normal glycoprotein synthesis. In a preferred embodiment, 
during active cell growth the growth conditions of the culture are 
regulated to prevent the heterologous gene from being expressed. When the 
cells reach optimal density, the growth conditions are selectively 
altered, thereby blocking the expression of the MNN gene, and allowing the 
heterologous protein product with modified core glycosylation to be 
synthesized. Heterologous proteins and polypeptides which may be produced 
according the present invention include growth factors (e.g., 
platelet-derived growth factor), tissue plasminogen activator, urokinase, 
immunoglobulins, plasminogen, thrombin, factor XIII and analogs thereof. 
According to the present invention, another method for controlling the 
addition of outer chain oligosaccharides to secretion-directed 
glycoproteins involves the isolation of a unique and unexpected mnn9 
disruption mutant. This mutant provides a yeast host which is able to 
produce heterologous glycoproteins containing modified core glycosylation, 
without the need to manipulate culture conditions. A mnn9 disruption was 
made as previously described. Briefly, a DNA construct, comprising the 
MNN9 coding sequence which has been disrupted with a selectable marker 
(URA3 gene), was introduced into strain SEY2101. Transformants were 
selected for their ability to grow on synthetic medium lacking uracil. 
Transformants were assayed for the presence of the Mnn9.sup.- phenotype. 
Southern analysis was done to confirm the disruption of the MNN9 gene. A 
positive clone was identified which retained the URA3 marker and the 
Mnn9.sup.- phenotype and exhibited a pattern on Southern analysis 
(Southern, J. Mol. Bio. 98 503-517, 1975) showing that the MNN9 gene is 
intact. Pulsed-field gel electrophoresis (Southern et al., Nuc. Acids Res. 
15:5925-5943, 1987) on genomic DNA derived from this strain has shown that 
the mnn9 disruption isolate has undergone chromosome aberrations involving 
at least chromosomes V and VIII. The strain, designated ZY300 (ATCC 
Accession No. 20870), grows faster than the mnn9 point mutation isolated 
by Ballou (ibid., 1980) or other confirmed mnn9 deletion strains. Analysis 
of the strain shows that it is apparently able to grow without osmotic 
support. Transformation of this strain with certain yeast plasmids (e.g., 
YEp13), which contain REP3 and the replication origin, but not REp or 
REP2, has shown that the plasmids are unstable due to the variant 2 micron 
plasmid present in the parent strain. Yeast vectors which contain REP1, 
REP2. REP3 and a replication origin or which utilize a centromere fragment 
and a replication origin are stable in the strain. It is preferable to 
cure the strain of the variant 2 micron plasmid and replace it with a 
wild-type 2 micron plasmid to allow the strain to utilize yeast vectors of 
the YEp13 type. For production of foreign proteins, a DNA construct 
comprising a promoter and a sequence encoding a secretion signal followed 
by a sequence encoding a polypeptide or protein of interest is introduced 
into strain ZY300. The promoter may be a regulated or constitutive 
promoter. The resultant proteins are homogeneous in nature and lack the 
characteristic yeast hyperglycosylation. It is preferable to introduce 
both a pep4 disruption and a mnn1 disruption in ZY300. Disruptions of 
these cloned genes are carried out in a manner similar to the gene 
disruption described previously. 
Techniques for transforming fungi are well known in the literature and have 
been described, for instance, by Beggs (ibid.), Hinnen et al. (Proc. Natl. 
Acad. Sci. USA 75:1929-1933, 1978), Russell (Nature 301:167-169, 1983) and 
Yelton et al. (Proc. Natl. Acad. Sci. USA 81:1740-1747, 1984). Host 
strains may contain genetic defects in genes which are complemented by the 
selectable marker present on the vector. Such genetic defects include 
nutritional auxotrophies, for example, leu2, which may be complemented by 
the LEU2 gene and defects in genes required for carbon source utilization, 
for example, tpi1, which may be complemented by the POT1 gene of 
Schizosaccharomyces pombe. Choice of a particular host and selectable 
marker is well within the level of ordinary skill in the art. 
Proteins produced according to the present invention may be purified by 
conventional methods. Particular purification protocols will be determined 
by the nature of the specific protein to be purified. Such determination 
is within the ordinary level of skill in the art. Generally, the cell 
culture medium will be separated from the cells and the protein will be 
isolated from the medium. Useful isolation techniques include 
precipitation, immunoadsorption and fractionation or a variety of 
chromatographic methods. 
EXAMPLES 
Example 1 
Cloning of the S. cerevisiae MNN9 Gene 
TABLE 1 
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YEAST GENOTYPES 
______________________________________ 
LB347-1C MAT.alpha. mnn9 gal2 
ZA447 MATa lea2-3,112 barl-1 gal2 ura3 
XV732-1-9A MAT.alpha. leu2-3,112 ura3 mnn9 gal2 
XP660-2A MATa leu2-3,112 barl-1 trp1 gal2 
XCYl-5D MATa leu2-3,112 ura3 trp1 mnn9 
SEY2101 MATa leu2-3,112 ade2-101 ura3-52 suc2-.DELTA.9 
gal2 .DELTA.pep4::CAT 
ZY100 MATa leu2-3,112 ade2-101 ura3-52 suc2-.DELTA.9 
gal2 .DELTA.pep4::CAT 
ZY300 MATa leu2-3,112 ade2-101 ura3-52 suc2-.DELTA.9 
gal2 mnn9::URA3::tl-1 
ZY400 MATa leu2-3,112 ade2-101 ura3-52 suc2-.DELTA.9 
gal2 .DELTA.pep4::CAT mnn9::URA3 
381G-59a MATa sir3-8 SUP4-3 ade2-1 his4-580 lys2 
trpl-1 tyrl cryl 
A2 MAT.alpha. leu2-3,112 his3-11,15 canl 
XLl-4B MATa leu2-3,112 trpl-1 ade2-1 lys2 
sir3-8 
XCY15-3C MAT.alpha. ade2-1 leu2-3,112 .DELTA.mnn9::URA3 
XCY42-28B MATa sir3-8 .DELTA.mnn9::URA3 leu2-3,112 
trpl-1 ade2-1 lys2 .DELTA.pep4::CAT 
LBl-22D MAT.alpha. mnn1 gal2 SUC2 mal CUP1 
______________________________________ 
A. Construction of Strain XCY1-5D 
A S. cerevisiae strain having the mnn9 mutation and genetic defects in the 
URA3, LEU2, and TRP1 genes was constructed using parent strains listed in 
the table. Genetic methods and media used are generally known in the art. 
(See, for example: R. K. Mortimer and D. C. Hawthorne, in Yeast Genetics, 
A. H. Rose and J. S. Harrison, eds., London:Academic Press, Inc., ltd., p. 
385-460; and Hartig et al., ibid.) Strain LB347-1C (Tsai et al., J. Biol. 
Chem. 259:3805-3811, 1984) was crossed with ZA447. Zygotes were pulled 
from the mating mixture to isolate diploids. A diploid colony designated 
XV732 was sporulated and dissected. Tetrad analysis of the spores showed a 
2:2 segregation for small colonies when the spores were grown on medium 
without osmotic stabilization. (Small colony size on non-osmotically 
stabilized medium correlated with the presence of the mnn9 gene.) A spore 
which developed into a very small colony with leucine and uracil 
auxotrophies was chosen and designated XV732-1-9A. This spore was crossed 
with XP660-2A. Diploids were selected on minimal medium (Table 2) 
supplemented with 80 mg/l leucine to yield the diploid XCY1. XCY1 was 
sporulated and dissected. Tetrad analysis was carried out on the spores. 
Spore XCY1-5D (MAT.alpha. mnn9 leu2-3 leu2-112 trp1 ura3 gall) was 
selected as the host strain for cloning the MNN9 gene. 
TABLE 2 
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MinD 
______________________________________ 
20 g glucose 
6.7 g Yeast Nitrogen Base without amino acids (Difco 
Laboratories, Detroit, Mich.) 
18 g Agar 
______________________________________ 
Mix all the ingredients in distilled water. Add distilled water to a final 
volume of 1 liter. Autoclave 15 minutes. Pour plates and allow to 
solidify. 
-LeuDS plates 
20 g glucose 
6.7 g Yeast Nitrogen Base without amino acids (Difco Laboratories, Detroit, 
Mich.) 
40 mg adenine 
30 mg L-arginine 
50 mg L-aspartic acid 
20 mg L-histidine free base 
60 mg L-isoleucine 
40 mg L-lysine-mono hydrochloride 
20 mg L-methionine 
60 mg L-phenylalanine 
50 mg L-serine 
50 mg L-tyrosine 
40 mg uracil 
60 mg L-valine 
60.75 g sorbitol 
18 g Agar 
Mix all the ingredients in distilled water. Add distilled water to a final 
volume of 1 liter. Autoclave 15 minutes. After autoclaving add 150 mg 
L-threonine and 40 mg L-tryptophan. Pour plates and allow to solidify. 
-LeuDS 
Use the recipe for -LeuDS pates, but omit the agar. 
-LeuD plates 
Use the recipe for -LeuDS plates, but omit the sorbitol. 
-LeuD 
Use the recipe for -LeuDS plates, but omit the sorbitol and agar. 
-TrpDS plates 
20 g glucose 
6.7 g Yeast Nitrogen Base without amino acids (Difco Laboratories, Detroit, 
Mich.) 
40 mg adenine 
30 mg L-arginine 
50 mg L-aspartic acid 
20 mg L-histidine free base 
60 mg L-isoleucine 
80 mg L-leucine 
40 mg L-lysine-mono hydrochloride 
20 mg L-methionine 
60 mg L-phenylalanine 
50 mg L-serine 
50 mg L-tyrosine 
40 mg uracil 
60 mg L-valine 
60.75 g sorbitol 
18 g Agar 
Mix all the ingredients in distilled water. Add distilled water to a final 
volume of 1 liter. Autoclave 15 minutes. After autoclaving add 150 mg 
L-threonine. Pour plates and allow to solidify. 
-TrpD 
Use the recipe for -TrpDS, but omit the sorbitol and agar. 
YEPDS plates 
20 g glucose 
10 g Bacto-peptone (Difco) 
20 g yeast extract (Difco) 
60.75 g sorbitol 
18 g Agar 
Mix all ingredients in distilled water. Add distilled water to a total 
volume of 1 liter. Autoclave 25 minutes. Pour plates and allow to 
solidify. 
YEPDS 
Use the recipe for YEPDS plates, but omit the agar. 
YEPD plates 
20 g glucose 
10 g Bacto-peptone 
20 g yeast extract 
18 g agar 
Mix all ingredients in distilled water. Add distilled water to a total 
volume of 1 liter. Autoclave 25 minutes. Pour plates and allow to 
solidify. 
YEPD 
Use the recipe for YEPD plates but omit the agar. 
-UraDS plates 
20 g glucose 
6.7 g Yeast Nitrogen Base without amino acids (Difco Laboratories, Detroit, 
Mich.) 
40 mg adenine 
30 mg L-arginine 
50 mg L-aspartic acid 
20 mg L-histidine free base 
60 mg L-isoleucine 
80 mg L-leucine 
40 mg L-lysine-mono hydrochloride 
20 mg L-methionine 
60 mg L-phenylalanine 
50 mg L-serine 
50 mg L-tyrosine 
60 mg L-valine 
60.75 g sorbitol 
18 g Agar 
Mix all the ingredients in distilled water. Add distilled water to a final 
volume of 1 liter. Autoclave 15 minutes. After autoclaving add 150 mg 
L-threonine and 40 mg L-tryptophan. Pour plates and allow to solidify. 
-Leu-TrpDS 
20 g glucose 
6.7 g Yeast Nitrogen Base without amino acids (Difco Laboratories, Detroit, 
Mich.) 
40 mg adenine 
30 mg L-arginine 
50 mg L-aspartic acid 
20 mg L-histidine free base 
60 mg L-isoleucine 
40 mg L-lysine-mono hydrochloride 
20 mg L-methionine 
60 mg L-phenylalanine 
50 mg L-serine 
50 mg L-tyrosine 
40 mg uracil 
60 mg L-valine 60.75 g sorbitol 
18 g Agar 
Mix all the ingredients in distilled water. Add distilled water to a final 
volume of 1 liter. Autoclave 15 minutes. After autoclaving add 150 mg 
L-threonine. Pour plates and allow to solidify. 
M9+CA+amp+W 
6 g Na.sub.2 HPO.sub.4 .multidot.H.sub.2 O 
3 g KH.sub.2 PO.sub.4 
0.5 g NaCl 
1 g NH.sub.4 Cl 
5 g casamino acids 
1 ml 1 M MgSO.sub.4 
0.2 ml 0.5 M CaCl.sub.2 
5 ml 40% glucose 
10 ml 1 mg/ml thiamine B1 
2 ml 10 mg/ml L-tryptophan 
Dissolve ingredients in distilled water. Add distilled water to a final 
volume of one liter. Autoclave 25 minutes. After autoclaving, add 100 mg 
ampicillin. 
M9+CA+amp 
Use the recipe for M9+CA+amp+W, but omit the tryptophan. 
B. Construction of the plasmid pM111 
As illustrated in FIG. 2, a yeast shuttle vector was constructed which 
contained YRp7 (Stinchcomb et al., .ibid.) vector sequences and the yeast 
centromere CEN3. A 630 bp Bam HI-Sau 3A fragment, comprising the yeast 
CEN3 sequences derived from pYe(CEN3)41 (Clarke and Carbon, Nature 
287:504-509, 1980), was ligated into pUC8 which had been linearized by 
digestion with Bam HI and dephosphorylated with bacterial alkaline 
phosphatase. The ligation mixture was transformed in E. coli strain JM83. 
Plasmid DNA was made from the resultant transformants and cut with Bam HI 
to determine the presence of the CEN3 fragment. Positive clones were 
digested with Eco RI and Bam HI to determine the orientation of the 
insert. A clone with the CEN3 fragment in the proper orientation was 
designated pM101B. plasmid pM101B was linearized by digestion with Bam HI 
and treated with DNA polymerase I Klenow fragment to blunt the cohesive 
ends. The linearized plasmid was recircularized. The resultant plasmid, 
pM102A, was linearized by digestion with Hinc II and then cut with Eco RI 
to isolate the 0.6 kb CEN3 fragment. The Hinc II-Eco RI fragment was 
treated with DNA polymerase I Klenow fragment to fill in the Eco RI 
cohesive end, resulting in a 0.6 kb CEN3 fragment with blunt ends. Plasmid 
pFRT-1, comprising YRp7 which has had the Eco RI site distal to the 5' end 
of the TRP1 gene destroyed, was linearized by digestion with Pvu II. The 
pFRT-1 linear fragment was ligated with the 0.6 kb CEN3 fragment and the 
ligation mixture was transformed into E. coli strain RR1. DNA made from 
the resulting transformants was digested with Eco RI to confirm the 
presence of the insert and to determine the orientation of the CEN3 
insert. (In one orientation, the Eco RI site is regenerated by ligation to 
the Pvu II blunt end.) The resultant plasmid was designated pM111. 
C. Cloning the MNN9 gene 
A pool of yeast genomic fragments from strain X2180 (ATCC 26109) cloned 
into the vector pM111 was used as the starting material for isolating the 
MNN9 gene. Briefly, genomic DNA was partially cut with Sau 3A and the 
resulting genomic fragments were cloned into the Bam HI site of the vector 
pM111. The average size of the inserts was 8 kb. 
The pool of genomic DNA in pM111 was transformed into strain XCY1-5D 
essentially as described by Beggs (Nature 275:104-108, 1978). 
Transformants were selected for their ability to grow on -TrpDS plates 
(Table 2). 
The transformant colonies were resuspended and replated using the method 
described by MacKay (Methods In Enzymology 101:325-343, 1983). The 
transformant colonies, suspended in top agar, were mixed and resuspended 
in -TrpD (Table 2)+0.5 M KCl to free the cells from the top agar. This 
mixture was grown for 2 hours at 30.degree. C. and plated on -TrpDS 
plates. Colonies were allowed to grow on the -TrpDS plates at 30.degree. 
C. Colonies were then picked to master -TrpDS plates in a grid formation. 
Replicas of the master plates were made onto -TrpDS plates and allowed to 
grow before MNN9 phenotype was determined. 
Approximately 3,000 positive colonies were assayed for the presence of the 
MNN9 phenotype using the method described in Section D below. Sixteen 
colonies were found to consistently complement the mnn9 mutation present 
in the host strain and their ability to do so was linked to the presence 
of the plasmid. Plasmid DNA was isolated from the sixteen positive 
colonies as described by Hartig et al (Mol Cell. Biol. 6:2106-2114, 1986) 
and transformed into E. coli strain RR1. Plasmid DNA was isolated from the 
E. coli transformants and was subjected to restriction enzyme analysis. 
Fifteen of the plasmids showed two common Xba I sites. 
The plasmid with the smallest insert that restored the Mnn+ phenotype when 
transformed into mnn9 strains was designated pZY23. Plasmid pZY23 
comprised a 6 kb yeast genomic DNA insert in pM111. Subclones of the 
genomic DNA insert present in pZY23 were made and used to transform strain 
XCY1-5D to check for complementation. As illustrated in FIG. 3, a subclone 
of pZY23 was made by digesting pZY23 with Cla I and Bgl 11 to isolate the 
3.1 kb fragment comprising the MNN9 gene. The fragment was then ligated 
into pM111 which had been linearized by digestion with Cla I and Bg1 II. 
The resultant plasmid pZY37 has been deposited as an E. coli strain RR1 
transformant with the American Type Culture Collection (ATCC No. 67550). A 
2.4 kb Bgl II-Sst I fragment of the cloned insert was found to be 
sufficient for complementation. This fragment was subcloned into pIC9H 
(Marsh et al., Gene 32:481-486, 1984; ATCC 37408) which had been 
linearized by digestion with Bam HI and Sst I. The resultant plasmid was 
designated pZY48 (FIG. 3). 
D. Assay Methods. 
Preparation of colonies 
Appropriately grown cells were lysed by one of two methods. In the first 
method, colonies grown in YEPDS (Table 2) were treated with chloroform to 
permeabilize the cells. The plates were inverted (for 5 minutes at room 
temperature) onto paper towels which had been saturated with chloroform. 
The plates were then placed upright for 30 minutes to allow the chloroform 
to evaporate before assaying. 
The second method was employed for colonies which required selective growth 
conditions on synthetic medium to maintain plasmids. Colonies that were 
grown on synthetic medium+1 M sorbitol were first transferred to 
nitrocellulose filters (Schleicher & Schuell, Keene, N.H.). Circular 
nitrocellulose filters were laid on top of colonies grown on synthetic 
medium +1 M sorbitol, until the filters were completely wetted. The 
filters were then carefully peeled away from the surface of the agar and 
dipped into liquid nitrogen for 30 seconds. This effectively lysed the 
cells. The filters were then paced cell-side up on YEPD plates (Table 2) 
for assaying. 
Assay Method 
Substrate was prepared as described below: 
______________________________________ 
per plate: 
2 ml 2% agar, melted, held at 55.degree. C. 
1 ml 0.5M NaH.sub.2 PO.sub.4 pH 7.0, 55.degree. C. 
0.1 ml 20% sodium dodecylsulfate, 55.degree. C. 
6.4 ml dH.sub.2 O, 55.degree. C. 
0.5 ml 2 mg/ml cycloheximide (Sigma, St. Louis, Mo.) 
100 mg azocoll (Sigma) 
______________________________________ 
The azocoll does not dissolve. The mixture was swirled and quickly poured 
as an overlay over the colonies on the plate or filter. 
The plates were incubated at 37.degree. C. for 5-8 hours. Colonies 
exhibiting the Mnn9.sup.- phenotype were able to break down the azocoll 
immediately surrounding the colony resulting in a clear halo around mnn9 
colonies. 
EXAMPLE 2 
Disruption of the MNN9 gene 
In order to disrupt the MNN9 gene, a plasmid was constructed in which the 
URA3 gene replaced the coding region between the unique Hind III and Eco 
RI sites present in the MNN9 gene as illustrated in FIG. 3. Plasmid p1148, 
comprising the 1.3 kb Hind III fragment encoding the URA3 gene (derived 
from YEp24; Botstein et al., Gene 8:17, 1979) in plasmid pIC19R, was 
digested with Hind III and Xma I to isolate the 1.1 kb URA3 fragment. This 
fragment was ligated into pIC19R which had been linearized by digestion 
with Hind III and Xma I. The resultant plasmid, pZY61, was digested with H 
nd III and Eco RI to isolate the 1.1 kb URA3 fragment. Plasmid pZY48 was 
digested with Eco RI and Sal I to isolate the 1.2 kb fragment encoding the 
3' portion of MNN9. The fragment was joined with the URA3 fragment and 
pUC13, which had been linearized by digestion with Hind III and Sal I, n a 
three-part ligation. The resultant plasmid, pZY62, was digested with Hind 
III and Sal I to isolate the 2.3 kb fragment comprising the URA3 gene 
fused to the 3' portion of the MNN9 gene. Plasmid pZY48 was digested with 
Sst I and Hind III to isolate the 0.44 kb MNN9 fragment. This fragment was 
joined with the fragment from pZY63 and pUC13, which had been linearized 
with Sst I and Sal in a three-part ligation. The resultant plasmid, pZY63, 
comprised the MNN9 gene disrupted with the URA3 gene (FIG. 3). 
The genomic MNN9 was disrupted in strains SEY2101 and ZY100 (Table 1) using 
the method described by Rothstein (ibid.). Plasmid pZY63 was digested with 
Sst I and Sal 1 to isolate the 2.7 kb fragment comprising the MNN9 coding 
region which has been disrupted with the URA3 gene. This fragment was 
transformed into yeast strains SEY2101 and ZY100. The transformants were 
selected for their ability to grow on UraDS plates (Table 2). 
Transformants were then assayed for the presence of a Mnn9.sup.- phenotype 
(Example 1.D.) which indicated the integration of the linear DNA fragment 
at the MNN9 locus. Positive clones were tested for the stability of the 
URA3 marker by growth on nonselective medium. Positive clones were 
inoculated into 5 ml YEPDS (Table 2) and grown overnight at 30.degree. C. 
The overnight cultures were diluted 1 ul into 5 ml fresh YEPDS and were 
grown overnight at 30.degree. C. The second overnight cultures were 
diluted 1 ul in 10 ml 1 M sorbitol. Ten ul of the mixture, added to 100 ul 
1 M sorbitol, was plated on a YEPDS plate. These plates were incubated at 
30.degree. for 24 hours. The colonies were replica plated onto -UraDS to 
test for the stability of the URA3 marker. All the clones were stable. 
Southern blot analysis was carried out on the transformants to confirm the 
integration event. Genomic DNA was prepared by the method described by 
Davis et al. (Proc. Natl. Acad. Sci. USA 802432-2436, 1983) and cut with 
Eco R1 and Sst I. The digests were electrophoresed in a 0.7% agarose gel 
and blotted onto a nitrocellulose filter according to the method described 
by Southern (ibid., 1975). The filter was probed with the 2.3 kb Hind 
III-Hind III fragment from pZY48, comprising the coding region of MNN9 
(Example 1.C.) which was random primed with an Amersham random priming kit 
(Amersham, Arlington Hts., Ill.). A disruption in strain ZY100, designated 
ZY400, was confirmed by the presence of 1.5 and 1.55 kb labeled fragments 
on the Southern blot. A clone was isolated from the disruption in strain 
SEY2101, designated ZY300 (ATCC Accession No. 20870), which showed no gene 
disruption. Further experimentation confirmed the presence of a Mnn9.sup.- 
phenotype. 
Pulsed-field gel electrophoresis (Southern et al., ibid., 1987 was carried 
out on genomic DNA derived from ZY300 and ZY400 and their parent strains. 
Genomic DNA was prepared using a method modified from the agarose bead 
method reported by Overhauser and Radic (BRL Focus 9:8-9, 1987). Briefly, 
overnight cultures were grown in 15 ml YEPDS at 30.degree. C. The cultures 
were centrifuged, the supernatants were discarded and the pellets were 
resuspended in 5 mls SCE (1 M sorbitol, 0.1 M Na.sub.2 Citrate pH 5.8, 
0.01 M Na.sub.2 EDTA pH 8.0). The cell suspensions were transferred to 50 
ml Erlenmeyer flasks. 10 ml paraffin oil held at 55.degree. C. and 1 ml 
2.5% low-gelling agarose (Sea Plaque Agarose, FMC Corp. Bioproducts, 
Rockland, Me.) held at 55.degree. C. were added to each flask. The cell 
slurries were mixed vigorously on a vortex at maximum speed for 1 minute 
until a fine emulsion was obtained. The emulsions were cooled rapidly, 
with swirling, in an ice-water bath. After cooling, the emulsions were 
transferred to 50 ml polystyrene tubes and 20 ml TE8 (10 mM Tris-HCl, 1 mM 
EDTA, pH 8.0) was added. The solutions were centrifuged at 2500 rpm for 5 
minutes after which the paraffin oil and supernatants were discarded. The 
pellets, comprising the agarose beads, were resuspended n 30 m TE8 and 
centrifuged as described in the previous step. The supernatants were 
discarded and 5 ml spheroplasting buffer (for 2 ml of beads: 3 ml SCE, 2 
ml 0.5 M EDTA (pH 9.0), 1 mg zymolyase 60,000 (Miles, Elkhart, Ind.), 0.25 
ml .beta.-mercaptoethanol (Sigma, St. Louis, Mo.) was added. The solutions 
were incubated at 37.degree. C. for 1 hour on a rotating drum after which 
the solutions were centrifuged as previously described. The supernatants 
were discarded and replaced with 1 ml 0.5 M EDTA (pH 9.0) and stored at 
4.degree. C. 
The yeast chromosomes were separated essentially as described by Southern 
et al. (ibid., 1987). Pulsed-field gel electrophoresis, in a 1% agarose 
ge, (Seakem Agarose, FMC Corp. Bioproducts, Rockland, Me.) was performed 
using a Rotogel (Moonlight Cat Door Company, Seattle, Wash.). Yeast DNA 
was visualized by staining with ethidium bromide. Analysis of the stained 
gel revealed that strain ZY300 had undergone chromosome rearrangement 
involving at least chromosomes V and VIII. A Southern blot was made of the 
gel as previously described, and probed first with the 2.3 kb Hind 
III-Hind III MNN9 fragment derived from pZY48 and then with the 1.3 kb 
Hind III-Hind III URA3 fragment derived from p1148 (Example 2). The probes 
were labeled using the Amersham random priming kit (Amersham, Arlington 
Hts., Ill.). Results of the Southern blot showed that in both ZY300 and 
ZY400, all of the MNN9 coding region, mapped to chromosome XVI (the 
natural site for MNN9) and URA3 mapped to chromosomes XVI and V as 
expected. 
EXAMPLE 3 
Expression of Barrier in mnn9 deletion strains 
A DNA construct comprising the BAR1 gene was transformed into the mnn9 
deletion strains generated in Example 2 and their parent strains to 
examine the glycosylation of the Barrier protein. The BAR1 gene product, 
Barrier, is an exported protein which has been shown to be highly 
glycosylated. Plasmid pSW24, comprising the ADH1 promoter, the BAR1 coding 
region fused to the coding region of the C-terminal portion of substance p 
(Munro and Pelham, EMBO J. 3:3087-3093, 1984) and the TPI1 terminator, was 
constructed as follows (FIG. 5). Plasmid pZV9, comprising the entire BAR1 
coding region and its associated flanking regions, was cut with Sal I and 
Bam HI to isolate the 1.3 kb BAR1 fragment. This fragment was subcloned 
into pUC13 cut with Sal I and Bam HI to generate the plasmid designated 
pZV17 (FIG. 4). plasmid pZV17 was digested with Eco RI to remove the 
3'-most 0.5 kb of the BAR1 coding region. The vector-BAR1 fragment was 
religated to create the plasmid designated pJH66. Plasmid pJH66 was 
linearized with Eco RI and blund-ended with DNA polymerase I (Klenow 
fragment). Kinased Bam H linkers (.sup.5 'CCGGATCCGG.sup.3 ') were added, 
and excess linkers were removed by digestion with Bam HI before 
re-ligation. The resultant plasmid was designated pSW8. Plasmid pSW8 was 
cut with Sal I and Bam HI to isolate the 824 bp fragment encoding amino 
acids 252 through 526 of Barrier. Plasmid pPM2, containing the synthetic 
oligonucleotide sequence encoding amino acids 252 through 526 of Barrier. 
Plasmid pPM2, containing the synthetic oligonucleotide sequence encoding 
the dimer form of the C-terminal portion of substance P in M13mp8, was 
obtained from Munro and Pelham. Plasmid pPM2 was linearized by digestion 
with Bam HI and Sal I and ligated with 824 bp BAR1 fragment from pSW8. The 
resultant plasmid, pSW14, was digested with Sal I and Sma I to isolate the 
871 bp BAR1-substance P fragment. Plasmid pZV16, comprising a fragment of 
BAR1 encoding amino acids 1 through 250, was cust with Xba I and Sal I to 
isolate the 767 bp BAR1 fragment. This fragment was ligated with the 871 
bp BAR1-substance P fragment in a three-part ligation with pUC18 cut with 
Xba I and Sma 1. The resultant plasmid, designated pSW15, was digested 
with Xba I and Sma I to isolate the 1.64 kb BAR1-substance P fragment. The 
ADH1 promoter was obtained from pRL029, comprising the ADH1 promoter and 
the 116 bp of the BAR1 5' coding region in pUC18 (MacKay, WO 87/02670). 
Plasmid pRL029 was digested with Sph I and Xba I to isolate the 0.42 kb 
ADH1 promoter fragment. The TPI1 terminator (Alber and Kawasaki, J. Mol. 
Appl. Genet. 1:410-434, 1982) was provided as a blunted Xba I-Sph I 
fragment comprising 0.7 kb of the TPI1 terminator (blunted Xba I to Eco 
R1) linked to pUC18 (Eco RI-Sph I). This fragment was ligated with the 
0.42 kb ADH1 promoter fragment and the 1.64 kb BAR1-substance P fragment 
in a three-part ligation to produce plasmid pSW22. Plasmid pSW22 was 
digested with Sph I and Sma I to isolate the 2.8 kb expression unit which 
was 1 gated into YEp13 which had been linearized by digestion with Sph I 
and Pvu II. The resultant plasmid was designated pSW24 (FIG. 5). 
Plasmid pSW24 was transformed into the mnn9 deletion strains pZY300 and 
pZY400 and their parent strains SEY2101 and pZY100. Transformants were 
selected for their ability to grow on -LeuDS plates (Table 2). 
Transformants were inoculated into 5 ml -LeuDS (Table 2) and incubated 
overnight at 30.degree. C. Five hundred ul of the overnight cultures were 
inoculated into 50 ml -LeuDS and incubated for 48 hours at 30.degree. C. 
The cultures were centrifuged, and the supernatants were decanted into 250 
ml centrifuge bottles. An equal volume of 95% ethanol, held at -20.degree. 
C., was added and the mixtures were vortexed and incubated at -20.degree. 
C. for 30 minutes. The mixtures were then centrifuged at 9,000 rpm for 30 
minutes in a SA (Sorvali) rotor. The supernatants were discarded and the 
protein pellets were allowed to dry overnight at room temperature. The 
dried pellets were resuspended in 500 ul dH.sub.2 O. 
Fifty ul of 2X sample buffer (Table 3) was added to each of the resuspended 
samples and the samples were electrophoresed in a 10% polyacrylamide gel 
and transferred to nitrocellulose using the method described by Towbin et 
al. (Proc. Natl. Acad. Sci. USA 76:4350-4353, 1979). The nitrocellulose 
filter was probed with rat anti-substance P (Capell, Malvern, Pa.) and 
visualized using horseradish peroxidase-conjugated goat anti-rat 
antibodies. The immunoblot showed a homogeneous species recognized by the 
anti-substance P antibody in the mnn9 disruption strains ZY300 and ZY400, 
indicating that the Barrier protein produced by these strains is 
homogeneous. Parental strains showed a heterogeneous, hyperglycosylated 
species which was recognized by the anti-substance P antibody. 
The pSW24 transformants were assayed for Barrier activity as follows. The 
assay used for detection of Barrier production by transformed yeast cells 
relies on the ability of Barrier to reverse the inhibition of growth of 
sensitive a cells exposed to .alpha.-factor. A lawn was prepared using a 
test strain, such as strain RC629 (MATa -bar1) which is abnormally 
sensitive to a-factor, in a soft agar overlay on an agar plate. A 
sufficient quantity of .alpha.-factor (0.05-0.1 unit, as assayed by 
Manney, J. Cell Biol. 96:1592-1600, 1983) was added to the overlay to 
inhibit growth of the cells. Transformants to be screened for Barrier 
production were spotted onto the lawn. Secretion of Barrier by the 
transformed cells reversed the .alpha.-factor growth inhibition 
immediately surrounding the spot, thereby allowing the sensitive cells to 
recover. The recovered cells were observed as a fringe of growth around 
the normally smooth edge of the colony of transformed cells. The presence 
of this fringe indicated that the plasmid in the transformed strain 
directed the expression and secretion of Barrier protein. The 
transformants were shown to make active Barrier protein. 
EXAMPLE 4 
Expression of tissue plasminogen activator from mnn9 deletion strains 
A DNA construct comprising the tissue plasminogen activator (tPA) cDNA was 
transformed into the mnn9 deletion strain ZY400 to examine the 
glycosylation of the protein produced. Plasmid pDR1498 (deposited as a 
yeast transformant in strain E8-11C, ATCC #20730), comprising the TPI1 
promoter, the MF.alpha.1 signal sequence fused to the serine codon of the 
mature tPA cDNA sequence and the TPI1 terminator, was transformed into 
strain ZY400 and its parent, ZY100. Transformants were selected for their 
ability to grow on -LeuDS plates (Table 2). 
Transformants were grown as described in Example 3. After 48 hours of 
growth at 30.degree. C., the cultures were split into 25 ml aliquots and 
centrifuged. The supernatants from one set of 25 ml aliquots were decanted 
and saved at -70.degree. C. Their respective pellets were also saved at 
-70.degree. C. 
Cell extracts were made on the remaining cell pellets in the following 
manner. One ml Phosphate Buffered Saline (PBS; obtained from Sigma, St. 
Louis, Mo.)+1 mM EDTA was added to one-half the total volume. The mixtures 
were vortexed at full speed for 2.5 minutes, three times with the samples 
cooled on ice between vortex bursts. The lysates were centrifuged in an 
Eppendorf microfuge (Brinkmann, Westbury, N.Y.) at top speed for 10 
minutes at 4.degree. C. The supernatants, comprising soluble cell 
proteins, were removed and stored at -70.degree. C. The pellets were 
washed with 1 ml 2X TNEN (100 mM Tris-Base, 200 mM NaCl, 1 mM EDTA, 0.5% 
NP-40, adjusted to pH 8.0). The mixtures were vortexed and centrifuged as 
previously described. The supernatant, comprising the membrane protein 
fraction, was removed and stored at -70.degree. C. 
EXAMPLE 5 
Temperature-Regulated MNN9 gene 
The TPI1 promoter was obtained from plasmid pTPIC10 (Alder and Kawasaki, J. 
Mol. Appl. Genet. 1:410-434, 1982), and plasmid pFATPOT (Kawasaki and 
Bell, EP 171,142; ATCC 20699). Plasmid pTP1C10 was cut at the unique Kpn I 
site, the TP11 coding region was removed with Ba1-31 exonuclease, and an 
Eco RI linker (sequence: GGAATTCC) was added to the 3' end of the 
promoter. Digestion with Bg1 II and Eco RI yielded a TPI1 promoter 
fragment having Bg1 II and Eco RI sticky ends. This fragment was then 
joined to plasmid YRp7" (Stinchcomb et al., Nature 282:39-43, 1979) which 
had been cut with Bg1 II and Eco RI (partial). The resulting plasmid, 
TE32, was cleaved with Eco RI (partial). The resulting plasmid, TE32, was 
cleaved with Eco RI (partial) and the Bam HI to remove a portion of the 
tetracycline resistance gene. The linearized plasmid was then 
recircularized by the addition of an Eco RI-Bam HI linker to produce 
plasmid TEA32. Plasmid TEA32 was digested with Bg1 II and Eco RI, and the 
.about.900 bp partial TPI1 promoter fragment was gel-purified. Plasmid 
pIC19H (Marsh et al., Gene 32:481-486, 1984) was cut with Bg1 II and Eco 
RI and the vector fragment was gel-purified. The TPI1 promoter fragment 
was then ligated to the linearized PIC19H and the mixture was used to 
transform E. coli RR1. Plasmid DNA was prepared and screened for the 
presence of a .about.900 bp Bg1 II-Eco RI fragment. A correct plasmid was 
selected and designated pICTPIP. 
The TPI1 promoter was then subcloned to place convenient restriction sites 
at its ends. Plasmid pIC7 (Marsh et al., ibid.) was digested with Eco RI, 
the fragment ends were blunted with DNA polymerase I (Klenow fragment), 
and the linear DNA was recircularized using T.sub.4 DNA ligase. The 
resulting ligation mixture was used to transform E. coli RR1. Plasmid DNA 
was prepared from the transformants and screened for the loss of the Eco 
RI site. A plasmid having the correct restriction pattern was designated 
pIC7RI*. Plasmid pIC7RI* was digested with Hind III and Nar I, and the 
2500 bp fragment was gel-purified. The partial TPI1 promoter fragment (ca. 
900 bp) was removed from pICTPIP using Nar I and Sph I and was 
gel-purified. The remainder of the TPI1 promoter was obtained from plasmid 
pFATPOT, by digesting the plasmid with Sph I and Hind III and a 1750 bp 
fragment, which included a portion of the TPI1 promoter, was gel-purified. 
The pIC7RI* fragment, the partial TPI1 promoter fragment from pICTPIP, and 
the fragment from pFATPOT were then combined in a triple ligation to 
produce pMVR1 (FIG. 6). 
As shown in FIG. 6, the MAT.alpha.2 operator sequence was then inserted 
into the TPI1 promoter. Plasmid pSXR101 was constructed by ligating the 
2.7 kb Sal I-Bam HI fragment of pUC9 with 0.9 kb Xho *-Bam HI fragment of 
the TPI1 promoter derived from plasmid pMVR1. The Sph I site of the TPI1 
promoter in plasmid pSXR101 was then changed to a unique Xho I site. 
pSXR101 DNA was cleaved with Sph I and dephosphorylated according to 
standard procedure (Maniatis et al., eds., Molecular Cloning: A Laboratory 
Manual, Cold Spring, N.Y., 1982). An Sph I-Xho I adaptor (GCTCGAGCCATG) 
was kinased in a separate reaction containing 20 pmoles of the 
oligonucleotide, 50 mM Tris-Hcl, pH 7.6, 10 mM MgCl2, 5 mM DTT, 0.1 mM 
spermidine, 1 mM ATP, and 5 units of polynucleotide kinase in a reaction 
volume of 20 ul for 30 minutes at 37.degree. C. The kinased Sph I-Xho I 
adaptor was ligated with Sph I-cut pSXR101, and the ligation mixture was 
used to transform E. coli RR1. Plasmids with inserted adaptor were 
identified by restriction analysis and named pSXR102 (FIG. 6). The 
oligonucleotides specifying the MAT.alpha.2 operator (element 609: 5' 
TCGAG TCA TGT ACT TAC CCA ATT AGG AAA TTT ACA TGG 3' and 5' TCGA CCA TGT 
AAA TTT CCT AAT TGG GTA AGT ACA TGA C 3') were kinased as described above. 
Plasmid pSXR102 was cut with XHO I and dephosphorylated according to 
standard procedures. Three independent ligations were set up, with molar 
ratios of plasmid DNA to oligonucleotide of 1:1, 1:3 and 1:6, 
respectively. The resultant ligation mixtures were used to transform E. 
coli RR1. Plasmids with inserted oligonucleotide(s) were identified by 
colony hybridization and restriction analysis. Subsequent DNA sequencing 
showed the pSXR104 contained two copies of the MAT.alpha.2 operator. 
In the next step, plasmid pSXR104 was cut with Bam HI, dephosphorylated 
according to standard procedure (Maniatis et al., eds. in Molecular 
Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1982), and ligated 
with a 3.2 kb Bam Hi-Bam Hi fragment comprising the E. coli lacZ gene. The 
ligation mixture was used to transform E. coli strain RR1. A plasmid 
containing the lacZ fragment in the appropriate orientation was designated 
pSXR111. 
As illustrated in FIG. 7, the MNN9 gene was placed under the regulation of 
the hybrid promoter present in plasmid pSXR111. Plasmid pZY48 was digested 
with Hind III and Pst I to isolate the 0.56 kb MNN9 fragment. This 
fragment was cut with Dde I to isolate the 0.36 kb MNN9 fragment. 
Oligonucleotides ZC1429 (5' TTA GGC GGT ACG ATA CAA GAG AAA GTG ACA TTG 
TTT CCT G 3') and ZC1430 (5' AAT TCA GGA AAC AAT GTC ACT TTC TCT TGT ATC 
GTA CCG CC 3') were kinased and annealed using methods essentially 
described by Maniatis et al. (ibid.). The kinased, annealed 
oligonucleotides create an adaptor with an Eco RI cohesive end followed by 
37 bp of MNN9 coding region and a Dde I cohesive end. The ZC1429/ZC1430 
adaptor was joined to the Dde I-Pst I fragment from pZY48 in a three-part 
ligation with pUC13 which ad been linearized by digestion with Eco RI and 
Pst I. The resultant plasmid, comprising the ZC1429/ZC1430 adapter fused 
to the MNN9 gene, was designated pZY64. 
Plasmid pZY64 was digested with Eco RI and Pst 1 to isolate the 0.4 MNN9 
fragment. Plasmid pZY38, comprising the 1.5 kb Pst I-Bgl II fragment from 
pZY23 and YEp13 vector sequences, was digested with Pst I and Bgl II to 
isolate the 1.5 kb MNN9 fragment. Plasmid pSXR111 was digested with Hind 
III and Eco RI to isolate the 0.9 kb hybrid promoter fragment. This 
fragment was ligated in a four-part ligation with the 1.5 kb Pst I-Bgl II 
fragment from pZY38, the Eco RI-Pst I fragment from pZY64 and pIC19R which 
had been linearized by digestion with Hind III and Bgl II. The resultant 
plasmid was designated pZY65. Plasmid pZY65 was digested with Bgl II and 
Pvu 1. The 2.8 kb Bgl II-Bgl II fragment comprising the expression unit 
was isolated. Plasmid pM111 was linearized by digestion with Bam HI and 
ligated with the 2.8 kb Bgl II fragment comprising the expression unit 
from pZY65. The resultant ligation mix was transformed into E. coli strain 
RR1 . Plasmid DNA was made from the transformants and cut with Hind III 
and Eco RI to determine the orientation of the insert. A plasmid with the 
expression unit in the correct orientation was designated pZY66. 
EXAMPLE 6 
Expression of the Temperature-Regulated MNN9 gene. 
A. Construction of Strain XCY42-28B. 
A S. cerevisiae strain having the sir3-8 mutation, a deletion in the MNN9 
gene and genetic defects in at least LEU2 and TRP1 genes was constructed 
as follows. (Genotypes of all strains are listed in Table 1.) Strain 
381G-59A (Hartwell, J. Cell Biol 85:811-822, 1980) was crossed with strain 
A2 (Ruby et al., Meth. Enzymo. 101:253-269, 1983) and diploids were 
selected and sporulated. Asci were dissected and a spore with the genotype 
MATa leu2-3,112 trp1-1 ade2-1 lys2 sir3-8 was designated XL1-4B. Strains 
ZY400[pSW24]and ZA447 were crossed and diploid cells were selected. 
Diploid cells were sporulated using conventional methods and asci were 
dissected. Tetrad analysis was carried out on the resultant spores. A 
spore was selected having the genotype MAT.alpha. ade2-1 leu2-3,112 
.DELTA.mnn9::URA3. The spore was designated XCY15-3C. 
Strains XCY15-3C and XL1-4B were crossed to generate the diploid XCY42. 
This diploid strain was sporulated and asci were dissected. A spore was 
chosen which had the genotype of MAT.alpha. sir3-8 .DELTA.mnn9::URA3 
leu2-3,112 trp1-1 ade2-1 lys2. This spore was designated XCY42-28B. Strain 
XCY42-28B has been deposited with the American Type Culture Collection 
under Accession No. 20877. 
B. Production of polyclonal antibodies directed against a trpE-BAR1 fusion. 
Polyclonal antibodies were raised against a trpE-Barrier protein. The 
trpE-Barrier protein was produced from E. coli RR1 which had been 
transformed with pSW242. Plasmid pSW242 was constructed as follows. 
Plasmid pSW22 (Example 3) was digested with Eco RI to isolate the 1.47 kb 
BAR1 fragment. Plasmid pATH11 (Morin et al., Proc. Natl. Acad. Sci. USA 
82:7025-7029, 1985; a variant of pATH2 [Dieckmann and Tzagoloff, J. Biol. 
Chem. 260:1513-1520, 1985], in which a portion of the E. coli trpE gene is 
followed by a multiple cloning region and vector sequences of a pUC type 
plasmid) was linearized by digestion with Eco RI. The two Eco RI fragments 
were joined by ligation and transformed into E. coli strain RR1. Plasmid 
DNA made from the transformants was screened by restriction analysis and a 
clone containing the BAR1 fragment in the appropriate orientation was 
designated pSW242. 
A transformant colony of E. coli RR1 harboring plasmid pSW242 was 
inoculated into 4 ml of M9+CA+amp+W (Table 2) and grown overnight at 
37.degree. C. The overnight culture was diluted 1:10 in 30 ml M9+CA+amp 
(Table 2) and grown for 1 hour at 30.degree. C. with great aeration. After 
1 hour 150 ul of 10 mg/ml indoleacrylic acid (Sigma, St. Louis, Mo.) in 
100% ethanol was added to the culture and it was grown for an additional 2 
hours at 30.degree. C. 
TABLE 3 
______________________________________ 
2 .times. Sample Buffer 
36 ml 0.5M Tris-HCl, pH 6.8 
16 ml glycerol 
16 ml 20% SDS 
4 ml 0.5% Bromopheno1 Blue in 0.5M Tris-HCl, pH 6.8 
Mix all ingredients. Immediately before use, add 
100 ul .beta.-mercaptoethanol to each 900 ul dye mix. 
Cracking Buffer 
0.01M sodium phosphate, pH 7.2 
1% .beta.-mercaptoethanol 
1% sodium dodecylsulfate 
6M urea 
Western Transfer Buffer 
25 mM Tris, pH 8.3 
19 mM glycine, pH 8.3 
20% methanol 
Western Buffer A 
50 ml 1M Tris, pH 7.4 
20 ml 0.25 mM EDTA, pH 7.0 
5 ml 10% Np-40 
37.5 ml 4M NaCl 
2.5 g gelatin 
______________________________________ 
The Tris, EDTA, NP-40 and NaCl were diluted to a final volume of one liter 
with distilled water. The gelatin was added to 300 ml of this solution and 
the solution was heated in a microwave oven until the gelatin was 
dissolved into solution. The gelatin solution was added back to the 
remainder of the first solution and stirred at 4.degree. C. until cool. 
The buffer was stored at 4.degree. C. 
Western Buffer B 
50 ml 1 M Tris, pH 7.4 
20 ml 0.25 M EDTA, pH 7.0 
5 ml 10% NP-40 
58.4 g NaCl 
2.5 g gelatin 
4 g N-lauroyl sarcosine 
The Tris, EDTA, NP-40 and NaCl were mixed and diluted to a final volume of 
one liter. The gelatin was added to 30 ml of this solution and heated in a 
microwave oven until the gelatin was dissolved into solution. The gelatin 
solution was added back to the original solution and the N-lauroyl 
sarcosine was added. The final mixture was stirred at 4.degree. C. until 
the solids were complete)y dissolved. This buffer was stored at 4.degree. 
C. 
The culture was pelleted by centrifugation and the supernatant was 
discarded. The cell pellet was resuspended in 50 ul cracking buffer (Table 
3) and incubated at 37.degree. C. for 0.5-3 hours. An equal volume of 
2.times.sample buffer (Table 3) was added and the sample was heated in 
boiling water bath for 3-5 minutes. The sample was electrophoresed in a 
10% SDS-polyacrylamide gel. The proteins were transferred to 
nitrocellulose using the method essentially described by Towbin et al. 
(ibid.). The nitrocellulose filter was stained by immersion in a solution 
of 100 ml distilled water, 4 ml glacial acetic acid and 4 drops Schilling 
green food coloring (McCormick and Co., Inc., Baltimore, Md.). The band 
corresponding to Barrier protein was cut out of the filter and the stain 
was removed by a distilled water wash. The de-stained nitrocellulose 
filter containing the Barrier protein was dried at 37.degree. C. for one 
hour and was subsequently mixed with Freund's adjuvant (ICN Biochemicals, 
Costa Mesa, Calif.) and dimethyl sulfoxide (DMSO). The mixture was 
injected subcutaneously at three sites into New Zealand White rabbits. The 
injections were repeated 2.5 months after the first injection. Ten days 
after the final injection, whole blood was removed from the rabbit and 
allowed to coagulate. The blood clot was separated from the serum by 
centrifugation. The serum was removed to a fresh tube and stored at 
-20.degree. C. These polyclonal antibodies, designated C-2465, recognized 
the Barrier protein. 
C. Expression of BAR1 in a temperature regulated MNN9 strain 
S. cerevisiae strain XCY42-28B was transformed with the temperature 
regulated MNN9 expression vector pZY66 (Example 5) and pSW24 (Example 3) 
or with pSW24 and pM111 using methods known in the literature, see for 
example, Beggs (ibid.). Transformants were selected for their ability to 
grow on -Leu-TrpDS (Table 2) at 25.degree. C. 
Transformants were streaked for single colonies on -Leu-TrpDS plates and 
were grown at 25.degree. C., 30.degree. C. or 35.degree. C. Transformant 
colonies were inoculated into 5 ml -Leu-TrpDS and were grown overnight at 
25.degree. C., 30.degree. C. or 35.degree. C., depending on the growth 
temperature of the inocula. The overnight cultures were diluted 1:100 in 
50 m -Leu-TrpDS and grown for approximately 48 hours at 25.degree. C., 
30.degree. C. or 35.degree. C. 
The cells were removed from the culture by centrifugation and the 
supernatants were decanted and saved. An equal volume of 95% ethanol, held 
at -20.degree. C., was added to each supernatant and the mixtures were 
kept at -20.degree. C. for 45 minutes. The ethanol mixtures were spun in a 
GSA (Sorval) rotor at 9,0000 rpm for 30 minutes at 4.degree. C. to pellet 
the precipitate. The supernatants were decanted and the pellets were 
allowed to dry. The pellets were resuspended in 500 ul distilled water. 
Fifty ul of 2.times.sample buffer (Table 3) was added to 50 ul of each 
resuspended sample and the mixture was electrophoresed in a 10% 
polyacrylamide gel and transferred to nitrocellulose using the method 
essentially described by Towbin et al. (ibid.). The nitrocellulose filter 
was probed with the rabbit polyclonal C-2465 and visualized using 
horseradish peroxidase-conjugated goat anti-rabbit antibodies. The 
immunoblot showed that at 35.degree. C., the Barrier-substance P protein 
made by XCY42-28B[pSW24, pZY66] was present as a homogeneous species which 
carried the same amount of glycosylation as XCY42-28B[pSW24, pM111] grown 
at all temperatures. This indicated that at 35.degree. C. the MNN9 gene is 
turned off and protein glycosylation is carried out as is found in a 
similarly transformed mnn9 strain. At 30.degree. C., the Barrier-substance 
P protein produced from XCY42-28B[pSW24, pZY66]was mostly 
hyperglycosylated and at 25.degree. C., the Barrier-substance P protein 
produced from XCY42-28B[pSW24, pZY66]was a very heterogeneous 
hyperglycosylated species. 
EXAMPLE 7 
A Method to Detect mnn1 Mutants 
Rabbit polyclonal antibodies were raised against Barrier protein which was 
produced from a mnn9 strain. Barrier protein was produced from XV732-1-9A 
(Example 1.A.) which had been transformed with pZV100, comprising the TPI1 
promoter, MF.alpha.1 signal sequence, and the BAR1 coding sequence. 
Plasmid pZV100 was constructed as follows. 
The TPI1 promoter was derived from plasmid pM210 (also known as pM220, 
which has been deposited with ATCC Accession No. 39853). plasmid pM210 was 
digested with Bgl II and Hind III to isolate the 0.47 kb fragment 
(fragment 1). 
A Hind III-Eco RI adaptor encoding the MF.alpha.1 signal peptide was 
subcloned with a portion of the 5' coding sequence of the BAR1 gene 
deleted for the putative BAR1 signal sequence into the cloning vector 
pUC13. Plasmid pZV16 (Example 3) was digested with Eco RI and Sal I to 
isolate the 0.67 kb BAR1 fragment. Oligonucleotides ZC566 (5' AGC TTT AAC 
AAA CGA TGG CAC TGG TCA CTT AG 3') and ZC567 (5' AAT TCT AAG TGA CCA GTG 
CCA TCG TTT GTT AA 3') were kinased and annealed essentially as described 
in Maniatis et al. (ibid.). The kinased, annealed ZC566/ZC567 adaptor was 
joined with the 0.67 kb BAR1 fragment in a three-part ligation with pUC13 
which had been linearized by digestion with Hind III and Sal I. The 
resultant ligation mixture was transformed into E. coli strain JM83. 
plasmid DNA made from the resultant transformants were screened by 
digestion with Hind III and Sal 1. A positive clone was designated plasmid 
pZV96. Plasmid pZV96 was digested with Hind III and Sal I to isolate the 
0.67 kb fragment comprising the ZV566/ZC567 adaptor-BAR1 fragment 
(fragment 2). 
The remainder of the BAR1 gene was derived from pZV9 (Example 3). Plasmid 
pZV9 was digested with Sal I and Bam HI to isolate the 1.25 kb BAR1 
fragment (fragment 3). Fragments 1 and 2 (comprising the TPI1 
promoter-MF.alpha.1 signal sequence and the ZC566/ZC567-BAR1 fragment, 
respectively) were joined with fragment 3 (1.25 kb BAR1 fragment) and 
YEp13 which had been linearized by digestion with Bam HI. The resultant 
ligation mixture was transformed into E. coli RR1. Plasmid DNA made from 
the resultant transformants was digested with Bam HI+Hind III and Bam 
HI+Sal I to confirm the construction and to determine the orientation of 
the insert. A positive clone having the TPI1 promoter proximal to the Hind 
III sites on the YEp13 vector was designated pZV100. 
S. cerevisiae strain XV732-1-9A was transformed with pZV100 and 
transformants were selected for their ability to grow on -LeuDS plates 
(Table 2). A transformant colony was inoculated into 10 m -LeuDS (Table 2) 
and was grown overnight at 30.degree. C. The overnight culture was diluted 
1:100 into 978 ml -LeuDS and the culture was grown for 43 hours at 
30.degree. C. The culture was centrifuged and the supernatants were 
decanted into 250 ml centrifuge bottles. An equal volume of 95% ethanol, 
held at -20.degree. C., was added and the mixtures were incubated at 
-20.degree. C. for approximately 2 hours. The mixtures were centrifuged in 
a GSA (Sorval) rotor at 9,000 rpm for 30 minutes at 4.degree. C. The 
supernatants were discarded and the protein pellets were allowed to air 
dry. The pellets were resuspended in a total volume of 6 ml of 1.times. 
sample buffer (3 ml dH.sub.2 O and 3 ml 2.times.sample buffer [Table 2]). 
The sample was electrophoresed in a 10% polyacrylamide gel and was 
transferred to nitrocellulose using the method described by Towbin et al. 
(ibid). The nitrocellulose filter was stained by immersion in a solution 
of 100 ml distilled water, 4 ml glacial acetic acid, and 4 drops Schilling 
green food coloring. The band corresponding to Barrier protein was cut out 
of the filter and the stain was removed by a distilled water wash. The 
de-stained nitrocellulose-Barrier band was dried at 37.degree. C. for one 
hour and was subsequently mixed with Freund's adjuvant (ICN Biochemicals, 
Costa Mesa, Calif.) and dimethyl sulfoxide (DMSO). The mixture was 
injected subcutaneously at three sites into New Zealand White rabbits. The 
injections were repeated a total of three times at approximately one-month 
intervals. Ten days after the final injection, whole blood was removed 
from the rabbit and allowed to coagulate. The blood clot was separated 
from the serum by centrifugation. The serum was removed to a fresh tube 
and stored at -20.degree. C. These polyclonal antibodies recognized the 
Barrier protein and the sugar moieties present on the protein. 
Colonies of test strains were grown on YEPDS and the resultant colonies 
were replica plated onto nitrocelluose filters. The filters were subjected 
to three fifteen-minute washes in Western Transfer Buffer A (Table 3). The 
filters were then washed in Western Buffer A (Table 3) for five minutes. 
The filters were transferred to fresh Western Buffer A and incubated for 
one hour. The filters were then washed with Western Buffer A for five 
minutes. A 1:500 dilution of the rabbit polyclonal anti-Barrier (mnn9) 
antibody was added to the filters and incubated for longer than one hour. 
Excess antibody was removed by three fifteen-minute washes in Western 
Buffer A. A 1:1000 dilution of goat anti-rabbit horseradish 
peroxidase-conjugated antibody was added to the filters, which were 
incubated for at least one hour. Excess conjugated antibody was removed 
with a distilled water rinse followed by three ten-minute washes with 
Western Buffer B (Table 3) and a final distilled water rinse. The assay 
was developed by the addition of horseradish peroxidase substrate (BioRad, 
Richmond, Calif.) which was allowed to develop until there was sufficient 
color generation. Colonies which were lightly stained with the antibodies 
were mnn1 colonies. 
EXAMPLE 8 
Construction of mnn1 and mnn1 mnn9 strains 
S. cerevisiae strains carrying mnn1 and mnn1 mnn9 mutations were 
constructed as follows. ZY400 (Table 1) was crossed with LB1-22D (Table 1, 
Yeast Genetic Stock Center, Berkeley, Calif.), and a diploid was selected 
and designated XV803. XV803 diploid cells were sporulated and asci were 
dissected. Spores were screened for the presence of the mnn1 mutation 
using the mnn1 screening method. The .DELTA.mnn9::URA3 marker was scored 
by the growth of the spores on YEPD (mnn9 mutants grow poorly on medium 
without osmotic support). A spore whose genotype was MAT.alpha. leu2-3,112 
.DELTA.mnn9::URA3 mnn1 was designated XV803-18. Another spore whose 
genotype was MAT.alpha. leu2-3,112 mnn1 .DELTA.pep4::CAT was designated 
XV803-16C. 
EXAMPLE 9 
Expression of BAR1 in a mnn1 mnn9 strain 
The expression of the BAR1 gene was examined in mnn1 mnn9 strains. Strains 
XV803-16C, XV803-16C, XY100 and ZY400 were transformed with pSW24. The 
transformants were selected for their ability to grow on -LeuDS plates 
(Table 2). Transformant colonies were streaked for single colonies onto 
fresh -LeuDS plates and allowed to grow at 30.degree. C. Transformant 
colonies were inoculated into 50 ml -LeuDS (Table 2) and grown at 
30.degree. C. for approximately 48 hours. The cultures were harvested and 
the cells were removed from the culture media by centrifugation. The 
supernatants were decanted into GSA bottles and equal volumes of 95% 
ethanol, held at -20.degree. C., were added. The mixtures were incubated 
at -20.degree. C. followed by centrifugation in a GSA rotor at 9000 rpm 
for 30 minutes at 4.degree. C. The supernatants were discarded and the 
precipitates were allowed to air dry. The precipitates were resuspended in 
4 ml distilled water and were re-precipitated by the addition of 4 ml of 
cold 95% ethanol. The mixtures were incubated and centrifuged as describe 
above. The supernatants were discarded and the pellets were allowed to air 
dry. 
The protein precipitates were resuspended in 150 ul distilled water. The 
samples were diluted with 150 ul 2.times. sample buffer (Table 3), and 100 
ul of each sample was then electrophoresed in a 10% polyacrylamide gel. 
The proteins were transferred to nitrocellulose by the method of Towbin et 
al. (ibid.) and the Barrier protein was visualized using the substance P 
antibody, as described in Example 3. The results showed that the Barrier 
protein made from the mnn1 mnn9 double mutant ran faster than the Barrier 
protein isolated from a mnn9 or mnn1 mutant, indicating that the Barrier 
protein made from the double mutant contained fewer sugar moieties than 
the protein made from the mnn9 mutant. 
EXAMPLE 10 
Cloning the MNN1 gene 
The MNN1 gene is cloned using the antibody screening method described 
above. A library of plasmids containing a random mixture or total yeast 
DNA fragments cloned into the vector YEp13 (Nasmyth and Reed, Proc. Natl. 
Acad. Sci. USA 77:2119-2123, 1980) is transformed into strain XV803-16C, 
and transformants are selected for their ability to grow on -LeuDS plates 
(Table 2). Transformants are resuspended in -LeuD (Table 2) by the method 
of MacKay (ibid., 1983) and counted. The pools are diluted and plated on 
-LeuD plates (Table 2) at a density of approximately 1200 cells/plate (if 
all the cells are viable). The plates are incubated at 30.degree. C. until 
colonies are grown. The colonies are replica-plated onto nitrocellulose 
filters and screened by the assay method described Example 7. Colonies 
which exhibit dark staining with the rabbit polyclonal antibodies will 
contain plasmids which complement the mnn1 mutation and allow the host 
cell to make wild-type glycosylated proteins. Plasmid DNA is isolated from 
the positive clones by methods known in the literature (e.g., Hartig et 
al., ibid.) and is transformed into E. coli transformants. Plasmid DNA is 
isolated from E. Coli transformants and is subjected to restriction 
enzyme analysis. 
From the foregoing, it will be appreciated that, although specific 
embodiments of the invention have been described herein for purposes of 
illustration, various modifications may be made without deviating from the 
spirit and scope of the invention. Accordingly, the invention is not 
limited except as by the appended claims.