The isolation and nudeotlde sequence of the complex AROM locus of Aspergillus nidulam
Ian G.Charles1, John W.Keyte1, William J.Brammar1, Melanie Smith1 and Alastair R.Hawkins1-2*
'Department of Biochemistry, University of Leicester, Leicester LEI 7RH and 'Department ofGenetics, Ridley Building, University of Newcastle upon Tyne, Newcastle upon Tyne NE1 7RU, UK
Received 30 December 1985; Revised and Accepted 10 February 1986
ABSTRACT.The AROH locus of A.nldulans, which governs five consecutive step3 in
pre-chorismate aromatic amino acid biosynthesis, ha3 been cloned in abacterlophage vector. The nucleotlde sequence of the locus reveals a single,open reading-frame of 4,812 base-pairs, apparently without lntrons. AnInternal segment of the A.nldulan3 AROH sequence has extensive homology withthe E.coll aroA gene that encodes the 5-enolpyruvylshlkimate 3-phosphatesynthase.
INTRODUCTION
Pre-chorismate aromatic amlno acid biosynthesis has been the subject
of intensive genetlcal and biochemical study in a variety of microorganisms.
The enzymes catalysing steps two to six in the pathway (see Fig 1, ref. 1)
have been shown to co-sediment within a number of genera (2, 3. t)• Genetic
analysis of the AROH locus in Neurospora crassa has indicated the presence
of a "cluster gene", containing five, distinct genetic units corresponding
to the five enzymatic functions (5). Biochemical analysis of the N.crassa
AROM-speclfled protein complex reveals a single, pentafunctlonal polypeptide
of 165 KDa which is active as a homodlmer (6,7,8). A similar conclusion has
arisen from genetic studies in Schlzosaccharomycea pombe, where a k.5 kb
mRNA species has been 3hown to be specified by the aro-3 locus (9, 10).
These findings contrast with the situation In E.coll, where the corres-
ponding blosynthetic genes are unlinked (11).
The genes governing aromatic amlno acid biosynthesis in several micro-
organisms are now being subjected to molecular analysis. A DNA fragment
containing the biosynthetic dehydroqulnase function of the A.nldulans AROH
locus has been cloned (12) and sequenced (13)- The entire aro loci from
S.cerevlslae (11) and S.pombe (10) and part of the N.crassa AROH locus (15)
Downloaded from https://academic.oup.com/nar/article-abstract/14/5/2201/2382022by gueston 11 April 2018
Nucleic Acids Research
These continuing studies should provide answers to a number of important
questions, lncludlng:-
(I) Is the AROM locus a single large gene, with or without lntrons,
or Is it a cluster of five distinct genes?
(II) What is the nature of the AROM-speclfled messenger RNA?
(Hi) What are the evolutionary relationships between the unlinked
aro genes of E.coll and the eukaryotic AROM loci, and between
the blosynthetlc and catabollc dehydroqulnase lso- enzymes in
A.nldulans?
(iv) How many functional domains within the AROM-speclfled
polypeptlde can be detected by molecular analysis and how do
they relate to the sequence of the AROM locus?
We have previously (13) addressed questions (11) and (Hi) for
A.nldulans. This communication describes the Isolation and presents the
coding sequence of the complete AROM locus of that organism.
MATERIALS AND METHODS
Strains
A white spored, pyrldoxlne-requirlng strain R153 (wA3; pyroAl) of
Asperglllus nldulans was used for the preparation of genomio DNA. The geno-
types of the bacterial strains used are shown In Table 1 of reference 16.
Media
Defined minimal medium and provision of nutritional supplements for
auxotrophlc strains of A.nldulans were those previously described (16).
Media for liquid culture contained the wetting agent Tween 80 (diluted 10~5
v/v) and MgSOi| and carbon source (glucose) added aseptically after
sterilization. Bacterial minimal medium and provision of nutritional
supplements were as previously described (17,18).
Growth of mycelium
Conditions for the growth of mycelium were those previously described
(19).
DMA preparation
A.nldulana chromosomal DNA was prepared from freshly grown mycelium
rapidly frozen In liquid nitrogen. Frozen mycelium was powdered in a Waring
Blender, resuspended in 10 mH Tris-HCl pH 8.1, 1 mM EDTA, 1J (w/v) SDS, 25J
sucrose at 20 ml g"1, shaken with an equal volume of phenol, chloroform,
lsoamylalcohol (PCA; 1)8:18:1 v/v) and centrlfuged to remove debris. The
resulting supernatant was made 1 H In potassium acetate, the precipitate
2202
Downloaded from https://academic.oup.com/nar/article-abstract/14/5/2201/2382022by gueston 11 April 2018
Nucleic Acids Research
removed by centrlfugation, the procedure repeated twice, and the nucleic
acids In the supernatant precipitated by the addition of 2 volumes of cold
ethanol. The precipitated nucleic acids were resuspended in TE (10 mM
Trls-HCl pH 8.1, 1 mM EDTA) and further purified by buoyant density centrl-
fugatlon in CsCl. DNA prepared in this manner was free of RNA contamination
and was greater than 50 Kb in length. Plasmld DNA was prepared by a scaled-
up version of the method of Blrnbolm and Doly (20).
The vector XDB286 (21) was propagated by heat-lnductlon of a lyaogenic
bacterial strain, precipitated by polyethylene glycol and purified on a CsCl
block-gradient as previously described (22). Vector DNA was prepared from
the phage particles by treatment with RNAse and pronase followed by phenol-
extraction. Recombinant phage3 derived from XDB286 were grown by the method
of Blattner et al (23) U3ing E.coll 3train C600 as the host.
Construction of a gene-bank
The 'freeze thaw' and 'sonicated' extracts for in vitro X packaging
were prepared by the method of Scalenghe et al. (21) and found to have an
efficiency of packaging of 1.5"3.Ox108 pfu ug"1 with XcI857 control DNA.
Gene-bank3 were prepared following standard protocols (22), using 0.5 ug
of XDB286 'arms' and a 3-fold molar excess of fungal DNA in the packaging
reaction. The XDB286 vector 'arms' were purified from potassium acetate
gradients following digestion with endonucleases BamHI and Sail. A.nldulans
DNA fragments In the size range 8-16 Kb were prepared from sucrose gradients
as previously described (22) following partial digestion with endonuclease
Sau3A.
DNA probes.
DNA fragments and plasmld DNAs were labelled with a-32P-dATP as
previously described (25).
Purification of DNA fragments
DNA fragments produced by endonuclease action were purified using
DE-81 paper as previously described (26), following separation by gel
electrophoresis.
Southern blotting and plaque-hybrldlsatlon
DNA fragments separated by gel electrophoresis and DNA from X phage
particles were transferred to nitrocellulose, denatured and fixed by stan-
dard methods (27, 28). Filters were screened with specific radioactive DNA
probes using the method of Jeffreys et al. (29).
2203
Downloaded from https://academic.oup.com/nar/article-abstract/14/5/2201/2382022by gueston 11 April 2018
Nucleic Acids Research
jjjjlflJJi transformation
E.coll s t r a in s were transformed using the method of Kushner (30) with
modifications as previously described (18).
Construction of recomblnant plasmlds
Plasmids were subjected to a ten-fold over-digestion with the appro-
pr ia te endonuclease in the presence of calf i n t e s t ina l phosphatase (0.5
units u«~1). Digested plaamld DNA (20 ng) was mixed with a three-fold molar
excess of fungal DNA in a buffer containing 50 mM Tr i s - HC1 pH 7 .5 , 10 mM
MgCl2, 1 mM rATP, 1 mM DTT in a volume of 10-30 p i . Paral lel react ions
containing half and double the proposed amount of fungal DNA (to allow for
errors in DNA estimations from ethldium bromide-stained fragments in
agarose), and no fungal DNA ( to check the effectiveness of the phosphatase
treatment) were set up and included in the bac ter ia l transformations.
Plasmld and Ml 3 RF DNA purif icat ion
The methods used for preparing Ml3 RF DNA and plasmld DNA have been
detai led previously (13).
DNA sequencing
The DNA sequencing methods using 35s-dATpp dldeoxynucleoside t r l -
phosphates, and buffer-gradient gels have been previously described (31.32).
Synthesis of specif ic ollgonucleotldes
Oligodeoxyrlbonucleotides were made by the method of Matthes et a l .
(33) with modifications to the wash-cycle as described by Sproat and Gait
Sources of material
Restr ic t ion endonucleases, DNA polymerase I , nuclease-free sucrose,
u l t ra pure urea and SDS were from BRL Ltd. (Glbco) or NBL Ltd; T4 DNA
l igase , calf In tes t ina l phosphatase, large fragment DNA polymerase were from
BCL Ltd. or Pharmacia Ltd.; Fujlmax X-Ray film was from Hannlmex U.K. Ltd.;
Nitrocel lulose was from Sartorius; DE81 paper was from Whatman, BBL-
trypt lcase powder was from BBL Microbiology Systems; Agar was from Dlfco
Laboratories; Agarose was from Miles Ltd. ; Tryptone, peptone, and yeast ext-
ract powder were from Oxold Ltd . ; Salmon sperm DNA; polyvinyl pyrollldone,
bovine serum albumin, f icol l 100, polyethylene glycol 6000, d i t h l o t h r e i t o l ,
amplci l l ln , t e t r acyc l ine , chloramphenlcol, nucleoslde trlphosphates and
dideoxynucleoside trlphosphates were from Sigma Ltd. , 35s-dATP, and a32pdCTP
were from Amersham, chemicals for oligonuoleotide synthesis were from
Cruachem Chemical Co.; a l l other reagents were of Analar or greater pur i ty .
2204
Downloaded from https://academic.oup.com/nar/article-abstract/14/5/2201/2382022by gueston 11 April 2018
Nucleic Acids Research
RESULTS AND DISCUSSION
Cloning the ARQH locus
The blosynthetic dehydroqulnase function of the AROM locus of
A.nldulans Is located on a 1.9 Kb Hlndlll fragment, previously cloned on
pla3mld pHK 29 (12). The isolated Hlndlll fragment was used a3 a DNA probe
to locate AROM-contalnlng recomblnant phages in a gene-bank of A.nldulans
DNA cloned in the vector ADB286.
Approximately 10^ recombinant phage, Identified by their Spi"
phenotype on P2 ly30genic strain Q359, were plated on the recB, £" E.coll
strain ED8910 on ten 9 cm plates. The phages were transferred to nitro-
cellulose, the DNAs were denatured and screened for the presence of AROM
sequences U3lng the 32p-iat,elled 1.9 Kb Hlndlll fragment as probe.
Positively hybridising phages were plaque-purified through several cycles,
checked for purity, propagated in bulk and their DNAs subjected to restric-
tion enzyme mapping. Ten AROM-posltlve isolates analysed contained
approximately 19 Kb of unique A.nldulans DNA. The physical maps of two 3uch
isolates, AARH 3.1 and XARH 8.1, which cover the entire region, are shown in
Figure 1 together with the position of the 1.9 Kb Hlndlll fragment used as
probe.
DNA sequence of the AROM locus.
The nucleotlde sequence of the 1.9 Kb Hlndlll fragment that contains
the coding sequence for the biosynthetic dehydroqulnase function has been
determined (13), allowing us to deduce its reading-frame and the orientation
of the AROM locus. Assuming similarity with the structure of the AROM locus
of N.crassa, where the sequence encoding the dehydroquinase function has
been localised to the 3'-end of the locus (8), we concentrated our search
for the AROM locus of A.nldulans to the region extending from 1 Kb to the
right to 5 Kb to the left of the 1.9 Kb Hlndlll fragment. To facilitate the
determination of the entire AROM DNA sequence, appropriate restriction
endonuclease-generated fragments were subcloned into the RF form of the DNA
sequencing vectors M13mp8 and 9 (35). Fragments were inserted in both
orientations and sequences determined from both ends using universal primer.
Gaps in the sequence were filled by using synthetic oligonucleotldes as
specific primers to extend and overlap the sequences on both strands. The
overall sequencing Strategy is summarized lnTTg.~1.
Analysis of 6.5 Kb of unique sequence revealed a single, open reading-
frame of 1,812 bp in the same orientation and phase as previously determined
for that encoding the dehydroqulnase function (13). This open reading-frame
2205
Downloaded from https://academic.oup.com/nar/article-abstract/14/5/2201/2382022by gueston 11 April 2018
Nucleic Acids Research
HK29
B B E H H E B H B H B E EI I L J I I I I I I I I I
s D '- 1 '
f. H E B X H X P X H B E"I I I I I I I ill I I I
XARH31
I — XARH81
*«*<
O L I O O N U C L E O T I O E I
Figure 1. Restriction enzyme map of the cloned AROM DNA. The positions ofthe cloned DNAs within two recombinant phages, XARH3.1 and AARH8.1, areshown above the restriction map derived using the endonucleases BamHI (B),EcoRI (E) and Hlndlll (H). The 1.9 Kb Hlndlll fragment used as probe,previously cloned in plasmid pHK29, is indicated. A section of 7 Kb illus-trated at a relative scale expansion of two is shown with the recognitionsites for Xhol (X) and PstI (P) Included. The arrows beneath this sectionshow the direction and extent of sequencing reactions using universal primer(restriction sites) or specific oligonucleotlde primers (ollgonucleotides).
begins 217 b.p. downstream from the leftward Xhol site, Incorporates the
previously determined sequence (13) and extends it rightwards for a further
388 b.p. The sequence of the open reading- frame is shown in Fig. 2 with
the deduced amlno acid sequence. The inferred molecular weight of the AROM-
polypeptlde encoded by this open reading-frame is 175,101, which is in close
agreement (6J) with that of 165,000 for the arom polypeptide of N.crassa
measured by its mobility in SDS-polyacrylamlde gels (8).
The observation that the AROM locus has a single open reading-frame of
a size that is in close agreement with that required to specify the multi-
functional polypeptide product suggests that the AROM locus contains a
single structural gene lacking lntrons. This conclusion is supported by the
finding that segments of the AROH locus can be expressed in E.coll to
provide the blosynthetlc activities missing in aroD or aroA mutants of the
bacterium (12 and Hawkins, unpublished). Other structural genes of
A.nldulans have been shown to contain lntrons (36,37), which are flanked by
the eukaryotlc consensus boundary sequences. Inspection of the A.nldulans
AROM sequence does not reveal any likely lntron sequences. Together these
findings strongly suggest the absence of introns in the A.nldulans AROM
2206
Downloaded from https://academic.oup.com/nar/article-abstract/14/5/2201/2382022by gueston 11 April 2018
Figure 2. The nuoleotlde sequence of the protein-coding region of the AROMlocus. The DNA strand shown has the same orientation as the messenger RNA,and the deduced amlno acid sequence is translated from a single openreading-frame.
locus, although the possibility of small, ln-phase introns containing no
translation-termination codons cannot be excluded.
Codon-usage in the AROM sequence.
The amlno acid composition of the AROM-speclfled protein, together
with the corresponding codon-usage, are shown in Table i. The marked bias
in codon-utllisatlon is evident, with strong preference for G or C and the
diminution of A in the third position. The overall G + C content of the
AROH coding sequence is 51J, compared with a value of 53). for A.nldulans DNA
(38). A similar bias in codon-usage has been observed for other genes in
A.nldulans, Including PGK (10) and QutE (41).
Evolutionary relationships with E.coll aro genes and implications for domain
structure within the Aj ft polypeptlde.
It has previously been reported that the sequences encoding the two
dehydroqulnase iso-enzymes of A.nldulans, the bio3ynthetlc enzyme specified
within The AROM" Jrocus and the cataboiicr dehydroqulnase <leterHiine<i by the
qulnlc acld-^utillsation (Qut) gene-cluster, show no sequence homology,
strongly suggesting convergent evolutionary pathways (13). I t Is therefore
of interest to determine whether there is any sequence homology between the
2209
Downloaded from https://academic.oup.com/nar/article-abstract/14/5/2201/2382022by gueston 11 April 2018
Nucleic Acids Research
Table 1. Codon-usage within the AROM mesoenger RHA of A.nidulans.
u
c
A
c
u
PHEPHELEULEU
LEULEULEULEU
ILEILEILEMET
VALVAI,VALVAL
1831525
2834847
4556439
35391124
C
SEHSERSERSER
PROPROPROPRO
THRTHRTHRTHR
ALAALAALAALA
26291727
23361418
32292912
45492620
A
TYRTYRTERTER
HISHISGLNGLN
ASNASNLYSLYS
ASPASPGLUGLU
132710
8211625
14422161
35374453
G
CYSCYSTERTRP
ARCARGARCARG
SERSERARGARG
GLYGLYGLYGLY
1314019
27291317
142387
31442917
UcAG
UCAG
UCAG
UCAG
A.nidulans AROM locus and the corresponding aro genes of E.coll. The
sequence of the E.coll aroA gene, encoding the 5-enolpyruvylshlklmate 3-
phosphate (EPSP) synthase, is available (39) and this was compared with the
A.nidulans AROM sequence using a conventional dot-matrix programme at 70>
stringency. This matrix showed distinct homology between the two sequences
from nucleotlde 1005 to 2361 in the AROM sequence. The Inferred amino acid
sequence from the appropriate section of AROM DNA is shown in Fig. 3, alig-
ned with the amino acid sequence of the E.coll aroA gene. The relatedness
of the two sequences Is evident: overall there is 36J homology, though this
is considerably higher In discrete regions (v. residues 511 to 530). In
addition, there are many examples of conservative amino acid substitutions.
The clear homology between the A.nidulans AROM sequence and the E.coll
aroA gene has implications concerning the evolution of the complex locus and
the domain structure of the AROM polypeptide. The data suggest that an
ancestral EPSP synthase gene has been fused into a complex gene encoding a
multifunctional polypeptide In A.nidulans. Alternatively, though Ies3
likely, an ancestral, multifunctional locus or group of clustered genes has
become dispersed in E.coll. This observation suggests the likelihood that
homology will be found between other E.coll aro genes and corresponding
regions of the A.nidulans AROM locus.
The homology between AROM and the E.coll aro A gene locates the
sequence encoding the EPSP synthase function within the overall AROM locus
sequence. Overlapping sections of A.nidulans DNA containing this region of
2210
Downloaded from https://academic.oup.com/nar/article-abstract/14/5/2201/2382022by gueston 11 April 2018
Figure 3. Sequence alignment between the E.coll EPSP synthase and theA.nldulans AROM-product. Both amlno acid sequences are shown in the singleletter code; the upper sequence is that deduced for the A.nldulans AROM-product from residues 392 to 813 and the lower the E.coll EPSP synthasesequence. The alignment is by computer analysis using the Beckman Micro-Genie suite of programmes; stars indicate positions of Identical residues.
AROM have been expressed In E.coll to suppress the auxotrophy of aroA
mutants (A.R.H., unpublished), suggesting that the relevant segment of the
AROH Polypeptide is independently capable of folding Into a catalytically
active structure.
The 1.9 Kb Hlndlll fragment of the A.nldulans AROM locus can also be
expressed to give an enzymatlcally active polypeptide, the biosynthetic
dehydroquinase (12,13). The Hlndlll site at the 5' end of this DNA fragment
lies exactly at the 3"end of the sequence encoding the EPSP synthaae
activity and may be fortuitously located In a nucleotide sequence encoding a
peptide link between adjacent domains. The ability of the 1.9 Kb Hlndlll
fragment from the 3' end of the AROM gene to encode an active dehydroquinase
demonstrates that the folding of this polypeptide into a catalytically
active configuration can occur quite Independently of the rest of the
protein structure.
In many fungi qulnate catabollsm and aromatic amino acid synthesis
2211
Downloaded from https://academic.oup.com/nar/article-abstract/14/5/2201/2382022by gueston 11 April 2018
Nucleic Acids Research
share two intermediates, dehydroquinate and dehydroshlkimate, and have two
distinct dehydroqulnase lao-enzymes (12, 42). Enzymes encoded by the AROM
locus have been implicated in the channelling of Intermediates in aromatic
biosynthesis, keeping the levels of the common intermediates low enough to
prevent induction of the catabolic pathway in wild-type strains (1). The
existence of adjacent catalytic domains on a single, multifunctional enzyme
might be well suited to achieve that end.
With the complete sequence of the AROH locus now available, we are in
a stronger position to probe the relationship between domain-3tructure and
function in the complex enzyme, using molecular genetic dissection and in
vitro mutagenesls.
ACKNOWLEDGEMENT
We are grateful to the SERC for financial support through Project
Grants GR/C/21144 and GR/C/94711, to the Society for General Microbiology
for a grant partially funding computing facilities in Newcastle, and to
Anne Bates for typing the manuscript.
*To whom correspondence should be sent at the University of Newcastle upon Tyne
REFERENCES1. Giles, N.H. (1978). The American Naturalist. _1ji2, 641-657.2. Ahmed, S.I. and Giles, N.H. (1969). J. Bacteriol. 99_, 231^237.3. Burgoyne, L.M., Case, M.E. and Giles, N.H. (1969). Blochlm. Blophys.
Acta. J_91_, 452-462.4. Patel, V.B. and Giles, N.H. (1979). Blochim. Blophys. Acta. 576,
24-34.5. Case, M.E. and Giles, N.H. (1968). Genetics 60, 49-58.6. Gaertner, F.H. and Cole, K.W. (1977). Blochem. Blophys. Res. Comm.
75., 259-264.7. Lumsden, J . and Coggins , J . R . ( 1977 ) . Biochem. J . ^61_, 599-607 .8. Smi th , D.D.S. and Coggins , J . R . ( 1983 ) . Biochem. J . 21_3. 405-415 .9. S t r a u s s , A. ( 1 9 7 9 ) . Ho lec . Gen. Gene t . 1_7 , 233-241 .
10. N a k a n i s h l , N. and Yamamoto, M. (1984 ) . H o l e c . Gen. Gene t . 195,164-0 6 9 .
11. Bachman, B.J. (1983). Microbiol. Revs. k]_, 180-230.12. Kinghorn, J.R. and Hawkins, A.R. (1982) Molec. Gen. Genet. 186,
145-152.13. C h a r l e s , I . G . , Keyte , J . W . , Brammar, W.J. and Hawkins, A.R. ( 1 9 8 5 ) .
Nucle ic Acids Res . 1_3, 8119-8128.14. Lar imer , F.W., Morse, C . C . , Beck, A.K., C o l e , K.W. and G a e r t n e r , F.H.
( 1 9 8 3 ) . Mol. C e l l . B t o l . 2 - 1609-1614.15 . C a t c h e s i d e , D.E.A. , S t o r e r , P . J . and K l e i n , B. ( 1 9 8 5 ) . Molec. Gen.
Gene t . 222- 446-451.16. Hawkins, A.R. , Da S l l v a , A . J . F . and R o b e r t s , C .F . ( 1 9 8 5 ) . C u r r e n t
Genet . 9 , 305"311 .
2212
Downloaded from https://academic.oup.com/nar/article-abstract/14/5/2201/2382022by gueston 11 April 2018
Nucleic Acids Research
17.
18.
19.
20.21 .
22.
23.
21).
25.
26.
27.28.29.
30.
31 .
32.
33.
34.35.36.
37.38.39.
10.11 .42.
A r m i t t , S . , McCollough, W. and Robe r t a , C .F . ( 1 9 7 6 ) . J . Gen.M l c r o b l o l . 9£, 263-282.
W l l l e t t s , N . S . , C l a r k , A . J . and Low, B. ( 1 9 6 9 ) . J . B a c t e r i o l 97^2l)l)-259.
Hawkins, A.R. , Da S l l v a , A . J . F . and R o b e r t s , C.R. ( 1 9 0 4 ) . J . Gen.M i c r o b l o l . 21£'. 567-574.
Birnbolm, H.C. and Doly, J . (1979) . Nuc le ic Acid R e s . ]_, 1513-1523.Brammar, H . J . (1982) . In Gene t i c E n g i n e e r i n g , vol 3 , 53-82
Ed. R. Wi l l i amson , Acad. P r e s s , London.M a n l a t i s , T . , F r i t s c h , E .F . and Sanbrook, J . ( 1982 ) . I n Molecular
C lon ing , a l a b o r a t o r y manual . Cold Spr ing Harbor l a b o r a t o r y .B l a t t n e r , F . R . , W i l l i a m s , B.G., B l e c h l , A . E . , Dennlston-Thomson, K.,
Faber , H .E . , Fur long , L . , Grunwald, D . J . , K l e f e r , D.O. ,Moore, D.D. , Schumm, J .W. , Sheldon, E.L. and S m i t h i e s , 0 . (1977) .Sc ience 2£4_. 161-169.
Sca lenghe , F . , Turco , E . , Edstrom, J . E .(1981) . Chromosana, 82_, 205-216.
Rigby, P .W.J . , Diekmann, M., Rhodes, C.J . Mol. B l o l . 2 1 3 , 237-251 .
Dre tzen , G. , B e l l a r d , M., S a r a o n n e - C o r r i , P. and Chambon, P. ( 1981 ) .Anal . Biochem. 112, 295-298.
Sou the rn , E.M. (1975T. J . Mol. B i o l . 98_, 503"517.Benton, W.D. and Davie3 , R.W. (1977 ) . S c i e n c e , 1_96.. 180-182.J e f f r e y s , A . J . , Wilson, V. , Wood, D . , Simons, J . P . , Kay, R.M. and
W i l l i a m s , J . G . ( 1980 ) . Ce l l 21_, 555-564.Kushner, S.R. (1978) . I n Gene t i c Eng inee r ing e d s . Boyer, H.W. and
N i c o s i a , S. North Holland Biomedlcal P r e s s , Amsterdam, 17-23 .Sanger , F . , N lck l en , S. and Coul3on, A.R. (1979) . P r o c . N a t l . Acad.
S c l . USA 74^, 5463-5467.Bigg in , M.D., Gibson, T .S . and Hong, C .F .
S c i . USA 8£, 3963-3965.M a t t h e s , M.W.D., Zenke, W.M., Grundstrom, T . , S t a u b , A.,
W i n t z e r i t h , M. and Chambon, P. ( 1 9 8 4 ) . EMBO. J . 3., 801^805.S p r o a t , B.S. and G a i t , M.J. (1985) . Nuc le ic Acid R e s . 2 1 . 2959-2978.Messing, J . ( 1 9 8 3 ) . Methods i n Enzymol. 2 2 1 . 2 0 - 7 8 .McKnight, G . L . , Kato , H. , U p s h a l l , A. , P a r k e r , M.D., S a a r i , G. and
O'Hara, P . J . ( 1985 ) . EMBO J . 4_, 2093-2099.Clements , J .M. and R o b e r t s , C.F. ( 1 9 8 5 ) . Cur ren t Gene t . £ , 293-298.S t o r c k , R. and Alexopolous , C . J . ( 1970 ) . Bac t . Rev3. 3 ^ , 2 , 126-154.Duncan, K., Lewendon, A. and Cogglns , J .R . ( 1 9 8 4 ) . FEBS L e t t . 170,
5 9 - 6 3 .Clements, J.M. (1985). Ph.D. Thesis, University of Leicester, U.K.Da Silva, A.J.F. (1985). Ph.D. Theaia, University of Leicester, U.K.Hawkina, A.R., Giles, N.H. and Klnghorn, J.R. (1982). Biochem. Genet.
20, 271-286.
Pirotta, V., and Melll, M.
and Berg, P. (1977).
(1983). Proc. Natl. Acad.
2213
Downloaded from https://academic.oup.com/nar/article-abstract/14/5/2201/2382022by gueston 11 April 2018
Downloaded from https://academic.oup.com/nar/article-abstract/14/5/2201/2382022by gueston 11 April 2018
