Tel

Left arm has selectable marker X

Right arm has „,f selectable marker Y

^ ^S^ Fragments of genomic DNA generated by light Ligate digestion with EcoRI

Yeast cell

Enzymatic digestion of cell wall

YAC Transform

Select for X and Y

Select for X and Y

Yeast with YAC clone

Yeast cell

Yeast spheroplast

Yeast with YAC clone

FIGURE 9-8 Construction of a yeast artificial chromosome (YAC). A

YAC vector includes an origin of replication (ori), a centromere (CEN), two telomeres (TEL), and selectable markers (X and Y). Digestion with BamH! and EcoRI generates two separate DNA arms, each with a telomeric end and one selectable marker. A large segment of DNA (e.g., up to 2 X 106 bp from the human genome) is ligated to the two arms to create a yeast artificial chromosome. The YAC transforms yeast cells (prepared by removal of the cell wall to form spheroplasts), and the cells are selected for X and Y; the surviving cells propagate the DNA insert.

proper segregation of the chromosomes at cell division. Before being used in cloning, the vector is propagated as a circular bacterial plasmid. Cleavage with a restriction endonuclease (.BamHI in Fig. 9-8) removes a length of DNA between two telomere sequences (TEL), leaving the telomeres at the ends of the linearized DNA. Cleavage at another internal site (EcoRI in Fig. 9-8) divides the vector into two DNA segments, referred to as vector arms, each with a different selectable marker.

The genomic DNA is prepared by partial digestion with restriction endonucleases (EcoRI in Fig. 9-8) to obtain a suitable fragment size. Genomic fragments are then separated by pulsed field gel electrophoresis, a variation of gel electrophoresis (see Fig. 3-19) that allows the separation of very large DNA segments. The DNA fragments of appropriate size (up to about 2 X 106 bp) are mixed with the prepared vector arms and ligated. The ligation mixture is then used to transform treated yeast cells with very large DNA molecules. Culture on a medium that requires the presence of both selectable marker genes ensures the growth of only those yeast cells that contain an artificial chromosome with a large insert sandwiched between the two vector arms (Fig. 9-8). The stability of YAC clones increases with size (up to a point). Those with inserts of more than 150,000 bp are nearly as stable as normal cellular chromosomes, whereas those with inserts of less than 100,000 bp are gradually lost during mitosis (so generally there are no yeast cell clones carrying only the two vector ends ligated together or with only short inserts). YACs that lack a telomere at either end are rapidly degraded.

Specific DNA Sequences Are Detectable by Hybridization

DNA hybridization, a process outlined in Chapter 8 (see Fig. 8-32), is the most common sequence-based process for detecting a particular gene or segment of nucleic acid. There are many variations of the basic method, most making use of a labeled (such as radioactive) DNA or RNA fragment, known as a probe, complementary to the DNA being sought. In one classic approach to detect a particular DNA sequence within a DNA library (a collection of DNA clones), nitrocellulose paper is pressed onto an agar plate containing many individual bacterial colonies from the library, each colony with a different recombinant DNA. Some cells from each colony adhere to the paper, forming a replica of the plate. The paper is treated with alkali to disrupt the cells and denature the DNA within, which remains bound to the region of the paper around the colony from which it came. Added radioactive DNA probe anneals only to its complementary DNA. After any unannealed probe DNA is washed away, the hybridized DNA can be detected by autoradiography (Fig. 9-9).

Agar plate with transformed bacterial colonies

Press nitrocellulose paper onto the agar plate. Some cells from each colony stick to the paper.

Nitrocellulose paper

Treat with alkali to disrupt cells and expose denatured DNA.

Agar plate with transformed bacterial colonies

Press nitrocellulose paper onto the agar plate. Some cells from each colony stick to the paper.

DNA bound to paper

Radiolabeled DNA probe

Incubate the paper with the radiolabeled probe, then wash.

Probe annealed to colonies of interest

Expose x-ray film to paper.

FIGURE 9-9 Use of hybridization to identify a clone with a particular DNA segment. The radioactive DNA probe hybridizes to complementary DNA and is revealed by autoradiography. Once the labeled colonies have been identified, the corresponding colonies on the original agar plate can be used as a source of cloned DNA for further study.

Nitrocellulose paper

Treat with alkali to disrupt cells and expose denatured DNA.

DNA bound to paper

Radiolabeled DNA probe

Incubate the paper with the radiolabeled probe, then wash.

Probe annealed to colonies of interest

Expose x-ray film to paper.

FIGURE 9-9 Use of hybridization to identify a clone with a particular DNA segment. The radioactive DNA probe hybridizes to complementary DNA and is revealed by autoradiography. Once the labeled colonies have been identified, the corresponding colonies on the original agar plate can be used as a source of cloned DNA for further study.

A common limiting step in detecting and cloning a gene is the generation of a complementary strand of nucleic acid to use as a probe. The origin of a probe depends on what is known about the gene under investigation. Sometimes a homologous gene cloned from another species makes a suitable probe. Or, if the protein product of a gene has been purified, probes can be designed and synthesized by working backward from the amino acid sequence, deducing the DNA sequence that would code for it (Fig. 9-10). Now, researchers typically obtain the necessary DNA sequence information from sequence databases that detail the structure of millions of genes from a wide range of organisms.

Expression of Cloned Genes Produces Large Quantities of Protein

Frequently it is the product of the cloned gene, rather than the gene itself, that is of primary interest—particularly when the protein has commercial, therapeutic, or research value. With an increased understanding of the fundamentals of DNA, RNA, and protein metabolism and their regulation in E. coli, investigators can now manipulate cells to express cloned genes in order to study their protein products.

Most eukaryotic genes lack the DNA sequence elements—such as promoters, sequences that instruct RNA polymerase where to bind—required for their expression in E. coli cells, so bacterial regulatory sequences for transcription and translation must be inserted at appropriate positions relative to the eukaryotic gene in the vector DNA. (Promoters, regulatory sequences, and other aspects of the regulation of gene expression are discussed in Chapter 28.) In some cases cloned genes are so efficiently expressed that their protein product represents 10% or more of the cellular protein; they are said to be overexpressed. At these concentrations some foreign proteins can kill an E. coli cell, so gene expression must be limited to the few hours before the planned harvest of the cells.

Cloning vectors with the transcription and translation signals needed for the regulated expression of a cloned gene are often called expression vectors. The rate of expression of the cloned gene is controlled by replacing the gene's own promoter and regulatory sequences with more efficient and convenient versions supplied by the vector. Generally, a well-characterized promoter and its regulatory elements are positioned near several unique restriction sites for cloning, so that genes inserted at the restriction sites will be expressed from the regulated promoter element (Fig. 9-11). Some of these vectors incorporate other features, such as a bacterial ribosome binding site to enhance translation of the mRNA derived from the gene, or a transcription termination sequence.

Genes can similarly be cloned and expressed in eu-karyotic cells, with various species of yeast as the usual hosts. A eukaryotic host can sometimes promote post-translational modifications (changes in protein structure made after synthesis on the ribosomes) that might be required for the function of a cloned eukaryotic protein.

Known amino acid sequence H3N Possible codons

Gly

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