Post-transcriptional heme regulated heterologous gene expression in yeast using the leg hemoglobin leader sequence

A method for controlling the level of heterologous gene expression in yeast using a soybean leghemoglobin leader sequence and a suitable promotor. Levels of expression are controlled at the post-transcriptional level by adjusting the amount of heme in the yeast culture medium.

The invention relates to a novel method for the expression of genes in 
yeast, and DNA fragments as well as plasmids comprising said DNA fragments 
to be used when carrying out the method. 
In the description i.a. the following terms are used: 
Promoter region: A DNA fragment containing a promoter and target sequences 
for RNA polymerase as well as possible activation regions comprising 
target sequences for transcriptional effector substances. 
Effector substance: Substances exerting or mediating a regulator function. 
Thus the effector substances also include substances influencing the 
concentration of substances exerting or mediating a regulatory function. 
Leader sequence: Generally is meant a DNA sequence being transcribed into a 
mRNA, but not further translated into protein. The leader sequence 
comprises thus the DNA fragment from the transcription start to the ATG 
translation start condon. 
Leader sequence: In relation to the present invention is meant a short DNA 
fragment typically having 40-70 bp and comprising target sequences for a 
post transcriptional regulation exerted or mediated by intracellular heme. 
Furthermore the following terms generally known to persons skilled in the 
art of molecular biology are used. 
CAP addition site: The site where 7-methyl-GTP is added. 
DNA sequence or DNA segment: A linear array of nucleotides interconnected 
through phosphodiester bonds between the 3' and 5' carbon atoms of 
adjacent pentoses. 
Expression: The process undergone by a structural gene to produce a 
polypeptide. It is a combination of transcription and translation as well 
as possible posttranslational modifications. 
Flanking regions: DNA sequences surrounding coding regions. 5' flanking 
regions contain a promoter. 3' flanking regions may contain 
transcriptional terminator signals etc. 
Gene: A DNA sequence composed of three of four parts (1) the coding 
sequence for the gene product, (2) the sequences in the promoter region 
which control whether or not the gene will be expressed, (3) those 
sequences in the 3' end conditioning the transcriptional termination and 
optional polyadenylation, as well as (4) intervening sequences, if any. 
Intervening sequences: DNA sequences within a gene which are not coding for 
any peptide fragment. The intervening sequences are transcribed into 
pre-mRNA and are eliminated by modification of precursorRNA into mRNA. 
Cloning: The process of obtaining a population of organisms or DNA 
sequences deriving from one such organism or sequence by asexual 
reproduction, or more particular: 
the process of isolating a particular organism or part thereof, and the 
propagation of this subfraction as a homogeneous population. 
Coding sequence: DNA sequence determining the amino acid sequence of a 
polypeptide. 
Messenger-RNA (mRNA): RNA molecule produced by transcription of a gene and 
possible modification of precursorRNA. The mRNA molecule mediates the 
genetic message determining the amino acid sequence of a polypeptide by 
part of the mRNA molecule being translated into said peptide. Nucleotide: 
A monomeric unit of DNA or RNA consisting of a sugar moiety (pentose), a 
phosphate, and a nitrogeneous heterocyclic base. The base is linked to the 
sugar moiety via a glucosidic bond (1' carbon of the pentose), and this 
combination of base and sugar is a nucleoside. The base characterizes the 
nucleotide. The four DNA bases are adenine (A), guanine (G), cytosine (C), 
and thymine (T). The four RNA bases are A, G, C, and uracil (U). 
Plasmid: A nonchromosomal double-stranded DNA sequence comprising an intact 
replicon such that the plasmid is replicated in a host cell. When the 
plasmid is placed within a unicellular organism, the characteristics of 
that organism are changed or transformed as a result of the DNA of the 
plasmid. For instance a plasmid carrying the gene for tetracycline 
resistance (Tc.sup.R) transforms a cell previously sensitive to 
tetracycline into one which is resistant to it. A cell transformed by a 
plasmid is called a transformant. 
Polypeptide: A linear array of amino acids interconnected by means of 
peptide bonds between the .alpha.-amino and carboxy groups of adjacent 
amino acids. 
Recombination: The creation of a new DNA molecule by combining DNA 
fragments of different origin. 
Replication: A process reproducing DNA molecules. 
Repilcon: A self-replicating genetic element possessing an origin for the 
initiation of DNA replication and genes specifying the functions necessary 
for controlling the replication. 
Restriction fragment: A DNA fragment resulting from double-stranded 
cleavage by an enzyme recognizing a specific target DNA sequence. 
RNA polymerase: Enzyme exerting the transcription of DNA into RNA. 
Transformation: The process whereby a cell is incorporating a plasmid. 
Translation: The process of producing a polypeptide from mRNA or: 
the process whereby the genetic information present in an mRNA molecule 
directs the order of specific amino acids during the synthesis of a 
polypeptide. 
Transcription: The method of synthesizing a complementary RNA sequence from 
a DNA sequence. 
Vector: A plasmid, phage DNA or other DNA sequences capable of replication 
in a host cell and having one or a small number of endonuclease 
recognition sites at which DNA sequences may be cleaved in a determinable 
manner without attendant loss of an essential biological function of the 
DNA, e.g. replication, production of coat proteins or loss of promoter or 
binding sites, and which contain a marker suitable for use in the 
identification of transformed cells in the form of for instance 
tetracycline resistance or ampicillin resistance. A vector is often called 
a cloning vehicle. 
The production of a biologically active product by means of recombinant DNA 
technology is a complex matter which involves many process steps from the 
initiation of the transcription to the final achievement of the 
biologically active molecule. 
Many of these process steps do not appear in procaryotic organisms the 
reason why eucaryotic production organisms must be used in many cases. 
Yeast is an eukaryotic organism, the synthesis apparatus of which comprises 
many of the processes and regulating mechanisms characteristic of higher 
organisms. In addition yeast cells have a short generation time and a 
thousand-year old experience basis exists for the use of yeast as a 
culture organism. 
Completely decisive factors for a biological synthesis of a desired gene 
product are a possibility and improvement of transcriptional initiation as 
well as transcriptional and posttranscriptional regulation of the gene 
expression. 
These functions are mainly carried out by 5' flanking regions. A wide range 
of 5' flanking regions of prokaryotic and eukaryotic genes has been 
sequenced, and inter alia based thereon a comprehensive knowledge has been 
provided of the regulation of gene expression and of the subregions and 
sequences being of importance for the regulation of expression of the 
gene. Great differences exist in the regulatory mechanism in procaryotic 
and eucaryotic organisms, but within these two groups there are many 
common features. 
The regulation so the gene expression may take place on the transcriptional 
level and then it is preferably exerted by regulation of the initiation 
frequency of transcription. The latter is well known and described inter 
alia by Benjamin Lewin, Gene Expression, John Weily & Sons, vol. I. 1974, 
vol. II, Second Edition 1980, vol. III, 1977. Alternatively the regulation 
may be exerted at the posttranscriptional level e.g. the regulation of the 
frequency of the translation initiation at the rate of translation and of 
the termination of translation. 
Leghemoglobins are monomeric hemoproteins exclusively synthesized in the 
root nodules which develop through the symbiotic association of Rhizobia 
with leguminous plants. A logical candidate for an effector substance 
activating the leghemoglobin genes is heme produced in Rhizobia and 
constituting the prostetic group of the leghemoglobins. The synthesis of 
several hemoproteins in the yeast Saccharomyces cerevisiae is also 
regulated by the level of intracellular heme which also forms the 
prostetic group of these proteins. Thus the transcription of the 
isocytochrome c gene is heme dependant while in the case of catalase 
T.sub.1 the heme control is exerted both at the transcriptional and the 
posttranscriptional level.

In accordance with the present invention is presented the sequence of 5' 
flanking regions of the four soybean leghemoglobin genes Lba, Lbc1, Lbc2, 
and Lbc3. The sequences are presented in the enclosed sequence scheme, 
FIG. 1, where the sequences are aligned in such a manner that the homology 
appears clearly. 
In the sequence scheme "-" indicates that no base is present in the 
position in question. The names of the genes and the base position counted 
upstream from the ATG start condon are indicated to the right of the 
sequence scheme. Furthermore the important sequences have been underlined. 
As it appears from the sequence scheme a distinct degree of homology exists 
between the four 5' flanking regions, and in the position 23-24 bp 
upstream from the CAP addition site they all contain a TATATAAA sequence 
corresponding to the "TATA" box which is eucaryotic cells usually are 
located a corresponding number of bp upstream from the CAP addition site. 
Furthermore a CCAAG sequence is present 64-72 bp upstream from the CAP 
addition site, said sequence corresponding to the "CCAAT" box usually 
located 70-90 bp upstream from the CAP addition site. From the CAP 
addition site to the translation start codon, ATG, leader sequences of 
52-59 bp are present and show a distinct degree of homology of approx. 
75-80%. 
In accordance with the present invention it has furthermore been proved, 
exemplified by Lbc3, that the 5' flanking regions of the soybean 
leghemoglobin genes are functionally active in yeast. The latter has been 
proved by fusing the E. coli chloroamphenicol acetyl transferase (CAT) 
gene with the 5' and 3' flanking regions of the soybean Lbc3 gene in such 
a manner that the expression of the CAT gene is controlled by the Lb 
promoter. The fusion fragment was inserted in the yeast plasmid vector, 
YEP 24, comprising the yeast URA-3 gene as a selectable marker. The yeast 
strain S. cerevisiae TM1 which is URA-3 and unable to synthesize heme due 
to a mutation in the .delta.-amino levulinic acid synthetase gene, 
.delta.-ALA, was subsequently transformed with the resulting construction. 
The transformed yeast cells showed a CAT activity under all growth 
conditions tested. The conclusion can therefore be made that the 5' 
flanking regions of soybean leg hemglobin genes are functional in yeast. 
In accordance with the present invention it has furthermore been proved 
that the 5' flanking regions act as target for a regulation exerted or 
mediated by intracellular heme. The CAT activity is thus 20-40 fold higher 
in the yeast S. cerevisiae TM1 grown in the presence of .delta.-ALA than 
the CAT activity in yeast grown without this heme precursor in the growth 
medium. Similar high CAT activities were present in the yeast S. 
cerevisiae TM1 grown in the presence of heme, protoporphyrin IX, or the 
heme analog deuteroporphyrin IX. The effect of heme on the CAT activity is 
specific since the amount of the URA-3 gene product remains constant under 
all the conditions tested. Furthermore the transcriptional level does not 
apparently change because of the presence of heme as the CAT-mRNA level 
remains constant independent of changes in the intracellular heme 
concentration. It can therefore be concluded that the regulatory mechanism 
exerted or mediated by heme occurs on the posttranscriptional level. 
The observed increase of CAT activity is dependent on protein synthesis. 
The half life of the CAT enzyme is furthermore independent of the presence 
of heme, and in vitro CAT activity is not stimulated by heme. Therefore 
heme most likely regulates the gene expression on the translational level. 
A fusion of the 5' flanking region of Lbc3 with coding region of the 
neomycine phospho transferase (neo) gene, is controlled by heme is a 
completely similar manner as the CAT gene fused with a 5' flanking region 
of the Lbc3 gene. The effect of heme is thus not mediated by heme 
interacting with the coding sequence, but rather by heme interacting with 
the 5' or 3' flanking Lbc3 sequences present in the CATmRNA. The 
expression of a gene only containing the Lbc3 5' flanking region and the 
neo gene is controlled in a similar manner by heme. The effect of 
intracellular heme on the gene expression can thus be mediated by an 
interaction with the leader sequence. 
The short leader sequences do not contain translation start codons. The 
regulatory mechanism exerted or mediated by intracellular heme is 
therefore not related to regulatory mechanisms involving false start 
codons, cf. the disclosure of Hunt, T. Nature, Vol. 316, 580-581, (1985). 
The regulatory mechanism described in relation to the present invention is 
exerted or mediated by heme interacting with a leader sequence and is 
therefore a novel regulatory mechanism. 
The presence of plasmids in a cell present in a natural environment 
provides the cell with a property which is only advantageous for the cell 
under certain circumstances. Plasmid encoded properties may for instance 
be resistance to an antibiotic present in the surrounding environment. The 
presence of a plasmid and synthesis of the plasmid encoded gene products 
do, however, also load the energy metabolism of the cell and the protein 
synthesis apparatus, and a cell containing a plasmid is therefore ousted 
and lost in an environment not needing the plasmid encoded properties. 
The above mentioned instability is additionally increased by using plasmids 
as vectors for synthesis of a desired gene product not usually produced by 
the cell in question. The latter implies tha cells synthesizing such 
products must be subjected to a selection pressure in order to ensure that 
the desired gene product can still by synthesized. The previous method of 
achieving a high expresion of a certain gene product is that the 
expression is controlled by a strong promoter causing a high concentration 
of the mRNA being translated into the gene product in question. 
A high concentration of gene product can, however, also be obtained by a 
more efficient translation of the mRNA in question. The latter implies 
that the gene product synthesis is controlled both at the transcriptional 
and on the translation level, which means that the genetic load on a cell 
synthesizing a certain gene product can be distributed on two activities 
instead of one as usually. 
The two activities can therefore be manipulated in such a manner that the 
same result concerning concentration of gene product can be obtained 
though the promoter is not as strong as the promoters usually employed. 
Such a distribution of the two activities implies that the cell is not as 
genetically loaded as when the gene product synthesis is only controlled 
by one strong promoter. As a result the selection pressure on the cell in 
question can be reduced. 
It is furthermore of importance to obtain the utilization most rational for 
the cell of the energy metabolism and of the protein synthesis apparatus 
in the phase where the synthesis of the desired gene product occurs. Such 
a rational utilization of the energy metabolism and of the protein 
synthesis apparatus is improved preferably by optimizing the late steps of 
the synthesis of a gene product rather than optimating the early steps. 
An important feature of a gene expression system is therefore that the 
expression of the desired product can be increased from an initially low 
expression to an overproduction by a manipulation of the external 
environment of the cell as disclosed by the present invention. Furthermore 
an inducible optimizing of the posttranscriptional synthesis steps--which 
has been disclosed by the present invention--is more advantageous than an 
induced optimization of the transcription. 
Previous methods for the expression of genes in yeast employ a range of 
promoters and expression vectors, cf. for instance EP 120 551 A2, in which 
the use of GAPDH- or PyK-yeast promoters is disclosed, as well as of 
expression vectors comprising these promoters. 
GB No. 2,137,208 A discloses furthermore the use of the promoter GAL1 in 
several expression vectors. 
When using these previously known promoters, the expression can only be 
increased by increasing the transcription-initiation frequency. The use of 
these promoters involves consequently a high genetic load on the cell, an 
irrational utilization of the energy metabolism and the synthesis 
apparatus, as well as a resulting instability necessitating a high 
selection pressure on the host organism when using these promoters. 
The object of the present invention is therefore to disclose a method of 
using novel promoters active in yeast, as well as leader sequences 
subjecting in a novel manner the expression of the following gene to a 
regulation at the posttranscriptional level. Further objects of the 
invention are to provide combinations of the promoter and leader sequence, 
whereby these combinations have been obtained from 5' flanking regions of 
plant leghemoglobin genes and proved to be functional in yeast, as well as 
it is an object of the invention to provide plasmids comprising the above 
combination of promoter and leader sequences. 
The method according to the present invention is characterized by using a 
first DNA fragment comprising a leader sequence, in combination with a 
second DNA fragment comprising a promoter sequence, said combination 
increasing at high intracellular concentrations of heme the expression of 
a desired gene by increasing the translation efficiency. In this manner it 
is possible by inserting a gene downstream from the combination and in a 
suitable vector being able to replicate in yeast to obtain a synthesis of 
a biologically active product. This method allows an increase of the 
expression of a desired gene by a novel regulatory mechanism acting at the 
posttranscriptional level. As a particular result a reduced genetic strain 
of the host cell and an optimal utilization of the protein synthesis 
apparatus and the energy metabolism of the host cell is obtained and 
consequently an increased stability of the expression vector in the host 
cell. 
A particular embodiment of the method according to the invention uses as a 
first DNA fragment an isolated or synthesized leader sequence to be 
combined with a second isolated or synthesized DNA fragment. In this 
manner it is possible to combine any leader sequence from yeast, plants or 
animals under natural conditions being subjected to a posttranscriptional 
regulation with any suited yeast promoter, plant promoter or another 
promoter being functional in yeast. 
According to a particular embodiment of the method according to the 
invention the intracellular concentration of heme is increased by adding 
to the growth medium such carbon sources, especially non-fermentable 
carbon sources, which cause increased intracellular concentrations of 
heme. In this manner an induction of the expression of the desired gene is 
obtained by adding a carbon source to the growth medium. Examples of such 
carbon sources are glycerol and succinate which are particularly preferred 
because they are inexpensive and easily available carbon sources. 
Furthermore ethanol can be used. Under certain circumstances the yeast 
itself can produce this ethanol while growing. After termination of the 
growth the ethanol is utilized whereby the translation is increased. 
According to a special embodiment the same effect can be obtained by the 
intracellular concentration of heme being increased by adding to the 
growth medium one or several substances selected from the group consisting 
of heme, heme analogs, and heme precursors. An example of a heme analog is 
deuteroporphyrin IX, and an example of a heme precursor is .alpha.-amino 
levulinic acid. 
A special embodiment of the method according to the invention uses a DNA 
fragment comprising a promoter sequence and a leader sequence, said DNA 
fragment being identical with, derived from or comprising 5' flanking 
regions of plant leghemoglobin genes, yeast genes or other genes subjected 
to an expression regulation under natural circumstances, said expression 
regulation being exerted or mediated by intracellular heme. In this manner 
a simple access to a combination of leader sequence and promoter sequence 
is obtained, said combination being proved according to the present 
invention to be functional in yeast. Examples of such DNA fragments are 
the four 5' flanking regions of the soybean leghemoglobin genes, viz. 
__________________________________________________________________________ 
Lba with the sequence: 
GAGATACATT 
ATAATAATCT 
CTCTAGTGTC 
TATTTATTAT 
TTTATCTGGT 
GATATATACC 
TTCTCGTATA 
CTGTTATTTT 
TTCAATCTTG 
TAGATTTACT 
TCTTTTATTT 
TTATAAAAAA 
GACTTTATTT 
TTTTAAAAAA 
AATAAAGTGA 
ATTTTGAAAA 
CATGCTCTTT 
GACAATTTTC 
TGTTTCCTTT 
TTCATCATTG 
GGTTAAATCT 
CATAGTGCCT 
CTATTCAATA 
ATTTGGGCTC 
AATTTAATTA 
GTAGAGTCTA 
CATAAAATTT 
ACCTTAATAG 
TAGAGAATAG 
AGAGTCTTGG 
AAAGTTGGTT 
TTTCTCGAGG 
AAGAAAGGAA 
ATGTTAAAAA 
CTGTGATATT 
TTTTTTTTGG 
ATTAATAGTT 
ATGTTTATAT 
GAAAACTGAA 
AATAAATAAA 
CTAACCATAT 
TAAATTTAGA 
ACAACACTTC 
AATTATTTTT 
TTAATTTGAT 
TAATTAAAAA 
ATTATTTGAT 
TAAATTTTTT 
AAAAGATCGT 
TGTTTCTTCT 
TCATCATGCT 
GATTGACACC 
CTCCACAAGC 
CAAGAGAAAC 
ACATAAGCTT 
TGGTTTTCTC 
ACTCTCCAAG 
CCCTCTATAT 
AAACAAATAT 
TGGAGTGAAG 
TTGTTGCATA 
ACTTGCATCG 
AACAATTAAT 
AGAAATAACA 
GAAAATTAAA 
AAAGAAATAT 
G, 
Lbc1 with the sequence: 
TTCTCTTAAT 
ACAATGGAGT 
TTTTGTTGAA 
CATACATACA 
TTTAAAAAAA 
AATCTCTAGT 
GTCTATTTAC 
CCGGTGAGAA 
GCCTTCTCGT 
GTTTTACACA 
CTTTAATATT 
ATTATATCCT 
CAACCCCACA 
AAAAAGAATA 
CTGTTATATC 
TTTCCAAACC 
TGTAGATTTA 
TTTATTTATT 
TATTTATTTT 
TACAAAGGAG 
ACTTCAGAAA 
AGTAATTACA 
TAAAGATAGT 
GAACATCATT 
TTATTTATTA 
TAATAAACTT 
TAAAATCAAA 
CTTTTTTATA 
TTTTTTGTTA 
CCCTTTTCAT 
TATTGGGTGA 
AATCTCATAG 
TGAAGCCATT 
AAATAATTTG 
GGCTCAAGTT 
TTATTAGTAA 
AGTCTGCATG 
AAATTTAACT 
TAACAATAGA 
GAGAGTTTTC 
GAAAGGGAGC 
GAATGTTAAA 
AAGTGTGATA 
TTATATTTTA 
TTTCGATTAA 
TAATTATGTT 
TACATGAAAA 
CATACAAAAA 
AATACTTTTA 
AATTCAGAAT 
AATACTTAAA 
ATATTTATTT 
GCTTAATTGA 
TTAACTGAAA 
ATTATTTGAT 
TAGGATTTTG 
AAAAGATCAT 
TGGCTCTTCG 
TCATGCCGAT 
TGACACCCTC 
CACAAGCCAA 
GAGAAACTTA 
AGTTGTAAAC 
TTTCTCACTC 
CAAGCCTTCT 
ATATAAACAT 
GTATTGGATG 
TGAAGTTATT 
GCATAACTTG 
CATTGAACAA 
TAGAAAATAA 
CAAAAAAAAG 
TAAAAAAGTA 
GAAAAGAAAT 
ATG, 
Lbc2 with the sequence: 
TCGAGTTTTT 
ACTGAACATA 
CATTTATTAA 
AAAAAACTCT 
CTAGTGTCCA 
TTTATTCGGC 
GAGAAGCCTT 
CTCGTGCTTT 
ACACACTTTA 
ATATTATTAT 
ATCCCCACCC 
CCACCAAAAA 
AAAAAAAACT 
GTTATATCTT 
TCCAGTACAT 
TTATTTCTTA 
TTTTTACAAA 
GGAAACTTCA 
CGAAAGTAAT 
TACAAAAAAG 
ATAGTGAACA 
TCATTTTTTT 
AGTTAAGATG 
AATTTTAAAA 
TCACACTTTT 
TTATATTTTT 
TTGTTACCCT 
TTTCATTATT 
GGGTGAAATC 
TCATAGTGAA 
ACTATTAAAT 
AGTTTGGGCT 
CAAGTTTTAT 
TAGTAAAGTC 
TGCATGAAAT 
TTAACTTAAT 
AATAGAGAGA 
GTTTTGGAAA 
GGTAACGAAT 
GTTAGAAAGT 
GTGATATTAT 
TATAGTTTTA 
TTTAGATTAA 
TAATTATGTT 
TACATGAAAA 
TTGACAATTT 
ATTTTTAAAA 
TTCAGAGTAA 
TACTTAAATT 
ACTTATTTAC 
TTTAAGATTT 
TGAAAAGATC 
ATTTGGCTCT 
TCATCATGCC 
GATTGACACC 
CTCCACAAGC 
CAAGAGAAAC 
TTAAGTTGTA 
ATTTTTCTAA 
CTCCAAGCCT 
TCTATATAAA 
CACGTATTGG 
ATGTGAAGTT 
GTTGCATAAC 
TTGCATTGAA 
CAATAGAAAT 
AACAACAAAG 
AAAATAAGTG 
AAAAAAGAAA 
TATG, 
and Lbc3 with the sequence: 
TATGAAGATT 
AAAAAATACA 
CTCATATATA 
TGCCATAAGA 
ACCAACAAAA 
GTACTATTTA 
AGAAAAGAAA 
AAAAAAACCT 
GCTACATAAT 
TTCCAATCTT 
GTAGATTTAT 
TTCTTTTATT 
TTTATAAAGG 
AGAGTTAAAA 
AAATTACAAA 
ATAAAAATAG 
TGAACATCGT 
CTAAGCATTT 
TTATATAAGA 
TGAATTTTAA 
AAATATAATT 
TTTTTGTCTA 
AATCGTATGT 
ATCTTGTCTT 
AGAGCCATTT 
TTGTTTAAAT 
TGGATAAGAT 
CACACTATAA 
AGTTCTTCCT 
CCGAGTTTGA 
TATAAAAAAA 
ATTGTTTCCC 
TTTTGATTAT 
TGGATAAAAT 
CTCGTAGTGA 
CATTATATTA 
AAAAAATTAG 
GGCTCAATTT 
TTATTAGTAT 
AGTTTGCATA 
AATTTTAACT 
TAAAAATAGA 
GAAAATCTGG 
AAAAGGGACT 
GTTAAAAAGT 
GTGATATTAG 
AAATTTGTCG 
GATATATTAA 
TATTTTATTT 
TATATGGAAA 
CTAAAAAAAT 
ATATATTAAA 
ATTTTAAATT 
CAGAATAATA 
CTTAAATTAT 
TTATTTACTG 
AAAATGAGTT 
GATTTAAGTT 
TTTGAAAAGA 
TGATTGTCTC 
TTCACCATAC 
CAATTGATCA 
CCCTCCTCCA 
ACAAGCCAAG 
AGAGACATAA 
GTTTTATTAG 
TTATTCTGAT 
CACTCTTCAA 
GCCTTCTATA 
TAAATAAGTA 
TTGGATGTGA 
AGTTGTTGCA 
TAACTTGCAT 
TGAACAATTA 
ATAGAAATAA 
CAGAAAAGTA 
GAAAAGAAAT 
ATG. 
__________________________________________________________________________ 
The present invention deals furthermore with a novel DNA fragment to be 
used as a first DNA fragment when carrying out the method according to the 
invention, said fragment being charactreized in that it is a short DNA 
fragment transcribed into a messenger RNA strand which is a target for a 
regulation exerted or mediated by intracellular heme. Examples of such DNA 
fragments are DNA fragments identical with, derived from or comprising a 
leader sequence from plant leghemoglobin genes, yeast genes or other genes 
in which said leader sequence under natural circumstances is a target for 
a regulation exerted or mediated by intracellular heme. Examples thereof 
are according to the invention DNA fragments which are identical with, 
derived from or which comprise a leader sequence from the soybean 
leghemoglobin genes, viz. 
__________________________________________________________________________ 
Lba with the sequence: 
AACTTGCATC 
GAACAATTAA 
TAGTAATAAC 
AGAAAATTAA 
AAAAGAAATA 
TG 
Lbc1 with the sequence: 
AACTTGCATT 
GAACAATAGA 
AAATAACAAA 
AAAAAGTAAA 
AAAGTAGAAA 
AGAAATATG, 
Lbc2 with the sequence: 
AACTTGCATT 
GAACAATAGA 
AATAACAACA 
AAGAAAATAA 
GTGAAAAAAG 
AAATATG, 
and Lbc3 with the sequence: 
AACTTGCATT 
GAACAATTAA 
TAGAAATAAC 
AGAAAAGTAG 
AAAAGAAATA 
TG. 
__________________________________________________________________________ 
The present invention deals furthermore with novel promoter sequences 
applicable as the second DNA fragment when carrying out the method 
according to the invention. 
This second DNA fragment is characterised in that it is identical with, 
derived from or comprises a promoter sequence from plant leghemoglobin 
genes. Examples of such a second DNA fragment are according to the 
invention DNA fragments identical with, derived from or comprising a 
promoter sequence from soybean leghemoglobin genes, viz. 
__________________________________________________________________________ 
Lba with the sequence 
GAGATACATT 
ATAATAATCT 
CTCTAGTGTC 
TATTTATTAT 
TTTATCTGGT 
GATATATACC 
TTCTCGTATA 
CTGTTATTTT 
TTCAATCTTG 
TAGATTTACT 
TCTTTTATTT 
TTATAAAAAA 
GACTTTATTT 
TTTTAAAAAA 
AATAAAGTGA 
ATTTTGAAAA 
CATGCTCTTT 
GACAATTTTC 
TGTTTCCTTT 
TTCATCATTG 
GGTTAAATCT 
CATAGTGCCT 
CTATTCAATA 
ATTTGGGCTC 
AATTTAATTA 
GTAGAGTCTA 
CATAAAATTT 
ACCTTAATAG 
TAGAGAATAG 
AGAGTCTTGG 
AAAGTTGGTT 
TTTCTCGAGG 
AAGAAAGGAA 
ATGTTAAAAA 
CTGTGATATT 
TTTTTTTTGG 
ATTAATAGTT 
ATGTTTATAT 
GAAAACTGAA 
AATAAATAAA 
CTAACCATAT 
TAAATTTAGA 
ACAACACTTC 
AATTATTTTT 
TTAATTTGAT 
TAATTAAAAA 
ATTATTTGAT 
TAAATTTTTT 
AAAAGATCGT 
TGTTTCTTCT 
TCATCATGCT 
GATTGACACC 
CTCCACAAGC 
CAAGAGAAAC 
ACATAAGCTT 
TGGTTTTCTC 
ACTCTCCAAG 
CCCTCTATAT 
AAACAAATAT 
TGGAGTGAAG 
TTGTTGCAT, 
Lbc1 with the sequence: 
TTCTCTTAAT 
ACAATGGAGT 
TTTTGTTGAA 
CATACATACA 
TTTAAAAAAA 
AATCTCTAGT 
GTCTATTTAC 
CCGGTGAGAA 
GCCTTCTCGT 
GTTTTACACA 
CTTTAATATT 
ATTATATCCT 
CAACCCCACA 
AAAAAGAATA 
CTGTTATATC 
TTTCCAAACC 
TGTAGATTTA 
TTTATTTATT 
TATTTATTTT 
TACAAAGGAG 
ACTTCAGAAA 
AGTAATTACA 
TAAAGATAGT 
GAACATCATT 
TTATTTATTA 
TAATAAACTT 
TAAAATCAAA 
CTTTTTTATA 
TTTTTTGTTA 
CCCTTTTCAT 
TATTGGGTGA 
AATCTCATAG 
TGAAGCCATT 
AAATAATTTG 
GGCTCAAGTT 
TTATTAGTAA 
AGTCTGCATG 
AAATTTAACT 
TAACAATAGA 
GAGAGTTTTC 
GAAAGGGAGC 
GAATGTTAAA 
AAGTGTGATA 
TTATATTTTA 
TTTCGATTAA 
TAATTATGTT 
TACATGAAAA 
CATACAAAAA 
AATACTTTTA 
AATTCAGAAT 
AATACTTAAA 
ATATTTATTT 
GCTTAATTGA 
TTAACTGAAA 
ATTATTTGAT 
TAGGATTTTG 
AAAAGATCAT 
TGGCTCTTCG 
TCATGCCGAT 
TGACACCCTC 
CACAAGCCAA 
GAGAAACTTA 
AGTTGTAAAC 
TTTCTCACTC 
CAAGCCTTCT 
ATATAAACAT 
GTATTGGATG 
TGAAGTTATT 
GCAT, 
Lbc2 with the sequence: 
TCGAGTTTTT 
ACTGAACATA 
CATTTATTAA 
AAAAAACTCT 
CTAGTGTCCA 
TTTATTCGGC 
GAGAAGCCTT 
CTCGTGCTTT 
ACACACTTTA 
ATATTATTAT 
ATCCCCACCC 
CCACCAAAAA 
AAAAAAAACT 
GTTATATCTT 
TCCAGTACAT 
TTATTTCTTA 
TTTTTACAAA 
GGAAACTTCA 
CGAAAGTAAT 
TACAAAAAAG 
ATAGTGAACA 
TCATTTTTTT 
AGTTAAGATG 
AATTTTAAAA 
TCACACTTTT 
TTATATTTTT 
TTGTTACCCT 
TTTCATTATT 
GGGTGAAATC 
TCATAGTGAA 
ACTATTAAAT 
AGTTTGGGCT 
CAAGTTTTAT 
TAGTAAAGTC 
TGCATGAAAT 
TTAACTTAAT 
AATAGAGAGA 
GTTTTGGAAA 
GGTAACGAAT 
GTTAGAAAGT 
GTGATATTAT 
TATAGTTTTA 
TTTAGATTAA 
TAATTATGTT 
TACATGAAAA 
TTGACAATTT 
ATTTTTAAAA 
TTCAGAGTAA 
TACTTAAATT 
ACTTATTTAC 
TTTAAGATTT 
TGAAAAGATC 
ATTTGGCTCT 
TCATCATGCC 
GATTGACACC 
CTCCACAAGC 
CAAGAGAAAC 
TTAAGTTGTA 
ATTTTTCTAA 
CTCCAAGCCT 
TCTATATAAA 
CACGTATTGG 
ATGTGAAGTT 
GTTGCAT, 
and Lbc3 with the sequence: 
TAACTTGCAT 
AAAAAATACA 
CTCATATATA 
TGCCATAAGA 
ACCAACAAAA 
GTACTATTTA 
AGAAAAGAAA 
AAAAAAACCT 
GCTACATAAT 
TTCCAATCTT 
GTAGATTTAT 
TTCTTTTATT 
TTTATAAAGG 
AGAGTTAAAA 
AAATTACAAA 
ATAAAAATAG 
TGAACATCGT 
CTAAGCATTT 
TTATATAAGA 
TGAATTTTAA 
AAATATAATT 
TTTTTGTCTA 
AATCGTATGT 
ATCTTGTCTT 
AGAGCCATTT 
TTGTTTAAAT 
TGGATAAGAT 
CACACTATAA 
AGTTCTTCCT 
CCGAGTTTGA 
TATAAAAAAA 
ATTGTTTCCC 
TTTTGATTAT 
TGGATAAAAT 
CTCGTAGTGA 
CATTATATTA 
AAAAAATTAG 
GGCTCAATTT 
TTATTAGTAT 
AGTTTGCATA 
AATTTTAACT 
TAAAAATAGA 
GAAAATCTGG 
AAAAGGGACT 
GTTAAAAAGT 
GTGATATTAG 
AAATTTGTCG 
GATATATTAA 
TATTTTATTT 
TATATGGAAA 
CTAAAAAAAT 
ATATATTAAA 
ATTTTAAATT 
CAGAATAATA 
CTTAAATTAT 
TTATTTACTG 
AAAATGAGTT 
GATTTAAGTT 
TTTGAAAAGA 
TGATTGTCTC 
TTCACCATAC 
CAATTGATCA 
CCCTCCTCCA 
ACAAGCCAAG 
AGAGACATAA 
GTTTTATTAG 
TTATTCTGAT 
CACTCTTCAA 
GCCTTCTATA 
TAAATAAGTA 
TTGGATGTGA 
AGTTGTTGCA 
T. 
__________________________________________________________________________ 
The present invention deals furthermore with novel DNA fragments comprising 
the combination of a first DNA fragment and a second DNA fragment and to 
be used when carrying out the method according to the invention. 
These DNA fragments are characterized by comprising a promoter sequence and 
a leader sequence and by being identical with, derived from or comprising 
5' flanking regions of plant leghemoglobin genes. 
Examples of such DNA fragments according to the invention are DNA fragments 
comprising a promoter sequence and a leader sequence, and which are 
identical with, derived from or comprise 5' flanking regions of the 
soybean leghemoglobin genes, viz. 
__________________________________________________________________________ 
Lba with the sequence: 
GAGATACATT 
ATAATAATCT 
CTCTAGTGTC 
TATTTATTAT 
TTTATCTGGT 
GATATATACC 
TTCTCGTATA 
CTGTTATTTT 
TTCAATCTTG 
TAGATTTACT 
TCTTTTATTT 
TTATAAAAAA 
GACTTTATTT 
TTTTAAAAAA 
AATAAAGTGA 
ATTTTGAAAA 
CATGCTCTTT 
GACAATTTTC 
TGTTTCCTTT 
TTCATCATTG 
GGTTAAATCT 
CATAGTGCCT 
CTATTCAATA 
ATTTGGGCTC 
AATTTAATTA 
GTAGAGTCTA 
CATAAAATTT 
ACCTTAATAG 
TAGAGAATAG 
AGAGTCTTGG 
AAAGTTGGTT 
TTTCTCGAGG 
AAGAAAGGAA 
ATGTTAAAAA 
CTGTGATATT 
TTTTTTTTGG 
ATTAATAGTT 
ATGTTTATAT 
GAAAACTGAA 
AATAAATAAA 
CTAACCATAT 
TAAATTTAGA 
ACAACACTTC 
AATTATTTTT 
TTAATTTGAT 
TAATTAAAAA 
ATTATTTGAT 
TAAATTTTTT 
AAAAGATCGT 
TGTTTCTTCT 
TCATCATGCT 
GATTGACACC 
CTCCACAAGC 
CAAGAGAAAC 
ACATAAGCTT 
TGGTTTTCTC 
ACTCTCCAAG 
CCCTCCTATA 
AAACAAATAT 
TGGAGTGAAG 
TTGTTGCATA 
ACTTGCATCG 
AACAATTAAT 
AGAAATAACA 
GAAAATTAAA 
AAAGAAATAT 
G, 
Lbc1 with the sequence: 
TTCTCTTAAT 
ACAATGGAGT 
TTTTGTTGAA 
CATACATACA 
TTTAAAAAAA 
AATCTCTAGT 
GTCTATTTAC 
CCGGTGAGAA 
GCCTTCTCGT 
GTTTTACACA 
CTTTAATATT 
ATTATATCCT 
CAACCCCACA 
AAAAAGAATA 
CTGTTATATC 
TTTCCAAACC 
TGTAGATTTA 
TTTATTTATT 
TATTTATTTT 
TACAAAGGAG 
ACTTCAGAAA 
AGTAATTACA 
TAAAGATAGT 
GAACATCATT 
TTATTTATTA 
TAATAAACTT 
TAAAATCAAA 
CTTTTTTATA 
TTTTTTGTTA 
CCCTTTTCAT 
TATTGGGTGA 
AATCTCATAG 
TGAAGCCATT 
AAATAATTTG 
GGCTCAAGTT 
TTATTAGTAA 
AGTCTGCATG 
AAATTTAACT 
TAACAATAGA 
GAGAGTTTTC 
GAAAGGGAGC 
GAATGTTAAA 
AAGTGTGATA 
TTATATTTTA 
TTTCGATTAA 
TAATTATGTT 
TACATGAAAA 
CATACAAAAA 
AATACTTTTA 
AATTCAGAAT 
AATACTTAAA 
ATATTTATTT 
GCTTAATTGA 
TTAACTGAAA 
ATTATTTGAT 
TAGGATTTTG 
AAAAGATCAT 
TGGCTCTTCG 
TCATGCCGAT 
TGACACCCTC 
CACAAGCCAA 
GAGAAACTTA 
AGTTGTAAAC 
TTTCTCACTC 
CAAGCCTTC 
ATATAAACAT 
GTATTGGATG 
TGAAGTTATT 
GCATAACTTG 
CATTGAACAA 
TAGAAAATAA 
CAAAAAAAAG 
TAAAAAAGTA 
GAAAAGAAAT 
ATG, 
Lbc2 with the sequence: 
TCGAGTTTTT 
ACTGAACATA 
CATTTATTAA 
AAAAAACTCT 
CTAGTGTCCA 
TTTATTCGGC 
GAGAAGCCTT 
CTCGTGCTTT 
ACACACTTTA 
ATATTATTAT 
ATCCCCACCC 
CCACCAAAAA 
AAAAAAAACT 
GTTATATCTT 
TCCATTACAT 
TTATTTCTTA 
TTTTTACAAA 
GGAAACTTCA 
CGAAAGTAAT 
TACAAAAAAG 
ATAGTGAACA 
TCATTTTTTT 
AGTTAAGATG 
AATTTTAAAA 
TCACACTTTT 
TTATATTTTT 
TTGTTACCCT 
TTTCATTATT 
GGGTGAAATC 
TCATAGTGAA 
ACTATTAAAT 
AGTTTGGGCT 
CAAGTTTTAT 
TAGTAAAGTC 
TGCATGAAAT 
TTAACTTAAT 
AATAGAGAGA 
GTTTTGGAAA 
GGTAACGAAT 
GTTAGAAAGT 
GTGATATTAT 
TATAGTTTTA 
TTTAGATTAA 
TAATTATGTT 
TACATGAAAA 
TTGACAATTT 
ATTTTTAAAA 
TTCAGAGTAA 
TACTTAAATT 
ACTTATTTAC 
TTTAAGATTT 
TGAAAAGATC 
ATTTGGCTCT 
TCATCATGCC 
GATTGACACC 
CTCCACAAGC 
CAAGAGAAAC 
TTAAGTTGTA 
ATTTTTCTAA 
CTCCAAGCCT 
TCTATATAAA 
CACGTATTGG 
ATGTGAAGTT 
GTTGCATAAC 
TTGCATTGAA 
CAATAGAAAT 
AACAACAAAG 
AAAATAAGTG 
AAAAAAGAAA 
TATG, 
and Lbc3 with the sequence: 
TATGAAGATT 
AAAAAATACA 
CTCATATATA 
TGCCATAAGA 
ACCAACAAAA 
GTACTATTTA 
AGAAAAGAAA 
AAAAAAACCT 
GCTACATAAT 
TTCCAATCTT 
GTAGATTTAT 
TTCTTTTATT 
TTTATAAAGG 
AGAGTTAAAA 
AAATTACAAA 
ATAAAAATAG 
TGAACATCGT 
CTAAGCATTT 
TTATATAAGA 
TGAATTTTAA 
AAATATAATT 
TTTTTGTCTA 
AATCGTATGT 
ATCTTGTCTT 
AGAGCCATTT 
TTGTTTAAAT 
TGGATAAGAT 
CACACTATAA 
AGTTCTTCCT 
CCGAGTTTGA 
TATAAAAAAA 
ATTGTTTCCC 
TTTTGATTAT 
TGGATAAAAT 
CTCGTAGTGA 
CATTATATTA 
AAAAAATTAG 
GGCTCAATTT 
TTATTAGTAT 
AGTTTGCATA 
AATTTTAACT 
TAAAAATAGA 
GAAAATCTGG 
AAAAGGGACT 
GTTAAAAAGT 
GTGATATTAG 
AAATTTGTCG 
GATATATTAA 
TATTTTATTT 
TATATGGAAA 
CTAAAAAAAT 
ATATATTAAA 
ATTTTAAATT 
CAGAATAATA 
CTTAAATTAT 
TTATTTACTG 
AAAATGAGTT 
GATTTAAGTT 
TTTGAAAAGA 
TGATTGTCTC 
TTCACCATAC 
CAATTGATCA 
CCCTCCTCCA 
ACAAGCCAAG 
AGAGACATAA 
GTTTTATTAG 
TTATTCTGAT 
CACTCTTCAA 
GCCTTCTATA 
TAAATAAGTA 
TTGGATGTGA 
AGTTGTTGCA 
TAACTTGCAT 
TGAACAATTA 
ATAGAAATAA 
CAGAAAAGTA 
GAAAAGAAAT 
ATG. 
__________________________________________________________________________ 
In addition the invention relates to any plasmid to be used when carrying 
out the method according to the invention and characterized by comprising 
a first DNA fragment as previously defined, and a second DNA fragment, 
also as previously defined. Suitable plasmids according to the invention 
are YEP Lb CAT and YEP 5 Lb Km. The plasmids according to the invention 
allow a high expression of a desired gene product by inserting coding 
sequences for these gene products. 
EXAMPLE 1 
Sequence Determination Of 5' Flanking Regions Of Soybean Leghemoglobin 
Genes 
From a soybean gene library the four soybean leghemoglobin genes Lba, Lbc1, 
Lbc2, and Lbc3 are provided as described by Jensen, E.O. et al., Nature 
Vol. 291, No. 3817, 677-679 (1981). The 5' flanking regions of the four 
soybean leghemoglobin genes are isolated, as described by Jensen, E.O., Ph 
D Thesis, Institut for Molekylaer Biologi, Arhus Universitet (1985), and 
the sequences of the four 5' flanking regions are determined by the use of 
the dideoxy chain-termination method as described by Sanger, F., J. Mol. 
Bio. 143, 161 (1980) and indicated in the sequence scheme. 
EXAMPLE 2 
Construction of YEP Lb CAT 
The construction has been carried out in a sequence of process sections as 
described below: 
Sub-cloning the Lbc3 Gene 
The Lbc3 gene was isolated on a 12kb EcoRI restriction fragment from a 
soybean DNA library, which has been described by Wiborg et al., in Nucl. 
Acids Res. 10, 3487. A section of the fragment is shown at the top of FIG. 
2. This fragment was digested by the enzymes stated so as subsequently to 
be ligated to pBR322 as indicated in the Figure. The resulting plasmids 
Lbc3HH and Lbc3HX were subsequently digested by PvuII and religated, which 
resulted in two plasmids called pLpHH and pLpHX. 
Sub-Cloning 5' Flanking Sequences From The Lbc3 Gene 
For this purpose pLpHH was used as shown in FIG. 3. This plasmid was opened 
by means of PvuII and treated with exonuclease Ba131. The reaction was 
stopped at various times and the shortened plasmids were ligated into 
fragments from pBR322. These fragments had been treated in advance as 
shown in FIG. 3, in such a manner that in one end they had a DNA sequence 
______________________________________ 
TTC -- 
AAG -- 
. 
______________________________________ 
After the ligation a digestion with EcoRI took place, and the fragments 
containing 5' flanking sequences were ligated into EcoRI digested pBR322. 
These plasmids were transformed into E. coli K803, and the plasmids in the 
transformants were tested by sequence analysis. A plasmid, p213 5'Lb, 
isolated from one of the transformants contained a 5' flanking sequence 
terminating 7 bp before the Lb ATG start condon in such a manner that the 
sequence is as follows: 
______________________________________ 
2kb 
______________________________________ 
5' flanking AAAGTAGAATTC 
Lbc3 sequence 
______________________________________ 
Sub-Cloning 3' Flanking Region Of The Lbc3 Gene 
For this purpose pLpHX was used which was digested by XhoII. The ends were 
partially filled out and excess DNA was removed, as shown in FIG. 4. The 
fragment shown was ligated into pBR322 which had been pretreated, as shown 
in the Figure. The construction was transformed into E. coli K803. One of 
the transformants contained a plasmid called Xho2a-3'Lb. As the XhoII 
recognition sequence is positioned immediately after the Lb stop codon, 
cf. FIG. 2, the plasmid contained about 900 bp of the 3' flanking region, 
and the sequence started with GAATTCTACAA---. 
The Construction Of Lb Promoter Cassette 
An EcoRI/SphI fragment from Xho2a-3'Lb was mixed with a BamHI/EcoRI 
fragment from p 213-5'Lb. These two fragments were ligated via the 
BamHI/SphI cleaving points into a pBR322 derivative where the EcoRI 
recognition sequence had been removed, cf. FIG. 4. The ligated plasmids 
were transformed into E. coli K803. A plasmid in one of the transformants 
contained the correct fragments, and it was called pEJLb 5'-3'-1. 
Construction Of Chimeric Lb/CAT Gene 
The CAT gene of pBR322 was isolated on several smaller restriction 
fragments, as shown in FIG. 5. The 5' coding region was isolated on an 
A1uI fragment which was subsequently ligated into pBR322 and treated as 
stated in the Scheme. This was transformed into E. coli K803, and a 
selected transformant contained a plasmid called A1u11. The 3' coding 
region was isolated on a TaqI fragment. This fragment was treated with 
exonuclease Ba13, whereafter EcoRI linkers were added. Then followed a 
digestion with EcoRI and a ligation into EcoRI digested pBR322. The latter 
was transformed into E. coli K803 and the transformants were analyzed. A 
plasmid, Taq 12, contained the 3' coding region of the CAT gene plus 23 bp 
3' flanking sequences so as subsequently to terminated in the following 
sequence. 
##STR1## 
Subsequently the following fragments were ligated together into EcoRI 
digested pEJLb5'-3'-1: EcoRI/PvuII fragment from A1u11, PvuII/DouI 
fragment from pBR322 and DdeI/EcoRI fragment from Taq 12. The latter was 
transformed into E. coli K803. A selected transformant contained the 
correct plasmid called pEJLb 5'-3' CAT 15. 
Cloning Chimeric Lb/CAT Gene In Yeast Plasmid 
This chimeric gene was isolated on a BamHI/Sa1I fragment from pEJLb 5'-3' 
CAT 15 and ligated into the yeast plasmid YEP24 cut with the same enzymes. 
After the transformation into E. coli K803 a selected transformant was 
examined. It contained the plasmid YEP LbCAT shown in FIG. 6. This plasmid 
was further transformed into the yeast strains Saccharomyces cerevisiae 
DBY747 and TM1. 
EXAMPLE 3 
Construction of YEP 5Lb Km 
The neomycine phosphotransferase (NPTII) gene was isolated from pKM2 (Beck. 
et al., Gene 19, 327). The 5' coding region was isolated on a Sau3A 
fragment, as shown in FIG. 7, and subsequently ligated into pBR322 
resulting in a plasmid called Sau 13. The 3' coding and flanking region 
was isolated on a PvuII fragment. The latter was together with a 
EcoRI/PvuIi fragment from Sau 13 ligated into EcoRI/PvuII digested pEJLB 
5'-3'-1. Upon transformation into E. coli K803 a transformant with the 
correct plasmid, pEJLb 5' Km 1, was selected. This plasmid was later on 
digested by means of BamHI and partially by means of PvuII in such a 
manner that the entire 5' flanking Lb sequence +the coding NPTII sequence 
were present on a BamHI/PvuII fragment. This fragment was ligated into 
BamHI/PvuII digested YEP24 resulting in the plasmid YEP 5Lb Km shown in 
FIG. 8. This plasmid was transformed into the yeast strain Saccharomyces 
cerevisiae Tm1. 
EXAMPLE 4 
The Effect Of Carbon Source On Expression of CAT 
Saccharomyces cerevisiae DBY747 containing the plasmid YEP Lb CAT is grown 
in minimum medium plus 2% of a carbon source. The cells are harvested at a 
cellular density of 5.times.10.sup.6 cells per m1. The CAT activity is 
measured as described by Walker, Edlund, Boulet & Rutter, Nature, 306, 557 
(1983). 
In Table 1 the CAT activity has been indicated as a function of the carbon 
source. The highest activity obtained by growing on succinate and glycerol 
has been arbitrarily set to 100%. 
TABLE 1 
______________________________________ 
Carbon Source Activity 
______________________________________ 
Succinate 100 
Glycerol 100 
Glucose 28 
Sucrose 18 
______________________________________ 
EXAMPLE 5 
The effect of heme precursors and heme analogs on the induction of gene 
expression. 
Saccharomyces cerevisieae TM1 containing the plasmid YEP Lb CAT is grown in 
a minimum medium plus 2% glucose plus 0.1% Tween.RTM. plus 20 .mu.g/ml 
ergosterol plus 50 .mu.g/ml methionine as well as a heme analog or a heme 
precursor. Deuteroporphyrin IX (dp) and protoporphyrin (pp), respectively, 
is added to a final concentration of 5 .mu.g/ml. Hemin is added to a final 
concentration of 5 .mu.g/ml. .alpha.-amino levulinic acid, .delta.-ALA, is 
added to a final concentration of 50 .mu.g/ml. 
In table 2 the CAT activity has been indicated as the activity of heme 
precursor and heme analog, respectively. the highest activity obtained by 
adding .delta.-ALA or dp has arbitrarily been set to 100%. 
TABLE 2 
______________________________________ 
Inducer CAT activity 
______________________________________ 
ALAcose + .delta. 
100 
Glucose + dp 100 
Glucose + pp 50 
Glucose + hemin 25 
Glucose 5 
______________________________________ 
It is obvious that the patent protection of the present invention is not 
restricted to the Examples indicated here. 
Thus the invention does not exclusively use 5' flanking regions of soybean 
leghemoglobin genes. It is well known that the leghemoglobin genes of all 
leguminous plants have the same activity, cf. Appleby (1974) in The 
Biology of Nitrogen Fixation, Quispel. A. Ed. North-Holland Publishing 
Company, Amsterdam Oxford, pages 499-554, and furthermore it has proved 
for the kidney bean PvLb1 gene that a distinct degree of homology exists 
with the sequences of soybean Lbc3. Thus the invention comprises the use 
of 5' flanking regions of leghemoglobin genes from all plants. 
According to the invention it is also possible to use such fragments from 
plants, animals or yeast which under natural conditions exert or mediate 
the novel regulatory activity described according to the present 
invention. The latter applies especially to such fragments which can be 
isolated from DNA fragements from gene libraries by hybridization with 
labelled sequences from 5' flanking regions of soybean leghemoglobin 
genes. 
It is well known that it is possible to change nucleotide sequences in 
non-essential subregions of 5' flanking regions without the latter causing 
a changed promoter activity and regulation. It is also well known that by 
changing the sequences of important subregions of 5' flanking regions it 
is possible to change binding affinities between nucleotide sequences and 
the factors or effector substances necessary for the transcriptional 
initiation and the translation initiation, and consequently that it is 
possible to improve the promoter activity and/or regulation. The present 
invention covers, of course, also the use of such changed sequences of 5' 
flanking regions. In particular the use of leader sequences can be 
mentioned which have been extended beyond the natural length provided the 
use of a such fragments makes the expression of a desired gene product the 
subject of the novel regulation according to the present invention.