THE ESTROGEN RECEPTOR PROTEIN OF PARACOCCIDIOIDES BRASILIENSIS

 

Its role in paracoccidioidomycosis and the molecular mechanism by which it inhibits mycelium to yeast transformation

 

 

 

 

 

 

Blanca I. Restrepo A.

 

 

Microbiology

 

 

 

ABSTRACT

 

 

Paracoccidioidomycosis, the most common systemic mycosis in Latin America is caused by the dimorphic fungus Paracoccidioides brasiliensis.  Although it is 13-87 times more common in men than women, contact with the fungus is essentially the same for both sexes.  Transformation of mycelia to yeast, a process essential for disease to occur, is inhibited by estrogens (E2) in vitro.  A cytosolic estrogen recepetor protein has been described in this fungus.  The objective of this proposal is to determine the role of this estrogen receptor protein (ERP) in influencing resistance to disease in females, and to study the molecular mechanism by which estrogens inhibit mycelium to yeast transformation.  I will design a mouse model of paracoccidioidomycosis which shows a difference in disease susceptiblity dependent on the sex of the mouse in order to establish the role of the ERP in determining resistance to disease.  To identify the molecular mechanism by which the ERP determines susceptibility to disease, I will clone the ERP gene and synthesize enough recombinant protein to identify a DNA element to which the E2*ERP complex binds.  Then, the relationship between in vitro binding of the E2*ERP complex to the ERE and its effect on gene expression will be evaluated in P. brasiliensis and Saccharomyces cerevisiae.  These studies will contribute to understanding the low incidence of paracoccidioidomycosis in females, the interactions between fungi and mammalian hormones,  the molecular events underlying the process of dimorphism in pathogenic fungi and the E2-dependent events which occur during the development of breast cancer.
A.        OBJECTIVES AND SPECIFIC AIMS

 

            The objective of my proposal is to test the role of the estrogen receptor protein (ERP) ofParacoccidioides brasiliensis  in resistance to disease in females, and to determine the molecular mechanism by which estrogens inhibit the mycelium to yeast transformation.  My hypothesis is that 17-b-estradiol (E2) enters the mycelial cell and binds to the cytoplasmic ERP.  The resulting E2*ERP complex can now bind a specific estrogen responsive DNA element (ERE) and influence the expression of a nearby gene.   The E2-induced events result in the inhibition of mycelium to yeast transformation, a process essential for disease to occur.   Gene expression may be altered by increasing the expression of a gene product which represses the expression of one or several proteins important for dimorphism.  This hypothesis is based on the mechanism of action of E2 on other E2-dependent systems (39), and also on the observed general repression in protein expression of P. brasiliensis  72h after exposure of mycelia to E2 at 37oC (8).   This does not exclude the possibility that estrogens induce a direct inhibitory effect on protein expression in P. brasiliensis.

 

            Specifically, the following mayor points will be addressed:

 

1.        Design a mouse model of paracoccidioidomycosis which exhibits a difference in susceptiblity to disease dependent on the sex of the mouse

2.        Establish the role of the ERP in determining resistance to disease using the mouse animal model

3.        Purification, partial amino acid sequencing and development of monoclonal antibodies against the ERP

4.        Cloning of the ERP gene and characterization of the recombinant gene product

5.        Identification of the estrogen responsive element (ERE)

6.        Determination of the relationship between in vitro binding of E2*ERP to the ERE and its effect on transcription in vivo

 

B.        BACKGROUND AND SIGNIFICANCE

 

            Paracoccidioides brasiliensis, is the etiologic agent of paracoccidioidomycosis, the most common systemic mycosis in Latin America.  It is striking that the disease is 13-87 times more common in men than women, although skin test surveys indicate that exposure to the fungus occurs with about equal frequency in both sexes (10).  Also, there is no sex-based difference in those who acquire the disease before puberty.  A microculture system was established to study the in vitro transformation from mycelia (environmental form) to yeast (tissue form), as would occur in vivo during infection.  In these studies transformation was inhibited by 17-b-estradiol (E2) (at physiological concentrations), diethylstilbesterol (DES) a synthetic estrogen, while other steroid hormones were inactive.  This transition must occur during the initial establishemnt of infection after the fungus gains access to the host through its portal of entry, the lungs.  In contrast, yeast-to-mycelial transformation and yeast growth and budding were unaffected by E2  (reviewed in ref. 29,34).  Similar results were obtained for the transformation of conidia (spore produced during the mycelial stage, presumed to be the infectious propagule in nature) to yeast (32).

            An estrogen receptor protein (ERP) was identified in the cytosol of P. brasiliensis yeast cells (20).  Scatchard analysis of [3H]-estradiol binding showed an apparent dissociation constant (Kd) of 1.7 x 10-8 M and a maximal binding capacity (Nmax) of 235 fmol/mg of protein.  The plot produced a straight line indicating a single class of binding sites. Competition studies revealed that estrone, estriol, DES and progesterone had 25% of the affinity of E2 while binding of other steroid hormones was negligable.  Binding activity was inhibited by trypsin and redued by N-ethylmaleimide suggesting the binder is a protein containing sulfhydryl groups.  Liquid chromatography (HPLC) indicated that the binding protein has a relative molecular mass of 60,000 daltons and sucrose gradient centrifugation indicated a sedimentation coefficient of 4.4S.  Later, an ERP in the mycelial cytosol was also found (38). 

            Analysis of cytosol proteins by one-dimensional SDS-PAGE revealed numerous differences between the mycelial and yeast forms as well as alterations induced by E2 (8).  The synthesis of several proteins which normally appear during the transition and those which are associated specifically with the yeast form was apparently blocked upon treatment of mycelial cultures with 2.6 x 10-7 M E2.  Methionine uptake by the fungus was also increased by E2.  In conjunction with these steroid-induced alterations on protein expression, little or no morphological transformation of mycelium to yeast occurred.  This regulation of protein expression by E2 is consistent with the hypothesis that the mechanism of action of E2 on P. brasiliensis is analogous to steroid-receptor mediated action in mammalian cells.

            These studies have led to the hypothesis that after infection in the female E2 inhibits or delays the transformation step necessary for fungal pathogenicity, possibly allowing sufficient time for the host to mount a protective immune response.  The cytosolic ERP of the fungus may be the molecular site of action of this hormone. Although a fungal ligand has not been identified, the "accidental" molecular interaction of the cytosolic protein with the mammalian hormone may have an important role in determining resistance to paracoccidioidomycosis in the female host. 

            Since other factors in the female may also affect resistance to disease (sex hormones may influence immune responses and killing by toxic oxygen metabolities), I want to verify the role of the ERP in  determining resistance to paracoccidioidomycosis in females.   My findings will be important for understanding better the molecular events associated with the dimorphism of pathogenic fungi.   The transformation from an environmental form (mycelia) to a tissue form (yeast or spherula in Coccidioides immitis) in dimorphic fungi like P. brasiliensis, Histoplasma capsulatum, Blastomyces dermatitidis and C. immitis represents an adaptation of the fungus for survival inside the host, allowing it to proliferate and cause disease (34).  Little is known about the molecular events required for transformation, or the advantages the tissular form provides for survival.   The identification of target genes whose expression is directly altered by the cytosolic ERP of P. brasiliensis could lead to the identification of key proteins required for dimorphism.   These molecules could be targets for the design of very specific prophylactic drugs to prevent disease in males which have been infected with the fungus (positive cutaneous test but no disease symptoms).   The interaction of mammalian estrogens with P. brasiliensis' ERP may provide a simple eukaryotic model system to study the mechanisms by which the E2*ERP complex act at the cell level.  This findings will contribute to understanding the interaction between mammalian hormones with other fungal species and to preventing or treating an afflicting problem in women; breast cancer.

 

C.        EXPERIMENTAL DESIGN

 

Specific aim 1:  Design a paracoccidioidomycosis mouse model in which susceptibilty to the disease is sex dependent

Rationale and hypothesis:  A variety of murine experimental paracoccidioidomycosis models have been described in the literature.  Different mouse strains have varying degrees of susceptibility to this mycoses but no study has shown a significant difference in susceptibility to disease in males compared to females.  The intranasal inhalation of conidia is the model that more closely mimics natural infection in men (21), but in this study, a high dose of infectious conidia was used to infect  Balb/c mice, a strain with shows intermediate resistance to paracoccidioidomycosis (7).  Both of these factors may contribute to hinder a sex distinction in the susceptibility this mycoses.  The combination of a more suceptible mouse strain (B10A, B10.D2/oSn, or B10.D2/nSn) and a lower dose of conidia may allow a sex distinction in susceptibility to paracoccidioidomycosis.

Experimental design:  Following the experimental design for intranasal infection with conidia (21),  but using lower doses of the propagule (105/mouse), 5 male and 5 female mice from the susceptible strains B10A, B10.D2/oSn, or B10.D2/nSn will be infected.  Evaluation of susceptiblity to paracoccidioidomycosis will be done by hystopathology and colony counts of lung, liver and spleen cultures, performed at the time periods described(21). 

Alternative approach:  If the previous approach fails to show a difference in susceptibility to paracoccidioidomycosis based on sex, I will follow the same experimental design, but in this case male and female animals will be castrated prior to puberty and then treated with either androgens or estrogens as previously described (31).  Then, mice will be infected.

Expected results:  In males or androgen treated mice, I expect to see a greater susceptibility to disease, in vivo transformation of mycelia to yeast and higher colony counts on the cultured organs . Females or estrogen-treated mice should be more resistant to disease and histopathology should reveal inhibition of mycelia to yeast transformation and reduced (if any) colony counts on cultured organs.

 

Specific aim 2:  Evaluate the role of the ERP in the determining disease susceptibility in male and female mice

Rationale and hypothesis:   Paracoccidioidomycosis is 48 times more common in men than women.  In vitro studies have shown that mycelia to yeast transformation of P. brasiliensis at 37oC is inhibited by E2 (29).  A cytosolic ERP has been identified in the yeast and mycelial forms of the fungus (20,38).   These observations have led to the hypothesis that females are more resistant to the disease because E2, acting through the ERP of P. brasiliensis inhibits the mycelia to yeast transformation.  To determine the role of the ERP in disease resistance I will select mutant P. brasiliensis cells resistant to the inhibition of transformation by E2, and use the conidia from the cells unable to bind E2 in the animal model described under specific aim 1.

Experimental design:  P. brasiliensis conidia will be exposed to the chemical mutagen nitrosoguanidine (26) and cells resistant to E2 transformation inhibition will be selected by plating the surviving conidia at 37oC with E2.  I expect to see growth only in conidia able to transform to yeast in the presence of E2.  The cytosol extracts of mutant cells will be tested for the capacity to bind E2.  Conidia from mutants unable to bind E2 (due to a mutation in the ERP) and the parent wild type P. brasiliensis strain (control) will be used to inoculate mice.   Susceptibility to disease in male and female mice will be determined as described above.  

Expected results: If mutant fungal strains resistant to inhibition of transformation by E2 cause infection and disease equally well in males and females, (compared to wild type strain where males should have been more susceptible to disease) this suggests the ERP plays a role in determining susceptibility to paracoccidioidomycosis.  Since chemical mutagens can cause several mutations in the same cell, another mutation in these cells (besides the ERP) could influence susceptibility to disease. To make this possibility unlikely, different mutant strains unable to bind E2 in vitro will be tested, and I expect to see essentially the same results in all of them, if the ERP is the main molecule mediating resistance to infection in females. 

 

Specific aim 3:  Purification, determination of partial amino acid sequence and development of monoclonal antibodies against the ERP.

Purification of the ERP from P. brasiliensis:  The ERP will be purified by affinity chromatography using the method described previously to purify ERP from MCF-7 human breast cancer cells (9).  Essentially, the cytosol of P. brasiliensis cells will be passed through a column of estradiol linked to Sepharose 6B.  Elution of the receptor will be facilitated with 50 uM [3H]-estradiol in 10% (vol/vol) dimethyl formamide/0.5M sodium thiocyanate.  This method should allow good elution of the bound receptor under conditions compatible with its stability, yielding a steroid-receptor complex (E2*ERP). 

An alternative approach is to purify the ERP by affinity chromatography (35) using anti human E2*ERP monoclonal antibodies D547Sp-g, D75P3-g and D58P3u, (kindly recieved from Goeffrey L. Greene) (9) linked to a sepharose 4B column.  These monoclonals crossreacted with monkey endometrium and showed varied crossreactivity with calf and rat cytosol E2*ERP complex and may therefore react also with P. brasiliensis ERP.  The ERP will be eluted from the antibody by increasing the salt concentration of the buffer.  SDS-PAGE and Coomasie blue staining will be performed to check the purity of the protein eluted from the column.  I expect to see a single protein with Mr of 60,000 daltons.  The E2*ERP complex may show a single band of a higher molecular weight.

Partial amino acid sequence of the whole protein or component peptides will be determined on an Edman gas-phase sequencer (12), after cyanogen bromide fragmentation of the protein (5).   Two peptide sequences (aprox. 15 amino acids long) will be chosen to design a mixture of antisense oligonucleotide probes using an automated oligonucleotide synthesizer. 

Monoclonal antibodies against the ERP and some of its component peptides will be produced by standard techniques (9) and the specificity of the antibodies will be determined by Western blot. 

 

Specific aim 4:  Cloning and sequencing of the ERP gene and characterization of the recombinant ERP

Experiment 1: ( Cloning of the cDNA for the ERP into lgt11):  RNA will be isolated from mycelial cells of P. brasiliensis  (17) and poly(A)+ RNA will be purified by oligo(dT)-cellulose chromatography (27).  To enrich for mRNA coding for the ERP,  mRNA will be fractionated on a 5-20% sucrose gradient and aliquots of each fraction will be in vitro translated in the presence of [35S]methionine.  Samples will be precipitated with a mixture of anti-ERP monoclonal antibodies (described above) and the antibody-precipitated proteins will be separated on SDS-PAGE (3) and displayed by fluorography(6).   As a control total mRNA will also be translated in vitro, immunoprecipitated and run in the same gel.  The mRNA from the sucrose gradient fractions equivalent to the lanes containing the ERP will be used to prepare cDNA (18) and cloned into the expression vector lgt11(16).  Monoclonal antibodies will be used to screen the expression cDNA library (25) and the positive clones will be rescreened with the oligonucleotide probes designed above.  The positive clones will be further characterized.

Experiment 2:  (expression of the ERP in E. coli):  The lgt11 positive clones will be subcloned into pUC18 and lysates from the transformed E. coli (white colonies) will be subjected to Western blot using anti-ERP monoclonal antibodies.  Clones coding for a protein of about 60,000 daltons, reactive with anti-ERP monoclonal antibodies will be selected and cell extracts from these clones will be assayed for E2-binding activity (20).   The clones capable of binding E2 will be used to produce recombinant ERP (rERP) for further studies.  To characterize the binding constant and binding capacity of the rERP, extracts from cells synthesizing the whole gene or only pUC18 (negative control), and P. brasiliensis yeast cell extracts (positive control) will be prepared and binding to E2, DES, and other hormones will be determined as described (20). The ERP will be purified by affinity chromatography (35).

Expected results and alternative approaches:  I expect to see similar binding properties between P. brasiliensis ERP and the E. coli rERP.  If no clones contain the whole 60,000 daltons protein, I may have to screen a genomic DNA library using the lgt11 positive clones as probes.  If the binding properties of the E. coli rERP are different from the fungal ERP, we will subclone the pUC18 inserts in front of the 3-phosphoglycerate kinase (PGK) promoter of the yeast/E. coli shuttle vector pTG848 (Transgene SA), and S. cerevisiae strain TGY14 (MATa, ura3-251-373-328, leu2, pep4.3) will be transformed.  Expression of the ERP and its binding capacities will be determined as described above.

Experiment 3:(Sequencing of the ERP gene):  The clones used to express the rERP will be sequenced using Sequenase (United States Biochemical Corporation, Ohio).  The nucleotide sequence will be compared with the amino acid sequence of the ERP peptides to confirm we are working with the ERP gene.  A gene bank sequence analysis will be done (Genetics Computer Group Version 5).

Expected results: A high degree of homology between the E2-binding domain of the human and fungal ERP would help explain the "accidental" molecular interaction observed between the fungal ERP and mammalian estrogens. Homology at the estrogen responsive element (ERE)-recognizing domain may depend on how conserved the ERE sequence of P. brasiliensis is compared to the ERE sequences from other species.  Although the EREs identified so far are very conserved, it is also possible that the ERE of P. brasiliensis is very different.  

 

Specific aim 5:  Identification of the estrogen responsive element (ERE)

Rationale and hypothesis:  In E2-responsive systems, estrogen enters the cell and binds the cytosolic ERP.  This complex has the capacity to bind a DNA element (ERE) present upstream of an open reading frame and modulate the expression of this gene.  One to three homologous 13 bp palindromic sequences 5' to various estrogen regulated genes has been proved to be essential for estrogen-regulated expression (23). Except for the three central nucleotides, this sequence is conserved in the estrogen-regulated genes of xenopus and chicken and also mediates an E2-dependent effect in humans (19).  To determine if E2 affects gene expression thru an ERE in P. brasiliensis, I will try to identify a DNA sequence to which E2*ERP binds.

Experimental design:  To search for an ERE, I will extract total DNA from P. brasiliensis  (11) and construct a genomic DNA library in the l vector EMBL3 (28).  My first option will be to probe this library with an oligonucleotide constructed on the basis of the consensus sequence for ERE of other systems.  DNA from positive clones will be subcloned into pUC18 and subjected to gel retardation assays (11) in the presence of purified rERP and E2.  As controls, DNA alone or DNA with ERP will be incubated.  The ERE from clones which exhibited retarded migration when compared to the controls will be mapped to individual restriction fragments of the insert by incubating E2*ERP in the presence of whole plasmid DNA digested with a restriction enzyme. The restriction fragments containing the ERE will be subcloned into pUC18 and sequenced with Sequenase.  DNase protection assays will be done to determine the boundaries of the ERE (11).

Expected results:  If positive clones are obtained by this approach, then it is likely that the ERE sequence is very conserved in P. brasiliensis also.  If I do not get any positive clones, it is possible that the ERE of P. brasiliensis has a different consensus sequence from that of other systems or that technical problems (probe is a palindromic sequence) may hinder the detection of it.   If such is the case I will follow alternative approaches.

An alternative approach is to identify proteins whose regulation is directly influenced by E2.  For this 2-dimensional gel electrophoresis protein patterns of mycelia incubated at 37oC for 24 h with and without E2 (1) will be compared.  Proteins whose expression is influenced (increase or decrease) by E2 will be electroeluted from the gel (14) and their partial amino acid sequence will be determined (12).  The amino acid sequence will be used to design oligonucleotide mixtures to use as probes to screen the lEMBL3 genomic library.  DNA from positive clones will be treated as described above. 

Expected results and alternative approaches: It is likely to identify the ERE by this approach if, like in other systems, the ERE of P. brasiliensis is located near the E2-dependent genes (the nearest ERE element has been found at about -300 nucletides upstream of the transcription initiation site of E2-dependent gene).  This approach may be clear cut and helpful if one or a few proteins show altered expression but if none or too many proteins have altered expression, it may be confusing .

A 3rd alternative is to do an equilibrium binding method that can detect DNA:protein interactions.  However, all these methods work better when the putative specific EREs are enriched relative to the excess of non-specfic DNA (11).  The filter binding assay is perhaps the most adequate approach to detect unknown DNA binding elements from a genomic DNA library.  If no ERE is detected by any of the described approaches, I will look for additional factors from a P. brasiliensis  cell extract required for biniding of E2*ERP to DNA.

 

Specific aim 6:  Correlation between in vitro binding of E2*ERP to the ERE and its effect on gene expression

Experiment 1:  (ERE mutants and correlation with in vitro binding)

Rationale and hypothesis:  To correlate in vitro binding capacity of E2*ERP to the ERE with in vivo biological responses, I will generate specific mutations in the ERE, test their in vitro binding capacity and correlate with its effect in vivo.  In other E2-dependent systems, mutagenesis in the conserved nucleotides of the ERE has shown to alter binding of the E2*ERP complex in vitro and to decrease the E2-modulated activity in vivo (39).

Experimental design:  Oligonucleotides mutated at desired sites of the ERE will be synthesized, cloned into a plasmid (19) and their binding to E2*ERP will be evaluated by filter binding assays (11).  Mutations in nucleotides which inhibit binding of E2*ERP will be chosen for further studies.

Experiment 2: (experiments in P. brasiliensis)

Rationale and hypothesis: The direct effect of E2 on gene expression in P. brasiliensis is not known.  In a previous study(8), a general repression in protein expression was observed 72 h after incubation of mycelia with E2 at 37oC.  It is possible that E2 stimulates transcription of a regulator protein which represses expression of other proteins required for transformation ( a negative global regulation).  However, it is also possible that E2 exerts a direct negative regulation on gene expression.  To evaluate the effect of E2 on gene expression I will introduce chimeric plasmids into P. brasiliensis.  These plasmids will contain a reporter gene whose expression depends on the wild type or mutant ERE cloned upstream from it.  Expression of the reporter gene will be evaluated after addition of E2 to the cultures.  This approach may have some caveats because no molecular biology has been described in P. brasiliensis.

Experimental design: P. brasiliensis conidia will be subjected to 1-step gene disruption of the ERE (30). For this, I will subclone into the ERE, a yeast ura3 gene creating a disrutption of the ERE element.  The altered ERE-ura3 containing fragment will be used to transform P. brasiliensis conidia auxotroph for uracil (ura3).   This mutagenesis will be done in conidia because apparently 80% of them have one nucleus while yeast cells are multinucleate (22).   Presumable mutants in the genomic ERE will be selected as uracil prototrophs capable of transforming from mycelia to yeast when cultured at 37oC in the presence of E2.  Such strains will be used as hosts for transformation.  I will construct a parent shuttle vector which can replicate stably in P. brasiliensis, S. cerevisiae and E. coli.  This vector will contain the E. coli LacZ  gene under the control of test promoters.  These promoters will consist of wild type or mutant or no ERE sequences upstream of the yeast GAL1 promoter (contains the TATA sequence, but not the upstream activator sequence).  In the 3 types of plasmids, the GAL1 promoter should only produce basal levels of b-galactosidase in the absence of E2 (24).  The resulting "pBIR-1" plasmids are shown in figure 1.  The exact positions and distances of the regulatory elements will be based on the in vivo observations.  P. brasiliensis mycelia (ERE-) auxotrophic for leucine (leu2) will be transformed with pBIR-1.  The transformants will be incubated at 37oC in the presence or absence of E2 and b-galactosidase levels will be measured (33) twenty four hours later . 

Expected results and alternative approaches:  Upon E2 addition, I may see an increase in b-galactosidase expression (as in other estrogen-regulated genes) in cells containing pBIR1/ERE, while constructs containing a mutant ERE (no binding in vitro to E2*ERP) or no ERE (pBIR1) should not show any noticeable change in gene expression.  Even though a negative regulation by estrogens has not been described, it is possible that E2 exerts a repressive effect on transcription.  If this were the case, I would put LacZ under the control of a constitutively expressed promoter like the PGK promoter in order to detect the repression induced by E2.

Experiment 3: (experiments in S. cerevisiae)

Rationale:  Regardless of my success or failure with P. brasiliensis, I will also do in vivo studies in S. cerevisiae in order to characterize better the molecules and DNA elements required for E2-dependent events.  Cloning and characterization of the human ERP, as well as the evaluation of an E2-dependent response has been previously done in S. cerevisiae using an experimental approach similar to the one we are proposing.  Therefore,  S. cerevisiea is likely to be an adequate yeast host cell to study the elements required for an E2-dependent response in P. brasiliensis. 

Experimental design:  Since S. cerevisiae has no ERP, I will insert the ERP gene under the control of the PGK promoter (13) into the pBIR1-series plasmids containing wild type or mutant or no ERE.  Then I will transform the yeast TGY14 with the resulting plasmids (pBIR2, figure 1) and compare  the levels of b-galactosidase in the presence or absence of exogenous E2. 

Expected results:  The advantage of studies on S. cerevisiae is that an E2 response is not naturally present, so it allows us to evaluate the role of the ERE and the ERP in the expression of the reporter gene.  If we obtain results similar to those observed in P. brasiliensis, it will suggest E2, ERP and ERE are the essential elements required to alter gene expression.  If we do not get any response to E2, it is possible that additional factors or DNA elements are required for this response.  Further experiments should be done in this case to account for a lack of response to E2.

FIGURE LEGENDS:

 

Figure 1.  Vectors used to study the E2-dependent modulation of transcription.  These E. coli-S. cerevisiae -P. brasiliensis  shuttle vectors are derived from pYERE1/HER (24). a) pBIR1 contains the the E. coli LacZ gene under control of test promoters which contain wild type (pBIR-1/ERE) or mutant (pBIR-1/EREM) or no ERE sequences (pBIR-1) of P. brasiliensis  upstream of the TATA box of the yeast GAL1 promoter.  The human estrogen receptor protein gene was deleted and an origin of replication for P. brasiliensis  (pBRAS ori) was inserted. The transformation markers for S. cerevisiae or P. brasiliensis  are LEU2 and URA3, and ampicillin resistance forE. coli.  b) pBIR-2 plasmids were constructed from pBIR-1 by inserting the ERP of P. brasiliensis  under the control of the PGK promoter.

 

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