Isolation and Functional Studies of Genes

Abstract

Determining and analyzing the functional roles of genes is a prerequisite for genetic engineering. A wide variety of strategies are employed to isolate genes and to characterize their functions. This chapter describes hybridization techniques, such as Southern, Northern, and Western blotting; tools for mutant analysis, including mutant selection and phenotypic analysis; and the analytical methods of activation tagging and overexpression of target genes. Besides in planta biological approaches, a variety of biochemical methods have been described in previous research, such as heterologous protein expression, protein–protein (and other ligands) interactions, domain analysis, and protein structure. Meanwhile, the cloning of a gene controlling the targeted trait using linkage maps is a fundamental tool in gene functional studies and allows in vitro manipulation of the gene. A helpful guide with step-by-step procedures for map-based cloning, from the selection of a target gene to the complementation test, and a case study in rice are also described.

Glossary

Antigen

Any substance that causes the immune system to produce antibodies against it.

Callus

Undifferentiated or unorganized mass of cells.

CentiMorgan (cM)

The unit for measuring genetic linkage. It is defined as the distance between chromosome positions (loci or markers) for which the expected average number of intervening chromosomal crossovers in a single generation is 0.01. It is often used to infer distance along a chromosome.

Chromatography

Method of separating compounds in a mixture based on movement speed.

Cis-acting element

Region of non-coding DNA that binds a nucleotide sequence to regulate the transcription of nearby genes. These main regulate gene transcription.

Cleaved Amplified Polymorphic Sequences (CAPS)

An amplified DNA fragment is digested by restriction enzymes. Sequence differences in the DNA fragments of different individuals can be revealed by electrophoresis.

Derived Cleaved Amplified Polymorphic Sequences (dCAPS)

A modification of CAPS (or alternatively, PCR-RFLP) for detecting single nucleotide polymorphisms (SNPs). In a dCAPS assay, mismatches in PCR primers are used to create restriction endonuclease (RE)-sensitive polymorphisms based on the target mutation. dCAPS is useful for genotyping known mutations and for genetic mapping of isolated DNAs.

Detergent

Surfactants, compounds with a hydrophobic (water repellent) and a hydrophilic (water loving) part.

Electroporation

Method by which DNA is introduced into cells using a pulse of high voltage current.

F1 hybrid

The first filial generation of offspring of distinctly different parental types.

Freund adjuvant emulsion

A medium developed by J. Freund and used to cause a strong immune response in experimental animals.

Gene gun

Method developed to introduce biological material into living cells. DNA is coated with 1 μm tungsten or gold microparticles and accelerated through the cell wall or cell membrane. This technique is useful to study gene regulation.

Genetic marker

A gene or DNA sequence with a known location on a chromosome that can be used to identify individuals or species. It is an observable variation that may arise from mutation or alterations in the genomic loci. A genetic marker may be a short DNA sequence, such as the region near a single nucleotide polymorphism, or a long one, like a minisatellite.

Glycosylation

Attachment of a glucose molecule to proteins that alter the protein’s function by changing its structure. The complex is called a glycoprotein.

Heterozygous

Having two different alleles at a given locus.

Homozygous

Having identical alleles at a given locus.

Hydrophobic interaction

Interaction between a polar solution such as water and a non-polar substance such as alcohol.

In vitro

Latin term meaning an experiment performed in a controlled environment, such as a test tube or Petri dish, rather than in living cells.

Inclusion body

Insoluble protein commonly formed by excess expression of a foreign protein in a host cell. Must be activated by folding (refolding) but loss of active protein occurs.

Isoelectric point (pI)

The pH of a solution or dispersion at which the net charge on the molecules or colloidal particles (polyprotic acid) is zero.

Ligand

A small molecule or ion that binds to a protein to form a complex.

Linkage

The tendency of certain genes to be inherited together.

Multiple cloning sites (MCS)

A genetically engineered collection of several different restriction enzyme cleavage sites that allow convenient insertion of DNA fragments into a plasmid.

Nucleoplasm

A general term for the plasma within the nuclear membrane.

Pedigree

A line of ancestors.

Peroxisome

An organelle found in the liver and kidneys of vertebrate cells; in plant leaves and seeds; and in protozoa, yeast and fungi.

Post-translational modification

A generic term for any modification of a protein that occurs after translation and release from ribosomes. Adding a carbohydrate or acid to the protein deforms it.

Promoter

Particular sites on the DNA template that bind RNA polymerase to initiate transcription. A promoter has a regular common base sequence (consensus sequence). In prokaryotes, promoters present 10 base pairs before the pribnow box and 35 base pairs before a representative base sequence from the transcription initiation site TTGACA.

Reporter gene

A gene used to label a target gene so that its expression level and intracellular location can be easily measured. LacZ, GFP, GUS, and luciferase are commonly used marker genes that can be seen easily.

Ribosome

A complex of proteins and RNA that carries out translation.

Sequence Tagged Site (STS)

A short (200 to 500 base pair) DNA sequence that has a single occurrence in the genome and whose location and base sequence are known.

Simple Sequence Repeat (SSR)

The repeating sequences of 2–5 base pairs of DNA. This is also known as Microsatellites or Short Tandem Repeats (STRs).

Trans-acting element

Proteins that normally bind to DNA sequences in different regions of the genome. The main function is to regulate transcription.

Vacuoles

A membrane-bound organelle that is present in all plants. It has no common shape or size, and its structure varies according to the needs of the cell.

Vector

A DNA molecule that carries foreign DNA into a host cell, replicates inside a bacterial (or yeast) cell, and produces many copies of itself. Also called a cloning vehicle.