2.2 Selection and isolation of genes
Genetic information is carried in the linear sequence of nucleotides in DNA. Its
expression involves the translation of the linear sequence of specific regions of
DNA existing in the nucleus of the cell (called coding regions or genes) into a
colinear sequence of amino acids (proteins). As an intermediate step, however,
DNA must be copied into a different type of polynucleotide known as
ribonucleic acid (RNA) which retains all the information of the DNA sequence
from which it was copied. Single-stranded RNA molecules are synthesized by a
process known as DNA transcription which is regulated by interactions between
DNA sequences located upstream of the gene (promoters) and proteins
(transcription factors). Thousands of RNA transcripts can be made from the
same DNA segment in a given cell. Many of these RNA molecules undergo
major chemical changes before they leave the nucleus to serve as the messenger
RNA (mRNA) molecules that direct the synthesis of proteins in the cytosol.
Fragments of DNA can be amplified by a process called DNA cloning which
consists in inserting the DNA into a plasmid or a bacterial virus and then
growing these in bacterial (or yeast) cells. Plasmids are small circular molecules
of DNA that occur naturally in bacteria, where they replicate as independent
units. As these bacteria divide, the plasmid also replicates to produce an
enormous number of copies of the cloned DNA fragment. Although restricted
genomic DNA fragments can be cloned to produce genomic libraries, cDNA
libraries are most frequently used to isolate and characterize genes necessary for
the production of genetically engineered plants.
cDNA libraries represent the
information encoded in the mRNA of a particular tissue or organism. mRNA
molecules are exceptionally labile and difficult to amplify in their natural form.
For this reason, the information encoded by the mRNA is converted into a stable
DNA duplex (cDNA) via enzymatic reactions catalyzed by reverse transcriptase
and DNA polymerase I, and then is inserted into a self-replicating plasmid. The
resulting heterogeneous population of cDNA molecules collectively encodes
virtually all of the mRNAs sysnthesized by the cell. Once the information is
available in the form of a cDNA library, individual processed segments of the
original genetic information can be isolated and examined with relative ease.
A representative cDNA library should contain full-length copies of the
original population of mRNA. cDNA libraries provide a method by which the
transcription and processing of mRNA can be examined and interpreted to
produce models for the flow of information responsible for the fundamental
characteristics of each organism and tissue type. Comprehensive cDNA libraries
can be routinely established from small quantities of mRNA, and a variety of
reliable methods are available to identify cDNA clones corresponding to
extremely rare species of mRNA. As the enzymatic reactions used to synthesize cDNA have improved, the sizes of cloned cDNAs have increased, and it is often
Genetic information is carried in the linear sequence of nucleotides in DNA. Its
expression involves the translation of the linear sequence of specific regions of
DNA existing in the nucleus of the cell (called coding regions or genes) into a
colinear sequence of amino acids (proteins). As an intermediate step, however,
DNA must be copied into a different type of polynucleotide known as
ribonucleic acid (RNA) which retains all the information of the DNA sequence
from which it was copied. Single-stranded RNA molecules are synthesized by a
process known as DNA transcription which is regulated by interactions between
DNA sequences located upstream of the gene (promoters) and proteins
(transcription factors). Thousands of RNA transcripts can be made from the
same DNA segment in a given cell. Many of these RNA molecules undergo
major chemical changes before they leave the nucleus to serve as the messenger
RNA (mRNA) molecules that direct the synthesis of proteins in the cytosol.
Fragments of DNA can be amplified by a process called DNA cloning which
consists in inserting the DNA into a plasmid or a bacterial virus and then
growing these in bacterial (or yeast) cells. Plasmids are small circular molecules
of DNA that occur naturally in bacteria, where they replicate as independent
units. As these bacteria divide, the plasmid also replicates to produce an
enormous number of copies of the cloned DNA fragment. Although restricted
genomic DNA fragments can be cloned to produce genomic libraries, cDNA
libraries are most frequently used to isolate and characterize genes necessary for
the production of genetically engineered plants.
cDNA libraries represent the
information encoded in the mRNA of a particular tissue or organism. mRNA
molecules are exceptionally labile and difficult to amplify in their natural form.
For this reason, the information encoded by the mRNA is converted into a stable
DNA duplex (cDNA) via enzymatic reactions catalyzed by reverse transcriptase
and DNA polymerase I, and then is inserted into a self-replicating plasmid. The
resulting heterogeneous population of cDNA molecules collectively encodes
virtually all of the mRNAs sysnthesized by the cell. Once the information is
available in the form of a cDNA library, individual processed segments of the
original genetic information can be isolated and examined with relative ease.
A representative cDNA library should contain full-length copies of the
original population of mRNA. cDNA libraries provide a method by which the
transcription and processing of mRNA can be examined and interpreted to
produce models for the flow of information responsible for the fundamental
characteristics of each organism and tissue type. Comprehensive cDNA libraries
can be routinely established from small quantities of mRNA, and a variety of
reliable methods are available to identify cDNA clones corresponding to
extremely rare species of mRNA. As the enzymatic reactions used to synthesize cDNA have improved, the sizes of cloned cDNAs have increased, and it is often
