ConceptsB

An expression vector contains a promoter, a ribosome-binding site, and other sequences that allow a cloned gene to be transcribed and translated in bacteria.

A 18.11 To ensure transcription and translation, a foreign gene may be inserted into an expression vector — in this example, an E. coli expression vector.

| Expression vectors contain operon sequences that allow inserted DNA to be transcribed and translated.

Operator (O)

Bacterial promoter (P ) sequences

^ They also include sequences that regulate—turn on or turn off the desired gene.

Restriction sites

Restriction sites

| Expression vectors contain operon sequences that allow inserted DNA to be transcribed and translated.

Operator (O)

Transcription termination sequence

Ribosome-binding site

Gene-encoding repressor that binds O and regulates P

Transcription termination sequence

Ribosome-binding site

Transcription initiation sequences

Selectable genetic marker (e.g., antibiotic resistance)

Cloning vectors for eukaryotes The vectors discussed so far allow genes to be cloned in bacterial cells. Other cloning vectors have been developed for transferring genes into eu-karyotic cells. Special plasmids, for example, have been developed for cloning in yeast, and retroviral vectors have been developed for cloning in mammals.

Shuttle vectors are used to shuttle genes back and forth between two hosts. For example, plasmids have been engineered that allow gene sequences to be cloned and manipulated in bacteria and then transferred to yeast cells for study. For this reason, they must contain replication origins and selectable markers that work in both hosts.

Yeast artificial chromosomes (YACs ) are DNA molecules with a yeast origin of replication, a pair of telomeres, and a centromere. Mitotic spindle fibers attach to the centromere, and YACs segregate in the same way as yeast chromosomes; the telomeres ensure that YACs remain stable within the cell; and the origin of replication allows YACs to be replicated. YACs are particularly useful because they can carry DNA fragments as large as 600 kb, and some special

YACs can carry inserts of more than 1000 kb. YACs have been modified so that they can be used in eukaryotic organisms other than yeast (see introduction to Chapter 11). Bacterial artificial chromosomes (BACs), constructed from F factors (see Chapter 8), are used to clone large fragments ranging in length from 100 to 500 kb in bacteria.

The soil bacterium Agrobacterium tumefaciens, which invades plants through wounds and induces crown galls (tumors), has been used to transfer genes to plants. This bacterium contains a large plasmid called the Ti plasmid, part of which is transferred to a plant cell when A. tumefa-ciens infects a plant. In the plant, the Ti plasmid DNA integrates into one of the plant chromosomes where it is transcribed and translated to produce several enzymes that help support the bacterium (IFigure 18.12a). Transfer of the DNA segment from the Ti plasmid to a plant chromosome requires two 25-bp sequences that flank the Ti DNA, as well as several genes located in the Ti plasmid.

Geneticists have engineered an Agrobacterium - E. coli shuttle vector that contains the flanking sequences required

I The helper Ti plasmid

Shuttle vector

Restriction site

^ Sequences required for transfer

Restriction site

^ Sequences required for transfer

Selectable

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