Table Of ContentPreface
Combinatorial chemistry is a field that did not exist five years ago but
is so vibrant today, especially in medicinal chemistry, that almost every
major pharmaceutical company has a group working in this area and many
start-up companies have been formed with combinatorial chemistry as their
raison d'etre. Like many other fast-breaking developments, this field had
its main origins in work done in academic research laboratories, and many
of the techniques were developed to solve specific problems in basic re-
search. The common feature of all combinatorial approaches is the genera-
tion of a complex mixture of molecules coupled to screens or selections
which can identify out of that mixture a single molecule with desired proper-
ties, e.g., as the ligand or inhibitor of an enzyme or as a macromolecule
with novel or enhanced properties. At the start most combinatorial libraries
were of biological molecules, mostly peptides or nucleic acids, but because
these molecules only rarely exhibit good pharmacological properties, in-
creasingly the libraries of interest to medicinal chemists are of small mole-
cules with a range of pharmacologically attractive properties.
Because of the rapid progress in this field, a follow-up to this volume
would not be possible in a single volume of Methods in Enzymology, but
at the time of the organization of this volume one could identify the main
themes that constitute this field and present the key technologies in a
single volume.
One of the earliest techniques for the generation and screening of a
diverse library of peptides was the display of random sequences in the coat
protein of single-strand DNA phages. The diversity of these libraries is
limited to the titers of phage one can obtain, typically >10 H particles/ml.
The phage coat protein can also accommodate entire proteins such as DNA
or RNA binding proteins that have been partially randomized so that
proteins with novel binding properties can be selected.
The techniques for the generation and screening of small molecule
libraries originated with peptides, and this volume contains a number of
early and still very useful techniques in this area. These libraries can be
screened by a number of very clever methods, including deconvolution of
different pools and the elegant and potentially very powerful encoded -il
braries.
The exploration of sequence space is most striking in the case of nucleic
acid libraries. Here, due to the power of the polymerase chain reaction,
libraries with diversities as high as 1016 different molecules have been
explored. This is an extremely exciting area in which we are continually
xiii
xiv ECAFERP
being surprised by the diversity of form and function possible within the
confines of the polynucleotide backbone. One can select RNA molecules
which can bind specifically to virtually any protein or small molecule and
also which can catalyze a diverse set of chemical reactions. And not only
can one explore sequence space in large libraries but as Tsang and Joyce
show in article 23 in this volume, one can expand that sequence space by
judicious mutagenesis during amplification between rounds of selection as
must have occurred during biological evolution.
It is clear, however, that much of the creative energy these days in this
field is being directed at inventing sophisticated methods for the generation
and screening of diverse kinds of small molecules, such as the pioneering
work by Ellman and colleagues on benzodiazepine libraries described in
this volume. Interestingly, the need to generate large diversity in these
libraries is not the key factor, and, instead, the ingenuity in the selection
of scaffolds and functional groups in generating the libraries will probably
be most important in generating interesting new pharmacological leads. In
this regard, one can expect interactions between computational chemistry
and combinatorial chemistry in which libraries are generated and screened
by computer methods in a search to find the most appropriate library for
a particular target. It is perhaps in that area that we should think now of
organizing a new volume in order to have something interesting for the
new millenium.
NHOJ N. NOSLEBA
Contributors to Volume 267
Article numbers are in parentheses following the names of contributors,
Affiliations listed are current.
NEVETS C. ELLIVNAB (25), Chiron Corpora- WERDNA D. NOTGNILLE (20), Department of
tion, Emeryville, California 80649 Chemistry, Indiana University, Blooming-
ton, Indiana 50474
LEOJ G. OCSALEB (9), Department of Microbi-
ology and Molecular Genetics, Harvard NAHTANOJ A. NAMLLE (26), Department of
Medical School, Boston, Massachusetts Chemistry, University of California, Berke-
51120 ley, Berkeley, California 02749
OCNARF FELICI (6, 7), IRBM .P Angeletti,
E1VLYS E. ELLEDNOLB (13), Torrey Pines In-
00040 Pomezia, Rome, Italy
stitute for Molecular Studies, San Diego,
California 12129 ENINAIG M. IZZOILGIF (25), Chiton Corpora-
tion, EmeryviUe, California 80649
YRRAB A. NINUB (26), Department of Chem-
MIT RETAWZTIF (17), NeXstar Pharmaceuti-
istry, University of California, Berkeley,
cals, Inc., Boulder, Colorado 10308
Berkeley, California 02749
INNAVOIG ~RFLAG (6, 7), IRBM .P Angeletti,
EILRAHC L. CHEN (12), Hoechst Marion
04000 Pomezia, Rome, Italy
Roussel, Tucson, Arizona 85737
KRAM POLLAG (16), Affymax Research Insti-
YEZREJ AKLOISEIC (19), Department of Mo- tute, Palo Alto, California 40349
lecular, Cellular, and Developmental Biol-
NAITSIRHC M. SETAG (10), Affymax Research
ogy, University of Colorado, Boulder, Colo-
Institute, Palo Alto, California 40349
rado 80309
IROL GIVER (20), Division of Chemistry and
DRAHCIR C. DARNOC (20), Department of Chemical Engineering, Californm Institute
Chemistry, Indiana University, Blooming- of Technology, Pasadena, California 52119
ton, Indiana 50474
DRAHCIR HTIMSDLOG (25), Chiron Corpora-
ODRACCIR ESETROC (6, 7), IRBM P. Angel- tion, Emveryville, California 80649
etti, 00040 Pomezia, Rome, Italy YEVRAH A. NAMSIERG (8), Department of Bi-
SELRAHC KIARC (3), Departments of Pharma- ology, Massachusetts Institute of Technol-
ceutical Chemistry, Pharmacology, and ogy, Cambridge, Massachusetts 93120
Biochemistry and Biophysics, University of OOSNUYH NAH (14), Departments of Molecu-
California, San Francisco, San Francisco, lar Biology and Chemistry, The Scripps Re-
California 34149 search Institute, La Jolla, California 92037
DRALLIM G. LLUC (10), Enzyco, Inc., Denver, ENILEUQCAJ L. NOSIRRAH (5), United States
Colorado 60208 Biochemicals Pharma Ltd. (Europe), War-
ford WD1 8YH, United Kingdom
YERFFEJ P. SIVAD (18), NeXstar Pharmaceuti-
REHPOTSIRHC P. SEMLOH (16), Affymax Re-
cals, Inc., Boulder, Colorado 10308
search Institute, Palo Alto, California 40349
REFINNEJ M. DIAS (11), Affymax Research
DRAHCIR A. NETHGUOH (13), Torrey Pines
Institute, PaiD Alto, California 40349
Institute for Molecular Studies, naS Diego,
ARABRAB RENROD (13), Torrey Pines Insti- California 12129
tute for Molecular Studies, San Diego, Cali-
MALI ERAKESAGNALLI (19), Department "fo
fornia 12129
Molecular, Cellular, and Developmental Bi-
MAILLIW J. REWOD (11), Affymax Research ology, University of Colorado, Boulder,
Institute, Palo Alto, California 40349 Colorado 80309
X CONTRIBUTORS TO VOLUME 267
NYRHTAK M. HCITENAVI (15), Biomolecular NOMIS C. NG (25), Chiron Corporation, Em-
Resource Center, University of California, eryville, California 94608
San Francisco, San Francisco, California ZHI-JIE NI (16), Affymax Research Institute,
34149 DiaP Alto, California 94304
MIK D. JANDA (14), Departments of Molecu- MIT SELKCIN (19), Department of Molecular,
lar Biology and Chemistry, The Scripps Re- Cellular, and Developmental Biology, Uni-
search Institute, La Jolla, California 92037 versity of Colorado, Boulder, Colorado
A~JOBEN JANJI¢ (18), NeXstar Pharmaceuti- 90308
,slac Inc., Boulder, Colorado 10308 ODERFLA AISOCIN (6, 7), IRBM P. Angeletti,
DLAREG F. JOYCE (23), Departments of 00040 Pomezia, Rome, Italy
Chemistry and Molecular Biology, The RETEP E. NESLmN (24), Department of Medi-
Scripps Research Institute, LaJolla, Califor- cal Biochemistry and Genetics, Center for
nia 92037 Biomolecular Recognition, The Panum In-
KCAJ D. ENEEK (21), Department of Microbi- stitute, DK-2200 N Copenhagen, Denmark
ology, Duke University Medical Center, AHUVA MISSIN (5), The Institute of Hematol-
Durham, North Carolina 27710 ogy, The Chaim Sheba Medical Centre,
TREBOR C. LADNER (2, 4), Protein Engi- Sachler School of Medicine, Tel Hashomer
neering Corporation, Cambridge, Massa- ,12625 Israel
chusetts 02138 NHOJ M. HSERTSO (13), Torrey Pines Institute
ETI A. AGNIRFFO-DR1AL (9), Departments of for Molecular Studies, San Diego, Califor-
Surgery and Biochemistry and Molecular nia 12129
Biology, University of Southern California CARL O. PABO (8), Department of Biology,
Medical School, Los Angeles, California Howard Hughes Medical Institute, Massa-
33009 chusetts Insitute of Technology, Cambridge,
KIT .S MAL (12), Departments of Medicine, Massachusetts 93120
Microbiology, and Immunology, Arizona WEHrTAM J. rTEKNULP (26), Department of
Cancer Center, University of Arizona, Col- Chemistry, University of California, Berke-
lege of Medicine, Tucson, Arizona 85724 ley, Berkeley, California 02749
EAHCIM LEBL (12), Hoechst Marion Roassel, YRRAB YKSILOP (17), NeXstar Pharmaceuti-
Tucson, Arizona 85737 cals, Inc., Boulder, Colorado 10308
ARDNASELLA OGAZZUL (6, 7), IRBM P. An- DRAWDE J. REBAR (8), Department of Biol-
geletti, 00040 Pomezia, Rome, Italy ogy, Massachusetts Institute of Technology,
DEREK NAELCAM (16), A ffymax Research In- Cambridge, Massachusetts 93120
stitute, Palo Alto, California 40349 ECURB L. STREBOR (2, 4), Genzyme Corpora-
ENERI DLEFREJAM (19), Department of Mo- tion, Framingham, Massachusetts 1071O
lecular, Cellular, and Developmental Biol- TERAGRAM E. SKAS (22), Division of Biology,
ogy, University of Colorado, Boulder, Colo- California Institute of Technology, Pasa-
rado 80309 dena, California 52119
MAILLIW DNALKRAM (2, 4), Vertex Pharma- YERFFEJ R. NOSPMAS (22), Division of Biol-
ceuticals, Inc., Cambridge, Massachusetts ogy, California Institute of Technology,
93120 Pasadena, California 52119
HTIDE L. NITRAM (10), Affymax Research In- LEINAD V. ITNAS (15), Department of Phar-
stitute, Palo Alto, California 40349 maceutical Chemistry, University of Cali-
LARRY C. SIKAEHFTAM (11), Affymax Re- fornia, San Francisco, San Francisco, Cali-
search Institute, DiaP Alto, California 40349 fornia 34149
OLOAP MONACI (6, 7), IRBM P. Angeletti, RETEP J. ZTAHCS (10), Affymax Research In-
04000 Pomezia, Rome, Italy stitute, Palo Alto, California 40349
CONTRIBUTORS TO VOLUME 762 xi
EGROEG P. HTIMS (1), Division of Biological GREG RETNIW (5), Medical Research Council
Sciences, University of Missouri, Columbia, Centre for Protein Engineering, and Labo-
Missouri 11256 ratory of Molecular Biology, Cambridge
CB2 2QH, United Kingdom
RETEP PORTS (12), Hoechst Marion Roussel,
Tucson, Arizona 85737 QING GNAY (3), Department of Pharmaceuti-
cal Chemistry, University of California, naS
Yu TIAN (20), Department of Chemistry, Indi- Francisco, San Francisco, California 34149
ana University, Bloomington, Indiana
50474 LEAHCIM YARUS (19), Department of Molec-
ular, Cellular, and Developmental Biology,
ECYOJ GNAST (23), Departments of Chemistry University of Colorado, Boulder, Colo-
and Molecular Biology, The Scripps Re- rado 90308
search Institute, La Jolla, California 92037
NANIJ YU (1), Department of Pharmacology,
I-GNEHC WANG (3), Department of Pharma- School of Medicine, University of -'sttiP
ceutical Chemistry, University of California, burgh, Pittsburgh, Pennsylvania 16251
San Francisco, San Francisco, California
CINIMOD A. IHC1Z (18), NeXstar Pharmaceu-
34149
ticals, Inc., Boulder, Colorado 10308
KRAM HCLEW (19), Department of Molecular, NWAHS ~rEN~rXZ (19), Department of Molecu-
Cellular, and Developmental Biology, Uni- lar, Cellular, and Developmental Biology,
versity of Colorado, Boulder, Colorado University of Colorado, Boulder, Colo-
90308 rado 80309
LEUMAS C. SMAILLIW (5), Medical Research DLANOR N. NAMREKCUZ (25), Drug Design
Council Centre for Protein Engineering, and Development, Chiton Corporation,
Cambridge CB2 2QH, United Kingdom Emeryville, California 80649
1 AFFINITY MATURATION OF PHAGE-BORNE LIGANDS 3
11 Affinity Maturation of Phage-Displayed
editpeP sdnagiL
yB
JINAN YU and GEORGE P. SMITH
Introduction
Many experiments in this volume start with large libraries of random
amino acid or nucleotide sequences of a certain length from which a tiny
subset is selected according to some criterion of "fitness"--most often,
affinity for a chosen target receptor. In most cases the library represents
sequences of the same length exceedingly sparsely. Even the very best
(fittest) sequence in a sparse initial library may be much inferior to the
globally best sequence of the same length.
If the sequences are capable of heritable mutation--phage display and
random RNA and DNA libraries fall into this category--the problem of
sparseness might be addressed by encouraging fitter sequences to "evolve"
from parent sequences in the initial library. 2'1 This sort of artificial evolution
is exemplified by the "greedy" strategy: Step A, from the initial library
select the very best sequence; call this the "initial champion." Step B,
mutagenize the initial champion randomly, producing a "clan" of closely
related mutants. Step C, from that clan select the mutant with the very
best fitness. Step D, repeat Steps B and C as needed until an optimal ligand
is found. Each round of selection thus selects "greedily" for the very best
sequence available in the current population.
A drawback of the greedy strategy is that it can only explore close
relatives of the initial champion--a tiny parish in the vast "space" of
possible sequences. Yet, for all we know, the best sequence in that neighbor-
hood may be far inferior to sequences lying totally elsewhere in sequence
space. Might it not then be worthwhile to explore the neighborhood of the
second-best sequence in the initial library? of the third best? of every
sequence with fitness above a certain threshold? In order thus to broaden
the search for fitter sequences, the stringency (fitness threshold) can be
reduced in the early rounds of selection, so as to include sequences some-
what inferior to the initial champion: Step A', from the initial library select
a mixture of sequences with diverse fitnesses (ideally, above a certain
threshold). Step B', mutagenize the entire population of selected sequences
1 D. J. Kenan, D. E. Tsai, and J. D. Keene, Trends Biochem. Sci. 19, 57 (1994).
2 j. W. Szostak, Trends Biochern. Sci. 17, 89 (1992).
thgirypoC © 6991 yb cimedacA ~sserP .cnI
SDOHTEM NI .YGOLOMYZNE .LOV 762 llA sthgir fo noitcudorper ni yna mrof .devreser
4 PHAGE DISPLAY LIBRARIES 1
to produce many clans of mutants. Step C', from those clans select a mixture
of sequences with diverse fitnesses (ideally, above a slightly higher threshold
than in Step A'). Step D', repeat steps B' and C' as often as desired,
possibly increasing the stringency of selection with succeeding rounds. Step
E', after the final round of mutagenesis, stringently select the very best
sequence in the current population.
Alternating nonstringent selection with mutagenesis in this way makes
it possible to discover "dark horses": sequences in the initial library that
are inferior to the initial champion, yet can be mutated to even higher
fitness than can that champion. A dark horse will usually lie in a different
neighborhood than the initial champion, since in most cases two sequences
in the same small neighborhood will be able to mutate to the same local
optimum. Even a well-implemented experiment may fail to reveal dark
horses in any particular case (see Discussion), most obviously because there
are none to reveal. Still, dark horses may appear sufficiently frequently to
make this an attractive alternative to the greedy strategy.
When the fitness being selected for is affinity for a target receptor
molecule, the foregoing program is called "affinity maturation," the term
coined by immunologists for the interspersed rounds of selective stimulation
by antigen and somatic mutation of antibody genes that is thought to
give rise to antibodies with increasing affinity in the course of an immune
response. 3 This chapter covers affinity maturation from random peptide
libraries displayed on phage. The procedures and underlying principles will
be discussed in the context of a specific exemplar experiment in which
ligands for a model receptor were selected from a library of random 15-
mers. 4 The model receptor was S-protein, a 104-residue fragment of bovine
ribonuclease prepared by partial digestion with subtilisin; the other frag-
ment, S-peptide, corresponds to the N-terminal 20 amino acids. 5 Neither
fragment alone is enzymatically active, but when they are mixed, S-peptide
binds strongly to S-protein, restoring enzyme activity. 6
Vector, Initial Library, and Overall Plan
The procedures in this article are tailored for libraries in fUSE5 7 and
related vectors, which have a tetracycline (Tc) resistance determinant in
3 Eisen, H. N., in "Molecular Evolution on Rugged Landscapes: Proteins, RNA and the
Immune System" (A. .S Perelson and S. A. Kauffman, eds.), p. 75. Addison-Wesley, New
York, 1991.
4 T. Nishi, H. Tsurui, and H. Saya, Exp. Med. 11, 1759 (1993).
s F. M. Richards and P. I. Vithayathil, I. Biol. Chem. 234, 1459 (1959).
6 H. C. Taylor, D. C. Richarson, I. S. Richardson, A. Wlodawer, A. Komoriya, and I. M.
Chaiken, J. Mot Biol. 149, 313 (1981).
7 j. K. Scott and G. P. Smith, Science 249, 386 (1990).
1 AFFINITY MATURATION OF PHAGE-BORNE LIGANDS 5
BglI BgII
CTATTCTCACTCC-GCCGACGIGGGCT(NNK) 15GGGC~CGCT~ GGGCCGAAAcTGTTGAA
Forward primer ~ --+ Reverse primer
A D G A X15 G A A G A E T V E
FIG. .1 Nucleotide sequence near the beginning of the pill gene in the random 15-mer
library. 4 Only the plus strand--the strand that is packaged in virions and that is anticomplemen-
tary to mRNA--is shown. In the initial library, before selection, positions designated N had
(theoretically) an equal mixture of all four nucleotides, K an equal mixture of G and T. The
corresponding amino acid sequence at the N terminus of mature pill is shown in the one-
letter code; 5~X stands for the random 15-mer encoded by the degenerate codons. The PCR
priming sites used in construction of mutant libraries (see Mutagenesis) are underlined.
Cleavage of the PCR product at the flanking BglI sites releases a degenerate 60-bp fragment
that can be spliced to the Sill-cleaved fUSE5 vector.
the minus-strand origin 8 (changes required for other vectors 9 are obvious
and do not materially affect the discussion). Although the resulting defect
in minus-strand replication reduces plaque size to near invisibility, the
phage can be cloned and propagated as plasmids by infecting a Tc-sensi-
tive host and growing in medium containing Tc (filamentous phage do not
kill the host cell); phage are titered as transducing units (TU) by counting
Tc-resistant colonies. Only cells bearing F-pili can be infected, but the
pilus is not required for phage production by transfected cells. Expression
of Tc resistance by newly infected or transfected cells is induced by
culturing them ~30 min in a subinhibitory concentration of Tc (0.2
/zg/ml).
Phage libraries, including the receptor-specific mutant libraries created
in the course of affinity maturation (see Mutagenesis), are constructed by
splicing foreign DNA inserts into the gene for coat protein plIl (five copies
at one tip of the virus) or pVIII (thousands of copies forming the tube
surrounding the DNA). The peptide encoded by the insert is displayed
on the virion surface fused to the coat protein and is available to bind
macromolecular target receptors for which it has affinity.
The fUSE5 vector has two SfiI cloning sites near the beginning of the
plII gene, 7 between which a synthetic BglI fragment with 51 degenerate
codons was inserted to create the initial library for the exemplar experiment 4
(Fig. 1). Each clone has a particular sequence of 51 codons and displays
the corresponding 15-residue peptide. There are 3.3 × 9101 possible -51
mers altogether, but only ~2 × 801 clones in the initial library--a sparse
library indeed.
8 G. P. Smith, Virology 167, 156 (1988).
9 G. P. Smith and J. K. Scott, Methods Enzymol. 217, 228.
6 PHAGE DISPLAY LIBRARIES 1
Affinity maturation begins with alternating rounds of affinity selection
and mutagenesis, the stringency of selection being kept low (see Introduc-
tion). The phage population resulting from these alternating rounds--
hopefully greatly enriched for receptor-binding clones--is then subjected
to additional rounds of stringent selection without mutagenesis in order to
identify the highest-affinity clones, which are analyzed by sequencing and
binding studies. Figure 2 outlines the sequential arrangement of selection
steps (producing Eluates 1-3, 4A-4F, and 5A-5F) and mutagenesis steps
(producing Mutant Libraries 1 and 2) in the exemplar experiment; also
Initial ~rarbil
~ pets-l noitceles
htiw 01 gal rotpecer
. 1
Conventional ~ sisenegatum
selection
Mutantlibrary 1 1
,~ pets-l noitceles
~" 01 gal htiw I gal rotpecer
Eluate 2' I Eluate 2 I
~ sisenegatum
~ pets-2 ~ pets-l noitceles
001 gn htiw I gal rotpecer
I Eluate 3' I I Eluate 3 I
gn ¢4~ pets-2
Ol gn
Eluate 4A I Eluate 4B Eluate 4C I Eluate 4D I LEluate 4E Eluate 4F I
pets-, ~ pets-1 ~ pets-1 ~ pets-2 ~ pets-2 % pets-2
I gal 001 gn 01 gn I gal 001 gn 01 gn
"Eluate5A Eluate5BI ~ Eluate5D I Eluate5E IEiuate5F
FIG. .2 Outline of the exemplar affinity maturation of ribonuclease S-protein ligands.
Arrows labeled "l-step selection" and "2-step selection" correspond to rounds of affinity
selection by the one- and two-step methods described under Affinity Selection; the amount
of receptor (biotinylated S-protein) used in each round is shown. All eluates but 3' and 5A-5F
were amplified (see gniyfitnauQ dleiY dna gniyfilpmA setaulE under Affinity Selection) before
being mutagenized or subjected to the next round of affinity selection. Arrows labeled "muta-
genesis" correspond to PCR mutagenesis and mutant library construction (see Mutagenesis).
Also shown is a conventional affinity selection experiment (without mutagenesis) that was
carried out in parallel with affinity maturation. °1 Thus, Eluate 2' was selected directly from
Eluate ,1 and Eluate 3' from Eluate 2', without mutagenesis.
l I YTINIFFA NOITARUTAM FO ENROB-EGAHP SDNAGIL 7
shown is a conventional selection experiment without mutagenesis (Eluates
2' and 3') that was carried out in parallel for comparison.
MT
In the sections that follow, the principles and practice of affinity matura-
tion will be discussed in detail, with the exemplar experiment serving
throughout as an illustration. Table I gives the formulas or recipes for
solutions and preparations, Table II describes standard procedures, and
Table III lists Escherichia coli strains.
Affinity Selection
Each affinity selection step starts with a mixture of phage and seeks to
select from that mixture phage whose displayed peptide binds the target
receptor. These phage are specifically "captured" by immobilizing the re-
ceptor on a solid surface (e.g., a plastic petri dish); unbound phage are
washed away and captured phage are eluted (still in infective form), yielding
a selected subset of the original phage mixture that is called an "eluate."
Stringency
The stringency of affinity selection is controllable in some degree by
the choice of conditions, as will be detailed later. The logic of affinity
maturation calls for low stringency (thus high yield) in the early rounds of
selection (see Introduction). There is an additional argument--even in
conventional selection without mutagenesis--for choosing high yield in the
very first round of selection, whose input consists of all clones in the initial
library. Because the library has many clones, each clone is represented by
few particles (-500 TU/clone on average in the exemplar experiment);
consequently, if the yield for a binding clone is not high in the first round
(>0.2% in the exemplar experiment), that clone has a good chance of being
lost, and of course can never be recovered. In later rounds, especially after
the last round of mutagenesis, stringency can be increased in order to select
for the tightest binder.
There is a limit to stringency, however. The reason is that there is always
a background yield of nonspecifically bound phage; if stringency is set
too high, the yield of specifically captured phage will fall far below the
background of nonspecifically bound phage, and all power of discrimination
in favor of high affinity is lost.
In practice, because the relationship between selection conditions and
stringency is unknown in advance, it is advisable to explore a range of
conditions in the final rounds of selection; those whose yields are close
01 .D .A ,ztluhcS .J .E ,yrubdaL .G .P ,htimS dna .R .O Fox, dehsilbupnu .)5991(
