Chapter 20 Sexual Reproduction, Meiosis, and Genetic Recombination.

Chapter 20 Sexual Reproduction, Meiosis, and Genetic Recombination

Sexual Reproduction, Meiosis, and Genetic Recombination
During asexual reproduction new (genetically identical) individuals are generated by mitosis It can be efficient as long as environmental conditions don’t change However, under changing environmental conditions, organisms that undergo sexual reproduction usually have an advantage 2

Sexual Reproduction Sexual reproduction allows genetic information from two parents to be mixed together, producing genetically novel offspring Most plants and animals, and many eukaryotic microorganisms, reproduce sexually 3

Sexual Reproduction Produces Genetic Variety by Bringing Together Chromosomes from Two Different Parents Sexual reproduction allows for production of an enormous variety among individuals in a population Genetic variety depends on mutations, unpredictable alterations in DNA base sequence They are rare events; beneficial mutations are rarer 4

Homologous chromosomes
Sexually reproducing organisms have cells with two copies of each type of chromosome, one from each parent The two members of the chromosome pair are called homologous chromosomes, which look alike under the microscope They carry the same lineup of genes, but these may vary slightly in base sequence 5

Sex chromosomes There are two kinds of sex chromosomes, which determine the gender of the individual carrying them They are generally called X and Y chromosomes, and differ in appearance, and genetic makeup (XX  female; XY  male) During sexual reproduction the X and Y chromosomes behave as homologues 6

Diploid and haploid A cell or organism with two sets of chromosomes is said to be diploid (2n) A cell or organism with one set of chromosomes is called haploid (n) In humans, with 23 pairs of chromosomes, n  23 and 2n  46 7

Diploid Cells May Be Homozygous or Heterozyygous for Each Gene
A gene locus is the place on a chromosome that contains the DNA for a particular gene, which controls a character (trait) in the organism Slight variations in the sequence of a gene are called alleles The combination of alleles determines how the organism will express the character controlled by the gene 8

Figure 20-1

Heterozygous and homozygous
Homozygous individuals have the same two alleles of a particular gene Heterozygous individuals carry two different alleles of the gene; the appearance of the organism depends on the relationship between the alleles

Dominant and recessive alleles
In a heterozygous individual, the dominant allele determines the trait that appears in the individual The recessive trait does not show up unless the individual is homozygous for that allele Genotype is the whole genetic makeup of an individual, whereas the phenotype is the physical expression of the genotype

Figure 20-2

Gametes Are Haploid Cells Specialized for Sexual Reproduction
Gametes are the haploid cells from each parent that fuse to form a new individual In animals and plants, males make sperm and females make ova (eggs) Fertilization, the union of sperm and egg, creates a zygote

Variations on gametes Parthenogenesis, in which females reproduce without males, is known but rare Unicellular eukaryotes and fungi produce gametes of identical size rather than sperm and ova; these gametes are said to differ in mating type

Meiosis Gametes produced by mitosis would be diploid and would fuse to form a tetraploid (four sets of chromosomes) offspring A different type of cell division is used to produce gametes with a haploid chromosome content This process is meiosis and involves DNA replication followed by two divisions

Figure 20-3

The Life Cycles of Sexual Organisms Have Diploid and Haploid Phases
The life cycles of sexually reproducing organisms is divided into a diploid (2n) and haploid (1n) phase The diploid phase begins at fertilization and extends to meiosis, whereas the haploid phase begins at meiosis and ends at fertilization Organisms vary greatly in the relative prominence of haploid and diploid phases 17

Fungi are primarily haploid
Fungi are predominantly haploid, but include a brief diploid phase that begins with gamete fusion and ends at meiosis Meiosis usually takes place almost immediately after gamete fusion, so the diploid phase is very short 18

Mosses and ferns have prominent haploid and diploid phases
For mosses, the haploid form is larger and more prominent; in ferns it is the opposite In both, gametes develop from preexisting haploid cells, whereas haploid spores are produced by meiosis This alternation of forms is alternation of generations 19

Alternation of generations
Haploid spores germinate to give rise to the haploid form of the plant or alga (gametophyte) The haploid form produces gametes by mitosis Gametes fuse by fertilization to form the diploid form, called the sporophyte In most plants, the sporophyte generation predominates 20

Alternation of generations in flowering plants
In flowering plants, the gametophyte is inside the flower The female gametophyte is called the carpel and the male gametophyte is the anther Meiosis in plants is called sporic meiosis, whereas in animals it is called gametic meiosis 21

Figure 20-4 22

Meiosis Converts One Diploid Cell into Four Haploid Cells
Meiosis is preceded by chromosome duplication and involves two successive divisions A diploid nucleus is converted into four haploid nuclei Meiosis I is called the reduction division because it reduces the chromosome number from diploid to haploid

Meiosis I Early during meiosis I the chromosomes of each homologous pair bind together during prophase to exchange some of their genetic information This pairing is called synapsis The two chromosomes behave as a unit called a bivalent (or tetrad) that aligns at the spindle equator

Meiosis I and II After lining up at the equator, the bivalent splits so that each member of the pair moves to the opposite pole of the cell Each pole receives only one of each pair, so the resulting cell is considered haploid In meiosis II, the chromatids separate just as in mitosis

Meiosis I Produces Two Haploid Cells That Have Chromosomes Composed of Sister Chromatids
The first meiotic division segregates homologues (and thus the alleles on those homologues) into different daughter cells This separation makes possible the eventual remixing of different pairs of alleles at fertilization This and the exchange of DNA segments is called genetic recombination

Prophase I: Homologous Chromosomes Become Paired and Exchange DNA
Prophase I is a particularly long and complex phase It can be divided into five stages: leptotene, zygotene, pachytene, diplotene, and diakinesis

Leptotene and zygotene
The leptotene stage begins with condensation of chromatin fibers into long, threadlike structures At the zygotene stage, condensation continues to make individual chromosomes distinguishable and homologues undergo synapsis to form bivalents

Figure 20-6

Pachytene and diplotene
At the pachytene stage chromosomes condense dramatically DNA segments are exchanged by crossing over At the diplotene stage, the homologous chromosomes begin to separate but remain attached by connections called chiasmata—the positions of crossovers

Figure 20-6

Diakinesis In diakinesis, chromosomes recondense to their compacted state In some organisms, chromosomes decondense during diplotene and cells take a break from meiosis In diakinesis chromosomes continue to separate from their homologues and are only connected by chiasmata, nucleoli disappear, and the spindle forms

Figure 20-6

Figure 20-5A

Figure 20-6

The synaptonemal complex
Homologous chromosomes are held together by the synaptonemal complex, an elaborate protein structure resembling a zipper The lateral elements begin to attach to chromosomes during leptotene The central element, which actually joins the chromosomes together, does not form until zygotene 36

Figure 20-7

Figure 20-7A

Figure 20-7B

Metaphase I: Bivalents Align at the Spindle Equator
During metaphase I the bivalents attach via their kinetochores to spindle microtubules, and migrate to the spindle equator The presence of paired homologues at the equator is a feature specific to meiosis The bivalents are randomly oriented, and homologues are held together only by chiasmata

Anaphase I: Homologous Chromosomes Move to Opposite Spindle Poles
As anaphase I begins, homologues separate from each other and start to migrate toward opposite spindle poles Homologue separation is a fundamental feature of meiosis A protein called shugoshin protects the cohesins at the centromeres from degradation

Telophase I and Cytokinesis: Two Haploid Cells Are Produced
Telophase I begins when the haploid set of chromosomes arrives at each spindle pole In some cases nuclear envelopes form around the chromosomes prior to cytokinesis, generating two haploid cells Usually the chromosomes do not decondense before meiosis II begins

Figure 20-5 b-d 43

Meiosis II Resembles a Mitotic Division
A brief interphase may intervene before meiosis II begins Each cell contains one set of replicated chromosomes, each with two sister chromatids The purpose of meiosis II is to divide the sister chromatids into two newly forming cells 44

Meiosis II resembles mitosis
Prophase II is very brief, and resembles prophase of mitosis In metaphase II chromosomes line up at the spindle equator as in mitosis, except that there are only half the normal number of chromosomes In anaphase II sister chromatids move to opposite poles of the cell 45

Nondisjunction Occasionally an error in segregation called nondisjunction occurs This produces cells that have an extra chromosome or are missing one, a condition called aneuploidy If aneuploid gametes fuse with normal gametes, defective embryos are produced that usually die before birth 46

C value Through the stages of meiosis, the DNA content of a cell changes The amount of DNA present in a cell is expressed as C value, where one haploid set of chromosomes is 1C In a diploid cell before replication the ploidy is 2n and the DNA content is 2C 47

C value after replication
After replication, the DNA is doubled to 4C because each chromosome consists of two chromatids After meiosis I, the chromosome number (ploidy) is 1n and the DNA content is 2C After meiosis II, the chromosome number is still 1n, and the DNA content is reduced to 1C 48

Figure 20-8

Sperm and Egg Cells Are Generated by Meiosis Accompanied by Cell Differentiation
In males meiosis converts a diploid spermatocyte into four haploid spermatids After meiosis is complete, the spermatids differentiate into sperm cells by discarding most of the cytoplasm, and developing flagella 51

Figure 20-9A

Video: Meiosis I in Sperm Formation

Oocyte development In females meiosis converts a diploid ooctye into four haploid cells but only one of the four survives and gives rise to an egg cell The two meiotic divisions divide the cytoplasm unequally, with one daughter cell receiving the bulk of the cytoplasm The other three very small cells are called polar bodies

Figure 20-9B

Developing egg cells acquire special features during meiosis
Many special features of the egg are acquired during prophase I, when meiosis I is temporarily halted to allow for growth During this growth phase, the cell develops special coatings to protect the egg from injury Oocytes remain in prophase I until resumption of meiosis is triggered by a signal

MPF In amphibians, resumption of meiosis is triggered by progesterone, which causes an increase in MPF activity Progesterone stimulates production of Mos, a protein kinase that activates a series of kinases, leading to MPF activation In some organisms the second meiotic division does not occur until fertilization

Metaphase II arrest Metaphase II arrest in vertebrate eggs is triggered by cytostatic factor (CSF), an inhibitor of the anaphase-promoting complex CSF is inactivated when the egg is fertilized The mature egg contains everything needed for early stages of embryonic development

Meiosis Generates Genetic Diversity
Meiosis plays a role in generating genetic diversity in sexually reproducing populations Various combinations of chromosomes are assembled into gametes to be passed on to the next generation Also, crossing over leads to more combinations of alleles, generating additional diversity

Genetic Variability: Segregation and Assortment of Alleles
The work of Gregor Mendel laid the foundation for what is now called Mendelian genetics Mendel worked with garden peas and studied seven readily identifiable characters He began by establishing that each of his plant strains was true-breeding, meaning that plants produced the same phenotype generation after generation 60

Figure 20-10A

Information Specifying Recessive Traits Can Be Present Without Being Displayed
In his first set of experiments Mendel cross-fertilized the true-breeding parental plants (P1 generation) to produce hybrid strains The resulting offspring (F1 generation) showed only one of the parental traits, the dominant trait Next, Mendel allowed the F1 hybrids to self-fertilize and looked at the offspring (F2 generation) 62

Figure 20-10B

The F2 generation For each trait, the F2 generation showed a 3:1 ratio of dominant to recessive phenotypes Mendel allowed the F2 plants to self-fertilize The F2 plants with the recessive trait always bred true, and one-third of the dominant plants bred true The remaining F2 plants produced a 3:1 ratio 64

Figure 20-11

Mendel’s conclusions Mendel concluded that information for the recessive trait must be present in the F1 hybrid plants even though it was not visible An additional experiment, in which F1 hybrids were backcrossed (crossed to one of the parental strains), supported this

Figure 20-12

The Law of Segregation States That the Alleles of Each Gene Separate from Each Other During Gamete Formation Mendel formulated principles, known as Mendel’s laws of inheritance The first was that traits are determined by factors (genes) present as pairs of determinants (alleles) Most scientists believed in a blending theory of inheritance 69

The law of segregation Mendel’s law of segregation states that the two alleles of a gene are distinct entities that separate from one another during gamete formation 70

The Law of Independent Assortment States That the Alleles of Each Gene Separate Independently of the Alleles of Other Genes Mendel studied multifactor crosses between plants that differed in several characters He generated F1 hybrid plants and allowed them to self fertilize He found all combinations of traits in the F2 71

The Law of Independent Assortment
Mendel deduced that all combinations of alleles occurred in the gametes with equal frequency The two alleles of each gene segregate independently of other genes, the law of independent assortment 72

Early Microscopic Evidence Suggested That Chromosomes Might Carry Genetic Information
By 1875 microscopists had identified chromosomes and fertilization was shown to involve fusion of sperm and egg nuclei The first proposal that chromosomes carry genetic information was made in 1883 It was soon realized that the chromosome number remains constant throughout the development of an organism 73

Rediscovery of Mendel Montgomery, Boveri, and Sutton made crucial observations following the rediscovery of Mendel’s paper Montgomery recognized the existence of homologous chromosomes Boveri observed that each chromosome plays a unique genetic role with experiments involving abnormal chromosome numbers in sea urchin eggs 74

Rediscovery of Mendel Sutton observed that the orientation of each pair of homologous chromosomes (bivalent) at the spindle equator during metaphase I is random The work of these three scientists helped make the connection between chromosome behavior during meiosis and the inheritance of genetic information 75

Chromosome Behavior Explains the Laws of Segregation and Independent Assortment
The chromosome theory of inheritance can be summarized as follows: 1. Nuclei of all cells except the germ line (sperm and eggs) contain a paternal and a maternal set of chromosomes 2. Chromosomes retain their individuality and are genetically continuous throughout the life cycle of an organism 76

Chromosome behavior and Mendel’s laws (continued)
The chromosome theory of inheritance 3. The two sets of homologous chromosomes carry a similar set of genes 4. Maternal and paternal homologues synapse during meiosis and move to opposite poles of the spindle 5. The maternal and paternal members of different pairs segregate independently during meiosis 77

Figure 20-13

Figure 20-14

The DNA Molecules of Homologous Chromosomes Have Similar Base Sequences
Homologous chromosomes have DNA molecules whose sequences are nearly identical The minor sequence differences create allele differences and arise from mutations DNA homology, the similarity in base sequences, explains the ability of homologues to undergo synapsis

Chromosome pairing Chromosomes of some organisms possess DNA sequences called pairing sites that promote synapsis between chromosomes Proteins in the synaptonemal complex also play a role in facilitating pairing

Genetic Variability: Recombination and Crossing Over
Segregation and independent assortment of homologues in meiosis I lead to random assortment of alleles In a diploid organism of genotype Aa Bb, meiosis will produce gametes in which A assorts with B as often as with b In a case where two genes reside on the same chromosome, alleles will not assort randomly 82

Figure 20-15A

Figure 20-15B

Genes on the same chromosome
Alleles on the same chromosome tend to segregate together But even in this case, some scrambling of alleles occurs because of crossing over Crossing over involves the exchange of material during meiosis I when homologues are synapsed

Figure 20-15C

Chromosomes Contain Groups of Linked Genes That Are Usually Inherited Together
Morgan and colleagues first discovered linkage in the fruit fly They began with wild type, the “normal” type of fly They had to generate mutations for their genetic experiments, naturally occurring and X-ray induced mutations

Linkage An early observation was that not all fruit fly genes assorted independently; instead some behaved as though they were physically connected In these cases, new combinations of alleles were rare Fruit fly genes can be classified into four linkage groups

Linkage groups Linkage groups are collections of linked genes that are usually inherited together Morgan realized that the number of linkage groups was the same as the number of chromosomes in the fly (n  4) He concluded that each linkage group corresponded to a chromosome

Homologous Chromosomes Exchange Segments During Crossing Over
Morgan found that linkage of linked genes was not complete Though genes assorted together most of the time, sometimes nonparental combinations would appear in offspring This was called recombination

Crossing over Morgan proposed that homologous chromosomes can exchange segments through crossing over Non-crossover chromosomes are called parental and those that have crossed over are called recombinant Crossing over takes place during the pachytene stage of meiotic prophase I; the resulting chiasmata hold homologues together at metaphase I

Gene Locations Can Be Mapped by Measuring Recombination Frequencies
Morgan and others noticed that recombination frequency differed for different pairs of genes This suggested that crossover frequency was related to distance between the genes They used recombination frequency to determine the distance between pairs of genes This is called genetic mapping

Figure 20-16

Figure 20-16A

Figure 20-16B

Genetic mapping Recombination frequency is used to determine distances between genes in map units (centimorgans) The percent of nonparental offspring corresponds to map distance where 1% crossover  1 mu

Genetic Recombination in Bacteria and Viruses
Crossing over is not restricted to sexually reproducing organisms Viruses and bacteria are capable of genetic recombination

Co-infection of Bacterial Cells with Related Bacteriophages Can Lead to Genetic Recombination
Genetic recombination between related phages takes place when bacterial cells are infected by both types of phage simultaneously As the phages replicate in the bacterium, DNA segments can be exchanged between homologous regions Recombinant phages arise at a frequency dependent on the distance between the genes being studied

Figure 20-17

Transformation and Transduction Involve Recombination with Free DNA or DNA Brought into Bacterial Cells by Bacteriophages In bacteria, several mechanisms exist for recombining genetic information The ability of bacteria to take up DNA molecules and incorporate DNA into their genomes is called transformation Transduction involves DNA that has been brought into a bacterium by a bacteriophage

Figure 20-18A

Transducing phages Phages that occasionally incorporate some bacterial DNA into their progeny phages are called transducing phages Cotransductional mapping involves determining how frequently two genes are transduced together The closer two genes are on a chromosome, the more likely they will be transduced together

Figure 20-18B

Conjugation Is a Modified Sexual Activity That Facilitates Genetic Recombination in Bacteria
Bacteria also transfer DNA from one cell to another by conjugation It resembles mating in that one bacterium is the donor (often called “male”) and the other is the recipient (“female”) It usually involves only a portion of the genome and so does not qualify as true sexual reproduction

The F Factor The F factor enables E. coli cells to act as donors during conjugation It is either an independent replicating plasmid, or a part of the bacterial chromosome Donor cells develop long, hairlike projections called sex pili, that selectively bind to recipient cells to form a transient cytoplasmic mating bridge through which DNA is transferred

Figure 20-19

Figure 20-19A

Figure 20-19B

DNA transfer The donor cell quickly transfers a copy of the plasmid into the recipient cell (F), transforming the recipient into a donor cell (F) Transfer begins at the origin of transfer The donor cell remains F because it retains one copy of the plasmid

Figure 20-20A

Hfr Cells and Bacterial Chromosome Transfer
The F factor can sometimes integrate into the bacterial chromosome This converts the F cell into an Hfr (high frequency of recombination) cell When mated with an F cell, an Hfr cell transfers a copy of its chromosomal DNA (or part of it) starting at the origin of transfer

Figure 20-20b

Bacterial chromosome transfer
Transfer of the entire bacterial chromosome is rare because it takes about 90 minutes Genes located close to the origin of transfer are most likely to be transferred to the recipient cell Once inside the cell, the donor DNA can recombine with the recipient DNA

Mapping bacterial chromosomes
The correlation between the position of a gene on the chromosome and its likelihood of transfer can be used to map genes A cross is made between Hfr and F strains that differ in several genetic properties After conjugation, cells are plated to allow growth of recombinant but not parent cells, and frequency of recombination is calculated

Figure 20-20C

Molecular Mechanism of Homologous Recombination
Homologous recombination involves exchange of genetic information between DNA molecules with extensive sequence similarity 116

DNA Breakage and Exchange Underlies Homologous Recombination
Two theories were proposed to explain how homologous recombination occurs The breakage-and-exchange model postulated that breaks occur in the DNA followed by exchange and rejoining of the broken segments In the copy-choice model genetic recombination occurs when DNA replication switches from one homologue to the other 117

Experimental evidence for the breakage-and-exchange model
In 1961 Meselson and Weigle used phages of the same type labeled with heavy (15N) or light (14N) nitrogen Co-infecting bacteria with both phages resulted in some progeny phages containing genes from both original phages The progeny phages contained both isotopes of nitrogen 118

Figure 20-21

More evidence in eukaryotes
Taylor exposed eukaryotic cells to 3H-thymidine during S phase in the last mitosis prior to meiosis, then allowed the next S phase to proceed without 3H thymidines generating chromosomes with one labeled and one unlabeled chromatid In the subsequent meiosis chromatids contained a mixture of radioactive and nonradioactive segments

Figure 20-22

Homologous Recombination Can Lead to Gene Conversion
A simple breakage-exchange model would predict that genetic recombination should be reciprocal This is usually observed, but not always Nonreciprocal recombination is called gene conversion, because one allele appears to be converted into the other 122

Homologous Recombination Is Initiated by Single-Strand DNA Exchanges (Holliday Junctions)
Recombination is not accomplished by cleaving two double-stranded molecules and then exchanging and rejoining the cut ends Holliday was the first to propose that recombination is based on the exchange of single DNA strands between two double-stranded DNA molecules 123

Steps of recombination
One or both strands of the DNA double helix are cleaved (1) A single strand from one molecule invaded a complementary region of a homologous DNA double helix, displacing one of the strands (2) Strand invasion is catalyzed by the RecA protein in bacteria and Rad51 in eukaryotes

Figure 20-23

Steps of recombination (continued)
Localized DNA synthesis and repair generate a Holliday junction (3, 4) Electron microscopy has provided direct evidence for the existence of Holliday junctions

Steps of recombination (continued)
Once the Holliday junction is formed, unwinding and rewinding the DNA double helix causes movement of the crossover point (5) This is branch migration and can increase the amount of DNA exchanged

Cleaving of the Holliday junction
After branch migration the junction is cleaved and the broken strands rejoined If it is cleaved in one plane, the two DNA molecules will exhibit crossing over (6a) If the junction is cut in the other plane, there is no crossing over, but there is a noncomplementary region near the site of the Holliday junction (6b) 128

Fate of noncomplementary DNA
Noncomplementary DNA may be corrected by repair or left intact The net effect of repair can be to convert genes from one allele to the other

The Synaptonemal Complex Facilitates Homologous Recombination During Meiosis
The synaptonemal complex appears at the time when recombination takes place Its location between the opposed homologues is the region where crossover takes place Synaptonemal complexes are absent from organisms that fail to carry out meiotic recombination

Homology searching Cells ensure that the synaptonemal complex forms only between homologues In homology searching, a single-strand break in one DNA molecule produces a free strand that invades another and checks for complementarity Only then does the synaptonemal complex develop

Recombinant DNA Technology and Gene Cloning
Recombinant DNA technology has enabled researchers to isolate and study genes from any source with greater ease than was thought possible A central feature of the technology is the ability to produce specific pieces of DNA in large quantities—this is DNA cloning 132

DNA cloning DNA cloning is accomplished by the splicing of the DNA of interest into an element called a cloning vector that can replicate inside a cell grown in culture Usually the vector is a plasmid or virus, grown in bacteria

The Discovery of Restriction Enzymes Paved the Way for Recombinant DNA Technology
Much of recombinant DNA technology is made possible by restriction enzymes They cleave DNA molecules at specific sequences called restriction sites Those that make staggered cuts in the DNA are especially useful, because they generate sticky ends that make it easy to join DNA fragments

Generating recombinant DNA molecules
DNA molecules from two sources are treated with a restriction enzyme that generates sticky ends (1) The fragments are mixed together under conditions that favor base pairing (2) The fragments are sealed together by DNA ligase (3) to produce a recombinant molecule

Figure 20-24

DNA Cloning Techniques Permit Individual Gene Sequences to Be Produced in Large Quantities
Recombinant DNA molecules can be inserted into a cloning vector that can replicate itself when introduced into bacteria Five steps are typically involved in the process

1. Insertion of DNA into a Cloning Vector
DNA is inserted into a cloning vector, usually a plasmid or bacteriophage, most of which are engineered molecules designed for cloning Plasmids used as cloning vectors have a variety of restriction sites and carry antibiotic resistance genes These allow for selection of bacteria containing the plasmids

Use of the -galactosidase gene
Plasmids such as pUC19 have a number of restriction enzyme sites in a region containing the lacZ gene, which encodes -galactosidase Integration of foreign DNA at one of these sites disrupts the lacZ gene 139

Figure 20-26A

How a gene is inserted into a plasmid vector
Incubation with the restriction enzyme cuts the plasmid at a single site in the lacZ gene, making the DNA linear (1) The same restriction enzyme is used to cleave the DNA to be cloned (2) 141

Inserting a gene into a plasmid (continued)
The cut molecules are incubated under conditions that favor base pairing (3) They are treated with DNA ligase to link the molecules covalently (4) 142

Figure 20-26B

Activity: DNA Cloning in a Plasmid Vector

2. Introduction of the Recombinant Vector into Bacterial Cells
If the vector is phage DNA, it is incorporated into phage particles that are used to infect an appropriate cell population Plasmids are introduced into the medium with target cells, which take up the plasmid after the appropriate treatment 145

3. Amplification of the Recombinant Vector in Bacteria
After cells take up the vector, they are plated on a medium so that the vector can be replicated or amplified As the bacteria divide, the plasmids also replicate Billions can be produced in a short time (less than half a day) 146

Phages In the case of phages, the particles with the recombinant DNA are mixed with bacteria and placed on a culture medium A lawn of bacteria grows on the plate; some cells are infected by phage, which replicate and cause lysis of the host cell The cycle repeats with nearby cells, producing a plaque in the lawn

4. Selection of Cells Containing Recombinant DNA
During amplification of the cloning vector, selection is used to preferentially isolate the cells that have incorporated the vector E.g., bacterial cells that have incorporated pUC19 acquire ampicillin resistance because the plasmid contains the ampR gene This is called a selectable marker 148

Selection of cells with recombinant plasmids
Not all cells that have acquired plasmids contain recombinant plasmids (plasmids containing the DNA insert) Cells with recombinant plasmids can be recognized because the insert disrupts the lacZ gene, and prevents production of -galactosidase Cells with normal pUC19 plasmid stain blue with a simple color test—with recombinant pUC19, the cells are white 149

Selection of cells with recombinant phages
Phage cloning vectors are about 70% as long as normal phage DNA These are too small to be packaged into functional phage particles Only vectors that have the insert DNA added will be large enough to produce progeny phage particles 150

Figure 20-27

5. Identification of Clones Containing the DNA of Interest
Many cells may be generated that produce many different types of recombinant DNA Recombinant colonies or plaques can be screened to identify those containing the DNA of interest For bacterial clones, DNA is isolated and restriction enzymes are used to confirm the identity of the insert DNA 152

Genomic and cDNA Libraries Are Both Useful for DNA Cloning
In one approach to cloning, the shotgun approach, an organism’s entire genome is cleaved into a large number of restriction fragments and inserted into cloning vectors The resulting group of clones, a genomic library, contains fragments representing most or all of the genome A partial DNA digestion is used to produce overlapping fragments 153

cDNA libraries In a second approach, mRNA is copied with reverse transcriptase, which makes cDNA (complementary DNA) If the entire population of mRNA in a cell is isolated and copied into cDNA, the result is a cDNA library The value of the cDNA library is that it contains only the sequences that are actively transcribed in the cell or tissue used to make the library 154

Figure 20-28

Another advantage to cDNA libraries
cDNA libraries contain only the gene coding sequences; there are no introns Introns can be large and can make cloned genes too long for easy DNA manipulation Bacteria cannot make the correct protein product from a gene unless the introns have been removed 156

Screening libraries Once a library has been constructed, several approaches to screening for identification of plasmids or phages that contain genes of interest can be used The type of technique used depends on the prior knowledge of the gene and the type of library If the DNA sequence of the gene is known, a nucleic acid probe (single stranded DNA or RNA specific for the desired sequence) can be used 157

Screening libraries (continued )
The nucleic acid probe is labeled with radioactivity of some other chemical group that allows the probe to be easily visualized Colonies containing sequences complementary to the probe can be identified by this technique, called colony hybridization DNA is recovered from colonies by isolating the vector and restriction enzyme digestion 158

Figure 20-29

Screening approaches based on function
If the protein product of the gene is known and has been purified, antibodies that recognize it can be prepared as probes to check bacteria for the presence of the protein Or, the function of the protein can be measured, e.g., an enzyme’s activity can be tested Techniques like this only work if bacteria are able to produce the protein; special expression vectors make this more likely 160

Large DNA Segments Can Be Cloned in YACs and BACs
Eukaryotic genes are often larger than 30,000 bp, the upper limit for phage vectors In mapping projects, larger clones mean fewer are needed to cover the whole genome Yeast artificial chromosomes (YACs) can accommodate large inserts; they are “minimal” chromosomes that contain centromeres, telomeres, and DNA origins of replication 161

YACs When the components of the YAC are combined with foreign DNA, the resulting chromosome will replicate in yeast and sequence into daughter cells with each round of cell division YACs contain genes that function as selectable markers and restriction sites for cloning foreign DNA 162

Figure 20-30

BACs Bacterial artificial chromosomes (BACs) are F factor derivatives that can hold up to 350,000 bp of foreign DNA They contain a bacterial origin of replication, antibiotic resistance genes, and insertion sites for foreign DNA One type contains a gene, the product of which converts sucrose into a toxic substance 164

Selection of a BAC with a DNA insert
Foreign DNA is inserted into the gene (called SacB) Bacteria grown in the presence of sucrose will die unless the BAC they contain has foreign DNA inserted into SacB, disrupting its function 165

PCR Is Widely Used to Clone Genes from Sequenced Genomes
In cases where a genome has been sequenced, the polymerase chain reaction (PCR) is used to clone genes from libraries Gene-specific primers, complementary to the gene of interest, are used to amplify the sequence It is also possible to modify genes by adding desired base sequences to the DNA primers used in amplification 166

Epitope tagging Epitope tagging adds nucleotides to the amplified sequence These encode a stretch of amino acids recognized by commercially available antibodies When the amplified gene is expressed in cells, the protein product can be detected by the antibody (e.g., polyhistidine tagging, or His tagging) 167

Genetic Engineering Genetic engineering involves the application of recombinant DNA technology to practical problems, especially in medicine and agriculture 168

Genetic Engineering Can Produce Valuable Proteins That Are Otherwise Difficult to Obtain
Among the first proteins to be produced by genetic engineering was human insulin, required by diabetics There are several ways to produce human insulin from genetically engineered bacteria Other proteins produced this way are blood-clotting factors, growth hormone, tissue plasminogen activator, and other 169

The Ti Plasmid Is a Useful Vector for Introducing Foreign Genes into Plants
Cloned genes can be transferred into plants by first inserting them into Ti plasmid, which naturally occurs in Agrobacterium tumefaciens A small part of the plasmid, the T DNA region, usually inserts into the plant chromosomal DNA It causes uncontrolled growth of tissue called a crown gall tumor 170

Modification of the Ti plasmid for cloning
In the laboratory, the sequences of the Ti plasmid that cause tumor formation have been removed Inserting genes of interest into the modified plasmids allows transfer of foreign genes into cells The gene is put into the plasmid, which is then put into Agrobacterium cells 171

Figure 20-31

Producing transgenic plants
Transformed Agrobacterium are used to infect plant cells growing in culture These cells are used to generate plants containing the foreign gene Such plants are said to be transgenic; a term to describe any type of organism that carries one or more genes from another organism (also called GM, genetically modified) 173

Genetic Modification Can Improve the Traits of Food Crops
Scientists have created many new GM crops exhibiting a variety of new traits Plants can be made resistant to insect damage by introducing a gene cloned from soil bacteria, Bacillus thuringiensis (Bt); this gene produces a protein very toxic to some insects “Golden rice” that has high-carotene content has been produced to help address vitamin A deficiency 174

Concerns Have Been Raised About the Safety and Environmental Risks of GM Crops
For consumers, the main focus has been on safety, especially concerning possible allergic reactions However, a single gene inserted into a crop can be easily assessed for safety hazards Environmental concerns have also been raised, such as the possibility that plants containing the Bt gene might be hazardous to beneficial insects 175

Concerns about GM crops
Despite legitimate concerns there has so far been little evidence of significant environmental or health risks GM crops have allowed for reduced use of pesticides and may be beneficial in that regard 176

Animals Can Be Genetically Modified by Adding or Knocking Out Specific Genetic Elements
Techniques for genetically engineering animals varies among different animals but often includes microinjection of engineered DNA Palmiter and Brinster transferred the gene for growth hormone into a fertilized mouse egg to produce a transgenic mouse Proteins can be fused with GFP so that their locations can be followed in living cells 177

Figure 20A-1

Practical applications of genetic engineering in animals
Genetic engineering can produce farm animals that synthesize medically important proteins These can be produced, for instance, in the milk of female mammals Engineered livestock are produced as a food source

Removal of genes Removing a gene of interest or inactivation of it, is referred to as “knock out” Homologous recombination can be used for this; hundreds of stains of knockout mice have been created, each defective in a single gene

Production of knockout mice
DNA is synthesized that is similar in base sequence to the target gene and flanking sequences but with two changes (1) An antibiotic resistance gene is inserted into the target sequence DNA encoding the enzyme thymidine kinase is attached to the end of the DNA; cells containing this DNA will die if treated with an antiviral drug

Figure 20-32

Production of a knockout mouse
The engineered DNA is introduced into embryonic stem cells (2) In rare cases the DNA enters the nucleus and the artificial DNA aligns with complementary sequences flanking the target gene Homologous recombination replaces the target gene with the engineered gene 183

Selection of ES cells containing the engineered gene
If homologous recombination occurs, the engineered gene confers antibiotic resistance to the ES cells (3) In addition, the thymidine kinase gene is removed from the engineered DNA and degraded; cells treated with antiviral drugs will survive (4)

Producing adult mice with the knocked out gene
ES cells identified using the double drug selection are introduced into mouse embryos, which develop into adult mice Some tissues in the mice have the inactivated gene (5) Crossbreeding these animals eventually produces strains of pure knockout mice (6) 185

Gene Therapies Are Being Developed for the Treatment of Human Diseases
Gene transplantation techniques might be used to repair defective genes in humans, referred to as gene therapy A candidate for this is severe combined immunodeficiency (SCID) The first person to be treated with gene therapy was a girl with SCID caused by a defect in the adenosine deaminase (ADA) gene 186

Gene therapies The girl suffered from frequent, life-threatening infections In 1990 she underwent treatments whereby a cloned ADA gene was inserted into a virus, which was used to infect T lymphocytes from her blood, with the lymphocytes then injected into her bloodstream She experienced improvement, but the effect diminished over time 187

Gene therapies—improvements
In 2000, scientists reported a successful treatment for SCID using a more efficient virus and better conditions for culturing cells during gene transfer Immune function was restored to the children; but later some developed leukemia, due to the virus causing insertional mutagenesis of a normal gene The virus used was a retrovirus, whereas adeno-associated virus is less likely to inactivate host genes 188

Gene therapies—clotting factor
Patients with hemophilia have deficiencies in blood-clotting factors Hemophilia patients have been injected with adeno-associated virus containing a gene coding for the blood-clotting factor they require The cure was short lived, but promising 189

Chapter 20 Sexual Reproduction, Meiosis, and Genetic Recombination.

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Chapter 20 Sexual Reproduction, Meiosis, and Genetic Recombination.

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Presentation on theme: "Chapter 20 Sexual Reproduction, Meiosis, and Genetic Recombination."— Presentation transcript:

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