Note: Descriptions are shown in the official language in which they were submitted.
1 30967~
~OE R4/F 109
The invention relates to a signal peptide whic
is a constituent of a propeptide which, in a Streptomyces
cell ~hich contains a signal peptidase, is cleaved into
the signal peptide and a polypeptide, the latter being
removed from the cell and excreted into the culture medium.
The invention further relates to DNA sequences which code
for this signal peptide, gene structures which contain this
DNA sequence in the reading frame with a structural gene,
plasmids which contain a gene structure of this type, and
host organisms containing plasmids of this type. Further
aspects of the invention and its preferred embodiments
are illustrated in detail below.
A process for the preparatîon of tendamistat by
fermentation of Str ~ o~ces ten~ae~ha~Oa ~e3~d~ n pro-
~15 posed, in ~erman ~ , ~hichprocess comprises using S~ tendae strains which produce
tendamistat and have been treated ~i~h sublethal doses of
acriflavine. A DNA fragment containing the gene ~or ten-
damistat was isolated from strains thus obtained, namely a
2.3 kb Pst I fragment. It was possible, by incorporation
of this fragment in pBR 322 which had been cut ~ith Pst
I to amplify this DNA in E~ coli and to re-isolate this
DNA in pure form.
It has now been found that the signal peptide
(prepeptide) of the formula I
Ar~-Val-Ar~-Ala~Leu-Arg-Y.-Ala-S~r-Ala
(I)
;n wh;ch X represents a hydrophob;c region compr;s;ng 10
to 25, preferably 17 to ZO am;no acids (most likely 20
am;no ac;ds)~ ;s coded on this 2.3 kb fr3gment ;mmed;-
ately upstream of the structural gene for tendam;stat.
It has also been found tha~ by use of th;s signalpept;de other peptides are excreted from host cells which
conta;n an appropriate s;gnal pep~idase~ Thus the inven-
~'ij9
'~
1 3~961~
-- 3 --tion also relates to propeptides of the formuLa II
Sig-R (II)
in ~hich Sig denotes the amino acid sequence of the formula
I, and R represents the residue of a genetically c~dable
peptide linked by its amino terminal end, in ~hich an
acidic amino ac;d, ;n part;cular aspart;c acid, is prefer-
ably located at the am;no terminal end.
Thus, one aspect of the invention relates to pep-
tides of the formula II in which R denotes hydrogen or a
peptide residue, for example the tendamistat residue~
Another aspect of the invention relates to the correspon-
ding DNA sequence which can be obtained from Streptomyces
tendae strains which produce tendamistat and which have
preferably been treated with sublethal doses of acri-
flavine, by isolation of the total DNA, digestion ~ithPst I, Southern hybridization ~ith the DNA sequence A,
5'-(32P-)CCT TCh GmG TCG T~T T~G r~ 3' !A)
isolation of the 2.3 kb Pst I fragment, cutting ~ith BamHI,
Southern hybridi~ation with the sequence A, isolation of
the 0.94 kb PstI BamHI subfragment, cutting with Sau 3a,
Southern hybridization with the sequence A, isolation of
the 0.~25 kb BamHI Sau 3a subfragment and sequencing of
the DNA, and which has the following features:
a) it is located immediately upstream of the tendamistat
structural gene,
b) it codes at the amino terminal end ~or
Me~-Arg-V~l-Arg-~la-Leu-Ar~,
c) it codes at the carboxy terminal end for
Ala-Ser-~la
3~ and
d) it codes in the middle for a hydrophobic region which
comprises 10 to 25, preferably 17 to 20 amino acids.
This DNA sequence is called sequence 8 or signal
peptide in the following textu
The kb figures wh;ch were determined by compari-
son with standard markers have the customary accuracy.
In place of the sequence A, it is possible to
select for the Southern hybridization any desired sequence
~hich is complementary to the tendamistat gene or the
1 30967~
-- 4 --
counterpart strand.
For the various steps for the characterization of
the DNA sequence B, in each case in practice the DNA is
introduced into a suitable vector, the latter is trans-
formed into a host cell, amplified there, the transfor-
mants determined by colony hybridi~ation with the sequence
A, and the DNA reisolated. These steps are known per se.
The DNA sequence C, whose coding strand is repre-
sented in the attachment, has the nucleotide sequence of
10 the tendamistat structural gene from S. tendae.
Thus, the gene structures mentioned contain the
DNA sequence B in the reading frame with a structural gene
which codes, for example, for tendamistat, preferably the
DNA sequence C. ~
The invention also relates to plasmids which com-
prise the DNA sequence 9 in the reading frame with a
structural gene which codes, for example~ for tendamistat,
in particular for the DNA sequence C. These plasmids can
contain a replicon which is effective in E. coli, and are
20 then able to amplify and, possibly, also to express the
DNA in E. coli.
Preferred plasmids additionally contain a replicon
which is effective in Streptomycetes. If a Streptomyces
is transformed with a plasmid of this type~ it becomes
25 able to express the peptide, which is determined by the
structural gene~ in the form of the propeptide of the
formula II, which is then cleaved by a signal peptidase
during the processing, and the desired peptide is excreted
into the culture medium.
So-called shuttle plasmids which contain both a
replicon which is effective in E. coli and one which is
effective in Streptomycetes are also advantageous. These
shuttle vectors can be amplified in E. coli and, after
re-isolation~ transformed into Streptomycetes, where
35 production of the desired polypeptide then takes place.
The invention also relates to host organisms which
have been trar,sformed with the plasmids mentioned, in
particular host organisms of the genus Streptomyces,
especially of the species S. tendae or, in particular,
1 30967~
S. l;vidans~
Furthermore, the invention relates to a process
for the preparation of a polypeptide of the general for-
mula III
H2N-R (III)
in which R has the meaning indicated for formula II, in
which is used one of the transformed host organisms men-
tioned, which contains a signal peptidase which splits off
the propeptide of the formula II and secretes the desired
10 polypeptide.
While a plethora of vectors are available for Gram-
negative bacteria, only a few vectors have been described
for Gram-positive bacteria, in particular for Strepto-
mycetes. Vectors for bacteria of the species S. tendae
15 have not hitherto been disclosed~ Thus, an approach for the
utilization of S. tendae as host organisms is made possible
by the invention.
A particular advantage of the inven~ion is that
transformed Streptomyces strains, in particular S. lividans
20 strains, sporulate optimally, that is to say the content
of recombinant plasmid does not adversely affect these
strains in their generative phase. Thus, the transformed
organisms are also suitable for further strain improve-
ments, for example for the production and selection of
25 metabolic mutants involving the use of spores.
Compared with untransformed strains of S. tendae,
the transformed strains, in particular S. lividans, do not
form melanin. Thus, there is no necessity to remove it,
and this makes the isolation of the desired peptide, for
30 example tendamistat, considerably easier and it prevents
losses in yield.
Another advantage oF the invention is that foreign
genes are also expressed in S. lividans, and the corres-
ponding polypeptides are excreted, which likewise offers
35 a variety of possibilities for strain improvement and for
modification of the polypeptide thus produced.
~ lowever, it is also possible according to the
invention to transform other species of Streptomyces,
for example S. ghanaensis or aureofaciens. When strains
l 3ns67~
which contain no plasmid and are able to synthesize a
specific signal peptidase are transformed with ~he hybrid
plasmids according to the invention, then stable trans-
formants ~hich express and secrete the coded peptide are
5 obtained.
Particularly preferred embodiments of the inven-
tion are illustrated in detail in the examples which
follow. In these examples, percentage data relate to
weight unless other~ise specified. The figures repre-
10 senting the hybrid plasmids show the restriction sitestrue to scale.
The following vectors, known from the literature,
were used in the examples: single-strand phages M 13 mp 8
and M 13 mp 9: Messing et al., 6ene 19 (1982) 269; pUC 8:
15 Vierra et al., Gene 19 (1982) 259; pAC 177 and 184:
Chang et al., J. Bacteriology 134 (1978) 1141; pIJ 102
and 350: Kieser et al., Mol. Gen. Genet. 185 (1982) Z23.
Maintenance of the S. tendae strain is described
;n U.S. Patent 4,226,764. In principle, the tendamistat
20 gene can be isolated from any strain which produces ten-
damistat. However, the procedure of German ~sh
3 ~ ~6~'c~ ~ ~is particularly advantageous~ the iso-
lation of the DNA being described ;n Example 3 therein.
This isolated complete DNA is the starting material for
25 Example 1 below.
Example 1
5 ~ug of DNA is completely digested with the res-
triction enzyme Pst I and, after fractionation in a 0.8%
agarose gel, transferred to nitrocellulose filters
30 (Southern transfer). The filter with the bound, denatured
DNA is prehybridized for 6 hours in 5 ml of prehybridiza-
tion medium (0.6 M NaCl, 0.06 M Na EDTA, 0.1X sodium
dodecyl sulfate solution, 100 ug/ml sonicated calf thymus
DNA and 4-fold concentrated Denhardt's solution). It is
35 then again treated with 5 ml of the prehybridization medium
to which, however, 500,000 cpm/ml of radiolabeled DNA have
been added. This radiolabeled probe is obtained as
follows:
The DNA sequence A is synthesized chemically by
1 30~67~
7 --
the phosph;te process. It contains 20 nucleotides (mole-
cular ~eight about 13,000) and is complementary to the
putative 9NA sequence for tendamistat~ deriYed from the
amino acid sequence of tendamistat beQinniny from amino
acid 37 of tendamistat, using the triplets preferred by E.
coli. This DNA sequence A is radiolabeled at the ~' end
using r-32P-ATP and nucleotide kinase.
For the hybridization of this radioactive probe
to the complementary DNA sequence in the complete DNA, the
10 mixture is allo~ed to stand at 37C for 24 hours. Then the
non-bound radioactive DNA is removed, and the filter is
washed at 37C with 5 x 200 ml of hybridization medium
for 30 minutes each time, and then subjected to auto-
radiography. After exposure for 24 hours, the hybridiza-
15 tion signals show that the gene is located on the 2.3 kbPst I fragment. This fragment is obtained by electro-
elution of a section corresponding to this fragment size,
cut out of a preparative agarose gel on which the Pst I
digested total DNA had been fractionated. The eluted DNA
20 is cloned in the Pst I restriction site of the plasmid pUC 8.
These hybrid plasmids are transformed into E. coli
JM 103 and are amplified. The clones which carry the
insertion with the desired tendamistat gene are detected by
colony hybridization using the radioactive DNA probe A. The
25 hybrid plasmids pKAI 1a and 1 b thus obtained are represen-
ted in Figures 1 a and b.
The localization of the gene can be determined
exactly by further Southern hybr;dization steps against
the isolated 2.3 kb Pst I fragment and its subfragments
30 (Figures 2a to 2c).
Example 2
The 2.3 kb Pst I fragment from S. tendae is cloned
in the unique Pst I restriction site in the plasmid pIJ 102.
The hybrid plasmid pAX 1 a and 1 b thus obtained, ~hich
35 differ in their orientation of the insertion, confer the
ability to produce tendamistat on S. lividans strains
after hav;ng been transformed into them. Figure 3 shows the
plasmid pAX 1 a.
1 30~67~
- 8 -
Example 3
The commercially available strain S. lividans
TK 24 (John Innes Institute, Norwich, England) is converted
;nto protoplasts in known manner, and 108 protoplasts
5 are added to 1 ~9 of hybrid plasmid pAX 1 a in the pre-
sence of 20% polyethylene glycol 6000. The transformed
protoplasts are incubated on regeneration medium R2YE
(Thompson et al., Nature 286 (1980) 525) at 30C for
5 days.
The formation of an extracellular amylase inacti-
vator can be demonstrated by a plate test:
5 ml of an aqueous solution containing 0.4 to
1.0 mgtml pancreatin is poured over the regenerated colo-
nies, and the mixture is incubated at 37C for 1 hour.
15 The solution is then removed and replaced by 5 ml of a
2% starch agar. AFter incubation of the plates at 37C
for 2 hours, 5 ml of an iodine/potassium iodide solution
is poured over them to develop them. Colonies with a blue
halo indicate that the clones synthesi~e and excrete ten-
20 dam;stat.
As a check, the plasmid DNA of strains which pro-
duce tendamistat and sporulate well can be isolated and
mapped. All strains which produce tendamistat carry
pAX 1 plasmid DNA.
25 _ample 4
The process is carried out in accordance with
Example 2, but the plasmid pIJ 350 which carries a thio-
strepton-resistance gene as a selectable marker in Strepto-
mycetes is used. The hybrid plasmids pAX 350 a and b are
30 thus obtained (which differ in the orientation of the
insertion). Figure 4 shows the plasmid pAX 350 a.
After transformation in accordance with Example 3,
resistant clones are selected on minimum medium (Hopwood,
Bacteriological Reviews 31 (1967) 373 - 403) in the pre-
35 sence of 50 ~g/ml thiostrepton, and are tested for pro-
duction of tendamistat either directly on minimum medium
or after being transferred to non-selective R2YE agar.
9 ~ 7 aT
xample 5
Hybrid plasmids ~hich contain the 2~3 kb Pst I
fragment and, in addition to the Streptomyces replicon~
contain an E. coli replicon have a number of advantages as
shuttle vectors; because of thç E. coli replicon and re-
sistance markers effective in E~ coli~ they are able to be
well amplified in these organisms. After isolation and
transformation into Streptomycetes, in particular into S.
lividans, they have high stability~ As a consequence of
their selection markers uhich are effective in Strepto-
mycetes, and of the Streptomyces replicon, they can also be
well amplified in these organisms and can express and
secrete tendamistat.
The plasmid pAC 184 is completely digested with
the restriction enzyme Sal I, and the en2yme is removed by
extraction with phenol/chloroform. The protruding 5' ends
are filled using the enzyme DNA polymerase (Klenow frag-
ment) in the presence of ATP, CTP, GTP and TTP. A Pst I
linker of the structure
5 ' TCG AGC TGC AGC TCG A 3 '
3 ' AGC TCG AC~ TC5 AGC T 5 '
is ligated to the blunt ends in a ligase reaction at 22C
(2 ~9 of linker to 0.4 ~g o~ PNA). The DNA is extracted
with phenol/chloroform and, after precipitation, is diges-
ted with the enzyme PSt I to obtain Pst I ends which can be
25 ligated~ The Pst I ends of the vector are then dephos-
phorylated, again extracted with phenol/chloroform, and
l;gated with plasmid pAX 1 a which has been partially
digested with Pst I. The ligation mixture is transformed in-
to E. coli (HB 101 or MC 1061). Clones resistant to chlor-
amphenicol are rinsed off the plate, and the plasmid DNA isisolated. S. lividans TK 24 is transformed with 1 to 2 ~g
of plasmid DNA and tested for production of tendamistat~
Clones with a positive reaction in the tendamistat
test are isolated, the plasmid DNA is isolated by rapid
alkaline lysis and introduced into E. coli HB 101 or
MC 1061 by back-transformation. Plasmids re-isolated
after amplification do not differ from the plasmids isola-
ted from S. lividans strains. The recombinant plasmids
,
`1 ~0967~
- 10 -
are designated pSA 2 a or b (Figures 5a and b) depending
on the orientation o~ the insertion which carries the
tendamistat gene.
Example 6
The process ;s carried out in accordance with
Example 5, but starting from plasmid pAX 350 a, selecting
in E. coli for chloramphenicol resistance and in S~ livi-
dans for thiostrepton resistance and production of ten-
damistat, and the plasmids pSA 351 a and b ~Figures 6a/b)
are obtained~
Example 7
The process is carried out in accordance w;th
Example 5, but starting from the plasmid pAC 177 in place
of pAC 184, and the plasmids pSA 3 a and b (Figures 7 a/b)
are obtained.
For this purpose, the plasmid pAX 1 a is par-
tially cut with Pst I, and the enzyme is heat-inactivated
by heating at o8C for 15 minutes. The DNA is ligated into
the plasmid pAC 177 which has been cut w;th Pst I, dephos-
phorylated and deproteinized. After transformation ofE~ coli HB 101 or MC 1061~ clones resistant to kanamycin
are rinsed off the plate, the plasmid DNA is isolated,
and S. lividans TK Z4 is transformed ~ith 1 to 2 ~9 of
this DNA. Clones which produce tendamistat are selected
and the plasmids are characterized.
Example 8
The process is carried out in accordance with
Example 7, but the plasmid pAX 350 a is used ;n place of
the plasmid pAX 1, selection in S. lividans for thio-
strepton resistance is carried out, and the plasmids pSA
352 a and b ~Figures 8 a/b) are obtained.
Example 9
As is evident from Figure 2, the gene which codesfor tendamistat and the signal sequence is ~v 0.3 kb long.
The 2.3 kb fragment used in the examples detailed above
can thus be used in a shortened form for the construction
of hybrid plasmids wh;ch br;ng about the product;on of
tendamistat:
The plasmid pKAI 1 a is d;gested with Sal I and
'I 30q67~
rel;gated. In th;s manner, the plasmid pKAI 2 which has
been shortened by about 750 base-pairs is obtained. It is
cloned, isolated and cut with Pst I~ The DNA is dephos-
phorylated using alkaline phosphatase from calf intestines,
and is deproteinized with phenol/chloroform.
The plasmid pIJ 102 is completely cut ~ith Pst I
and, after heat-inactivation of the enzyme, the fragments
are ligated in the Pst I restriction site of pKAI 2. The
ligation mixture is transformed into E. coli H8 101 or MC
1061. The plasmid DNA from clones which are resistant to
ampicillin is isolated by the rapid alkaline lysis process,
and S. lividans TK 24 is transformed with 1 to 2 ~9 of th;s
DNA. Clones wh;ch produce tendam;stat are seLected and the
plasm;d DNA from them ;s isolated by rapid alkaline lysis.
After re-transformation into E. coli HB 101 or MC 1061 and
after isolation of the plasmid DNA from the transformed E.
coli strains, the plasmid pSA 1 is obtained and charac-
terized by restriction analysis (Figure 9). The plasmid
shows no difference from the plasmids isolated from the S.
lividans strains, but the DNA work-up from E. coli is more
productive and possible in a shorter time.
The process is carried out in accordance with
Example 9, but the plasmid pIJ 350 is used in place of
the plasmid pIJ 102~ it then being possible additionally
to select for thiostrepton resistance in S. lividans, an d
the plasmids pSA 350 a and b are obtained. Figure 10
shows the plasmid pSA 350 a. In shake cultures, this
plasm;d leads to higher yields of tendamistat than does
the plasmid pSA 350 b which contains the insertion with
the tendamistat gene in the reverse orientation. In con-
trast, the reduction in size of the insertion to 1.5 kb
has no significant effect on the formation of product.
Example 11
To determine the structure and nucleotide sequence
of the tendamistat gene, the 0.94 kb Pst I/Bam HI sub-
fragment tFigures 2a and b) and 295 bp Sau 3a/8am HI sub-
fragment (Figure 2c) were cloned in the single-strand
phages M 13 mp 8 and M 13 mp 9. The primer used for the
`I 30967~
- 12 -
dideoxy sequencing reaction was the 20 nucleotide DNA
sequence A and a commercially available 15 bp primer
(Bethesda Research Laboratories GmbH, Neu-Isenburg). The
DNA sequence C ~as found.
Example 12
Upstream of the structural gene of tendamistat~
there is on ~he DNA an open read1ng frame up ~o the start
codon ATG (Met) for a protein ~hich is located immediately
upstream of the amino terminal end of tendamistat. This
s;gnal peptide corresponds to DNA sequence B.
Appendix:
DNA sequence C ~coding strand)
5'-GAC ACG ACC GTC TCC GAG CCC GCA CCC TCC TGC GTG
NH2-Asp Thr Tilr ~al Ser Glu Pro Ala Prc Ser Cys Val
ACG CTC TAC CAG AGC TGG CGG TAC TCA CAG GCC GAC
Thr Leu Tyr Gln Ser Trp Arg Tyr Ser Gln Ala ~sp
3s
AAC GGC TGT GCC GAG ACG GTG ACC GTG AAG GTC GTC
Asn Gly Cys Ala Glu Thr Val Thr ~Tal Lys Val Val
4~
TAC GAG GAC GAC ACC GAA GGC CTG TGC TAC GCC GTC
Tyr Glu Asp Asp Thr Glu Gly Leu Cys Tyr Ala '~al
sO 55 ~o
GCh CCG GGC CAG ATC ACC ACC GTC GGC 5AC GGC TAC
Ala Pro Gly Gln Ile Thr Thr Val Gly Asp Gly Tyr
ATC GG5 TCG ChC GGC CAC GCG CGC TAC CTG GCT CGC
Ile Gly Ser His Gly His Ala Ar~ Tyr Leu Ala Ar~
TGC CTT TAG-3'
Cys Le~ Stp
,,,,~,~ o~nle~ s~lri~
V ~ ~ 1 and 3 of German Pa~ pp~sc~4=--
Example 1
Mutat;on procedure: About 0.25 cm2 of sporulated
1 309~74
- 13 -
mycelium of the strain DSM 2727, which has been cultured
on oatmeal agar tEuropean Patent Application A 49,847), is
transferred into 100 ml conical flasks which contain 25 ml
of the following culture med;um:
Soybean flour Z %
Glucose 3
Corn starch 0.2 X
Urea 0.1 %
Ammonium citrate 0.1 %
Malt extract 0.5 %
KH2Po4 0.2 %
The medium is autoclaved at 121C and 1 bar for
30 minutes, and the pH is about 7Ø The inoculated
flasks are incubated at 30C on a shaking machine for 2
days. Then 2 ml of the precul~ure which has grown are
transferred into 20 ml of main culture medium in 100 ml
conical flasks (European Patent Application A 49,847):
Soluble starch 2-~ %
Soybean flour 0.4 %
Cornsteep liquor 0.4 %
Skim milk powder 0.7 X
Glucose 1.0 %
(NH4) ~PO4 1.2 X
After autoclaving, the pH of the medium is 7.6.
Acriflavine is added at a concentration of 10-25 ~gt
ml to this medium as the mutagenic agent. The culture is
shaken at 220 rpm for 5 days at 28C. The culture solution
is then centrifuged (1,300 x g, 5 minutes) and the cell
pellet is washed in main culture medium~ The washed cells
are fra~mented using a glass homogenizer and are plated
out on agar plates containing the following medium:
Sucrose 0.3 %
Dextrin 1.5 X
NaCl 0.05 %
K2HP04 0.05 %
FeS04 x 7 ~l2 0.001%
Tryptone soya broth 0.5 X
(Oxoid)
1 3()967~1
~ 14 -
Meat extract 0.1
(Difco)
Yeast extract 0.2 YO
(Difco)
Agar (Merck) 1.5 X
The pH of the solution is 7~1 (autoclaving con-
ditions as above).
The plates are ;ncubated at 2~C for 5 days, and
individual colonies are transferred into slant tubes con~
tain;ng oatmeal agar. These are incubated as described
until the mycelium has sporulated. The sporulated mycelium
is propagated as described above in the preculture and
main culture, and the culture filtrate after a 5-day main
culture is tested for its content of tendamistat tcF.
European Patent Application A 49,847). It ~as possible in
this manner to select the strains I-9353, I-9362, I-9417
and I-9418 which produce, during their period of growth
under the conditions described, about 90,000-100,000 IU
tendamistat/l culture solution~ corresponding to 1.5-1~ 9
inactivator/l.
Example 3
Isolation of the deoxyribonucleic acid from S.
tendae and determination of its molecular biological
characteristics.
The cells are cultured in accordance with Example
1. After growth for 3 days in the main culture medium,
the cells in a conical flask are harvested by centrifuga-
tion at 3~000 x 9 and 4C for 5 minutes and are washed
twice with 50 ml of a solution of 50 mM Tris/HCl buffer,
30 100 mM NaCl and 25 mM EDTA. In a typical preparation,
3 9 of cells which have been compacted by centrifugation
are instantaneously deep-frozen in liquid nitrogen at
-198C and then homo~enized to a fine powder in the pre-
sence of N2 using a blade-homogenizer tWarring Commercial
~lender). This powder is taken up in 10 ml of the above-
mentioned bufFer, dissolved on ice and incubated with
0.8 ml of lysozyme solution ~30 mg/Ml) at 2~C for 20 min-
utes, shaking gently~ 0.~ ml of a proteinase K soluticn
1 3 n~67/1,
- 15 -
tl5 mg/ml) and 0.8 ml of a 2~% solution of the sodium salt
of N-lauroylsarcos;ne are then added to the suspension.
After careful mixing, the mixture is ;ncubated on ice
for 30 minutes and at room temperature for a further
5 15 minutes. After addition and dissolut;on of 22 g of
solid cesium chloride, the volume is made up to 22 mL
using the abovementioned buffer, and the suspension is
centrifuged at 12,000 x 9 and 4C for 25 minutes. Ethidium
bromide is added to the clear centrifugate and the refrac-
10 tive index is adjusted with CsCl to n = 1.3920 using arefractometer. After preparative ultracentrifugation
(120,000 x 9, 36 hours, 15C), the band of chromosomal
DNA is removed from the centrifuge tubes and is recen-
trifuged under the same conditions. The ethidium bromide
15 is removed from the isolated band by extraction with n-
butanol, and the band ;s dialyzed to remove all the
CsCl. The DNA thus obtained can be cut with, for example,
the enzymes Pst I, Bam H I, Sau 3A, Cla I, Bgl II and ~ho
I, and cannot be cut with, for example, the endo-nucleases
2a Eco R I and Hind III.
Compared with the DNA from ATCC 31210 and DSM 2727
the DNA obtained from, for example, the mutants I-9353 and
I-9362 has an amplified genetic element, the individual
fragments of which, after staining with the fluorescent
25 dyestuff ethidium bromide, are clearly visible and stand
out from the background of fragments. The size of the
amplified element and the characteristic individual
fragments after digestion with restriction endonucleases
are shown below:
1 30q674
~ 16 -
FragmentFragment si2e (kbp)
No. Pst IXho I _ Bam_H I
1 9.0 13.5 7.2
2 7.0 11.5 6.1
3 6.8 9.3 S.3
4 4.6 2.8 2,9
3.4 0.7 2~6
6 3.1 2.5
7 2.3 2.15
8 1.1 2.1
~ 1 a 5
1 O 1 ~ 2
1 1 1 . 1
12 1aO
13 0.8
14 0.5
0.48
Total 37.3 3708 37.43
