Microbiology and Immunology

WORLD OF MICROBIOLOGY AND IMMUNOLOGY Cryoprotection

141

•

which led to Ingram’s 1956 demonstration that sickle cell

hemoglobin differed from normal hemoglobin by a single

amino acid. Ingram’s research presented evidence that a

molecular genetic disease, caused by a Mendelian mutation,

could be connected to a DNA-protein relationship. The

importance of this work to Crick’s thinking about the func-

tion of DNA cannot be underestimated. It established the

first function of “the genetic substance” in determining the

specificity of proteins.

About this time, South African-born English geneticist

and molecular biologist Sydney Brennerjoined Crick at the

Cavendish Laboratory. They began to work on the coding

problem, that is, how the sequence of DNA bases would spec-

ify the amino acid sequence in a protein. This work was first

presented in 1957, in a paper given by Crick to the

Symposium of the Society for Experimental Biology and

entitled “On Protein Synthesis.” Judson states in The Eighth

Day of Creationthat “the paper permanently altered the logic

of biology.” While the events of the transcriptionof DNA and

the synthesis of protein were not clearly understood, this

paper succinctly states “The Sequence Hypothesis... assumes

that the specificity of a piece of nucleic acid is expressed

solely by the sequence of its bases, and that this sequence is

a (simple) code for the amino acid sequence of a particular

protein.” Further, Crick articulated what he termed “The

Central Dogma” of molecular biology, “that once ‘informa-

tion’ has passed into protein, it cannot get out again. In more

detail, the transfer of information from nucleic acid to nucleic

acid, or from nucleic acid to protein may be possible, but

transfer from protein to protein, or from protein to nucleic

acid is impossible.” In this important theoretical paper, Crick

establishes not only the basis of the genetic code but predicts

the mechanism for protein synthesis. The first step, tran-

scription, would be the transfer of information in DNA to

ribonucleic acid(RNA), and the second step, translation,

would be the transfer of information from RNA to protein.

Hence, the genetic message is transcribed to a messenger, and

that message is eventually translated into action in the syn-

thesis of a protein. Crick is credited with developing the term

“codon” as it applies to the set of three bases that code for one

specific amino acid. These codons are used as “signs” to

guide protein synthesis within the cell.

A few years later, American geneticist Marshall Warren

Nirenberg and others discovered that the nucleic acid

sequence U-U-U (polyuracil) encodes for the amino acid

phenylalanine, and thus began the construction of the

DNA/RNA dictionary. By 1966, the DNA triplet code for

twenty amino acids had been worked out by Nirenberg and

others, along with details of protein synthesis and an elegant

example of the control of protein synthesis by French geneti-

cist François Jacob, Arthur Pardée, and French biochemist

Jacques Lucien Monod. Brenner and Crick themselves turned

to problems in developmental biology in the 1960s, eventually

studying the structure and possible function of histones, the

class of proteins associated with chromosomes.

In 1976, while on sabbatical from the Cavendish, Crick

was offered a permanent position at the Salk Institute for

Biological Studies in La Jolla, California. He accepted an

endowed chair as Kieckhefer Professor and has been at the

Salk Institute ever since. At the Salk Institute, Crick began to

study the workings of the brain, a subject that he had been

interested in from the beginning of his scientific career. While

his primary interest was consciousness, he attempted to

approach this subject through the study of vision. He pub-

lished several speculative papers on the mechanisms of

dreams and of attention, but, as he stated in his autobiogra-

phy, “I have yet to produce any theory that is both novel and

also explains many disconnected experimental facts in a con-

vincing way.”

During his career as an energetic theorist of modern

biology, Francis Crick has accumulated, refined, and synthe-

sized the experimental work of others, and has brought his

unusual insights to fundamental problems in science.

See alsoCell cycle (eukaryotic), genetic regulation of; Cell

cycle (prokaryotic), genetic regulation of; Genetic identifica-

tion of microorganisms; Genetic mapping; Genetic regulation

of eukaryotic cells; Genetic regulation of prokaryotic cells;

Genotype and phenotype; Immunogenetics

CCryoprotectionRYOPROTECTION

Cryopreservation refers to the use of a very low temperature

(below approximately –130° C [–202° F]) to store a living

organism. Organisms (including many types of bacteria,

yeast, fungi, and algae) can be frozen for long periods of time

and then recovered for subsequent use.

This form of long-term storage minimizes the chances

of change to the microorganism during storage. Even at refrig-

eration temperature, many microorganismscan grow slowly

and so might become altered during storage. This behavior has

been described for strains of Pseudomonas aeruginosathat

produce an external slime layer. When grown on a solid agar

surface, the colonies of such strains appear like mucous drops.

However, when recovered from refrigeration storage, the

mucoid appearance can be lost. Cryopreservation of mucoid

strains maintains the mucoid characteristic.

Cryostorage of bacteria must be done at or below the

temperature of –130° C [–202° F], as it is at this temperature

that frozen water can form crystals. Because much of the inte-

rior of a bacterium and much of the surrounding membrane(s)

are made of water, crystal formation would be disastrous to the

cell. The formation of crystals would destroy structure, which

would in turn destroy function.

Ultralow temperature freezers have been developed that

achieve a temperature of –130° C. Another popular option for

cryopreservation is to immerse the sample in a compound

called liquid nitrogen. Using liquid nitrogen, a temperature of

–196° C [–320.8° F] can be achieved.

Another feature of bacteria that must be taken into

account during cryopreservation is called osmotic pressure.

This refers to the balance of ions on the outside versus the

inside of the cell. An imbalance in osmotic pressure can cause

water to flow out of or into a bacterium. The resulting shrink-

age or ballooning of the bacterium can be lethal.

womi_C 5/6/03 2:05 PM Page 141