Overview

To reproduce, a cell must first copy its genome via DNA replication. In DNA replication, double-stranded DNA (dsDNA) is copied to yield two daughter molecules, each identical to the parent molecule. As in transcription, the new polymer grows 5′→3′. Unlike transcription, whose product is a free, single-stranded RNA molecule, replication yields two double-stranded B-form DNA helices. DNA replication is semiconservative, as each daughter helix consists of one parental strand and one newly synthesized strand.

In cells, a large complex of proteins, the replisome, replicates DNA. The replisome includes DNA-dependent DNA polymerase, which, unlike RNA polymerase, cannot begin with a single nucleotide, but rather adds nucleotides to the 3′ end of a primer, a short chain of RNA synthesized by primase. DNA synthesis is faster and much more accurate than RNA synthesis.

Chain Growth

In DNA synthesis, as in RNA synthesis, the polymer is built by making a phosphodiester bond between the α phosphate of a nucleotide triphosphate (dNTP) and the 3′ oxygen of the growing polynucleotide chain, lengthening the chain by one and producing diphosphate. Required are the four dNTPs (dATP, dGTP, dCTP, and dTTP), a template strand, a primer (a short nucleotide chain that base pairs with the template) to which nucleotides are added, and DNA polymerase. Each of the two strands of a DNA double helix is a template for the synthesis of a new, complementary strand.

I wrote this book both for the student learning genetics for the first time and for the biologist or health professional looking for background information. Fundamental Genetics is a brief account of the basic facts, theories, and experimental approaches of genetics. The book is unconventional. The organizing principle is to progress from structure to function, from simple to complex, and from molecular events to epiphenomena, such as how genes are inherited from parent to child. The history of genetics is scrupulously avoided, mainly to save space but also to avoid the trap of genuflecting to famous geneticists rather than discussing their experiments in the detail necessary for a full understanding – such a discussion, properly done, would fill several large volumes. This is a short book, and each chapter is focused.

A friend of mine says, “It's not how long life is that matters, but how thick.” The chapters of Fundamental Genetics are short but thick, so if you are a student using the book as a text, it is probably best to read one and only one chapter at a sitting – any less, and you will have trouble getting the whole picture of the chapter; any more, and you will have mental indigestion. If you are using the book as a reference, the glossary may often be a helpful starting point. The teacher who chooses Fundamental Genetics as a textbook can skip chapters that are not appropriate for the course; it will be easy to use the chapters in a different order.

Overview

Mutations are essential tools for the science of genetics. Mutations are required to discover a gene and establish its identity, to learn what genes affect a particular phenotype, and to determine the relationship between a gene's structure and its function. Mutations have practical applications in studying genetic disease and in improving organisms used for food, fiber, or valuable molecules.

A mutation is not usable until a strain of organisms carrying that mutation is established. The selection and isolation of mutant strains of any organism require special procedures; examples are described in this chapter.

Because mutation is a fundamental genetic process, geneticists need sensitive, reliable, efficient methods of measuring mutation rates. One such method, devised to screen chemicals for mutagenicity, is the Ames test.

Sources of New Mutations

Natural populations are treasure troves of mutations. Most natural mutations are rare but can be recovered by selection screens, experiments designed to separate organisms that carry mutations in a specific gene or locus.

A second way to get novel mutations is to induce them in organisms, using chemical mutagens, radiation, or genetically engineered transposons. Mutagens increase the rates of random mutation, often by 1000-fold or more. As with natural mutations, a selection screen must be used.

When a gene has been cloned, new mutations can be induced in a controlled way by in vitro mutagenesis. The idea here is to target specific DNA sequences within the gene for change, rather than to make random changes.

Overview

To understand genes, one must first consider nucleic acid, for nucleic acid is the stuff that genes are made of. Inasmuch as function follows structure, a clear picture of nucleic acid will illuminate all genetic processes.

The genetic material of all life forms is nucleic acid, either deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). DNA and RNA are linear chains made of subunits called nucleotides. Two strands of DNA typically associate in a two-stranded helix. This chapter describes the structure of nucleotides, the way nucleotides are connected to make chains, and a bit about the shapes of RNA and DNA, including some properties of double helices.

Polymers

Nucleic acids, the coding molecules of life, are linear polymers. A linear polymer is an unbranched chain made of many subunits connected by covalent chemical bonds; short polymers are known as oligomers (Figure 2.1). The subunit of a nucleic acid polymer is the nucleotide. Energy [typically about 400 kJ/mol] is required to make or to break a covalent bond between subunits of a polymer.

In this simplified representation of an RNA segment, the circles and triangles stand for nucleotides and each line stands for a covalent bond joining adjacent nucleotides. A = adenosine, G = guanidine, U = uridine, and C = cytidine.

Nucleotides

Every nucleotide has three parts: sugar, nitrogenous base, and one to three phosphates; the subunits of nucleic acid are monophosphates. A nucleoside is a nucleotide minus the phosphate (nucleoside = sugar + nitrogenous base) (Figure 2.2).

Overview

Natural selection is differential reproduction of genotypes in the absence of human intervention. The relative reproductive ability of a genotype is its fitness. Selection is a potentially strong force that can change both the population mean and its genetic variation; it is the principal agent of directional genetic change. The speed of evolution under natural selection is a function of fitness and allele frequencies. Rare deleterious alleles are eliminated very slowly from a population. One way genetic variation is maintained in a population is balancing selection, which favors heterozygotes.

Kinds of Selection

Selection occurs whenever one genotype reproduces at a higher or lower rate than other genotypes, on average, causing that genotype to contribute more or fewer copies of its genes to the population than other genotypes do. For example, a recessive lethal allele that acts at a prereproductive stage will be selected against because homozygotes will die before they can reproduce. Selection acts on all life forms, regardless of the mode of reproduction or the nature of the life cycle. It is the principal driving force of evolution. The speed of selection and its effect on equilibrium frequencies of alleles and genotypes can be quantified. Selection that acts on survival, fertility, and fecundity is called natural selection, while selection that acts on the mating success of sexual organisms is called sexual selection.

Overview

Genomes in organisms evolve primarily by microevolutionary changes in DNA sequences – substitutions, deletions, and insertions. Genomes can expand by gene duplication.

Genes evolve at different rates, depending on how the functions of RNAs and proteins are constrained by structure. The more a protein's structure is constrained, the more slowly does its gene evolve. Any given protein tends to evolve at a more or less characteristic rate, in extreme cases for hundreds of millions of years, making each protein useful in estimating when evolutionary lineages diverged.

All organisms arose from ancestral organisms, implying a single ancestor at the beginning. The organisms living today can be arranged as the twigs at the ends of a tree-like diagram whose trunk represents the original ancestor. The pattern of branching in the evolutionary tree is phylogeny. Much can be learned about phylogeny by comparing the nucleotide sequence of homologous genes – genes that descended from a common ancestral gene.

Evolution of Homologous Genes

Genes in different species that descended from a common ancestral gene are said to be homologous. There are two kinds of homology, orthology and paralogy. Orthologs are copies of a gene, present in two species and inferred to have existed in their most recent common ancestor – e.g., the β-hemoglobin genes of mice and humans. Paralogs are copies of a gene that arose by gene duplication – e.g., α-hemoglobin and β-hemoglobin.

Overview

The chromosomes of all life forms naturally undergo recombination, a physical exchange between two DNA molecules that results in new, recombinant DNA molecules. Recombination plays many roles: producing new combinations of gene copies, introducing novel sequences into chromosomes, making novel genes, regulating gene expression, and facilitating some DNA repair. Recombination has a huge impact on the biology of sexually reproducing eukarya.

There are three main kinds of recombination in nature: (1) general recombination, an exchange between homologous DNA molecules, in which the location of the exchange site is not restricted, (2) site-specific recombination, an exchange between nonhomologous DNA molecules and occurring only at specific short sequences, and (3) transposition, the movement of a transposon.

This chapter focuses on the molecular events of recombination, not on its biological consequences, analysis, or practical uses, all very important in genetics.

General Recombination

Homologous DNA molecules (different copies of a double helix, such as the two copies of chromosome 1 that you received from your mother and father), when they are together inside a cell, exchange parts by general recombination (Figure 14.1). In general recombination, portions of double-stranded DNA (dsDNA) chromosomes having extensive homology pair with the help of DNA-binding proteins. At least some homologous sequences in the two DNA molecules are aligned precisely. The two DNA molecules exchange strands in a multistep process catalyzed by many enzymes.

The Aviemore Model of General Recombination

General recombination is not identical in all life forms, and there are many theories of molecular mechanisms.

Overview

This chapter introduces several basic genetic concepts, without going into detail about any of them. These genetic concepts are as follows:

• life form

• nucleic acid

• gene

• chromosome

• organism

• virus

• semiautonomous organelle

The origin of life and the evolution of the three domains of life are described briefly.

Life Forms Are Genetic Systems

Two essential components of every life form are proteins and nucleic acids. Nucleic acids (DNA and RNA) are thread-like coding molecules, the building material of genes and chromosomes. Genetics is about genes and chromosomes – their structure and function, their behavior and misbehavior, their evolution, and methods of studying them. Because genes are the coding molecules of life, they are complicated and varied. It is difficult to pin down the term “gene” in a simple definition, but, to a first approximation, a gene is a segment of nucleic acid whose immediate function is to encode a piece of RNA (Figure 1.1). The key concepts here are replication (copying) of genes and coding. The replication of genes and their coding properties are described in detail in later chapters.

From a genetic point of view, a life form is an assemblage of large molecules capable of reproducing itself and including at least one chromosome. A chromosome is a long, thin thread made of DNA or, in some cases, RNA and may also contain proteins.

Overview

Eukarya, like bacteria, regulate the amounts of rRNA, tRNA, and mRNA by controlling rates of synthesis and degradation. On the synthesis side, control is exerted in transcription and splicing. Degradation is controlled by RNases, and is influenced by RNA processing.

The rate-limiting binding step of transcription is complicated both by the inaccessibility of DNA in chromatin and by large genome size. Transcription goes hand in hand with the disassembly of chromatin, and a large genome requires a large number of regulatory DNA sequences and transcription factors. Chromatin disassembly is effected by the chemical modification of histones and by the actions of chromatin remodeling complexes.

RNA Degradation

Stable versus Unstable RNAs

Like bacteria, eukarya have much more stable RNA than unstable RNA. The cytoplasmic RNA of a human cell is ≈80% rRNA, ≈15% tRNA and other small RNAs, and ≈1% to 5% mRNA. In growing eukaryal cells, 50% to 70% of transcription is from class I genes (big rRNAs), 20% to 40% of transcription is from mRNA genes, and the remaining 10% is from class III genes (tRNA and other small RNAs). The rarity of mRNA compared with the rate of transcription by RNA polymerase II reflects the short half-life of eukaryal mRNAs.

mRNA Degradation

Capping and polyadenylation increase RNA's resistance to degradation. These modifications are significant in mRNA, because virtually all mRNAs are capped, and most are polyadenylated.

Overview

During reproductive cycles genetic information is transferred vertically from parent to offspring, and from progenitor to descendant. Genes can also be transferred horizontally between contemporaneous individuals. In this book, horizontal gene transfer is defined in a broad, inclusive way: the incorporation of DNA from any external source into the genome of the recipient life form – a cell, a virus, a mitochondrion, but excluding genomes of parasites that invade a cell. This chapter focuses on horizontal gene transfer in bacteria.

Types of Horizontal Transfer

In bacteria, there are three kinds of transfer of genes between cells:

• Transformation – the cell takes up exogenous DNA directly from the surrounding medium

• Conjugation – two cells join and a donor cell transfers DNA, usually a plasmid, to a recipient cell

• Transduction – bacteriophages transport DNA from one cell to another

Mitochondria transfer genes horizontally infrequently, when two genetically different mitochondria fuse and their chromosomes undergo recombination. The same is true of chloroplasts. Viruses can exchange genes by recombination, following infection of the host cell by two or more genetically different virions.

Transformation

Transformation is the alteration of a cell's genome by acquiring exogenous DNA. In nature, the acquired DNA is integrated into a main chromosome. The three major events of natural transformation are as follows:

• Binding of double-stranded DNA to receptor proteins

• Uptake of a single strand of the bound DNA

• Integration of that DNA into the genome

Transformation does not include viral infection and does not occur by cell contact.

Overview

The mode of chromosome replication varies from life form to life form and fits with chromosome structure and with the course and tempo of reproduction.

Chromosome replication begins at origins of replication and stops at termination sequences; small chromosomes typically have a single origin. Replication may be bidirectional or unidirectional. Linear chromosomes may have interior or terminal origins. Most linear chromosomes have telomeres, which are terminal sequences that are replicated in special ways. The large, linear chromosomes of eukarya are divided into many replicons (simultaneously replicated regions), each having an origin at its center.

Chromosomes made of RNA or single-stranded DNA replicate by exceptional mechanisms.

Replication of Circular Chromosomes

Theta Replication

Many circular chromosomes – bacterial chromosomes, most plasmids, and some dsDNA viral chromosomes – replicate by the theta mechanism, so named because the shape of partly replicated chromosomes resembles the Greek letter theta. Theta replication is usually bidirectional, but in some chromosomes it is unidirectional – e.g., colE1 phage.

Replication begins at the chromosome's origin (Figure 13.1). In Escherichia coli the origin is called oriC, which consists of 245 bp and contains short repeated sequences: four copies of a 9-bp sequence and three copies of a 13-bp sequence. First, about 30 copies of initiator protein (dnaA) bind to the four 9-bp sequences of oriC. DnaA helps the DNA to melt, making a single-stranded region. Helicase + dnac bind to the three 13-bp sequences and open up the double-stranded DNA (dsDNA) further, after which two primosomes are assembled and bidirectional replication starts (Figure 13.2).

Overview

The two kinds of final gene products, RNA and protein (polypeptides and peptides), are encoded polymers, and just as a gene's nucleotide sequence specifies an RNA molecule's nucleotide sequence during transcription, so does an RNA molecule's nucleotide sequence specify a polypeptide's amino acid sequence during translation, the synthesis of a polypeptide in the ribosome. The newly made polymer often undergoes enzymatic processing.

Coding in translation is less direct than in transcription, for amino acids do not pair with the nucleotides that encode them. Instead, tRNA is an intermediary, base-pairing with mRNA as it carries an amino acid to a growing polypeptide. Outside the ribosome, amino acids bond covalently to the appropriate tRNAs (aminoacylation); inside the ribosome, peptidyltransferase adds one amino acid at a time to a polypeptide chain.

This chapter breaks down protein synthesis into three phases: (1) the enzymatic aminoacylation of tRNAs; (2) translation, accomplished by ribosomes, mRNA, tRNA, and various protein factors; and (3) posttranslational processing of polypeptides (cutting, folding, covalent modification, and sometimes splicing).

Transfer of Amino Acids: A Cellular “Bucket Brigade”

A polypeptide grows by successive addition of amino acids to its carboxyl end, through the formation of peptide bonds. However, because peptides are not complementary to nucleotide chains, polypeptide synthesis requires special machinery. Each incoming amino acid is “handed off” from molecule to molecule before the peptide bond is formed. An amino acid first makes a covalent bond between its carboxyl carbon and the 5′ α phosphate of ATP.

Overview

Viruses are acellular, intracellular parasites whose reproduction depends in part on the host's genetic machinery. Viral infection may kill a cell; at the very least viruses are a burden to their host. All organisms are susceptible to viral infection, and by conservative estimate there are 108 different kinds of virus. The ubiquity, variety, and destructiveness of viruses have motivated study of their reproduction. In addition, viruses are extremely useful per se in the study of genetics (replication, mutation, recombination, and gene expression), as tools in molecular biology, medicine, and agriculture, and as “windows” into the biology of the host organisms.

This chapter summarizes viral life cycles. A virus's host and genetic material strongly influence its mode of reproduction; accordingly, the chapter is organized both by host and by genetic material – RNA or DNA, single-or double-stranded.

A Little Terminology

The (+) strand of an RNA virus genome can be translated into proteins, and the (-) strand is complementary to the (+) strand; essentially, the (+) strand is mRNA. The (+) strand of a DNA virus corresponds to the mRNA sequence, while the (-) strand is the template for RNA synthesis. The chromosome, which may be circular or linear, may contain a small amount of protein.

Bacteriophages, the Viruses of Bacteria

Bacteriophages (also simply phages) are viruses that infect bacteria. Phages consist of a chromosome contained within a protein capsid, ranging in length between 10 and 300 nm.

Overview

Genetics is a quantitative science. Many genetic processes (mutation, recombination, assortment of genes) are not deterministic but are based on random events. The outcome of a random process cannot be known; its frequency of occurrence is predicted by its probability, or expected frequency. Thus, one needs statistics, the mathematical analysis of random variables. To design an experiment in genetics and to interpret the results correctly – or at all – requires knowledge of probability and statistics.

This chapter introduces a few rules of probability and gives the bare minimum of statistics for describing genetic phenomena and for testing genetic hypotheses. It is just a little toolkit, to be hauled out when you need it.

Rules of Probability

A probability is a number between 0 and 1 that predicts the frequency of a random event; the sum of the probabilities of all possible outcomes is 1. During meiosis in a heterozygote A1/A2, a gamete is equally likely to receive A1 or A2, and the probability of each is ½. In general:

If event E happens in m of n equally likely events, then the probability of event E is P[E] = m/n.

Solved Problem. In a cross between two heterozygotes, A1/A2 × A1/A2, what is the probability of a heterozygous offspring? Because alleles segregate 1:1, half the eggs and sperm are A1 and half are A2.

Overview

An important task for geneticists is to determine individuals' genotypes – genetic make-up. Most hereditary analysis requires some knowledge about the genotypes of closely related individuals. When reproduction is purely sexual (excluding clones, twins, and the like), individuals tend to differ from each other in genomic DNA sequence. In sexually reproducing species, each individual is genetically unique. Two kinds of individual differences in DNA sequence are nucleotide substitution and variation in sequence length.

This chapter describes a few of the many approaches to genotyping. One valuable method is determining the nucleotide sequence of DNA (DNA sequencing). Other, easier, more rapid methods provide partial information about genotypes. Four such methods are described, all based on polymerase chain reaction (PCR).

Sequencing DNA

Most DNA sequencing entails synthesis of DNA by the dideoxy method, which is based on the fact that 2′,3′-dideoxynucleotides terminate DNA synthesis. The deoxyribose of DNA lacks a hydroxyl group on the 2′ sugar; dideoxyribose lacks the 3′ hydroxyl group as well. Recall that DNA chain growth is 5′→3′; the α phosphate of the incoming nucleotide makes a covalent bond with the oxygen of the 3′-OH group of the growing polymer (Figure 28.1). If the 3′ nucleotide of the polymer lacks a 3′-OH group, then a nucleotide cannot be added and chain growth stops. A dideoxynucleotide can add to a growing polynucleotide chain, but, after it is incorporated into the polymer, it blocks further DNA synthesis.

Overview

To learn anything meaningful about a gene – its identity, its effects on an organism's traits, its interaction with other genes – one must study its behavior and inheritance in vivo. It is alarming how common are the patently false beliefs that a gene is a piece of DNA divorced from the organism and that a gene's existence, characteristics, functioning, and genetic interactions can be known from molecular analysis alone.

The inheritance of genes in complex organisms has two hallmarks: (1) it is inextricably tied to sex, in which chromosomes segregate, assort, and recombine, and (2) it works according to quantitative rules. This chapter introduces the rules of inheritance and ties them to the behavior of chromosomes in sexual reproduction.

Genetic Terminology

A diploid organism's genetic makeup is its genotype; depending on the context, this can refer to a single gene or to many genes (Figure 31.1). An organism's traits are its phenotype(s). Genotype and environment both contribute to the phenotype. Variant forms of a gene (variant DNA sequences) are alleles. Functional alleles that occur in nature and make for a normal phenotype are wild type (this term is not applied to humans); abnormal alleles, especially those induced experimentally, are mutant. A gene's name usually relates to its phenotypic effects, and that name is abbreviated with one or a few letters; alleles are ideally designated by meaningful superscripts.

Overview

When a multicellular organism develops from a fertilized egg, four genetically regulated processes are at work: cell proliferation, programmed cell death, differentiation, and association of functionally related cells.

Genetic mechanisms of development are of two kinds, differential gene expression and changes in genome structure. Differential gene expression – spatial and temporal variation in the rates of synthesis of gene products – is by far the more prevalent. Gene expression, often triggered by signals from outside the cell, is regulated at the level of transcription, RNA processing, or protein synthesis.

Basic Developmental Processes

Cell Proliferation

Cell proliferation via cell cycling (Chapter 19) is universal. In plants and animals, cells proliferate when the organism grows, replaces dead cells, metamorphoses (remodels the body during development), or regenerates lost body parts. In the first 270 days or so of human development, cell number increases exponentially from 1 to ~1014; the rate of cell proliferation far exceeds the rate of cell death. By conservative estimate, 99.9% of human cells die and are replaced, making a person's lifetime cell number ~1017, although the true figure may be orders of magnitude greater than this.

The G1→S transition, blocked by RB-E2F, is critical to cell proliferation (Chapter 19); once a cell passes the R point it is committed to enter S phase, and passage through a full cycle normally occurs. External signaling molecules influence the G1→S transition: mitogens stimulate the transition and growth inhibitors block it.

Overview

The essence of sex is alternation between a haploid phase, when cell nuclei possess one set of chromosomes, and a diploid phase, when they possess two. Each set of chromosomes is homologous and usually nonidentical, having come from unrelated haploid sex cells. The transition from diploid to haploid requires meiosis, a process in which precisely one copy of each chromosome is apportioned to each haploid cell.

The events of meiosis determine quantitative, predictable patterns of genetic transmission from parent to offspring in sexual species. Two hallmarks of meiosis are the 1:1 segregation of gene copies and recombination of genes and chromosomes. In recombination, chromosomes and chromosome segments shuffle to make a virtually limitless number of new genetic combinations.

The first part of the chapter describes meiosis as a formal dance of chromosomes. The second part of the chapter explains the genetic consequences of meiosis – the segregation of homologous chromosomes and recombination. The third part of the chapter describes exceptional patterns of meiosis.

Recap of Ploidy and DNA Content

To recap what was explained in Chapter 19, for any eukaryon with sexual reproduction the haploid number of nuclear chromosomes is N and the diploid number is 2N; in a diploid cell the two sets of chromosomes are nonidentical and homologous. In asexually reproducing cells, chromosome number is constant through the life cycle. In sexual organisms, gametes (sperm and eggs, or their equivalents) are haploid, while zygotes (cells formed by the union of gametes) are diploid.