Some Additional Tools for the Molecular Biologist - Insect Molecular Genetics - page 211

including cosmids or phagemids. Cloning into single-stranded M13 phage results
in single-stranded DNA suitable for sequencing.
Cloning is feasible because a diverse array of enzymes is available to synthe-
size, ligate, and modify the ends of DNA molecules. Nucleic-acid or antibody
hybridizations, chromosome walking, and sequencing can be used to screen
libraries for genes of interest.
Cloning provides the basis for identifying specific genes, producing DNA cop-
ies of mRNA ( = cDNA), and, in some cases, producing gene products (proteins)
in E. coli , yeast, or insect cells by incorporating the DNA into expression vectors.
cDNA libraries are used to determine which genes are being transcribed in a
particular tissue or cell at a particular time and to provide clues important in
understanding the development of insects. Two insect baculoviruses have been
genetically engineered to express proteins (expression vectors), and they are
used to produce proteins in insect cells in tissue culture or in intact lepidopteran
larvae. Microarrays are used to determine the expression of hundreds or thou-
sands of genes at a time in expression microarrays.
6.2 Introduction
The term cloning has multiple meanings. For example, Dolly the sheep was a
“clone” because a nucleus of a donor somatic cell was inserted into an egg from
which the original nucleus was removed. That egg was implanted in a host ewe
and produced a lamb, Dolly, that was genetically identical to the sheep donat-
ing the nucleus. In this chapter, cloning also means that a single vector molecule
(plasmid or phage or engineered versions of these) containing exogenous DNA
is multiplied in cells so that multiple identical copies (clones) are produced. A
vector is the agent used to replicate, or multiply, the exogenous DNA. Vectors
are segments of DNA with an origin of replication so that it can be replicated
after it is introduced into a host cell (origins of replication are essential for clon-
ing). Vectors can be plasmids, bacteriophage, baculoviruses, or hybrid engi-
neered molecules called cosmids and phagemids.
Chapter 5 introduced the use of plasmid vectors. This chapter introduces vec-
tors derived from the Escherichia coli bacteriophage λ ; the single-stranded DNA
bacteriophage M13; and engineered, hybrid vectors combining components
from bacterial plasmids and λ called cosmids. Phagemids are engineered hybrid
molecules that combine elements of plasmids and M13 vectors. Bacterial arti-
ficial chromosomes (BACs) are used to clone very large segments of DNA. The
most commonly used host cell for cloning is E. coli , but others are used including
the bacterium Bacillus subtilis or the yeast Saccharomyces cerevisiae . Insect cells