Note: Descriptions are shown in the official language in which they were submitted.
W096/02640 .~ 5l~165l
21 ~35 1 3
1 "Alpha-LactAlh~lm;n Gene Constructs"
3 The present invention is concerned with recombinant
4 gene constructs for expressing the protein ~-
lactalbumin, or functional equivalents or parts
6 thereof.
8 Human milk has been shown to be superior over other
9 milk types, notably cow, sheep, camel and goat milk,
for human infant nutrition. ~owever, many mothers find
11 breast feeding difficult or inconvenient. Moreover, in
12 countries where infant food supplements are in great
13 demand, it would be highly desirable to be able to
14 supply a milk product with the nutritional benefits of
human milk.
16
17 One of the major differences of human milk over milk
18 from other mammals (for example cows or sheep) is the
19 presence of ~-lac~Alh~lm;n as the major whey protein.
Whilst ~-lactalbumin is present in other milk types,
21 the concentration is relatively low and instead the
22 major whey protein is ~-lactoglobulin. The level of ~-
23 lac~lh~lmin varies from species to species, with human
24 milk containing about 2.5 mg/ml, cow milk O.S-1.0 mg~ml
and mouse milk 0.1-0.8 mg/ml.
SUBSTITUTE SHEET ~RVLE 26)
WO'~/02640 PCT/GB9~/01651
2 ( q 3,5 j 3
1 The gene sequences encoding for the bovine ~-
2 lactA1h~;n and for the human ~-lactalbumin proteins
3 have been elucidated and the sequence information
4 published by ~ilotte et al, in Biochemie 69: 609-620
(1987) and by Hall et al, in Biochem J 242: 735-742
6 ~1987), respectively.
8 The present invention seeks to utilise genetic
9 engineering technique5 to provide a recombinant gene
construct capable of producing an ~-lactalbumin
11 concentration of greater than 1.0 mg/ml, for example
12 1.2 mg/ml or above, in milk when expressed in mammalian
13 cells. Generally said construct is adapted to be
14 expressed in non-humarl animal, especially bovine,
cells.
16
17 In one aspect, the present invention provides a
18 recombinant expression system adapted to express ~-
19 lactalbumin, or a functional equivalent or part thereof
in cells of a non-human, preferably bovine, animal.
21 Preferably, the recombinant expression system of the
22 present invention is adapted to express the human ~-
23 lact~lh~7~in protein, or a functional equivalent or part
24 thereof.
26 The term "expression system" is used herein to refer to
27 a gerretic sequence which includes a protein-encoding
28 region and is operably linked to all of the genetic
29 signals necessary to achieve expression of the protein
encoding region. Optionally, the expression system may
31 also include a regulatory element, such as a promoter
32 or enhancer, to increaSe transcription and/or
33 translation of the protein-encoding region, or to
34 provide control over expression. The regulatory
element may be located upstream or downstream of the
36 protein-encoding region, or may be located at an intron
WO9~102C40 1 ~ I / ~D~. C 1651
~ 3 ~ ~ 9 3 5 1 3
1 (non-coding portion) interrupting the protein encoding
2 region. Alternatively it i5 also possible for the
3 sequence of the protein-encoding region itself to
4 comprise a regulatory ability.
6 The term "functional equi~alent~ refers to any
~ 7 derivative which is functionally substantially similar
8 to the reference sequence or protein. In particular
9 the term '~functional equivalent'~ includes derivatives
in which nucleotide base(s) and/or amino acid(s) have
11 been added, deleted or replaced without a significantly
12 adverse effect on biological function, especially
13 biological function in milk production.
14
Genetic engineering has been recognised as a powerful
16 technique not only in research but also for commercial
17 purposes. Thus, by using genetic engineering
18 techniques (see Maniatis et al Molecular Cloning, a
19 Laboratory Manual Cold Spring, ~arbor Laboratory, Cold
Spring ~arbor, New York 1982 and "Principle of Genetic
21 Engineering", Old and Primrose, 5th edition, 1994, both
22 incorporated herein by reference) exogenous genetic
23 material can be transferred to a host cell and the
24 protein or polypeptide encoded by the exogenous genetic
material may be replicated by and/or expressed within
26 the host. For the purposes of simplicity genetic
27 engineering is normally carried out with prokaryotic
23 micro-organisms, for example bacteria such as E. coli,
29 as host. However, use has also been made of eukaryotic
organisms, in particular yeasts or algae, and in
31 certain applications eukaryotic cell cultures may also
32 be used.
33
34 Genetic alterations to mammalian species by micro-
injection of genes into the pro-nuclei of single-cell
36 embryos has been described by Brinster et al, in Cell
W096~02640 ~ 7~ 16~1
~ . 4 ~i 7JJIJ
1 27: 223-231, 1981. Here the foreign genetlc material
2 i5 introduced into the fertilised egg of an animal
3 whish then proceeds to develop into an embryo in the
4 normal manner having been transplanted into a foster
S mother. Truly transgenic animals contain copies of the
6 exogenous DNA in each cell.
8 Where the injected genetic material i5 successfully
9 incorporated into the host chromosome the animal is
termed "transgenic" and the transgene is inherited in
11 the normal Mendelian manner. However, only a low
12 proportion of gene transfer operations are successful,
13 especially for large domestic animals such as pigs,
14 sheep and cattle. To date it has not been possible tu
control the location at which the transgene integrates
16 into the host chromosome for such animals.
17
18 For the purpose of the present invention it may, in
19 certain circumstances, be sufficient simply to produce
a ~mosaic'' donor animal. In this situation the
21 transgene is incorporated into the chromosome copies of
22 only certain body organs. Mosaic animals are generally
23 produced by introducing exogenous ~NA into an embryo at
24 a later developmental stage.
26 One of the most promising application of transgenesis
27 in livestock aims to utilise the mammary gland as a
28 ~-bioreactor" to produce recombinant proteins of
29 pharmaceutical or nutritional interest in milk. The
mammary gland is an attractive organ in which to
31 express heterologous proteins due to its capacity to
32 produce large quantities of protein in an exocrine
33 manner. Recombinant DNA techniques may be used to
34 alter the protein composition of cow's mil}: used for
human or animal consumption. Por example, the
36 expression of human milk proteins in cow's milk could
W096~02640 ~ ~ r~ 5lC1~51
~ i ~ s ~ 5 ~3
1 improve its nutritional value in infant formula
2 applications ~Strijker et al, in ~larnessing
3 Biotechnology for the 21st Century, ed Ladisch and
4 Boser, American Chemical Society, Pages 38-21 (1992~).
Both applications would benefit from ability to
6 increase production capacity inexpensively by
7 multiplying producer animals with conventional and
8 advanced breeding techniques
The first step in developlng a transgene to be
11 expressed in the mammary gland is to clone the gene for
12 the protein of interest. To direct expression into
13 milk, the promoter gene for a major milk protein
14 expressed in milk is employed. Milk protein genes are
tightly regulated and are not expressed in tissues
16 other than the mammary gland, a characteristic that
17 minimises the possibility of negative effects on the
18 animal from inappropriate expression in other tissues.
19 Among the regulatory genes used to express heterologous
proteins in the mammary gland are alpha-SI-casein
21 (Strijker et., 1992, supra), beta-lactoglobulin (~right
22 et al., sio~Technology 9:831-834 (1991)) whey acidic
23 protein (Ebert and Schindler, Transgenic Farm animals:
24 Progress Report (1993)) and beta-casein (Ebert and
Schindler, 1993, supra).
26
27 Newly-made gene constructs are normally tested in
28 transgenic mice before adopting them for use in cattle.
29 ~ilk obtained from the transgenic mice is assayed for
quantity of recombinant protein. If enough milk is
31 available, the protein may be isolated to examine its
32 structural characteristics and bioactivity. The
33 selection of a particular construct for use in cattle
34 will depend primarily on consideration of both
expression level and authenticity of the resultant
36 recombinant protein.
W09610~640 .,~ 3 5 1 3 r~ 65l
1 Transgenesis in cattle is normally initiated by
2 microinjecting several hundred copies of gene construct
3 into one of the two pronuclei in a zygote. ~ygotes may
4 be obtained in vivo from the oviducts (Roschlau et al~,
Arch Tierz. ~erlin 31:3-8 (1988); Roschlau et al., in
6 J. Reprod. Fertil. (suppl 38), Cell Biology of
7 Mammalian Egg ~anipulation, ed Greve et al (1989~; Hill
8 et al., Theriogenology 37:222 (1992); ~3Owen et al. ~iol
9 Reprod. ~Q:664-448 (1994)~ or by in vitro fertilisation
of in vitro matured oocytes (Krimpenforth et al.,
11 Biotechnology 9:844-847 (1991); Hill et al., 1992,
12 supra; Bowen et al., 1993 supra). Bovine zyyotes must
13 be centrifuged at 15,000 x g for several minutes to
14 displace opaque lipid in order to visualise the
pronuclei with phase contrast, Nomars~i or Hoffrnan
16 interference contrast optics. 2-4 pl of buffer
17 containing several hundred copies of DNA construct are
18 injected into a pronucleus. Transgenes are thought to
19 integrate into random breaks in chromosomal DNA that
result from mechanical disruption during the
21 microinjection proce5s. Ideally, the transgenes
22 integrate at the zygote stage prior to DNA replication
23 to ensure that eYery cell in the adult contains the
24 transgene. In general, several "copies" of the
transgene, linked together linearly, integrate in a
26 single site on a single chromosome. The site of
27 integration is random. Integration probably occurs
28 after the first round of DNA replication, and perhaps
29 as late as the 2- or 4- cell stage (Wall and Seidel,
1992), resulting in animals that are mosaic with
31 respect to the transgene. Indeed, up to 30~ of animal~
32 in which transgenes are detected in somatic tissues do
33 not transmit the transgenes to their offspring (or
34 transmit to less than the expected so~).
36 After microinjection, embryos are either transferred
WO ~J6/0264~ 2 ~ 9 3 5 1 3 r~ C 16sl
1 directly into the oviducts of recipients or cultured
2 for a few days and transferred to the uterus of
3 recipient cattle. Confirmation of transgene
4 integration is obtained hy Southern blot analysis of
tissues sampled from the calf after birth. Transgene
6 expression is measured by assaying for the gene product
~ 7 in appropriate tissues, or in this case milk. Embryo
8 survival after microinjection, transgene integration
9 frequency, frequency of expression and expression
level, and frequency of germline transmission vary
11 according to quantity and quality of DNA construct
12 injected, strain of mice used (Brinster et al., Proc.
13 Natl. Acad Sci. USA 82:4438-4442 (1985)) and skill and
14 technique of the operator performing microinjection.
This basic approach has been routinely applied to
16 produce transgenic sheep (Wright et al., 1991, supra),
17 goats (~bert and Schindler, 1993, supra) pigs (Rexroad
18 and Purcel, Proc 11th Intl. Congr. Anim. Reprod. A.I.
19 Dublin 5:29-35 (1988)) and cattle (Krimpenfort et al.,
1991, supra; Hill et al., 1992, supra; Bowen et al.,
21 1994, supra).
22
23 Reference is also made to WO-A-88/01648 (of Immunex
24 Corporation), to WO-A-83~00239 and to WO-A-90~05188
(both of Pharmaceutical Proteins Limited) for
26 describing suitable techniques and methodologies for
27 production of recombinant gene constructs, production
28 of transgenic animals incorporating such constructs and
29 expression of the protein encoded ir the mammary gland
of the lactating adult female mammal. The disclosures
31 of these references and the references recited above
32 are incorporated herein by reference.
33
34 Reference is further made to Stacey et al in molecular
and Cellular Biology 14(21: 1009-1016 (~ebruary 1994)
36 which describes a knock-out experiment in which the
W09hlO2640 ~ 93~ 3 ~~1.~.S 1651
1 mouse ~-lactalbumin qene i5 replaced with a human ~-
2 lactalbumin gene. This paper (incorporated herein by
3 reference) does not however report expression of the ~-
4 lactalbumin protein.
6 In one embodiment the present invention provides an
7 expression system which has been produced by techniques
8 other than by knock-out of the gene naturally present.
In a further aspect, the present invention provides a
ll transgenic mammalian animal, said animal having cells
12 incorporating a recombinant expression system adapted
13 to express ~-lactalbumin ~preferably human ~-
14 lactalbumin) or a functional equivalent or part
thereof. Generally the recombinant expression system
16 will be integrated into the genome of the transgenic
17 animal and will thus be heritable so that the offspring
18 of such a transgenic animal may themselves contain the
l9 transgene and thus also be covered by the present
invention. Suitable transgenic animals include (but
21 are not limited to~ sheep, pigs, cattle and goats.
22 Cattle are especially preferred.
23
24 Additionally, the present invention comprises a vector
containing such a recombinant expression system and
26 host cells transformed with such a recombinant
27 exprèssion system (optionally in the form of a vector).
28
29 In a yet further aspect the present invention provides
~-lactalbumin produced by expression of a recombinant
31 expression system of the present invention, desirably
32 sucil ~-lactalbumin produced in a transgenic ma11lmal.
33 The ~-lactalbumin gene is naturally activated in the
34 mammary glands of the lactating female mammal. Thus
the protein expressed by the recombinant expression
36 system of the present invention would be produced at
W096l02640 ~ i 9 ~ 5 1 3 ~ s.[ 1651
1 such a time and would be excreted as a milk component.
2 It may also be possible for the protein of interest to
3 be produced by inducing lactation through hormonal or
4 other treatment. Processed milk products comprising
such ~-lactalbumin are also covered by the present
6 invention.
8 In one preferred embodiment, the recombinant expression
9 system comprises a construct designated pHAl, pHA2,
pBB~A, pOBHA, pBAHA, pBo~a-A or pBova-B. The
11 constructs pHAl, pHA2, p~BHA, pOBHA and pBAHA express
12 human ~-lactalbumin and are thus preferred,
13 particularly pHA2. The construct pHA2 was deposited on
14 15 February 1955 at NCI~B under Accession No NCIMB
40709.
16
17 Likewise transgenic mammals comprising the specific
18 constructs listed above are preferred.
19
It has further been found t~-~t, in addition to
21 increased concentrations of ~-lactalbumin per unit
22 volume of milk achieved by he present invention, where
23 a human ~-laotalbumin gene is present the volume of
24 milk produced increases also. This finding is totally
unexpected and for this reason constructs containing
26 the human ~-lactalbumin gene (or functional equivalents
27 or parts thereof) and transgenic animals (especially
28 cattle) are preferred Pmho~ir Ls of the invention.
29
Whilst we do not wish to be bound by theoretical
31 considerations, it is further believed that the
32 promoter region of the human ~-lactalbumin gene is only
33 partially responsible for the enhanced natural
34 expression of ~-lactalbum n by humans. It is believed
that enhanced expression may be obtained by including
36 within the recombinant expression system of the present
37 invention the 3' sequence flanking the protein-encoding
38 region and/or the 5' sequence flan~ing the protein-
SUI~STI~UT' SHEET II~ULE 26~
.. .. .. . , _, .. _, . , . , _ _
W0961~2640 ~ 9 ~ 5 ~ 3 P~ . ~L C. _ 1651
1 encoding regiQn itself.
3 The flanking sequences 3~ and 5' to the protein-
4 encoding region of the human ~-lac gene have been
sequenced for the first time. Partial sequences
6 (nucleotides 1-264, 1331-2131, 2259-2496, 251g-2680 and
7 3481-3952) of the 3' flanking region are presented in
8 SEQ ID Nos. 16 to 20 whilst the full sequence of the 5-
9 flanking region is presented in SEQ ID No. 21. In
experiments it has been observed that inclusion of
11 either or both of these sequences give a surprisingly
12 marked increase in expression levels of the a-
13 lactalbumin protein. This increase in expression may
14 be observed when the protein-encoding region is non-
human ~-lactalbumin as well as human a-lactalbumin.
16
17 Both the sequences of SEQ ID Nos. 16-20 and 21 are now
18 believed to contribute towards the higher levels of
19 expression of a-lactalbumin in human milk, and
therefore comprise a further aspect of the present
21 invention.
22
23 In a further aspect, the present invention provides a
24 polynucleotide having a sequence substantially as set
out in any one of SEQ ID Nos. 16-20 or SEQ ID No. 21 or
26 a portion or functional equivalent thereof.
27
28 The polynucleotides may be in any form (for example DNA
29 or RNA, double or single stranded), but generally
double stranded DNA is the most convenient. Likewise
31 the polynucleotides according to the present invention
32 may be present as part of a recombinant genetic
33 construct, which itself may be included in a vector
34 (for example an expression vector) or may be
incorporated into a chromosome of a transgenic animal.
36 Any vectors or transgenic animals comprising a
W096l02640 ~ / } ~1 935 ~ 3 P ., ~C1~51
11
1 polynucleotide as described above form a further aspect
2 of the present invention.
4 Viewed from a yet further aspect the present invention
- 5 provides a recombinant expression system ~preferably
6 adapted to express ~-lactalbumin ~preferably human ~-
7 lactalbumin) or a portion or functional equivalent
8 thereof), said recombinant expression system comprising
9 a polynucleotide selected from the polynucleotide
located between the EcoRI and XhoI restriction sites of
11 the wild-type ~-lactalbumin gene and the polynucleotide
12 located between the BamHI and EcoRI restriction sites
13 of the wild-type human ~-lactalbumin gene, or a portion
14 or functional equivalents thereof
16 In one preferred embodiment, the recombinant expression
17 sequence of the present invention comprises both
18 polynucleotides as defined above, portions and
19 functional equivalents thereof.
21 The invention also encompasses vectors containing the
22 recombinant expression systems defined above and cells
23 transformed with such vectors. Further, the present
24 invention comprises transgenic animals wherein the
transgene contains the recombinant expression system.
26
27 Figu~e 1 as discussed in Example 1 and shows the
28 sequence of bovine ~-lactalbumin PCR primers.
29
Figure 2 is discussed in Example 1 and 4 shows the
31 posltion of bovine ~-lactalbumin PCR primers and
32 products.
33
34 Figure 3 is discussed in Example 2 and shows a
restriction map of two overlapping genomic A clones for
36 the human ~-lactalbumin gene (pHA-2 and pHA-l).
WO96~)26~ ,,,.~ q 35 1 3 ~ 5.'C16~
1 Fiyure 4 is discussed in Example 3 and shows a
2 restriction map of three overlapping genomic A clones
3 for the bovine beta-lactaglobulin gene.
Figure 5 is discussed in Example 4 and shows SDS-PAGE
6 analysis of skimmed milk from bovine a-lactalbumin
7 transgenic mice run against non transgenic mouse milk.
9 Figure 6 is discussed in Example S and shows human a-
lactalbumin transgene constructs.
11
12 Figure 7 is discussed in Example 5 and shows SDS-PAGE
13 analysis of skimmed milk from human a-lactalbumin
14 transgenic mice run against non transgenic mouse milk.
16 Figure 8 is discussed in Example 5 and shows a Western
17 analysis of the milk from human a-lactalbumin
18 transgenic mice run against human a-lactalbumin
19 standard.
21 Pigure 9 shows the PCR cloning strategy for transgene
22 constructs P~l to PKU4 as discussed in Example 6.
23
2g Pigure 10 gives the se~uences of P~U primers 1 to 10 as
2S discussed in Example 6.
26
27 Figure 11 shows the structure of null and humanised a-
28 lactalbumin alleles.
2~
Figure 12 is a Northern analysis of total RNA from a-
31 lactalbumin-deficient lactating mammmary glands.
32
33 Figure 13 illustrates a Western analysis of a-
34 lactalbumin from targeted mouse strains.
36 Figure 14 is a histological analysis of wild type and
W096/0264~ 3 ~. 1 9 ~ 5 ~ 3 r~ ,l651
1 ~-lac~ lactating mammary glands.
3 Figure 15A shows an RNase protection assay used to
4 distinguish human replacement and mouse ~-lactalbumin
- 5 mRNA and Figure 15~ shows an RNase protection assay of
6 mouse and human replacement ~-lactalbumin mRNA,
8 Figure 16 gi~es the quantification of ~-lactalbumin by
9 hydrophobic interaction chromatography.
11 SEQ ID Nos. 16 - 20 give parts of the sequence from the
12 BamHI site to the vector restriction sites (including
13 EcoRI sites) 3' of the protein-encoding region of the
14 endogenous human ~-lactalbumin gene, as set out below:
16 SEQ ID No. 16 : Nucleotides 1 to 264 (inclusive)
17 SEQ I3 No. 17 : Nucleotides 1331 to 2131 (inclusive)
18 SEQ ID No. 18 : Nucleotides 2259 to 24g6 (inclusive~
19 SEQ IC No. 19 : Nucleotides 2519 to 2680 (inclusive)
SEQ ID No. 20 : Nucleotides 3481 to 3952 (inclusive)
21
22 SEQ ID No. Z1 gives the sequence Erom the EcoRI
23 restriction site to the XhoI restriction site which are
24 5' to the protein-encoding region of the endogenous
human a-lactalbumin gene.
26
27 In more detail, in Figure 11 the upper portion shows
28 the wild type murine ~-lactalbumin locus. The positio
29 and direction of the transcribed region is indicated by
the arrow. The translational stop site and RNA
31 polyadenylation sites are also indicated. The middle
32 portion shows the structure of the null allele. The
33 striped bar indictes the HPRT selectable cassette. The
34 lower portion shows the structure of the human
replacement allele. The checkered bar shows the human
36 ~-lact.albumin fragment. The transcription initiation,
wo g6/n2640 ~ 3 5 ~ 3 PCrlGB9S~ GSI
14 ~
1 translat.ional 8tOp and polyadenylation sites are shown.
2 Restriction enzyme sites shown are: HindIII (H); BamllI
3 (B); XbaI (Xl.
In Figure 12, the two autoradiographs shown are .repeat
6 hybridLsations of the 5ame membrane filter using a
7 human a-lactalbumin probe followed by a rat ~-casein
8 probe. The probes used are indicated under each
9 autoradiograph. The source of RNA in each lane is
indicated above the lane mar~.ers.
11
12 In Figure 13 Lane A contains purified human o-
13 lactalbumin. Lanes B-F show samples of mil~ from
14 targeted mice, genotypes are indicated abo~e the lane
markers. Lanes ~ and H are a shorter exposure of Lanes
16 C and D.
17
18 The light micrographs shown in Figure 14 are
19 haemtoxylin~eosin stained sections of mammary tissue
(original magnification lOOx). The genotypes of each
21 gland are indicated.
22
23 In Fiqure 15A, the 3- junction between mouse and human
24 DNA in the a-lach allele lies between the translational
stop site and the polyadenylation signal. Human ~-
26 lactalbumin mRNA contains 120bp of mouse sequences in
27 the 3' untranslated end. ~uman replacement and mouse
28 ~-lactalbumin mRNA were detected by hybridisation with
2~ a mouse RNA probe and distinguished by the size of RNA
3C fragments protected from ribonuclease digestion. Human
31 sequences are indicated by the chequered bar and mouse
32 sequences by the shaded bar. Restriction enzyme sites
33 shown are: HindIII (H); BaI (~3); XbaI (X).
34
3S In ~igure 15B the autoradiograph shown is of a 5~
36 polyacrylamide urea thin layer gel. ~he source of RNA
W09Cl02610 ~ ~ t~ L i 935 t 3 PCT/GB95101651
~ 15
1 is indicated above the lane markers. Lane A shows a
2 wild-type RNA hybridised t.o the mouse RNA proe
3 undigested with ribonuclease. ~anes D to J show RNA
4 samples from ~-lac~ lach heterozygotes, the numbers
indicate inùividual mice and are the source of the
6 quantitative estimates given in Figure 15. The
7 predicted size of protected fragments are inùicated.
9 The upper portion of Figure 16 shows phenyl-Sepharose
elution profiles oE three milk samples. 1, a-lacb/~-lach
11 homozygote fmouse #22); 2, ~-lac~/~-lach heterozygote
12 (mouse #76); 3, ~-lac'/~-lacm wild type. The lower
13 portion shows a standard curve of known quantities of
14 human ~-lactalbumin plotted aaainst integrated peak
area.
16
17 The present invention will now be further described
18 with reference to the following, non-limiting,
19 examples.
21 Examole 1 - Clonina of Bovine ~-lactalb:lmin aene
22
23 There are three known variants of bovine ~-lactalbumin,
24 of which the B form is the most common. The A variant
from Bos (Bos) nomadicus f.d. indicus differs from the
26 B variant at residue 10: ClU in A i.s substituted for
27 Arg in B. The sequence difference for the C variant
28 from Bos (Bibos) javanicus has not been established
29 (McKenzie ~ White, Advances in Protein Chemistry 41,
173-315 (1991). The bovine ~-lactalbumin gene
31 (encoding the B form~ was cloned from genomic DNA using
32 the PCR primers indicated in Figure 1. The primers
33 have been given the following sequence ID Nos:
34 Ba-2 SEQ ID No 1
~ 35 Ba-7 SEQ ID No 2
36 Ba-8 SEQ ID No 3
~o ~6fn264o ~ r ~ i 9 3 5 1 3 r~ 6sl
1 sa-9 SEQ ID No 4
2 The source of DNA in all the PCR reactions was blood
3 from a Holstein-Friesian cow.
The length of the amplified promoter region using
6 primer Ba-9 in combination with primer Ba-8 is a . 72kb.
7 This BamHI/EcoRI fragment was cloned into Bluescript
8 ~pBA-P0.71.
The length of the ampli~ied promoter region using
11 primer Ba-7 in combination with primer Ba-8 is 2.05kb.
12 This BamHI~EcoRI fragment was cloned into Bluescript
13 (pBA-P2).
14
The entire bovine a-lactalbumin gene including 0.72kb
16 of 5' and 0.3kb of 3' flanking region was amplified
17 using primer Ba-9 in combination with primer Ba-2.
18 These primers include BamHI restriction enzyme
19 recognition sites, which allowed direct subcloning of
the amplified 3kb fragment into the BamHI site of
21 pUC18, yiving rise to construct pBova-A (see Figure 2).
22
23 Ligation of the BamHI/EcoRv fragment from clone pBA-P2
24 to the EcoRV~amHI fragment of pBOVA-a gave rise to
construct pBOYA-b (see Figure 2).
26
27 Since TAO polymerase lacks proof-reading activity, it
28 was essential to ensure that the amplified bovlne ~-
29 lactalbumin DNA was identical to the published bovine
a-lactalbumin gene. Sequence analysis was carried out
31 across all the exons and the two promoter fragments.
32 Comparison of the bovine a-lactalbumin exons with those
33 published by Vilotte showed 3 changes:
34
(i) Exon I at ~759 C to A. 5~ non-coding region;
36 (ii) Exon I at +792 CTA to C~G. Both code Eor Leucine
W096/02640 ' :-.'.p; 17 ~) 935 1 3 p~ .SCl651
1 (iii) Exon II at +1231 GCG to ACG. Alanine to
2 Threonine
3 This i5 indicative of the more common "B" form of the
4 protein.
6 Although misreading of sequence during the PCR
7 amplification cannot be ruled out, the above mismatches
8 were probably due to the difference in the source of
9 bovine DNA.
11 ExamPle 2 - Clonina of Human a-lactalbumin qene
12
13 The DNA sequences of human a-lactalbumin has been
14 published (Hall et al, Biochem. J., 242 : 735-742
(1987)). Using the iluman sequence, PCR primers were
16 designed to clone two small fragments from human
17 genomic DNA, one at the 5' end of the gene and the
18 other at the 3' end. These were suhcloned into the
19 pUC18 vector and used as probes to screen a commercial
(Stratagene) A genomic library. Two recombinant
21 bacteriophages, 4a and 5b.1, which contained the a-
22 lactalbumin gene, were isolated by established methods
23 (Sambrook et al, ~olecular Cloning 2nd ed., Cold Spring
24 Harbor Laboratory (1989)). Restriction mapping
demonstrated that both clones contained the complete
26 coding sequence for the human a-lac gene but differed
27 in the amount of 5' and 3' sequences present (Figure
28 3). Sequence analysis of exons from clone 5b.1 and
29 exons and 5' flanking region of clone 4a showed that
these were identical to the published sequence.
31
32 Portions of the 3' sequence are given in SEQ ID Nos. 16
33 to 20 and the 5' sequence is given as SEQ ID No. 21.
34
EY~ le 3 - Cloninq of bovine beta lactoalobulin gene
36 rbBLG)
W096102640 ~ 935~ 3 rc~ . S.~1651
1 ~'he DN~ sequence of bovine BLG (bBLG! has been
2 published (Jamieson et al; Gene, 61; 85-90, (1987);
3 Wagrler, unpublished, EMBL Data Library: BTBLACE~
4 ~1591)). Using the bovine sequence, PCR primers were
designed to clone a fragment from the 5' portion of the
6 bovine BLG gene. This was subcloned into the pUC18
7 vector and used as probes to screen a commercial bovir.e
8 (Stratagene) A genomic library- Three genomic A clones
9 were isolated and characterised by restriction enzyme
analysis (see ~ig. 4). Two of the clone5 (BB13, BB17)
11 contain the complete bBLG coding region plus various
12 amounts of flar-king regions, while clone BB25 lacks the
13 coding region and consists entirely of 5' flanking
14 region. Sequence analysis showed that the end of this
clone lies 12 bp upstream of the ATG translation start
16 site. Sal I fragments containing the entire insert of
17 clone BB13 and BB17 were subcloned into pUC18, as well
18 as EcoR I fragments from clone BB25 (the latter was
19 cloned into pBluescript (Figure 4).
21 ExamDle 4 - Assemblv ~n~ eXDression of bovine a-
22 lactalbumin constructs
23
24 TransGene constructs (FiG. 2)
26 pBova-A consists of the bovine a-lactalbumin coding
27 region, -0.72kb of 5' flank and 0.3kb of 3' flank,
28 cloned as a 3kb BamHI fragment into Bluescript vector.
pBova-B consists of 3 fragments:
31 1. The 1.47kb BamHI to EcoRV fragment from clone pBA-
32 P2.
33 2. The 2.78kb EcoRV to BamHI fragment from clone
34 pBova-A.
3. The cloning vector Bluescript digested Bam~
36
WOg~/026~0 ~ ; 19 l~~ 65
1 Bovine ~-lactalbumin exrression in transaenic mice
3 The two constructs pBova-A and pBova-B (Figure 2) were
4 injected into mouse embryos and gave rise to transgenic
S animals. Milk analysis by SDS-PAGE gel stained with
6 Coomasie blue (referred to as "Coomassie gels~) and
7 comparison to standard amounts of ~-lactalbumin showed
8 expression levels of bovine ~-lactalbumin varied from
non detectable for pBova-A and up to ~O.S-lmg~ml for
pBova-B (see Figure S and Table 1).
WO 96/0264(~ . 20 ~ f 9 3 5 1 3 PCT/GB9!;/01651
1Table 1
2Bovine a-lactalbumin expression in transgenic mice
3 Mouse Coomassie
4 244.12 BOVA-a
244.14 BOVA-a
6 244.15 BOVA-a
8 245.23 BOVA-b
9 245.8 BOVA-b
245.4 BOVA-b
11 245.7 BOVA-b +
12 245.21 BOVA-b
13 245.13 BOVA-b +
lg 249.13 BOVA-b
246.15 BOVA-b
16 249.18 BOVA-b ++
17 249.23.1 BOVA-b ++
18 249.23.5 BOVA-b ++
19 249.25.3 BOVA-b
249.25.7 BOVA-b
21 249.30.3 BOVA-b - - = < 0.5mg~ml
22 249.30.4 BOVA-b - + = =0.5-lmg/ml
23 249.33.2 BOVA-b +/++ ++ = -1-2mg/ml
24 249.33.3 BOVA-b +/++
26 Table 1 shows the relative levels of bovine a-
27 lactâlbumin in transgenic mouse milk as estimated by
28 comparison to protein standards on Coomassie gels.
~ WOg6102640 ,~ 21 2 ~ 935 ~ 3 r~ . tl651
1 Example 5 - Assembl~ and exoression of human a-
2 lactalbumin constructs
4 a-lactalbumin is the major whey protein in humans,
beta-lactoglobulin the major whey protein in sheep and
6 cow. The level of a-lactalbumin expression varies from
7 species to species, human milk contains about 2.5mg/ml,
8 cow milk 0.5-l.Omg/ml, and mouse milk O.l-O.Bmg/ml. To
9 define sequences which allow maximum expression of the
human a-lactalbumin gene several different constructs
11 were designed. These contain a) different amounts of
12 5' and 3' flanking regions derived from the human a-
13 lactalbumin locus, b) 5' flanking regions derived from
14 the bovine a-lactalbumin locus, or c) 5' flanking
regions derived from the bovine or ovine beta-
16 lactaglobulin gene. The ovine beta-lactoglobulin gene
17 promoter has been successfully used to allow high
18 expression (>lOmg/ml) of heterologous genes in mouse
19 milk.
21 Transoene constructs (Ficure 6
22
23 pHA-1 consists of the human a-lactalbumin coding
24 region, =1.8kb of 5' flank and 3kb of 3~ flank derived
from A clone 5b.1 cloned as a 7kb EcoRI/SalI fragment
26 into puc18.
27
28 pHA-2 consists of the human a-lactalbumin coding
29 region, =3.7kb of 5' flank and ~13kb of 3~ flank
deri-ed from A clone 4a cloned as a ~19kb SalI fragment
31 into puc18.
32
33 pOBHA (ovine beta-lactaglobulin, human a-lactalbumin3
34 was constructed from 4 DNA fragments:
1. a 4.2kb SalI~EcoRV fragment containing the ovine
36 beta-lactoglobulin promoter (see WO-A-90~05188);
W096~02fi40 ~ 9 3 ~ 1 3 rc~lGBg5Jol~sl
~ ' 22
I 2. a 74bp synthet;c oli~onucleotide corresponding to
2 a 8bp BclI linker and bases 15-77 of the human a-
3 lactalbumin sequence used as a bluntiBglI
4 fragment;
3. a 6.2kb BglI~PstI human a-lactalbumin fragment
6 derived from A clone 4a comprising a region
7 between a BglI site at base 77 and a XhoI site in
8 the 3' flank;
9 4. pSL1180 (Pharmacia) cut with PstI and SalI.
11 pBBHA (bovine beta-lactoglobulin, human a-lactalbumin)
12 was constructod from 4 DNA fragments:
13 1. a 3.Okb EcoRI fragment containing the bovine beta-
14 lactoglobulin promoter derived from clone BB25-3
and used as a EcoRI/EcoRV fragment;
16 2. a 74bp synthetic oligonucleotide corresponding to
17 a 8bp BclI linker and bases 15-77 of the human a-
18 lactalbumin sequence used as a blunt/BglI
19 fragment;
3. a 6.2kb BglI/PstI human a-lactalbumin fragment
21 derived from A clone 4a comprising a region
22 between a Bgll site at base 77 and a XhoI site in
23 the 3' flank;
24 4. Bluescript vector cut with EcoRI and PstI.
26 pBAHA (bovine a-lac~lh~min~ human a-lactalbumin) was
27 constructed from 4 DNA fragments:
28 1. a 0.72kb samHI to StuI fragment containing the
29 bovine ~-lactalbumin promoter derived from clone
pBA-PO.7;
31 2. a 62bp synthetic oligonucleotide corresponding to
32 bases 15-77 of the human ~-lactalbumin sequence
33 used as a blunt/BglI fragment;
34 3. a 6.2kb BglIJPstI human a-lactalbumin fragment
derived from A clone 4a comprising a region
36 between a BglI site at base 77 and a XhoI site in
:
W0~6/02640 j ~ F~ 5.~i65t
1the 3' flank;
2 4. Bluescript vector cut with BamHI and PstI.
4 ~luman a-lactalbumin ex~ression in transcenic mice
6 5 constructs were injected into mouse embryos and gave
7 rise to transgenic animals- All constructs expressed
8 human a-lactalbumin in the milk of mice. pHA-1 and
9 pHA-2, which contain the human a-lactalbumin gene and
various amounts of flanking regions expressed between 1
11 to ~18mg/ml (213.5 pHA-2) in the majority of animals.
12 pOBHA and pBBHA containing the human a-lactalbumin gene
13 driven by either the ovine or bovine BLG promoter had
14 slightly lower levels of expression. pBAHA containing
the human a-lactalbumin gene driven by the 0.72kb
16 bovine a-lactalbumin promoter had expression levels
17 similar to pHA-l or pHA-2 but a lower percentage of
18 transgenic animals expressed detectable levels of
19 protein. This finding is surprising as the same bovine
promoter sequence driving the bovine a-lactalbumin gene
21 gave very poor results (see Example 4 and Vilotte et
22 al; FEBS, ~ol. 297, 1.2. 13-18 ~1992)).
23
24 Table 2 gives a summary of the relative amount of the
transgenic protein. Skimmed milk from these animals
26 was analysed by SDS-PAGE stained with Coomasie blue,
27 isoeIectric focusing, Western blots visualised with a
28 commercial anti-human a-lactalbumin antibody (Sigma)
29 and chromatofocusing. The results from these analyses
showed that the transgenic protein was of the correct
31 size, pI and antigenicity when compared to a human a-
32 lactalbumin standard (Sigma).
W096/02640 ,~ i 935 1 3 PCT/GB9~01GSI
1 Table 2
3Human a-lactalbumin expression in transgenic mice
Mouse Coomassie Western
6 205.19 pHAl
7 204.10 pHAl ++ ++
8 204.7 pHAl +++ +++
9 230.1S.3 pHA1 +++ n.d.
230.15.5 pHAl +++ n.d.
11 230.15.6 pHA1 +++ n.d.
12 230.21.5 pHA1 +++ n.d.
13 230.21.1 pHA1 ++ n.d.
14
211.18 pHA2 + ++
16 211.17 pHA2 - -
17 211.lfi pHA2 +++ +++
18 212.11 pHA2 + n.d.
19 213.5 pHA2 ++++ ++++
212.13 pHA2 ++ n.d.
21 212.19 pHA2 - n.d.
22 213.4 pHA2 +++ n.d.
23 212.7 pHA2 - n.d.
24
232.10 B5HA
26 233.1 BBHA
27 231.4 BBHA ++ ++
28 232.9 BBHA + +
29 231.9 BBHA - n.d.
232.5 BBHA + +
31 231.3 BBHA + +
32 232.6 BBHA - n.d.
33 237.6 BBHA - n.d.
34
235.15 OBHA - n.d.
36 235.19 OBHA ++ ++
37 236.6 OBHA ++ ++
38 234.1 OBHA + +
39 234.4 OBHA ++ ++
234.~4 OBHA + +
41
42 239.~4 BAHA +++ +++
43 239.4 BAHA - n.d.
44 240.7 BAHA - n.d.
239.3 BAHA - n.d.
46 239.6 BAHA +~- ++
47 239.12 BAHA - n.d.
48 243.1 BAHA ++ n.d.
49 242.9 BAHA +++ n.d.
241.16 BAHA + n.d.
51 234.14 BAHA - n.d.
52 243.13 BAHA + n.d.
53 243.10 BAHA - n.d.
54 243.4 BAHA - n.d.
W0~6/02~0 ~193~13 r~ 75,~l65l
~ Z5
2 Table 2 shows the relative levels of human a-
3 lactalbumlrl in transgenic mouse milk as estimsted by
4 comparison to proteil- standards on Coomassie gels and
Western Blots.
7 - = < 0.5 mg/ml
8 + = ~0.5-lmg/ml
9 ++ = ~1-2 mg~ml
++~ = ~2-3 mg/ml
11 ++++ = ~5 mg/ml
12 n.d. = not determined
13
14 The results from several mice are shown in Figs. 7 and
8. Fig. 7 shows an SDS-PAGE analysis of skimmed
16 transgenic mouse milk run against a non-transgenic
17 control mouse milk. Fig. 8 shows a Western blot of
18 human a-lactalbumin transgenic milks run against a
lg human a-lactalbumin standard.
21 Ex~r~le 6 - Exr~ression of ~utacenised Bovino a-
22 Lact.album;n under the control of the Human a-
23 Lactalbumin Promoter in vivo
24
Expression of the human a-lactalbumin transgene is
26 considerably higher than that of the native bovine a-
27 lactalbumin transgene, reflecting the difference in
28 expression levels of the endogenous bovine and human
29 genes. As this might be caused by differences in the
5- control regior, the 5' region of the bovine a-
31 lactalbumin transcriptional start sit.e was substituted
32 with sequences from the human a-lactalbumin gene.
33
34 Two constructs were made, namely PKU-5 and PKU-l~,
which incorporate the amino acid substitutions shown in
36 Table 3.
37
~ 1 935 1 ~
~'096102640 ~ JI~5I
J ~ 26
1 The follo~ing SEQ ID Nos. have beer. assiqned to the PCR
2 primers used.
4 PKU-1 SRQ ID No. 5
PKU-2 SEQ ID No. 6
6 PKU-2L SEQ ID No. 7
7 PKU-3 SEQ ID No. 8
8 PKU-4 SEQ ID No. 9
9 PKU-5 SEQ ID No. 10
PKU-6 SEQ ID No. 11
11 PRU-7 SEQ ID No. 12
12 PKU-8 SEQ ID No. 13
13 PKU-9 SEQ ID No. 14
14 PKU-10 SEQ ID No. 15
16 PRU-5
17
18 In a first cloning step three frayments were subcl.oned
19 into the EcoRI/Eam~I site of pUC18:
21 tl) the Eco~I to PvuI fragment derived by PCR
22 amplification using PKU-primer 7 in combination
23 wit.h 8 (see Figure 10);
24
2S (2~ the PvuI to ~saBI frayment derived by PCR
26 amplification using PKU-primer 9 in combination
27 'with 10 (see Fiyure 10); and
28
29 (3) The ~saBI to HindIII fragment derived from pBA.
31 The final construct included 6 DNA fra~ments:
32
33 (1) the 3.7kb SalI to KpnI fragment contai.ning the
34 human a-lactalbumin promoter derived from ~ clone
4a (Figure 3);
36
W096/02640 ' '~ r.~ 1f'1
27 ,~l j 935 ~ 3
1 (2~ the 152bp synthetic oligorlucleotide containing
2 human ~-lactalbumin sequences from the KpnI site
3 to the AUG and bovine a-lactalbumin sequences from
4 the AUG to the ~apI site;
6 ~3) the 1.25kb HpaI to ~lindIII fragment from the first
7 subcloning step;
9 ~4) the 0.95 kb HindIII to BglII fragment derived from
pBA;
11
12 (5) the 3.7kb BamHI to X~oI fragment from the 3' flank
13 of the human ~-lactalbumin gene derived from A
14 clone 4a (Figure 3) used as a BamHI fragment; and
16 (6) a Bluescript KS- vector cut with SalI and BamHI.
17
18 PKU-lH was constructed in the same way as PKU-5 with
19 the exception of fragment (3), which was derived from
PKU-1.
21
22 PKU-] was constructed from six DNA fragments (see
23 Figure 9):
24
(1) a 2.04kb SstI to HpaI fragment derived from
26 pBOVA-6;
27
28 (2) a 0.46kb HpaI to PvuI fragment derived from PCR
29 product A (PKU-primer pair 1 and 2; see Figure
10);
31
32 (3) a 0.60kb PvuI to BsaB-r fragment derived from PCR
33 product B (primer pair 3 and 4; see Figure 10);
34
(4) a 0.22kb BsaBI to HindIII fragment derived from
36 pBoVA-6;
W096f0264(1 ~j . 2 1 ~ 3 5 1 3 r~
~ A 0.95kb HindIII to BglII frayment derived from
2 pBO~A-6;
4 (6~ the vector pSL1180 digested wlth SstI and BglII.
6 Table 3
8 Amino Acid Substitutions present in Transgene
9 Constructs
11 Substi.tutions Human promoter Plasmid
12 Human 3' flank
13 pos~n 9 30 53 80
14 Tyr, Tyr, Tyr, Tyr + pPKU-lll
Ser, Tyr, Leu, Leu + pPKU-5
16
17 Expression in transgenic mice
18
19 The two constructs PK~-lH and P~U-5 have been injected
20 into mouse embryos. So far transgenic animals were
21 derived ~or the PKU-5 construct. These animals are set
22 up for breeding to allow milk analysis.
23
24 E~Amnle 7 - Effect on Lactation bv disruption of ~-
Lactalbu~in deficiency and insertion of human a-
26 Lactalbumin cene re~lacement in mice
27
28 Materials and Methods
29 Mouse lines
31 Mice carrying the null ~-lactalbumin allele and the
32 humanised ~-lactalbumin replacement allele were derived
33 by breeding chimeras produced from the targeted
34 embryonic stem cell clones M2 and Fh respectively
against Balb/c mates, as described previously
36 (Fitzgerald et al J. Biol. Chem 245:2103-2108). During
W096/02640 .~ 29 2 93 r~ .l651
1 the breeding of these strains, ~-lactalbumin genotypes
2 were detenDined by Southern analysis o~ genomic DNA
3 prepared from tail biopsies.
RNA analysis
7 Total RNA was prepared by the method of Auffray and
8 Rougeon (Eur. J. 8iochem 107:303-14) from ~h~l in~l
9 mammary glands of female mice 506 days postpartum.
Northern analysis was carried out according to standard
11 procedures ~Sambrook et al, Molecular cloning). Probes
12 used for hybridisation were: a 3.5kb Bam~l fragment
13 containing the complete mouse ~-lactalbumin gene; and a
14 l.lkb rat ~-casein cDNA (Blackburn et al Nucl. Acids
Res 10:2295-2307).
16
17 RNAse protection analysis
18
19 32P-CTP radiolabelled antisense RNA was transcribed by
T7 RNA polymerase (Promega) from a 455 bp ~indIII-BalI
21 mouse ~-lactalbumin fragment ~Figure 15A) cloned in
22 Bluescript ~S. The conditions for the transcription
23 reaction, solution hybridisation and RNAse digestion
24 were as rec~ ied by Promega. Protected fragments
were separated by polyacrylamide gel electrophoresis
26 and visualised by autoradiography.
27
28 Nilk composition and yield analysis
,29
Milk samples were collected between days 3-7 of
31 lactation under Hypnorm (~oche)~Hypnovel (Janssen)
32 anaesthesia. 150mU of oxyt.ocin ~Intervet) was
33 administered by intraperitoneal injection and milk
34 expelled by gentle massage. Milk fat content was
measured as described by Fleet and Linzell (J. Physiol
36 175:15). Defatted milk waa assayed for protein
W096102640 .~ l651
~ 30 ~ ~ 9 ~ 5 7 3
1 (Bradford Analyt. Biochem 72:24a-54) and lactose was
2 measured enzymatically by sequential incubation with l~-
3 galactosidase, (Boehringer) glucose oxidase and
4 peroxidase (Sigma) by a method adapted Erom that of
Bergmeyer and Bernt (Methods in Enzyme Analysis 3:1205-
6 1212).
8 Milk yield was estimated using a titrated water
3 technique described by Knight et al., (Comp. Biochem.
Physiol ~ 127-133) in mice suckling young over a 43
11 hour period between days 3 and 6 of lactation.
12
13 Milk ~-lactAl' in analysis and quantification
14
Milk samples were analysed on 16% of SDS-PAGE gels
16 (Novex) and western blotted onto lmmobilon P membrane.
17 a-lactalbumin was detected by absorption with rabbLt
18 anti-human ~-lactalbumin antiserum (Dako), followed by
19 goat anti-rabbit LgG peroxidase antibody conjugate and
visualised with an enhanced chemiluminescence system
21 (Amersham).
22
23 ~-lactalbumin in milk samples was quantified by a
24 modification of the method of Lindahl et al., (Analyt.
Biochem 140:394-402) for calcium dependant purification
26 of ~-lactalbumin by phenyl-Sepharose chronatography.
27 Milk samples were diluted 1:10 with 27% w/v ammonium
28 sulphate solution, incubated at room temperature for 10
29 minutes and centrifuged. Supernatant was mixed with an
equal volume of lOOmM Tris~Cl, pH 7.5, 70mM EDT~ and
31 loaded onto a column (200~1 packed volume) of phenyl-
32 Sepharose (Pharmacia) pre-equilibrated with 50mM
33 Tris/Cl, pH 7.5, lmM E~TA. The column was washed with
34 the same buffer and ~-lactalbumin eluted with 50mM
Tris~Cl, pH 7.5, lmM CaCl~. The optical absorbance at
36 280nm of the column was monitored and integrated peak
W096/02640 r~ ;. 1651
~i935~3
1 areas corresponding to the a-lactalbumin fraction
2 computed. A standard curve was constructed using known
3 quantities of purified human a-lactalbumin from 0 to
4 2.46 mg~ml (Figure 16).
6 .-iiistology
8 Pups were removed for two hours from lactating mothers
9 on the sixth day postpartum, mothers sacrificed and
thoracic mammary glands were dissected, preserved in
11 neutral buffered formalin, paraffin embedded and
12 stained with haematoxylin/eosin by standard methods.
13
14 Results
16 Nouse a-lactalbumin gene deletion
17
18 A line of mice in which a 2.7kb fragment covering the
19 complete mouse a-lactalbumin coding region and a 0.57kb
of promoter has been deleted and replaced with a 2.7kb
21 fragment containing a hypoxanthine phosphoribosyl-
22 transferase (HPRT) selectable marker gene was
23 established as described in Stacey et al, 1994 suDra
24 (see Figure 11). Animals carrying this allele are
designated a-lac-. The wild type mouse a-lactalbumin
26 allele is designated ~-lac~.
27
28 Northern analysis of RNA from mammary glands taken on
29 the fifth day of lactation showed that a-lactalbumin
mRNA was absent in a-lac-/a-lac- homosygotes (see
31 Figure 12) confirming that the targeted a-lactalbumin
32 gene has been removed and that no other source of ~-
33 lactalbumin mRNA exists. Hybridisation of the same RNA
34 samples with a ~-casein RNA in all samples (see Figure
12).
36
~ 3Z ~ 5 1 3 . ~ ~ 1651
1 ~-Lactalbumin deflciency has no apparent effect in mice
2 other than during lactation. ~-lac-/a-lac- homozygotes
3 and ~-lacm/~-lac-heterozygotes of both sexes are normal
4 in appearance, behaviour and fertility. However, ~-
S lac-/~-lac- homozygous females cannot rear litters
6 successfully. Their pups fail to thrive and die within
7 the first 5-10 days of life. Off5pring of homozygous
8 ~-lac-/~-lac- females do survive normally when
g transferred to wild type foster mothers. Conversely,
offspring from wild type mothers transferred to
11 homozygous a-lac-/a-lac- mothers are not sustained.
12 Table 4 shows that pups raised by ~-lac-/a-lac- mothers
13 are approximately half the weight of those raised by ~-
14 lac=/Q-lac~ wild type mice. Estimates of milk yield are
consistent with this, ~-lacm/~-lac- heterozygotes
16 produce similar quantities of milk as wild type, but
17 the yield of a-lac-/~-lac- hl ~yyuLes was severely
18 reduced (Table 4).
' Table 4
I Milk composition, pup weight, mammary tissue weight and milk yield in targeted mouse
lines
Genotype ~-lac=/~-lac' ~-lac=/~-lac- ~-lac-/~-lac- ~-lac=~~-lact' ~-lact'/o-lac~
Fat (~ 28.23 + 1.65(7) 29.6 + 1.3(6) 45.25 + 2.15(6) 25.25 + 1.36(7) 21.2 + 0.23(4)*
v/v )
Protein 87.52 ~ 5.82(7) 95.81 + 9.5(5) 164.63 + 13.92(8)~i 94.51 + 5.97(7) 77.07 + 1.05(4)
(mg/ml) - -
Lactose 62.44 + 9.27(7) 42.7 + 4.2(6) 0.7 + 0.34(3) 42.40 +1.93(7)56.85 + 3.8(g) ;~-
(mM)
Single 2.82 + 0.25(8) 3.14 + 0.1(7) 1.52 ~ 0.12(10)~ 2.9 + 0.15(8) 3.4 + 0-75(4)
pup
weight
(g)
~lamnialy 0.34 ~ 0.06(7) 0.4 + 0.1(7) 0.35 ~ 0.05(8)0.31 + 0.04(6) 0.51 + 0.09(4)
tissue
weight w
per pup
(g) ,~.
Milk 7.51 +- 0.44(4) 6.7 + 0.38(6) 1.37 + 0.48(4) n.t. 9.94 + 0.65(5)*
yield
(g/day)
Statistical analysis by unpaired t-test, * p<0.05; **p<0.01; ***p<0.001;
Values are means + S~.
Figures in brackets indicate the num~er of mothers analysed.
n.t., not tested
WO~61U264~ 34 ~o~ 93~1 3 P~JI~ III6SI
l Milk was obtained from each genotype hy manual milking
2 and the composition of key components analysed. ~lilk
3 from a-lac=/~-lac-heterozygote5 was indistinguishabLe in
4 appearance from wild type milk and showed fat and
protein contents similar to wild type (Table 4). ~hile
6 lactose concentration appeared to be slightly reduced
7 in a-lac~/a-lac- heterozygotes, statistical analysis
8 showed that the difference was not significant. In
9 contrast, milk from a-lac-~a-lac- homozygotes w~s
viscous, difficult to express from the teats and was
ll markedly different in composition to wild type. Fat
12 content was -60% greater than wild type, protein content
13 was -88~ greater, and lactose was effectively absent.
14 The apparent 0.7mM lactose detected in a-lac-/a-lac-
females represents milk glucose content, since the
16 lactose assay used involved the enzymatic conversion of
17 lactose to glucose. Direct assay of glucose in wild
18 type milk indicated a concentration of 1.8mM.
19
Western analysis of milk protein failed to detect a-
21 lactalbumin in milk from a-lac-/a-lac- homozygotes (see
22 Figure 13, Lane F). This was confirmed by phenyl-
23 Sepharose chromatography, a technique used to
24 specifically identify a-lactalbumin which has been
adapt.ed to obtain quantitative estimates of milk a-
26 lactalbumin content (Table 5; see also Figure 16).
27 ~hen~applied to milk from ~-lac-/a-lac-homozygotes no
28 a-lactalbumin was detected. In contrast, a-lactalbumin
29 concentration in ~-lac=/a-lac- heterozygote milk was
estimated as 0.043mg/ml, appro~imately half that of
31 wild type (Table 5).
~ W096~026~ 35 ~ t 935 ~ 3 r~ ,~C~l6~l
1 Table 5
2 Milk ~-lactalbumin con~en~.
4 Source a-lactalbumin (mc/ml)
Human 2.9 + 0.1(2)
6 a-lac=/a-lac~ mice 0.09 + 0.005(6
7 a-lac-/a-lac-mice 0 (3)
8 a-lacm/a-lac-mice 0.043 + 0.004(5)
9 a-la~=/Q-lach mice 0.65 + 0.07(4)
a-lacb/a-lach mice 1.38 + 0.12(5)
11
12 a-Lactalbumin content of milk samples were estimated by
13 phenyl-Sepharose chromatography.
14
Values are means t SE.
16
17 Figures in brackets indicate the number of mothers
18 analysed.
19
a-Lactalbumin deficiency has no apparent effect on
21 mammary gland development. Table 4 shows that total
22 mammary tissue weights of wild type, heterozygous a-
23 lac=/a-lac- and homozygous a-lac-/a-lac- lactating
24 mothers were not significantly different. ~ight
microscopic analysis of mammary glands (Figure 14)
26 revealed that heterozygous and homozygous a-lac-/a-lac-
27 glands were hi5tologically normal. However, the
28 alveoli and ducts of homozygous glands were distended
29 and clogged with material rich in lipid droplets.
31 Replacement of mouse a-lactalbumin by human ~-
32 lac~l' in
33
34 We have generated mice carrying the human a-lactalbumin
gene at the mouse a-lactalbumin locus. The 2.7kb mouse
36 a-lactalbumin fragment deleted at the a-lac- null
37 allele was replaced by a 2.97kb fragment containing the
38 complete human a-lactalbumin coding region and 5'
WOg61(1~640 ~ r . ' 36 ~7 ~ 9351 3 ~ '.ilt6~1
1 flanking se~uences. The human fragment stretches from
2 0.77kb upst:ream of the human transcription initiation
3 site to an Eco~I site 136bp 3' of the human
4 translational stop site. Junctions with mouse
sequences were made at a Bam~I site 0.57~.b upstream of
6 the mouse transcription initiation site and at an Xbal
7 site 147bp 3' of the mouse translational stop site (see
8 Stacey et al, 1994, ~Y2E~; see also Figure 11). Here
9 we describe our analysis of animals carrying this
allele, designated ~-lach.
11
12 Deletion of the murine ~-lactalbumln gene established
13 that ~-lactalbumin deficiency blocks lactose synthesis
14 and severely disrupts milk production. We have used
the ~-lacb allele to test the ability of human ~-
16 lactalbumin to restore milk production in the absence
17 of mouse ~-lactalbumin. ~-lac~/~-lach heterozygous and
18 ~-lach/~-lach homozygous mice were normal in appearance,
19 fertility and behaviour.
21 In contrast to ~-lac-J~-lac- mice, ~-lac~ lach
22 homozygous mothers produce apparently normal. milk and
23 rear oftspring successfully. Table 4 shows that pups
24 raised by ~-lac~-lach heterozygous and ~-lach/a-lach
hr Gy~ous females are similar in weight to those of
26 wild type mothers. This is supported by our
27 observation that these animals raised successive
28 litters of pups entirely normally. These data
29 constitute clear evidence that the human gene can
functionally replace the mouse gene. Analysis of milk
31 composition (Table 4) shows that lactose concentration
3Z is similar in all genotypes. Although both protein and
33 fat concentrations seem reduced in ~-lach/~-lach
34 homozygous animals, only the fat reduction was judged
statist.ically siqnificant by unpaired t-test. These
36 animals show an lncrease in milk volume over wild type
37 (Table 4).
38
~ wOg6/0264n J~ 37 2 1 ~351 3 r~ 3~l651
1 Relative quantification of human and mouse a-
2 lactalbumin RNA.
4 ~luman milk contains considerably more ~-lactalbumin
(2.5mg/ml) than murine milk (O.lmg/ml). We wished to
6 determine whether the human a-lactalbumin fragment
7 retained a high level of expression when placed at the
8 mouse locus, or assumed a lower level more
9 characteristic of the rrlouse gene. ~-lacm/a-lach
heterozygous mice provided an ideal means of addressing
11 this question, as the expression of the human gene
12 could be directly compared with its mouse counterpart
13 in the same animal.
14
Figure 15A shows the strategy used to compare levels of
16 mouse and human ~-lactalbumin mRNA. Because the
17 junction between human and mouse a-lactalbumin
18 sequences lies upstream of the polyadenylation site, ~-
19 lach mRNA contains a "tag~' of 120 bases of untranslated
mouse sequences at the 3' end. A uniformly
21 radiolabelled mouse RNA probe was u.sed in a
22 ribonuclease protection assay to detect and distinguish
23 human and mouse a-lactalbumin mRNA in the same RNA
24 sample. The relative abundance of each mRNA was
calculated from the amount of label in fragments
26 protected by human and mouse mRNAs.
27
28 A ribonuclease protection assay was performed and the
29 results are shown in Figure 15B. Lane A shows the
undigested 455 base probe and Lane ~ shows that yeast
31 tRNA did not protect any fragments. Wild type mouse
32 RNA protected a fragment consistent with the predicted
33 305 base RNA from endogenous mouse ~-lactalbumin RNA
34 ~see Lane B). Homozygous ~-lact/~-lac}' gland RNA
protected a smaller band consistent with the predicted
36 120 base RNA protected by human ~-lactalbumin mRNA (see
37 Lane C). Lanes D-J show results consistent with a
38 series of hetero7ygous a-lac~-lach animals were
W096/02640 ~ ; 38 2 i 9 3 5 7 3 r~~ c 6!,
1 obtained ~see Lanes D to J). Small and large protected
2 iragments in each sample indicate thc presence c~ both
3 human and mouse ~-lactalbumin mRNA. Protected
4 fraqments were excised from the gel, radioisotope
content measured, adjusted for the size difference and
6 the ratio of human to mouse ~-lactalbumin mRNA
7 estimated. Table 6 shows the amount of radioisotope
8 present in the 305 base and 120 base fragments excised
~ from Lanes D to J of the gel shown in Figure 15B, and
the calculated ratio of human to mouse ~-lactalbumin
11 mRNA in each ~-lac=/~-lac~ heterozygote. It is apparent
12 that, although there was variation between individual
13 animals, human a-lactalbumin mRNA was significantly
14 more abundant than mouse mRNA. Averaging the seven ~-
lS lac~l~-lac~ heterozygotes gives a value of 15-fold
16 greater expression for human ~-lactalbumin mRNA.
';
WOg6l02640 ~ ~ 3g ~ 93513 r~ J~il01651
1 Table 6
3 Relat.ive quantification of human and mouse ~-
4 lactalbumin mRNA in ~-iacm/~-lach mammary glands
S
6 Lane Mouse# 120 base 305 base human/mouse
7 fragment' fragment' RNA ratiob
9 D 2 5957 1000 15:1
11 E 3 5770 547 26:1
12
13 F 4 4825 810 15:1
14
G 76 6018 1077 14:1
16
17 H 98 5206 1452 9:1
18
19 I 99 5481 1117 12:1
21 J 79 26858 3561 19:1
22
23
24
26 Lane designations indicate the source of protected
27 frag~ents and correspond to those shown in Figure 15B.
28
29 a. numbers are expressed in counts per minute
(c.p.m.)
31
32 b. Ratio between c.p.m. of 120 ba.se fragment
33 multiplied by 2.54 (to adjust for size difference
34 and c.p.m. of 305 base fragment.
9 ~ 5 1 3
W0 ~2640
1 Human a-lactalbumin protein expression
3 A Western analysis of ~-lactalbumin in targeted mouse
4 lines was conducted. Human ~-lactalbumin can be
distinguished irom mouse ~-lactalbumin boy its faster
6 electrophoretic mobility (see Lanes A, L). A prominent
7 lower band in a-lach/~-lach homozygotes and ~-lach/~-lacm
8 heterozygotes was observed (see Lanes C, D, G, H), and
9 corresponds to the position of the human ~-lactalbumin
standard and was only observed in mice which e~press
11 human a-lactalbumin generated either by gene targeting
12 or by pronuclear microinjection ~data not shown). This
13 identity was confirmed by phenyl-Sepharose
14 chromatography (See Figure 16) and analysis of peptides
lS released by cyanogen bromide cleavage (data not shown).
16 The band with slower mobility, similar to mouse a-
17 lactalbumin, is also a human ~-lactalbumin gene product
18 the nature of which is unknown. This species varied in
19 intensity with ~-lacb gene dosage (see Lanes G, H) and
was also present in milk from human ~-lactalbu~in
21 transgenic mice generated by pronuclear microinjection
22 (data not shown).
23
24 The ~-lact~lh~lmin content of milk samples was
quantified by phenyl-Sepharose chromatography. Figure
26 16 shows superimposed absorbance profiles of column
27 eluates of three illustrative milk samples including
28 the ~-lac=~-lach heterozygote and ~-lac~/~-lac~
2Y h~ z~ote shown in Figure 13. The peaks corresponding
to eluted ~-lactalbumin are marked. ~-Lactalbumin
31 contents were estimated by comparing the integrated
32 peak areas with the human ~-lactalbumin standard curve
33 shown. The relationship between integrated peak area
34 and ~-lactalbumin quantity was linear and highly
reproducible. ~-Lactalbumin content for the samples
36 shown in Figure 16 were estimated as follows: ~-lacm/~-
37 lacm wild-type O.lmg/ml; ~-lacmi~-lach heterozygote #76
38 0.45mg~ml; ~-lachi~-lach homozygote #22 8.9mg~ml. Table
~ W096~26~ ~ 41 ~ 7 935 ~ 3 r~ Jl~l65l
1 S shows the concentration of a-lactalbumin in milk
2 samples from targeted mouse lines an~ lactating women.
3 It is clear that the concentration of a-lactalbumin in
4 milk is directly related to gene dosage, eg a-laP/a-
lac- heterozygotes shown an a-lactalbumin concentration
6 half that of wild type- Given that the volumes of milk
7 produced by these mice are similar (Table 4), the
8 concentration of a-lactalbumin provides a reasonable
9 indication of the quantity synthesised. The relative
proportions of human and mouse a-lactalbumin c, o.lents
11 in a-lac=~a-lach heterozygote milk were estimated by
12 assuming that a-lactalbumin expression from a single
13 mouse allele was 0.043mg/ml and the rest represented
14 human a-lactalbumin. This is consistent with the
amounts of a-lactalbumin expressed by a-lac~/a-lac-
16 heterozygotes and wild type mice. Therefore, a-lac=/a-
17 lac-heterozygotes were estimated as expressing
18 0.61mg/ml human and 0.043mg/ml mouse a-lactalbumin.
19 Thus, human a-lactalbumin is approximately 14-fold more
abundant than mouse a-lactalbumin in a-lac~/a-lach
21 heterozygote milk. This is remarkably consistent with
22 the relative proportions of mRNA.
23
24 ~Y~le 8 Enhanced ex~ression of a heteroloaous cene.
26 These data confirm that the upstream promoter region
27 (AUG to about -3.7 kb) which is included in the pHA-2
28 construct enhances expression of a heterologous gene.
29 Table 7 shows the results of milk analysis from pHA-2
transgenic founder females. Out of 10 females, 6
31 animals expressed high levels of human a-lac. 3
32 animals failed to express detectable levels of human a-
33 lac (less than 0.2 mg/ml in this assay~, all 3 also
34 failed to transmit the transgene. ~e can neither be
certain whether they were low expressors or not
36 transgenic.
37
W096l02640 ~ 4~ 2 I q3~ l 3 P~ 1651
1 Table 8: Constructs PKU-O to BALT-B all contain the
2 Lovine ~-lac promoter ~about 2kb). Constructs PKU~
3 to PRU-16 all contain the human ~-lac promoter (3.7kb).
4 Using the human ~-lac promoter increased the expression
of the transgene to almost 100%.
7 These data show that the use of the human ~-lac
8 promoter achieves a higher level of expression than the
9 use of the bovine promoter and inducefi expression in
more animals than the bovine promoter.
TABLE 7
CONSTRUCT NOUSE SEX C mg/ml TRANS. MI FREQ.
MILK
~ALYSIS
pHA2 210-17 M - - 1~21=5%
211-12 F ~1 - - 14/54
=26
211-16 F 10 5 3~4
211-17 F ~>10 ND 0/8
211-24 27 M
29 31 36
37 42 46
47 54
212-7 F 10 ND 0/5 8~77=10
212-11 F <10 1 0~4
212/13 F ~1 1-5 2~4
212-19 F >10 ND 0~3
212-36 44 M
45 46
213-4 F C10 5 0~2 2~14=14&
213-5 F 10 10 2~8
OVERALL MIF 25/166=15%
ND = Not detectable
TBA = To be analysed
TBO = To bred on
C = Copy number
TRANS. = Transmission
MI FR~Q. = Integration frequency
WO96101640 ~ 43 Z 1935 1 3 r~ l016SI
T~3LE 8
Constr. Transgenics Expres5ers max. expr.
PRU-0 6F/5M 3~5 500
PKU-l 18F~23M 5/18 200
PRU-2 8F/7M 3/8 goo*
PKU-3 6F/5M 2/6 100*
PKU-4 7F/3M 5/18 30U*
8ALT-A 34F/24M n.t. n.t,
BALT-B lOF/18M n.t. n.t.
PKU-lH 6F/5M 4/5 100
PKU-5 13F/14M 13/13 800
PKU-6 3F/4M 2/2 100
PKU-7 4F/llM 1/1 <20
PKU-16 13F/12M n.t. n.t.
HaPKU-l n.t. n.t. n.t.
HaPKU-2 n.t. n.t. n.t.
n.t. = not tested
* estimate
W09610264~ ? 1 935 ~ 3 PCI'IGB9~1016$1
44
SEQUEtlCE !,}STII; .
~1) GENER~L INFOY~ATION:
~1) AppLIcAr3T
A) NAME: PPL THERAPEUTICS ~SCOTLAtlDj LIMITED
~Bj STREET: RO5LIN
IC~ CITY: EDINBUFGH
~E) COUNTYY D'NITED ~INGDOM
~F) POSTAL CODE. ~ZIPj: EH25 9PP
iij TITLE OF IN;ENTIO?~: Alph2-Lactalbumin Gene Cor.structs
~li.) NUMEER OE SEQUENCES: 21
~iv) COMPUTER READABLE FORM:
~A) ~SEDIUM TYPE: Flcppy disk
~B) COMPUTER: I8.V. PC compatihle
~') OPERAT;tlG SYSTE!~: PC-DOS¦~S-DOS
~ù~ SOFTWAP.E: Pasor.~Ir Release 1 0 version 1.30 ~EPO)
~2) INFOR~T;ON FOR SEQ ID NO: 1:
111 SEQ~E?3CE cHA~cTERrsTIcs
~A) LENGTH: 27 base pairs
~E) TYPE: nucleic ac id
}C! STRANDED?3ES5: sinsle
~Dj ropoLoGv: 'incar
~is) VOLECUI.FA TYPE: cDNA
(xi) 52QUENCE DESCRIPTrO?!: SEO ID NO: 1:
GCGGATCCAC AACTGAAGTG ACTTAGC 27
~2j IriFo~yATIorl FOP. SEQ ID NO: 2:
~i; SEOUENCE CHAP~CTERISTICS:
~A) LENGTH: 35 base pairs
~B) TYPE: nUcle~c acld
~C~ STRANDEDNESS: single
~D~ TOPOLOGY: lsr.Oar
(ii) MOLECULE TYPE: cDtA
(xi) SEQUENCE DEscRrpTIo?! SE~ ID NO: 2:
SUBSTITUTE ~HEET ~ULE 26J
~ W 096/02640 ' '~ r~ .'O1651
4~ ~ j93~1 3
CATGGATCCT GGGTGGTC,.T TGAA~CGACT GATCC 35
12) }NFOQ~ATIo~ FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43 base pairs
(B) TYPE: nucleic acid
(C1 STRANDEDNESS: sinyle
(D) TOPOLOGY: llnear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCr DESCRIPTION: Si'.Q ID NO: 3:
GCAGGCGAAT TCCTCAAGAT TCTGAAATGG GGTCACCACA CTG 43
(2) INFOR~ATION FOR SEQ ID NO: 4:
(i~ SEQUENCE CHAQACTER}STICS:
(A) LENGT.H: 33 Dase pa.rs
(a) TYPE: nucleic acid
(C) STQANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQDENCE DESCRIPTION: SEQ ID NO: 4:
GAGGATCCAA TGTGGTATCT GGCTATTTAG TGG 33
(2) INFORMATIoN FOR S-Q ID NO: S:
(i) SEQUENCE CHAQACTERISTICS:
(A) LE~GTH: 46 base pai-s
(B) TYP2: nucleic acid
(C) STQANDEDNESS: sinyle
~D)-TOPOLOGY: linear
(ii) MOLECULE Typr: cONA
(xi) SEQUENCE DESCRIPTTON: S--Q ID NO: S:
GCTGAATTCG TTAACAAAAT GTGAGGTGTP. TCGGGAGCTG AAAGAC 46
(2) INFOP~ATION FOR SEQ ID NO: 6:
SUBSTITUTE 5HEET (RULE 263
W0 9~l02640 ' ~ ~s~ ; 4~ 9~5 ~ 3 r~ ;5~ol~sl
EQuEr~cE CHAPP~CTrP.I ST rcs:
~A; LENGTH 58 base pairs
~Bj TYPE nucleic acLd
~c) STRANCEDN_~S~S: singLe
(D) TOPOLOGY- iinear
~Lij MOLECULE TYPE cDNA
~xi) SEQUENCE DESCRIPTION: SEQ ID NO 6:
GCGGATCCCA TCGCTTGTC.T GrcATAAcc~ CTGGTATGGT ACGCGGTACA GACCCCTG S8
(2j INFOP~L~TION FOR SEQ ID r:o: 7:
(l~ SEQUENCE cHARp~cTERIsTrcs
(A~ LENGTH: 58 bAse pairs
(~ TYPE nucleic aclrl
~Cj sT~p-ANDEDr1Ess slngle
~D) TOPOLOGY linear
(ii) UOLECULE Typr cDriA
(~i) SEQUEN~;E DESCRI2TION SEQ ID ~lo
GCGGATCCGA TCGCTTGTGT GTCATAACCA CTGC-.. TGGA GCGCGGTACA GACCCCTG 58
~2) IrlFoRuATIoN FOR SEQ ID UO: 8:
~i) SEQUENCE CHAP~CTERISTrcs
~A) LErlGTH: 69 Dase pairs
~Ej TYPE ruclele acid
~Cj sTpAriDED~Ess single
ID) TOPOLOGY line~r
(li) UOLECULE TYPE: cDNA
~xi) SEQuEricE DEscRIpTroN SEQ rs NC a
GCGGATCCGA TCGTACA~AA CAATGACAGC ACAG.;.;T.A~G GACTCTACC.; GP.T.~AT.~P.T 60
AAAATTTGG s9
(2~ INFO~ ATION FOR SEQ ID NO 9
~L) SEQUENCE cHAp-AcrERIsTIcs
SUBSTITUTE SHEET (RULE 26)
W096~2640 ;'' ,~ 1651
4~ ~- I q~5 ~ 3
(~) LENCTk: 34 b_se pairr
(3) TYPE: nucle~c ~cid
(C) STRANDEDNESS: single
(D) TOPOLOCY: linear
~ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
GCTCTAGATC ATCATCCAGG TACTCTGGCA GGAG ~4
(2) INFORMATION FOR SEQ ID No 10
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(S) TYPE: nuclelc acid
IC~ STRANDEDNESS: single
ID~ TOPOLOGY: l~near
(ii) MOLECULE TYPE: cDNA
(xl) SEQUENCE DESCRIPTION: SEQ ID rlo: lo:
GCTGA,.GCTT C~CTTACTTC AC-C 24
(2) INFOP~ATION FOR SEQ I3 NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(.~l LENGTH: 65 base pairs
(31 TYPE: nuc!e:c acid
(C) STRANDEDNESS: sinyle
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DZSCRIPTION: SEQ ID NO: 11:
GCGGATCCAA AGACAGCAGG TGTTCACCGT CGACGACGCC TACGT.3ACTT CTCACAGAGC 60
CACTG 65
(2~ INFORMATION FOR SEQ ID NO: 12:
(i) SEQ"ENCE CHARACTEP.ISTICS.
(Al LENGTH: 46 base pairs
SU~STITUTE SHEET (RULE 26)
W 096/02640 . .' 2 i ~3~ 1 3 PCT/GB95/ot6Cl
~ ~ 48
lB ! TY?E: nucLeic ac~d
~CI STRANDEDNESS: s~ngle
~D) TOPOLOGY: linear
(LiJ HOLECULE TYPE: cDNA
(xi) SEQUENCE DESCR~PTIOI: SEQ ID NO: 2:
GCTGP~.TICG TT.~.caia.AT GTGAGGTG.~G CCGGGAGC-G AAAGAC :5
(2) rNFoR~lATloN FOR SEQ ID NO: 13:
(i1 SEQUENCE CHARACTEP~ISTICS:
~A) LENGTH: 54 base ralrs
(B1 TYPE: nucLeic acid
~C~ STRANDEDNESS: s:ngle
(D! TOPOLOGY: linesr
(ii) l~OLECUL~ TY:-_: cDNA
(XL) SEQUENCE DESCRIPTION: SEO I3 l30: 13:
GCGGATCCGP mCGCTTGTC.T GTCAT.~CC. CTGGTPTG.~.? P.CGCGGTP.CA GACC 54
(21 }NFOR.~TION FOR SEQ ID :IO: 14:
(i1 SEQUENCE CHAR~CTERISTICS:
(al LENGTH: 6g basc pairs
(?) TYPE: r.ucieic acld
(~I STRANDEDNESS: s!ngle
~D! TO?OLOGY: linear
( Li 1 HOLECULE TYPE: cDNA
(xi1 SEQUENCE OESCP.IPTION: SEQ ID NO: 14:
CCGGATCCGA TCGTACA~A CAATGACAGC ACAGAATAmG GACTCCTCCA GATAAATAAT 60
A~AATTTGG 69
~2) TlJFoRl~ATIoN FOR SEQ ID NO: iS:
(i) SEQUE~JCE C~:P.RACTERISTICS:
~A! LENGTH: 34 base raLrs
(B) TYPE: nucieic acid
SUBSTI~UTE SHEET lRVLE 261
W 096/02640 '~ 5 ~ 3 r~,5. 1651
49
(Cj sTRArJDEGtJEss singLe
l~) TOPOLOGY: linear
(ilj MOLECULE TYPE: cDNP,
(xl1 SEQUENCE DESCP~IPTION: SEQ rD rlo: lS:
GCTCTAGATC ATCATCCAGC AGCTCTGGCA GGAG 34
(2j INFORMATrON FOR SEQ ID rlo: 16:
(:) SEQUENCE C.:APiCTERISTrCS:
(A~ L-NGT~: 264 base Fairs
(E) TYPE: nucleic acid
(C) STRANDEDNESS: double
(Dl TOPOLOGY: llnear
(il) /MOL.ECULE Typr: cnNA
(Xl) SEOUENCE DESC?.IPT}ON: SEQ ID NO: 16:
GGATCCAAAG TTGGCT.~AAC ACTGGCCGGG TGCAGTGCTT CCACCTGTAA TTCCAGCACT 60
TTGGAAGGCT GAGGTGGGCA Gr.TTGCTTGA GGTC.'!GG.i_r T-GP.G~CCAG C?TGGCTAAC 12C
AGCAAPACCC TGTCTCTACC .~ AGTACAA P.AP.T-ATCTG GGI~ ~u CAGGCGCCTG 13G
TAATCCCAGC TACTCGGGAG GCTGAGGCAG AAGAATTGTT TGAACCTGGG AGCCAGAGGT 240
TGTAGTGAGC TGAGATCGCT CATT 264
~2) IrJFoR~ATIoN FOR SEQ ID NO: 17:
(l) SEQUENCE C~ARACTERISTICS:
~A) LENGT.-': 303 base p2Lr~
(B~' TYPE: nucleic acid
(C) STRP.NDEDNE~SS do~bL~
(D) TOPOLOGY: li~ear
(ii) MOLECULE TYPE: cDrJA
(xi) sEQuErJcE D~5CP.LPTION SEQ Ir~ NO 17:
TCTTTTTCAA TTATTCATTT GTTACAGTGG GTTATGATAC .iAATGTTTAT AGATGCCTAC 6C
SUBSTITUTE SHEET (RULE 26)
W 0~6~2640 ,; ,~ , 2 1 93~13 r~ 7."~165
5'~
TCTGTACTAG TACT.ACACAG CAC.-TT.CT GTG,TTT.- - .;T T5ACTTCAA- TGT,;CICrC: 12~.
TGAGTTCTAT W-v.~r.r~T'_CAT GTATTAAATC, AAATAAAcr~r~ hCAAAATGCC ATGTTCTTTG 180
GTACAAGCAA CACTCACCh~; AGGCATTTGG CGTCTGCATT TGCAATTCTC ACCC,AAACTC 240
L ~1 U CTACTCTCTA C.TATTTTCC CCACACTACC TT.rTCTATAT ATATTTTTCA 300
GATTGGAGTI ~UOU~I~LL~ CCCACGCTC~G AGTCCAGTGG CACCATTCTT GGCTCACGAG 360
ACCTCCACGT CTTGGGTI'AA AGCGTTTC-C CTCCCTCACC CTCCTGACTA CTCCGATTAC 420
AGGCGCC-GC CACCATGCCC GGCTP~;rTrT TGTATTTTT.r. GTAGAGATGG GCTTTCACCA 460
TGTTGCTC~C CCTGCTCTTG A~;CTCC,CCP. CCTCGGCCCT TCCCA~-GC GCTCCGATTA Sq0
CAGGTGTGAG CCACACTGCC TCGCCTGTP.C ATTTTTTAAA TTTCP.ATGTC TA.rTATGGTG 600
TCCACTGP'~T TAAGAATTCT TTTGAGAAAA TGAATCAAT.r. PATCTATACA OL~1UUII 660
TArCCAGTGA GGTATGGCTG G.;TC.'.GCTTC ATGACAT.~C.r. -GCCAG.ACT TcTcCrcCTC 72C
~ .L~ l ACAAATAAhA AT-GTATP.TG TTGAACG-CT .'.CAACTTG.riT GTTTGTTATP. 730
TGTATACACT TPUAATGTCAC CAC 803
~2) INFOR~rTION FOR SEQ D IIO: la:
(i) SEQUE~ICE C'rlARACTE~ISTICS:
(A) LENGT~: 233 base prlirS
~3) TYPE: r,uc lo ! C acLd
(C) STPUJDEDNESS double
~D) TOPOLOGY: llnear
(li) MOLECUEE T'YPE: cD~lA
(~i) SEQUENCE DESCPI?TrOrl: SEQ ID NO: lB:
TTTGCGTP.GA ACrCAGACAGT AAACTTGCTG Il_l~LI~_~ C.. G.ArCTTTT GTTGAGP.TGC 60
TGAATAGGAG GCAGCATGGC AGCTGAGCTA TCTGTTCT5C ---CTCTACC ru~rrl~i~ i2D
TCCCTTAGGC cT~r~AAATGAA GCTCTAAGCC PAGCA.~CGT C-GAAGTCAT CCAGAcTAr.~ lB0
IGGGAAGCGG GTAGGCTCCA GGGAGTGGCT CTCAGAGA:C AGACCP.TTTA CTGAGCTC 338
r2) INFOI~ATION FOP SEO ID NO: 19:
~-! SEQUENCE Cr.A'.~CTEP.ISTICS:
'A) LENGTP:: 162 ba66 pa~rs
(B) TYPE: nucl~c acid
SUBSTITUTE SHEET (RULE 26)
W 096/02640 . ~ - ' r ~,5!01651
~ 5~ 3 5 ~ 3
~C1 STRAr~DED~IESS ioubl.
(rj TOPOLOCY: llnear
~il) MOLEC~LE TYPE: cDNA
(X1) SEQUENCE 3ESCPIPTION: SEQ ID NO: 19:
AATACAGACT l L1~1 L~l - - ACTCLTATCC I~CL L I~L-;~ TCCCTCCTAC lLlL.~ ~A 60
CACCTATCTT GTTGTGAAGA CAGG~TTGC ATT.~CATAAA ATCA~ TCTT TTTTATTTTT 120
TTTTGAGATG GAATCTTGCT CTGTTTCCAG CCTC.GAGTGC TG162
(2) INFORMATION FOR SEQ ID NO: 2D:
(i) SEQUEN'CE CHARACTERrSTICS:
A) LENGTH: 472 base pairs
~3 j TYPE: nuc!eic aciù
~C) STRANDEDNESS: doub ie
~D) TOPOLOGY: !:~ear
(ii) MOLECULE TYPE: cDN.'.
(x~) SEQ'uENCE 3ESCRrPTrO';: S~Q I3 NO: 2G:
ATCTGGTCAG CAGTGAAGCT CAGTGTACAC ATTCP.TTCCT TCCTI'CACTG CTTGATTTGT 60
CACCAAGTGG TTATTGAGGA TATGCTG-TT GCT.9GGTACT ACTTTACTTA TTTATTTGTT 120
TATTTAGAGA TGGGGTCTCr CAATGTTGC;' CAGTCTACAG GAC.;GTGGCT P.TTCACAGGT 180
GTGAGCACAG CACACTACAG CCTCAAACTC CTGA5TTCPA GAGATCCTCC TGCCTCAGTC 240
TCTCGAGTAG CTGGGACTAC AGGGP.TGTGC CACCACACAT GGCTTAGGCT CTACTTTAGC 300
TGCTACTTGA AGGATGAAC.A T.;GGAGGAGA CACTCTTATT TT.;TTTGATT Ll_Ll L 1 ~1 1 1 36G
111~L~IL. L TTGACAGAGT TTTGCTCTGT TGCCAGGCTG GAGTGCTCAC TGCAACCTCC 42D
ACCTCCAGGT CAAGCPATTC TCCTGCTCAC CCTCCGAG-A GT''5GACCAA GG 472
~2j INFORA~ATI3N FOR SEQ ID NO: 2!:
~i) SEQUENCE CHAP..;CTERIS-ICS:
(A) LENGTH: 2}1~ base pairs
~31 TYPE: nucLe1c acLd
(Cj STRANDEDNESS: double
(D~ TOPOLOGY: llnear
SUBSTITUTE SHEET ~RULE 26~'
WO 9~il0264 , " t ~ 52 ~ I 5 3 ~ ~ 3 ~ ~ "~ O~
~ MOLEGULE TLPE: C:DI~P.
(x13 SEQUE~CE DESC~I~TlOII: SEC ID NO: 21:
GGAATTCCCA LL~-L~I-L~I GTACCCTTGC AGTGCCTCTG GGTGCAATGC GGAGA'~ATGG 60
AGTGGCTCC.A Oll~L~IL~I GTTTCTGP-~C ATG?~TCTCT TGCTATC.~GA ACTTTCTGCT lZ0
CATCCCTTCT GGCACACC-3~- G~TCCTCCAC A~TCCCTTCA CTCATGCC.;C TTCATATACT 180
GGTTATCCAT GGTACAGA.~G ACAGG~TTT.3'. ACTGAGAGGA ~111 - -L IG ACI'CTGAATA 240
CATGTAGGAG hT.3'ACGATAT GG~AGACCTT CAGTATGTPA GTC?TAAAT.~ GATTGGTTGG 3GO
GATAAATGTT CCCTGAA9CA TAAGPAACAG CGCAC~CGGCT CCTGTCTGTA ATCTAGCACT 360
TTGGGAGGGC CGAGGCCAGG C.'LGGCAAATT GCCTGAGCTC AGAAGTTTGA GACCAGCCTG 420
GCCAACATGC AGAAACTCCG TCTCTACTAA AAATACATA.9 ATT.3ACCGC.G CATGGTAACA 480
CGTGCCTGTA GTCCCAGCTA CTCGGGAGGC TGP.GCCAGGA GAATCACTTG AGCCTGGGAG 545
GCAGAGGTTG CAGTGAGCCA AGATCGCGCC r.CTGCATTCC AGCCTGGGCA ACAG.'.GTGAG 60Q
ACTTGGTCAA AAAAAAAA.AA A,3AA.3'A3LAAA AAP.LGGAAGA AGAAGAAGAA P.TCAGGTTTA 660
GAG.;TGAGG.A CP9AGAAG.'C GAATCGGTGG CATGAAGG;LG CTAAGAGC?A C-TGTCACCA 720
TGACATGAAG CTTCATGCCA GC.3'..AATTAA.'. GGAGCTA?TC P.GAACTAGTA TCCTC.3U~CTC 780
TACTTGCTC.i GGGGCACTGA CCTTATAGhG ATTCCAGACA TAAGCTTGTT CAGCCTTAAG 840
TCCALTCTTT CCACTGGCTT ~L - _I L~'~ ACTT?CTGTG GCCAACTCTG AGGTTGTCTA 900
CAAGTTATTG GTCTTAGATT TATGTAATGT CTCAATGCCA GTGTAGTAT? TGCTTATTTA 960
CGGTAGGAGT GGTTAGGGGT GGGGAATCTG ATAATAGCTC GTAGGATAGC TAGATTCTTT 1020
LLLLlLI L ! TTTTTTTT.~A AGATAGGC-TC TCACTTTGTC TCCCAGGATC- GA?GGATGGA lG80
GTGCAGTGGA GTGPACATGG C?CACTGCAG CCTCGP.CCTC CTGTGCTCAA ~ ~ ~OL~ 1140
TGCCTCAGCC CCTCAAGTAG CTGGGACTA.C AGGC.AC;L.LC? CACCATGC.-C AGCTA.3rTTT lZ50
TTTTGTAG.3G ATCGC-P.TT?T ACCArGT?GC CCP.GGCTGG- CTCGAGCTCC TGGGCTCAAG 126;J
TGATCCACCA GACTCGGCCT CCCA.4AATGC CGGGATT.~CA GCTGTGAGCC ACTGTGCCTG 1320
GCCTAGATGC TTTCATACAG GCTTTTCA.AT T.'TGCAT'rrr CCTTAAGTAG GL~GTCTTAA 13a5
GATCCAAGTT ATATCGGATT GTTGTAGTCT ACGTTCCCAT ATTCTATTCC TP.TTTCTGAG 144.l
SUBSTITUTE SHEET ~RULE 2ti)
~ W 096~02640 2 t 9 3 5 1 3 1~ a~OlG .
CCTTC,jGTC.i TGAGCTAC ~ TT ~;Gr~ CT.'_;~.T.'.~ C_TTC...'.C .'TGG_TGG.:T i;OJ
TGGTTGGACA P.GTGCCAGCT CTC~A~CCTGG GACTCTGGC.; TGTGATGACA TACACCCCCT 1560
CTCCACATTC TGCATGTC C TAGG'GGGAA GGGGGA~C. '_GG.ATAGAA CCTT;.iTT'? 7620
ATTTTCTGAT TGCCTCAC.T CTTA~--TTGC CCCC..TGCCC ~.L L !~L ! - CTCA'.GTAAC 1680.
CAGAGACAGT GCTTC.CC.9G; ACCA.CCCT.~ C.~.~GA.;.~C.;. .~GCGCTP.AAC A.iAGCC,'u~iT 1740
GGGAAGCAGG ATC.iTGG-TT GA-~C~C-TTC TGGCC.iGiGr rC..~ATACCTG CTATGGAcTA 1800
GAT.;CTGGG.i GL.GGG.~.iGG .~i.~AC ;GGG TG---}.T-'TGG ;';GGA.iGCTG GCAGGCTCAG 1860
CGTTTCTGT_ TTGGCATG.':C C.~GTC CTCA TCA ICTCTT C'T.'.G.'TGIA GGGCTTGGTA 19~0
CCAGAGCCCC TGAGGCTTTC -GCATG.~.'; A T.~'T.~A~TG AA~'TGAGrG ATGCTTCCAT 1980
TTCAGGTTCT TGGGGGT.~GC C.'Ar..-.~.iGC TT'.TTG C~ CTCTCT-CCT CC'TGGGCA-C '04G
~ C_,;TCCTC~ .'C.~.................... '.. :~~~~ . ~_~-.; C~GC-G.~ 0
GAC,iTAGA.C. G-!.i-GG.i~ -L119
SUBSTITUTE SHEET (RULE 26)
W096/02640 ~ ~ r P~ Iru._ !rnl651
j4 2 ~ ~3 ~ ~ 3
lNDlCAI lONS }3~:LA~ING TO A DEPOSITED hnCROORGANIShl
~PCI Rule 13bis)
A . Thc ind~ ons rrl~de below telu le to the ~ relerred to in ~be descripBon
on p~ge 9 , iine 13-1 S
B. }DENT~iCATlON OF DEPOSIT Furtber deposits 9rc idtnrified on ~n ~ddi~ional shce~ iO
Name of deposiary institutiun NATIONAL COLLECTIONS OF INDUSTRIAL AND
MARINE BACTERIA LTD
Address of deposiurry insdtution (i~:tlulinl~partalco/tcarutcou~urr)
23 ST MACHAR DRIVE
ABER,DEEN AB2 lRY
SCOTLAND
UNITED KINGDOM
D~e of deposil ¦ Accession Nutnber
15 FEBRUARY 1995 ¦ NCIi'~B 40709
C. ADDITIONAL ~DICATIONS flaavcbtariifrclapplicablcl This inform~tion isi continued on~n ~dditionrl sheet O
~.~rH~RTrHTA COLI DH50~ F' (K12)
CONTAINING PLASHID pHA-2
D. DE.SlGl~ATErl STATES FOR WEIICII INDICATIONS ARE .5~DE fiJrtusirulicuiorlrarcr~tforaUreri~atct~r~s
ALL STATES
E. SEPARATE FURNlSnTNG OF INDICATIONS ItarYc bu~u4 ilnGI applieablc~
Thcindirttionsiisledbeiowwillbesubminedtotb-lnt-rnalionslBuraul~ter(.~ b~ln~uurcofrhc~ri~ucarior~e~ Aecr-rion
NuntbsrrfDapcrf~ j
AC OE SSION ~iO : 40709
DATE OF DEPOSII' : 15 FEBRUARY 1995
CONSTRUCT DEPOSITED : pHA-2
i'or receivin~ Of ~ce use only For Intetlurionul Bure-u use only
Thi5 sbee~ WIS reaived wilh Ihe intern~lion~l IppliGttion O This shect wss rcceivcd by the Intern~tion~l Burau ort:
~11 SEPTEMBER 1995
Authori~cd of ~Iccr AutboriIcd of Bs es
D. J. MAcKERNEss tMRSl
ortn PCI/ROtl3~ (Julv i992)
