Part III Organization of Cell Populations Chapter The earth is believed to house over 10 million species of organisms that have prospered by reproducing individuals of the same species. Thus, one of the most distinct characteristics of organisms is their creation of progeny while replicating their own genomes; this mechanism is called reproduction. To perform reproduction, organisms, including humans, have created and utilized sexes. However, many organisms do not use sexes for reproduction. As an example, unicellular organisms essentially multiply through somatic cell division, which replicates the same cells. Among multicellular organisms, some animals multiply by budding, and some plants multiply through regeneration from vegetative organs (e.g., potatoes). What, then, is the purpose of reproduction using sexes? The key to answering this question lies in meiosis and fertilization. This chapter discusses the roles that sexes play by focusing on these two processes. I. Sexual and Asexual Reproduction Reproduction is roughly classified into sexual and asexual types. Asexual reproduction is a mechanism by which individuals produce multiple equivalent progeny by division or other means, and is not accompanied by gene-level changes. This mechanism is commonly found in bacteria (Fig. -1A) and protista*1, but is also found in animals (jellyfish, hydras, etc.; Fig. -1B) and plants (regeneration through vegetative organs (as seen in potatoes) or by cuttage; Fig. -1C). Eukaryotic genomes are generally diploid, and have n pairs of homologous chromosomes (n: the ploidy for each cell), generally expressed as 2n. In sexual reproduction, a haploid gamete derived from a paternal source (in humans, a sperm) and one derived from a maternal source (in humans, an ovum) fuse together to form a diploid zygote (in humans, a fertilized egg). In multicellular organisms, this zygote performs repeated cell division to create new individual (Fig. -1D). Unicellular organisms such as yeast may also perform sexual reproduction depending on their environment, and in such cases, a zygote creates new progeny. Meiosis is the mechanism by which haploid cells are created from diploid cells. *1 Protista: The term protista is used to collectively describe unicellular eukaryotes, and includes flagellata and ciliates. Their reproduction methods include binary fission, multiple fission and budding. CSLS / THE UNIVERSITY OF TOKYO 229
(A) (B) (C) (D) Figure -1 Sexual and asexual reproduction II. Somatic Cell Division and Meiosis In somatic cell division, a 2n parent cell doubles its DNA in accordance with the cell cycle and distributes it to two daughter cells (Fig. -2A). In meiosis, on the other hand, a 2n cell doubles its DNA and undergoes two successive divisions to become four 1n cells (Fig. -2B). These two cell division types are different in that, with meiosis, DNA distribution occurs after the first cell division without DNA replication. However, this is not the only difference between the two types. Let s look at the meiotic process in detail (Fig. -3). A 2n cell has a pair of paternal and maternal chromosomes known as homologous chromosomes. Each chromosome is doubled by DNA replication and become two sister chromatids. During the somatic cell division process, each homologous chromosome moves independently, and two sister chromatids of the single chromosome are divided into two cells during the fission process. In meiosis, on the other hand, homologous chromosomes form pairs (synapsis). This pairing also occurs between sex chromosomes (in humans, X and Y), and genetic crossover takes place between paternal and maternal homologous chromosomes. Figure -4 shows formation of multiple crossover points between homologous chromosomes and chromatids; chromosome transfer takes place at crossover points, resulting in a change in gene combinations in a process known as genetic recombination. This process occurs randomly between homologous chromosomes, and involves the creation of a variety of chromosomes in which paternal and maternal regions are mixed. CSLS / THE UNIVERSITY OF TOKYO 230
Chapter (A) (B) Figure -2 Somatic cell division and meiosis (A) The somatic cell division cycle. A cell doubles its chromosomes in the S phase (the DNA synthesis phase) and enters the M phase (the karyokinesis phase, which is followed by cytokinesis) via the G2 phase. In the M phase, the doubled chromosomes are evenly distributed to two daughter cells, which then go through the G1 phase and enter the S phase, thus repeating the cell cycle. (B) The meiosis cycle. A meiosis-induced cell performs one DNA replication (premeiotic DNA synthesis) and then successively goes through the first and second meiotic divisions, thereby creating 1n cells. In the case of an ovum, however, four equal ova are not necessarily created. Figure -3 Meiosis and somatic cell division processes C S L S / T H E U N IV E R S IT Y OF T OK YO 231
*2 Kinetochores: Kinetochores are regions found in chromosomes. They contain highly repetitive DNA sequences, and are bound to by many proteins. During cell division, microtubules are attached to these regions for chromosome segregation (kinetochore). Kinetochores are equivalent to the primary constriction sites of chromosomes in higher eukaryote. The point at which paternal and maternal chromosomes cross and attach is called chiasma, and these paired chromosomes are lined up in the center of a pair of mitotic spindles the chromosome segregation apparatus. Then, following the degradation of proteins that connect the homologous chromosomes, they are segregated and distributed by the mitotic spindle to two cells (representing the first division). The second division then occurs, in which the sister chromatids that constitute the homologous chromosomes are segregated, and each is distributed to one cell. The microtubules that constitute the mitotic spindle bind to chromosomes with their kinetochores*2, pushing and pulling them (see Chapter 6). The directions of kinetochores are different in somatic cell division and in meiosis. In somatic cell division (in which paired chromatids are carried in opposite directions), kinetochores are positioned facing opposite directions, and in meiosis I (in which paired chromatids are carried in the same direction), kinetochores are positioned facing the same way (Fig. -5). Figure -4 Genetic Crossover Each chromatid (1 or 2) can cross with either sister chromatid (3 or 4). Figure -5 Directions of kinetochores In the first meiosis, the kinetochores of the two chromatids face the same direction (left), but in somatic cell division they face opposite directions (right). CSLS / THE UNIVERSITY OF TOKYO 232
Column Sex Determination and Reversal The sex of mammals, including humans, is determined by the combination of X and Y chromosomes. Males have one X and one Y chromosome, while females have two X chromosomes. The SRY (i.e., the sex-determining region of Y) of the Y chromosome plays an important role in forming male organs. On the other hand, birds with heterozygous sex chromosomes become females, and those with homozygous sex chromosomes become males. In such cases, Z and W are used to express the sex chromosomes; females have ZW chromosomes, and males have ZZ chromosomes. In addition, there are many organisms, including fruitflies (Drosophila melanogaster), in which the sex is determined by the ratio of sex chromosomes to autosomes*3. Some plant species, such as the evening campion, use sex chromosomes to determine their sex. On the other hand, the sex of many organisms is changed by environmental factors. The sex of some reptile species is determined by their thermal environment. By way of example, turtles tend to become male in lowtemperature conditions and female in high-temperature conditions. Conversely, alligators tend to become female under low temperatures and male under high temperatures. Sexual reversal is also found in fish; black porgies change from male to female as they age, while giltheads change from female to male. Sex determination mechanisms are therefore diverse, and are believed to have evolved as a survival strategy that enables organisms to create progeny effectively in the natural environment. *3 Sex chromosomes and autosomes: Chromosomes that differ by sex and have genes that are involved in sex determination are called sex chromosomes. Other chromosomes are collectively called autosomes. III. The Purpose of Meiosis Meiosis exists to provide gametes the origins of next-generation progeny with diverse gene combinations by mixing paternal and maternal genes. In this process, homologous chromosomes form pairs, which are distributed independently to separate gametes (Fig. -6A). Gametes can therefore have many combinations of homologous chromosomes. As an example, in humans (which have 23 pairs of homologous chromosomes), the number of CSLS / THE UNIVERSITY OF TOKYO 233
(A) (B) Figure -6 Models explaining the consequence of meiosis the possible combinations is 2 23 (8.4 x 10 6 ). Genetic crossover also occurs between paired homologous chromosomes, resulting in gene recombination. Since crossover occurs independently in each sister chromatid, all four resultant chromatids have different gene combinations (Fig. -6B). In this way, new chromosomes with a mix of paternal and maternal chromosomes are created. In chromosomal recombination, the greater the distance between two genes, the more likely recombination is to occur between them. Recombination rarely takes place if two genes are close to each other. The distance between genes can therefore be estimated by measuring the gene recombination rate (known as genetic mapping). As discussed above, intraspecies genetic diversity is increased during the meiotic process through the formation of many homologous-chromosome combinations and gene recombination by crossover. This diversity is believed to be advantageous in creating progeny that can expand its habitat to a variety of environments and adapt to rapidly changing circumstances. IV. Genetic Recombination We have discussed how DNA in chromosomes is rearranged via genetic recombination during the meiotic process. However, genetic recombination occurs CSLS / THE UNIVERSITY OF TOKYO 234
not only in meiosis but also in somatic cell division and viral infection. The resulting DNA rearrangement brings about genetic diversity, allowing organisms to survive in various environments. In this section, genetic recombination is more broadly discussed, including general recombination and site-specific recombination. General Recombination General recombination occurs between homologous DNA regions, and includes the type that takes place between homologous chromosomes during meiosis. In recombination between such chromosomes, a double-strand break occurs in one of the paired homologous DNA strands followed by the initiation of partial degradation of the 5 ends at the break point, thereby exposing the 3 ends (Fig. -7). The 3 ends recognize the similar DNA sequence in the paired DNA strand, and bind to it through the action of proteins that mediate recombination. The partial synthesis of complementary DNA then proceeds, and DNA recombination is finally completed after the breaking of the DNA strands and the repair of the chromosomes. Site-specific Recombination Site-specific recombination is caused by specific short sequences. These sequences are known as movable genetic elements, and move not only within the same chromosome but also to other chromosomes, where they cause recombination. Figure -8 shows the movement of a movable genetic element called transposon and the rearrangement of chromosomes that accompanies it. In this figure, the deletion of the movable genetic element and its insertion to a new site are shown. There are many types of movable genetic element, which are roughly classified into those that move as DNA and those that move as RNA (despite being incorporated into chromosomes as DNA). All organisms have large amounts of movable genetic elements in their chromosomes, although element types vary by organism. As an example, in the human genome, regions that encode proteins account for less than 5% of the total, whereas the proportion of movable-geneticelements-like sequences is 45%. Although it is thought that many of the sequences have been mutated and are no longer transposable, some are capable of movement. On the other hand, as confirmed in a number of viruses, foreign genes can enter host chromosomes using site-specific recombination. Figure -7 Molecular process of general recombination during the meiotic process CSLS / THE UNIVERSITY OF TOKYO 235
Figure -8 Molecular process of site-specific recombination by transposition Transposase recognizes and binds to short repetitive sequences in transposons. Transposases that bind to the repetitive sequences at both ends are paired to form a complex consisting of a dimer and a DNA loop. This DNA region is cut out from donor chromosome A and is incorporated randomly into target chromosome B. In chromosome B, two DNA strands are nicked at separate sites, forming a gap through which the transposon enters. This gap is then repaired using the complementary strand as a template, thus creating short parallel repetitive sequences. Chromosome A, broken by the removal of the transposon, is reconnected. V. Gametogenesis *4 Syncytium: a coenocyte created by the fusion of multiple cells that share the cytoplasm. Haploid cells created by meiosis do not immediately become gametes such as ova and sperms. As an example, in angiosperms, a female haploid cell undergoes three more divisions to create eight cells, one of which becomes an egg cell (Fig. -9A). Through a single division, a male haploid cell becomes a reproductive cell (generative cell) and a vegetative cell to support a generative cell, and the generative cell is further divided into two cells (Fig. -9B). The two resulting sperm cells later fertilize an egg cell and a central cell of the female, respectively (double fertilization). Interestingly, in lower plant forms such as moss, the generation time of haploid cells is very long, while that of diploid cells is very short (Fig. -10). Compared with plants, higher animal forms such as mammals have a very short haploid generation time. In mammals, although meiosis for oogenesis is initiated in the early stages, the process is arrested at the primary oocyte stage in the prophase of meiosis I (Fig. -11). In humans, the process then remains dormant for many years. Once individuals mature and hormone secretion is initiated, meiosis is resumed and ova are rapidly formed. Unfertilized ova are then promptly removed. In mammalian males, spermatogenesis is initiated after sexual maturation. In humans, it takes 24 days for a spermatocyte to complete meiosis and become four spermatids, and approximately 9 weeks for a spermatid to become a mature sperm (Fig. -). Unlike ova, much of the differentiation process for sperms occurs after they become haploids. Sperms compensate for the disadvantage of being haploids by forming a special structure called a syncytium*4. In other CSLS / THE UNIVERSITY OF TOKYO 236
(A) (B) Figure -9 Gametogenesis in plants (A) Female gametes. The figure shows a pattern common to many angiosperms. A megaspore mother cell is divided into four haploid cells by meiosis I and II. Of these cells, only one matures into a megaspore. Through three mitoses, this megaspore becomes an embryo sac consisting of eight cells. (B) Male gametes. A pollen mother cell undergoes meiosis to become a pollen tetrad, which becomes dissociated and produces four microspores. The nucleus of each microspore moves to the side wall before mitosis I. This mitosis has unequal cell division, producing a large vegetative cell and a small generative cell having a nucleus with condensed chromatin structure. The generative cell moves into the vegetative cell and divides into two spermatids via mitosis II. Figure -10 Life cycle of moss CSLS / THE UNIVERSITY OF TOKYO 237
Chapter words, a spermatogonia does not undergo cytokinesis during the first somatic cell division and the subsequent meiosis, and the resulting cells continue to share the cytoplasm. Haploid spermatids therefore inherit the cytoplasm from diploid cells, and this cytoplasm controls the differentiation of sperms. The syncytium also synchronously contributes to spermatogenesis. Figure -11 Oogenesis in mammals A primordial germ cell that moves into an ovary in early embryogenesis, becomes an oogonium. After performing several mitoses, the oogonium starts meiosis I and becomes a primary oocyte. In mammals, primary oocytes are formed in the very early stages, and their development is arrested in the early stage of the first division until the individual becomes sexually mature. Once this happens, a small number of cells periodically mature under the influence of hormones, complete meiosis I to become secondary oocytes, and become mature ova via meiosis II. During this process, two polar bodies are released. The stage at which ova are released from the ovary for fertilization differs by species. Figure - Spermatogenesis in mammals Progeny cells derived from the same spermatogonium are connected through the cytoplasmic bridge until they are differentiated into mature sperms. The structure is called syncytium. To aid understanding, the figure shows how two connected spermatogonia become eight connected haploid spermatids through meiosis. The actual number of connected cells simultaneously differentiated through meiose is much higher than shown in the figure. C SLS / THE UNIVERSITY OF TOKYO 23 8
Column Agrobacteria and Genetically Modified Plants It was discovered in 1974 that the agrobacteria-related swelling in plants is caused by the circular DNA of bacteria. Subsequent studies showed that part of this circular DNA is incorporated into the plant genomic DNA and is replicated along with DNA replication. It was also found that the inserted DNA contains plant hormone synthesis genes that promote the growth of plant cells, as well as synthesis genes for special amino acids that bacteria feed on. Indeed, bacteria cause the host plant to produce large amounts of plant cells on which they feed. In other words, bacteria use the host plant as a factory to produce their food. Based on these findings, this system was proposed for use in artificially introducing various genes to plant cells. It is currently common practice to introduce only target genes to plant cells via agrobacteria by removing the genes that cause swelling. One somatic plant cell can be directly differentiated to form a whole plant; this ability is known as totipotency, and makes it easy to regenerate a plant from a plant cell with introduced genes. The plants with artificially introduced genes are called transgenic plants. Many transgenic plants have already been created, and crops with a pest-resistance gene as well as pesticide-resistant plants are widely cultivated. Since environmental destruction, including desertification, is predicted to progress in the future, the creation of genetically modified crops that can be grown under poor conditions is an urgent issue. VI. Specialization of Gametes A particular characteristic of male gametes is that they exclude most organelles (other than the nucleus) and the cytoplasm. The same applies to spermatids in the pollen tube and mammalian sperms. This may be because the main task of male gametes is to pass nuclear DNA on to ovum, and other intracellular components may adversely affect embryogenesis in the female body. Indeed, it has been reported that male mitochondrial DNA is actively destroyed in egg after fertilization. In the sperm nucleus, nuclear proteins are replaced by different kinds of nuclear proteins and DNA is condensed during the maturation process, which inactivates the nucleus. In addition to the nucleus, sperm have a flagellum, mitochondria and CSLS / THE UNIVERSITY OF TOKYO 239
Figure -13 Sperm structure an acrosomal vesicle. All of these are devices to introduce the male nucleus into an ovum; the flagellum is a strong propellant device enabling the sperm to swim to the ovum, mitochondria are devices that supply the energy for the swim, and the acrosomal vesicle is a sac containing hydrolases that allow the sperm to penetrate the oolemma (Fig. -13). However, these devices become unnecessary once fertilization takes place. Ova are specialized in a number of ways. First, they are large (Table -1), since they contain the nutrients, organelles, protein synthesis devices, etc. needed after fertilization for embryogenesis. Second, animal ova have a zona pellucida on the surface; this layer physically supports the bulk of the ovum from the outside, protects it from physical damage, and functions as a species-specific barrier that only particular sperms (i.e., those of the same species) can penetrate. VII. Fertilization Preparations for Fertilization Table -1 Ovum sizes The fusion of female and male gametes is called fertilization. For this process to occur species-specifically without multiple fertilizations, close interaction between male and female cells is necessary. Prior to fertilization, male gametes or gametophytes need to be activated. In human sperms, this activation (known as sperm capacitation) is caused by bicarbonate ions in the genital duct of females. The bicarbonate ions enter the sperm and promote the production of camp, which changes the composition of lipids and glycoproteins in the plasma membrane, lowers membrane potential and increases intracellular metabolic activity and motor activity, thereby making the sperm fertile. Since spermatids (male gametes) in plants do not have motor ability, they are carried by pollen tubes (male gametophytes) (Fig. -14A). The activation of pollen tubes and their extension to the embryo sac (a female gametophyte) have been studied in torenia, whose embryo sac is denuded, making experimental procedures easy. Pollen tubes acquire the ability to extend toward an egg cell by passing through the style a female tissue (Fig. -14B). This alone, however, does not guide pollen tubes correctly to the ovum; such tubes can send spermatids to the embryo sac only in the presence of species-specific elements secreted from synergids in the embryo sac (Fig. -14C). The pollen tube enters the embryo sac via a synergid and sends one spermatid to the egg cell and the other CSLS / THE UNIVERSITY OF TOKYO 240
spermatid to the diploid central cell, thus allowing fertilization (double fertilization). Studies on species related to torenia have shown that the response of pollen tubes to the elements secreted from synergids is species-specific, indicating that these elements lead to the species-specificity of fertilization. Fertilization Process When activated animal sperms approach the ovum surface, they are blocked by special matrices called the zona pellucida or the vitelline membrane. The zona pellucida mainly consists of three glycoproteins, Z1, Z2 and Z3. The Z3 protein species-specifically binds to Z3 receptors on the plasma membrane of sperms (Fig. -15). This binding triggers the acrosome reaction (Fig. -15), in which hydrolase stored in the acrosomal vesicle of a sperm is released to the zona pellucida and degrades it, allowing the sperm to penetrate. Fusion of the ovum plasma membrane and the sperm plasma membrane then occurs, and the sperm nucleus enters the ovum. (A) (B) Figure -14 Pollen-tube guidance and fertilization in plants (A) Pollens attached to the stigma (1) extend pollen tubes (male gametophytes). These tubes reach the embryo sac (a female gametophyte) after passing through the style (2), the placenta (3), the funiculus (4) and the micropyle (5). Since pollen tubes sometimes stray in the regions circled by the broken lines, it is believed that signals that guide the tubes exist in these areas. (B) The extension of pollen tubes is promoted by elements contained in the style. Pollen tubes that do not pass through the style therefore do not elongate significantly. (C) A pollen tube that has reached the embryo sac enters one synergid via the filiform apparatus in response to inducers secreted from the synergid. The pollen tube breaks down in the synergid, and two spermatids fuse with the central cell and the egg cell, respectively (double fertilization). (C) CSLS / THE UNIVERSITY OF TOKYO 241
*5 Asteroid body: Structure of microtubules growing radially from both poles of the mitotic spindle during cell division In addition to introducing a paternal gene set to an ovum, the sperm also activates the ovum. In mammals, when a sperm fuses with the surface of an ovum, there is a local increase in the level of intracellular Ca 2+, which is then transmitted to the entire ovum in a wave-like motion (known as a calcium wave; see Figure 9-4A in Chapter 9) and activates the ovum. In the activated ovum, hydrolase is secreted from the cortical granules in the cell to the zona pellucida, thus changing its nature to block the entry of other sperms. On the other hand, the Ca 2+ signal drives the developmental program of the ovum, leading the fertilized egg to the next stages of development. These sperm roles can be replaced with physical or chemical treatment. As an example, a frog ovum can be activated by a pinprick. In some organisms, the ovum is activated without a sperm and develops into a complete organism. This type of development is called parthenogenesis. When only the sperm involved in fertilization enters the ovum, the sperm nucleus undergoes nuclear protein conversion and swells, thus forming the male pronucleus (Fig. -16). The centrosome located at the base of the flagellum also plays a pivotal role in creating the asteroid body*5 in the ovum. Using the microtubules of the asteroid body as rails, the female pronucleus is then carried to the center of the asteroid body. In sea urchins, the male and female pronuclei fuse together, forming a 2n fertilized nucleus. In mammals, ovulation occurs in the metaphase of meiosis II, leading to fertilization. After fertilization, meiosis II resumes, and the male and female pronuclei approach each other using the same mechanism seen in sea urchins. However, they do not fuse together, and chromosomes derived from the two nuclei start to exhibit the same behavior following the breakdown of the nuclear membrane during division. The cell that thus becomes the 2n type again performs cleavage repeatedly and undergoes the developmental process. VIII. Species and Sexes This book started with the diversity and uniformity of organisms in Chapter 1, and now ends with the passing of genetic information from parents to children in this chapter. All the knowledge presented demonstrates that organisms are amazing entities that, while sharing similar basic apparatus, have evolved in hugely diverse ways. An important function of organisms is their self-replication. However, if organisms that emerged in ancient times had simply continued to replicate themselves, the diversity of organisms we see today would not have developed. The biodiversity that currently exists on the earth is proof that CSLS / THE UNIVERSITY OF TOKYO 242
organisms have not only replicated but have also modified their genes during the course of their evolution. Organisms originating in the sea altered the global environment and found their way onto land and to the sea bottom, where they used various strategies to secure species-specific niches (or habitats). To expand a niche in manifold environments, it is advantageous for organisms to increase their intraspecies diversity, and one strategy for it is to acquire sexes and develop sexual reproduction. The existence of sexes allows the mixing of genes among many individuals of the same species, while sexual reproduction allows genetic Figure -15 The process of fertilization involving a sperm and an ovum in mammals The membrane receptors of a mammalian sperm bind to the glycoprotein Z3, which is located in the zona pellucida, after which the acrosome reaction of the sperm occurs in the order of (1) to (5). Figure. -16 Formation of the asteroid body and fusion of the nuclei in the ovum (sea urchin) CSLS / THE UNIVERSITY OF TOKYO 243
progeny, in which the genes of both parents are mixed through many combinations of homologous chromosomes and through genetic recombination by crossover. Here in the 21st century, we are fortunate to have genetic blueprints created by decoding the complete genome sequences of many organisms, and such genetic information for many more species will be published in the future. By comparing the blueprints of multiple organisms, strategies to transfer specific genes to progeny using sexes may be decoded in the not-too-distant future. Column Clone Animals Clones are groups of organisms with identical DNA sequences. It is easy to produce clones in plants (as demonstrated in potatoes and by grafting), and bees clone themselves by parthenogenesis. Is it then possible to clone humans? An adult human consists of 60 trillion cells; these include somatic cells, which last only for one generation, and germ-line cells, which survive to the next generation by producing ova and sperms. Gametes are not clones, since different gene sets are created through meiosis (as already discussed). Is it possible to produce clones using somatic cells without going through these specialized reproduction cells? It has long been known that individual animals can be produced by transplanting the somatic nucleus to a nucleus-removed, unfertilized ovum in frogs. In February 1997, the birth of a cloned sheep named Dolly was widely publicized. In this case, the nucleus of a mammary cell (a somatic cell) from a female sheep was implanted into a nucleus-removed, unfertilized ovum, which was then implanted into a surrogate female sheep, thus producing a cloned sheep with a gene composition identical to that of its mother (Column Fig. -1). Clones have now been successfully created in many animals including cattle and pigs, demonstrating that cloning is possible in mammals and indicating that there are few biological barriers to human cloning. Another important conclusion from these results is that even somatic cells have all the information necessary in their nucleus to produce a complete mammal. In other words, the various cells that make up an organism have all the information needed to produce the organism, but they express only part of this information, and the expression of other genes is masked (or suppressed) (see Chapter 4). CSLS / THE UNIVERSITY OF TOKYO 244
Column Figure -1 The cloning of a sheep CSLS / THE UNIVERSITY OF TOKYO 245
Column Knockout Mice A knockout mouse is one in which certain gene functions have been genetically disrupted through a method called targeting. As shown in Column Figure -2, using the characteristic of the recombination that tends to occur between DNA regions with similar gene sequences (homologous recombination), a normal gene in an ES cell (embryonic stem cell) is replaced with a gene in the knockout vector (targeting vector) prepared in advance. In the following cell selection, cells that did not perform homologous recombination and those in which the vector was incorporated into the wrong chromosome are removed, and only ES cells that had the gene inserted correctly and underwent homologous recombination are obtained. These cells are implanted into the early embryo, and it is then implanted into the uterus of a pseudopregnant mouse, thereby producing chimeric mice consisting of cells with and without the target gene. Homozygous knockout mice are then produced by crossing these mice with other mice. The functions of the removed gene are identified by observing the phenotypes (traits) of the mice thus created. Recently, a method known as conditional knockout has also been used, in which gene functions are disrupted only during certain stages or in certain organs. This technique enables the production of mice that cannot be created using the constitutive knockout method (in other words, the target gene participates in development) as well as the observation of the gene functions in adult mice. CSLS / THE UNIVERSITY OF TOKYO 246
Column Figure -2 Method to select cells that have performed homologous recombination Cells that have not performed recombination die as a result of adding the antibiotic neomycin. Cells in which the targeting vector has entered the wrong chromosome also die from the addition of ganciclovir (gcv), which inhibits DNA polymerase, due to the existence of the thymidine kinase (TK) gene in the vector. Only cells that performed homologous recombination survive. CSLS / THE UNIVERSITY OF TOKYO 247
Summary Chapter Reproduction is classified into asexual and sexual types. In asexual reproduction, equivalent progeny is created by cytokinesis or other means, and no change occurs at the gene level. In sexual reproduction, paternal and maternal haploid gametes fuse together to form a diploid zygote. In multicellular organisms, this zygote divides repeatedly and develops into a new individual. The mechanism of creating haploid cells from diploid cells is called meiosis. During the meiotic process, crossover occurs between homologous paternal and maternal chromosomes. Gene recombination takes place at the crossover points, which changes the combination of genes. Many combinations of homologous chromosomes are distributed to gametes. As a result, diverse gametes with varied mixtures of paternal and maternal genes are produced. In higher organisms, gametes become increasingly specialized for fertilization. As an example, mammalian male gametes differentiate into sperms with a flagellum, spermatogenesis and well-developed mitochondria. Fertilization occurs through the fusion of specialized male and female gametes. Fertilization is precisely controlled to avoid multiple fertilizations and to make the process species-specific. As an example, for the fertilization of a sperm and an ovum to occur in mammals, the glycoproteins in the zona pellucida on the ovum surface must species-specifically bind to the receptors on the sperm membrane. CSLS / THE UNIVERSITY OF TOKYO 248
Problems [1] Explain the mechanism that guarantees the species-specificity observed during mammalian fertilization. [2] Describe the biological significance of meiosis. [3] Outline the advantages and disadvantages of both asexual and sexual reproduction in terms of species survival and propagation. [4] Determine whether the following statements are correct and, if incorrect, provide the reasons. A) The mechanism of producing haploid cells from diploid cells is called meiosis. B) In meiosis, only one copy of each chromosome type is allocated to a germ cell. C) Variations that occur during meiosis are not passed on to the next generation. D) Since a germinated pollen has two spermatids, two embryos are produced by fertilization. E) Multicellular organisms do not perform asexual reproduction under natural conditions. (Answers on p.259) CSLS / THE UNIVERSITY OF TOKYO 249