 Detection

of Nucleic Acids and

 Gene

Function in Eukaryotes

Recombinant DNA

Bacteriophage λ vectors can accommodate larger fragments of insert DNA. Sequences that aren’t 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 λ 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.

. coli (the F factor).Recombinant DNA 4. 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. 5. Bacterial artificial chromosome (BAC) vectors are derived from a naturally occurring plasmid of E.

.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 λ vector.

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

26 Screening a recombinant library by hybridization (Part 2) .Figure 4.

Mutants that have specific nutrient requirements can be easily isolated. .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.

simply on the basis of its functional activity. Yeast genes encoding a wide variety of essential proteins have been identified in this manner.Gene Function in Eukaryotes A gene corresponding to any yeast mutation can be cloned. . 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) .

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

Figure 4.35 Production of transgenic mice .

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

36 Introduction of genes into mice via embryonic stem cells (Part 1) .Figure 4.

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

since a mutation is introduced into a gene first and its functional consequence is determined second. . Sometimes called reverse genetics.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.

.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.

38 Mutagenesis with synthetic oligonucleotides .Figure 4.

the activity of the normal gene copy must be eliminated. .Gene Function in Eukaryotes To determine the role of a cloned gene. 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.

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

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.

40 Production of mutant mice by homologous recombination in ES cells .Figure 4.

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 .

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 Homologous recombination has been used to systematically inactivate (knockout) every gene in yeast. .

Antisense nucleic acids are RNA or single-stranded DNA complementary to the mRNA of the gene of interest (antisense).Gene Function in Eukaryotes Other approaches are used to interfere with gene expression or function. They hybridize with the mRNA and block its translation into protein. .

Figure 4.41 Inhibition of gene expression by antisense RNA or DNA .

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

Key Experiment 4.2 RNA Interference: .

The filter is then incubated with a labeled probe. The gel is then overlaid with a nitrocellulose or nylon membrane to which the DNA fragments are transferred (blotted).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. .

25 Southern blotting .Figure 4.

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

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

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.

. for example. a comparison of the genes expressed by two different types of cells.Detection of Nucleic Acids and Proteins One application of DNA microarrays is in studies of gene expression.

Figure 4.27 DNA microarrays .

chromosomes. or intact cells. Hybridization of fluorescent probes to specific cells or subcellular structures is analyzed by microscopic examination.Detection of Nucleic Acids and Proteins In situ hybridization can be used to detect homologous DNA or RNA sequences in cell extracts. .

Figure 4.28 Fluorescence in situ hybridization .

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