Of course, Mendel had no idea of the molecular nature of the concepts he was working with. In this section, we can begin putting some of Mendel’s concepts into a molecular context. Let’s begin with alleles. We have used the concept of alleles without defining them at the molecular level. What are the structural differences between wild-type and mutant alleles at the DNA level of a gene? What are the functional differences at the protein level? Mutant alleles can be used to study single-gene inheritance without needing to understand their structural or functional nature. However, because a primary reason for embarking on single-gene inheritance is ultimately to investigate a gene’s function, we must come to grips with the molecular nature of wild-type and mutant alleles at both the structural and the functional level.

Mendel proposed that genes come in different forms we now call alleles. What are alleles at the molecular level? When alleles such as A and a are examined at the DNA level by using modern technology, they are generally found to be identical in most of their sequences and differ only at one or several nucleotides of the hundreds or thousands of nucleotides that make up the gene. Therefore, we see that the alleles are truly different versions of the same gene. The following diagram represents the DNA of two alleles of one gene; the letter x represents a difference in the nucleotide sequence:

If the nucleotide sequence of an allele changes as the result of a rare chemical “accident,” a new mutant allele is created. Such changes can occur anywhere along the nucleotide sequence of a gene. For example, a mutation could be a change in the identity of a single nucleotide or the deletion of one or more nucleotides or even the addition of one or more nucleotides.

There are many ways that a gene can be changed by mutation. For one thing, the mutational damage can occur at any one of many different sites. We can represent the situation as follows, where dark blue indicates the normal wild-type DNA sequence and red with the letter x represents the altered sequence:

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Replication of alleles during the S phase What happens to alleles at the molecular level during cell division? We know that the primary genomic component of each chromosome is a DNA molecule. This DNA molecule is replicated during the S phase, which precedes both mitosis and meiosis. As we will see in Chapter 7, replication is an accurate process and so all the genetic information is duplicated, whether wild type or mutant. For example, if a mutation is the result of a change in a single nucleotide pair–say, from GC (wild type) to AT (mutant)–then in a heterozygote, replication will be as follows:

DNA replication before mitosis in a haploid and a diploid are shown in Figure 2-10. This type of illustration serves to remind us that, in our considerations of the mechanisms of inheritance, it is essentially DNA molecules that are being moved around in the dividing cells.

Meiosis and mitosis at the molecular level The replication of DNA during the S phase produces two copies of each allele, A and a, that can now be segregated into separate cells. Nuclear division visualized at the DNA level is shown in Figure 2-11.

Demonstrating chromosome segregation at the molecular level We have interpreted single-gene phenotypic inheritance patterns in relation to the segregation of chromosomal DNA at meiosis. Is there any way to show DNA segregation directly (as opposed to phenotypic segregation)? The most straightforward approach would be to sequence the alleles (say, A and a) in the parents and the meiotic products: the result would be that one-half of the products would have the A DNA sequence and one-half would have the a DNA sequence. The same would be true for any DNA sequence that differed in the inherited chromosomes, including those not necessarily inside alleles correlated with known phenotypes such as red and white flowers. Thus, we see the rules of segregation enunciated by Mendel apply not only to genes but to any stretch of DNA along a chromosome.

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At the molecular level, the primary phenotype of a gene is the protein it produces. What are the functional differences between proteins that explain the different effects of wild-type and mutant alleles on the properties of an organism?

Let’s explore the topic by using the human disease phenylketonuria (PKU). We shall see in a later section on pedigree analysis that the PKU phenotype is inherited as a Mendelian recessive. The disease is caused by a defective allele of the gene that encodes the liver enzyme phenylalanine hydroxylase (PAH). This enzyme normally converts phenylalanine in food into the amino acid tyrosine:

However, a mutation in the gene encoding this enzyme may alter the amino acid sequence in the vicinity of the enzyme’s active site. In this case, the enzyme cannot bind phenylalanine (its substrate) or convert it into tyrosine. Therefore, phenylalanine builds up in the body and is converted instead into phenylpyruvic acid. This compound interferes with the development of the nervous system, leading to mental retardation.

Babies are now routinely tested for this processing deficiency at birth. If the deficiency is detected, phenylalanine can be withheld with the use of a special diet and the development of the disease arrested.

The PAH enzyme is made up of a single type of protein. What changes have occurred in the mutant form of the PKU gene’s DNA, and how can such change at the DNA level affect protein function and produce the disease phenotype? Sequencing of the mutant alleles from many PKU patients has revealed a plethora of mutations at different sites along the gene, mainly in the protein-encoding regions, or the exons; the results are summarized in Figure 2-12. They represent a range of DNA changes, but most are small changes affecting only one nucleotide pair among the thousands that constitute the gene. What these alleles have in common is that they encode a defective protein that no longer has normal PAH activity. By changing one or more amino acids, the mutations all inactivate some essential part of the protein encoded by the gene. The effect of the mutation on the function of the gene depends on where within the gene the mutation occurs. An important functional region of the gene is that encoding an enzyme’s active site; so this region is very sensitive to mutation. In addition, a minority of mutations are found to be in introns, and these mutations often prevent the normal processing of the primary RNA transcript. Some of the general consequences of mutation at the protein level are shown in Figure 2-13. Many of the mutant alleles are of a type generally called null alleles: the proteins encoded by them completely lack PAH function. Other mutant alleles reduce the level of enzyme function; they are sometimes called leaky mutations, because some wild-type function seems to “leak” into the mutant phenotype. DNA sequencing often detects changes that have no functional impact at all, so they are functionally wild type. Hence, we see that the terms wild type and mutant sometimes have to be used carefully.

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We have been pursuing the idea that finding a set of genes that impinge on the biological property under investigation is an important goal of genetics, because it defines the components of the system. However, finding the precise way in which mutant alleles lead to mutant phenotypes is often challenging, requiring not only the identification of the protein products of these genes, but also detailed cellular and physiological studies to measure the effects of the mutations. Furthermore, finding how the set of genes interacts is a second level of challenge and a topic that we will pursue later, starting in Chapter 6.

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Dominance and recessiveness With an understanding of how genes function through their protein products, we can better understand dominance and recessiveness. Dominance was defined earlier in this chapter as the phenotype shown by a heterozygote. Hence, formally, it is the phenotype that is dominant or recessive, but, in practice, geneticists more often apply the term to alleles. This formal definition has no molecular content, but both dominance and recessiveness can have simple explanations at the molecular level. We introduce the topic here, to be revisited in Chapter 6.

How can alleles be dominant? How can they be recessive? Recessiveness is observed in null mutations in genes that are functionally haplosufficient, loosely meaning that one gene copy has enough function to produce a wild-type phenotype. Although a wild-type diploid cell normally has two fully functional copies of a gene, one copy of a haplosufficient gene provides enough gene product (generally a protein) to carry out the normal transactions of the cell. In a heterozygote (say, +/m, where m is a null), the single functional copy encoded by the + allele provides enough protein product for normal cellular function. In a simple example, assume a cell needs a minimum of 10 protein units to function normally. Each wild-type allele can produce 12 units. Hence, a homozygous wild type +/+ will produce 24 units. The heterozygote +/m will produce 12 units, in excess of the 10-unit minimum, and hence the mutant allele is recessive as it has no impact in the heterozygote.

Other genes are haploinsufficient. In such cases, a null mutant allele will be dominant because, in a heterozygote (+/P), the single wild-type allele cannot provide enough product for normal function. As another example, let’s assume the cell needs a minimum of 20 units of this protein, and the wild-type allele produces only 12 units. A homozygous wild type +/+ makes 24 units, which is over the minimum. However, a heterozygote involving a null mutation (+/P) produces only 12; hence, the presence of the mutant allele in the heterozygote results in an inadequate supply of product and a mutant phenotype ensues.

In some cases, mutation results in a new function for the gene. Such mutations can be dominant because, in a heterozygote, the wild-type allele cannot mask this new function.

From the above brief considerations, we see that phenotype, the description or measurement that we track during Mendelian inheritance, is an emergent property based on the nature of alleles and the way in which the gene functions normally and abnormally. The same can be said for the descriptions dominant and recessive that we apply to a phenotype.