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Eur. J. Biochem. 203,33 -42 (1992)
a FEBS 1992

Review
Protein interaction with ice
Choy L. HEW and Daniel S. C. YANG2
Research Institute, The Hospital for Sick Children and Departments of Clinical Biochemistry
and Biochemistry University of Toronto, Canada
Department of Biochemistry, McMaster University, Hamilton, Canada

(Received May 28,1991) - EJB 91 0694

Many organisms have evolved novel mechanisms to minimize freezing injury due to extracellular
ice formation. This article reviews our present knowledge on the structure and mode of action of two
types of proteins capable of ice interaction. The antifreeze proteins inhibit ice crystal formation and
alter ice growth habits. The ice nucleation proteins, on the other hand, provide a proper template to
stimulate ice growth. The potential applications of these proteins in different industries are discussed.

Water is essential to all living cells. It serves as a medium
for biological reactions, solute transport and interaction, and
regulation of intracellular pH. It is also one of the reactants
in many biochemical reactions, and contributes to the
stabilization of various macromolecular structures. Any sig-
nificant deviation on the accessibility of water due to dehy-
dration, dessication and the alteration of its physical state
from aqueous phase to ice crystal will pose a severe threat to
the normal function and survival of organisms [l].For many
organisms, it is both desirable and important to have the
ability to counteract or minimize these threats. The production
of specific protein molecules to prevent the loss of water or to
inhibit extracellular ice crystal growth are the better known
examples.
In the past decade, significant progress has been made
in characterizing several classes of proteins capable of ice
interaction. These include, surprisingly, two functionally dis-
tinct and opposite classes of proteins. These are the antifreeze
proteins which inhibit ice crystal growth and ice-nucleation
proteins, which on the other side, promote ice growth. The
present review will only highlight some of the recent findings.
Several excellent reviews are available [2 - 81.
ICE STRUCTURE AND ICE CRYSTAL GROWTH
When pure liquid water is cooled at atmospheric pressure it
does not freeze spontaneously at 0 “C, the equilibrium freezing
point of water; instead it continues to remain a liquid well
below O‘C. The water is then said to be ‘supercooled’ or
‘undercooled’. Due to density fluctuations in liquid water,
water molecules form clusters with the same molecular ar-
rangement as in the ice crystal. This process is energetically
Correspondence to C. L. Hew, Department of Clinical Biochemis-
try, University of Toronto, 100 College Street, Toronto, Ontario M5G
1L5, Canada
Abbreviafions. INA, ice nucleation activators; INP, ice nucleation
proteins; AFP, antifreeze proteins or polypeptides; AFGP, antifreeze
glycoproteins; .f, cumulative ice nucleation frequency; z, number of
cells.
unfavourable, as it tends to increase the total energy of the
system; as a result, these clusters tend to be unstable and break
up rapidly. However, upon further cooling, likelihood of these
nuclei to grow in both number and size is increased. When
these ‘ice-embryos’ or ‘nuclei’ reach a certain dimension (of a
criticlal size/volume ratio), their further growth leads to the
decrease in the total energy (a favourable process) and ice
crystallization occurs rapidly. This process is called ‘homo-
geneous ice nucleation’. The temperature at which this occurs
(about - 40°C) is called the ‘supercooling threshold tempera-
ture’.
In practice, impurities or foreign particles usually present
in water act to attach water molecules on their surfaces. On
some surfaces, water molecules may be oriented in a way such
as to resemble an ice-nucleus. Upon further cooling, these
become compatible with the critical dimension of ice-nu-
cleation. These foreign particles are called ‘ice nucleation acti-
vators‘ (INA) and the process is called ‘heterogeneous ice
nucleation’. Since the formation of the ice embryo starts off
with the dimension of the INA, heterogeneous ice nucleation
always occurs at a temperature greater than the homogeneous
ice nucleation. Threshold temperatures of heterogeneous ice
nucleation usually lie between - 2 “C and - 15“C. Many INA
are known, including silver iodide and many inorganic clays.
Ice can exist in several crystalline polymorphic structures
and also in an amorphous or vitreous state of rather uncertain
structure. Of these, only the ordinary or the hexagonal ice, l h ,
is stable under normal pressure at 0°C. The structure of the
I,, is shown in Fig. 1 with the a and c axis indicated 19, lo].
The function of the antifreeze proteins is to inhibit ice
formation by suppressing the growth of ice nuclei. In doing
so, they alter the ice habit and growth rate and, consequently,
lower the freezing temperature. The ice nucleation proteins
stimulate ice growth by providing templates for ice crystals.
In several freeze-tolerant insects, both the antifreeze proteins
and ice-nucleation proteins are present together in the
hemolymphs 1111. These two proteins work cooperatively to
control the size and stability of small ice crystals.

34
c- f is
Fig. 1. Schematic representationof an hexagonal ice crystal. The basal
plane, prism faces and the c and three a axis ( a , , u 2 , u 3 ) are shown.
Normally, the ice growth takes place on each prism face.
STRUCTURE AND FUNCTION
OF ANTIFREEZE PROTEINS
Structural diversity of antifreeze proteins
Freezing is detrimental to most organisms. Many organ-
isms face the threat of freezing all year around or seasonally.
For example, in Antarctic sea waters, the year round tempera-
ture (- 2 "C) could be well below the freezing temperature of
most fishes (- 0.7"C), while in the polar regions, the sea water
temperature could lower to -1.7"C in the winter months.
Similarly, the overwintering larvae of many terrestial insects
encounter extreme fluctuation in temperatures. Many
adaptative mechanisms have been utilized by various species.
These include seasonal migration, hibernation, supercooling,
synthesis of small cryoprotectant molecules such as glycerol,
trehalose, mannitol and others, and lastly, the synthesis of
antifreeze proteins.
It is known that most fishes, due to the presence of electro-
lytes In their sera (mostly as Na', C1-, 0.14 M), have a freez-
ing temperature of around - 0.6 "C to - 0.7 "C. Therefore,
they cannot tolerate any lower temperatures. Scholander and
coworkers [I21 were the first to observe the unusually low
freezing temperatures (- 1.6"C) and the presence of
macromolecular antifreezes in Arctic sculpins in Baffin island
and in Hebron Fjord, Northern Labrador, Canada. Similar
observations were later confirmed by DeVries and coworkers
from several species of Antarctic fishes having freezing tem-
peratures of - 2°C [2]. More recently, several laboratories
[2 - 41 have demonstrated the presence of antifreeze proteins
in a variety of cold water inarine fish species. Based on their
primary structure, as well as the presence or absence of carbo-
hydrates, these macromolecular antifreeze can be classified
into two major groups: the antifreeze glycoproteins (AFGP)
and antifreeze proteins/polypeptides (AFP). The AFP can be
further subdivided into at least three subtypes [4]. The general
features of these AFGPiAFP is shown in Table 1. At present,
the tertiary structure of only the type I AFP is known [13].
Obviously, there are major structural differences among these
different antifreezes and, therefore, it is difficult to formulate
a general mechanism. Nevertheless, some understanding has
been gained concerning their mode of action and will be pre-
sented below.
Recently, the corrected primary structure of the sea raven
AFP with the disulfide bonds assigned has been determined
by Ng and Hew (unpublished results). Computer search of
the structure has revealed that the sea raven AFP is similar to
a wide range of lectin and lectin domains in larger proteins
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For example, it is similar to the galactose-binding lectin from
the acorn barnacle, echinoidin (a lectin from sea urchin), the
human pancreatic stone/thread protein, cartilage pro-
teoglycan core protein, lymphocyte IgE receptor and human
hepatic lectins (Hl, H2A). Several of the disulfide bonds are
conserved. A striking feature of one of these comparisons
comes from the apparent similarity in function between the
sea raven AFP and pancreatic stone protein: their ability to
inhibit crystallization. While sea raven AFP binds to ice to
prevent growth of ice crystals, pancreatic stone protein binds
to calcium to prevent growth of calcium carbonate cyrstals.
The elucidation of the tertiary structure of any of these two
proteins might provide some insight on their mode of action.
Antifreeze proteins have also been isolated from terrestrial
arthropods including several insects, spiders, a centipede and
an Antarctic mite. Most of these proteins have not been well
characterized. The antifreeze proteins from the larvae of the
beetle, Tenebrio molitor, are rich in half-cystine [14] and might
share structural similarities with the type I1 fish AFP. In
general, insect antifreeze proteins are more active than the fish
antifreeze proteins 1111. It will be interesting to study the
structure/function relationship of the insect antifreezes once
their structures are characterized.
In addition to fishes and insects, many plants are known to
withstand freezing temperatures. The plant hormone abscisic
acid has been shown to modulate the response to cold and
water stresses. Both of these stresses lower the water potential
of the cell and cause similar damage to plants. The freeze
resistance in plants is apparently due to the tolerance of cell
dehydration as a result of ice formation and growth in the
intracellular space 1151. Consistent with this thinking, Kurkela
and Franck [16] have isolated a cold-inducible gene, Kin1 from
Arahidosis thaliana. The gene is inducible by both water stress
and abscisic acid. The Kin1 gene codes for a small hydrophilic
polypeptide which is intracellularly located. The polypeptide
shares many similar features with the type I flounder AFP
and has prompted the authors to suggest that it functions as
an antifreeze protein.
Ice binding and alteration of ice morphology of antifreezes
It is generally believed that antifreeze proteins function at
the ice water interface; therefore, the binding of antifreeze
proteins to ice is requisite for their functions. The adsorption
of the proteins to the ice surface has been examined by a
variety of techniques. Raymond and DeVries [17] monitored
the changes in temperature during the freezing of pure water,
and solutions of antifreezes and NaCI. A broad transition
temperature range for NaCl solution was observed, suggesting
the exclusion of the ions from the ice phase. However, a sharp
transition temperature was observed for both pure water and
the antifreeze proteins indicating that the antifreeze proteins
were actually incorporated into the ice phase. Similar results
have also been observed by Tomimatsu et al. [18] and Kerr et
al. [39].
Direct evidence for AFGP adsorption onto ice surface
was provided by Brown et al. [20] using light spectroscopy.
Polarizable materials respond non-linearly to incoming light
of high intensity, and the signal transmitted by such a system
is a superposition of the fundamental and harmonic frequen-
cies. If the polarizability of the material possesses inversion
symmetry with respect to the light wave oscillation, then only
the odd harmonics of the polarizability would be induced by
intense incident light. Since a surface does not possess inver-
sion symmetry with respect to its normal, the reflected light

Table 1. Occurrence and classification of fish antifreeze proteins. Results are compiled from references in [4] unless indicated otherwise
Property AFGP AFP
~ ~ ~~~ ~ ~
General feature carbohydrate-rich alanine-rich
Molecular mass 2.6- 34 kDa 3.3 -4.5 kDa
Primary structure (Ala-Ala-Thr), (1 1 amino acid
dissacharide rcpeat)3 ~
Secondary structure expanded amphipathic
a-helical
Hiosynthetic precursor polyprotein 1691 preproAFP
Fish species antarctic cod winter flounder
Atlantic cod yellowtail flounder
frost fish shorthorn sculpin
polar cod grubby sculpin
will also induce harmonic, an effect termed ‘surface second
harmonic generation’. Pure ice-water interface exhibits no
such discernible effect. Consequently, an adsorbed antifreeze
layer on an ice crystal should cause surface inversion asym-
metry and an increase in detected intensity, which was verified
experimentally. These studies have provided the evidence for
a specific interaction of active antifreezes with ice crystals.
The antifreeze proteins, rather than binding uniformly to
all ice surfaces, adorb preferentially to specific planes of ice
crystals. As a result, they deter the morphology of the ice.
Scholander and Maggert [12] noticed that whereas normal
dendritic ice was formed in fish sera devoid of antifreeze, ice
grew as fine needles in those sera with the antifreeze proteins,
and the rate of ice growth was considerably faster in the
latter. The altered growth habits and growth rates of ice were
observed subsequently by many other workers. For example,
Raymond and DeVries [21] showed that in antifreeze solutions
(both AFGP and type I AFP), the needle-shaped crystals grew
along the crystallographic c axis of ice, as compared to normal
a axis growth in water. Therefore, antifreeze proteins appeared
to inhibit the normal growth direction of ice by preferentially
adsorbing to the prism faces of ice crystals.
The most definitive proof of adsorption of a type I AFP
on specific ice crystals planes was provided by Knight et al.
[22]. They grew a single ice crystal hemisphere into a dilute
solution of the AFP, thus presenting various crystallographic
planes to the protein for adsorption. Due to the low AFP
concentration employed, only the planes that had the highest
AFP affinities were coated with the protein. These planes were
later identified by evaporating the ice crystal in a - 10°C cold
room. During the sublimation process, most surfaces of the
ice crystal became glass-like except sites of adsorption of the
AFP which remained rough, resembling finely ground glass.
Knight et al. [22] identified these planes as the [20,21] pyrami-
dal planes of ice. The planes of adsorptions for other anti-
freezes appeared to be crystallographically different and were
much more complex. Based on this study, they have proposed
a mechanism for the observed preferential binding to the
pyramidal planes. We will summarize this in a later section.
In our own studies, the distinct morphology of the ice
crystal is routinely used as an indicator for the presence of
antifreeze activity during the fractionation of naturally occur-
ring and recombinant-derived AFP, i.e. bipyramid ice crystal
shape for the AFGP, type I and 111 AFP, multifaceted ice
crysfal for type I1 AFP [4]. Similarly, the inhibition of n or/
and r axis ice crystal growth by various AFP or their structural
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35
type I1 type I11
cy she-rich average
11.3 - 24 kDa 6 kDa
disulfide- average
linked
contains ordered but
/-sheet unclassified
preproAFP (?) preAFP
sea raven ocean pout
smelt [70] wolffish
Atlantic herring [70]
analogs has been used as a measurement of the relative ice
binding affinity [23].
Adsorption inhibition due to reversible antifreeze binding
While the growth of most crystals occurs by propagation
of steps across crystal faces, various studies have implied that
impurities can adsorb to crystal faces and interfere with the
step propagation. Further growth of the steps will have to
force through between the impurities and thus result in a
curved front. Such a process is energetically less favorable and
therefore the growth rate is retarded. This effect is known as
the ‘Kelvin effect’ [22]. Based essentially on the Kelvin effect
and on an earlier proposed theory on the inhibition of crystal
growth by adsorbants [24], Raymond and DeVries [21] have
described the action of antifreeze proteins in ierms of
adsorption-inhibition. This model predicts that the growth
of an ice crystal halts when the average separation between
adsorbed antifreezes approaches twice the critical radius of
curvature for that particular temperature.
Assuming that antifreeze proteins are evenly distributed
over the entire ice surface, the lowering of the freezing tem-
perature of water due to surface effects is:
AT = (~cJMT,/LQJ( ~ K ~ C N / M , ) ’ ~ ~
where AT is the freezing point depression (i.e. antifreeze ac-
tivity); (T is the surface tension; Tois the normal freezing point
(273 K); M is the molar mass of water; L is the molar heat of
fusion; ei is the density of ice; 2r is the diameter of the anti-
freeze protein; a is the relative amount of antifreeze protein
that is incorporated into the ice crystal; C is the antifreeze
concentration (mgiml); N is Avagadro’s number; and M , is
the molar mass of the antifreeze protein (in g/mol). Using
experimental values for CI and assuming that 2r was equal to
0.8 nm, the curves of AT versus C were calculated. Using this
model, Raymond and DeVries were able to fit experimental
data from a number of antifreezes.
Burcham et al. [25] have also analyzed curves of antifreeze
activity versus concentration. Their analysis assumes that anti-
freeze proteins bind reversibly to ice. They described the inter-
action between AFGP and ice as simple equilibrium binding:
AFGP + ICE 8 AFGP-ICE
where AFGP is one antifreeze molecule and ICE is one binding
site on an ice crystal. The reversible binding was treated like
a receptor-ligand interaction. It was assumed that an ice crys-

36
tal had a finite number of binding sites (which was determined
by the crystal size) to which antifreeze molecules bind with
equal affinity. The following equation was derived.
AT = AT,[AFGP]/(Kd + [AFGP])
After measuring the activity ( A T ) of different types of
AFGP as a function of concentration, the binding capacity
(AT,) and the binding constant (Kd) for individual AFGP
were calculated. These parameters varied in magnitude be-
tween the AFGP. If the calculated Kd represents the true
equilibrium constant for the reversible binding of the anti-
freeze proteins, then it should be possible to calculate the
enthalpy and entropy contributions to the binding process. As
pointed out by these authors, determination of the antifreeze
activity by the freezing point osmometer method or the micro-
scopic observation method yields different values of Kd and
AT,. Therefore, it is difficult to say whether these calculated
parameters are physically meaningful. Nevertheless, this
method may still be useful for comparing the antifreeze ac-
tivity curves of different antifreeze proteins using a single
technique of measuring antifreeze activity.
The two proposed models compliment each other in ad-
dressing two different aspects of the adsorption-inhibition
mechanism. While Burcham et al. [25]purposely neglected the
physical consequences of the binding of the protein to ice
surfaces, Raymond and DeVries [21] failed to consider the
distribution coefficient to be concentration-dependent. Gen-
eralizing from the two models, one obtains the following:
the binding of the protein molecules to the ice surfaces are
reversible and Kelvin effect is the main reason for the freezing
depressions observed. The observed plateau in activity might
correspond to the saturation of protein binding sites on the
ice crystal surface.
Mechanism of the preferential binding of antifreeze proteins
to ice prism faces
Although the above discussion provides plausible expla-
nations of how antifreeze proteins retard the growth of ice
crystals once they are adsorbed onto the ice crystal surfaces,
one of the central questions remains unanswered: why does
antifreeze protein adsorb preferentially to the prism faces of
ice? DeVries and Lin [26] attributed the preferential binding
of the winter flounder AFP to the apparent complimentarity
between the spatial orientation of the polar groups on the
AFP and that of the oxygen atoms on the ice surface. The
winter flounder AFP is a-helical (translation = 0.15 nm/resi-
due) and is composed of triplicated 11-residue segments of
Thr-Xaa,-(Asp or Asn)-Xaa,, where Xaa is mainly alanine
(see Table 1). Therefore, the distance separating Thr and Asp
or Asn residues is 0.45 nm. This distance matches the distances
separating adjacent oxygen atoms of the water molecules on
the ice surfaces. The presence of this lattice match prompted
DeVries and Lin to suggest that this AFP binds to the ice
crystal surfaces by hydrogen bond formation between the
sidechains of Thr, Asp, and Asn and the oxygen atoms of the
ice crystal surface.
This lattice match model fails to explain the preference of
binding to the prism faces because as the 0.45-nm separation
is not unique to prism faces, it can be found on a large family
of ice crystal planes including the basal plane. Therefore, the
lattice match model will predict AFP to bind to different ice
planes with similar affinity.
The question of the origin of binding preferences was
explored by Yang et al. [13] after obtaining the three-dimen-
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unordered
basal plane
I
1. AFP binds preferentially to prlsrn faces
through dipolar and hydrogen bond
interactions.
2. This results in ordering of waterdipoles
that lie within the field of AFP helix-dipoles
(unshaded area).
3. Ice still grows on unordered basal plane
(shaded area).
4. AFP binds to new ice fronts.
5. Continued ice growth on basal plane,
and continued binding of AFP to prism
faces results in bipyramidal ice crystals.
Fig. 2. Schematic representation of the interaction of the type I AFP
with ice.
sional structure of the type I AFP from winter flounder. After
extensive efforts to identify a match between the binding sur-
face on the crystal structure of the AFP molecule and the
prism face (which has so far been assumed to be the preferred
site of binding), Yang et al. [13] failed to find any matching
patterns which were significantly better for the prism faces
than for the basal planes. Consequently, they proposed that
the selection of prism face for binding might originate from
the dipolar nature of the AFP. It was argued that the dipole
field of the a helix would align dipole moments of individual
water molecules in the ice crystal, thus inducing a dipole-
dipole interaction between the protein molecule and the ice
crystal. It was observed that the direction for maximum dipole
induction would be along the < 221 > vectors. Since these
vectors can be found on the prism face but not on the basal
plane, the dipole-induced dipole interactions then confer the
‘specificity’for prism face binding (Fig. 2) [4, 131.
The dipolar model offers an attractive explanation for
preferential binding of a-helical AFPs but it cannot account
for the binding of other AFPs that are non-a-helical. Preli-
minary data using the program DELPHI [27] to calculate
the electrostatic potential around the a-helical type I AFP
indicates that the field effect around the AFP is relatively
small to account for the dipolar interaction (Sicheri and Yang,
unpublished). More work is needed to clarify its role in protein
ice interaction. The dipole moment, however, is important in
the stability of the a helix in the AFP [28].
Recently, Knight and coworkers [22] have demonstrated
that the prism faces are not necessarily the preferred site of
binding. They have found that AFP from winter flounder and
Alaskan plaice adsorb onto the [2021] pyramidal planes while
the sculpin AFP absorbs on [2770] (Fig. 3). They have also
indicated that the planes of adsorption are quite complex for

I I
c-axis c-axis
cl [OOOI] (d) [OOOI]
02
[izio]
o'f
[ziio]
1 X, L o r p t i o n plane
[z iio]
(2021)
Fig. 3. Tracings of photographsof the etched, single crystal hemispheres
grown from solutions of two types of AFP. (a, b) Tracings of photo-
graphs of the etched, single-crystal hemispheres grown from solutions
of (a) winter flounder and Alaskan plaice (F/P) and (b) sculpin (S).
(c, d) Perspectives (shaded area) of the adsorption planes of these
antifreezes with respect to the prism and basal planes in hexagonal
ice. (Reproduced with permission from [22]).
other AFPs and are crystallographically different from those
described. Interestingly, they have identified a common vector
on the [2021] and [2'f'fO] planes containing the repeating dis-
tance of 1.67 nm which matches with that of the a-helical AFP
(1.65 nm for 11 residues). They attributed the origin of binding
preferences to this matching of lattice distances. From their
study it is apparent that, at low protein concentrations, differ-
ent AFPs might have different preferred planes of binding.
However, it is not clear whether the same preferences hold
at high protein concentrations. The flexibility of the protein
molecule and the torsional movement of sidechains might
allow the AFP to bind to other faces of the ice crystal. This
is essential because at high protein concentrations (which is
physiologically relevant), most of the AFPs induce the forma-
tion of similar ice spicules, which argue against different
modes of binding for different AFPs. Thus, it is fair to con-
clude that the mechanism of AFP binding is still unresolved.
ICE NUCLEATION PROTEINS
In addition to the antifreeze proteins, another group of
proteins which can interact with ice, are the ice nucleation
proteins (INP) which are the integral components of various
types of ice nucleation activators (INA) of biogenic origin.
INA are present in a variety of plant bacteria [7], insects [5],
intertidal invertebrates [29],plants [30] and lichen [31,32]. The
JNA found in the frost-resistant frog, Rana sylvatica, have
also been shown to be composed of proteins [33].
Ice nucleating activators (INA) of bacterial origin
Various Gram-negative epiphytic bacteria have been
known to produce INA. These belong to genera Pseudomonas,
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37
Erwiniu and Xunthomonas. Interestingly, a strain of Pseudo-
monus Juorescens producing INA has also been reported to
be associated with the cultures of marine dinoflagellate He-
terocupsu niei [34]. In some species of these bacteria, not all
natural strains exhibit ice nucleation activity [35]. Those
strains which produce INA are called h a + , and those which
do not, Ina- phenotypes. The h a + strains can lead to frost
damage in some sensitive host plants 1351. This fact has led to
the recognition of the importance in ecology and agriculture
of the role played by the bacterial INA.
At present, the function of INA in bacteria is not all well
understood, although their effect on certain plants is well
known. In the laboratory, reducing the growth temperature
from 23 "C to 5 "C has been shown to stimulate the production
of INA. However, to date, frost susceptibility of the Ina-
strain has not been demonstrated. It has also been shown that
frost damage in the host plant leads to proliferation of the
a2
guest bacteria, possibly due to increase in available nutrients
[izio] [36]. Surely in the long term, the death of the host organism
sculpin adsorption cannot be beneficial to the guest bacterium. A suggestion has
plane (2110)
been made that h a f strains may have adapted to prolonged
dry weather conditions by using INA to capture water from
(ioio)
the surroundings 1371. A suggestion has also been made that
originally, in the temperate regions, h a ' bacteria may be
associated with native frost-resistant plants symbiotically and
that this relationship becomes pathogenic only in the case of
tropical plants imported into these regions [37]. The occur-
rence of Ina' and Ina- strains in nature and the role of the
INA in the guestlhost relationship between the bacteria and
the plant are fascinating topics of biology.
Measurement of ice nucleation activity
In the case of bacteria, ice nucleation activity is measured
directly on cells [38]. It is found that bacterial cells exhibit
varying threshold temperatures in the range of -2°C to
- 15"C. A procedure has been developed for performing the
analysis of ice nucleation activity [38]: a fixed volume (in a
drop of 10 - 1000 pl) of a bacterial suspension containing
a known number of cells (Z) is allowed to cool to a fixed
temperature (in the range -2°C to - 15°C). The number of
ice nuclei present, N , at this threshold temperature, t , are then
counted. This is repeated for each degree of the temperature
in this range. Cumulative ice nucleation frequency,,f, is defined
as the number of ice nuclei/cell, i.e. for the given t,
J'= N / Z .
This measurement can be repeated for each of a series of
10-fold dilutions and more statistically accurate values of ,f
can be obtained. Plotting logf against t gives the cumulative
ice nucleation spectrum (or simply, cumulative spectrum). In
some instances, another graph is obtained from the cumulative
spectrum by plotting the derivative of,fagainst t. The resultant
plot is called the differential ice nucleation spectrum. Usually,
the cumulative spectrum gives more information than the
differential spectrum, although the latter has been used to
advantage in certain studies [39]. After examining shapes of
cumulative spectra, it was suggested that bacterial nuclei could
be classified into three classes: types I, 11, and 111with respec-
tive threshold temperature ranges of - 5°C or warmer;
-5°C to -8°C; and -1O'C or colder [40]. Recent activity
measurements using D 2 0 has led to a revision of the classifi-
cation to types A, B, and C, with respective threshold tempera-
ture ranges of -4.4"C or greater, - 4 3 ° C to -5.7"C, and
-7.6"C or lower [41].

38
Another simpler analytical procedure used comonly by
insect physiologists is to measure the highest threshold tem-
perature ( t ) of the INA in the sample [42]. Usually this is done
for each of a series of 10-fold dilutions and the threshold
temperature is plotted against dilution. This procedure does
not assay for less active nucleators and is best suited for cases
where INA do not exhibit heterogeneity in their activity.
Structure of bacterial ice nucleation proteins
Comparison of DNA fragments from h a + and Ina-
phenotypes of Pseudoomonas syringae and Erwinia herbicola led
to identification of 4.5-kb and 5.7-kb fragments, respectively,
responsible for coding for INA from these two organisms [43].
Separate insertion of these two fragments into Escherichia coli
led to transformations of E. coli from the Ina- to the two
h a + phenotypes. A similar 7.5-kb DNA fragment was iso-
lated from an h a + strain of P.fluorescens [44].The cumulative
nucleation spectrum of E. coli transformed with the DNA
from P. syringae was almost identical to that of the h a f P.
.yyringae. The cumulative spectra obtained from the two
strains of E. coli, transformed with DNA fragments from E.
herhicola and P. fluorescens, respectively, were very similar in
shape, but contained higher frequencies at all temperatures,
when compared to those of the respective original bacteria.
Thus it can be concluded that h a + E. coli can perform equally
well all post-translational modifications required to manufac-
ture active INA. Complete DNA sequences of INA genes
from P. syringae, P.fluorrscens and E. herhicola genes have
been obtained. Primary protein sequences of these three re-
spective gene products (INP) have thus been deduced [44].
The three sequences appear to be highly similar, each with a
molecular mass of around 115 kDa and consisting of a large
number of eight-amino-acid repeats of a consensus sequence
(Ala-Gly-Tyr-Gly-Ser-Thr-Leu-Thr). In addition, every alter-
nate eight-residue peptide exhibits greater similarity than
neighbouring ones. Thus a 16-residue fragment also becomes
highly repetitive. Several repeats of 48-residue fragments (con-
taining three 16-residue fragment) are also observed. Thus,
bacterial INP are found to exhibit three orders of symmetry.
These features have also been found in the INP of Erwinia
ananus, bacteria thought to be responsible for frost damage
to tea plants and other crops in Japan [45]. Briefly, each of
these protein sequences may be represented as shown in Fig. 4.
Most of the structural studies carried out on bacterial INA
has been performed by measuring nucleation activities directly
on Inat cells and on recombinant products isolated from h a +
phenotypes of E. coli transformed by inserting ina genes from
Pseudonionas and Erwinia species. Extensive mutational stud-
ies and nucleation activity studies on mutant cells show that
any disruption of a 48-residues repeating fragment affects the
nucleation activity only when it disturbs the periodicity of 48-
residue repeats [44, 4.51. Deletions at the N-terminal non-
repeating region reduce activity at all temperatures, especially
at the higher temperatures (- 2°C to - 6°C). Progressive de-
letions at the C-terminal non-repeating region destroy activity
very rapidly. It is possible that in some cases low nucleation
activity in cells may be due to poor translocation of INP
across the cytoplasm and the cell membrane and not due to
functionally inferior INP. The conclusions from these studies
are that (a) N- and C-terminal regions may be involved in
assembly and stabilization of nucleation sites, and (b) the
main site of ice-interaction must lie in the repeating region.
14321033, 1992, 1-2, Downloaded from https://febs.onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1992.tb19824.x by CAPES, Wiley Online Library on [09/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
N- terminal Middle C- terminal
(non-repeating) (repeating section) (no-repeating)
A G Y G S T - T ~ A - - S- - L -
Fig. 4. Schematic representation of primary structure of a typical bac-
terial INP. The structure consists of three well-defined segments. The
N-terminal segment consists of approximately 160- 170 residues; the
middle repeating segment consists of about 900- 1100 residues; and
the C-terminal section consists of 40-50 residues. Consensus 8-, 16-
and 48-repeating amino acid residues which make up the repeating
section are shown. One letter code is used for amino acids; a dash
indicates a variable amino acid. Modified from 1461.
Since no experimental data on the tertiary structure of
INP is presently available, model buildings have mainly been
relying on the following considerations [46,47].
a) Secondary structure predictions show that both proteins
consist mainly of fi-sheets with strong tendency for turns at
Gly-Tyr-Gly positions in each of the 16-residue repeats.
b) Generally, antiparallel stacked P-sheets are thermo-
dynamically more stable.
c) By analogy to the highly repeating primary structure of
INP, it is reasonable to asume that the tertiary structure would
also be highly repetitive.
d) The non-repeating N- and C-terminal sequences do not
contribute to the determination of the conformation of the
main, highly repetitive section of the INP believed to be in-
volved in ice interaction.
e) Finally, the tertiary structure of INP should also consist
of a surface that is latice-matching with ice.
The above factors impose structural constraints and, thus,
simplify the problem of model construction for INP. Two
models of three-dimensional structures have been suggested
by Warren and coworkers and are shown in Fig. 5 [46]. The
first one (a) uses trigonal symmetry and consists of three pairs
of anti-parallel fl sheets per 48-residue repeat. The second one
(b) is hexagonally symmetric and conists of two antiparallel
helices, combining to form a double helix.
Other interesting models are based on computer calcu-
lations for minimum energy conformations on the INP mol-
ecule [47]. A theoretical protein of infinite number (to simplify
calculations) of repeats of the consensus eight-residue frag-
ment: Ala-Gly-Tyr-Gly-Ser-Thr-Leu-Thr was chosen as a
model for study. Upon stepwise energy minimizations, several
minimum energy conformation, all showing large right-
handed helical structures, were obtained. Ice-binding proper-
ties on surfaces of these were examined, and two motifs
capable of binding to ice were found. The first motif contained
a hexagonal symmetry; three conformations were found with
this motif. The second motif contained a pentagonal sym-
metry; only one conformation was found to contain this motif.
In all four cases, contacting crystal planes of ice were found
to be the basal plane [OOOl]. These results show the feasibility
of stable helical conformations capable of interacting with ice

a Table 2. Correlation between the extent of INP aggregation with ice
Fig. 5. Two models of INP as suggested by Warren and Wolber 1461. (a)
Trigonal model: a 48-residue repeating fragment forms three pairs of
8-sheets stacked in an antiparallel fashion on each of three symmetric
planes. (h) Antiparallel double helix model : two antiparallel helices,
one clockwise and the other anticlockwise, combine to form a double
helix.
at its basal [OOOZ] plane. Interestingly, no left-handed helical
conformations with minimum energies were found.
Assemhlv o f I N A
In the case of P. syringae [48] and E. herhicola [49], ice
nuclei have been found to be located on the outer cell mem-
branes. E. herhicola is known also to release its ice nuclei in
microvesicles [49]. Therefore it appears that INA consist of
INPs attached to the outer cell membrane; the nature of this
attachment has not been fully established. However, extensive
evidence has been presented to show that lipid phos-
phatidylinositol may play an important role in nucleation sites
[48, SO]. It has also been shown that Ina' strains exhibit
phosphatidylinositol synthase activity, while Ina- do not [50].
Also, E. coli does not show activity for this enzyme, while its
Ina' does. These findings have lead to a suggestion that, to
form ice nuclei, INP may associate with membrane
components, possibly through a covalent linkage to phos-
phatidylinositol.
Several different reports indicate that each bacterial
INA consist of multiple copies of INP. For example, data from
y ray inactivation analysis studies show that the threshold
temperature increases linearly with the log size 1391. The results
of recent theoretical calculations of dimensions of INA of
various activities also support arguments that INA are formed
by aggregations of INP residues [39, 511. Also, concentration/
activity analyses have shown that at least three copies of INP
must be involved in a co-operative fashion to form an ice
nucleus active at -8°C [52]. The relationship between the
extent of aggregation and ice nucleation activity is shown in
Table 2. It appears that INP, as a monomer, would have a
threshold temperature of - 12 "C to - 13°C. A large number
of copies may be involved in some sort of a co-operative
process to form more active nuclei. To produce a nucleus
active at - 2°C approximately 132 units INP may be required.
This may account for the rarity of these INA in the cells.
14321033, 1992, 1-2, Downloaded from https://febs.onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1992.tb19824.x by CAPES, Wiley Online Library on [09/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
39
nucleating activities. Results are compiled from [39, 521.
~
Supercooling Estimated No. of I N P
threshold molecular residues
temperature mass required to
form nucleus
'C kDa
-12 to -13 150 1
-3 870 60
-2 19800 132
-1 83 700 558
INP
Bilayer Membrane
Membrane anchor
Fig. 6. A cartoon of the proposed assembly of INA on the lipid bilayer
membrane by aggregation of multiple units of INP. See text for details.
Although no hard physical data on the three-dimensional
structure of INP and INA are available at this time, we would
like to use existing knowledge to formulate a scheme by which
INPs might be arranged to form INA. Such a model might
facilitate our understanding and designing of future exper-
iments. Based on the constraints listed previously, Warren et
al. and Mizuno have arrived at similar conclusions about the
three-dimensional structure of the INP: it has to have some
sort of helical arrangement. This conclusion is probably cor-
rect because if the INP does have a unique three-dimensional
structure, then this protein consisting of a large number (60 -
70) of repeats must adopt a helical conformation. We therefore
assume INP to be a cylindrical molecule, with the 48 residues
being the basic repeating unit along the cylindrical axis. The
next piece of information is the requirement of a large number
of INP in forming the warm-temperature INA. This aggre-
gation of INP will most likely form a hexagonal packing
arrangement such that the resulting aggregate will be compat-
ible with the ice lattice. To maximize the intermolecular inter-
actions among INPs, the external surfaces of the cylindrical
INP molecule may also have to adopt an hexagonal shape (see
Fig. 6). In such an arrangement, the surfaces of INA that
form the ice templates will also be those that are involved in
protein -protein interaction. Since these interactions are
likely to be hydrogen bonding in nature, the forces that are
holding the INPs together in the INA might be quite weak.
The anchoring of the INPs on a membrane surface will en-
hance the stability of the LNA by reducing the entropy gain

40
when an INP breaks apart from the INA. The membrane
anchor can also keep the INP in register for maximum or-
dering with the INA cluster. Since phosphatidylinositol has
been implicated as an important component in the INA as-
sembly, the membrane anchoring of INP might be mediated
by it. However, no experimental evidence for this involvement
in the anchoring of proteins to prokaryotic membranes has
appeared in the literature. In addition, most of the phos-
phatidylinositol anchor in eukaryotic cells require stretches of
hydrophobic C-terminal sequences for the glycan phos-
phatidylinositol processing; no such hydrophobic stretches
exist in the C-terminal sequence of the INP. These might seem
to preclude the involvement of phosphatidylinositol in the
membrane anchoring of INP; however, we cannot rule out
the possibility that this might be the first observed incidence
of such anchoring in prokaryotic cells, although it is possible
that the actual mechanism might be different from that in
eukaryotes. The other alternative mechanism for the mem-
brane anchoring must involve the N-terminal domain which
itself is quite hydrophobic; if so, then the N-terminal sequence
would be serving roles both as a sequence targeting INP to
the outer membrane, as well as an anchoring domain. What-
ever the mode of anchoring, the membrane can provide the
fluidity that would allow the trial and error process in forming
the optimal arrangement of INP. This same fluidity, however,
becomes a disadvantage when cells are grown at higher tem-
peratures. The INPs will tend to break apart from the larger
INA to form smaller INAs resulting in lower threshold tem-
peratures. It is important to establish firmly the relationship
between phosphatidylinositol, INP and INA before the struc-
ture of INA and the mechanism of ice interaction can be
elucidated completely.
An understanding of the assembly of INA might also shed
light on an interesting puzzle presented by the h a + bacteria.
In a given population of the h a C strain (even within the
same clonal population), every cell does not possess the same
nucleation activity. A very small population (about 1 in lo6)
show the activity of type A. Almost all cells show threshold
temperatures greater than - 13"C. This is the reason why
cumulative nucleation spectra have to be obtained in order to
analyze activity. The reason for this variability is not very
clear. Expression studies with recombinant E. coli using vari-
ous promoters have shown that cumulative spectra do not
vary except in absolute frequencies; relative shapes remain the
same [53]. This rules out the possibility that heterogeneous
activity is a result of varying levels of expression of INP among
cells. Another possibility that has also been suggested involves
'endergonic metabolism' [54]. According to this postulation,
the reversible transformations of the less active INA to the
most active ones are controlled by metabolic processes. Since
these processes can vary among individuals, the occurrence of
an INA of a specific activity can also be expected to vary
among individuals. Another possibility also suggested is that
each nucleus may be interacting with some 'impurities' to a
different extent; this would lead to varying extents of 'doping'
of the nuclei and, thus, to varying threshold temperatures [46].
It is also suggested that the INA assembly process would
require an involvement of a very large number of INP in a
highly precise arrangement to produce an effective ice nucle-
ation template, this may not be entirely controllable and there-
fore may be a hit-or-miss affair among cells [7]. Solving this
puzzle could prove extremely useful in the design and pro-
duction in bacteria of INA with known homogeneous
threshold temperatures. Alternatively, and in v i b o process will
14321033, 1992, 1-2, Downloaded from https://febs.onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1992.tb19824.x by CAPES, Wiley Online Library on [09/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
have to be developed to convert monomeric INP (or small
aggregates of INP) to stable forms of INA of specific activity.
Ice nucleators from non-bacterial organisms
Several frost-resistant insects have been known to produce
ice nucleators [5]. Most of them are thought to be composed
of proteins or lipoproteins. However, the INA from insects
have not yet been characterized as well as those from bacteria,
although it is known that threshold temperatures of insect
INA are usually much lower than those of bacterial ones.
Recently, a lipoprotein with ice nucleating activity isolated
from the overwintering larvae of the cranefly, Tipula trivittata,
has been partially characterized [55]. This lipoprotein ice nu-
cleator has been shown to consist of phosphatidylinositol,
lipids and two apoprotein chains, apo-I (molecular mass
265 kDa) and apo-I1 (molecular mass 81 kDa). The molecular
mass of the lipoprotein was estimated to be about 800 kDa.
Approximately 13 phosphatidylinositol residues were esti-
mated to be associated with apo I. Also, in this case, both
phosphatidylinositol residues and apo-I were postulated to be
involved in ice interaction. Amino acid compositions of the
lipoprotein ice nucleators vary considerably from bacterial
INP. The threshold temperature of the cranefly one was found
to be concentration-dependent and varying between - 6°C
and - 10°C. This suggests that they may also undergo aggre-
gation in the hemolymph to form INA with higher activity. It
appears that, as compared to the bacterial INA, insect INA
may have a different mechanism to make the ice nucleator
sufficiently large for increased activity.
Biotechnology application
As discused in this review, protein-ice interaction plays
an extremely important role in determining the survival of
many organisms. Both the antifreeze proteins and ice-nu-
cleation proteins obviously have many unique and interesting
properties which will have potential biotechnology appli-
cations.
Application of antifreeze proteins
As discussed in the earlier section, the freezing point of
most fish is around -0.6"C to -0.7"C. The Atlantic salmon,
for example, lacks any of the AFGP or AFP genes and is
vulnerable to freezing to death when cultured in sea pens.
Earlier experiments by Fletcher et al. [56]have demonstrated
the direct application of AFP in the freezing protection of
other fish species incapable of producing their own antifreeze
proteins. Purified AFP from the winter flounder was injected
into sea-water-acclimated rainbow trout. The ability of inject-
ed rainbow trout to withstand the low temperature correlated
with the amount of AFP in its circulation. It was concluded
that the AFP alone was sufficient and effective in providing
freeze tolerance. The acquisition and expression of the anti-
freeze protein gene by the Atlantic salmon might help to
overcome this danger and expand many coastal regions in
Northern Atlantic regions for salmon farming. As a part of
the Canadian strategic grant program, we have successfully
incorporated the AFP gene in Atlantic salmon by gene transfer
technology [57]. However, the level of AFP expressed in these
transgenic fish, approximately 2 - 30 pg/ml, is relatively low.
More work is needed to improve the transgene expression in
order to achieve its goal of providing freeze resistance.

In a similar fashion, several laboratories are trying to
transfer the AFP genes into plants [58,59]. The freeze damage
to vegetable and citrus plants are a recurring and severe threat
facing the agricultural industries in many parts of the world.
The justification for the AFP gene transfer was demonstrated
by the vacuum infiltration of the protein into the leaves of
potato and other plants which resulted in a significant de-
pression of the spontaneous freezing temperature relative to
water-infiltrated control [60]. These results demonstrate the
feasibility of improving cold hardiness in plants by AFP gene
transfer.
In addition to the experiments on gene transfer, McKeown
and Warren [61] have recently demonstrated that a fusion
protein containing the flounder AFP expressed intracellularly
in yeast can significantly improve the cell viability after freez-
ing and thawing. This is consistent with the recent finding
from Rubinisky et al. [62] that the AFGP facilitate the survival
of a variety of cells at cryogenic temperatures after rapid
cooling and vitrification. Using pig oocyte, which is sensitive
to hypothermic temperatures, Rubinisky et al. [62] have dem-
onstrated that AFGP protects the structural integrity of the
oolemma and inhibits ion leakage across the oolemma at
hypothermic temperatures. All these studies indicate the po-
tential application of antifreeze proteins for the cryogenic and
hypothermic preservation of cell and organs. Other direct
applications of the antifreeze proteins include the inhibition
of recrystallization of ice in dairy products such as ice cream
and deicing agents.
Applicutions of bacterial ice nucleating agents
In addition to the production of artificial snow, the im-
portant role of bacterial INA in ecology and agriculture have
been recognised ever since it was first demonstrated that h a f
bacteria can cause frost damage to plants. The discovery of
natural Ina- phenotypes and success in engineering of h a -
mutations have made it possible to examine these Ina- pheno-
types as a means to control Ina’ populations and thus to
reduce frost damage in agriculture [63]. Several field test have
been carried out and many more are under way. Results,
available so far from literature, indicate that Ina- strains
may satisfy most environmental concerns and the criteria for
‘fitness’ and that they can successfully dislodge h a + popu-
lations from certain plants. However, we have not as yet come
across any documentation demonstrating the effectiveness in
the field against frost damage. Also, it appears that frequent
spraying with h a - bacteria may be required for prolonged
frost control.
In cryopreservation of tissues and cells, it is important to
minimize supercooling in order to control damage by intra-
cellular ice [64]. It will be interesting to test each of types A,
B and C of INA separately, or together, for their effectiveness
against cellular damage. Similarly, feasibility of the use of
INA in controlling both the rate of freezing and the texture
of frozen foods have been examined [65, 661. It is essential to
make cell-free INA of homogeneous activity commercially
available so that their suitability can be studied properly in
the food industry.
The use of INA as a signal transmitter has been suggested
[67]. As very small concentrations (0.1 pM) of the most active
INA can be detected and quantitated accurately by a unique
assay method (viz. the measurement of cumulative ice-nu-
cleation frequency), their use in immunoassay, for example,
offers a very attractive and a novel alternative to the usual tags
such as radioisotopes, fluorescent groups, linked enzymes, etc.
14321033, 1992, 1-2, Downloaded from https://febs.onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1992.tb19824.x by CAPES, Wiley Online Library on [09/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
41
This technique could find a widespread application when an
INA with the threshold temperature of -2°C to -5°C be-
comes available commercially and when procedures have been
established for the conjugating of INA to proteins and other
molecules. Recently, a number of recombinant h a + bacterio-
phages each showing specificity against a specific bacterium
sp. have been developed by Warren and coworkers [68].These
have been used to develop very sensitive assays for bacterial
contamination in food stuff.
SUMMARY
It is both exciting and rewarding to notice that both the
antifreeze proteins and ice-nucleation proteins, in addition to
their biological importance, show a high potential for appli-
cations in a wide variety of economically important fields.
At present, intact, free INA are not available commercially,
except perhaps as dead cells. Also, it is desirable that each of
the three types, A, B, and C, of INA be available so that their
performances in many potential applications can be evaluated
properly. The environmental and ecological concerns arising
out of some of these potential applications will need to be
discussed and addressed before these applications become a
reality.
We wish to thank Mr S. Joshi for helpful discussions, and Elaine
Vorvis and Linda Gardiner for the preparation of the manuscript.
This work is supported by grants from the Medical Research Council
of Canada to C.L.H. and D.Y.
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