C h a p t e r 5
Genetic Control of Cell Function and Inheritance
101
chromosomes of both species. Because these hybrid cells
are unstable, they begin to lose chromosomes of both
species during subsequent cell divisions. This makes it
possible to obtain cells with different partial combina-
tions of human chromosomes. The enzymes of these
cells are then studied with the understanding that for an
enzyme to be produced, a certain chromosome must be
present and, therefore, the coding for the enzyme must
be located on that chromosome.
In situ hybridization
involves the use of specific
sequences of DNA or RNA to locate genes that do not
express themselves in cell culture. Deoxyribonucleic acid
and RNA can be chemically tagged with radioactive or
fluorescent markers. These chemically tagged DNA or
RNA sequences are used as probes to detect gene loca-
tion. The probe is added to a chromosome spread after the
DNA strands have been separated. If the probe matches
the complementary DNA of a chromosome segment, it
hybridizes and remains at the precise location (therefore
the term
in situ
) on a chromosome. Radioactive or fluo-
rescent markers are used to find the location of the probe.
Haplotype Mapping
As work on the Human Genome Project progressed,
many researchers reasoned that identifying the com-
mon patterns of DNA sequence variations in the human
genome would be possible. An international project,
known as the
International HapMap Project,
was orga-
nized with the intent of developing a haplotype map
of these variations. One of the findings of the Human
Genome Project was that the genome sequence was
99.9% identical for all people. It is anticipated that
the 0.1% variation may greatly affect an individual’s
response to drugs, toxins, and predisposition to various
diseases. Sites in the DNA sequence where individuals
differ at a single DNA base are called
single-nucleotide
polymorphisms
(SNPs, pronounced “snips”). A haplo-
type consists of the many closely linked SNPs on a single
chromosome that generally are passed as a block from
one generation to another in a particular population.
One of the motivating factors behind the HapMap proj-
ect was the realization that the identification of a few
SNPs was enough to uniquely identify the haplotypes in
a block. The specific SNPs that identify the haplotypes
are called
tag SNPs
. A HapMap is a map of these hap-
lotype blocks and their tag SNPs. This approach should
prove useful in reducing the number of SNPs required to
examine an entire genome and make genome scanning
methods much more efficient in finding regions with
genes that contribute to disease development.
Recent improvements in sequencing technology
(“Next-Gen” sequencing platforms) have dramati-
cally reduced the cost of sequencing. The goal of the
1000 Genomes Project is to sequence the genomes of
a large number of people and provide a comprehen-
sive high resolution resource for human genetic varia-
tion
. It is anticipated that the
HapMap Project and the 1000 Genomes Project will
provide a useful tool for disease diagnosis and manage-
ment. Much attention has focused on the use of SNPs
to decide whether a genetic variant is associated with
a higher risk of disease susceptibility in one population
versus another. Pharmacogenetics addresses the variabil-
ity of drug response due to inherited characteristics in
individuals. With the availability of SNPs, it may soon
be possible to identify persons who can be expected
to respond favorably to a drug and those who can be
expected to experience adverse reactions. This would
result in safer, more effective, and more cost-efficient use
of medications.
Recombinant DNATechnology
The term
recombinant DNA
refers to a combination of
DNA molecules that are not found together in nature.
Recombinant DNA technology makes it possible to iden-
tify the DNA sequence in a gene and produce the pro-
tein product encoded by a gene. The specific nucleotide
sequence of a DNA fragment can often be identified by
analyzing the amino acid sequence and mRNA codon of its
protein product. Short sequences of base pairs can be syn-
thesized, radioactively labeled, and subsequently used to
identify their complementary sequence. In this way, iden-
tifying normal and abnormal gene structures is possible.
Gene Isolation and Cloning
The gene isolation and cloning methods used in recom-
binant DNA technology rely on the fact that the genes
of all organisms, from bacteria through mammals, are
based on a similar molecular organization. Gene cloning
requires cutting a DNA molecule apart, modifying and
reassembling its fragments, and producing copies of the
modified DNA, its mRNA, and its gene product. The
DNA molecule is cut apart using a bacterial enzyme,
called a
restriction enzyme,
that binds to DNA wherever
a particular short sequence of base pairs is found and
cleaves the molecule at a specific nucleotide site. In this
way, a long DNA molecule can be broken down into
smaller, discrete fragments, one of which presumably
contains the gene of interest. Many restriction enzymes
are commercially available that cut DNA at different
recognition sites.
The restrictive fragments of DNA can often be
replicated through insertion into a unicellular organism,
such as a bacterium (Fig. 5-12). To do this a
plasmid
,
which is a cloning vector such as a bacterial virus or a
small DNA circle that is found in most bacteria, is used.
Viral and plasmid vectors replicate autonomously in the
host bacterial cell. During gene cloning, a bacterial vec-
tor and the DNA fragment are mixed and joined by a
special enzyme called a
DNA ligase
. The recombinant
vectors formed are then introduced into a suitable culture
of bacteria, and the bacteria are allowed to replicate and
express the recombinant vector gene. Sometimes, mRNA
taken from a tissue that expresses a high level of the gene
is used to produce a complementary DNA molecule that
can be used in the cloning process. Because the fragments
of the entire DNA molecule are used in the cloning pro-
cess, additional steps are taken to identify and separate
the clone that contains the gene of interest.
