Selection marker gene free recombinant strains: method for obtaining them and the use of these strains

The present invention discloses a selection marker free system which can be used to introduce genetic modifications in bacteria, yeasts and fungi. The system can be employed to introduce or delete desired genes or DNA fragments in the genome of the indicated host species without leaving any undesired DNA i.e. the selection marker used for selection of transformants or other DNA used for cloning. In this way strains have been developed containing only desired genes introduced at desired chromosomal sites. Similarly, desired DNA fragments have been deleted or replaced at desired sites.

TECHNICAL FIELD 
The present invention discloses selection marker gene free recombinant 
strains, a method for obtaining these strains and the use of these 
strains. Furthermore, the method of the present invention is used for 
performing strain improvement. 
BACKGROUND OF THE INVENTION 
There is an increasing social concern about the use of recombinant DNA 
technology. One of the promising application areas of recombinant DNA 
technology is strain improvement. Starting from the early days of 
fermentative production processes there has been a demand for the 
improvement of the productivity of the strains used for production. 
Classical strain improvement programs for industrially employed 
microorganisms are primarily based on random mutagenesis followed by 
selection. Mutagenesis methods have been described extensively; they 
include the use of UV light, NTG or EMS as mutagens. These methods have 
been described extensively for example in "Biotechnology: a comprehensive 
treatise in 8 vol." Volume I, Microbial fundamentals, Chapter 5b, Verlag 
Chemie GmbH, Weinheim, Germany. 
Selection methods are generally developed around a suitable assay and are 
of major importance in the discrimination between wild type and mutant 
strains. 
It has turned out that these classical methods are limited in their 
potential for improvement. Generally speaking consecutive rounds of strain 
improvement yield diminishing increases in yield of desired products. This 
is at least partially due to the random character of the mutagenesis 
methods employed. Apart from desired mutations these methods also give 
rise to mutations which are undesirable and which may negatively influence 
other characteristics of the strains. 
In view of these drawbacks it can be understood that the use of recombinant 
DNA methods was hailed as a considerable improvement. In general, 
recombinant DNA methods used in strain improvement programs aim at the 
increased expression of desired gene products. 
The gene products may be proteins that are of interest themselves, on the 
other hand it is also possible that the encoded gene products serve as 
regulatory proteins in the synthesis of other products. 
Strains can be improved by introducing multiple copies of desired protein 
encoding genes into specific host organisms. However, it is also possible 
to increase expression levels by introducing regulatory genes. 
Genes are introduced using vectors that serve as vehicles for introduction 
of the genes. Such vectors may be plasmids, cosmids or phages. The vector 
may be capable of expression of the genes in which case the vector 
generally is self-replicating. The vector may however also only be capable 
of integration. Another characteristic of the vector is that, when the 
expression product cannot be selected easily based on altered phenotypic 
properties, the vector is equipped with a marker that can easily be 
selected for. 
Vectors have not been isolated from all known microorganisms either since 
no vector could be found in the organism or since available vectors from 
other organisms could be used with little or no modification. The same 
applies to selection marker genes. 
Widespread use and the subsequent spreading of specific marker genes has 
recently become debatable. This is especially due to the finding that the 
use of antibiotics and antibiotic selection markers gives rise to an 
undesired spread of strains that have become antibiotic resistant. This 
necessitates the continued development of novel ever more potent 
antibiotics. 
It is therefore not surprising that there is a general tendency in large 
scale production to use recombinant microorganisms containing no 
antibiotic resistance genes or more generally as little as possible of 
foreign DNA. 
Ideally the transformed microorganism would contain only the desired 
gene(s), fragments thereof or modifications in the gene and as little as 
possible or no further remnants of the DNA used for cloning. 
SUMMARY OF THE INVENTION 
The present invention discloses a selection marker gene that can easily be 
deleted again from the recombinant host organism. The deletion of the said 
marker gene is based on dominant selection. 
The marker is used in species so diverse as bacteria, filamentous fungi and 
yeasts. 
The advantageous activity of the selection markers used herein is based on 
the following two step principle: 
a) the gene is integrated into the genome of the host organism and 
recombinant cells are selected, 
b) the transformed cell is grown on a substrate, which is converted by the 
marker gene encoded activity to a product that is lethal to the cell. 
Selected cells will be recombinant and will have deleted the selection 
marker gene. 
In general terms the present invention discloses cells, that may be animal 
or plant cells, and microorganisms that have a modification in the genome 
characterized in that the alteration is introduced using the amdS gene or 
the cDNA derived therefrom. 
An example of a selection marker gene that can be used in this way is the 
acetamidase gene. Preferably, this gene is obtainable from filamentous 
fungi, more preferably from Aspergilli, most preferably from Aspergillus 
nidulans. 
The invention further shows the introduction, deletion or modification of 
desired heterologous or homologous genes or DNA elements in the host 
organisms of choice using the acetamidase (amdS) gene as a marker. 
Subsequently the amdS gene is deleted. Preferably, the amdS and the 
desired genes are introduced site-specifically. 
The invention discloses a vector containing: 
a) a desired DNA fragment destined for introduction into the host genome, 
b) optionally a DNA sequence that enables the vector to integrate 
(site-specifically) into the genome of the host strain, 
c) a gene encoding an acetamidase (e.g. the amdS gene from A.nidulans) 
between DNA repeats. 
The invention further discloses host organisms transformed with the said 
vector. 
The invention further discloses selection marker gene free recombinant 
microorganisms. 
Specifically, the invention discloses organisms containing 
site-specifically introduced genes without any further foreign DNA being 
present. The method is therefore also suited for repeated modifications of 
the host genome, e.g. the sequential introduction of multiple gene copies 
at predetermined loci. 
The invention provides a method for obtaining selection marker gene free 
recombinant strains comprising the following steps: 
a) integration into the genome of the strain of a desired DNA fragment and 
a selection marker, 
b) selection of the recombinants, 
c) deletion of the selection marker preferably using internal recombination 
between selection marker flanking repeats, 
d) counter-selection based on the absence of the selection marker. 
Although this is the preferred method for obtaining selection marker gene 
free recombinant strain, the invention also provides modifications of this 
method, for example: The desired DNA fragment and the selection marker may 
be present on two different DNA molecules which are co-transformed. The 
selection marker does not necessarily integrate into the genome of the 
strain but may be present on an episomal DNA molecule which can be cured. 
The present invention further illustrates that this marker gene can be 
deleted from the genome of the transformed organisms without leaving a 
trace i.e. DNA used for cloning. 
The present invention discloses the use of the amdS gene from Aspergillus 
as a marker in bacteria and yeast. 
The invention discloses also the use of the amdS gene for deleting a 
desired gene from the chromosome of a `host` organism. Such modification 
techniques may be applied to filamentous fungi, yeasts and bacteria. In 
specific embodiments the following strains are employed Aspergilli, 
Trichoderma, Penicillium, Bacilli, E.coli, Kluyveromyces and 
Saccharomyces. 
The method of the present invention provides recombinant strains with 
genomic modifications obtained by repeating the procedure with the same or 
other vectors.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention discloses the use of a marker for selecting 
transformed host strains. The selection marker gene can be used on an 
episomal DNA vector. However, in the present invention, the marker gene is 
preferably integrated into the genome of the host strain. The advantage of 
the selection marker of the present invention is that it is a 
non-antibiotic dominant selection marker. Another advantage of the 
selection marker of the present invention is that it can be easily deleted 
from the transformed host organism. The deletion of the marker is based on 
dominant selection. As such the selection marker of the present invention 
is a dominant and bi-directional selection marker. To our knowledge it is 
the only selection-marker available which is bidirectional and dominant in 
both directions. 
In the present description we use the term `selection marker gene`. With 
this term we mean the DNA coding for the marker protein in a functional 
form irrespective of whether it is the actual gene or the cDNA derived 
therefrom. The gene or cDNA is used dependent on the host organism and the 
expected splicing problems. 
In the present invention we use the term `vector`. By this is intended any 
DNA molecule that can be introduced into a selected host irrespective of 
whether the vector integrates into the genome of the host cell or remains 
episomal. The vector contains a selectable marker gene functional in the 
selected host or can be co-transformed with another DNA molecule 
containing such a selection marker gene. 
The present description uses the term `desired heterologous or homologous 
genes or DNA fragments`. By this is intended a DNA fragment that may be 
obtained from the host strain or from another species or strain. The 
desired DNA fragment may contain any genetic element, parts thereof or 
combinations thereof, such as a gene (coding part or complete locus), a 
cDNA, a promoter, a terminator, an intron, a signal sequence, any 
regulatory DNA sequence or recognition sequence of DNA-binding proteins. 
The fragment may also be a DNA sequence that has been modified i.e. 
contains one or more nucleotide alterations (e.g. insertions, deletions, 
substitutions). 
The present description further uses the term `introduction` of a desired 
gene or DNA fragment. By this is intended an insertion, deletion, 
substitution of desired DNA sequences in a selected host cell. 
The term `genetic modification` used in the present invention refers to any 
modification of DNA sequences in a selected host cell which is the result 
of the introduction of any one of the above mentioned desired DNA 
fragments into the host cell, preferably by transformation or 
co-transformation. 
In general all these genetic modifications can be performed using the 
method of the present invention with subsequent deletion of the selection 
marker gene. Due to the fact that the recombinant strain containing such a 
genetic modification does not contain the selection marker gene, the 
procedure of the present invention can be repeated, so that the 
modifications suggested above can be combined in the recombinant strain. 
Ultimately, the procedure of the present invention can be used repeatedly 
up to the point that a recombinant strain is obtained from which all 
undesired activities have been removed by deletion or inactivation of the 
corresponding genetic elements and which contains the desired activities 
at the desired levels by sequential introduction of the corresponding 
desired DNA fragments at desired copynumbers and preferably at desired and 
defined loci. 
The A. nidulans acetamidase (amdS) gene allows A. nidulans to grow on 
acetamide as the sole N-source. For microorganisms that lack the 
possibility or only have a very limited capacity to use acetamide as the 
sole N-source the acetamidase gene can in principle be used as a selection 
marker provided that acetamide is taken up by the cells. The amdS gene has 
successfully been employed as a marker gene in Aspergilli (Kelly and Hynes 
(1985) EMBO J. 4, 475-479; Christensen et al. (1988) Bio/technology 6, 
1419-1422), Penicillium (Beri and Turner (1987) Curr. Genet. 11, 639-641) 
and Trichoderma (Pentilla et al. (1987) Gene 61, 155-164). 
The present invention for the first time discloses the use of the amdS gene 
from A.nidulans as a selection marker in organisms other than filamentous 
fungi. The use of this selection marker is disclosed in bacteria and 
yeasts. Specifically, the use is demonstrated in S.cerevisiae, in K.lactis 
in B.subtilis, in B.licheniformis and in E.coli. In view of the disclosed 
applicability of the selection marker in species selected from such 
diverse groups as fungi, yeasts and bacteria it is to be expected that the 
marker will also be applicable in other species pertaining to these 
groups. Use of this marker is therefore not restricted to the disclosed 
species. 
The amdS gene from A.nidulans is capable of converting acetamide to ammonia 
and acetic acid. This property enables A.nidulans to grow on a medium 
containing acetamide as the sole N-source or C-source. 
Another property of the amdS gene is that it is also able to convert 
fluoracetamide to ammonia and fluoracetic acid. Fluoracetic acid however 
is toxic to the cell. It is this property that forms the basis for another 
aspect of the present invention i.e the production of marker gene free 
recombinant strains. The fluoracetamide converting property enables the 
counter-selection of transformed cells. The amdS gene is introduced into 
the host strain and integrated into the genome through homologous 
recombination. The transformed strains are selected on a medium containing 
acetamide as the sole N-source. Subsequently the selected strains are 
grown on a medium containing fluoracetamide and urea (or other preferably 
defined N-sources) as the sole N-sources. The surviving strains will have 
deleted the amdS gene. 
The present invention uses the A.nidulans amdS gene as acetamidase marker 
gene. The relevant properties provided by the acetamidase encoded by the 
A.nidulans amdS gene, i.e. the ability to hydrolyse acetamide into ammonia 
and acetate as well as the ability to liberate fluoracetic acid from 
fluoracetamide, can also be provided by acetamidases from other sources. 
Use of an acetamidase marker gene is therefore not restricted to the 
A.nidulans amdS gene but includes any DNA sequence encoding a functional 
acetamidase. 
The frequency of marker deletion is substantially increased by increasing 
the capacity of the gene for intrachromosomal homologous recombination. To 
achieve this the amdS gene is preferably placed between DNA repeats. These 
repeats are not necessarily both present in the vector but may also be 
created by a single cross-over integration. Alternatively, one may omit 
flanking repeats and rely on other mechanisms for removal or inactivation 
of the marker gene. In that case, however, the outcome may be less 
predictable and may not result in removal but rather in mere inactivation 
of the marker gene. 
The vector may be constructed in such a way that, after deletion of the 
marker gene, no extraneous foreign DNA (except the DNA of interest) 
remains in she chromosome of the host strain. The invention discloses a 
vector comprising: 
a) a desired DNA fragment destined for introduction into the host genome, 
b) optionally a DNA sequence that enables the vector to integrate 
(site-specifically) into the genome of the host strain, 
c) a gene encoding an acetamidase (e.g. the amdS gene from A.nidulans) 
between DNA repeats. 
Identical results may be obtained when the DNA-fragment destined for 
introduction into the host genome and the selectable marker gene (e.g. the 
acetamidase gene) are present on two different DNA molecules which are 
co-transformed, in which case the DNA molecule containing the selectable 
marker does not necessarily integrate into the host genome but may be 
present on an episomal DNA molecule which can be cured. 
The sequences used for integration as mentioned under b) are used if 
site-specific (or better locus specific) integration is desired. If such a 
sequence is not present the vector nevertheless may integrate into the 
genome. This does not influence the ability to delete the selection marker 
gene. 
The dominant counter-selection described above can be employed in the 
development of industrial production strains in various ways. The use of a 
dominant selection marker is especially advantageous in the development of 
improved production strains due to the fact that these strains are often 
diploid or polyploid. 
The vector used for integration of the amdS gene preferably contains 
another gene of interest. The invention thus further enables the 
introduction of desired foreign or homologous genes or DNA elements in the 
host organisms of choice using the amdS gene as a marker. Subsequently the 
amdS gene is deleted. Preferably, the amdS and the desired genes or DNA 
elements are introduced site-specifically, whereafter the amdS gene is 
deleted. 
Specifically, the invention discloses organisms containing 
site-specifically introduced genes without any further foreign DNA being 
present. The invention is used for integration of multiple copies of a 
desired gene or a DNA element at predetermined genomic loci. 
The invention provides a method for obtaining selection of marker gene free 
recombinant strains comprising the following steps: 
integration of a desired gene or DNA element and a selection marker by 
homologous recombination between sequences incorporated in an expression 
cassette and sequences on the host chromosome, 
selection using the selection marker gene that is dominant, 
deletion of the selection marker gene using selection marker gene flanking 
regions, 
selection based on the absence of the selection marker gene 
(counter-selection). 
The present invention further shows that this marker gene can be deleted 
from the chromosomes of the transformed organisms without leaving a trace 
i.e. DNA used for cloning. Moreover, the invention also shows that similar 
if not identical results can be obtained when the desired gene or DNA 
element and the selection marker are present on two different DNA 
molecules which are co-transformed. 
Finally the invention discloses the use of the amdS gene for deleting a 
desired gene from the chromosome of a `host` organism. 
In view of the above, the method of the present invention is ideally suited 
for, but not limited to the cloning and expression of genes coding for 
proteins used in food, feed or pharmaceutical applications or genes 
involved in biosynthesis of antibiotics and other bio-active compounds, 
i.e. recombinant proteins and/or hosts-organisms that are subject to 
strict registration requirements. 
Examples of such proteins are well known in the art and include chymosin, 
phytase, xylanases, amylases, cellulases and hemicellulases, cytokines and 
other pharmaceutical proteins, etc. 
The same method is employed for deletion of genes coding for proteins that 
influence production levels of desired proteins again without leaving a 
marker gene in the genome. Such proteins include proteases which actively 
digest the desired products that are highly expressed in the host strain 
and that therefore have a reduced potential of producing and or secreting 
the desired proteins. A preferred method for the deletion of a given gene 
would use a DNA construct containing the following elements in a 5' to 3' 
order: sequences 5' of the gene to be deleted, directly fused to sequences 
3' of the gene to be deleted, followed downstream by a functional 
selection marker gene (preferably an acetamidase gene), followed 
downstream by again sequences 3' of the gene to be deleted. In this case 
both sequences 3' of the gene to be deleted are chosen such that they form 
repeats flanking the selection marker gene. Transformation of this DNA 
construct and subsequent replacement of the chromosomla copy of the gene 
to be deleted by the DNA construct with cross-over points in the sequences 
5' and 3' of the gene to be deleted results in deletion of the given gene. 
Subsequent intrachromosomal recombination between the repeats flanking the 
selection marker gene and counter-selection for these recombinants finally 
results in a selection marker free strain with the given gene deleted. The 
DNA construct used for this deletion can be constructed such that no 
foreign DNA or other traces of the genetic modification are left in the 
strain carrying the deletion. 
The invention discloses selection marker gene free recombinant 
microorganisms. Such microorganisms can be organisms that, after the use 
of the disclosed technology, contain an extra copy of a desired gene 
(either homologous or heterologous). Such microorganisms can be 
re-transformed over and over by sequential application of the same 
technology to insert or delete additional copies of the same or other 
gene(s) of interest. 
The microorganisms may also be characterized in that they have (a) 
predetermined gene(s) deleted or altered in any desired way. 
The method of the present invention makes possible the fine-tuning of the 
production of desired proteins. This possibility is based on the ease with 
which repeated rounds of insertion and deletion can be performed. The 
method makes possible the insertion or deletion of a desired number of 
gene copies. Thus the proteins are produced in desired amounts and in 
desired ratios. This is especially useful for the production of mixtures 
of proteins or enzymes. 
Whereas it is known that the acetamidase gene is capable of conversion of 
acetamide as the sole N-source in Aspergillus it is here shown that the 
acetamidase gene is easily deleted from the genome of transformed 
Aspergilli. To achieve this the amdS gene is cloned between direct 
repeats. In principle any direct repeat which allows for internal 
recombination can be employed. In the present examples this is 
demonstrated by cloning the amdS gene between 3' amyloglucosidase (glaA) 
non-coding DNA sequences. 
It is shown that the amdS gene can be integrated and deleted upon plating 
on medium containing fluoracetamide and urea as N-sources. 
It is further demonstrated that the amyloglucosidase gene can be deleted 
from the genome of Aspergillus. A replacement vector is constructed 
containing a part of the glaA promoter, a synthetic DNA sequence 
containing stop codons in all three reading frames, the amdS gene from A. 
nidulans under the control of the A.nidulans glyceraldehyde-3-phosphate 
dehydrogenase promoter and wherein the amdS gene is flanked by 3' glaA 
non-coding sequences. After transformation of A.niger the vector is 
integrated by double crossing-over thereby effectively replacing the 
amyloglucosidase gene. After selection for amdS activity the transformed 
strains are plated on fluoracetamide and urea. Selection resulted in 
strains wherein the amdS gene was deleted. 
This example is an illustration of the possibility of using the amdS gene 
for deletion of a desired gene from the genome of an Aspergillus strain. 
Other genes can be eliminated or modified in a similar manner. 
In a further example it is demonstrated that a gene can be inserted marker 
free at a predetermined site in the genome. An integration vector is 
constructed containing the A.niger glaA locus and the amdS gene flanked by 
two 3' glaA non-coding repeats. 
The construct is shown to integrate at the amyloglucosidase locus. After 
selection on fluoracetamide the amdS gene is deleted. In this way a gene 
copy is integrated at a specific locus without leaving marker DNA. 
It is evident from the above that the procedures described herein enable 
one of skill in the art to integrate or delete desired genes at 
predetermined loci without leaving selection marker DNA behind. 
This method can be employed for gene amplification and gene replacement. 
An especially important application would be the integration of desired 
genes. Followed by classical strain improvement whereafter the genes that 
may be adversely affected by the classical strain improvement techniques 
are replaced with fresh unaffected copies of the gene of interest without 
loss of expression level. 
The system as described for Aspergillus above is expected to give the same 
results when other fungal strains are employed, which are known to be 
incapable of growth on acetamide as the sole N-source. The use of the amdS 
gene as a selection marker has been described for among others Penicillium 
and Trichoderma. Moreover, the amdS gene can even be used in filamentous 
fungi which are capable of using acetamide as sole N-source albeit poorly. 
In this case the background of poorly growing untransformed cells can be 
repressed by the inclusion of CsCl in the selection media (Tilburn, J. et 
al. (1983) Gene, 26, 205-221). Hence the system is expected to be 
applicable to filamentous fungi in general. 
In one embodiment of the present application it is surprisingly 
demonstrated that the A.nidulans amdS gene can be used as a selection 
marker in K.lactis. In this Example it is shown that two different 
K.lactis strains cannot grow on acetamide as the sole N-source. The two 
K.lactis strains are plated on YCB medium which is 
a) complete but without N-source, 
b) as a) but with acetamide, 
c) as a) but with ammonium sulphate. 
It is shown that the strains do not grow on the medium under b) but do grow 
on medium under c). Hence provided that the acetamide is taken up by the 
yeast cells and that the amdS gene can be expressed in K.lactis the system 
is applicable in yeasts also at least as a selection marker. Concerning 
the counter-selection using fluoracetamide some further requirements have 
to be met. Fluoracetate is toxic when activated by the enzyme 
acetyl-CoA-synthetase. Prerequisites for the fluoracetamide 
counter-selection to also work on amdS.sup.+ yeasts are therefore 
1) fluoracetamide should not be toxic for amdS.sup.- yeasts, 
2) the yeast cell wall and plasmamembrane should be permeable to 
fluoracetamide and 
3) the enzyme acetyl-CoA-synthetase should be active. 
To test this the amdS gene was cloned in K. lactis. 
To avoid any potential splicing problems of the A.nidulans amdS gene in 
K.lactis the amdS cDNA from A.nidulans was cloned as shown in the 
Experimental section. 
Subsequently the amdS was cloned downstream of a yeast promoter (LAC4, 
ADH1, KlEF) in a vector containing another marker 
(phosphotransferase-G418). This cloning is described in Example 8. The 
vectors containing both the G418 marker and the amdS gene were selected 
using the G418 marker and were then used to optimize selection conditions 
for the amdS+ phenotype. 
Direct selection of K.lactis is shown in another embodiment of the present 
invention and for S.cerevisiae direct selection is shown in Example 11. 
Subsequently it is demonstrated that counter-selection can be employed on 
the transformed yeast strains to remove the amdS gene. 
The amdS gene system is used for both marker gene free insertion and marker 
gene free deletion of a gene in yeast. 
In a further embodiment the lactase gene is deleted from K.lactis whereas 
in Example 14 a copy of the chymosin gene is inserted into the K.lactis 
genome. 
The genes used here for insertion and deletion are only used as examples. 
The same technology can be applied using other genes or DNA elements. As 
mentioned before the DNA fragments used for insertion or deletion can be 
mutated genes, promoter sequences, regulatory sequences etc. In all cases 
it is possible to insert or delete these sequences at desired genomic 
sites and in desired numbers, without leaving a marker gene behind. 
The feasibility of the use of this system in other yeast strains is 
evident. 
As a first step for use of the system of the present invention in bacteria 
it is shown in Example 15 that Bacillus subtilis and E.coli cannot grow on 
acetamide as the sole N-source. 
Example 16 describes the vectors that have been constructed for use in 
Bacillus and E.coli. 
It is demonstrated in Examples 17 and 18 that the amdS gene can be 
effectively used in Bacillus and E.coli as selection marker, whereas 
Example 19 demonstrate the fluoracetamide counter-selection of bacterial 
amdS.sup.+ transformants. 
The advantages of the system of the present invention are manifold. The 
most striking advantages are given below: 
It is demonstrated that the amdS system is universally applicable (plant 
cells, animal cell, yeasts, bacteria and filamentous fungi etc.), 
requiring only that the host in question cannot or only poorly grow on 
acetamide as sole C- or N-source but can utilize either acetate or ammonia 
as sole C- or N-source, respectively. 
The amdS system represents the only bi-directional and dominant selection 
system. This feature is extremely convenient for use in poly- or aneuploid 
strains which often is the case with natural isolates and/or industrial 
strains. 
After classical strain improvement any mutated copies of the desired gene 
can be easily replaced by unmutated copies by gene replacement due to the 
fact that the desired genes have been integrated at well-defined loci. The 
genes are thus replaced with unmutated genes without affecting the 
expression level. 
Due to the ability to introduce multiple integrations at well-defined and 
therefore non-random loci one can be assured that no undesirable traits 
arise in the strain upon gene amplification. 
The growing concern about the release of various selection markers in the 
environment is overcome by the presented system. No selection marker gene 
or other unnecessary or undesired DNA sequences need to present in the 
production strains after introduction of the desired genes or other 
genetic modifications. 
Experimental 
General molecular cloning techniques 
In the examples described herein, standard molecular cloning techniques 
such as isolation and purification of nucleic acids, electrophoresis of 
nucleic acids, enzymatic modification, cleavage and/or amplification of 
nucleic acids, transformation of E.coli, etc., were performed as described 
in the literature (Sambrook et al. (1989) "Molecular Cloning: a laboratory 
manual", Cold Spring Harbour Laboratories, Cold Spring Harbour, N.Y.; 
Innis et al. (eds.) (1990) "PCR protocols, a guide to methods and 
applications" Academic Press, San Diego). Synthesis of 
oligo-deoxynucleotides and DNA sequence analysis were performed on an 
Applied Biosystems 380B DNA synthesizer and 373A DNA sequencer, 
respectively, according to the user manuals supplied by the manufacturer. 
Transformation of A.niger 
Transformation of A.niger was performed according to the method described 
by Tilburn, J. et.al. (1983) Gene 26, 205-221 and Kelly, J. & Hynes, M. 
(1985) EMBO J., 4, 475-479 with the following modifications: 
spores were grown for 16 hours at 30.degree. C. in a rotary shaker at 300 
rpm in Aspergillus minimal medium. 
Aspergillus minimal medium consists of the following components: Per liter: 
6 g NaNO.sub.3 ; 0.52 g KCl; 1.52 g KH.sub.2 PO.sub.4 ; 1.12 ml 4M KOH; 
0.52 g MgSO.sub.4.7H.sub.2 O; 10 g glucose; 1 g casamino acids; 22 mg 
ZnSO.sub.4.7H.sub.2 O; 11 mg H.sub.3 BO.sub.3 ; 5 mg FeSO.sub.4.7H.sub.2 
O; 1.7 mg CoCl.sub.2.6H.sub.2 O; 1.6 mg CuSO.sub.4.5H.sub.2 O; 5 mg 
MnCl.sub.2.4H.sub.2 O; 1.5 mg Na.sub.2 MoO.sub.4.2H.sub.2 O; 50 mg EDTA; 2 
mg riboflavin; 2 mg thiamine.HCl; 2 mg nicotinamide; 1 mg pyridoxine.HCl; 
0.2 mg panthotenic acid; 4 .mu.g biotin; 10 ml Penicillin (5000 
IU/ml)/Streptomycin (5000 UG/ml) solution (Gibco). 
only Novozym 234 (Novo Industri), and no helicase, was used for formation 
of protoplasts; 
after protoplast formation (60-90 minutes), KC buffer (0.8M kCl, 9.5 mM 
citric acid, pH6.2) was added to a volume of 45 ml. and the protoplast 
suspension was centrifuged at 2500 g at 4.degree. C. for 10 minutes in a 
swinging-bucket rotor. The protoplasts were resuspended in 20 ml. KC 
buffer. Then, 25 ml of STC buffer (1.2M sorbitol, 10 mM Tris-HCl pH7.5, 50 
mM CaCl.sub.2) was added and subsequently the protoplast suspension was 
centrifuged at 2500 g at 4.degree. C. for 10 minutes in a swinging-bucket 
rotor, washed in STC-buffer and resuspended in STC-buffer at a 
concentration of 10.sup.8 protoplasts/ml; 
to 200 .mu.l of the protoplast suspension the DNA fragment, in a volume of 
10 .mu.l in TE buffer (10 mM Tris-HCl pH7.5, 0.1 mM EDTA), was added and 
subsequently 100 .mu.l of a PEG solution (20% PEG 4000 (Merck), 0.8M 
sorbitol, 10 mM Tris-HCl pH7.5, 50 mM CaCl.sub.2); 
after incubation of the DNA-protoplast suspension at room temperature for 
10 minutes, 1.5 ml PEG solution (60% PEG 4000 (Merck), 10 mM Tris-HCl 
pH7.5, 50 mM CaCl.sub.2) was added slowly, with repeated mixing of the 
tubes. After incubation at room temperature for 20 minutes, the 
suspensions were diluted with 5 ml STC buffer, mixed by inversion and 
centrifuged at 2000 g at room temperature for 10 minutes. The protoplasts 
were resuspended gently in 1 ml 1.2M sorbitol and plated onto selective 
regeneration medium consisting of Aspergillus minimal medium without 
riboflavin, thiamine.HCl, nicotinamide, pyridoxine.HCl, panthotenic acid, 
biotin, casamino acids and glucose but with 10 mM acetamide as the sole 
nitrogen source, 1M sucrose, solidified with 2% bacteriological agar #1 
(Oxoid, England). Following growth for 6-10 days at 30.degree. C., the 
plates were replica plated onto selective acetamide plates consisting of 
Aspergillus selective regeneration medium with 2% glucose instead of 
sucrose and 1.5% agarose instead of agar. Single transformants were 
isolated after 5-10 days of growth at 30.degree. C. 
Transformation of A. oryzae 
Transformation of A. oryzae was performed according to the method described 
by Christensen, T. et al. in European Patent Application 0 238 023 A2. 
Transformation of T. reesei 
Transformation of T. reesei was performed according to the method described 
by Penttilla M., Knowles, J. (1987) Gene 61 155-164. 
Transformation of P. chrysogenum 
The Ca-PEG mediated protoplast transformation procedure is used. 
Preparation of protoplasts and transformation of P.chrysogenum was 
performed according to the method described by Gouka et al., Journal of 
Biotechnology 20(1991), 189-200 with the following modifications: 
After transformation, the protoplasts were plated onto selective 
regeneration medium plates consisting of Aspergillus minimal medium, 
osmotically stabilized with 1.2M sucrose, containing 0.1% acetamide as 
sole nitrogen source and solidified with 1.5% bacteriological agar #1 
(Oxoid, England). 
After 5-8 days of incubation at 25.degree. C. transformants appeared. 
Transformation of K.lactis 
The yeast K.lactis was transformed using the lithium acetate procedure 
described by Ito H. et al. (1983) J. Bacteriol. 153, 163-168 with the 
following modifications: 
For transformation a K.lactis culture was taken with an OD.sub.610 between 
0.5 and 1.0. 
After the 5 minutes heatshock of the transformed cell suspensions, 1 ml 
YEPD/YNB (1% yeast-extract, 2% Bacto-peptone, 2% glucose and 0.17% Yeast 
Nitrogen Base w/o amino acids (YNB; Difco) was added and the 
cell-suspensions were incubated at 30.degree. C. in a shaker incubator for 
150-180 minutes. 
After the above mentioned incubation (at 30.degree. C. for 150-180 
minutes), the cell-suspensions were centrifuged at 2000 g at room 
temperature for 5 minutes and plated on YEPD/G418 double layer medium 
solidified with 2% Bacto-agar (Difco). YEPD/G418 double layer plates were 
prepared as followed: 10 minutes prior to plating of the cell-suspensions 
15 ml YEPD agar (1% yeast-extract, 2% Bacto-peptone, 2% glucose solidified 
with 2% Bacto-agar (Difco)) without G418 was poured onto 15 ml YEPD agar, 
which contained 50 .mu.g G418/ml. This results in YEPD/G418 double layer 
plates which contain 25 .mu.g G418/ml after diffusion of the antibiotic. 
The YEPD/G418 double layer plates contained 25 .mu.g G418/ml or 100 .mu.g 
G418/ml in case of strains K.lactis CBS 683 or CBS 2360, respectively. 
Isolation of DNA from Aspergillus, Trichoderma, Penicillium and yeast 
The isolation of DNA from Aspergillus and Trichoderma was performed 
according to the procedure as described by Yelton, et al. (1984), Proc. 
Natl. Acad. Sci. 81, 1470-1474. 
The isolation of DNA from Penicillium was performed according to the 
procedure described by Kolar et al., Gene 62 (1988), 127-134. 
The isolation of DNA from K.lactis or S.cerevisiae was performed according 
to the procedures described by Fujimura and Sakuma (1993), Biotechniques 
14, 538. 
Bacillus transformation and DNA-isolation 
Transformation of the different Bacillus species as well as isolation of 
plasmid or chromosomal DNA from these species was performed as described 
by Bron (1990) "Plasmids" In: Molecular Biological Methods for Bacillus, 
Harwood, CR and Cutting, SM, eds., series Modern Microbiological Methods, 
John Wiley & Sons, Chichester, UK. 
For the transformation of B.subtilis BS-154 (CBS 363.94) competent cells 
were used and for the transformation of B.licheniformis T5 (CBS 470.83) 
protoplast transformation was used. In the case of neomycin selection a 
concentration of 20 .mu.g/ml was used. For acetamide selection of 
B.subtilis transformants, minimal medium agar was used in which casamino 
acids and yeast extract were replaced by 20 mM acetamide. For acetamide 
selection of B.licheniformis transformants, protoplast regeneration medium 
was used in which ammonium sulphate was replaced by 20 mM acetamide. 
Removal of the amdS selection marker 
The amdS marker in most examples relating to Aspergillus, Trichoderma and 
Penicillium is cloned between repeats consisting of a part of the 3' 
non-coding region of amyloglucosidase gene. Removal of the amdS selection 
marker is achieved either by internal recombination between the 3' glaA 
non-coding repeats that flank the amdS selection marker or by homologous 
recombination between the repeats that are created by integration via a 
single cross-over event. Selection of cells that have lost the amdS 
selection marker is achieved by growth on plates containing 
fluoracetamide. Cells harbouring the amdS gene metabolize fluoracetamide 
to ammonium and fluoracetate which is toxic to the cell. Consequently, 
only cells that have lost the amdS gene are able to grow on plates 
containing fluoracetamide. 
In case of removal of the amdS marker from Aspergillus transformants, 
spores from these transformants were plated onto selective regeneration 
medium (described above) containing 32 mM fluoracetamide and 5 mM ureum 
instead of 10 mM acetamide, 1.1% glucose instead of 1M sucrose and 1.1% 
instead of 2% bacteriological agar #1 (Oxoid, England). After 7-10 days of 
growth at 35.degree. C. single colonies were harvested and plated onto 
0.4% potato dextrose agar (Oxoid, England). In case of removal of the amdS 
marker from Trichoderma transformants, spores of these transformants were 
plated onto non selective minimal medium plates (per liter: 20 g. glucose, 
5 g. (NH.sub.4).sub.2 SO.sub.4, 15 g. KH.sub.2 PO.sub.4, 0.6 g. 
MgSO.sub.4, 0.6 g. CaCl.sub.2, 0.005 g. FeSO.sub.4.7H.sub.2 O, 0.0016 g. 
MnSO.sub.4.H.sub.2 O, 0.0014 g. ZnSO.sub.4.7H.sub.2 O, 0.002 g. CoCl.sub.2 
; pH5.5) supplemented with 10 mM fluoracetamide. After 5-10 days at 
30.degree. C., colonies were harvested and plated onto 0.4% potato 
dextrose agar (Oxoid, England). 
In case of removal of the amdS marker from Penicillium transformants, 
spores from these transformants were plated on selective medium plates 
consisting of Aspergillus minimal medium with 10 mM fluor-acetamide and 5% 
glucose, solidified with 1.5% bacteriological agar #1 (Oxoid, England). 
After 5-10 days of growth at 25.degree. C. resistant colonies appeared. 
Determination of glucoamylase production by A.niger transformants 
Of recombinant and control A.niger strains spores were collected by plating 
spores or mycelia onto PDA-plates (Potato Dextrose Agar, Oxoid), prepared 
according to the supplier's instructions. After growth for 3-7 days at 
30.degree. C. spores were collected after adding 0,01% Triton X-100 to the 
plates. After washing with sterile water approximately 10.sup.7 spores of 
selected transformants and control strains were inoculated into shake 
flasks, containing 20 ml of liquid pre-culture medium containing per 
liter: 30 g maltose.H.sub.2 O; 5 g yeast extract; 10 g hydrolysed casein; 
1 g KH.sub.2 PO.sub.4 ; 0.5 g MgSO.sub.4.7H.sub.2 O; 3 g Tween 80; 10 ml 
Penicillin (5000 IU/ml)/Streptomycin (5000 UG/ml); pH 5.5. These cultures 
were grown at 34.degree. C. for 20-24 hours. 5-10 ml of this culture was 
inoculated into 100 ml of fermentation medium containing per liter: 70 g 
maltodextrines; 25 g hydrolysed casein; 12.5 g yeast extract; 1 g KH.sub.2 
PO.sub.4 ; 2 g K.sub.2 SO.sub.4 ; 0.5 g MgSO.sub.4.7H.sub.2 O; 0.03 g 
ZnCl.sub.2 ; 0.02 g CaCl.sub.2 ; 0.01 g MnSO.sub.4.4H.sub.2 O; 0.3 g 
FeSO.sub.4.7H.sub.2 O; 10 ml penicillin (5000 IU/ml)/Streptomycin (5000 
UG/ml); adjusted to pH 5.6 with 4N H.sub.2 SO.sub.4. These cultures were 
grown at 34.degree. C. for 5-10 days. Samples were taken for the analysis 
of the glucoamylase production at different time points during 
fermentation. Fermentation broth samples were centrifuged (10 minutes, 
10.000.times.g) and supernatants collected. 
The glucoamylase activity was determined by incubating 10 .mu.l of a six 
times diluted sample of the culture supernatant in 0.032M NaAC/HAC pH4.05 
with 115 .mu.l of 0.2% (w/v) p-Nitrophenyl .alpha.-D-glucopyranoside 
(Sigma) in 0.032M NaAc/HAc pH 4.05. After a 30 min incubation at room 
temperature, 50 .mu.l of 0.3M Na.sub.2 CO.sub.3 was added and the 
absorption at a wavelength of 405 nm was measured. The A.sub.405nm is a 
measure for the AG production. 
Cloning of the amdS cDNA 
The A.nidulans amdS gene contains three small introns (Corrick et al. 
(1987) Gene 53, 63-71). In order to avoid problems caused by incorrect 
splicing of these introns in yeast or lack of splicing in bacteria, we 
have used an amdS cDNA for expression in yeasts and bacteria. Cloning of 
the amdS cDNA from an A.nidulans polyA.sup.+ RNA preparation has been 
described by Corrick et al. ((1987), Gene 53, 63-71). In this example we 
have used the A.niger NRRL 3135 transformant #4, which is transformed by 
multiple copies of the A.nidulans amdS gene containing plasmid pAF2-2S 
(van Hartingsveld et al (1993). Gene 127, 87-94). Total RNA was isolated 
by a direct LiCl precipitation according to a procedure modified from 
Auffray et al. ((1980) Eur.J.Biochem. 107, 303-314). A.niger spores were 
allowed to germinate and were grown overnight at 37.degree. C. in a 
minimal medium (Cove (1966) Biochim. Biophys. Acta 113, 51-56) 
supplemented with glucose as carbon source and with acetamide as sole 
nitrogen source. Mycelium was obtained and dried by filtration and 
subsequently frozen with liquid nitrogen to be grounded. The powder was 
dispersed in 3M LiCL, 6M urea at 0.degree. C. and maintained overnight at 
4.degree. C. Total cellular RNA was obtained after centrifugation at 
16.000 g for 30 minutes and two successive extractions with 
phenol/chloroform/isoamylalcohol (50:48:2). The RNA was precipitated with 
ethanol and dissolved in 1 ml 10 mM Tris-HCL (pH7.4), 0.5% SDS. For 
polyA.sup.+ selection the total RNA sample was heated for 5 minutes at 
65.degree. C. and subsequently applied to an oligo(dT)-cellulose column. 
After several washes with a solution containing 10 mM Tris-HCl pH 7.4, 
0.5% SDS and 0.5M NaCl, the polyA.sup.+ RNA was collected by elution with 
10 mM Tris-HCl pH 7.4 and 0.5% SDS and precipitated with ethanol. 
Approximately 5 .mu.g of the polyA.sup.+ mRNA was used as template for 
reverse transcription primed with oligo(dT) primers. The reaction mixture 
(50 mM Tris-HCl pH 7.6, 10 mM DTT, 6 mM MgCl.sub.2, 80 mM KCl, 0.2 mM each 
dNTP and 0.1 mg BSA/ml) was incubated for 30 minutes at 37.degree. C. with 
500 units Murine MLV reverse transcriptase (BRL) and 75 units RNase 
inhibitor (Promega) in a volume of 100 .mu.l. Another 200 units of reverse 
transcriptase were added and the reaction was continued for 30 minutes. 
The mixture was extracted with chloroform and precipitated with ethanol in 
the presence of 0.25M ammonium acetate. This mixture of first strand cDNAs 
was used as template in a subsequent Polymerase Chain Reaction (PCR) to 
amplify the amdS cDNA. The genomic amdS sequence was used to design 2 
synthetic oligonucleotides that were used as primers in this PCR: 
AB3100 (SEQ ID NO: 1) 
5'-CTAATCTAGAATGCCTCAATCCTGAA-3' (an amdS-specific sequence from nucleotide 
-3 to +16 preceded by an XbaI site and 4 additional nucleotides). 
AB3101 (SEQ ID NO: 2) 
5'-GACAGTCGACAGCTATGGAGTCACCACA-3' (an amdS-specific sequence positioned 
downstream of the amdS stopcodon from nucleotides 1911 to 1884 flanked by 
an additional SalI site). 
The PCR reaction was performed using 10% of the cDNA mixture as template 
and 0.1 .mu.g of each of the oligos AB3100 SEQ ID NO: 1) and AB3101 (SEQ 
ID NO: 2) as primer. After denaturation (7 minutes at 100.degree. C.) and 
addition of 1.3 units Taq-polymerase the reaction mixture was subjected to 
25 amplification cycles (each cycle: 2 minutes at 94.degree. C., 2 minutes 
at 55.degree. C. and 3 minutes at 72.degree. C.). In the last cycle the 
extension step was longer (7 min.) to allow synthesis of full-length 
fragments. The obtained DNA fragment was digested with XbaI and SalI and 
subcloned into the XbaI/SalI sites of pUC18. The resulting plasmid was 
designated pamdS-1 (see FIG. 1). Restriction analysis of the plasmid 
pamdS-1 confirmed the absence of introns and the correct fusion of exons 
in the amdS cDNA. 
EXAMPLE 1 
Marker Gene Free Deletion of an A.niger Gene by Using the amdS Gene 
In this example a genomic target gene in A.niger will be replaced by 
transforming A.niger with a replacement vector which integrates into the 
A.niger genome via a double cross-over homologous recombination. The 
replacement vector comprises a DNA region homologous to the target locus 
interrupted by a selectable marker gene flanked by DNA repeats. 
In this example plasmid pGBDEL4L is used to delete the glaA coding region 
and a (proximal) part of the glaA promoter region. This vector comprises a 
part of the A.niger glaA genomic locus, wherein the glaA coding sequences 
as well as a part of the glaA promoter sequences are replaced by the 
A.nidulans amdS gene under the control of A.nidulans gpdA promoter as 
selection marker flanked by 3'-untranslated glaA sequences as direct 
repeats. Transformation of A.niger with this vector directs replacement of 
the glaA gene by the amdS marker gene. By performing the fluoracetamide 
counter-selection on these transformants as described in the experimental 
procedures, the amdS marker gene will be deleted properly by an internal 
recombination event between the 3'glaA DNA repeats, resulting in a marker 
gene free .DELTA.glaA recombinant strain, possessing finally no foreign 
DNA sequences at all (for a schematic view, see FIG. 2). 
Short description of the glaA gene replacement vector pGBDEL4L 
The gene replacement vector pGBDEL4L contains 5'-part of the A.niger 
amyloglucosidase (glaA) promoter region, a synthetic DNA sequence of 16 bp 
providing stopcodons in all three reading frames, the A.nidulans 
acetamidase (amdS) gene under control of the A.nidulans 
glyceraldehyde-3-phosphate dehydrogenase (gpdA) promoter, flanked at both 
sides by 3' glaA non-coding sequences. 
Construction pathway of pGBDEL4L 
In order to obtain the final deletion vector pGBDEL4L several subclones of 
the glaA locus were derived first. A schematic view is presented in FIG. 
3. The glaA locus of A.niger was molecular cloned and described previously 
(EP 0 463 706 A1). Plasmid pAB6-1 contains the entire glaA locus from 
A.niger on a 15.5 kb HindIII fragment cloned in the HindIII site of pUC19 
(Yanisch-Perron et al., Gene 33 (1985) 103-119, and is obtainable from 
e.g. Boehringer Mannheim, Germany). pAB6-1 was digested with EcoRI and the 
1.8 kb EcoRI DNA fragment just upstream of the glaA gene was isolated by 
agarose gel electrophoresis and ligated into pUC19 digested with EcoRI and 
subsequently transferred to E. coli and molecular cloned. The resulting 
plasmid was designated pAB6-3 (FIG. 3A). To construct plasmid pAB6-4, 
which is another subclone of pAB6-1, pAB6-1 was digested with HindIII and 
BglII. The 4.6 kb sized DNA fragment comprising the glaA promoter and a 
part of the glaA coding sequence was isolated by agarose gel 
electrophoresis and ligated into pUC19 which was digested prior with 
HindIII and BamHI (FIG. 3B). As a result the BamHI as well as the BglII 
sites in pAB6-4 were destroyed appropriately by this cloning procedure. 
Subsequently, after digesting plasmid pAB6-4 with HindIII and EcoRI and 
filling in the 5' sticky ends using E. coli DNA polymerase, the 1.8 kb 
glaA promoter DNA fragment was isolated by agarose gel electrophoresis, 
ligated into pAB6-3 which was partially digested with EcoRI and treated 
with E. coli DNA polymerase to generate blunt ends, the ligation mixture 
was transferred to E. coli for molecular cloning. The derived plasmid 
(designated pAB6-31) contains a 3.6 kb glaA promoter fragment with a 
destroyed EcoRI site in the middle, but still possessing the EcoRI site 
(now unique in this DNA fragment) just upstream of the glaA ATG initiation 
site (FIG. 4). 
The A.nidulans amdS gene used herein is located on an approximately 4 kb 
sized EcoRI-KPnI fragment in plasmid pGW325 (Wernars et al., thesis (1986) 
Agricultural University, Wageningen, The Netherlands). This EcoRI-KpnI DNA 
fragment containing the amdS gene, flanked by its own regulatory 
sequences, was molecular cloned into the appropriate sites of pUC19 as 
described by Verdoes et al. (Transgenic Res. 2 pp 84-92, 1993) resulting 
in pAN4-1. pAN4-1 was digested with EcoRI and KpnI, the 4 kb sized DNA 
fragment containing the amdS gene was isolated by agarose gel 
electrophoresis, ligated into pAB6-31 digested with EcoRI and KpnI and the 
ligation mixture was transferred to E. coli for molecular cloning. The 
obtained plasmid was designated pAB6S (FIG. 5) and contains a 3.8 kb glaA 
promoter DNA fragment and the 4 kb amdS fragment. 
Plasmid pAB6S was first partially digested with SalI, and ligated to the 
synthetic derived oligonucleotide TN0001 (SEQ ID NO: 3) having the 
following sequence: 
TN0001 (SEQ ID NO: 3): 5' TCGATTAACTAGTTAA 3' and secondly digested with 
EcoRI. The DNA fragment comprising the pUC19, the glaA promoter and the 
amdS gene sequences was purified and isolated by agarose gel 
electrophoresis. From plasmid pAB6-1, digested with SalI, the 2.2 kb 3' 
flanking glaA DNA fragment was isolated as well by agarose gel 
electrophoresis and ligated to the above mentioned synthetic 
oligonucleotide, treated with T4 polynucleotide kinase, subsequently 
digested with EcoRI and ligated to the above mentioned DNA fragment 
isolated of pAB6S. The DNA ligation mixture was transferred to E. coli and 
molecular cloned. The derived plasmid was designated pGBDEL1 and is shown 
in FIG. 6. By this procedure simultaneously the SalI restriction site was 
destroyed and stopcodons in all reading frames were introduced. 
To obtain an approximately 1 kb large DNA fragment, containing 3' glaA 
non-coding DNA sequences positioned just downstream the stop codon of the 
glaA gene and flanked by suitable restriction sites, a PCR amplification 
was performed. In this PCR amplification, the plasmid pAB6-1 was used as 
template and as primers two synthetical derived oligonucleotides: 
Oligo AB2154 (SEQ ID NO: 4) 
5'AACCATAGGGTCGACTAGACAATCAATCCATTTCG 3' (a 3'glaA non-coding sequence just 
downstream of the stopcodon) and 
Oligo AB2155 (SEQ ID NO: 5) 
5'GCTATTCGAAAGCTTATTCATCCGGAGATCCTGAT 3' (a 3'glaA non-coding sequence 
around the EcoRI site approx. 1 kb downstream of the stopcodon). 
The PCR was performed as described by Saiki et al. (Science 239, 487-491, 
1988) and according to the supplier of TAQ-polymerase (Cetus). Twenty five 
amplification cycles (each 2 minutes at 55.degree. C.; 3 minutes at 
72.degree. C. and 2 minutes at 94.degree. C.) were performed in a 
DNA-amplifier (Perkin-Elmer/Cetus). The 1 kb amplified DNA fragment was 
digested with HindIII and SalI, purified by agarose gel electrophoresis, 
ethanol precipitated and subsequently cloned into the HindIII and SalI 
restriction sites of pGBDEL1. The thus obtained plasmid was designated 
pGBDEL2 (FIGS. 7A,B). 
To obtain the final glaA gene replacement vector pGBDEL4L, the amdS 
promoter region in pGBDEL2 was exchanged by the stronger A.nidulans gpdA 
promoter. Fusion of the gpdA promoter sequence to the coding sequence of 
the amdS gene was performed by the Polymerase Chain Reaction (PCR) method. 
For this PCR fusion two different templates were used: plasmid pAN7-1 
(Punt et al., Gene 56, 117-124, 1987) containing the E.coli hph gene under 
control of the A.nidulans gpdA promoter and the A.nidulans trpC terminator 
and plasmid pAN4-1, containing the A.nidulans amdS gene under control of 
its own regulatory sequences. As primers four synthetic oligonucleotides 
were used, possessing the following sequences: 
Oligo AB 2977 (SEQ ID NO: 6) 
5' TATCAGGAATTCGAGCTCTGTACAGTGACC 3' (a 5' gpdA promoter specific oligo 
nucleotide, positioned at approximately 880 bp upstream of the ATG 
startcodon of the E. coli hph gene) 
Oligo AB2992 (SEQ ID NO: 7) 
5' GCTTGAGCAGACATCACCATGCCTCAATCCTGGGAA 3' 
Oligo AB2993 (SEQ ID NO: 8) 
5' TTCCCAGGATTGAGGCATGGTGATGTCTGCTCAAGC 3' (both sequences are 
complementary to each other and contain 18 bp of the 3' end of the gpdA 
promoter and 18 bp of the 5' part of the amdS coding region) 
Oligo AB2994 (SEQ ID NO: 9) 
5' CTGATAGAATTCAGATCTGCAGCGGAGGCCTCTGTG 3' (an amdS specific sequence 
around the BglII site approximately 175 bp downstream of the ATG 
initiation codon) 
To fuse the 880 bp gpdA promoter region to the amdS coding sequence two 
separate PCR's were carried out: the first amplification with pAN7-1 as 
template and the oligo nucleotides AB 2977 (SEQ ID NO: 6) and AB2993 (SEQ 
ID NO: 8) as primers to amplify the 880 bp DNA fragment comprising the 
gpdA promoter flanked at the 3' border by 18 nucleotides complementary to 
the 5' end of the amdS gene, and the second PCR reaction with pAN4-1 as 
template and the oligo nucleotides AB2992 (SEQ ID NO: 7) and AB2994 (SEQ 
ID NO: 9) as primers to amplify a 200 bp sized DNA fragment comprising the 
5' part of the amdS gene flanked at the 5' border by 18 nucleotides 
complementary to the 3' end of the gpdA promoter. A schematic view of 
these amplifications is presented in FIG. 8A. The two fragments generated 
were subsequently purified by agarose gel electrophoresis, ethanol 
precipitated and used as templates in a third PCR reaction with oligo 
nucleotides AB 2977 (SEQ ID NO: 6) and AB2994 (SEQ ID NO: 9) as primers. 
The resulting DNA fragment was digested with EcoRI, purified by agarose 
gel electrophoresis and ethanol precipitation, and cloned into the EcoRI 
site of pTZ18R (United States Biochemicals). The resulting plasmid was 
designated pGBGLA24 (FIG. 8B). 
To exchange the amdS promoter sequence in pGBDEL2 by the gpdA promoter 
sequence, the approximately 1 kb sized EcoRI/BglII DNA fragment of 
pGBGLA24 was isolated by agarose gel electrophoresis after digestion with 
the appropriate restriction enzymes and ligated into the EcoRI and BglII 
sites of pGBDEL2. The resulting glaA gene replacement vector was 
designated pGBDEL4L (FIG. 9). 
Deletion of glaA promoter and coding sequences in A.niger 
Prior to transformation of A.niger with pGBDEL4L, the E.coli sequences were 
removed by HindIII and XhoI digestion and agarose gel electrophoresis. The 
A.niger strain CBS 513.88 (deposited Oct. 10, 1988) was transformed with 
either 2.5, 5 or 10 .mu.g DNA fragment by procedures as described in 
experimental procedures using acetamide as sole N-source in selective 
plates. Single A.niger transformants were purified several times onto 
selective acetamide containing minimal plates. Spores of individual 
transformants were collected by growing for about 5 days at 30.degree. C. 
on 0.4% potato-dextrose (Oxoid, England) agar plates. Southern analyses 
were performed to verify the presence of the truncated glaA locus. High 
molecular weight DNA of several transformants was isolated, digested with 
BamHI and KpnI and subsequently fractionated by electrophoresis on a 0.7% 
agarose gel. After transfer to nitrocellulose filters, hybridization was 
performed according to standard procedures using two .sup.32 P-labelled 
probes: a XhoI/SalI glaA promoter fragment isolated from plasmid pAB6-4 
(described above, FIG. 3A) and a probe recognizing endogenous xylanase 
sequences (European Patent Application. 0 463 706 A). The results of only 
4 transformants (#19, #23, #24, #41) and the control strain A.niger CBS 
531.88 are shown as examples in FIG. 10A. For a better understanding of 
this autoradiograph, a schematic presentation is presented in FIG. 11 
showing the size of the hybridizing fragments in intact and truncated glaA 
loci. 
Characteristic for the intact glaA locus is a 3.5 kb hybridizing fragment 
in a BamHI digest and a 4.5 kb hybridizing fragment in a KpnI digest (see 
FIG. 11A). In a truncated glaA locus, the 3.5 kb BamHI hybridizing 
fragment and the 4.5 kb KpnI hybridizing fragment are absent and replaced 
by a 5.5 kb BamHI hybridizing fragment and a 6.3 kb KpnI hybridizing 
fragment. In this example, as can be seen in FIG. 10A, transformant #19 
shows the expected pattern of a truncated glaA locus (FIG. 11B). This 
transformant was designated GBA-102. 
No replacement of the glaA gene had occurred in the other transformants. 
The poorly hybridizing bands: 4, 8 and 15 kb in the KpnI digest and 7 and 
12 kb in the BamHI digest, refer to the xylanase sequences as internal 
control. 
Removal of the amdS gene from A.niger GBA-102 by counter-selection on 
fluoracetamide containing plates 
The amdS gene in the transformant A.niger GBA-102 was removed again as 
described in the Experimental section. The removal of the amdS selection 
marker gene in only 2 surviving recombinant strains was verified by 
Southern analysis of the chromosomal DNA. High molecular weight DNA was 
isolated, digested with BamHI and KpnI and subsequently separated by 
electrophoresis on a 0.7% agarose gel. Following transfer to 
nitrocellulose hybridization was performed according to standard 
procedures using the probes described in the previous section. A schematic 
presentation of the hybridizing fragments is shown in FIG. 11C. The 
results of the Southern analyses are presented in FIG. 10B. The presence 
of a 5.2 kb hybridizing BamHI fragment and a 3.4 kb hybridizing KpnI 
fragment, with the concomitant loss of the 5.5 kb BamHI and the 6.3 kb 
hybridizing KpnI fragments is specific for the absence of the amdS 
selection marker. The weaker hybridizing 7 and 12 kb fragments in a BamHI 
digest and the 4, 8 and 15 kb KpnI fragments again refer to the endogenous 
xylanase locus. Both strains show the expected pattern. In these 
recombinant strains, which were designated GBA-107 and GBA108, the 
preferred glaA sequences are removed correctly and that possess finally no 
selection marker gene at all. Both strains can be reused again to delete 
or insert other genes or DNA elements by using the same type of vector. 
EXAMPLE 2 
Marker Gene Free Introduction of the glaA Gene Targeted at the 3'glaA 
Non-coding Region of the Truncated glaA Locus in A.niger GBA-107 
In this example the introduction of a gene into the genome of A.niger is 
described by using approximately the same approach and procedures as 
described in the previous example. Besides the desired gene or DNA element 
the vector contains DNA sequences homologous to the host genome to target 
the vector at a predefined genomic locus of the host, by a single 
cross-over event. This type of vector comprises a selection marker gene 
flanked by DNA repeats as well. The selection marker gene in transformants 
derived with this vector can be removed properly again by applying the 
counter-selection procedure. As an example the introduction of a glaA gene 
copy is described which becomes integrated at the truncated glaA locus in 
the recombinant .DELTA.glaA A.niger GBA-107 strain derived in Example I 
(for a schematic drawing see FIG. 12) 
Description of the glaA integration vector: pGBGLA30 
The integration vector pGBGLA30 consists of the A.niger amyloglucosidase 
(glaA) gene under control of the native promoter and the A.nidulans amdS 
gene under control of the A.nidulans gpdA promoter flanked by 3'glaA 
non-coding sequences to direct integration at the 3' glaA non-coding 
region and to remove the amdS selection marker gene via the 
counter-selection. 
Construction of the integration vector 
A 1.8 kb XhoI/EcoRI glaA promoter fragment from pAB6-1 (FIG. 13) was 
subcloned into the SmaI and EcoRI sites of pTZ19R (United States 
Biochemicals). The protruding 5' end of the XhoI site of the glaA promoter 
fragment was filled in using the Klenow fragment of E.coli DNA polymerase 
I prior to cloning in pTZ19R. The SmaI site is destroyed and the XhoI site 
is restored by this cloning procedure. The thus obtained plasmid was 
designated pGBGLA5 (FIG. 13). 
To introduce appropriate restriction sites (AatII, SnaBI, AsnI and NotI) 
and to destroy the XhoI site in the glaA promoter, the synthetic fragment 
consisting of the two oligonucleotides AB3657 (SEQ ID NO: 10) and AB3658 
(SEQ ID NO: 11): 
##STR1## 
was inserted into the HindIII and XhoI sites of pGBGLA5. The thus obtained 
plasmid was designated pGBGLA26 (FIG. 14). 
Next, the 3.4 kb EcoRI fragment from pAB6-1 containing the remaining 3' 
part of the glaA promoter, the glaA coding sequence and part of the 3' 
glaA non-coding sequence, was cloned into the EcoRI site of pGBGLA26. This 
new plasmid was designated pGBGLA27 (FIG. 15). This plasmid was partially 
digested with EcoRI and the synthetic fragment consisting of the 
oligonucleotides AB3779 (SEQ ID NO: 12) and AB3780 (SEQ ID NO: 13): 
##STR2## 
was inserted into the EcoRI site at the end of the 3' glaA non-coding 
sequence from the glaA gene. By this cloning step, the EcoRI site was 
destroyed and an ApaI and XhoI restriction site were introduced. The 
resultant plasmid was designated pGBGLA42 (FIG. 16). 
Amplification of the 2.2 kb 3' glaA non-coding sequences and concomitant 
adjustment of appropriate restriction sites was performed by the 
Polymerase Chain Reaction (PCR) method. 
In these PCR reactions, plasmid pAB6-1 containing the entire glaA locus was 
used as template and as primers four synthetic oligo nucleotides were 
designed possessing the following sequence: 
Oligo AB3448 (SEQ ID NO: 14) 
5' GTGCGAGGTACCACAATCAATCCATTTCGC 3' (a 3' glaA non-coding specific 
sequence just downstream the stopcodon of the glaA gene) 
Oligo AB3449 (SEQ ID NO: 15) 
5' ATGGTTCAAGAACTCGGTAGCCTTTTCCTTGATTCT 3' (a 3' glaA non-coding specific 
sequence around the KpnI site approx. 1 kb downstream of the stop codon) 
Oligo AB3450 (SEQ ID NO: 16) 
5' AGAATCAAGGAAAAGGCTACCGAGTTCTTGAACCAT 3' (a 3' glaA non-coding specific 
sequence around the KpnI site approx. 1 kb downstream of the stop codon) 
Oligo AB3520 (SEQ ID NO: 17) 
5'ATCAATCAGAAGCTTTCTCTCGAGACGGGCATCGGAGTCCCG 3' (a 3' glaA non-coding 
specific sequence approx. 2.2 kb downstream of the stopcodon) 
To destroy the KpnI site approximately 1 kb downstream of the stop codon 
from the glaA gene and to alter the SalI site approximately 2.2 kb 
downstream the stop codon from the glaA gene into a XhoI site two separate 
polymerase chain reactions were performed: the first reaction with 
oligonucleotides AB3448 (SEQ ID NO: 14) and AB3449 (SEQ ID NO: 15) as 
primers to amplify an approximately 1 kb DNA fragment just downstream the 
stopcodon of the glaA gene, and the second reaction with oligonucleotides 
AB3450 (SEQ ID NO: 16) and AB3520 (SEQ ID NO: 17) as primers to amplify an 
approximately 1.2 kb DNA fragment just downstream the KpnI site in the 3' 
glaA non-coding region both with pAB6-1 as template. A schematic view of 
these amplifications is presented in FIG. 17A. The PCR was performed as 
described in example I. Twenty-five amplification cycles (each 1 minute at 
55.degree. C.; 1.5 minutes at 72.degree. C. and 1 minute at 94.degree. C.) 
were carried out. 
The two generated PCR DNA fragments were purified by agarose gel 
electrophoresis and ethanol precipitation and subsequently used as 
template in the third PCR with oligonucleotides AB3448 (SEQ ID NO: 14) and 
AB3520 (SEQ ID NO: 17) as primers to generate the fusion fragment. 
Twenty-five amplification cycles (each: 2 minutes at 55.degree. C.; 3 
minutes at 72.degree. C.; 2 minutes at 94.degree. C.) were carried out in 
a DNA-amplifier (Perkin-Elmer/Cetus). The amplified DNA fragment was 
purified by agarose gel electrophoresis and ethanol precipitation and 
subsequently subcloned in the SmaI site of pTZ18R. The obtained plasmid 
was designated pGBGLA17 (FIG. 17B). 
To fuse this adjusted 3' glaA non-coding region to the amdS gene, a part of 
the amdS gene was subcloned from pGBDEL4L into pSP73 (Promega). For this 
construction, pGBDEL4L was digested with BglII and HindIII, the 3.4 kb 
amdS/3'glaA non-coding fragment was isolated by agarose gel 
electrophoresis and subcloned into the appropriate sites of pSP73 
(Promega). The resulting plasmid was designated pGBGLA21 (FIG. 18). 
The approximately 1 kb sized 3' glaA non-coding region in this plasmid was 
exchanged by the 2.2 kb 3' glaA non-coding region of pGBGLA17. pGBGLA17 
and pGBGLA21 were digested with KpnI and HindIII. The 2.2 kb 3' glaA 
non-coding region DNA fragment from pGBGLA17 and the 4.9 kb DNA fragment 
of pGBGLA21 were isolated by agarose gel electrophoresis, ligated and 
subsequently molecular cloned by transferring the ligation mixture to E. 
coli. The thus derived plasmid was designated pGBGLA22 (FIG. 19). 
The amdS gene with the extended 3'glaA non-coding region was completed with 
the gpdA promoter and fused to the remaining part of the amdS gene. 
pGBGLA22 was digested with BglII and HindIII, the 4.4 kb amdS/3'glaA 
non-coding region DNA fragment isolated by agarose gel electrophoresis, 
subsequently ligated with plasmid pGBGLA24 digested with BglII and HindIII 
and transferred to E. coli. The thus derived plasmid was designated 
pGBGLA25 (FIG. 20). 
pGBGLA25 was partially digested with EcoRI and in the EcoRI site of the 
gpdA promoter the synthetic fragment consisting of the two 
oligonucleotides AB3781 (SEQ ID NO: 18) and AB3782 (SEQ ID NO: 19): 
##STR3## 
was inserted. This new plasmid was designated pGBGLA43 (FIG. 21). Due to 
this cloning step, the EcoRI restriction site just in front of the gpdA 
promoter was destroyed by the introduction of an ApaI restriction site. 
The plasmid pGBGLA43 was digested with ApaI and XhoI, and the 5.3 kb DNA 
fragment comprising the gpdA promoter/amdS gene/3'glaA non-coding region 
was isolated by agarose gel electrophoresis, subsequently ligated with 
pGBGLA42 digested with ApaI and XhoI, and transferred to E.coli. The 
derived plasmid was designated pGBGLA28 (FIG. 22). 
Prior to cloning, the 3'glaA non-coding region DNA fragment (positioned at 
approximately 2.2 kb downstream the stop codon of the glaA gene, 
designated 3"glaA non-coding region), was amplified and provided with 
suitable restriction sites using the PCR method. 
For this PCR reaction, the plasmid pAB6-1 was used as template and as 
primers two synthetic oligonucleotides were designed possessing the 
following sequence: 
Oligo AB3746 (SEQ ID NO: 20) 
5' TGACCAATAAAGCTTCTCGAGTAGCAAGAAGACCCAGTCAATC 3' (a partly 3"glaA 
non-coding specific sequence around the SalI site positioned at about 2.2 
kb downstream the stop codon of the glaA gene) 
Oligo AB3747 (SEQ ID NO: 21) 
5'CTACAAACGGCCACGCTGGAGATCCGCCGGCGTTCGAAATAACCAGT3' (a partly 3"glaA 
non-coding specific sequence around the XhoI site located at about 4.4 kb 
downstream the stop codon of the glaA gene) 
Twenty-five amplification cycles (each: 1 minute 55.degree. C.; 1.5 minutes 
72.degree. C.; 1 minute 94.degree. C.) were carried out in a DNA-amplifier 
(Perkin-Elmer/Cetus). A schematic representation of this amplification is 
shown in FIG. 23A. The thus obtained DNA fragment was digested with 
HindIII, purified by agarose gel electrophoresis and ethanol precipitation 
and subcloned in both orientations into the HindIII site of pTZ19R. The 
resulting plasmids were designated pGBGLA29A and pGBGLA29B (FIG. 23). 
The final step comprises the insertion of the 3"glaA non-coding sequence 
from pGBGLA29A into the plasmid pGBGLA28. To achieve this, pGBGLA29A was 
digested with HindIII and NotI. The 2.2 kb sized 3'glaA non-coding region 
fragment was isolated by agarose gel electrophoresis, subsequently ligated 
to pGBGLA28 digested with HindIII and NotI and transferred to E. coli. The 
derived integration vector was designated pGBGLA30 (FIG. 24). 
Transformation of A.niger GBA-107 with the integration vector pGBGLA30 
Prior to transformation, E.coli sequences were removed from the integration 
vector pGBGLA30 by XhoI digestion and agarose gel electrophoresis. The 
A.niger strain GBA-107 was transformed with either 5 or 10 .mu.g DNA 
fragment by procedures as described in the experimental section. Single 
A.niger transformants were purified several times on selective acetamide 
containing plates. Spores of individual transformants were collected 
following growth for about 5 days at 30.degree. C. on 0.4% potato dextrose 
agar (Oxoid, England) plates. Southern analyses were performed to verify 
whether integration into the 3' glaA non coding region of the endogenous 
truncated glaA locus had occurred. High molecular weight DNA of several 
transformants was isolated, digested with either KpnI, or BglII and 
subsequently fractionated by electrophoresis on a 0.7% agarose gel. After 
transfer to nitrocellulose filters, hybridization was performed according 
to standard procedures. As probe a .sup.32 P-labelled approx. 0.7 kb 
XhoI/SalI glaA promoter fragment isolated from plasmid pAB6-4 (described 
in example 1) was used. The results of only 3 transformants (#107-5, 
#107-9 and #107-7) and the reference strain A.niger GBA107 and its 
ancestor A.niger CBS 531.88 are shown as example in FIG. 25. For a better 
understanding of the autoradiograph, a schematic presentation is given in 
FIGS. 26A,B,C showing the sizes of the hybridizing fragments of the intact 
glaA locus, the truncated glaA locus and of the truncated glaA locus with 
a single pGBGLA30 copy integrated into the predefined 3' glaA non-coding 
region. 
Characteristic for the intact glaA locus is a 4.5 kb hybridizing fragment 
in a KpnI digest and a 10 kb hybridizing fragment in a BglII digest. 
Characteristic for the truncated glaA locus of A.niger GBA-107 is a 3.4 kb 
hybridizing fragment in a KpnI digest and a 13 kb hybridizing fragment in 
a BglII digest. In case of integration of the pGBGLA30 vector into the 3' 
region of the truncated glaA locus, in a KpnI digest an additional 6.7 kb 
hybridizing fragment is expected besides the 3.4 kb hybridizing fragment 
and in a BglII digest the 13 kb hybridizing fragment is absent and 
replaced by a 14.5 kb hybridizing fragment. As can be seen in FIG. 25, 
transformants #107-5 and #107-9 show the expected hybridization pattern of 
a single pGBGLA30 copy integrated into the predefined 3' non-coding region 
of the truncated glaA locus. The hybridization pattern of transformant 
#107-7 indicates integration of the pGBGLA30 copy elsewhere into the 
genome of A.niger GBA-107. The transformants with the correctly integrated 
pGBGLA30 copy were designated GBA-119 and GBA-122 and were used to remove 
subsequently the amdS selection marker gene properly. 
Removal of the amdS selection marker gene from A.niger GBA-119 and GBA-122 
by counter-selection on fluoracetamide containing plates 
The amdS selection marker gene in the transformants A.niger GBA-119 and 
GBA-122 was removed again as described in the experimental section. The 
removal of the amdS selection marker gene in several surviving recombinant 
strains was verified by Southern analysis of the chromosomal DNA. High 
molecular weight DNA was isolated, digested either with KpnI or BglII and 
subsequently separated by electrophoresis on a 0.7% agarose gel. Following 
transfer to nitrocellulose, hybridization was performed according to 
standard procedures. As probe the .sup.32 P labelled 2.2 kb HindIII/NotI 
3"glaA non-coding fragment isolated from plasmid pGBGLA29A (described 
previously, FIG. 24) was used. 
A schematic presentation of the hybridizing fragments is shown in FIG. 26. 
The results of only 3 surviving recombinant strains from A.niger GBA-119 
(#AG5-5, #AG5-6 and #AG5-7) as well as 3 surviving recombinant strains 
from A.niger GBA-122 (#AG9-1, #AG9-2 and #AG9-4) and the reference strains 
A.niger CBS 531.88 and A.niger GBA-107 are shown in FIGS. 27A,B. 
In strain A.niger CBS 531.88 a 6.9 kb hybridizing fragment is present in a 
KpnI digest and a 6.9 kb hybridizing fragment in a BglII digest. In the 
A.niger GBA-107 strain a 6.9 kb hybridizing fragment is present in a KpnI 
digest and a 13 kb hybridizing fragment in a BglII digest. In the A.niger 
strains GBA-119 and GBA-122 with a single pGBGLA30 copy integrated into 
the 3' glaA non-coding region an 8 kb and a 6.7 kb hybridizing band are 
present in a KpnI digest and a 14.5 kb and a 7.6 kb hybridizing band are 
present in a BglII digest. 
Specific for correct removal of the amdS selection marker gene is the 
presence of a 6.7 kb and a 8.5 kb hybridizing fragment in a KpnI digest 
and concomitant loss of the 8 kb hybridizing fragment. In a BglII digest, 
a 14.5 kb and a 6.9 kb hybridizing fragment with concomitant loss of the 
7.6 kb hybridizing fragment is specific for the absence of the amdS 
selection marker gene. As can be seen in FIG. 27, strains #AG5-7, #AG5-5, 
#AG9-1 and #AG9-4 show the expected hybridizing pattern of the correctly 
removed amdS selection marker gene. These strains were designated GBA-120, 
GBA-121, GBA-123 and GBA-124 respectively. The hybridizing patterns of 
strains #AG5-6 and #AG9-2 indicate loss of the entire pGBGLA30 copy 
resulting in the parental A.niger GBA-107 strain with only a truncated 
glaA locus. 
Strains A.niger GBA-120, GBA-121, GBA-121 and GBA-124 were tested in shake 
flask fermentations for the ability to produce glucoamylase. As reference 
strains A.niger CBS 531.88, GBA-107, GBA-119 and GBA-122 were tested. 
Shake flask fermentations and the glucoamylase assay were performed as 
described in the experimental section. In the strains GBA-119 till GBA-124 
levels varying between 150-200 U/ml could be measured. These glucoamylase 
levels were to be expected and comparable to levels obtained with the 
parental untransformed wild-type strain A.niger CBS 531.88. 
EXAMPLE 3 
Marker Gene Free Introduction of the Phytase Gene Targeted at the 3'glaA 
Non-coding Region of the Truncated glaA Locus in A.niger GBA-107 
In this example describes the introduction of a gene into the genome of 
A.niger by using approximately the same approach and procedures as 
described in the previous example. The main difference is that the gene of 
interest and the selection marker gene are located on two separate vectors 
and that these vectors are co-transformed to A.niger. Besides the gene of 
interest or the marker gene, the vectors contain DNA sequences homologous 
to the host genome to target the vectors at a predefined genomic locus of 
the host, by a single cross-over event. By performing the fluoracetamide 
counter-selection on these (co)-transformants (as described in the 
experimental procedures), the amdS marker gene will be deleted properly by 
an internal recombination event between the DNA repeats that are created 
by integration via a single cross-over event. 
Description of the vectors used for co-transformation 
The vector with the gene of interest pGBGLA53 consists of the A.ficuum 
phytase gene under control of the A.niger glucoamylase (glaA) promoter 
flanked by 3'glaA non-coding sequences to direct integration at the 3'glaA 
non-coding region. The vector with the selection marker gene pGBGLA50 
consists of the A.nidulans amdS gene under control of the A.nidulans gpdA 
promoter flanked by 3'glaA non-coding sequences to direct integration at 
the 3'glaA non-coding region. 
Construction pathway of pGBGLA50 
The construction of pGBGLA50 comprises one cloning step. Plasmid pGBGLA29A 
was digested with HindIII and the sticky ends were filled in using the 
Klenow fragment of E.coli DNA polymerase. Next, the 2.2 kb 3"glaA 
non-coding region fragment was isolated by agarose gel-electrophoresis, 
subsequently ligated into pGBGLA43 digested with ApaI and treated with T4 
DNA polymerase to generate blunt ends, and transferred to E.coli. The 
derived plasmid with the 3"glaA non-coding region DNA fragment in the 
correct orientation was designated pGBGLA50 (FIG. 28). 
Construction pathway of pGBGLA53 
The first step in the construction pathway of pGBGLA53 is the subcloning of 
two fragments, comprising the glaA promoter fused to almost entire coding 
sequence of the A.ficuum phytase gene. To achieve this, plasmid pGBGLA42 
was digested with HindIII and EcoRI and the 1.8 kb HindIII/EcoRI 5'glaA 
promoter fragment was isolated by agarose gel-electrophoresis. Plasmid 
pFYT3 (European Patent Application 0 420 358 A1) was digested with EcoRI 
and BglII and the 1.6 kb EcoRI/BglII fragment comprising the 3'part of the 
glaA promoter fused to the 5' part of the phytase gene was isolated by 
agarose gel-electrophoresis and ligated together with the 1.8 kb 
HindIII/EcoRI 5'glaA promoter fragment isolated from pGBGLA42 into the 
HindIII and BglII sites of pSp73 (Promega). The resulting plasmid was 
designated pGBGLA49 (FIG. 29). 
The next step is the cloning of a 3'glaA non-coding region DNA fragment 
into pGBGLA49. Prior to cloning, this 3'glaA non-coding region DNA 
fragment (positioned at approximately 2.2 kb downstream the stop codon of 
the glaA gene) was amplified and provided with suitable restriction sites 
using the PCR method. 
For this PCR reaction, the plasmid pAB6-1 was used as template and as 
primers two synthetic oligonucleotides with the following sequence were 
designed: 
Oligo AB4234 (SEQ ID NO: 22) 
5' GAAGACCCAGTCAAGCTTGCATGAGC 3' (a 3'glaA non-coding sequence located 
approximately 2.2 kb downstream the stopcodon of the glaA gene) 
Oligo AB 4235 (SEQ ID NO: 23) 
5'TGACCAATTAAGCTTGCGGCCGCTCGAGGTCGCACCGGCAAAC 3' (a 3'glaA non-coding 
sequence located approximately 4.4 kb downstream the stopcodon of the glaA 
gene) 
Twenty-five amplification cycles (each: 1 minute 94.degree. C.; 1 minute 
55.degree. C.; 1.5 minutes 72.degree. C.) were carried out in a 
DNA-amplifier (Perkin-Elmer). A schematic representation of this 
amplification is shown in FIG. 30A. The thus obtained fragment was 
digested with HindIII, purified by agarose gel-electrophoresis and 
subcloned into the HindIII site of pTZ19R. The resulting plasmid was 
designated pGBGLA47 (FIG. 30). 
Plasmid pGBGLA47 was digested with HindIII en NotI, the 2.2 kb 3"glaA 
non-coding DNA fragment was isolated by agarose gel-electrophoresis and 
cloned into the HindIII and NotI sites of pGBGLA49. The resulting plasmid 
was designated pGBGLA51 (FIG. 31). 
The last step in the construction pathway of pGBGLA53 is the cloning of the 
DNA fragment comprising the remaining part of the phytase coding sequence 
fused to the 3'glaA non-coding DNA fragment located just downstream the 
stop codon of the glaA gene. Prior to cloning, the remaining part of the 
phytase gene and the 3'glaA non-coding DNA fragment located just 
downstream the stopcodon of the glaA gene were fused and provided with 
suitable restriction sites using the PCR method. In the PCR, plasmid 
pAB6-1 was used as template and as primers two synthetic oligonucleotides 
were used, having the following sequences: 
Oligo AB4236 (SEQ ID NO: 24) 
5' TGACCAATAAAGCTTAGATCTGGGGGTGATTGGGCGGAGTGTTTTGCTT AGACAATCAATCCATTTCGC 
3' (36 bp of the phytase coding sequence, starting at the BglII site until 
the stopcodon fused to the 3'glaA non-coding region, starting just 
downstream the stopcodon of the glaA gene) 
Oligo AB4233 (SEQ ID NO: 25) 
5' TGACCAATAGATCTAAGCTTGACTGGGTCTTCTTGC 3' (a 3'glaA non-coding sequence 
located approximately 2.2 kb downstream the stopcodon of the glaA gene) 
Twenty-five amplification cycles (each: 1 minute 94.degree. C.; 1 minute 
55.degree. C.; 1.5 minutes 72.degree. C.) were carried out in a 
DNA-amplifier (Perkin-Elmer). A schematic representation of this 
amplification is shown in FIG. 32A. The thus obtained fragment was 
digested with HindIII, purified by agarose gel-electrophoresis and 
subcloned in both orientations into the HindIII site of pTZ19R. The 
resulting plasmids were designated pGBGLA48 and pGBGLA52 (FIG. 32B). 
Plasmid pGBGLA52 was digested with BglII and partially digested with BamHI, 
the 2.2 kb phytase/3'glaA non-coding DNA fragment was isolated by agarose 
gel-electrophoresis and cloned into the BglII site of pGBGLA51. The 
derived plasmid with the 2.2 kb phytase/3'glaA non-coding DNA fragment in 
the correct orientation was designated pGBGLA53 (FIG. 33). 
Transformation of A.niger GBA-107 with the vectors pGBGLA50 and pGBGLA53 
Prior to transformation, E.coli sequences were removed from pGBGLA50 and 
pGBGLA53 by respectively XhoI or HindIII digestion followed by agarose 
gel-electrophoresis. The A.niger GBA-107 strain was transformed with 
respectively 1 .mu.g pGBGLA50 fragment plus 1 .mu.g pGBGLA53 fragment, 1 
.mu.g pGBGLA50 fragment plus 5 .mu.g pGBGLA53 fragment, or 1 .mu.g 
pGBGLA50 fragment plus 10 .mu.g pGBGLA53 fragment using the transformation 
procedure described in the experimental section. 
Single transformants were isolated, purified and Southern analysis was 
performed, using the same digests and probes as described in example 2, to 
verify integration of both pGBGLA50 and pGBGLA53. In about 10-20% of the 
analyzed transformants both pGBGLA50 and pGBGLA53 were integrated into the 
genome of the A.niger GBA-107 host strain. The transformant showing the 
correct integration pattern of a single copy pGBGLA50 and a single copy 
pGBGLA53, both integrated at the predefined 3'glaA non-coding region of 
the truncated glaA locus was used to remove subsequently the amdS 
selection marker gene. 
Removal of the amdS marker gene by counter-selection on fluoracetamide 
containing plates 
By performing the fluoracetamide counter-selection (as described in the 
experimental procedures), the amdS marker gene was deleted by an internal 
recombination event between the DNA repeats that were created by 
integration via a single cross-over event (i.e. the 3'glaA non-coding 
sequences). Proper removal of only the amdS marker gene was verified by 
Southern analysis using the same digests and probes as in example 2. 
EXAMPLE 4 
Marker Gene Free Introduction of the glaA Gene and the Phytase Gene in 
A.oryzae 
This example describes the marker gene free introduction of the glaA gene 
or the phytase gene in A.oryzae NRRL3485. A.oryzae NRRL3485 was 
transformed as described in the experimental section using the same 
vectors and approach as described in examples 2 and 3. Single 
transformants were isolated, purified and Southern analysis of chromosomal 
DNA of several transformants was performed to verify integrations of 
respectively the pGBGLA30 vector or the pGBGLA50 and pGBGLA53 vectors. In 
the Southern analysis, the same digests and probes were used as described 
in example 2. 
Removal of the amdS gene by counter-selection on fluoracetamide containing 
plates 
In case of integration of the pGBGLA30 vector, a transformant with a single 
copy of the pGBGLA30 integrated into the genome of the host strain 
A.oryzae NRRL3485 was used to remove the amdS gene properly. The 
counter-selection on fluoracetamide containing plates was performed as 
described in the experimental section. Correct removal of the amdS gene 
was verified by Southern analysis of chromosomal DNA of several 
fluoracetamide resistant strains. The same digests and probes were used as 
described in Example 2. 
In case of co-transformation of the pGBGLA50 and pGBGLA53 vector, a 
transformant with a single copy of both pGBGLA50 and pGBGLA53 integrated 
into the host genome was used to remove the amdS marker gene properly. The 
counter-selection using fluoracetamide plates was performed as described 
in the experimental section. Correct removal of the amdS marker gene (e.g. 
the pGBGLA50 vector) was verified by Southern analysis of chromosomal DNA 
of several fluoracetamide resistant strains using the same digests and 
probes as described in example 2. 
EXAMPLE 5 
Marker Gene Free Introduction of the glaA Gene and the Phytase Gene in 
T.reesei 
This example describes the marker gene free introduction of the glaA gene 
or the phytase gene in Trichoderma reesei strain QM9414 (ATCC 26921). 
T.reesei QM9414 was transformed as described in the experimental section 
using the same vectors and approach as described in examples 2 and 3. 
Single transformants were isolated, purified and Southern analysis of 
chromosomal DNA of several transformants was performed to verify whether 
integration of respectively the pGBGLA30 vector or the pGBGLA50 and 
pGBGLA53 vectors. In the Southern analysis, the same digests and probes 
were used as described in example 2. 
Removal of the amdS gene by counter-selection on fluoracetamide containing 
plates 
In case of integration of the pGBGLA30 vector, a transformant with a single 
copy of the pGBGLA30 integrated into the genome of the host strain 
T.reesei QM9414 was used to remove the amdS gene properly. The 
counter-selection on fluoracetamide containing plates was performed as 
described in the experimental section. Correct removal of the amdS gene 
was verified by Southern analysis of chromosomal DNA of several 
fluoracetamide resistant strains. 
In case of co-transformation of the pGBGLA50 and pGBGLA53 vector, a 
transformant with a single copy of both pGBGLA50 and pGBGLA53 integrated 
into the host genome was used to remove the amdS marker gene properly. The 
counter-selection using fluoracetamide plates was performed as described 
in the experimental section. Correct removal of the amdS marker gene (e.g. 
the pGBGLA50 vector) was verified by Southern analysis on chromosomal DNA 
of several fluoracetamide resistant strains using the same digests and 
probes as described in example 2. 
EXAMPLE 6 
Marker Gene Free Introduction into P.chrysogenum of a P.chrysogenum Gene by 
Co-transformation using the amdS-gene as a Selection Marker 
In this example the marker gene free introduction of a gene into the genome 
of P.chrysogenum by co-transformation is described. 
In the co-transformation procedure, 2 different pieces of DNA are offered 
to the protoplasts, one of them being the amdS-selection marker, on the 
presence of which the first transformant selection takes place, as 
described in the experimental section, the second being another piece of 
DNA of interest, e.g. encoding a particular enzyme of interest. In a 
certain number of transformants both pieces of DNA will integrate into the 
chromosomes and will be stably maintained and expressed. 
The amdS-selection marker gene can then be removed selectively from 
purified transformants by applying the counter-selection procedure as 
described in the experimental section, while the second piece of DNA will 
remain stably integrated into the chromosomes of the transformant. As an 
example to illustrate the general applicability of the method the stable, 
marker gene free introduction of a niaD-gene is described which enables a 
niaD.sup.- -host to grow on nitrate as sole nitrogen-source. 
Host for this co-transformation is a P.chrysogenum niaD.sup.- -strain which 
lacks nitrate reductase and therefore is unable to grow on plates 
containing nitrate as sole nitrogen source. These strains can be easily 
obtained by well known procedures (Gouka et al., Journal of Biotechnology 
20(1991), 189-200 and references there in). 
During the co-transformation (procedure described in experimental section), 
two pieces of DNA are simultaneously offered to the protoplasts: the 7.6 
kb EcoRI restriction fragment from pGBGLA28 containing the amdS selection 
marker gene and the 6.5 kb EcoRI restriction fragment from pPC1-1, 
containing the P.chrysogenum niaD-gene. Prior to transformation, both 
fragments have been separated from E.coli vector sequences by agarose 
gel-electrophoresis and purified from agarose gel by electro-elution. The 
first selection of transformants took place on selective plates containing 
acetamide as sole nitrogen source as described in the experimental 
section. 
Among the transformants, co-transformants are found by replica plating 
spores of purified transformants to plates containing nitrate as sole 
nitrogen source. 
Typically about 20-60% of the replica plated transformants were able to 
grow on this medium, indicating that in these transformants not only the 
amdS selection marker gene but also the niaD-gene has integrated into the 
genome and is expressed. 
Removal of the amdS gene by counter-selection on fluoracetamide containing 
plates 
The amdS selection marker gene is subsequently removed from the 
co-transformants by counter-selection on fluor-acetamide. 
For direct selection on the amdS.sup.- /niaD.sup.+ -phenotype the medium 
used contained 10 mM fluor-acetamide. Spores were plated at a density of 
10.sup.4 spores per plate. After 5-7 days of incubation at 25.degree. C., 
fluor-acetamide resistant colonies could be identified as solid colonies 
clearly distinct from the faint background. The niaD.sup.+ -phenotype of 
the recombinants is demonstrated by their growth on the 
fluoracetamide-medium containing nitrate as sole nitrogen source. The 
amdS.sup.- -phenotype of the recombinants was confirmed by lack of growth 
of the recombinants on plates containing acetamide as sole nitrogen 
source. Typically, 0.1-2% of the original number of plated spores 
exhibited the desired phenotype. 
Southern analysis on chromosomal DNA form several fluoracetamide resistant 
strains confirmed that the amdS selection marker gene was removed from the 
P.chrysogenum genome. 
EXAMPLE 7 
Test of the amdS-minus Phenotype of the Yeast Kluyveromyces lactis 
A prerequisite for the use of the amdS selection system in K.lactis is that 
this yeast does not contain any acetamidase activity. To test this we have 
plated the K.lactis strains CBS 683 and CBS 2360 on the following 3 
different solid media: 
I Yeast Carbon Base (YCB, Difco), containing all the essential nutritives 
and vitamins except a nitrogen-source. 
II YCB supplemented with 5 mM acetamide. 
III YCB supplemented with 0.1% (w/v) NH.sub.4 (SO.sub.4).sub.2. 
All 3 media contained 1.2% (w/v) Oxoid agar (Agar No. 1) and 30 mM Sodium 
Phosphate buffer at pH 7.0. Difco YCB was used at 1.17% (w/v). 
Full grown K.lactis colonies were only observed on medium III, containing 
ammonium as nitrogen source. In plates without nitrogen-source or with 
acetamide as sole nitrogen-source no growth or, occasionally slight 
background growth was observed, which is most likely caused by trace 
amounts of nitrogen contaminating the agar or other medium components. We 
conclude that both K.lactis strains lack sufficient acetamidase activity 
to sustain growth on acetamide as sole nitrogen source. This should allow 
for the A.nidulans amdS gene to be used as selection marker in the yeast 
K.lactis. 
EXAMPLE 8 
Construction of Plasmids for use of the amdS Gene in Yeasts 
Construction of pGBamdS1 
We have previously used pGBHSA20 for the expression of human serum albumin 
(HSA) in K.lactis (Swinkels et al. 1993, Antonie van Leeuwenhoek 64, 
187-201). In pGBHSA20 the HSA cDNA is driven from the K.lactis LAC4 
promoter (FIG. 34 for the physical map of the plasmid pGBHSA20). At the 
3'-end the HSA cDNA is flanked by LAC4 terminator sequences. For selection 
of transformants pGBHSA20 contains the Tn5 phosphotransferase gene which 
confers resistance to the antibiotic G418 (Geneticin, BRL) (Reiss et al. 
(1984) EMBO J. 3, 3317-3322) driven by the S.cerevisiae ADH1 promoter 
(Bennetzen and Hall (1982) J. Biol. Chem. 257, 3018-3025). In the unique 
SstII site of the LAC4 promoter pGBHSA20 contains the E.coli vector pTZ19R 
which is used for amplification in E.coli. Prior to transformation to 
K.lactis the pTZ19R sequences are removed from pGBHSA20 by SstII digestion 
and agarose gel purification. Transformation of pGBHSA20 linearized in the 
SstII site of the LAC4 promoter to K.lactis results in integration into 
the genomic LAC4 promoter by homologous recombination. pGBamdS1 is derived 
from pGBHSA20 by substitution of the HSA cDNA for the amdS cDNA from 
pamdS1. Using PCR, SalI sites were introduced at the 5' and 3' ends of the 
amdS cDNA. In this PCR pamdS1 was used as template and oligo's AB3514 (SEQ 
ID NO: 26) and AB3515 (SEQ ID NO: 27) were used as primers. 
Oligo AB3514 (SEQ ID NO: 26) 
5'-CTGCGAATTCGTCGACATGCCTCAATCCTGGG-3' (an 5'end amdS-specific sequence 
with the introduced SalI site) 
Oligo AB3515 (SEQ ID NO: 27) 
5'-GGCAGTCTAGAGTCGACCTATGGAGTCACCACATTTC-3' (an 3' end amdS-specific 
sequence with the introduced SalI site). 
The PCR fragment thus obtained was digested with SalI and cloned into the 
SalI/XhoI sites of pGBHSA20. Several clones were obtained containing 
either of the 2 possible orientations of the amdS cDNA as judged by 
restriction analysis. One of the clones with the amdS cDNA in the correct 
orientation is pGBamdS1, the physical map of which is shown in FIG. 34. 
Construction of pGBamdS3 
By heterologous hybridization using a probe derived from the S.cerevisiae 
elongation factor 1-.alpha. gene (EF1-.alpha.; Nagata et al. (1984) EMBO 
J. 3, 1825-1830), we have isolated a genomic clone containing the K.lactis 
homologue of the EF1-.alpha. gene, which we call KlEF1. In this example we 
have used a 813 bp fragment containing the KlEF1 promoter to express the 
amdS cDNA in K.lactis. Using oligonucleotides AB3701 (SEQ ID NO: 28) and 
AB3700 (SEQ ID NO: 29), this fragment was amplified in a PCR using genomic 
DNA from K.lactis strain CBS 683 as template. AB3700 (SEQ ID NO: 29) is 
designed such that it contains 21 nucleotides of the KlEF promoter and 38 
nucleotides upstream the ATG initiation codon of the amdS gene. The 
sequence of AB3701 (SEQ ID NO: 28) and AB3700 (SEQ ID NO: 29) is as shown: 
Oligo AB3701 (SEQ ID NO: 28) 
5'-CTGCGAATTCGTCGACACTAGTGGTACCATTATAGCCATAGGACAGCAAG 3' (a 5' 
KlEF1-specific promoter sequence with the additional restriction sites 
EcoRI, SalI, SpeI and KpnI at the 5' end of the promoter) 
Oligo AB3700 (SEQ ID NO: 29) 
5'-GCTCTAGAGCGCGCTTATCAGCTTCCAGTTCTTCCCAGGATTGAGGCATTTTTAATGTTACTTCTCTTGC-3 
' (3' KlEF1-specific promoter sequence fused to the 5'-sequences of the 
amdS cDNA with the restriction sites BssH2 and additional site XbaI). 
The PCR was performed using standard conditions and the PCR-fragment 
obtained was digested with EcoRI and XbaI and subcloned into EcoRI/XbaI 
digested pTZ19R. The physical map of the resulting plasmid pTZKlEF1 is 
shown in FIG. 35. The remaining part of the amdS cDNA as well as part of 
the LAC4 terminator sequences were obtained from pGBamdS1 by digestion 
with BssH2 and SphI. This BssH2-SphI fragment was cloned into the BssH2 
and SphI digested pTZKlEF1 and the resulting plasmid was designated 
pGBamdS2 (FIG. 35). For the final step in the construction of pGBamdS3, 
both pGBamdS2 and pTY75LAC4 (Das and Hollenberg (1982) Current Genetics 6, 
123-128) were digested were digested with SphI and HindIII. The 5.7 kb DNA 
fragment from pGBamdS2 and the 1.2 kb DNA fragment from pTY75LAC4, which 
contains the remaining LAC4 terminator sequences, were purified from 
agarose gels after fractionation and subsequently ligated and used to 
transform E.coli. The resulting expression vector, in which the amdS cDNA 
is driven from the K.lactis KlEF1 promoter, was designated pGBamdS3 (FIG. 
36). 
Construction of pGBamdS5 
Fusion of the S.cerevisiae alcohol dehydrogenase I (ADH1) promoter to the 
amdS cDNA was performed in a PCR using pGBHSA20 as template. One of the 
primers (AB3703; SEQ ID NO: 31) contains sequences complimentary to the 
3'-end of the ADH1-promoter sequence which are fused to sequences of the 
amdS cDNA. The other primer (AB3702; SEQ ID NO: 30) contains the 5'-end of 
the ADH1 promoter: 
Oligo AB3702 (SEQ ID NO: 30) 
5'-CTGCGAATTCGTCGACACTAGTGGTACCATCCTTTTGTTGTTTCCGGGTG-3' (a 5' 
ADH1-specific promoter sequence with the additional restriction sites 
EcoRI, SalI, SpeI and KpnI at the 5' end of the promoter). 
Oligo AB3703 (SEQ ID NO: 31) 
5'-GCTCTAGAGCGCGCTTATCAGCGGCCAGTTCTTCCCAGGATTGAGGCATTGTATATGAGATAGTTGATTG-3 
' (a 3' ADH1-specific promoter sequence fused to the 5' amdS sequence with 
additional restriction sites BssH2 and XbaI). 
The PCR reaction was performed using a "touchdown" protocol (Don et al., 
(1991) Nucleic Acids Res. 19, 4008). The reaction mixtures were subjected 
to 30 amplification cycles, while the annealing temperature was decreased 
1.degree. C. every two cycles, starting 55.degree. C. down to a 
"touchdown" at 40.degree. C., at which temperature 10 more cycles were 
carried out (cycles: 2' at 94.degree. C., 2' annealing, 3' at 72.degree. 
C.). The PCR-fragment obtained was digested with EcoRI and XbaI and 
subcloned into pTZ19R. The resulting plasmid pTZs.c.ADH1 is shown in FIG. 
37. pTZs.c.ADH1 and pGBamdS3 were digested with KPnI and BssH2. The 6.8 kb 
fragment from pGBamdS3 and the 750 bp fragment from pTZs.c.ADH1 were 
purified by gel electrophoresis, ligated and used to transform E.coli 
JM109. The resulting expression vector was designated pGBamdS5 (FIG. 37). 
Construction of pGBamdS6 
Plasmid pGBamdS3 contains the amdS cDNA under control of the KlEF1 promoter 
and flanked at the 3' end by 1.5 kb of LAC4 terminator sequences (FIG. 
36). pGBamdS6 is constructed by cloning a fragment which contains a fusion 
of the LAC4 promoter and terminator sequences upstream of the amdS 
expression-cassette in pGBamdS3 (FIG. 38). In order to fuse the LAC4 
promoter and terminator sequences we have first constructed pPTLAC4 (FIG. 
39). Using a PCR, additional restriction sites are introduced at the 5' 
and 3' end of a 600 bp LAC4 terminator fragment. In the PCR K.lactis CBS 
683 chromosomal DNA was used as template and oligonucleotides AB3704 (SEQ 
ID NO: 32) and AB3705 (SEQ ID NO: 33) were used as primers: 
Oligo AB3704 (SEQ ID NO: 32) 
5'-GCTCTAGAAGTCGACACTAGTCTGCTACGTACTCGAGAATTTATACTTAGATAAG-3' (a LAC4 
terminator-specific sequence starting at the LAC4 stop codon with the 
additional restriction sites XbaI, SalI, SpeI, SnaBI and XhoI). 
Oligo AB3705 (SEQ ID NO: 33) 
5'-TGCTCTAGATCTCAAGCCACAATTC-3' (3' LAC4 terminator-specific sequence with 
the additional restriction site XbaI). 
The PCR was performed using standard conditions and the resulting DNA 
fragment was digested with XbaI and subcloned into the XbaI site of pTZ19R 
to give pTLAC4 (FIG. 39). The LAC4 promoter sequence is obtained by 
digestion of pKS105 van den Berg et al. (1990) Bio/Technology 8, 135-139) 
with XbaI and SnaBI. The XbaI-SnaBI LAC4 promoter fragment was cloned into 
the SpeI/SnaBI sites of pTLAC4 and designated pPTLAC4 (FIG. 39). For the 
final step in the construction of pGBamdS6, the plasmid pPTLAC4 was 
digested with XbaI. The 4.1 kb DNA fragment from pPTLAC4 was purified by 
gel-electrophoresis and cloned into SpeI site of pGBamdS3. The obtained 
gene-replacement vector was designated pGBamdS6 (FIG. 38). 
Construction of pGBamdS8 
pGBamdS7 was constructed by cloning a fragment, which contains part of the 
LAC4 promoter as well as the chymosin expression-cassette, in between the 
LAC4 promoter and terminator sequences as present in pGBamdS6 (FIG. 40). 
Plasmid pKS105 contains the prochymosin cDNA fused to the prepro-region of 
S.cerevisiae .alpha.-factor under control of the LAC4 promoter (van den 
Berg et al. (1990) Bio/Technology 8, 135-139). Using a PCR, additional 
restriction sites were introduced at the 5' and 3' end of the fusion LAC4 
promoter and chymosin expression-cassette. In the PCR pKS105 DNA was used 
as template and oligonucleotides AB3965 (SEQ ID NO: 34) and AB3966 (SEQ ID 
NO: 35) were used as primers: 
Oligo AB3965 (SEQ ID NO: 34) 
5'-CTGCTACGTAATGTTTTCATTGCTGTTTTAC-3' (a LAC4 promoter-specific sequence 
starting at the restriction site SnaB1) 
Oligo AB3966 (SEQ ID NO: 35) 
5'-CCGCCCAGTCTCGAGTCAGATGGCTTTGGCCAGCCCC-3' (chymosin-specific sequence 
with the additional restriction site Xho1). 
The PCR was performed using standard conditions and the obtained PCR 
fragment was digested with SnaB1 and Xho1. The plasmid pGBamdS6 was 
partially digested with XhoI and subsequently digested with SnaBI and the 
10.9 kb DNA fragment was isolated and purified by gel-electrophoresis. The 
SnaB1-Xho1 fusion fragment LAC4 promoter/chymosin expression-cassette was 
cloned into the SnaB1/Xho1 sites of pGBamdS6. The resulting plasmid was 
designated pGBamdS7 (FIG. 40). 
To destroy the HindIII site approximately 66 bp upstream the startcodon 
from the chymosin gene, pGBamdS7 was partially digested with HindIII and 
treated with the Klenow fragment of E.coli DNA polymerase I to generate 
blunt ends, subsequently ligated and transferred to E.coli for molecular 
cloning. The derived plasmid was designated pGBamdS8 and contains a LAC4 
promoter fragment with a destroyed HindIII site. 
EXAMPLE 9 
Expression of the amdS cDNA from the LAC4 Promoter in the Yeast K.lactis 
The expression vector, pGBamdS1, contains, apart from the amdS cDNA a 
second selection marker which confers resistance to the antibiotic G418. 
This allows to first select for transformants using G418 resistance which 
is a well established procedure (Sreekrishna et al. (1984) Gene 28, 
73-81). The transformants obtained this way can subsequently be used to 
verify expression of the amdS cDNA and to optimize conditions for 
selection of the amdS.sup.+ phenotype in K.lactis. Once these conditions 
have been established, direct selection for amdS.sup.+ transformants can 
be performed, e.g. using expression cassettes without additional selection 
markers. 
pGBamdS1 (FIG. 34) was linearized in the LAC4 promoter by SstII digestion. 
The pTZ19R sequences were removed by fractionation in and purification 
from agarose gels. 15 .mu.g's of this DNA fragment were used to transform 
to the K.lactis strains CBS 2360 and CBS 683 as described by Ito H. et al. 
(1983) J. Bacteriol. 153, 163-168 with the modifications described under 
Experimental. The transformation plates were incubated at 30.degree. C. 
for 3 days. G418-resistant transformants were obtained with both strains. 
Several independent transformants of both strains as well as the wild type 
strains were subsequently streaked onto plates containing different solid 
media (see Table 1). YEPD and YEPD/G418 have been described in 
Experimental. YCB, YCB/NH.sub.4 and YCB/acetamide have been described in 
example 7 as media I, II and III, respectively. YNB-lac/NH.sub.4 and 
YNB-lac/acetamide contain 0.17% (w/v) Yeast Nitrogen Base w/o Amino Acids 
and Ammonium Sulfate (Difco) supplemented with 1% (w/v) lactose, 30 mM 
Sodium Phosphate buffer at pH 7.0 and either 0.1% (w/v) NH.sub.4 
(SO.sub.4).sub.2 or 5 mM acetamide, respectively. 
The amdS.sup.+ phenotype of the CBS 683/pGBamdS1 transformants was obvious 
on YCB/acetamide (see Table 1). However, the CBS 2360 transformants 
containing the same expression vector did not show any growth on 
YCB/acetamide. We reasoned that this might be due to the lack of induction 
of the LAC4 promoter driving the amdS cDNA in the absence of lactose or 
galactose as carbon-source dependent differences in the regulation of the 
LAC4 promoter between different K.lactis strains have been described 
(Breunig (1989) Mol. Gen. Genet. 216, 422-427). Table 1 shows that this is 
indeed the case, on medium containing lactose as sole carbon-source and 
acetamide as sole nitrogen source the CBS 2360 transformants were able to 
grow. We can therefore conclude that, depending on the carbon-source used, 
these transformants sufficiently express the A.nidulans amdS cDNA in order 
to sustain growth of the yeast K.lactis on acetamide as sole 
nitrogen-source. 
Southern analyses were performed to verify whether integration in the LAC4 
promoter had occurred. High molecular weight DNA of several CBS 2360 and 
CBS 683 transformants was isolated, digested with HindIII and subsequently 
fractionated by electrophoresis on a 0.7% agarose gel. After transfer to 
nitrocellulose, hybridization was performed according to standard 
procedures. As probe a .sup.32 P-labelled approximately 1.5 kb 
SacII/HindIII LAC4 promoter fragment isolated from plasmid pGBHSA20 (FIG. 
34) was used. We identified CBS 683 and CBS 2360 transformants containing 
a single pGBamdS1 expression cassette integrated in the LAC4 locus, one 
example of each is shown in FIG. 41 and is designated KAM-1 and KAM-2, 
respectively. Single copy integration of pGBamdS1 in the LAC4 promoter 
produces two new HindIII fragments of 4.2 and 8.6 kb, both of which are 
present in transformants KAM-1 and KAM-2. Since CBS 683 contains two LAC4 
loci and pGBamdS1 has integrated in only one of them in KAM-1, the digest 
of KAM-1 also shows the 5.6 kb HindIII fragment derived from the second 
undisturbed LAC4 locus. 
TABLE 1 
______________________________________ 
Growth of K. lactis CBS 683 and CBS 2360 wild type and 
pGBamdS1 transformants on solid media containing different 
nitrogen- and/or carbon-sources. 
strain CBS 683 CBS 2360 
transforming DNA 
none pGBamdS1 none pGBamdS1 
______________________________________ 
YEPD + + + + 
YEPD-G418 - + - + 
YCB - - - - 
YCB/NH.sub.4 
+ + + + 
YCB/acetamide 
- + - - 
YNB-lac/NH.sub.4 
+ + + + 
YNB-lac/acetamide 
- + - + 
______________________________________ 
EXAMPLE 10 
Direct Selection of K.lactis CBS 683 and CBS 2360 Transformants Using 
Acetamide as Sole Nitrogen-source 
SstII linearized pGBamdS1 (15 .mu.g) was transformed into K.lactis CBS 683 
and CBS 2360 using the transformation procedure as described by Ito H. et 
al. ((1983). J. Bact. 153, 163-168.) with the following modifications: 
K.lactis cultures were harvested for transformation at OD.sub.610 =0.5-1.0. 
After the 5 minutes heatshock of the DNA-cell suspension, the phenotypic 
expression prior to plating was performed for 150-180 minutes at 
30.degree. C. in volumes of 1 ml. Different media were used for both 
strains. For CBS 683 a YEPD/YNB solution (1*YNB (Yeast Nitrogen Base, 
Difco), 1% bacto-peptone, 1% yeast extract and 2% glucose) or YNB-glu 
(1*YNB (Yeast Nitrogen Base w/o Amino Acids and Ammonium Sulphate, Difco) 
supplemented with 1% (w/v) glucose and 30 mM Sodium Phosphate buffer at pH 
7.0) were used. After this incubation the cells were centrifuged at 2000 g 
at room temperature for 5 minutes and subsequently plated on YCB/acetamide 
(see example 7). For CBS 2360, YNB-lac (1*YNB (Yeast Nitrogen Base w/o 
Amino Acids and Ammonium Sulphate, Difco) supplemented with 1% (w/v) 
lactose and 30 mM Sodium Phosphate buffer at pH 7.0) was used. After this 
incubation the cells were centrifuged at 2000 g at room temperature for 5 
minutes and subsequently plated on YNB-lac/acetamide (see example 9). 
Growth was performed at 30.degree. C. for 3 days. amdS.sup.+ transformants 
were obtained for both strains. The transformation frequencies found were 
comparable to that found when using the G418 selection. The correct 
identity of the transformants was confirmed by subsequent plating on 
YEPD-plates containing G418 and by Southern analysis. 
pGBamdS3 (FIG. 36), in which the amdS cDNA is driven from the KlEF1 
promoter, was linearized in the LAC4 terminator by digestion with XhoI and 
15 .mu.g of the gel-isolated fragment was subsequently transformed into 
the K.lactis strain CBS 683 using direct selection on YCB/acetamide plates 
as described above for the transformation of pGBamdS1 into CBS 683. Some 
of the transformants obtained were analyzed by Southern blotting. High 
molecular weight DNA was isolated, digested with BamHI and subsequently 
separated by electrophoresis on a 0.7% agarose gel. Following transfer to 
nitrocellulose, hybridization was performed according to standard 
procedures. As probe the .sup.32 P-labelled 1.2 kb SphI/HindIII LAC4 
terminator fragment isolated from plasmid pTY75LAC4 (described in Example 
8) was used. The results of several CBS 683 transformants, containing the 
pGBamdS3 plasmid and from several transformants containing the pGBamdS5 
plasmid are shown in FIGS. 42A and 42B respectively. The reference strain 
CBS 683 is shown in FIG. 42B. In the CBS 683 transformants an additional 
6.8 kb sized hybridizing fragment is present besides the 3.7 kb 
hybridizing fragment of the intact LAC4 terminator. This implicates a 
correct integration of the plasmids into the LAC4 terminator region. 
In all of these transformants pGBamdS3 was integrated in one or more copies 
into the LAC4 terminator (the intensity of the 6.8 kb hybridizing fragment 
is an indication for the number of integrated copies of the vector). We 
conclude that also the constitutive KlEF1 promoter can drive the amdS cDNA 
for use as selection marker. Similar results were obtained with pGBamdS5 
(FIG. 37), in which the amdS cDNA is driven from the S.cerevisiae ADH1 
promoter. 
EXAMPLE 11 
Transformation of S.cerevisiae With pGBamdS5 by Direct Selection on 
Acetamide 
In this example we have tested whether the amdS cDNA can also be used as 
selection marker in other yeasts, e.g. S.cerevisiae. We have first 
established the amdS.sup.- phenotype of S.cerevisiae strain D237-10B and 
its ability to use ammonium as sole nitrogen-source, using the same media 
and procedures as we have described for K.lactis in example 7. As observed 
in the case of K.lactis, full grown S.cerevisiae colonies were only 
observed on the plates containing ammonium as nitrogen source. In plates 
without nitrogen-source or with acetamide as sole nitrogen-source no 
growth or, occasionally a slight background growth was observed. Plasmid 
pGBamdS5 was linearized in the ADH1 promoter by partial digestion with 
SphI. The S.cerevisiae strain D273-10B (ATCC 25657) was transformed with 
15 .mu.g of gel-isolated linearized pGBamdS5 fragment, using 
transformation procedures as described in example 10 for the 
transformation of pGBamdS1 to K.lactis CBS 683. After transformation the 
cells were plated onto YCB/acetamide plates (see Example 9) and allowed to 
grow at 30.degree. C. for 3 days. Several amdS.sup.+ transformants were 
obtained with in this transformation. Subsequent Southern analysis of some 
of the amdS transformants confirmed that the amdS cDNA was stably 
integrated into the S.cerevisiae genome. 
High molecular weight DNA was isolated and digested with BamHI, 
subsequently separated by electrophoresis on a 0.7% agarose gel and 
blotted onto nitrocellulose. As probe the .sup.32 P labelled 750 bp EcoRV 
amdS fragment was used isolated from pGBamdS1. The results of several 
D273-10B/pGBamdS5 transformants as well as the reference strain D273-10B 
(ATCC 25657) are shown in FIG. 43. Two hybridizing fragments are present 
in the D273-10B transformants respectively, a 6.6 kb fragment that 
represents the multicopy fragment and a hybridizing fragment of unknown 
size that represents the flanking. The reference strain D273-10B (ATCC 
25657) as expected does not show any hybridizing fragment. 
EXAMPLE 12 
Removal of the amdS-marker From K.lactis and S.cerevisiae 
amdS.sup.+ Transformants Using Fluoracetamide Counter-selection 
In the above described examples the amdS containing expression cassettes 
are integrated by a single cross-over homologous recombination in the 
K.lactis and S.cerevisiae genomes. This means that the amdS cDNA is 
flanked by direct repeats in the genomes of these amdS yeast 
transformants. Consequently, the amdS cDNA will be deleted in a small 
fraction of the transformant population by intra-chromosomal mitotic 
recombination events occurring at low frequency between the direct repeats 
flanking the cDNA. It should be possible to select for these events using 
media containing fluoracetamide, a compound which is toxic for amdS.sup.+ 
cells but not for amdS.sup.- cells as has been shown for A.nidulans by 
Hynes and Pateman ((1970) Mol. Gen. Genet. 108, 107-116). In amdS.sup.+ 
cells fluoracetamide is converted into ammonium and fluoracetate, the 
latter being toxic when activated by the enzyme acetyl-CoA-synthetase. 
Prerequisites for the fluoracetamide counter-selection to also work on 
amdS.sup.+ yeasts are therefore 1) fluoracetamide should not be toxic for 
amdS.sup.- yeasts, 2) the yeast cellwall and plasmamembrane should be 
permeable to fluoracetamide and 3) the enzyme acetyl-CoA-synthetase should 
be active. To test this we have used a K.lactis CBS 683 transformant 
containing a single copy of pGBamdS1 integrated in the LAC4 promoter, 
designated KAM-1 and a S.cerevisiae D273-10B transformant containing a 
single copy of pGBamdS5 integrated in the ADH1 promoter, designated SAM-1. 
Both KAM-1 and SAM-1 were subjected to at least 3 rounds of genetic 
purification on selective medium (YCB/acetamide) to exclude contamination 
with the amdS.sup.- parental strains. KAM-1 and SAM-1 were each plated at 
a density of approximately 10.sup.3 CFU per plate onto YCB/NH.sub.4 
supplemented with 10 mM fluoracetamide. For both KAM-1 and SAM-1, 5 to 20 
fluoracetamide resistant colonies appeared after 3 to 6 days at 30.degree. 
C. Southern analysis on chromosomal DNA from several independent KAM-1 and 
SAM-1 derived amdS.sup.- colonies confirmed that the amdS cDNA was 
correctly removed from the K.lactis and S.cerevisiae genomes by homologous 
recombination between the flanking direct repeats (FIG. 41). In fact, in 
one of the KAM-1 amdS.sup.- recombinants the crossover-point of the 
recombination was located between a polymorphic HindIII site and the amdS 
cDNA. This polymorphic HindIII is present 92 bp upstream of the LAC4 
reading frame in the LAC4 promoter of pGBamdS1, however, this site is not 
present in the CBS 683 LAC4 promoter. The recombination event has left the 
HindIII site in the genome of this particular KAM-1 recombinant which 
could otherwise not be discriminated from the parent strain CBS 683 (see 
the extra 4.2 kb fragment in FIG. 41, lane 6). This KAM-1 recombinant 
therefore excludes the possibility the we would have isolated CBS 683 
contaminants in stead of KAM-1 amdS.sup.- recombinants. We conclude from 
the above that the amdS cDNA can be removed from yeast genomes when 
flanked by direct repeats using fluoracetamide counter-selection. In the 
present example the amdS.sup.- K.lactis and S.cerevisiae recombinants 
occur at a frequency of about 0.1%. 
We have noted that for some yeast strains efficient counter-selection on 
fluoracetamide cannot be performed on YCB/NH.sub.4, probably due to strong 
carbon-catabolite repression of the acetyl-CoA-synthetase. In those 
instances we have successfully used YNB-galactose/NH.sub.4 (this medium is 
identical to YNB-lac/NH.sub.4 described in example 9 but contains 1% 
galactose in stead of 1% lactose) supplemented with 10 mM fluoracetamide 
for counter-selection. 
EXAMPLE 13 
Marker Gene Free Deletion of a K.lactis Gene Using the amdS-marker 
A frequently used technique for the manipulation of yeast genomes is 
"one-step gene disruption", a method which allows to disrupt (or modify) 
genes in a single transformation step (Rothstein et al. (1983) Methods 
Enzymol. 101, 202-211). In this method a transforming plasmid with a copy 
of a target gene disrupted by a yeast selectable marker integrates into 
the yeast genome via a double cross-over homologous recombination, 
resulting in the replacement of the wild-type target gene by the disrupted 
copy. Combination of "one-step gene disruption" and the fluoracetamide 
counter-selection of amdS.sup.+ -yeast-transformants as we have described 
in Example 12, should enable the deletion of genes from yeast genomes 
without leaving selectable markers. In this example we have used this 
combination to delete the LAC4 gene from the K.lactis CBS 2360 genome. For 
one-step gene transplacement of the K.lactis LAC4 gene pGBamdS6 (FIG. 38) 
was constructed, which contains the amdS expression-cassette flanked by 
LAC4 promoter and terminator sequences. An additional LAC4 terminator 
fragment is present directly upstream of the amdS expression-cassette such 
that the amdS expression-cassette is flanked by direct repeats which will 
allow the excision of the amdS sequences from the K.lactis genome by 
intrachromosomal recombination between these direct repeats. Plasmid 
pGBamdS6 was digested with SpeI and HindIII and a 6.6 kb DNA fragment was 
isolated after gel electrophoresis. This SpeI-HindIII fragment, containing 
the gene replacement vector, was used to transform K.lactis CBS 2360 using 
transformation procedures described in Example 10. amdS.sup.+ 
transformants were plated onto on YEPD plates containing 0.008% X-gal 
(5-bromo-4-chloro-3-indolyl .beta.-D-galactopyranoside) in order to screen 
for transformants with a transplaced LAC4 gene. 
The amdS+ transformants were analyzed on Southern blot. High molecular 
weight DNA was isolated, digested with HindIII, subsequently separated by 
electrophoresis on a 0.7% agarose gel and blotted onto nitrocellulose. As 
probe a .sup.32 -P-labelled 600 bp XbaI LAC4 terminator fragment isolated 
from plasmid pPTLAC4 (described in example 8) was used. The results of an 
amdS+ CBS 2360 transformant with a transplaced LAC4 gene as well as the 
reference strain CBS 2360 are shown in FIG. 44. In case of the amdS+ CBS 
2360 transformant, a 7.4 kb hybridizing fragment is present that 
implicates a correctly transplaced LAC4 gene. The reference strain CBS 
2360 shows a 2.0 kb hybridizing fragment that represents the intact LAC4 
locus. 
Subsequent fluoracetamide counter-selection of these amdS.sup.+ 
transformants as described in Example 12, yielded recombinants with an 
amdS.sup.- phenotype. Southern analysis was performed on the chromosomal 
DNA of the amdS.sup.- recombinants. High molecular weight DNA was 
isolated, digested with HindIII, subsequently separated on a 0.7% agarose 
gel and blotted onto nitrocellulose. The same .sup.32 P-labelled probed as 
described above was used. The results of the amdS- CBS 2360 recombinants 
are shown in FIG. 44. In case of the amdS- recombinants, a 5.4 kb 
hybridizing fragment is present, which confirmed the absence of the LAC4 
gene as well as the correct removal of the amdS marker from the yeast 
genome. The absence of the amdS marker from these K.lactis LAC4.sup.- 
strains offers the possibility to reuse the amdS marker for additional 
deletions and/or modifications of genes. 
EXAMPLE 14 
Marker gene free insertion of a gene into the K.lactis genome using the 
amdS marker 
For the marker gene free insertion of genes into the yeast genome we have 
used the chymosin cDNA as a model-gene. In this example we have inserted 
the chymosin CDNA at the K.lactis LAC4 locus while replacing the LAC4 gene 
and without leaving a selection marker. The principle of marker-free gene 
insertion is the same as that for marker-free deletion of genes as 
described in example 13 except that in this case the transplacement vector 
pGBamdS8 contains a gene of interest, the chymosin cDNA (FIG. 40). Plasmid 
pGbamdS8 was digested with SpeI and HindIII and the 8.0 kb DNA fragment 
was gel-isolated. 10 .mu.g of this fragment was transformed to K.lactis 
CBS 2360 as described in Example 10. amdS.sup.+ transformants with a 
transplaced LAC4 gene and chymosin activity were obtained. Chymosin 
activity was measured as described (van den Berg et al. (1990) 
Bio/technology 8, 135-139). By subsequent counter-selection of these 
transformants on fluoracetamide as described in example 12 recombinants 
were isolated with an amdS.sup.- phenotype but which still produced 
chymosin. Southern analysis of the chromosomal DNA of the amdS.sup.-, 
Chymosin.sup.+ recombinants confirmed the replacement of the LAC4 gene by 
the chymosin cDNA as well as the correct removal of the amdS marker from 
the K.lactis genome. The amdS.sup.- /chymosin.sup.+ phenotype of these 
recombinants was also confirmed by lack of growth on YCB/acetamide plates 
and by the presence of chymosin activity (see above). The amdS.sup.- 
phenotype of these recombinants allows further manipulation of these 
strains using the amdS marker, e.g. integration of additional copies of 
the chymosin expression-cassette and/or deletion of K.lactis genes as 
described in example 13. 
EXAMPLE 15 
Test of the amdS-minus phenotype of Bacilli and E.coli 
A prerequisite for the use of the amdS selection system in Bacilli is that 
these Gram-positive bacteria do not contain any acetamidase activity. In 
order to test this we have plated the B.subtilis strain BS-154 (CBS 
363.94) on a minimal Bacillus medium containing all the essential 
nutritives and vitamins except a nitrogen-source (28.7 mM K.sub.2 
HPO.sub.4, 22 mM KH.sub.2 PO.sub.4, 1.7 mM sodium citrate, 0.4 mM 
MgSO.sub.4, 0.75 .mu.M MnSO.sub.4, 0.5% (w/v) glucose and 1.5% agar. No 
growth was observed on this medium as such or when supplemented with 20 mM 
acetamide as nitrogen-source. Growth was only observed in the case that 
the minimal medium was supplemented with either 20 mM (NH.sub.4).sub.2 
SO.sub.4 or 20 mM KNO.sub.3 as nitrogen source. We conclude that Bacillus 
BS-154 (CBS 363.94) lacks sufficient acetamidase activity to sustain 
growth on acetamide as sole nitrogen source. This phenomenon should allow 
for the A.nidulans amdS gene to be used as selection marker in 
Gram-positive bacteria. 
Similarly we have tested the lack of acetamidase activity in a 
Gram-negative bacterium, in this case E.coli, in order to establish 
whether the A.nidulans amdS gene can also be used as selection marker in 
these micro-organisms. In this case we used M9 minimal medium (Sambrook et 
al. (1989) "Molecular Cloning: a laboratory manual", Cold Spring Harbour 
Laboratories, Cold Spring Harbour, N.Y.) supplemented with 0.02 .mu.g 
(w/v) thiamine. Full grown colonies of E.coli JM109 were observed on when 
plated on M9 plates. No growth or only slight background growth was 
observed, however, when the NH.sub.4 Cl was omitted from the M9 plates or 
replaced by 20 mM acetamide. We conclude that the E.coli JM109 strain 
lacks sufficient acetamidase activity to sustain growth on acetamide as 
sole nitrogen source. This should allow for the A.nidulans amdS gene to be 
also used as selection marker in Gram-negative bacteria. 
EXAMPLE 16 
Construction of amdS expression-vectors for use in bacteria 
Construction of pGBamdS22 
To express the A.nidulans amdS gene in different Bacilli species, we have 
cloned the amdS cDNA from pamdS-1 into the basic Bacillus expression 
vector pBHA-1 (European Patent Application 89201173.5; FIG. 45 for 
physical map). At the ATG initiation-codon of the amdS cDNA gene an NdeI 
site was introduced in pamdS-1 using oligonucleotides AB3825 (SEQ ID NO: 
36) and AB3826 (SEQ ID NO: 37) with the following sequences: 
Oligo AB3825 (SEQ ID NO: 36) 
5'-CGCGCTTATCAGCGGCCAGTTCTTCCCAGGATTGAGGCATATGT-3' Oligo AB3826 (SEQ ID NO: 
37): 
5'-CTAGACATATGCCTCAATCCTGGGAAGAACTGGCCGCTGATAAG-3' 
Annealing of these oligonucleotides was carried out using standard 
procedures. The resulting double stranded DNA fragment was ligated into 
BssHII/XbaI digested pamdS-1 and transferred to E.coli. From one of the 
transformants pGBamdS21 was isolated and characterized by 
restriction-enzyme analysis (FIG. 47). pGBamdS21 was digested with KpnI 
and HindIII and the amdS cDNA containing fragment was cloned into pBHA-1 
digested with KpnI and HindIII. The resulting plasmid was designated 
pGBamdS22 (FIG. 48). 
Construction of pGBamdS25 
To demonstrate site specific integration of a desired DNA sequence into the 
B.licheniformis genome using amdS as selection marker, the amdS cDNA was 
cloned in the expression/integration-vector pLNF (FIG. 46). This vector 
containing the 5' and 3' non-coding sequences of the B.licheniformis 
amylase gene enables site specific integration at the corresponding 
chromosomal amylase locus. pGBamdS21 (described above, FIG. 47) was 
digested with NdeI and PvuII and the amdS cDNA containing fragment was 
ligated with pLNF digested with NdeI and ScaI. The ligation mixture was 
transformed to B.subtilis BS-154 (CBS 363.94). Transformants were selected 
on minimal medium supplemented with 20 .mu.g/ml neomycin. From one of the 
transformants, designated BAA-101, the plasmid pGBamdS25 (FIG. 50) was 
isolated. 
Construction of pGBamdS41 
For the expression of the A.nidulans amdS CDNA in E.coli we have used 
pTZ18R/N, a derivative of pTZ18R which is described in the European Patent 
Application 0 340 878 A1 pTZ18R/N differs from pTZ18R in that an NdeI site 
was created at the ATG start-codon of the lacZ reading frame in pTZ18R 
using in vitro site directed mutagenesis. pGBamdS21 was digested with NdeI 
and HindIII and the gel-isolated fragment containing the amdS CDNA was 
ligated into pTZ18R/N digested with NdeI and HindIII. This ligation 
mixture was used to transform E.coli JM109 and from one of the 
transformants pGBamdS41 (FIG. 51) was isolated. 
EXAMPLE 17 
Transformation of Bacilli using the amdS gene as selection marker 
In order to delete the E.coli sequences from pGBamdS22 and to place the 
"hpa2"-promoter immediately upstream of the amdS cDNA, pGBamdS22 was 
digested with NdeI, recircularized by ligation and used to transform 
B.subtilis BS-154 (CBS 363.94). Transformants were selected on acetamide 
minimal plates and checked for neomycin resistance. From one of these 
transformants expression-vector pGBamdS23 (FIG. 49) was isolated and 
characterized by restriction enzyme analysis. These results show that 1) 
the A.nidulans amdS cDNA under the control of a Bacillus promoter sequence 
is expressed well and 2) that the amdS gene can be used as a selection 
marker in the transformation of Bacilli. 
B.licheniformis T5 (CBS 470.83) was transformed with vector pGBamdS25. 
Transformation was performed as described in Experimental and amdS.sup.+ 
transformants were obtained by direct selection on modified protoplast 
regeneration plates supplemented with 20 mM acetamide as sole nitrogen 
source (described in Experimental). The presence of pGBamdS25 in the 
transformants was confirmed by their neomycin resistance phenotype as well 
as the fact that the plasmid could be reisolated from the transformants. 
One of these transformants designated BAA-103 was used to achieve 
integration of plasmid pGBamdS25 into the B.licheniformis genome targeted 
at the amylase locus. Plasmid integration was performed by growing 
transformants at 50.degree. C. on minimal medium agar containing acetamide 
as sole nitrogen source. Several colonies were transferred repeatedly (2 
to 3 times) to fresh plates followed by incubation at 50.degree. C. 
Isolated colonies were tested for their ability to grow on acetamide as 
sole nitrogen source and for resistance to neomycin at 1 .mu.g/ml. The 
absence of autonomously replicating plasmid DNA was established by 
re-transformation of DNA isolated from the integrants to the host strain. 
No neomycin resistant colonies could be obtained. 
This result is a clear evidence that the amdS gene is a suitable marker to 
select Bacillus species containing a single amdS gene copy. 
EXAMPLE 18 
Transformation of E.coli using the amdS gene as selection marker 
E.coli JM109 was transformed with the vector pGBamdS41 using standard 
procedures. Selections were performed on either M9 plates supplemented 
with 0.02 .mu.g/ml thiamine and 50.mu.g/ml ampicillin or M9 plates without 
ammonium but supplemented with 20 mM acetamide, 0.02 .mu.g/ml thiamine and 
0.05 mM IPTG. Several amdS.sup.+ /ampicillin resistant transformants were 
obtained from which pGBamdS41 could be reisolated. The transformation 
frequencies using selection on ampicillin or acetamide were comparable. 
This demonstrates that the A.nidulans amdS gene is functional as selection 
marker for the transformation of Gram-negative bacteria as well. 
EXAMPLE 19 
Fluoracetamide counter-selection of amdS.sup.+ bacterial transformants 
Counter-selection of bacterial amdS.sup.+ transformants using 
fluoracetamide requires the activity of the enzyme acetylCoA synthetase 
for the conversion of fluoracetate to fluoracetyl-CoA. To avoid catabolite 
repression of acetylCoA synthase, as has been observed in E.coli (Brown et 
al., 1977), bacterial amdS.sup.+ transformants or single copy integrants 
were grown on defined media containing NH.sub.4 Cl as nitrogen source and 
acetate as carbon and energy source. 
Many organisms including B. subtilis (Freese, E. and Fortnagel, P. (1969) 
J. Bacteriol 90, 745-756) lack a functional glyoxylate shunt and therefore 
metabolize acetate only when the medium is supplemented with a source of 
TCA cycle intermediates, such as glutamate or succinate. Bacillus 
amdS.sup.+ strains were grown on TSS medium with 0.01% glutamate and 50 mM 
acetate as described by Grundy, F. J. et.al. (1993) Molecular Microbiology 
10, 259-271. To this medium solidified with agar, fluoracetamide was added 
in concentrations ranging from 1 to 50 mM. B.subtilis BAA-101 or 
B.licheniformis BAA-103 (single copy integrant) were plated at a density 
of 10.sup.2 cells per plate. At a certain fluoracetamide concentration 
only a few colonies appeared. The absence of pGBamdS25 in these colonies 
was demonstrated by plasmid and chromosomal DNA analysis, sensitivity 
towards neomycin, and inability to grow on acetamide as sole 
nitrogen-source. Counter-selection of BAA-103 in some cases led to the 
loss of the amylase gene as indicated by activity assays and Southern 
blots. This shows that fluoracetamide counter-selection can be used to 
select amdS.sup.- cells from a population containing a majority of 
amdS.sup.+ Bacillus cells and the simultaneous deletion of a specific 
target gene. 
Similarly we have used minimal medium #132 as described by Vanderwinkel E. 
and De Vlieghere M, European J. Biochem, 5 (1968) 81-90 supplemented with 
fluoracetamide in concentrations ranging from 1 to 50 mM and 0.05 mM IPTG 
to select amdS.sup.- E.coli JM109 cells from a population of pGBamdS41 
transformants. Cells were plated at a density of 10.sup.2 cells per plate. 
At a certain fluoracetamide concentration only a few colonies appeared. 
The absence of pGBamdS41 from the fluoracetamide selected colonies was 
confirmed by isolation of DNA, sensitivity toward ampicillin and inability 
to grow on acetamide as sole nitrogen-source. This demonstrates that the 
fluoracetamide counter-selection can be used to select amdS.sup.- cells 
from a population containing a majority of amdS.sup.+ E.coli cells. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 37 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: AB3100 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
CTAATCTAGAATGCCTCAATCCTGAA26 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 28 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: YES 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: AB3101 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
GACAGTCGACAGCTATGGAGTCACCACA28 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: TN0001 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
TCGATTAACTAGTTAA16 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 35 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: AB2154 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
AACCATAGGGTCGACTAGACAATCAATCCATTTCG35 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 35 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: YES 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: AB2155 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
GCTATTCGAAAGCTTATTCATCCGGAGATCCTGAT35 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: AB2977 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
TATCAGGAATTCGAGCTCTGTACAGTGACC30 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 36 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: YES 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: AB2992 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
GCTTGAGCAGACATCACCATGCCTCAATCCTGGGAA36 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 36 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: AB2993 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
TTCCCAGGATTGAGGCATGGTGATGTCTGCTCAAGC36 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 36 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: YES 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: AB2994 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
CTGATAGAATTCAGATCTGCAGCGGAGGCCTCTGTG36 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 31 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: AB3657 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
AGCTTGACGTCTACGTATTAATGCGGCCGCT31 
(2) INFORMATION FOR SEQ ID NO:11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 31 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: YES 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: AB3658 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
TCGAAGCGGCCGCATTAATACGTAGACGTCA31 
(2) INFORMATION FOR SEQ ID NO:12: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: AB3779 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
AATTGGGGCCCATTAACTCGAGC23 
(2) INFORMATION FOR SEQ ID NO:13: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 22 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: YES 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: AB3780 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
AATTGCTCGAGTTAATGGGCCC22 
(2) INFORMATION FOR SEQ ID NO:14: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: AB3448 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
GTGCGAGGTACCACAATCAATCCATTTCGC30 
(2) INFORMATION FOR SEQ ID NO:15: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 36 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: YES 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: AB3449 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
ATGGTTCAAGAACTCGGTAGCCTTTTCCTTGATTCT36 
(2) INFORMATION FOR SEQ ID NO:16: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 36 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: AB3450 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: 
AGAATCAAGGAAAAGGCTACCGAGTTCTTGAACCAT36 
(2) INFORMATION FOR SEQ ID NO:17: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 42 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: YES 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: AB3520 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: 
ATCAATCAGAAGCTTTCTCTCGAGACGGGCATCGGAGTCCCG42 
(2) INFORMATION FOR SEQ ID NO:18: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: AB3781 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: 
AATTGGGGCCCAGCGTCC18 
(2) INFORMATION FOR SEQ ID NO:19: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: YES 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: AB3782 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: 
AATTGGACGCTGGGCCCC18 
(2) INFORMATION FOR SEQ ID NO:20: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 43 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: AB3746 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: 
TGACCAATAAAGCTTCTCGAGTAGCAAGAAGACCCAGTCAATC43 
(2) INFORMATION FOR SEQ ID NO:21: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 47 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: YES 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: AB3747 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: 
CTACAAACGGCCACGCTGGAGATCCGCCGGCGTTCGAAATAACCAGT47 
(2) INFORMATION FOR SEQ ID NO:22: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: AB4234 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: 
GAAGACCCAGTCAAGCTTGCATGAGC26 
(2) INFORMATION FOR SEQ ID NO:23: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 43 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: YES 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: AB4235 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: 
TGACCAATTAAGCTTGCGGCCGCTCGAGGTCGCACCGGCAAAC43 
(2) INFORMATION FOR SEQ ID NO:24: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 69 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: AB4236 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: 
TGACCAATAAAGCTTAGATCTGGGGGTGATTGGGCGGAGTGTTTTGCTTAGACAATCAAT60 
CCATTTCGC69 
(2) INFORMATION FOR SEQ ID NO:25: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 36 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: YES 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: AB4233 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: 
TGACCAATAGATCTAAGCTTGACTGGGTCTTCTTGC36 
(2) INFORMATION FOR SEQ ID NO:26: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 32 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: AB3514 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: 
CTGCGAATTCGTCGACATGCCTCAATCCTGGG32 
(2) INFORMATION FOR SEQ ID NO:27: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 37 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: YES 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: AB3515 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: 
GGCAGTCTAGAGTCGACCTATGGAGTCACCACATTTC37 
(2) INFORMATION FOR SEQ ID NO:28: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 50 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: AB3701 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: 
CTGCGAATTCGTCGACACTAGTGGTACCATTATAGCCATAGGACAGCAAG50 
(2) INFORMATION FOR SEQ ID NO:29: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 70 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: YES 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: AB3700 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: 
GCTCTAGAGCGCGCTTATCAGCTTCCAGTTCTTCCCAGGATTGAGGCATTTTTAATGTTA60 
CTTCTCTTGC70 
(2) INFORMATION FOR SEQ ID NO:30: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 50 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: AB3702 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: 
CTGCGAATTCGTCGACACTAGTGGTACCATCCTTTTGTTGTTTCCGGGTG50 
(2) INFORMATION FOR SEQ ID NO:31: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 70 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: YES 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: AB3704 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: 
GCTCTAGAGCGCGCTTATCAGCGGCCAGTTCTTCCCAGGATTGAGGCATTGTATATGAGA60 
TAGTTGATTG70 
(2) INFORMATION FOR SEQ ID NO:32: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 55 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: AB3704 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: 
GCTCTAGAAGTCGACACTAGTCTGCTACGTACTCCAGAATTTATACTTAGATAAG55 
(2) INFORMATION FOR SEQ ID NO:33: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: YES 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: AB3705 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: 
TGCTCTAGATCTCAAGCCACAATTC25 
(2) INFORMATION FOR SEQ ID NO:34: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 31 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: AB3965 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: 
CTGCTACGTAATGTTTTCATTGCTGTTTTAC31 
(2) INFORMATION FOR SEQ ID NO:35: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 37 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: YES 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: AB3966 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35: 
CCGCCCAGTCTCGAGTCAGATGGCTTTGGCCAGCCCC37 
(2) INFORMATION FOR SEQ ID NO:36: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 44 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: AB3825 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: 
CGCGCTTATCAGCGGCCAGTTCTTCCCAGGATTGAGGCATATGT44 
(2) INFORMATION FOR SEQ ID NO:37: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 44 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: YES 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: AB3826 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: 
CTAGACATATGCCTCAATCCTGGGAAGAACTGGCCGCTGATAAG44 
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