Fundamentals of Molecular Biology. Part 2: Molecular Genetic Mechanisms - A. N. Ogurtsov 2011

Alleles and Mutations
Mutant Alleles and Gene Functions

The discovery of a new protein always poses three fundamental questions for a researcher:

1. What is the function of this protein?

2. Where is this protein localized within The Cell?

3. What is its Structure?

To answer these questions, researchers employ three approaches:

(I) investigating the Gene whose expression synthesizes the protein in question,

(II) studying a mutant cell line or an Organism in which the given protein has been artificially "knocked out", and

(III) attempting to isolate the protein in pure form and in sufficient quantities for biochemical studies.

These three approaches can be implemented through two fundamentally different experimental strategies, which are schematically illustrated by the black and white arrows in Figure 73.

The first approach, commonly referred to as classical genetics (represented by the black arrows in Figure 73), begins with the identification and ISOLATION OF A mutant exhibiting a distinct phenotype in the biological process of interest. Genetic Methods are then used to identify the altered gene and subsequently isolate it from a DNA library—typically a vast "collection" of individual DNA sequences representing either the entire genome of the organism or a significant part of it.

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Figure 73 - Two approaches for determining the function, cellular localization, and structure of a protein

Next, experiments are conducted with the isolated gene to produce large quantities of the protein of interest for biochemical analyses, as well as to determine where and when this protein is expressed in the organism.

The second approach utilizes essentially the same research steps but in reverse order (represented by the white arrows in Figure 73), starting with the Isolation of the protein of interest or its Identification based on the Analysis of the organism's genomic sequence. Once the corresponding gene is found in the DNA library, it can be modified and

introduced back into the organism. By investigating the effects of the altered gene on the organism, the researcher draws Conclusions about the function of the given protein in a normal organism.

An essential step in both approaches to studying Proteins and their biological Functions is the identification of the corresponding gene.

Genomics is the branch of molecular biology dedicated to the molecular characterization of The Genome and the general mechanisms of Gene Expression.

Currently, researchers can selectively alter a given gene, thereby modifying all copies of the protein synthesized As a result of that gene's expression.

Genetic analysis of such site-directed Mutations can:

1) reveal new genes required for the process under investigation;

2) determine the order in which gene expression occurs during this process; and

3) ascertain whether the proteins encoded by different genes interact in this process.

Let us define a few terms that will be used in the subsequent Discussion.

Allele - one of the alternative structural states of a gene, a gene variant.

Mutation - a term generally used when it is known that a new allele has been generated, for example, as a result of the action of a mutagen.

A mutagen is an agent that causes a heritable change in the DNA chain.

Genotype — general definition: the genetic constitution of an organism, the sum total of all hereditary factors of a given cell or organism, including gene alleles and their physical linkage patterns in Chromosomes; more narrowly: the set of alleles of a gene (or a group of genes) controlling a trait under investigation in a given organism.

Wild type — a term used to characterize the standard genotype of an initial experimental organism (prior to mutagenic Treatment) that serves as a reference standard against which experimental results are compared.

Thus, the original, normal, non-mutant allele represents the wild type. Since the human population harbors an unimaginably vast number of naturally occurring alleles, the term wild type denotes all alleles present at a frequency significantly higher than any other possible alternative.

Phenotype — the set of all observable traits and characteristics of an individual, formed through the interaction between its genotype and the external environment.

The phenotype is a particular manifestation of the genotype under specific conditions.

In practice, the term phenotype is frequently used to describe those external manifestations determined by the alleles under investigation in a specific study. Clearly observable phenotypic characteristics are exceptionally crucial for the genetic analysis of mutations.

From the perspective of the genome, It is important to note the existence of two fundamentally distinct types of experimental organisms whose Cells contain either a single or a double set of chromosomes, termed haploids or diploids, respectively. Complex Multicellular Organisms are diploid, whereas simple single-celled organisms are haploid. Some organisms, such as Saccharomyces Yeast, can exist in both Haploid and Diploid forms.

Instances are also known where cells—both healthy and cancerous, in animals as well as plants—contain more than two copies of chromosomes. However, the following discussion will focus exclusively on diploid organisms.

If both copies of chromosomes in a diploid organism are identical, the organism is said to be homozygous; if the chromosomes carry different alleles, the organism is heterozygous.

A recessive mutant allele is one in which both alleles must be mutant for the mutant phenotype to manifest; i.e., the organism must be homozygous for the mutant allele for the mutant phenotype to appear (Table 4).

Table 4 — Effect of recessive and dominant alleles on the phenotype of a diploid organism

A dominant mutant allele, by contrast, manifests its phenotype in heterozygous organisms carrying one mutant allele and one original (wild-type) allele.

Whether a mutant allele is dominant or recessive sheds light on the function of the mutated gene and The Nature of the mutagenic effect.

Recessive alleles typically arise from mutations that inactivate a gene, leading to a partial or complete loss of function. Such recessive mutations may

1) delete a part of the gene or the entire gene from the chromosome,

2) disrupt gene expression,

3) alter The structure of the protein encoded by the gene, thereby modifying its function.

Dominant alleles, on the other hand, frequently result from mutations that cause a gain of function.

Such dominant mutations may

1) increase The activity of the encoded protein,

2) confer a new function upon it,

3) lead to a disproportionate upregulation of gene expression.

However, in certain genes, dominant mutations are associated with a loss of function. For example, some genes are haploinsufficient, meaning that both alleles are required for normal function. The deletion or inactivation of one of the alleles leads to the appearance of a mutant phenotype.

In other rare cases, a dominant mutation in one of the alleles can lead to structural alterations in the expressed protein, resulting in Interference with the function of another protein encoded by the other, unaltered allele. This type of mutation, known as a dominant-negative mutation, leads to phenotypic manifestations similar to those observed in loss-of-function mutations.

One of the most commonly used agents for inducing mutations (Mutagenic Factors) in experimental organisms is ethylmethane sulfonate (EMS). Although this mutagen can alter DNA sequences in several ways, one of the most prominent effects of its application is the chemical modification of guanine bases in DNA, which ultimately leads to The conversion of guanine-cytosine Base Pairs (G=C) into adenine-thymine pairs (A=T). Such A change in The base sequence of a DNA strand affecting only a single base pair is called a point mutation.

"Silent" (conservative) point mutations cause no Changes in the Amino Acid Sequence or the activity of the proteins encoded by the given gene.

Observed phenotypic changes resulting from altered protein activity may arise from the substitution of one amino acid for another, known as missense mutations, The formation of a premature stop codon, known as nonsense mutations, or a shift in the reading frame, known as frameshift mutations.

Since changes in the DNA sequence that lead to a decrease in protein activity are more probable than those that increase or qualitatively alter protein activity, mutagenesis typically generates far more recessive mutations than dominant ones.