Outline

Detection

of Nucleic Acids and

Proteins
Gene

Function in Eukaryotes

Recombinant DNA

Bacteriophage vectors can accommodate larger fragments of insert DNA. Sequences that arent needed for virus replication are removed and replaced with unique restriction sites for insertion of cloned DNA. The recombinant molecules are then put into E. coli, where they replicate to yield millions of progeny phages containing a single DNA insert.

Recombinant DNA

For even larger fragments of DNA, 5 major types of vectors are used. 1. Cosmid vectors contain bacteriophage sequences, origins of replication, and genes for antibiotic resistance, so they are able to replicate as plasmids in bacterial cells.

Recombinant DNA

2. Bacteriophage P1 vectors allow recombinant molecules to be packaged in vitro into P1 phage particles and be replicated as plasmids in E. coli.

3. P1 artificial chromosome (PAC) vectors also contain sequences of bacteriophage P1 but are introduced directly as plasmids into E. coli.

Recombinant DNA

4. Bacterial artificial chromosome (BAC) vectors are derived from a naturally occurring plasmid of E. coli (the F factor).

5. Yeast artificial chromosome (YAC) vectors contain yeast origins of replication and other sequences that allow them to replicate as linear chromosome-like molecules in yeast cells.

Detection of Nucleic Acids and Proteins

Recombinant DNA libraries are collections of clones that contain all the genomic or mRNA sequences of a particular cell type.

A genomic library of human DNA can be made by cloning random DNA fragments of about 15 kb in a vector.

Figure 4.26 Screening a recombinant library by hybridization (Part 1)

Figure 4.26 Screening a recombinant library by hybridization (Part 2)

Gene Function in Eukaryotes

In addition to traditional genetic screens for new mutations, cloned DNA can be used to create transgenic or gene knock-out systems Yeasts are used in studies of eukaryotic cells because they are easily grown in culture, reproduce rapidly, and have a small genome. Mutants that have specific nutrient requirements can be easily isolated.

Gene Function in Eukaryotes

A gene corresponding to any yeast mutation can be cloned, simply on the basis of its functional activity. Yeast genes encoding a wide variety of essential proteins have been identified in this manner. In many cases, such genes have also been useful in identifying and cloning related genes from mammalian cells.

Figure 4.33 Introduction of DNA into animal cells

Gene Function in Eukaryotes

Direct microinjection into the nucleus

Coprecipitation of DNA with calcium phosphate to form small particles that are taken up by the cells Incorporation of DNA into liposomes that fuse with the plasma membrane
Exposure of cells to a brief electric pulse that opens pores in the plasma membrane (electroporation) Viruses

Figure 4.34 Retroviral vectors (Part 1)

Figure 4.34 Retroviral vectors (Part 2)

Gene Function in Eukaryotes

Cloned genes can also be introduced into the germ line of multicellular organisms. Mice that carry foreign genes (transgenic mice) are produced by microinjection of cloned DNA into the pronucleus of a fertilized egg.

Figure 4.35 Production of transgenic mice

Gene Function in Eukaryotes

Embryonic stem (ES) cells are another way to introduce cloned genes into mice. Cloned DNA is introduced into ES cells in culture, then stably transformed cells are introduced back into mouse embryos.

Figure 4.36 Introduction of genes into mice via embryonic stem cells (Part 1)

Figure 4.36 Introduction of genes into mice via embryonic stem cells (Part 2)

Gene Function in Eukaryotes

The ability to introduce specific mutations into cloned DNAs (in vitro mutagenesis) is a powerful tool in studying the expression and function of eukaryotic genes. Sometimes called reverse genetics, since a mutation is introduced into a gene first and its functional consequence is determined second.

Gene Function in Eukaryotes

The most common method of in vitro mutagenesis uses synthetic oligonucleotides to generate changes in a DNA sequence.

In vitro mutagenesis allows detailed characterization of the functional roles of both regulatory and protein-coding sequences of cloned genes.

Figure 4.38 Mutagenesis with synthetic oligonucleotides

Gene Function in Eukaryotes

To determine the role of a cloned gene, the activity of the normal gene copy must be eliminated. Gene inactivation by homologous recombination: the mutated copy of the cloned gene replaces the normal gene copy in the chromosomal DNA.

This occurs frequently in yeast but is rare in mammalian cells.

Gene Function in Eukaryotes

Yeasts are used in studies of eukaryotic cells because they are easily grown in culture, reproduce rapidly, and have a small genome. Mutants that have specific nutrient requirements can be easily isolated.

Gene Function in Eukaryotes

Temperature-sensitive mutants encode proteins that are functional at one temperature (permissive temperature) but not another (nonpermissive temperature). The ability to isolate temperaturesensitive mutants has allowed identification of genes controlling many fundamental cell processes.

Figure 4.32 Screening for Temperature sensitive mutations in yeast

Gene Function in Eukaryotes

Genes can be readily inactivated by homologous recombination in mouse embryonic stem cells Stem cells can be introduced into embryo to make chimeric mice These mice can be bred to yield progeny with mutated copies of the gene on both homologous chromosomes. The effects of inactivation of a gene can then be investigated in the context of the intact animal.

Figure 4.40 Production of mutant mice by homologous recombination in ES cells

Ensuring homologous recombination

NeoR
HSVtk

Homologous recombination

Random integration

NeoR

NeoR+/ HSVtk-

NeoR+/ HSVtk+ HSVtk will convert gancyclovir into a toxic drug and kill HSVtk+ cells

Gene Function in Eukaryotes

Homologous recombination has been used to systematically inactivate (knockout) every gene in yeast. A collection of genome-wide yeast mutants is available for scientists to use to study the function of any desired gene.

Gene Function in Eukaryotes

Other approaches are used to interfere with gene expression or function. Antisense nucleic acids are RNA or single-stranded DNA complementary to the mRNA of the gene of interest (antisense).

They hybridize with the mRNA and block its translation into protein.

Figure 4.41 Inhibition of gene expression by antisense RNA or DNA

Gene Function in Eukaryotes

RNA interference (RNAi) was first discovered in C. elegans in 1998, when Fire and Mello found that injection of double-stranded RNA inhibited expression of a gene with a complementary mRNA sequence. Double-stranded RNA resulted in extensive degradation of the target mRNA, whereas single-stranded antisense RNA had only a minimal effect.

Key Experiment 4.2 RNA Interference:

Detection of Nucleic Acids and Proteins

Southern blotting is widely used for detection of specific genes. DNA is digested with a restriction endonuclease, and the fragments separated by gel electrophoresis. The gel is then overlaid with a nitrocellulose or nylon membrane to which the DNA fragments are transferred (blotted). The filter is then incubated with a labeled probe.

Figure 4.25 Southern blotting

Detection of Nucleic Acids and Proteins

Northern blotting, a variation of Southern blotting, is used for detection of RNA instead of DNA. It is often used in studies of gene expression, for example, to determine whether specific mRNAs are present.

Detection of Nucleic Acids and Proteins

Any gene for which a probe is available can then be isolated from such a recombinant library. cDNA clones can be used as probes to isolate the corresponding genomic clones, or a gene cloned from one species (e.g., mouse) can be used to isolate a related gene from a different species (e.g., human).

Detection of Nucleic Acids and Proteins

Hybridization to DNA microarrays allows tens of thousands of genes to be analyzed simultaneously. A DNA microarray is a glass slide or membrane filter onto which oligonucleotides or fragments of cDNAs are printed by a robotic system in small spots at a high density.

Detection of Nucleic Acids and Proteins

One application of DNA microarrays is in studies of gene expression; for example, a comparison of the genes expressed by two different types of cells.

Figure 4.27 DNA microarrays

Detection of Nucleic Acids and Proteins

In situ hybridization can be used to detect homologous DNA or RNA sequences in cell extracts, chromosomes, or intact cells.

Hybridization of fluorescent probes to specific cells or subcellular structures is analyzed by microscopic examination.

Figure 4.28 Fluorescence in situ hybridization