Molecular Genetics 2: Chromosomes
Learning Objectives
- Explain how DNA is packaged into chromosomes through the hierarchical organisation of chromatin
- Compare genome organisation and chromosome structure in eukaryotes, prokaryotes, and viruses
- Describe the structural features of chromosomes and explain the functions of chromatin, centromeres, telomeres, and homologous chromosomes
- Explain chromosomal ploidy and the role of meiosis and fertilisation in maintaining chromosome number across generations
- Evaluate the biological and clinical significance of chromosomal abnormalities, including aneuploidy and chromosomal rearrangements
- Describe the principles and applications of chromosomal testing techniques used in genetic diagnosis, prenatal screening, and biomedical research
Introduction
The gene is the physical unit of inheritance, and genes are arranged in a linear order on chromosomes. Chromosome behaviour and interaction during meiosis explain, at a cellular level, inheritance patterns that we observe in populations. Genetic disorders involving alterations in chromosome number or structure may have dramatic effects and can prevent a fertilised egg from developing.
DNA to Chromosomes: Structural Mechanisms
The genome of a cell is often significantly larger than the cell itself. For example, if the DNA from a human cell containing 46 chromosomes were stretched out in a line, it would extend more than 2 meters. How is it possible that the genetic information not only fits into the cell but fits into the cell nucleus? Eukaryotes solve this problem by a combination of supercoiling and packaging DNA around the histone family of proteins. Prokaryotes do not contain histones (with a few exceptions). Prokaryotes tend to compress their DNA using nucleoid-associated-proteins (NAPs) and supercoiling.
DNA supercoiling refers to the over- or under-winding of a DNA strand and is an expression of the strain on that strand. Supercoiling is important in a number of biological processes, such as compacting DNA and regulating access to the genetic code. DNA supercoiling strongly affects DNA metabolism and possibly gene expression.
Additionally, certain enzymes such as topoisomerases can change DNA topology to facilitate functions such as DNA replication or transcription.
In a “relaxed” double-helical segment of B-DNA, the two strands twist around the helical axis once every 10.4–10.5 base pairs of sequence. Adding or subtracting twists, as some enzymes can do, impose strain. If a DNA segment under twist strain were closed into a circle by joining its two ends and then allowed to move freely, the circular DNA would contort into a new shape, such as a simple figure-eight, as shown in Figure 8.2. Such a contortion is a supercoil. The noun form “supercoil” is often used in the context of DNA topology.
Positively supercoiled (overwound) DNA is transiently generated during DNA replication and transcription, and, if not promptly relaxed, inhibits (regulates) these processes. The simple figure eight is the simplest supercoil and is the shape a circular DNA assumes to accommodate one too many or one too few helical twists. The two lobes of the figure eight will appear rotated either clockwise or counterclockwise with respect to one another, depending on whether the helix is over- or underwound. For each additional helical twist being accommodated, the lobes will show one more rotation about their axis. As a general rule, the DNA of most organisms is negatively supercoiled.
Lobal contortions of a circular DNA, such as the rotation of the figure eight lobes above, are referred to as writhe. The above example illustrates that twisting and writhing are interconvertible. Supercoiling can be represented mathematically by the sum of twist and writhe, The twist is the number of helical turns in the DNA and the writhe is the number of times the double helix crosses over on itself (these are the supercoils). Extra helical twists are positive and lead to positive supercoiling, while subtractive twisting causes negative supercoiling. Many topoisomerase enzymes sense supercoiling and either generate or dissipate it as they change DNA topology.
In part, because chromosomes may be very large, segments in the middle may act as if their ends are anchored. As a result, they may be unable to distribute excess twist to the rest of the chromosome or to absorb twist to recover from underwinding — the segments may become supercoiled. In response to supercoiling, they will assume an amount of writhe, just as if their ends were joined.
Supercoiled circular DNA forms two major structures; a plectoneme or a toroid, or a combination of both. A negatively supercoiled DNA molecule will produce either a one-start left-handed helix, the toroid, or a two-start right-handed helix with terminal loops, the plectoneme. Plectonemes are typically more common in nature, and this is the shape most bacterial plasmids will take. For larger molecules, it is common for hybrid structures to form – a loop on a toroid can extend into a plectoneme as shown in Figure 8.3. DNA supercoiling is important for DNA packaging within all cells and seems to also play a role in gene expression.
In addition to forming supercoiled structures, circular chromosomes from bacteria have been shown to undergo the processes of catenation and knotting upon the inhibition of topoisomerase enzymes. Catenation is the process by which two circular DNA strands are linked together like chain links, whereas DNA knotting is the inter-looping structures occurring within a single circular DNA structure. These are illustrated in Figure 8.4 below. In vivo, the action of topoisomerase enzymes is critical to keep knots and catenoids from tangling the DNA structure.
Histones and Nucleosomes
Within eukaryotic chromosomes, chromatin proteins, known as histones, compact and organise DNA. These compacting structures guide the interactions between DNA and other proteins, helping control which parts of the DNA are transcribed.
Histones are highly basic proteins found in eukaryotic cell nuclei that package and order the DNA into structural units called nucleosomes. They are the chief protein components of chromatin, acting as spools around which DNA winds, and playing a role in gene regulation. Without histones, the unwound DNA in chromosomes would be very long (a length-to-width ratio of more than 10 million to 1 in human DNA). For example, each human diploid cell (containing 23 pairs of chromosomes) has about 1.8 meters of DNA wound on the histones, and the diploid cell has about 90 micrometers (0.09 mm) of chromatin.
There are five major families of histones, H1/H5, H2A, H2B, H3, and H4. Histones H2A, H2B, H3, and H4 are known as the core histones, while histones H1/H5 are known as the linker histones.
The core histones all exist as dimers, which are similar in that they all possess the histone fold domain: three alpha helices linked by two loops. It is this helical structure that allows for interaction between distinct dimers, particularly in a head-tail fashion (also called the handshake motif). The resulting four distinct dimers then come together to form one octameric nucleosome core, approximately 63 Angstroms in diameter. Around 146 base pairs (bp) of DNA wrap around this core particle 1.65 times in a left-handed super-helical turn to give a particle of around 100 Angstroms across, called a nucleosome as illustrated in Figure 8.5.
The linker histone H1 binds the nucleosome at the entry and exit sites of the DNA, thus locking the DNA into place and allowing the formation of higher order structure. The most basic such formation is the 10 nm fibre or beads on a string conformation. This involves the wrapping of DNA around nucleosomes with approximately 50 base pairs of DNA, separating each pair of nucleosomes (also referred to as linker DNA).
The nucleosome contains over 120 direct protein-DNA interactions and several hundred water-mediated ones. Direct protein–DNA interactions are not spread evenly about the octamer surface but rather located at discrete sites. These are due to the formation of two types of DNA binding sites within the octamer; the α1α1 site, which uses the α1 helix from two adjacent histones, and the L1L2 site formed by the L1 and L2 loops. Salt links and hydrogen bonding between both side-chain basic and hydroxyl groups and main-chain amides with the DNA backbone phosphates form the bulk of interactions with the DNA. This is important, given that the ubiquitous distribution of nucleosomes along genomes requires it to be a non-sequence-specific DNA-binding factor. Although nucleosomes tend to prefer some DNA sequences over others, they are capable of binding practically to any sequence, which is thought to be due to the flexibility in the formation of these water-mediated interactions.
In addition, non-polar interactions are made between protein side-chains and the deoxyribose groups, and an arginine side-chain intercalates into the DNA minor groove at all 14 sites where it faces the octamer surface. The spatial distribution and strength of DNA-binding sites about the octamer surface distort the DNA within the nucleosome core. The DNA is non-uniformly bent and also contains twist defects. The twist of free B-form DNA in solution is 10.5 bp per turn. However, the overall twist of nucleosomal DNA is only 10.2 bp per turn, varying from a value of 9.4 to 10.9 bp per turn.
The histone tail extensions constitute up to 30% by mass of histones but are not visible in the crystal structures of nucleosomes due to their high intrinsic flexibility and have been thought to be largely unstructured. The N-terminal tails of histones H3 and H2B pass through a channel formed by the minor grooves of the two DNA strands, protruding from the DNA every 20 bp. The N-terminal tail of histone H4, on the other hand, has a region of highly basic amino acids (16-25), which, in the crystal structure, forms an interaction with the highly acidic surface region of an H2A-H2B dimer of another nucleosome, being potentially relevant for the higher-order structure of nucleosomes. This interaction is thought to occur under physiological conditions also and suggests that acetylation of the H4 tail distorts the higher-order structure of chromatin.
The packing of DNA from dsDNA to the metaphase chromosomes is schematically shown in Figure 8.6. The formation of the DNA double helix represents the first-order packaging of the chromosome structure. The formation of nucleosomes represents the second level of packaging for eukaryotic chromosomes. In vitro data suggests that nucleosomes are then arranged into either a solenoid structure which consists of 6 nucleosomes linked together by the Histone H1 linker proteins or a zigzag structure that is similar to the solenoid construct. Both the solenoid and zigzag structures are approximately 30 nm in diameter. The solenoid and zigzag structures reported from in vitro data have not yet been confirmed to occur in vivo.
Chromatin Structure
During interphase, distinct chromosomes as shown in Figure 8.6 are not observed. Rather each chromosome occupies a spatially limited, roughly elliptical domain which is known as a chromosome territory (CT). Each chromosome territory is comprised of higher-order chromatin units of ~1 Mb each. These units are likely built up from smaller loop domains that contain the solenoid/zigzag structural motifs. On the other hand, 1Mb domains can themselves serve as smaller units in higher-order chromatin structures. With the development of high-throughput biochemical techniques, such as 3C (chromosome conformation capture) and 4C (chromosome conformation capture-on-chip and circular chromosome conformation capture), numerous spatial interactions between neighbouring chromatin territories have been described as shown in Figure 8.7.
Chromosome territories are known to be arranged radially around the nucleus. This arrangement is both cell and tissue-type specific and is also evolutionarily conserved. The radial organisation of chromosome territories was shown to correlate with their gene density and size. In this case, the gene-rich chromosomes occupy interior positions, whereas larger, gene-poor chromosomes, tend to be located around the periphery. Chromosome territories are also dynamic structures, with genes able to relocate from the periphery towards the interior once they have been ‘switched on’. In other cases, genes may move in the opposite direction or simply maintain their position.
DNA Forms
A cell’s DNA, packaged as a double-stranded DNA molecule, is called its genome. In eukaryotes, the genome consists of several double-stranded linear DNA molecules (Figure 8.6). Each species of eukaryotes has a characteristic number of chromosomes in the nuclei of its cells. Human body (somatic) cells have 46 chromosomes, while human gametes (sperm or eggs) have 23 chromosomes each. A typical body cell contains two matched or homologous sets of chromosomes (one set from each biological parent)—a configuration known as diploid. (Note: The letter n is used to represent a single set of chromosomes; therefore, a diploid organism is designated 2n.) Human cells that contain one set of chromosomes are called gametes, or sex cells; these are eggs and sperm, and are designated 1n, or haploid.
In prokaryotes, the genome is composed of a single, double-stranded DNA molecule in the form of a loop or circle (Figure 8.8). The region in the cell containing this genetic material is called a nucleoid. Some prokaryotes also have smaller loops of DNA called plasmids that are not essential for normal growth. Bacteria can exchange these plasmids with other bacteria, sometimes receiving beneficial new genes that the recipient can add to their chromosomal DNA. Antibiotic resistance is one trait that often spreads through a bacterial colony through plasmid exchange from resistant donors to recipient cells.
Prokaryotic organisms are single-celled organisms that lack a cell nucleus, and their DNA therefore floats freely in the cell cytoplasm. To synthesise a protein, the processes of transcription and translation occur almost simultaneously. When the resulting protein is no longer needed, transcription stops. As a result, the primary method to control what type of protein and how much of each protein is expressed in a prokaryotic cell is the regulation of DNA transcription (Figure 8.9). All subsequent steps occur automatically. When more protein is required, more transcription occurs. Therefore, in prokaryotic cells, the control of gene expression is mostly at the transcriptional level.
The DNA of prokaryotes is organised into a circular chromosome, supercoiled within the nucleoid region of the cell cytoplasm. Genes for proteins that are needed for a specific function, or that are involved in the same biochemical pathway, are clustered together in blocks called operons. For example, all of the genes needed to use lactose as an energy source are coded next to each other in the lactose (or lac) operon, and transcribed into a single mRNA.
Viruses typically have a simple structure: a protein coat, called the capsid, and a nucleic acid core that contains the genetic material (either DNA or RNA). The bacteriophage infects the host bacterial cell by attaching to its surface, and then it injects its nucleic acids inside the cell. The phage DNA makes multiple copies of itself using the host machinery, and eventually the host cell bursts, releasing a large number of bacteriophages.
Unlike nearly all living organisms that use DNA as their genetic material, viruses may use either DNA or RNA. The virus core contains the genome — the total genetic content of the virus. Viral genomes tend to be small, containing only those genes that encode proteins which the virus cannot get from the host cell. This genetic material may be single- or double-stranded. It may also be linear or circular. While most viruses contain a single nucleic acid, others have genomes divided into several segments. The RNA genome of the influenza virus is segmented, which contributes to its variability and continuous evolution, and explains why it is difficult to develop a vaccine against it.
In DNA viruses, the viral DNA directs the host cell’s replication proteins to synthesise new copies of the viral genome and to transcribe and translate that genome into viral proteins. Human diseases caused by DNA viruses include chickenpox, hepatitis B, and adenoviruses. Sexually transmitted DNA viruses include the herpes virus and the human papilloma virus (HPV), which has been associated with cervical cancer and genital warts.
RNA viruses contain only RNA as their genetic material. To replicate their genomes in the host cell, the RNA viruses must encode their own enzymes that can replicate RNA into RNA or, in the retroviruses, into DNA. These RNA polymerase enzymes are more likely to make copying errors than DNA polymerases, and therefore often make mistakes during transcription. For this reason, mutations in RNA viruses occur more frequently than in DNA viruses. This causes them to change and adapt more rapidly to their host. Human diseases caused by RNA viruses include influenza, hepatitis C, measles, and rabies. The HIV virus, which is sexually transmitted, is an RNA retrovirus.
Chromosomal Structure
Within eukaryotic cells, DNA is organised into long linear structures called chromosomes. A chromosome is a “thread-like” structure in the nucleus of animal and plant that consists of a single but long molecule of double-stranded DNA (so it’s two ss-DNAs) with a myriad of bound proteins. The proteins bind to and condense the DNA molecule to prevent it from becoming an unmanageable tangle.
Before typical cell division (mitosis), these chromosomes are replicated by DNA polymerase to make two identical chromosomes, one for each future daughter cell. The two identical chromosomes, called sister chromatids, bind to each other at a common structure called the centromere.
Each sister chromatid develops a protein structure called a kinetochore in its centromeric region (Figure 8.11). The proteins of the kinetochore attract and bind to the mitotic spindle microtubules during prometaphase.
Telomeres
At the ends of the linear eukaryotic chromosomes are specialised regions of DNA called telomeres. The main function of these regions is to allow the cell to replicate chromosome ends using the enzyme telomerase, as the enzymes that normally replicate DNA cannot copy the extreme 3′ ends of chromosomes (Figure 8.12).
These specialised chromosome caps also help protect the DNA ends and stop the DNA repair systems in the cell from treating them as damage to be corrected. In human cells, telomeres contain 300-8000 repeats of a simple TTAGGG sequence. The repetitive TTAGGG sequences in telomeric DNA can form unique higher-order structures called quadruplexes.
During DNA replication, the double-stranded DNA is unwound and DNA polymerase synthesises new strands. However, as DNA polymerase moves in a unidirectional manner (from 5’ to 3’), only the leading strand can be replicated continuously. For the complementary lagging strand, DNA replication is discontinuous. In humans, small RNA primers attach to the lagging strand DNA, and the DNA is synthesised in small 5′-3′ stretches of about 100-200 nucleotides, which are termed Okazaki fragments. The RNA primers are removed and replaced with DNA and the Okazaki DNA fragments are ligated together. At the end of the lagging strand, it is impossible to attach an RNA primer, meaning that there will be a small amount of DNA lost each time the cell divides. This ‘end replication problem’ has serious consequences for the cell as it means the DNA sequence cannot be replicated correctly, with the loss of genetic information. Hence most telomeres have 3´ overhangs. Bacteria DNA, which is circular, does not have the problem.
To prevent this, telomeres are repeated hundreds to thousands of times at the end of the chromosomes. Each time cell division occurs, a small section of telomeric sequences is lost to the end replication problem, thereby protecting the genetic information. At some point, the telomeres become critically short. This decrease leads to cell senescence, where the cell is unable to divide, or apoptotic cell death. Telomeres are the basis for the Hayflick limit, the number of times a cell can divide before reaching senescence.
Telomeres can be restored by the enzyme telomerase, which extends the telomere’s length. Telomerase activity is found in cells that undergo regular division, such as stem cells and lymphocyte cells of the immune system. The enzyme has two major subunits.
One is the catalytic enzyme named telomerase reverse transcriptase (TERT), which is an RNA-dependent DNA polymerase. Almost all DNA polymerases use DNA as a template for replication. Some RNA viruses that use RNA as their genetic information like HIV encode their own reverse transcriptase, which directs the polymerisation of a DNA copy of the viral RNA genome as part of its life cycle. The virus that causes the Covid-19 pandemic is called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It is also an RNA virus but in contrast to HIV it uses and encodes an RNA-dependent RNA polymerase.
The second subunit of telomerase is the telomerase RNA (TR), which contains a template from which the new telomer is made. The enzyme makes many DNA copies from this to create a multitude of DNA repeat in the telomeres. Telomeres can also be extended through the Alternative Lengthening of Telomeres (ALT) pathway. In this case, rather than being extended, telomeres are switched between chromosomes by homologous recombination. As a result of the telomere swap, one set of daughter cells will have shorter telomeres, and the other set will have longer telomeres.
A downside to telomere extension is the potential for uncontrolled cell division and cancer. Abnormally high telomerase activity has been found in the majority of cancer cells, and non-telomerase tumours often exhibit ALT pathway activation. As well as the potential for losing genetic information, cells with short telomeres are at a higher risk for improper chromosome recombination, which can lead to genetic instability and aneuploidy (an abnormal number of chromosomes).
Heterochromatin and Euchromatin
When stained and viewed in a microscope, eukaryotic nuclear DNA in nondividing cells is observed in two different states, heterochromatin (dark areas) and euchromatin (light areas), as shown in Figure 8.13
The heterochromatin is darkly stained and found along the inner side of the nuclear envelope as well as inside the nucleus. Euchromatin doesn’t stain well since it is more dispersed and is found throughout the nucleus. The image above is of a cell in interphase, a part of the cell cycle in which the cell is in-between cell divisions. Here are some differences between heterochromatin and euchromatin:
Heterochromatin:
- contains genes that are transcriptionally inactive and not expressed;
- is not found in prokaryotic cells;
- in humans, one of the X chromosomes is silenced in heterochromatin while the other is transcribable and in euchromatin;
- protects the tightly packed in it from nucleases.
Euchromatin:
- contains about 90% of genome DNA;
- contains chromosomes that are unfolded into nucleosomal DNA resembling beads on a string and is characterised by looser association with packaging histone proteins;
- is the only form of DNA in bacteria;
- is transcriptionally active so it is open to the binding of RNA polymerase and transcription factors;
- has some genes which can be transcriptionally silenced by moving them into heterochromatin.
Homologous Chromosomes
Upon fertilisation, each gamete contributes one set of chromosomes, creating a diploid cell containing matched pairs of chromosomes called homologous (“same knowledge”) chromosomes. Homologous chromosomes are the same length and have specific nucleotide segments called genes in exactly the same location, or locus. Genes, the functional units of chromosomes, determine specific characteristics by coding for specific proteins. Traits are the variations of those characteristics. For example, hair colour is a characteristic with traits that are blonde, brown, or black, and many colours in between.
Each copy of a homologous pair of chromosomes originates from a different parent; therefore, the different genes (alleles) themselves are not identical, although they code for the same traits such as “hair colour.” The variation of individuals within a species is due to the specific combination of the genes inherited from both parents. Even a slightly altered sequence of nucleotides within a gene can result in an alternative trait.
For example, there are three possible gene sequences on the human chromosome that code for blood type: sequence A, sequence B, and sequence O. Because all diploid human cells have two copies of the chromosome that determines blood type, the blood type (the trait) is determined by the two alleles of the marker gene that are inherited. It is possible to have two copies of the same gene sequence on both homologous chromosomes, with one on each (for example, AA, BB, or OO), or two different sequences, such as AB, AO, or BO.
Apparently minor variations of traits, such as blood type, eye colour, and handedness, contribute to the natural variation found within a species, but even though they seem minor, these traits may be connected with the expression of other traits as of yet unknown. However, if the entire DNA sequence from any pair of human homologous chromosomes is compared, the difference is much less than one percent.
The sex chromosomes, X and Y, are the single exception to the rule of homologous chromosome uniformity: Other than a small amount of homology that is necessary to accurately produce gametes, the genes found on the X and Y chromosomes are different.
Chromosomal Ploidy: Numbers
Sexual reproduction requires fertilisation, a union of two cells from two individual organisms. If those two cells each contain one set of chromosomes, then the resulting cell contains two sets of chromosomes. The number of sets of chromosomes in a cell is called its ploidy level. Haploid cells contain one set of chromosomes. Cells containing two sets of chromosomes are called diploid. If the reproductive cycle is to continue, the diploid cell must somehow reduce its number of chromosome sets before fertilisation can occur again, or there will be a continual doubling in the number of chromosome sets in every generation. So, in addition to fertilisation, sexual reproduction includes a nuclear division, known as meiosis, that reduces the number of chromosome sets.
Most animals and plants are diploid, containing two sets of chromosomes; in each somatic cell (the nonreproductive cells of a multicellular organism), the nucleus contains two copies of each chromosome that are referred to as homologous chromosomes. Somatic cells are sometimes referred to as “body” cells. Homologous chromosomes are matched pairs containing genes for the same traits in identical locations along their length. Diploid organisms inherit one copy of each homologous chromosome from each parent; all together, they are considered a full set of chromosomes. In animals, haploid cells containing a single copy of each homologous chromosome are found only within gametes. Gametes fuse with another haploid gamete to produce a diploid cell.
The nuclear division that forms haploid cells, which is called meiosis, is related to mitosis. As you have learned, mitosis is part of a cell reproduction cycle that results in identical daughter nuclei that are also genetically identical to the original parent nucleus. In mitosis, both the parent and the daughter nuclei contain the same number of chromosome sets — diploid for most plants and animals. Meiosis employs many of the same mechanisms as mitosis. However, the starting nucleus is always diploid and the nuclei that result at the end of a meiotic cell division are haploid. To achieve the reduction in chromosome number, meiosis consists of one round of chromosome duplication and two rounds of nuclear division. Because the events that occur during each of the division stages are analogous to the events of mitosis, the same stage names are assigned. However, because there are two rounds of division, the stages are designated with a “I” or “II.” Thus, meiosis I is the first round of meiotic division and consists of prophase I, prometaphase I, and so on. Meiosis I reduces the number of chromosome sets from two to one. The genetic information is also mixed during this division to create unique recombinant chromosomes. Meiosis II, in which the second round of meiotic division takes place in a way that is similar to mitosis, includes prophase II, prometaphase II, and so on.
In humans, each cell normally contains 23 pairs of chromosomes, for a total of 46. In twenty-two of these pairs, called autosomes, each member of the pair is similar. The 23rd pair, the sex chromosomes, differ between males and females. Females have two copies of the X chromosome, while males have one X and one Y chromosome. Figure 8.15 shows a DNA karyotype with 22 pairs of autosomes and one pair of sex chromosomes. Karyotypes are prepared using standardised staining procedures that reveal characteristic structural features for each chromosome, usually from white blood cells. The karyotype of human cells shown in Figure 8.16 below shows an extra copy of chromosome 21 (trisomy 21), which causes Down’s Syndrome.
Each species has a unique chromosomal complement. For example, chickens have 39 pairs of chromosomes (78 total) with 38 autosomal pairs and one pair of sex chromosomes (Z and W). In this species, ZW chickens are female and ZZ chickens are male. The total length of the human genome is over 3 billion base pairs. The total length of the human genome is over 3 billion base pairs. The genome also includes mitochondrial DNA.
Chromosomal sex-determination systems
Mammalian sex is determined genetically by the presence of X and Y chromosomes. Individuals homozygous for X (XX) are female, while heterozygous individuals (XY) are male. The presence of a Y chromosome causes the development of male characteristics, while its absence results in the development of female characteristics. The XY system is also found in some insects and plants (Figure 8.18).
Avian sex determination is dependent on the presence of Z and W chromosomes (Figure 8.17). Homozygous for Z (ZZ) results in a male, while heterozygous (ZW) results in a female. The W appears to be essential in determining the sex of the individual, similar to the Y chromosome in mammals. Some fish, crustaceans, insects (such as butterflies and moths), and reptiles use this system.
X and Y sex chromosomes
The XX/XY sex-determination system is likely the most familiar, as it is found in humans. The XX/XY system is found in most other mammals, as well as some insects. In this system, most females have two of the same kind of sex chromosome (XX), while most males have two distinct sex chromosomes (XY). The X and Y sex chromosomes are different in shape and size from each other, unlike the rest of the chromosomes (autosomes), and are sometimes called allosomes. In some species, such as humans, organisms remain sex indifferent for a period of time after fertilisation; in others, however, such as fruit flies, sexual differentiation occurs as soon as the egg is fertilised.
Some species, such as fruit flies, use the presence of two X chromosomes to determine femaleness, called the X-centered sex determination system. Species that use the number of Xs to determine sex are nonviable with an extra X chromosome.
In the XX/X0 sex chromosome system, a variant of the XY system, females have two copies of the sex chromosome (XX) but males have only one (X0). The 0 denotes the absence of a second sex chromosome. Generally in this method, the sex is determined by amount of genes expressed across the two chromosomes. This system is observed in a number of insects, including the grasshoppers and crickets of order Orthoptera and in cockroaches (order Blattodea). A small number of mammals also lack a Y chromosome. These include the Amami spiny rat (Tokudaia osimensis) and the Tokunoshima spiny rat (Tokudaia tokunoshimensis) and Sorex araneus, a shrew species. Transcaucasian mole voles (Ellobius lutescens) also have a form of XO determination, in which both sexes lack a second sex chromosome. This mechanism of sex determination is not currently well-understood.
ZW/ZZ sex chromosomes
The ZW sex-determination system is found in birds, some reptiles, and some insects and other organisms. The ZW sex-determination system is reversed compared to the XY system: females have two different kinds of chromosomes (ZW), and males have two of the same kind of chromosomes (ZZ). Like the X/Y system, there are variations on this system.
For example, there are moths and butterflies that are ZW, but some have been found female with Z0, as well as female with ZZW. Also, while mammals deactivate one of their extra X chromosomes when female, it appears that in the case of Lepidoptera (butterflies and moths), the males produce double the normal amount of enzymes, due to having two Z chromosomes. Because the use of ZW sex determination is varied, it is still unknown how exactly most species determine their sex.
Despite the similarities between the ZW and XY systems, these sex chromosomes evolved separately. In the case of the chicken, their Z chromosome is more similar to humans’ autosome 9. The chicken’s Z chromosome also seems to be related to the X chromosome of the platypus. When a ZW species, such as the Komodo dragon, reproduces parthenogenetically (asexually), usually only males are produced. This is due to the fact that the haploid eggs double their chromosomes, resulting in ZZ or WW. The ZZ become males, but the WW are not viable and are not brought to term.
In both XY and ZW sex determination systems, the sex chromosome carrying the critical factors is often significantly smaller, carrying little more than the genes necessary for triggering the development of a given sex.
Down Syndrome
The most common chromosome number abnormality is trisomy-21 or, as it is more commonly known, Down syndrome. Having an extra copy of the smallest human chromosome, chromosome 21, causes substantial health problems. It is present in about 1 in 800 births. Infants with this condition have three copies of chromosome 21 rather than the normal two. Don’t confuse trisomy - having three copies of one chromosome (i.e. 2n+1) with triploidy – having three entire chromosome sets (3x; see Section 2.6.) Females with trisomy-21 are 47,XX,+21 while males are 47,XY,+21. In general, people with Down syndrome are 47,sex,+21 where the word ‘sex’ signifies that the sex chromosomes may be XX or XY.
Trisomy-21 may arise from a nondisjunction event during meiosis in either parent or during mitosis very early during embryogenesis. However, most cases are due to a first division non-disjunction event occurring in the female parent (Figure 8.18).
Having an extra copy of the smallest human chromosome, chromosome 21, causes substantial health problems. People with Down syndrome have various degrees of intellectual disability and often have other health problems such as heart defects. The disease was first described by John Down in 1866 but it was not until 1959 when its chromosomal basis was discovered. Current research suggests that at least some of the mental problems are due to having three copies of the DYRK gene on chromosome 21. This gene is active in the brain and there is evidence from humans and from mice that neurons are damaged if there is too much DYRK protein synthesised.
XYY and XXX
While foetuses trisomic for one of the other larger autosomes seldom survive to term, the situation is quite different for the sex chromosomes. Approximately 1 in 1000 males have an extra Y chromosome and yet most are unaware of it! There is little harm in having two Y chromosomes because they have relatively few genes. Similarly, 1 in 1000 females have an extra X chromosome. This situation is also relatively harmless although for a different reason. Normally in female mammals one of the two X chromosomes is inactivated in each cell so that there can be genetic balance (Figure 8.19). In 46,XX females one of the X chromosomes is inactivated while in 47,XXX females two are inactivated.
Turner Syndrome
Monosomy (2n-1) for autosomal chromosomes does occur at conception, but these embryos almost never survive to term. Similarly, embryos that are 45,Y are also non-viable because they lack many essential genes found on the X chromosome. The only viable monosomy in humans is 45,X, also known as Turner syndrome. These people are phenotypically female because they do not have a Y chromosome. They are viable because in females only one X is active in most cells anyway. People with this condition do have health problems though: they are shorter than average, they have an elevated risk of heart defects, and they are infertile.
The reason for the health problems is that there are a few genes that are found on both the X and the Y chromosome. Because these genes are found in two copies in both XY males and XX females they are in what is called the pseudoautosomal region. This region escapes X chromosome inactivation. One of the genes in this region is called SHOX. It makes a protein that promotes bone growth. 46,XX and 46,XY people have two functioning copies and have average height.
People with 47,XYY and 47,XXX genomes have three copies and are taller than average. And people with 45,X have one copy and are short. It is the single copy of SHOX and a few of the other genes in the pseudoautosomal region that causes health problems for women with Turner syndrome.
The reason for the infertility is that the X chromosome inactivation system only acts in somatic cells – it is not needed in the germline cells. Ovaries naturally have two functional X chromosomes. Women with Turner syndrome cannot perform oogenesis because this process only works if there are two active X chromosomes. Recently, it has become possible for these women to become pregnant with donated eggs and in vitro fertilisation.
Klinefelter Syndrome
There are four common sex-chromosome aneuploidies: 47,XYY, 47,XXX, 45,X, and 47,XXY. This last situation is known as Klinefelter syndrome. These people are male (because they have a Y chromosome) and tall (because they have three SHOX genes). They do not have health problems because the X chromosome inactivation system is independent of sex. In the embryonic nuclei the X chromosome are counted and all but one are shut down. It does not matter whether the embryo is male or female. Men with Klinefelter syndrome have a Barr bodies in their nuclei, the same as 46,XX females. They do have fertility problems because there are two active X chromosome in their testes and this interferes with spermatogenesis. They make enough sperm to conceive children using intracytoplasmic sperm injection though.
Cri-du-Chat Syndrome
Cri-du-chat syndrome occurs when a child inherits a defective chromosome 5 from one parent (Figure 8.20). This condition is rare – it is present in only 1 in 20,000 to 1 in 50,000 births but it does account for 1% of cases of profound intellectual disability. The specific defect is a deletion that removes 2 Mb or more from the tip of the short arm of the chromosome. In most cases the deletion is the result of a chromosomal rearrangement in one of the parent’s germ line cells. People with cri-du-chat have a karyotype of 46,sex, deletion.
As with Down syndrome this condition is associated with intellectual disability and other health problems. These problems include an improperly formed larynx which leads to infants making high pitched cat-like crying sounds (hence the name “cry of the cat”). It is suspected that at least some of the intellectual disability phenotype is due to having only a single copy of the CTNND2 gene. This gene is active during embryogenesis and makes a protein essential for neuron migration.
Down syndrome and cri-du-chat syndrome are two examples of the need for genomes to contain the proper number of genes. Having too many copies of key genes (Down syndrome) or too few (cri-du-chat syndrome) can lead to substantial developmental problems.
Chromosomal Testing
Many tests are now available to detect genetic diseases such as sickle cell disease, cystic fibrosis and phenylketonuria (PKU). Most of these tests can not only be performed on cells removed from adults but also on cells removed from the foetus and even from a pre-implantation embryo.
When visualising a karyotype, the chromosomal images are aligned so that heterologous chromosomes are paired together and positioned such that the p-arm (short arm) is on top and the q-arm (long arm) points downward. Karyotypes can be used to quickly identify gross chromatic abnormalities that are larger than a few megabases in difference. This includes abnormalities such as aneuploidy (the addition or absence of an entire chromosome), or translocations (the transfer of part of a chromosome to a neighbouring chromosome), as shown in Figures 8.21 and 8.22.
The telomeric regions of chromosomes can also be identified using fluorescent staining techniques (Figure 8.22).
More recently, techniques such as chromosome painting, use fluorescently labelled probes to hybridise with specific chromosomes or even specific gene regions of a chromosome. Karyotypes originating from this technique are called spectral karyotypes. This technique can be especially useful in identifying translocations that have occurred in human cells.
Amniocentesis
During its development, the foetus sheds cells into the amniotic fluid. After 14–22 weeks of pregnancy, a small volume of this fluid can be removed (using a needle inserted through the abdominal wall).
Using ultrasound to locate the position of the placenta prior to amniocentesis. These sonograms are made by recording the echoes received from structures within the abdomen. A, amniotic cavity; B, urinary bladder; F, part of the foetus; P, placenta. Both longitudinal (left) and transverse (right) scans are needed for accurate localisation of the placenta. (Courtesy of the Downstate Medical Center of the State University of New York).
Separating the cells and culturing them enables the clinician to look for
- chromosome abnormalities (e.g., the three number 21 chromosomes of Down syndrome)
- certain enzymatic defects (e.g., an inability to metabolise galactose, hence milk)
- the sex of the foetus
Over 100 genetic abnormalities can be diagnosed by amniocentesis and the pregnancy deliberately ended if the parents wish it.
Chorionic Villus Sampling (CVS)
This is an alternate method of prenatal diagnosis. A small amount of placental tissue is sucked out by a tube inserted through the abdominal wall or through the vagina (the latter avoiding the need for an incision). For some tests the foetal cells can be examined immediately without the need to culture them. Another advantage of CVS is that it can be performed earlier in pregnancy (after only 10–12 weeks) than amniocentesis. If an abortion is to be performed, it is a simpler process early in pregnancy.
Real-World Applications
Geneticists Use Karyograms to Identify Chromosomal Aberrations
The karyotype is a method by which traits characterised by chromosomal abnormalities can be identified from a single cell. To observe an individual’s karyotype, a person’s cells (like white blood cells) are first collected from a blood sample or other tissue. In the laboratory, the isolated cells are stimulated to begin actively dividing. A chemical is then applied to the cells to arrest mitosis during metaphase. The cells are then fixed to a slide.
The geneticist then stains chromosomes with one of several dyes to better visualise the distinct and reproducible banding patterns of each chromosome pair. Following staining, chromosomes are viewed using bright-field microscopy. An experienced cytogeneticist can identify each band. In addition to the banding patterns, chromosomes are further identified on the basis of size and centromere location.
To obtain the classic depiction of the karyotype in which homologous pairs of chromosomes are aligned in numerical order from longest to shortest, the geneticist obtains a digital image, identifies each chromosome, and manually arranges the chromosomes into this pattern (Figure 8.15).
At its most basic, the karyogram may reveal genetic abnormalities in which an individual has too many or too few chromosomes per cell. Examples of this are Down syndrome, which is identified by a third copy of chromosome 21, and Turner syndrome, which is characterised by the presence of only one X chromosome in women instead of two. Geneticists can also identify large deletions or insertions of DNA.
For instance, Jacobsen syndrome, which involves distinctive facial features as well as heart and bleeding defects, is identified by a deletion on chromosome 11. Finally, the karyotype can pinpoint translocations, which occur when a segment of genetic material breaks from one chromosome and reattaches to another chromosome or to a different part of the same chromosome. Translocations are implicated in certain cancers, including chronic myelogenous leukemia.
By observing a karyogram, geneticists can actually visualise the chromosomal composition of an individual to confirm or predict genetic abnormalities in offspring even before birth.
Basic, structural proteins in prokaryotes that help package and organise the circular chromosome within the nucleoid region.
Enzymes that change the winding state of DNA (dissipating or introducing supercoiling strain) to facilitate replication and transcription.
A supercoiled DNA conformation in which the circular molecule forms a two-start right-handed helix with terminal loops.
A supercoiled DNA conformation in which the circular molecule forms a one-start left-handed helix.
The mechanical linking of two circular DNA molecules together like links in a chain.
The structural interlooping of a single circular DNA molecule.
Highly basic, conserved eukaryotic nuclear proteins around which DNA winds to form nucleosome complexes.
The fundamental subunit of eukaryotic chromatin, consisting of ~146 bp of DNA wrapped 1.65 times around an octamer of core histone proteins.
The entire collection of an organism's DNA, or its total genetic content.
Haploid sex cells (eggs and sperm) produced through meiosis that fuse during fertilisation to create a diploid zygote.
Possessing two complete, homologous sets of chromosomes in each somatic cell, designated 2n.
A cell or organism possessing a single set of unpaired chromosomes (designated as 1n); typical of gametes.
The central nucleic acid core of a virus containing its linear or circular, single- or double-stranded DNA or RNA genome.
Enzymes that synthesise complementary RNA transcripts from a DNA template strand during transcription.
Thread-like structures in the nucleus containing a single long molecule of double-stranded DNA compacted with structural proteins.
Two identical copies of a duplicated chromosome that are held together at the centromere prior to segregation.
The constricted chromosomal region where sister chromatids join and where the mitotic spindle kinetochores assemble.
A specialised protein complex assembled on the centromeric region of sister chromatids where mitotic spindle microtubules attach.
The highly conserved, repetitive hexanucleotide sequence that comprises the telomeric DNA caps of human chromosomes.
Unique, higher-order four-stranded structures formed by repetitive G-rich TTAGGG sequences in telomeric DNA.
The new DNA strand synthesised continuously in the same direction as the replication fork movement.
The new DNA strand synthesised discontinuously in short Okazaki fragments in the direction opposite to replication fork movement.
Short, discontinuously synthesised DNA fragments on the lagging strand during replication, which are subsequently ligated into a continuous strand.
The finite number of times a normal somatic cell population can divide in culture before entering senescence.
A specialised eukaryotic enzyme that replicates and maintains telomere ends using an internal RNA template.
The catalytic protein subunit of telomerase that acts as an RNA-dependent DNA polymerase.
An enzyme that synthesises a complementary DNA copy from an RNA template strand.
The RNA subunit of telomerase that provides the template sequence used to direct the synthesis of new telomeric repeats.
Any non-reproductive diploid body cell in a multicellular organism.
The first meiotic division, which reduces the chromosome sets from diploid to haploid and mixes genetic information via crossing over.
The second meiotic division, which segregates sister chromatids into four distinct haploid nuclei.
The standardised number, size, banding pattern, and centromere location of an individual's complete set of chromosomes.
The mammalian sex-determination system where XX individuals are female and XY individuals are male.
A sex-determination system (like in Drosophila) where the ratio or number of X chromosomes determines female development.
A variant sex-determination system where females are XX and males have only a single sex chromosome (X0).
The avian sex-determination system where females are heterozygous (ZW) and males are homozygous (ZZ).
A genetic disorder (trisomy 21) caused by having three copies of chromosome 21, leading to intellectual disability and developmental defects.
An aneuploid condition where a diploid cell possesses an extra copy of a chromosome, designated 2n+1 (e.g., trisomy 21).
An aneuploid condition where a diploid cell is missing one chromosome from a homologous pair, designated 2n-1.
A female genetic disorder (monosomy X0) characterised by short stature and sterility, caused by possessing only a single X sex chromosome.
A male genetic disorder (47,XXY) characterised by tall stature and fertility problems, caused by possessing an extra X chromosome.
A rare genetic disorder caused by a deletion on the short arm of chromosome 5, characterised by microcephaly, developmental delays, and a cat-like cry.
A chromosomal abnormality characterised by an incorrect number of chromosomes in a cell, such as possessing an extra chromosome (trisomy) or lacking one (monosomy).
A chromosomal rearrangement where a segment breaks from one chromosome and reattaches to a different, nonhomologous chromosome.
A photographic chart where an individual's homologous chromosomes are arranged and aligned in numerical order from longest to shortest.
