updated ch6

Chapter 6: Molecular Genetic Techniques in Cell Biology

Major Goal of Molecular Cell Biology

• To understand cell function in terms of chemical and molecular mechanisms.

• Proteins perform most of the work in a cell, therefore this involves studying proteins.

Three Questions about a Protein under Study

1. What is its function in the context of a living cell?

2. What is the biochemical function of the purified protein?

3. Where is it located in a living cell?

• Additional questions concerning expression, evolutionary relatedness, and evolutionary origin are also relevant.

Tools Commonly Employed to Study Proteins

1. The gene that encodes a protein of interest.

2. Mutant cell line or organism lacking the protein function.

3. Purified protein for biochemical studies.

Methods for Obtaining and Using These Tools

• Modern-day Cell Biology is a combination of:

  • Classic microscopy, including newer variations like confocal microscopy.

  • Biochemistry.

  • Molecular biology.

  • Genetics (the main focus of this chapter).

• A modern-day cell biologist is a "jack-of-all-trades."

Classic Genetics

Approaches

• Mutation with Observable Phenotype:

  • Utilizes a model organism or human.

• Genomics-Based Approach:

  • Involves genomic sequencing with homology to known function.

• Reverse Genetics Approach:

  • Examines protein with observable biochemical activity.

  • Involves gene inactivation and identifying mutant phenotypes.

  • Function is deduced from sequence analysis, expression profile, cellular localization, and protein production for biochemical activity and structure determination.

Genetics: Definitions

• Alleles: Different forms (or variants) of a given gene (e.g., wild type vs. mutant).

• Mutation: A variant allele that differs from wild type, usually a recent change.

• Wild Type: The standard or reference genotype of an organism or gene.

• Genotype: The genetic constitution of an individual.

• Phenotype: The function and physical appearance due to genotype.

• Haploid: A single set of chromosomes (e.g., maternal).

• Diploid: Two sets of chromosomes (maternal and paternal).

• Homozygous: A diploid organism has two identical alleles.

• Heterozygous: A diploid organism has two different alleles.

More Genetics: Definitions

• Recessive: Both copies of a gene must be mutant to see a phenotype; must be homozygous for a mutant allele.

• Dominant: Mutant phenotype is observed when individual contains one wild type allele and one mutant allele.

• Loss-of-Function: Associated with recessive mutant alleles (e.g., tumor suppressor genes); analogy: losing both front and back brakes on a car.

• Gain-of-Function: Associated with dominant mutant alleles (e.g., oncogenes, which are cancer-causing genes); analogy: gas pedal stuck wide open.

• Haplo-insufficient: Loss of one wild type gene has an adverse effect, as the remaining wild type gene is insufficient.

• Dominant-Negative: A mutant gene product adversely affects wild type gene product.

Example: Mutations in a Diploid Beast

• W (White) is recessive (mutant: w).

• Cu (Curly) is dominant (mutant: cu).

Results of Crosses

• Wild Type:

• Dominant and Recessive Phenotypes associated with respective dominant or recessive alleles.

• Fig. 6-2: Phenotypes associated with gain-of-function or loss-of-function mutations.

Review of Meiosis

Meiosis I

• Synapsis: The exchange of DNA between homologous chromosomes, known as "crossing-over."

• Homologous chromosomes then separate, while sister chromatids remain attached.

Meiosis II

• Sister chromatids that constitute one homologue then separate.

• After reductive division:

  • 4n: Has 2 identical copies of each maternal and paternal chromosome.

  • 2n: After division, retains 2 identical copies of each chromosome.

Fertilization and Segregation Analysis

Segregation of Dominant Mutation

• First Filial Generation (F₁): All offspring have mutant phenotype.

• Second Filial Generation (F₂): 3/4 of offspring have mutant phenotype.

• Classic 3:1 Ratio for Phenotypic Trait.

Mendel's Law of Segregation: Recessive Mutation

• First Filial Generation (F₁): No offspring express mutant phenotype.

• Second Filial Generation (F₂): 1/4 of offspring exhibit phenotype of the mutant.

Segregation of Alleles in Yeast

• Yeast serves as a model organism in cell biology.

Example

• Wild type (type a) haploid cells of opposite mating type, producing diploid cells that do not exhibit mutant phenotype if the mutation is recessive.

• Sporulation and Meiosis: Producing haploid spores in tetrad: 2 will be mutant, 2 being wild type.

Creating and Screening for Conditional Mutations

• Conditional mutations: Such as temperature-sensitive mutations, where growth is permissible at 23 °C but not at 36 °C.

• Fig. 6-6: Illustrating method of conditioning.

Complementation Analysis

• Purpose: To determine whether mutations are in the same or different genes.

• Example: In mating haploids carrying different recessive temperature-sensitive cdc mutations.

• Concept of conditional mutants: Including temperature sensitivity and permissive/non-permissive temperatures.

Other Types of Genetic Interactions

a. Suppression

• Functional interaction restores genotype, leading to wild-type phenotype or a suppressed mutant phenotype.

b. Synthetic Lethality

• Opposite of suppression; combinations of mutations lead to severe defects or lethality.

Recombinant DNA Technology

• Developed in the 1970s with the use of restriction enzymes, DNA ligase, and DNA sequencing for cloning.

• DNA vectors: Plasmids and bacteriophage lambda.

• Insert DNA sequences of interest into vectors for replication.

Cloning Genomic DNA

• Creating a Genomic Library: Thousands of EcoRI digested fragments ligated to plasmids and transformed into E. coli.

• Identification of specific clones containing the desired gene can follow.

cDNA Libraries

• cDNA (Complementary DNA) Libraries: Made from tissue-specific or cell-specific mRNAs, lacking introns.

• Isolation of mRNA using oligo-dT columns, reverse transcription to synthesize cDNA.

Polymerase Chain Reaction (PCR)

• Powerful molecular technique developed by Kary Mullis in 1983; Nobel Prize in 1993.

• Amplifies specific DNA sequences, with forward and reverse primers in excess compared to template.

• Exponential increase of target DNA - uses: Basic cell biology research, human genetics, forensic science.

DNA Sequencing

• Classic techniques include Maxam and Gilbert and Sanger's dideoxy method.

• Sanger's technique is now widely used for its efficiency and ability to sequence over 500 bases.

RNA Interference (RNAi)

• Mechanism for targeted degradation of RNA and gene regulation discovered by Andrew Fire and Craig Mello in 2006.

• Involves double-stranded RNA that specifically induces the degradation of complementary mRNA.

Gene Knockout Techniques

• Techniques for creating gene knockouts in model organisms (like mice) to study gene function and phenotypes.

• Advances such as CRISPR-Cas9 allow for precise genome editing with applications in both research and potential therapies.