604th MEETING, CAMBRIDGE
283
Putative high-mobility-group (HMG) non-histone chromosomal proteins from wheat germ
1
ELAINE L. V. MAYES* and J O H N M. WALKER?
*Division of Protein Chemistry, P.O. Box 123,
Imperial Cancer Research Fund, Lincoln’s Inn Fields,
London WCZA 3PX. U .K..and tBioIogica1 Sciences,
Hatjield Polytechnic, HatJield, Herts ALlO 9AB, U . K .
Chromatin contains a group of non-histone proteins called
the ‘high-mobility-group’ (HMG) proteins (Johns, 1982).
There are four main H M G proteins in calf thymus, namely
H M G 1, 2, 14 and 17. The complete amino acid sequences
of calf thymus H M G 1 and 2 (Walker et al., 1980a), calf
thymus H M G 14 (Walker et al., 1979) and calf thymus and
chicken erythrocyte H M G 17 (Walker et al., 1977, 19806)
have been determined. Considerable sequence data also
exists for proteins from trout testes (Watson er al., 1977,
1979), trout liver (Walker et al., 1980~)and chicken erythrocytes (Walker et al., 1980c), all of which show extensive
sequence homology with calf thymus H M G proteins
(Walker er al., 1980~).The presence of H M G proteins has
also been indicated in wheat (Spiker et al., 1978), yeast
(Spiker et al., 1978; Peterson et al., 1978), Tetrahymena
(Hamana & Iwai, 1979) and insects (Alfageme el al., 1976;
Franco et al., 1977). However it cannot be stated for certain
that these proteins are true H M G proteins, since their
characterization is based only on gel-electrophoretic mobilities and total amino acid analyses. Only when sequence
data are available for these proteins can they be rigorously
compared with the calf thymus H M G proteins. We have
been studying some HMG-like proteins from wheat germ
and report here some initial studies on the structure of these
proteins.
Wheat-germ nuclei were prepared by centrifugation of
homogenized tissue through Percoll gradients. Nuclei were
then homogenized in saline to recover chromatin, and the
chromatin extracted as described below.
H M G proteins are defined as those proteins which are
extractable from calf thymus chromatin with 0.35M-NaCl
and soluble in 2% trichloroacetic acid. SDS (sodium dodecyl
sulphate)/polyacrylamide-gelelectrophoresis of the proteins
obtained from such a fractionation of wheat-germ chromatin is shown in Fig. 1. As well as histone H 1 (which is known
to be partially extracted by this procedure), and traces of
high-molecular-weight material, five other major bands
(putative H M G proteins) can be identified. Total proteins
extractable from wheat-germ chromatin with 5% (w/v)
HCIO, (a standard procedure for isolating mammalian
H M G protein) are also shown in Fig. 1. As well as that of
histone HI ten other bands can be identified (A-J). Five of
these proteins (A, B, C, F and J) correspond to the proteins
extracted with 0.35wNaCI from wheat-germ chromatin
and are therefore candidates for being HMG proteins.
Proteins B, C, F and J have been isolated by ion-exchange
chromatography of 5% HCIO, extracts of wheat germ. The
2
3
4
5
6
HMG 1
HMG 2
HMG 1 7 - 4
C A
L B
Fig . 1 . SDSlpolyacry lamide-gel-electrophoresis pat terns
Track 1, calf thymus H M G proteins; 2 , 0 . 3 5 ~ - N a C extract
1
(2%-trichloroacetic acid-soluble) of wheat-germ chromatin ;
3, 5%-HC10, extract of wheat-germ chromatin; 4, not
relevant to the present work; 5 , 0 . 2 5 ~ - H Cextract
l
of wheat
germ chromatin; 6, total calf thymus histone (H).
amino acid analysis of protein B shows little similarity with
that of any of the H M G proteins. Although having a
relatively high basic amino acid content like the HMG
proteins, protein B does not have a high aspartic and
glutamic acid content which is the major characteristic
feature of the H M G proteins. Purely on the basis of amino
acid analysis, therefore, protein B does not appear to be an
H M G protein. (Insufficient of protein B was isolated to
allow sequence analysis.) In contrast with protein B,
proteins C, F and J all have amino acid analyses
characteristic of H M G proteins. On the basis of gel mobility
and amino acid analysis, therefore, proteins C, F and J are
good candidates for being H M G proteins. Unfortunately,
N-terminal sequence data does not support this suggestion.
The N-terminal sequences of proteins C , F and J are shown
in Fig. 2. N o sequence homology is apparent with any of the
calf thymus H M G proteins (the sequence of H M G 2 is not
Protein C
Met-Lys-Gly-Ala-Lys-Ser-Lys-Gly-Ala-Val-Lys-Ala-Asp-Thr-Lys-Leu-Ala-Val-Lys-Gly-
Protein F
Met-Lys-Gly-Lys-Ala-Asp-Thr-(
Protein J
Met-Ser-Glu-Lys-Ala-Asp-Gly-Glu-Thr-Ala-Phe-( )-Ala-( )-Gly-
HMG 1
Gly-Lys-Gly-Asp-Pro-Lys-Lys-Pro-Arg-Gly-Lys-Met-Ser-Ser-~r-Ala-Phe-Phe-Val-Gln-
H M G 14
Pro-Lys-Arg-Lys-Val-Ser-Ser-Ala-Glu-Gly-Ala-Ala-Lys-Glu-Glu-Pro-Lys-Arg-Arg-
HMG 17
Pro-Lys-Arg-Lys-Ala-Glu-Gly-Asp-Ala-Lys-Gly-Asp-Lys-Ala-Lys-Val-Lys-Asp-Glu
)-Lys-
Fig. 2. Terminalsequences of wheat-germproteins, C, F. and J . and calfthymusproteins
HMG I , 14 and 17
VOl. 12
284
BIOCHEMICAL SOCIETY TRANSACTIONS
shown since it only differs in one position from that of
HMG 1). This was surprising, since comparison of the Nterminal sequencesof putative trout HMG proteins with the
calf HMG proteins shows an average of 65% homology in
the N-terminal regions (Walker et al., 1980~).Our observations suggest that, in the absence of a functional assay for
HMG proteins, only detailed sequence analysis of these
wheat-germ proteins will reveal whether they are true HMG
proteins. The amino acid analysis and el mobility alone
does not at present appear to be su cient to warrant
classifying a protein as an HMG protein.
fl
Alfageme, C. R., Rudkin, G. T. & Cohen, L. (1976)Proc. Natl.
Acad. Sci. U.S.A. 73, 2038-2042
Franco, L., Montero, F. & Rodriguez-Molina, J. J. (1977)FEBS
Lett. 78, 317-320
Hamana, K. & Iwai, K. (1979) J . Biochem. (Tokyo) 86, 789794
Johns, E. W. (ed.) (1982) The HMC Chromosomal Proteins,
Academic Press, London and New York
Peterson, J. G . L &Sheridan, W. F. (1978)Carlsberg Res. Commun.
43,415422
Spiker, S., Mardian, J. K . W. & Isenberg. I. (1978)Biochem. Res.
Commun. 82, 129-135
Walker, J. M., Hastings. J. R. B. & Johns, E. W.(1977) Eur. J .
Biochem. 76, 461-468
Walker, J. M., Goodwin, G. H. &Johns, E. W. (1979)FEBS. Lett.
100, 394-398
Walker, J. M., Gooderham, K., Hastings, J. R. B., Mayes, E. &
Johns, E. W. (198Oa)FEBS Lett. 122, 264270
Walker, J. M., Steam, C. & Johns, E. W. (19806)FEBS Lett. 112,
207-210
Walker, J. M., Brown, E., Goodwin, 0.H., Steam, C. & Johns,
E. W. (1980~)FEBS Lett. 113, 253-257
Watson, D. C., Peters, E. H. & Dixon, G. H. (1977) Eur. J .
B k h e m . 74,5 3 4
Watson, D. C., Wong, N . C. W. & Dixon, G . H. (1979)Eur. J .
B k h e m . 95, 193-198
Restriction-endonuclease digestion of topisomer families
G. S. SNOUNOU and A. D. B. MALCOLM
Department of Biochemistry. St. Mary’s Hospital Medical
School, Norfolk Place, Paddington. London W2 IPG. U.K.
It is clear that the conformational perturbation associated
with supercoiling is not distributed evenly around the DNA
molecule (Lilley & Markham, 1983). Presumably the base
composition and sequence will affect this distribution.
We have previously shown that rates of restriction-endonuclease digestions vary with conformation of the DNA
and have also shown that some DNA ligands may exert
their inhibitory effect at some distance by conformational
changes in the DNA (Malcolm & Moffatt, 1981;Malcolm et
al., 1982; Snounou & Malcolm, 1982). Since the enzymes
studied cleave the DNA at sites that may be many hundreds
of base-pairs apart, we are unable to say whether or not the
DNA conformation at these sites is the same. In an attempt
to clarify this problem, we have prepared DNA species of
different superhelical densities and compared the rates of
digestion with a variety of restriction endonucleases.
We have recently developed methods that allow us to
generate between -45 and +7 supercoils in the 4362 basepairs of plasmid-pBR322 DNA (Malcolm & Snounou,
1983;Snounou & Malcolm, 1983). This involves incubating
the plasmid with DNA topoisomerase (Wang, 1981) in the
presence of various concentrations of ethidium bromide (for
the generation of negative supercoils) or netropsin (to
generate positive supercoils). Subsequent removal of the
ligand generates the superhelical substrate.
Rates of digestion by endonucleases BamHI (GGATCC
at positions 375-380) and by Avo1 (CTCGGG at positions
1424-1429) are almost unaffected by changes in the overall
superhelical density of the molecule. Digestion by EcoRI
(GAATTC at positions 4360-3), on the other hand, is at a
maximum for the relaxed circular molecule and decreases
significantly for either positive or negative supercoiling.
An examination of the sequences surrounding these
restriction sites shows that for either 10 or 20 base-pairs on
each side of the BamHI site this region is very GC-rich
(68%). This is even more so with the AVaI region (75%
GC). On the other hand the EcoRI flanking sequences are
very AT-rich (38% GC). It is clear that negative supercoiling has a tendency to ‘melt’ DNA and it therefore seems
likely that such structural changes will be more pronounced
in regions of a high AT/GC ratio. We cannot say how large
a region is required for such an effect to be manifest, but it
seems unlikely to be more than the two turns of the double
helix considered above.
None of the three enzymes is known to cleave singlestranded DNA. The above argument leads us to expect that
EcoRI would be more sensitive than AuaI or BamHI to
negative supercoiling. The lack of previous experiments on
positively supercoiled DNA makes it impossible to extrapolate such an argument to these DNA molecules.
Our results suggest that great care must be exercised when
using susceptibility to restriction-endonucleasedigestion to
study ‘free’ DNA in chromatin (McGhe et al., 1981). DNA
in the nucleosome is topologically under-wound (Wang,
1982) and the effect this has will vary from enzyme to
enzyme, and also from sequence to sequence.
Lilley, D. M. J. & Markham. A. F. (1983)EMBO J . 2, 527-533
McGhee, J. D., Wood, W. I., Dolan, M., Engel, J. D. & Felsenfeld,
G. (1981)Cell 27, 45-55
Malcolm, A. D. B. & Moffatt, J. R. (1981)B k h i m . Bwphys. Acta
655, 128-135
Malcolm, A. D. B., Moffatt, J. R., Fox, K . R. & Waring, M. J.
(1982)B k h i m . Biophys. Acta 699 211-216
Malcolm, A. D. B. & Snounou, G. S. (1983)Cold Spring Harbor
Symp. Quant. BwI. 47, 323-326
Snounou, G. S. & Malcolm, A. D. B. (1982)Biochem. SOC.Trans.
10,348-349
Snounou, G. S . & Malcolm, A. D . B. (1983)J . Mol. Bwl. 167.21 1216
Wang, J. C. (1981)Enzymes 4th Ed. 14,331-344
Wang, J. C. (1982)Cell 29,724-726
1984