Meiosis

FIGURE 2.22 Cycle

Yeast Life

FIGURE 2.22 Cycle

Yeast Life

The yeast cell alternates between haploid and diploid phases and is capable of growth and cell division in either phase.

h. Yeast can be readily stored at low temperatures.

i. Genetic analysis using recombination is much more powerful in yeast than in higher eukaryotes. Consequently, collections of yeast strains that each have one yeast gene deleted are available.

Yeast may grow as diploid or haploid cells (Fig. 2.22). Both haploid and diploid yeast cells grow by budding, rather than symmetrical cell division. In budding, a bulge, referred to as a bud, forms on the side of the mother cell. The bud gets larger and one of the nuclei resulting from nuclear division moves into the bud. Finally, the cross wall develops and the new cell buds off from the mother. Especially under conditions of nutritional deprivation, diploid yeast cells may divide by meiosis to form haploid cells, each with a different genetic constitution. This process is analogous to the formation of egg and sperm cells in higher eukaryotes. However, in yeast, the haploid cells appear identical and there is no way to tell the sexes apart and so we refer to mating types. In contrast to the haploid gametes of animals and plants, the haploid cells of yeast may grow and divide indefinitely in culture. Two haploid cells, of opposite mating types, may fuse to form a zygote.

In its haploid phase, Saccharomyces cerevisiae has 16 chromosomes and nearly three times as much DNA as E. coli. Despite this, it only has 1.5 times as many genes as E. coli. Thus a substantial portion of yeast DNA apparently lacks genetic information and so is non-coding DNA. It is easier to use the haploid phase of yeast for isolating mutations and analyzing their effects. Nonetheless, the diploid phase is also useful for studying how two alleles of the same gene interact in the same cell. Thus, yeast can be used as a model to study the diploid state and yet take advantage of its haploid phase for most of the genetic analysis.

manner.

Nematodes in oceanic mud or inland soils may all look the same. Nonetheless, they harbor colossal genetic diversity.

A Roundworm and a Fly Are Model Multicellular Animals

"If all the matter in the universe except the nematodes were swept away, our world would still be dimly recognizable. . ."

Ultimately, researchers have to study multicellular creatures. The most primitive of these that is widely used is the roundworm, Caenorhabditis elegans. Nematodes, or roundworms, are best known as parasites both of animals and plants. Although it is related to the "eelworms"—nematodes that attack the roots of crop plants—C. elegans, is a free-living and harmless soil inhabitant that lives by eating bacteria. A single acre budding Type of cell division seen in yeasts in which a new cell forms as a bulge on the mother cell, enlarges, and finally separates non-coding DNA DNA sequences that do not code for proteins or functional RNA molecules

FIGURE 2.23 Caenorhabditis elegans

False-color scanning optical micrograph of the soil-dwelling bisexual nematode Caenorhabditis elegans. The round internal structures are eggs. C. elegans is convenient for genetic analysis because of its tendency to reproduce by self-fertilization. This results in offspring that are all identical to the parent. It takes only three days to reach maturity and thousands of worms can be kept on a culture plate. Approximate magnification: x80C. Courtesy of: James King-Holmes, Science Photo Library.

of soil in arable land may contain as many as 3,000 million nematodes belonging to dozens of different species.

The haploid genome of Caenorhabditis elegans consists of 97 Mb of DNA carried on six chromosomes. This is about seven times as much total DNA as in a typical yeast genome. C. elegans has an estimated 20,000 genes and so contains a much greater proportion of non-coding DNA than lower eukaryotes such as yeast. Its genes contain an average of 4 intervening sequences each.

The adult C. elegans is about 1 mm long and has 959 cells and the lineage of each has been completely traced from the fertilized egg (i.e., the zygote). It is thus a useful model for the study of animal development. In particular, apoptosis, or programmed cell death, was first discovered and has since been analyzed genetically using C. elegans. Although very convenient in the special case of C. elegans, such a fixed number of cells in an adult multicellular animal is extremely rare. C. elegans, which lives about 2-3 weeks, is also used to study life span and the aging process. RNA interference, a gene-silencing technique that relies on double-stranded RNA, was discovered in C. elegans in 1998 and is now used to study gene function during development in worms and other higher animals. RNA interference is discussed in Ch. 11.

As noted in Chapter 1, the fruit fly, Drosophila melanogaster (usually called Drosophila) was chosen for genetic analysis in the early part of the 20th century. Fruit flies live on rotten fruit and have a 2 week life cycle, during which the female lays several hundred eggs. The adults are about 3 mm long and the eggs about 0.5 mm. Once molecular biology came into vogue it became worthwhile to investigate Drosophila at the molecular level, in order to take advantage of the wealth of genetic information already available. The haploid genome has 180 Mb of DNA carried on 4 chromosomes. Although we normally think of Drosophila as more advanced than a primitive round-worm, it has an estimated 14,000 genes—6,000 fewer than the roundworm, C. elegans. Genes from Drosophila contain approximately 3 intervening sequences each on average. Research on Drosophila has concentrated on cell differentiation, development, signal transduction and behavior.

Zebrafish are used to Study Vertebrate Development

Danio rerio, (previously Brachydanio rerio) the zebrafish, is increasingly being used as a model for studying genetic effects in vertebrate development. Zebrafish are native to the slow freshwater streams and rice paddies of East India and Burma, including the Ganges River. They are small, hardy fish, about an inch long that have been bred apoptosis Programmed suicide of unwanted cells during development or to fight infection

Zebrafish are used to Study Vertebrate Development 43

FIGURE 2.24 Drosophila melanogaster, the Fruit Fly

False-color scanning electron micrograph of the fruit fly Drosophila melanogaster. The fruit fly has many external characteristics that can reveal mutation events. This specimen is of the wild type, known as Oregon R. Magnification: x18. Courtesy of: Dr Jeremy Burgess, Science Photo Library.

FIGURE 2.24 Drosophila melanogaster, the Fruit Fly

False-color scanning electron micrograph of the fruit fly Drosophila melanogaster. The fruit fly has many external characteristics that can reveal mutation events. This specimen is of the wild type, known as Oregon R. Magnification: x18. Courtesy of: Dr Jeremy Burgess, Science Photo Library.

FIGURE 2.25 Danio rerio

Danio rerio, the zebrafish, has recently been adopted as a model for the genetic study of embryonic development in higher animals.

FIGURE 2.25 Danio rerio

Danio rerio, the zebrafish, has recently been adopted as a model for the genetic study of embryonic development in higher animals.

Late in 2003, zebrafish became the first commercially available genetically engineered pets. Fluorescent red zebrafish are marketed in the USA by Yorktown Technologies as GloFish™. They fluoresce red when illuminated with white light, or better, black light (i.e. near UV) due to the presence of a gene for a red fluorescent protein taken from a sea coral. The principle is similar to that of the widely used green fluorescent protein taken from jellyfish (see Ch. 25 for use of GFP in genetic analysis). The price of about $5 per fish makes GloFish™ about five times as expensive as normal zebrafish. The fish were developed at the National University of Singapore by researcher Zhiyuan Gong with the ultimate objective of monitoring pollution. A second generation of more specialized red fluorescent zebrafish will fluoresce in response to toxins or pollutants in the environment.

for many years by fish hobbyists in home aquariums where they may survive for about five years. The standard "wild-type" is clear-colored with black stripes that run lengthwise down its body (Fig. 2.25). Its eggs are laid in clutches of about 200. They are clear and develop outside the mother's body, so it is possible to watch a zebrafish egg grow into a newly formed fish under a microscope. Development from egg to adult takes about three months. Zebrafish are unusual in being nearly transparent so it is possible to observe the development of the internal organs.

Zebrafish have about 1,700 Mb of DNA on 25 chromosomes and show about 75% homology with the human genome. Genetic tagging is relatively easy and micro-injecting the eggs with DNA is straightforward. Consequently, the zebrafish has become a favorite model organism for studying the molecular genetics of embryonic development.

Only a few animals have been investigated intensively. The rest are assumed to be similar except for minor details.

Only a few animals have been investigated intensively. The rest are assumed to be similar except for minor details.

FIGURE 2.26 Transgenic Mice

The larger mouse contains an artificially introduced human gene, which causes a difference in growth. Mice with the human growth hormone gene grow larger compared to mice without this gene.

Flowering plants have more genes than any other type of organism. The function of most of these genes is still a mystery.

Mouse and Man

The ultimate aim of molecular medicine is to understand human physiology at the molecular level and to apply this knowledge in curing disease. As discussed in Chapter 24, science now has available the complete sequence of the human genome, but researchers have little idea of what the products of most of these genes actually do. Since direct experimentation with humans is greatly restricted, animal models are necessary. Although a range of animals has been used to investigate various topics, the rat and the mouse are the most widespread laboratory animals. Rats were favored in the early days of biochemistry when metabolic reactions were being characterized. Mice are smaller and breed faster than rats, and are easier to modify genetically. Consequently, the mouse is used more often for experiments involving genetics and molecular biology. Mice live from one to three years and become sexually mature after about 4 weeks. Pregnancy lasts about three weeks and may result in up to 10 offspring per birth.

Humans have two copies each of approximately 30,000 genes scattered over 23 pairs of chromosomes. Mice have a similar genome, of 2,600 Mb of DNA carried on 20 pairs of chromosomes. Less than 1% of mouse genes lack a homolog in the human genome. The average mouse (or human) gene extends over 40 kilobases of DNA that consists mostly of non-coding intervening sequences (approximately 7 per gene). Nowadays there are many strains of mutant mice in which one or more particular genes have been altered or disrupted. These are used to investigate gene function (Fig. 2.26).

Intact humans cannot be used for routine experiments for ethical reasons. However, it is possible to grow cells from both humans and other mammals in culture. Many cell lines from humans and monkeys are now available. Such cells are much more difficult to culture than genuine single-celled organisms. Cell lines from multicellular organisms allow fundamental investigations into the genome and other cell components. Historically, the most commonly used cell lines, e.g. HeLa cells, are actually cancer cells. Unlike cells that retain normal growth regulation, cancer cells are "immortalized", that is they are not limited to a fixed number of generations. In addition, cancer cell lines can often divide in culture in the absence of the complex growth factors needed to permit the division of normal cells.

Arabidopsis Serves as a Model for Plants

Historically, the molecular biology of plants has lagged behind other groups of organisms. Ironically, plants now hold the record for the highest number of genes (40,000 to 50,000 genes for rice—some 10,000 more than humans). If our criterion for superiority is gene number, then it is the plants who represent the height of evolution, not mammals. Why do plants have so many genes? One suggestion is that because plants are immobile they cannot avoid danger by moving. Instead they must stand and face it like a man—or rather like a vegetable. This means that plants have accumulated many genes involved in defense against predators and pests as well as for adapting to changing environmental conditions. One of the most active areas in biotechnology today is the further genetic improvement of crop plants. Genetic manipulation of plants is not hindered by the ethical considerations that apply to research on animals or humans. Moreover, crop farming is big business.

Arabidopsis thaliana, the mouse-ear cress, has become the model for the molecular genetics of higher plants. It is structurally simple and also has the smallest genome of any flowering plant, 125 Mb of DNA—just over 10 times as much DNA as yeast, yet carried on only five pairs of chromosomes. Arabidopsis has an estimated 25,000

Haploidy, Diploidy and the Eukaryote Cell Cycle 45

FIGURE 2.27 Arabidopsis thaliana, the Mouse-ear Cress

The plant most heavily used as a model for molecular biology research is Arabidopsis thaliana, a member of mustard family (Brassicaceae). Common names include Mouse-ear cress, Thale cress and Mustard weed. Courtesy of: Dr Jeremy Burgess, Science Photo Library.

Many eukaryotes alternate between haploid and diploid phases. However, the properties and relative importance of the two phases varies greatly with the organism.

The concept of germline versus somatic cells applies to animals but not to other higher organisms.

genes with an average of 4 intervening sequences per gene. Arabidopsis can be grown indoors and takes about 6-10 weeks to produce several thousand offspring from a single original plant. Though slow by bacterial standards, this is much faster than waiting a year for a new crop of peas, as Mendel did.

Arabidopsis shares with yeast the ability to grow in the haploid state, which greatly facilitates genetic analysis. Pollen grains are the male germ line cells of plants and are therefore haploid. When pollen from some plants, including Arabidopsis, is grown in tissue culture, the haploid cells grow and divide and may eventually develop into normal looking plants. These are haploid, and therefore sterile. Diploid plants may be reconstituted by fusion of cells from two haploid cell lines. Alternatively, diploidy can be artificially induced by agents such as colchicine that interfere with mitosis to cause a doubling of the chromosome number. In the latter case, the new diploid line will be homozygous for all genes.

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