Caballero B. (ed.) Encyclopaedia of Food Science, Food Technology and Nutrition. Ten-Volume Set

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Large Deformation and Failure Behavior

0042 As the requirements of users of gelatin have become

more complex, the gel properties are now commonly

determined in other ways. Gelatin gels are viscoelastic

and incompressible materials (Poisson’s ratio close to

0.5). When submitted to large strains, they always

present a strain-hardening behavior before failure.

The strain at failure is always greater than 50% of

strain and can reach 200% of strain in appropriate

conditions of concentration and deformation rate.

These properties can be linked to the dynamic nature

of the cross-links which are melting and reforming

under stress, leading to alignment of the elastically

active chains. As might be expected, the strain, stress

at failure, and the Young’s modulus increase when the

concentration increases.

Factors Affecting the Properties of Gelatine Gels

0043 Concentration and temperature The exact relation-

ship between concentration and gel strength depends

on the type and origin of the gelatin itself, but using

gelatins commonly found in the food industry, i.e.,

100–250 Bloom, the relationship becomes (C

)



) ¼(C

)

(B

) where C is the concentration of

the gel concerned; B, the Bloom of the gel concerned;

n is equal to 1.7 for high-Bloom gelatins and 1.8–

1.9 for lower-strength gelatins of 150–100 Bloom.

More precisely, for well-characterized gelatins, a cor-

respondence between the elastic modulus, the meas-

urement temperature, and the concentration in a- þ

b- þ g-chains has been introduced at any time of the

gelation process (master curve). Two equations are

necessary for this model:

ðC

ðT

ÞðG

Þ¼ðC

ðT

ÞðG

and ð C

ÞðT

Þðt

Þ¼ðC

ÞðT

Þðt

where G is the elastic modulus, T the temperature in

degrees Celsius, and t the time scale.

0044 In principle, this model, together with a single

elasticity measurement at one temperature and one

concentration, can be used to calculate the whole

gelation kinetics of a sample. This includes setting to

several months, storage, at concentrations between 3

and 10% w/w and temperatures between 5 and 20



The Bloom value is directly proportional to the elastic

modulus at a concentration of 6.66% after 16 h

at 10



0045 Effect of pH The gel strength is only seriously

affected by solution pH at extremes of the range;

from pH 4 to pH 9 it is not affected to any significant

extent. Dilute gels (< 2%) are more affected while

stronger gels (> 10%) are relatively insensitive to pH.

0046Time and temperature of set The gel strength will

depend on the time and temperature of set. This will

vary from gelatin to gelatin and is also dependent on

the proportions of molecular fractions present and

thus the viscosity. When a gel is in the process of

formation, the more the coils can align themselves,

before or during maturation, the greater the number

of junctions that will form. This becomes apparent if

gelatins are snap-chilled as they are found to have

significantly lower gel strengths than expected. The

sites involved in hydrogen bonding cannot align

themselves efficiently enough to produce the expected

number of junctions, whereas if gels are ‘tempered’ at

a temperature just above the setting point, the gel

strength is above that which could be expected.

0047Effect of melting point The unique organoleptic

qualities exhibited by gelatin are strongly dependent

on its melting point, which in turn is influenced by:

0048The bloom value and viscosity of the gelatin.

0049The concentration of the gel.

0050These in turn can be altered by other ingredients

incorporated in the food concerned e.g., salts, sugars,

and other gelling or thickening agents.

0051Effect of low-molecular-weight compounds The

effect of other dissolved ingredients in the gel can be

either to weaken the gel or to strengthen it. Most

simple sugars, glycerol, and other nonelectrolytes

can contribute to an increase in gel strength (fructose

and sorbitol being an exception) while the addition of

most electrolytes has the opposite effect. It has been

shown that these components affect the number and

size of the junction zone as they impact on the quan-

tity of the solvent (i.e., in sucrose, the number of

cross-links increases and the size of each junction

zone decreases).

0052Compatibility with other food biopolymers Gelatin

is incorporated as a functional ingredient in a wide

variety of foods. When doing so, it is important to

have a knowledge regarding its compatibility with

other substances with which it is likely to be blended

in order to form the final food product.

0053Food biopolymers can be classified into two main

groups depending on their structural features. Gelatin

solutions will either interact constructively or de-

structively depending on the environment.

0054Noncharged biopolymers, e.g., locust bean gum, guar

gum, starches, agarose This class of hydrocolloid

can be added in low concentrations (separately from

the gelatin) to most food systems without signifi-

cantly impairing the gelling properties of the gelatin.

2868 GELATIN

However, as the concentration is increased, phase

separation of the two will often occur.

0055 When composites are prepared in the liquid state at

high temperature, gelatin is compatible with the other

biopolymer molecules. This compatibility is reduced

when the temperature decreases. The incompatibility

between the two biopolymers is revealed by an in-

crease in turbidity of the solution and by the appear-

ance of two separated phases (droplets). The two

phases have different compositions: one phase is

enriched in gelatin while the other is enriched in the

other biopolymer and a phase diagram and tie-lines

can be constructed (Figure 1). Ultimately, when the

densities of the two phases are different, the hot solu-

tion evolves into two physically separate layers, with

creaming of one phase and sedimentation of the other

(Figure 2).

0056The composite gel formed after cooling of the

mixed solution has properties which depend on the

microstructure produced. For instance, whether

the gelatin forms the included or continuous phase

for the same initial system composition, different

trapped microstructures can be achieved depending

on the processing conditions (i.e., temperature pro-

file, shear, etc.). This can even result in phase inver-

sion of the two biopolymer phases.

0057Charged biopolymers, e.g., pectins and alginates,

agar, carrageenan, and gum arabic This class of

hydrocolloid with its negative charge will interact

with gelatin, coacervating in a joint phase from solu-

tion when the gelatin has a net positive charge and

acting synergistically when the gelatin has a negative

charge. The degree of synergism is dependent on the

negative charge density, and its distribution on

the hydrocolloid. However, when the saturation of

the charges has been reached, similar behavior as

above is reached involving compatibility, incompati-

bility, and microstructure trapping.

0058Effect of processing conditions Gelatin will degrade

and lose its gelling properties when subjected to con-

ditions of heat, extremes of pH, and exposure to

enzyme attack. This has been studied in some detail

and modeled.

0059Also, when gelatin gels under physical disruptions

(shear), the final properties of the gel can be very

different. For instance, constant shear stress condi-

tions which modify the linear rheological properties

at short times of the gelatin gels still result in gels with

similar linear properties at long times, but in which

the resistance against fracturing is weaker. The model

proposed involves particles of gelatin that connect

one to the other by cross-link formation to form a

network with the same characteristics as the quies-

cent gel. However, the last cross-links formed are

weaker than the earliest and therefore are the first to

melt when the gel is submitted to large strains. This

highlights the healing properties of the gelatin. More-

over, if constant shear strain is used, instead of a

rubber-like material, the final product is more fluid

or paste-like as fluid gel particles are produced.

Polyelectrolyte Properties

0060The gelatin molecule, with its mixture of acidic and

basic amino acid side chains, has polyelectrolyte char-

acteristics and is described as amphoteric. At pH

levels lower than the isoelectric point the molecule

will have a net positive charge, and above this point a

net negative charge. Since the isoelectric point in

gelatin can vary considerably, the extent of net charge

is dependent on the environment pH.

0 5 10 15

Maltodextrin SA2 (% w/w)

Gelatin (% w/w)

20 25 30

Compatible

domain

Phase separation domain

Tie-line

fig0001 Figure 1 Phase diagram of gelatin (lime hide), maltodextrin

SA2 (degraded potato starch), measured at 60



C. The binodal

line separates compatibility and incompatibility domains.

fig0002 Figure 2 Confocal laser scanning microscopy image of the

boundary between two phases in equilibrium. Gelatin is clear

and maltodextrin is dark. The composition of the system corres-

ponds to the solid square presented in Figure 1. The composition

of each phase is determined by the end points of the tie-line

(20 mm ¼ 100 mm).

GELATIN 2869

Surface Activity and Related Sol Properties

0061 Gelatin is often used for its surface-active properties,

altering the rate of crystal growth in supersaturated

solutions, e.g., marshmallow and icecream, where

sugar and ice crystal growth are suppressed, giving

the desired product. It also finds applications in sta-

bilizing other emulsions such as mayonnaise, pa

and meat homogenates. Gelatin is added to various

types of yogurt formulations, stabilizing the product.

It forms a weak gel structure, which imbibes free

water, thus inhibiting separation of whey, especially

after pasterurization.

0062 The polyelectrolyte behavior of gelatin solutions

is widely used for clearing wine and apple juice

musts of yeast suspensions, tannins, and other poly-

saccharides formed during fermentation. It can also

be used in the modern ‘hot fining process’ designed

for improved cider and apple juice production. These

properties are related on the molecular level to three

parameters:

0063 Overall charge, and its distribution along the

chain.

0064 Distribution of nonionic groups.

0065 Molecular weight (mean chain length).

Applications

0066 Uses of gelatin in food production Gelatin is widely

used as an ingredient in the food industry, due to the

following functional properties:

0067 It forms high-quality gels in dilute solution with

typical clean, melt-in-the-mouth textures.

0068 It forms elastic gum-type textures in the concen-

trated gel, slowly dissolving in the mouth.

0069 It forms stable gels on cold storage.

0070 It produces emulsification and stabilization of

immiscible liquid/liquid, liquid/air or liquid/solid

mixtures.

0071 It acts as a polyelectrolyte to flocculate suspended

particles or unstable colloids from dilute solutions.

0072 It acts as a water retainer (it swells easily and there

is no syneresis).

0073 It acts as an efficient tableting aid and binder.

0074 Gelatin is sold to many sections of the food indus-

try. The major areas of use are in table jellies, con-

fectionery, meat products, icecreams, low-fat and

very-low-fat spreads, and chilled dairy products.

0075 Other applications are in the pharmaceutical in-

dustry for hard and soft capsule manufacture, and in

the photographic industry, which exploits the unique

combination of gel formation and surface activity to

suspend particles of silver chloride or light-sensitive

dyes, without any agglomeration.

Gelatine Derivatives

Cold-Water-Soluble Gelatin

0076When gelatin solutions are dried without passing

through the gel phase, they are of amorphous struc-

ture, and do not show any crystalline character, as do

gelatins dried from the gel phase. When these dried

solutions are rehydrated at a temperature below the

gelling point, they will set and form a rigid gel identical

in every way to that of a normal gelatin. Unfortunately,

when dehydrated, gelatin is extremely hygroscopic and

it is difficult to form gels of moderate concentration. In

order to overcome this difficulty, mixtures of gelatin

and certain carrier substances are available. Glucose

syrup and starch are the most popular, as they are

ingredients in many of the products for which the

gelatin is intended. Drum-dried instant gelatin is also

available, but still has to be intimately mixed with

other ingredients to insure complete solution.

Hydrolyzed Gelatins

0077Hydrolyzed gelatins differ from normal gelatins in

that they are soluble in cold water, but possess no

gelling power.

0078The hydrolysis of gelatin is complex. There must

be strict control of the reaction to avoid the formation

of undesirable products such as bitter peptides.

Collagen itself is sometimes used, thereby avoiding

the gelatin extraction stage. Several manufacturers

market a range of hydrolyzed gelatins, each with

controlled molecular weight ranges, which can vary

from < 1000 to 15 000 molecular weight.

0079They can be used in tablet binding and granulation

agents replacing gelatin, which is not cold-water-

soluble under normal conditions of use. Hydrolyzed

gelatines give the tablet a better rate of dissolution

and disintegration. They are also used as emulsifiers

in meat/fat emulsion and as an encapsulation agent

for flavor concentrates, taking advantage of its low

carbohydrate content.

0080Hydrolyzed gelatin can be kept as a concentrated

solution in a bulk pack after ultrahigh-temperature

treatment, or more commonly with preservative

added. These are often supplied to the wine industry

as ready-to-use fining agents. Hydrolyzed gelatins

still possess many of the useful properties of gelatin,

e.g., emulsion stability, colloid protection, and

flocculation promotion. Much of the production is

destined for the cosmetic market where it is added to

shampoos as ‘protein’ and to creams and lotions as

‘hydrolyzed collagen,’ where use is made of its skin

and hair absorption characteristics.

0081The price of food-grade hydrolyzed gelatine is

roughly that of a high-grade gelatin, partially due

2870 GELATIN

to the cost incurred in spray-drying the processed

materials.

Analysis of Gelatin Content

0082 The gelatin content of foods can be determined in

several ways, each depending on other known con-

stituents present. Kjeldahl nitrogen digestions or

biuret reagent determinations can only be applied if

it is certain that there are no other proteinaceous

compounds present. The procedure for Kjeldahl de-

terminations is well known and suitable conditions

have been published. The conversion factor for

gelatin determinations is considerably lower than

for most other proteins (5.36 for collagen, 5.51

for type B gelatin, and 5.46 for type A gelatin).

Hydroxyproline analysis is the definitive method

which is applicable in nearly all situations with a

high degree of accuracy.

Nutritional Aspects

0083 Because gelatin does not contain any tryptophan, it

cannot be used as a ‘complete protein;’ however it has

increased proportions of certain amino acids (e.g.,

lysine). It can therefore be used to supplement other

proteins, to give a mixture with a higher protein value

than each component. When mixed with beef protein,

the net protein value can rise from 84% to 99%.

0084The calorific value of gelatin is only 3.5 kcal g

1

which explains its use in low-calorie and diet foods.

See also: Bone; Fish Oils: Dietary Importance

Further Reading

Amman-Brass H and Pouradier J (eds) (1989) Proceedings

of the Fifth IAG Conference. Fribourg, Switzerland.

Harris P (ed.) (1990) Food Gels. Essex: Elsevier Science.

Mitchell JR and Ledward DA (eds) (1986) Functional

Properties of Food Macromolecules. Essex: Elsevier

Science.

Pearson AM, Dutson TR and Bailey AJ (eds) (1987) Colla-

gen as a Food. Advances in Meat Research, vol. 4.

London: Van Nostrand Reinhold.

Ross-Murphy SB (1997) Structure and rheology of gelatin

gels. Imaging Science Journal 45: 205–209.

teNijenhuis K (1997) Thermoreversible networks – visco-

elastic properties and structure of gels – introduction.

Advances in Polymer Science 130: 1–12.

Veis A (ed.) (1964) The Macromolecular Chemistry of

Gelatin. New York: Academic Press.

Ward AG and Courts A (eds) (1977) The Science and

Technology of Gelatin. London: Academic Press.

GENE EXPRESSION AND NUTRITION

P Trayhurn, University of Liverpool, Liverpool, UK

J E Hesketh, University of Newcastle, Newcastle

upon Tyne, UK

Introduction

0001 Molecular biology has provided a detailed description

as to how genes are organized and expressed. A number

of technologies have been developed in the period since

the late 1970s which enable the factors which influence

the expression of genes to be readily investigated. In

particular, these techniques permit study of the inter-

action between nutrients and the genetic information in

an organism. This article summarizes some of the key

concepts and techniques of molecular biology, and

their application to nutritional science.

Structure of DNA and RNA

0002 The two fundamental molecules involved in the

transfer and translation of genetic information are

DNA and RNA. These polymeric macromolecules

are composed of a chain of nucleotides. Each nucleo-

tide consists of a sugar moiety, a phosphate group,

and either a purine or a pyrimidine base. In the case of

RNA the base is ribose, while in DNA it is deoxy-

ribose. The nucleotides in DNA contain one of four

bases – the purines adenine (A) and guanine (G), and

the pyrimidines cytosine (C), and thymine (T). Both

the purines A and G are also present in RNA, as is the

pyrimidine C. However, the second pyrimidine in

RNA is uracil (U) rather than T.

0003The nucleotides in DNA and RNA are linked by

phosphate bridges to form a sugar–phosphate back-

bone. Each base is attached to the sugar moiety. DNA

consists of two polynucleotide strands arranged in the

double helix structure elucidated by Watson and

Crick. The sugar–phosphate is on the outside of the

helix, with the bases in the center. The two strands of

the helix are held together by hydrogen bonding be-

tween specific base pairs. Each base pair consists of a

purine and a pyrimidine, the pairings being A–T (two

hydrogen bonds) and C–G (three hydrogen bonds).

GENE EXPRESSION AND NUTRITION 2871

0004 The information necessary for the synthesis of

each individual protein in an organism is encoded

by lengths of DNA, termed genes. Large numbers

of different genes are linked together linearly, and

assembled with specific histone proteins, to form

chromosomes, the number of which varies from

species to species (e.g., 46 in humans and 8 in fruit

flies). In eukaryotes, the chromosomes and the genes

within them are localized in the nucleus of the cell.

The number of human genes is thought to be between

30 000 and 50 000, but the full realization of the

Human Genome project will provide a definitive as-

sessment.

Gene Expression

0005 The precision of base pairing allows the nucleic acids

to act as templates for the synthesis of strands

containing complementary sequences (Figure 1).

This is the critical feature of the transfer of genetic

information in all organisms. The sequence of the

four bases in DNA is transcribed into a complemen-

tary strand of RNA, messenger RNA (mRNA). This

transcription process uses the same base pairings as in

the two strands of DNA, except that mRNA contains

U rather than T. Thus A in DNA codes for U in

mRNA.

0006 Genes are now recognized to consist of coding

regions (exons), regulatory sequences (promoters),

and internal noncoding regions (introns); only a

small part (*2%) of the mammalian genome actually

codes for protein. Transcription produces an RNA

molecule which is complementary to both coding

and noncoding regions. However, sequences corres-

ponding to the introns are removed from the primary

transcript to produce the functional mRNA molecule.

In eukaryotic cells mRNA is transferred from the

nucleus to the cytoplasm, where it becomes associ-

ated with ribosomes. The ribosomes, which consist

of ribosomal RNA (rRNA) and protein, read the

sequence of bases in mRNA and translate this into

a sequence of amino acids specific for each individual

protein. Not only does the ribosome read the se-

quence of bases, but it also assembles the amino

acids into the protein through the intervention of a

further form of RNA, namely transfer RNA (tRNA).

0007Each amino acid in a protein is coded by a sequence

of three bases, and this triplet code is the same in all

organisms (universality of the genetic code). The code

has some degeneracy in that certain amino acids are

coded by more than one triplet.

0008It is evident from the above that DNA sequences

direct the synthesis of protein through the medium of

mRNA. Modern molecular biological techniques

allow the detection of specific mRNAs, and in prac-

tice the expression of a particular gene in a tissue is

assessed by identifying the presence of the corres-

ponding mRNA. Strictly, the protein itself should

also be identified since an mRNA could, in principle,

remain untranslated.

Technology

0009The ability to detect specific mRNAs depends on two

broad types of methodology, one depending on the

preparation of DNA sequences which are comple-

mentary to part, or all, of the sequence of the

mRNA of interest. This is achieved through the ap-

plication of recombinant DNA technology. Once

obtained, a complementary DNA (cDNA) probe will

bind, or hybridize, with the corresponding mRNA

through the specificity of the base pairings. By tagging

the cDNA molecule with a marker, the cDNA–

mRNA complex can be readily detected.

0010The other methodology is dependent on the con-

version of the mRNA sequence to DNA using reverse

transcriptase (see below) and the subsequent amplifi-

cation of this DNA by the polymerase chain reaction

(PCR).

Isolation of RNA

0011In order to obtain RNA, tissues and cell membranes

must first be disrupted in a denaturing medium, and

nucleic acids selectively solubilized. Precautions

should be taken throughout to avoid degradation by

endogenous and exogenous ribonuclease enzymes.

Most procedures yield total RNA, from which

mRNA can be obtained by exploiting the characteris-

tic poly(A)

sequence; this sequence can be bound

by synthetic poly(T) oligonucleotides. By linking oli-

DNA

mRNA

cDNA

oligonucleotide

fig0001 Figure 1 Coding of messenger RNA (mRNA) and complemen-

tary DNA (cDNA) molecules.

2872 GENE EXPRESSION AND NUTRITION

go(T) to supports, such as cellulose or magnetic

beads, mRNA may be readily isolated.

cDNA cloning

0012 The production of a cDNA probe utilizes the total

mRNA complement of a tissue as the starting mater-

ial. The process requires the synthesis of complemen-

tary DNA sequences, their linking to DNA from a

plasmid (vector), multiplication of the plasmids in

host bacteria, and subsequent screening and selection

of clones containing the cDNA of interest (Figure 2).

0013 cDNA sequences are synthesized from mRNAs

using the prokaryotic enzyme reverse transcriptase;

this involves a fundamental reversal of the normal

flow of genetic information. Each cDNA will be an

exact replica of parts, or all, of the coding regions of

the original DNA from which the mRNA was derived

(Figure 1). Since the starting material is invariably a

complex mixture of some hundreds or thousands of

mRNAs, a wide range of cDNAs will be obtained by

the action of the reverse transcriptase.

0014In the next stage the cDNAs are joined (ligated) to a

plasmid DNA (e.g., pBR322), thus generating a re-

combinant DNA molecule. The plasmids are incorp-

orated into a host bacterium, such as Escherichia coli,

which is then grown using standard microbiological

procedures, resulting in the production of large quan-

tities of the cDNAs. Using appropriate vectors, the

cDNAs will direct the synthesis of encoded proteins.

Traditionally, clones containing the required cDNAs

are identified using antibodies raised against the pro-

tein product of the mRNA of interest. Appropriate

clones are maintained in culture, and the cDNA

excised from the plasmid DNA within the bacteria,

using selective restriction enzymes (endonucleases).

Hybridization

0015A cDNA will bind, or hybridize, specifically to its

corresponding mRNA, and this complex can be

detected if the cDNA is first tagged with a radioactive

(

P) or other label (e.g., enzyme-linked systems

which generate chemiluminescence). Hybridization

Mixture of mRNAs

from tissue

Reverse transcriptase

cDNA library

Cut with restriction enzyme

Ligation (DNA ligase)

Transfection

Plasmid DNA

Bacterium

Bacterial chromosomal DNA

Select by antibiotic resistance

and screen for clone of interest

Plasmid DNA-containing genes

conferring antibiotic resistance

fig0002 Figure 2 A schematic representation of the application of recombinant DNA technology to produce a cDNA probe.

GENE EXPRESSION AND NUTRITION 2873

is carried out on solid supports such as nitrocellulose

or nylon membranes. This can be achieved by direct

application of a mixture of total RNA, or purified

mRNA, on to a membrane (dot blot). Alternatively,

and more usually, the RNA species are first separated

electrophoretically according to size (using agarose

gels), followed by transfer to a membrane (Northern

blotting); see Figure 3.

0016 Identification of mRNA–cDNA complexes is made

by autoradiography, film, or colorimetry, according

to the nature of the label attached to the probe. In

each of these cases quantification can be achieved by

densitometry, or image analysis. A membrane may be

sequentially reprobed for the analysis of different

RNA species, and this is particularly important in

quantitative studies in that it allows the level of a

specific mRNA to be related to reference RNAs. In

addition, and of special relevance to nutritional

studies, the effects of a defined manipulation can be

assessed with respect to the expression of different

genes associated with linked metabolic processes.

Oligonucleotides

0017 Once the sequence of a gene, or a cDNA probe, is

known, computer databases of gene and cDNA

sequences can be scanned, so as to design an

oligonucleotide probe which will specifically recog-

nize a particular mRNA. Such oligonucleotides pro-

vide a convenient alternative to cDNA probes for the

detection and measurement of mRNAs (Figure 1).

Automated oligonucleotide synthesizers are widely

available, and in general an oligonucleotide of

25–30 bases has sufficient specificity to provide a

unique probe for each mRNA.

Polymerase Chain Reaction

0018An alternative to mRNA detection by Northern

hybridization is detection by a combination of reverse

transcriptase and polymerase chain reaction (RT-

PCR). This approach uses short, complementary se-

quences (primers) to make a cDNA sequence from the

mRNA and then, using PCR, to amplify the DNA to

produce copies of this DNA sequence. Since PCR uses

primers specific to the DNA sequence only the cDNA

produced from the original RNA is amplified and

produced in sufficient quantity to be detectable after

subsequent electrophoretic separation. The method is

more sensitive than Northern blotting and more suit-

able for dealing with large numbers of samples. How-

ever, care needs to be taken to make the method

quantitative.

In situ

hybridization

0019A further methodology for examining gene expres-

sion is in situ hybridization, which utilizes probes

(often oligonucleotides) for detecting mRNAs in

tissue sections. In situ hybridization is particularly

valuable for identifying the specific region(s) of a

complex organ, such as the brain, in which expression

of a particular gene occurs. Similarly, the cell types in

which expression is taking place within a tissue can be

determined.

DNA microarrays

0020A recent, and rapidly evolving innovation comes from

the development of DNA microarrays. One such

technique involves the use of arrays of single-stranded

cDNA targets immobilized on nylon or glass sup-

ports. Each array can contain thousands of cDNA

target sequences. Gene expression is monitored by

isolation of RNA from a given material, followed by

conversion of all the mRNA species in the sample to

fluorescently labeled single-stranded DNA probes

(using reverse transcriptase) and then hybridization

of the labeled probes with the immobilized target.

This methodology allows the analysis of the expres-

sion of hundreds – or even thousands – of genes in

parallel; it offers considerable potential for investi-

gating the integrated effects of a given stimulus or

intervention on gene expression.

Extract RNA

Plasmid with cDNA

Excise cDNA

cDNA

Label probe

Oligonucleotide

Incubate probe with RNA on

membrane (hybridization)

Detect specific mRNA

Quantify

Transfer to nylon membrane

(Northern blot)

Separate RNA

electrophoretically

by size

fig0003 Figure 3 A schematic representation of the detection and

measurement of mRNA.

2874 GENE EXPRESSION AND NUTRITION

Nutrition

0021 An appropriate quantity and balance of nutrients is

essential for the genetic potential of each individual to

be fully realized. Nutrition represents a major inter-

action with the environment, and an organism has

to exhibit both acute and long-term adjustments to

changes in nutrient availability. For mammals, these

changes are especially marked both at birth, when

feeding by the oral route is initiated with the periodic

consumption of milks that are characteristically of

high fat content, and at weaning when there is

normally a switch towards a diet with a lower ratio

of fat to carbohydrate.

Gross Nutritional Changes and Gene Expression

0022 Changes in diet induce alterations in the flux of sub-

strates through individual metabolic pathways. Such

adaptations are regulated by alterations in hormonal

status, and require, on a long-term basis, changes in

the amount of associated enzymes. Adjustments in

enzyme synthesis generally involve changes in gene

expression – either by switching genes ‘on’ or ‘off,’ or

by ‘up’ or ‘down’ regulation of genes that are already

being expressed. The genes that code for hormones

and their receptor proteins, as well as for transport

and carrier proteins, are often also subject to nutri-

tional modulation.

0023 The expression of individual genes is affected by

changes in both the quantity and quality of the diet.

Thus fasting and refeeding (which represent extreme

changes in quantity) lead to alterations in the expres-

sion of a variety of genes, including those coding for

enzymes of carbohydrate and lipid metabolism, and

for receptor and transporter proteins. For example,

fasting induces an increase in the level of the mRNAs

for both the insulin and the insulin-like growth factor

(IGF

), while subsequent refeeding leads to a rapid

fall. Such changes may be tissue-specific, as is the

case with the glucose transporters where the level of

the mRNA for the insulin-sensitive variety (GLUT 4)

alters reciprocally in skeletal muscle and white

adipose tissue in response to fasting.

0024 Major changes in the macronutrient composition

of the diet can provoke substantial alterations in

the expression of particular genes. For example,

switching from a high-fat to a high-carbohydrate

diet leads to greatly increased levels of the mRNAs

for lipogenic enzymes, such as fatty acid synthetase

and acetyl coenzyme A carboxylase; in contrast, the

levels of the mRNAs for enzymes associated with

gluconeogenesis (e.g., phosphoenolpyruvate carbox-

ykinase) are decreased.

0025 It should be noted that, although changes in the

level of an mRNA generally imply that the expression

of a particular gene has altered, this is not always the

case since variations in the stability of mRNAs may

also occur.

Transcriptional and Posttranscriptional Control of

Gene Expression

0026In many cases, such as those discussed above, dietary

components do not themselves directly affect gene

expression, the effects being mediated through

changes in hormonal signals. However, in certain

instances there is a more direct interaction between

specific nutrients and gene transcription. This is

especially clear in the case of some micronutrients.

Zinc, for example, is required for the loop, or finger-

like, structures present in a number of regulatory

transcription factors (e.g., nuclear receptors for

thyroxine and steroid hormones), and has also been

implicated in the regulation of the level of the mRNA

for thymidine kinase, an enzyme involved in DNA

synthesis.

0027In addition, the expression of certain genes,

notably those coding for proteins involved in the

transport, or storage, of trace elements is sensitive

to changes in the supply of specific micronutrients.

This regulation occurs both at transcriptional and

translational levels. Thus transcription of the

gene for the metal-binding protein, metallothionein,

is regulated by zinc. On the other hand, synthesis

of the iron storage protein, ferritin, is controlled

by binding of an iron-dependent protein to a specific

sequence in an untranslated region of the ferritin

mRNA, thereby inhibiting its translation into the

protein.

0028Increasingly, it is being realized that nutrient–gene

interactions involve not only the regulation of gene

transcription but also posttranscriptional controls

such as mRNA stability, translation, and mRNA

localization.

Transgenics

0029Transgenics involve the insertion of genes from one

species into the chromosomes of another. The use of

transgenic animals has opened substantial opportun-

ities both for the study of metabolic control and for

biotechnological applications in agriculture and

medicine. One of the earliest and most dramatic

examples of the application of transgenics came

from the insertion of the gene for growth hormone

into mice – such transgenic mice are substantially

larger than normal animals. From a nutritional stand-

point transgenic animals may have altered nutrient

requirements, which will depend in part on the nature

of the incorporated gene(s) and the physiological

consequences of the change.

GENE EXPRESSION AND NUTRITION 2875

0030 Biotechnological applications of transgenics to live-

stock production include the possibility of inserting

genes coding for enzymes associated with the synthe-

sis of essential amino acids, such as lysine, which may

be rate-limiting in protein deposition. In medicine,

transgenics allows the insertion of genes coding for

proteins of therapeutic or pharmaceutical interest.

Production of such proteins in milk, through expres-

sion in the mammary gland, is likely to provide a

particularly potent route for harvesting large quan-

tities of substances such as growth factors and

hormones.

Glossary

cDNA

A DNA sequence complementary to part, or all, of

the sequence of a particular messenger RNA

Gene Transcription

Conversion of the base sequence of a gene into

the corresponding complementary RNA sequence

(messenger RNA)

Gene Translation

Conversion of the base sequence in messenger RNA

to the corresponding sequence of amino acids in a

protein

Hybridization

Formation of duplexes between complementary RNA

and/or DNA sequences

Northern Blotting

Transfer of RNA, following separation by molecular

size using agarose gel electrophoresis, to a nylon or

nitrocellulose membrane

Plasmid

A piece of circular, extrachromsomal DNA which

replicates independently; used for the amplification

of inserted cDNA sequences

See also: Cells; Enzymes: Functions and Characteristics;

Nucleic Acids: Properties and Determination;

Physiology; Zinc: Physiology