Moving Genes between Organisms Shuttle Vectors

The plasmid vectors we have discussed so far are designed to work in bacteria. Even when investigating genes from animals or plants, they are normally cloned first onto bacterial plasmids. Eventually, however, cloned genes are often moved from one organism to another. This may be done using a shuttle vector. As its name implies, this is a vector that can survive in more than one type of host cell. Obviously, the detailed requirements for vectors will vary depending on the host organism, but the general ideas are the same.

The earliest shuttle vectors were designed to shuttle between bacteria, such as E. coli, and yeast, a simple eukaryote (Fig. 22.17). Starting with a bacterial plasmid vector, several extra components are needed to create such a shuttle vector:

A. An origin of replication that works in yeast. Prokaryotic replication origins do not work in eukaryotes or vice versa, since the required DNA sequences differ substantially. However, the sequences of replication origins are rather similar in different eukaryotes, and so the yeast origin will work in many other higher organisms, at least to some extent.

B. A centromere sequence to allow correct partition of the plasmid in yeast.When a yeast cell divides, the duplicated chromosomes are pulled apart by micro-

centromere (Cen) sequence Sequence at centromere of eukaryotic chromosome that is needed for correct partition of chromosomes during cell division shuttle vector A vector that can survive in and be moved between more than one type of host cell

FIGURE 22.17 Shuttle Vector for Yeast

In order for a shuttle vector to grow in both yeast and E. coli, it must have several essential elements: two origins of replication, one for E. coli and one for yeast; a yeast centromere sequence so that it is partitioned into the daughter cells during yeast replication ; selectable markers for both yeast and E. coli; and a multiple cloning site for inserting the gene of interest.

Origin and replication genes for bacteria

Antibiotic resistance gene

Yeast origin of replication

Yeast origin of replication

Antibiotic resistance gene

Leucine biosynthesis gene for selection in

^ yeast

Multiple cloning

Leucine biosynthesis gene for selection in

^ yeast

Yeast centromere sequence tubules attached to their centromeres, so that each daughter cell gets a full set. Shuttle vectors must be segregated correctly at cell division also. To achieve this, the shuttle vector must contain a segment of DNA from the centromere of the yeast chromosomes, the Cen sequence. This is recognized by the micro-tubules that drag new chromosomes apart.

C. A gene to select for the plasmid in yeast. The problem here is that yeast is not affected by most of the antibiotics that kill bacteria. In practice, a less satisfactory selection technique is used. A yeast host strain that has a defect in a gene for making an amino acid, say leucine, is used. The corresponding biosyn-thetic gene is present on the vector. In the absence of leucine the yeast will starve. Only if it obtains the plasmid carrying the leu+ gene will it survive.

Viruses may be used as vectors, especially if they can adopt a lysogenic or latent state where they replicate in step with the host cell.

If essential genes are removed from a virus vector, a helper virus may be needed to allow replication.

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