Microbiology and Immunology

Genetic regulation of eukaryotic cells WORLD OF MICROBIOLOGY AND IMMUNOLOGY

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genes. If a characteristic C turns out in breeding experiments

to have 9% crossover with B and 17% crossover with A, it

would then be located between A and B at a point 9 units from

B and 17 units from A. Compiling the information from many

such breeding experiments creates a chromosome map that

indicates the relative positions of the genes that code for cer-

tain characteristics. Accordingly, the further apart any two

genes are on the same chromosome, the greater the incidence

of crossing over between them.

A linkage map is limited because recombinationfre-

quencies can be distorted relative to the physical distance

between sites. As a result, the linkage map is not always the

best possible representation of genetic material.

While linkage maps only indicate relative positions of

genes, physical maps are more accurate and aim to show the

actual number of nucleotides between each gene. Restriction

maps are constructed by cleaving DNAinto fragments with

restriction enzymes. These enzymesrecognize specific short

DNA sequences and cut the duplex. The distances between the

sites of cleavage are then measured. The positions of the tar-

get restriction sites for these enzymes along the chromosome

can be used as DNA markers. Restriction sites generally exist

in the same positions on homologous chromosomes so the

positions of these target sites can be used rather like mile-

stones along a road and can act as reference points for locat-

ing significant features in the chromosome.

A map of the positions of restriction sites can be made

for a localized region of a chromosome. It is made by com-

paring the sizes of single enzyme breakages (digests) of the

region of interest with double digests of the same region.

This means that two different restriction enzymes are

applied, one to each of two separate chromosome extracts of

the region of interest, and subsequently the two enzymes are

used together in a third digestion with the chromosome

extract. The chromosome fragments resulting from the three

digestions are then subjected to a biochemical procedure

known as gel electrophoresis, which separates them and

gives an estimation of their size. Comparison of the sizes of

the chromosome fragments resulting from single and double

restriction enzyme digestions allows for an approximate

location of the target restriction sites. Thus, such maps rep-

resent linear sequences of restriction sites. As this procedure

determines the sizes of digested chromosome fragments, the

distances between sites in terms of the length of DNA can be

calculated, because the size of a fragment estimated from an

electrophoresis experiment is proportional to the number of

base pairs in that fragment.

A restriction map does not intrinsically identify sites if

genetic interest. For it to be of practical use, mutationshave to

be characterized in terms of their effects upon the restriction

sites. In the 1980s, it was shown how restriction fragment

length polymorphisms (RFLPs) could be used to map human

disease genes. RFLPs are inherited by Mendelian segregation

and are distributed in populations as classical examples of

common genetic polymorphisms. If such a DNA variant is

located close to a defective gene (which can not be tested

directly), the DNA variant can be used as a marker to detect

the presence of the disease-causing gene. The prenatal exami-

nation of DNA for particular enzyme sites associated with cer-

tain hereditary diseases has proved to be an important method

of diagnosis. Clinically useful polymorphic restriction enzyme

sites have been detected within the Beta-like globin gene clus-

ter. For example, the absence of a recognition site for the

restriction enzyme HpaI is frequently associated with the

allele for sickle-cell anemia, and this association has been use-

ful in prenatal diagnosis of this disease.

The ultimate genetic map is the complete nucleotide

sequence of the DNA in the whole chromosome complement,

or genome, of an organism. Today, several completed genome

maps already exist. Simple prokaryotic organisms, e.g., bacte-

ria, with their relatively small (one to two million base pairs)

chromosomes of one to two million base pairs were the first to

be mapped. Later, eukaryotic organisms such as the yeast,

Saccharomyces cerevisiae, and the nematode worm,

Caenorhabditis elegans, were mapped. In 2000, the Human

Genome Project produced the first draft of the human genome.

The project adopted two methods for mapping the 3 billion

nucleotides. The earlier approach was a “clone by clone”

method. In this, the entire genome was cut into fragments up

to several thousand base pairs long, and inserted into synthetic

chromosomes known as bacterial artificial chromosomes

(BACs). The subsequent mapping step involved positioning

the BACs on the genome’s chromosomes by looking for dis-

tinctive marker sequences called sequence tagged sites

(STSs), whose location had already been pinpointed. Clones

of the BACs are then broken into smaller fragments in a

process known as shotgun cloning. Each small fragment was

then sequenced and computer algorithms, that recognize

matching sequence information from overlapping fragments,

were used to reconstruct the complete sequence inserted into

each BAC. It was later argued that the first mapping step was

unnecessary and that the algorithms used to reassemble the

shotgunned DNA fragments could be applied to cloned ran-

dom fragments taken directly from the whole genome. In this

whole genome shotgun strategy, fragments were first assem-

bled by algorithms into larger scaffolds and the correct posi-

tion of these scaffolds on the genome was worked out by

STSs. The latter method speeded up the whole procedure con-

siderably and is currently being used to sequence genomes

from other organisms.

See alsoCloning, application of cloning to biological prob-

lems; Fungal genetics; Gene amplification; Gene; Genetic

code; Genetic identification of microorganisms; Genetic regu-

lation of eukaryotic cells; Genetic regulation of prokaryotic

cells; Genotype and phenotype; Microbial genetics

GENETIC REGULATION OF EUKARYOTIC

CELLSGenetic regulation of eukaryotic cells

Although prokaryotes (i.e., non-nucleated unicellular organ-

isms) divide through binary fission, eukaryotesundergo a

more complex process of cell division because DNAis packed

in several chromosomeslocated inside a cell nucleus. In

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