细胞学英文版第十章

C HAPTER 10
T HE N ATURE O F T HE G ENE A ND T HE G ENOME
O BJECTIVES
▪Review early discoveries in genetics that put us on the path to gain the knowledge we now have.
▪Review the significance of crossing over in the recombination of genes.
▪Describe the structure of DNA and the discoveries that led to its determination.
▪Summarize the importance of the Watson-Crick proposal of DNA structure.
▪Describe DNA supercoiling and the topoisomerases that are used to control it in living organisms.
▪Introduce students to the concept of genome complexity.
▪Describe the process of DNA denaturation and the factors that influence DNA melting temperature.
▪Describe the ability of DNA to renature and emphasize the usefulness of nucleic acid hybridization in a variety of biological studies.
▪Discuss the types of DNA sequences found in prokaryotic and eukaryotic cells and outline what is known about their function.
▪Elaborate on the mechanisms by which genes may be duplicated giving rise to gene families and superfamilies. ▪Describe transposons and the mechanisms by which they displace genes throughout the genome.
▪Explain the medical impact of transposons and describe their role in evolution.
▪Discuss the function and operation of restriction endonucleases.
▪Explain RFLPs and the role they have played in the production of molecular maps of the genome and DNA fingerprinting technology.
L ECTURE O UTLINE
The Concept of a Gene as a Unit of Inheritance
I. Mendel crossed pea plants (lab at Austrian monastery) & found the inheritance pattern of defined traits
A. Chose garden pea for a number of practical reasons, including that he could buy a variety of seeds that would
give rise to plants with distinct characteristics
B. Mendel focused on 7 clearly definable traits, like plant height & flower color, each of which occurred in 2
alternate & clearly identifiable forms
C. He crossbred plants through several generations & counted individuals with various traits
II. Mendel's conclusions - published findings in 1865 in journal of the Natural History Society of Brünn, Austria; lost until rediscovered independently in 1900 by 3 botanists who gave him credit
A. Characteristics of plants governed by distinct factors or units of inheritance (genes)
1. Each organism has 2 copies of gene that controls development for each trait, one from each parent
2. The two genes may be identical or nonidentical (may have alternate forms; alleles)
3. Dominant alleles mask recessive alleles when together in same organism
B. Gametes (reproductive cells) have only 1 copy of gene for each trait (dominant or recessive, not both); plants
arise from union of male & female gametes
C. Law of Segregation - an organism's 2 alleles separate (segregate) from one another during gamete formation
D. Law of Independent Assortment - segregation of allelic pair for one trait has no effect on segregation of alleles
for another trait; single gamete can get paternal gene for one trait & maternal gene for another
Chromosomes: the Physical Carriers of Genes
I. The discovery of chromosomes (colored bodies) - got their name in 1888
A. By 1880s, investigators realized that inherited characteristics must be passed from cell to cell & from generation
to generation, even though they knew nothing of Mendel's results
B. Walther Flemming, early 1880s – observed dividing cells; during mitosis, cytoplasmic contents were divided
randomly & nuclear material divided equally between the daughter cell
1. During cell division, nuclear material organized into visible threads called chromosomes
2. Chromosomes appear as doubled structures, split to single ones & doubled at next division
II. Fertilization and meiosis: Changing chromosome numbers
A. Sperm & egg are very different cells - egg large, sperm small, but nuclei essentially the same
1. Sperm & egg equally important genetically - nuclei & chromosomes apparently identical
2. Importance of chromosomes contributed by male demonstrated by Boveri
B. Theodore Boveri (German) - studied sea urchin eggs fertilized by 2 sperm (polyspermy)
1. Results in disruptive cell division & early death of embryo
2. Second sperm donates extra chromosome set & extra centriole, causing abnormal cell division
3. Daughter cells receive variable numbers of chromosomes
4. Concluded that normal development depends upon a particular combination of chromosomes & that each
chromosome possesses different qualities; first evidence for such qualitative differences
C. Edouard van Beneden (Belgian, 1883) discovered Ascaris eggs & sperm had 2 chromosomes each, but that
somatic cells had four chromosomes; about this time, the process of meiosis was described
D. August Weismann (German biologist, 1887) - said that meiosis involved reduction division where chromosome
number was cut in half before forming gametes
1. If not, chromosome number would increase with every succeeding generation
III. Chromosomes as carriers of genetic information – after the rediscovery of Mendel's work, researchers realized that genetic material must behave in a manner consistent with his principles
A. Walter Sutton (Columbia, 1903) - studied grasshopper sperm formation; saw 2 types of division by
spermatogonia; 11 homologous chromosome pairs (look alike) & extra accessory (X) chromosome
1. Mitotic divisions where spermatogonia make more spermatogonia
2. Meiotic division where spermatogonia differentiate into sperm
3. What he saw (he noticed homologous pairs) correlated with Mendel's inheritable pairs of factors
4. Hypothesized that chromosomes are physical carriers of Mendel's genetic factors
B. In cells just starting meiosis, Sutton saw the members of each pair associated with one another; form complex
called bivalent; 11 were visible, each with cleft along its length where they were associated
1. First meiotic division separated pair members (Weismann's reduction division from 15 yr before)
2. Explained Mendel's proposal that hereditary factors exist in pairs that are together until they separate with the
production of gametes & other findings; allelic pairs reconstituted at fertilization
3. Also explained how gametes contained 1 allele of each gene & how the number of gametes with 1 allele was
equal was equal to the number with other allele
IV. Chromosome as linkage group - many of Mendel's factors are grouped on a single chromosome; should act as if they are linked & not independently
A. How did Mendel's factors assort independently? - the 7 traits were on different linkage groups (chromosomes) or
were far enough apart on same chromosome to act independently
1. Mendel owed his results to good luck or lack of interest in traits that did not fit his predictions
B. Soon traits found on same chromosome in many organisms, like flower color & pollen shape in pea
V. Genetic analysis in Drosophila - Thomas Hunt Morgan (Columbia, 1909) and others
A. Fruit flies were ideal for genetic studies & became focus of them
1. Short generation time (14 days) & produce up to 1000 eggs in a lifetime
2. Very small so can keep large number on hand; easy to maintain & breed; inexpensive
3. Only one disadvantage; there was only strain of fly available, the wild type
B. Needed variants from wild type so bred flies & looked for mutants; finally found one with white eyes, not the
normal red eyes
C. Soon (by 1915) he & his students found many mutants (85) that were localized to 4 linkage groups, one with very
few mutants
1. On rare occasions, a spontaneous change or mutation occurred in gene altering it permanently; the change
could then be passed generation to generation
2. Suggests mechanism for the origin of variation that exists in populations & evolution
3. Natural selection can act on these mutants —> then new species slowly emerge
D. Linkage groups correlate to number of homologous Drosophila chromosome pairs (4), one very small
VI. Crossing over & recombination - linkage incomplete; alleles of 2 different genes originally present on same chromosome do not always stay together during gamete formation
A. Can reshuffle genes; could have alleles originally derived from both parents on same chromosome or alleles from
1 parent could end up on both members of homologous pair & in different gametes
B. F. A. Janssens (1909) – observed that bivalent homologous chromosomes wrap around each other early in
meiosis; hypothesized breakage & exchange of pieces of chromosomes
C. Morgan (1911) - thought that this crossing over (genetic recombination) could account for appearance of
offspring (recombinants) having unexpected genetic trait combinations & breakdown in linkage
1. Said Janssens right; homologous chromosomes break & rejoin, exchange pieces & separate alleles
D. Analyses of offspring from large number of offspring from many mutant parents indicated that:
1. Recombination percentage between given gene pair was constant from cross to cross
2. Recombination percentages between different gene pairs could be very different
E. These results suggest that position (locus) of genes on chromosomes fixed & does not vary fly to fly
1. Therefore, recombination percentage is a measure of distance between genes
2. Bigger distance between genes means more crossovers between genes
3. Alleles at opposite ends of chromosome have so many crossovers between them that they
assort independently (essentially like they are on separate chromosomes)
4. Genes very close to one another on chromosome are less likely to become unlinked
F. Alfred Sturtevant (Columbia undergraduate, 1911) realized recombination frequencies could be used to map
relative positions of individual genes along given chromosome
1. Sturtevant constructed detailed maps of the 4 chromosomes of the fruit fly
2. This principle has been used to map genes in many organisms (viruses, bacteria, eukaryotes)
VII. Mutagenesis & giant chromosomes – finding mutants was tedious at first but then X-rays were found to increase the rate of mutant appearance
A. Herman Muller (Indiana U., 1927) - genetic material can be damaged by X-rays
1. Sublethal X-ray dose raises spontaneous mutation frequency >100 times of nonirradiated controls
2. Later, other mutagenic agents, like UV irradiation, were demonstrated; increases available mutants for
research & points out hazards of radiation usage in industry & medicine
3. Revealed that the genetic material, whatever its chemical nature, had to be sensitive to electromagnetic
radiation
B. Theophilus Painter (U. of Texas, 1933) - rediscovers polytene chromosomes in Drosophila;arise in salivary
gland cells that grow without division; ~100 times thicker than those in most other organisms
1. DNA replication continues to support cell secretion activity; strands stay together in register
2. As much as 1,024 times the normal strands of chromosomes because cell stops dividing, but keeps growing;
allows cell to maintain its high rate of secretory activity of these enormous cells
3. Exhibit banding patterns (~5000 bands) constant in different flies & tissues; but different in different
species
4. Bands correlate with specific genes & genetic maps; prove validity of mapping procedure
C. Banding pattern comparisons of polytene chromosomes of different species allow investigation of evolutionary
changes at chromosome level; also can use them to visualize gene expression
1. Dynamic structures - active genes puff out at particular stages of development to make RNA
The Chemical Nature of the Gene
I. The structure of DNA (much of this is discussed in detail in outline for Chapter 2)
A. Base composition - DNA building blocks are nucleotides (5C-sugar deoxyribose, 5'-phosphate, nitrogenous
base [A, G, C, T] at 1'-position)
1. Types of bases - pyrimidines (cytosine [C], thymine [T]); purines (adenine [A], guanine [G])
2. Nucleotides covalently linked to form linear polymer (strand) with backbone of alternating sugar &
phosphate groups joined by 3'-5'-phosphodiester bond
3. Bases attached to sugar thought to project from backbone like column of stacked shelves
4. Nucleotide (& polymer) is polarized - 5' end (phosphate) & 3' end (3'-OH of sugar)
B. Some terminology
1. 1 of the 4 nitrogenous bases + a pentose sugar = a nucleoside; 4 of them: deoxyadenosine, deoxyguanosine,
deoxythymidine, deoxycytidine
2. If a phosphate is attached to a nucleoside —> nucleotide; add 1 phosphate —> deoxyadenosine 5'-
monophosphate, deoxyadenosine 5'-diphosphate, deoxyadenosine 5'-triphosphate
3. RNA nucleotides are often employed in energy metabolism like ATP
C. X-ray diffraction revealed dimensions - 3.4 Å between nucleotides in stack; large structural repeat every 34 Å
(3.4 nm)
D. Tetranucleotide Theory - original concept of DNA was a simple repeating tetranucleotide; could not carry much
information; ratio of bases should be 1:1:1:1
E. Erwin Chargaff (Columbia, 1950) - determined relative amounts of every base in DNA samples
1. Found relative base amounts varied, not always 1:1:1:1, but always constant within species
2. Purines always equaled pyrimidines: [A] = [T]; [G] = [C]; [A] + [T] ? [G] + [C]
3. Different species - different ratios: A:G ratio of tubercle bacillus = 0.4; human DNA - 1.56
4. Within species, different tissues had same ratio
5. Chargaff gave DNA molecule specificity & individuality from organism to organism
II. The Watson - Crick Proposal - used X-ray diffraction data [Rosalind Franklin & Maurice Wilkins, King's College, London] & cutout nucleotide models [Watson & Crick] —> proposed DNA structure
A. Composed of 2 nucleotide chains (Pauling had suggested three strands)
B. 2 chains spiral around each other & central axis to form pair of right-handed helices
1. Look down central axis, each strand follows clockwise path moving away from observer
2. Helical nature of DNA revealed by pattern of spots produced by X-ray diffraction
C. 2 chains of double helix run in opposite directions (antiparallel); one 5' —> 3', the other 3' —> 5'
D. The —sugar-phosphate-sugar-phosphate— backbone is on outside of molecule; bases project toward center;
Pauling's model had backbone in center; phosphate groups give negative charge (an acid)
E. Planes of bases are approximately perpendicular to long axis & stack on top of each other
1. Hydrophobic interactions & van der Waal forces between them give DNA added stability
2. Together helical turns & planar base pairs (bp) make it resemble spiral staircase
F. Chains held together by H bonds between each base of one strand & associated base on other strand
1. H bonds weak enough individually to separate under certain circumstances; so can strands
2. Added together, the large number of H bonds is strong, making the double helix a stable structure
G. Distance from backbone phosphorus atom to axis center is 10 Å (1 nm); DNA width thus 20 Å (2 nm)
H. Pyrimidine in one chain pairs with purine in the other chain (results in 20 Å or 2 nm width as well); 2 purines
would be wider, 2 pyrimidines narrower
I. Only A can bind to T; only G can bind to C (matches Chargaff's Rules); A—T pairs joined by 2 H bonds &
G—C pairs joined by 3 H bonds
1. Nitrogen atoms on cytosine C4 & adenine C6 are mostly in amino (NH2) not imino (NH) form
2. Oxygens on guanine C6 & thymine C4 mostly in keto (C=O), not enol (C—OH) form
3. These structural restrictions on base configurations are responsible for pairing restrictions
J. From outside of molecule, spaces between adjacent turns of helix form 2 grooves of different width
1. Wider major groove; more narrow minor groove; grooves spiral around double helix outer surface
2. DNA binding proteins often contain domains that fit into grooves; sometimes such a protein can read
sequence without separating strands
K. Double helix makes 1 complete turn every 10 residues (3.4 nm); 150 turns per million daltons MW
L. No restriction on given chain base sequence, but sequence of 1 chain specifies the other's sequence (complementarity); their nucleotide sequences are always fixed relative to each other
III. Importance of the Watson-Crick Proposal
A. Primary functions of genetic material
1. Storage of genetic information - determines heritable characteristics; amino acid sequence of all proteins in
organism must be contained within the DNA structure
2. Self-duplication and inheritance – DNA must contain the information for its own replication
3. Expression of genetic message - genes usually encode proteins; DNA must direct protein synthesis
B. Watson - Crick model explains first two functions & how they might be accomplished
1. Information resides in DNA base sequence; change DNA sequence —> change protein coded for
2. Chains can separate as H bonds break & serve as template for synthesis of new strand —> at the end, of
replication get 2 strands identical to each other & the original DNA molecule
C. According to the proposal, each new DNA double helix would contain one strand from original DNA molecule
& one newly synthesized strand
IV. DNA supercoiling
A. Jerome Vinograd et al. (Caltech, 1963) found that 2 closed DNA circles of identical molecular mass could show
very different sedimentation rates after density gradient centrifugation
1. Fast sedimenting DNA had more compact shape because molecule was twisted (supercoiled) upon itself,
much like rubber band with ends twisted in opposite directions or tangled phone cord
2. Occupies less volume; moves more rapidly in response to centrifugal force or electrical field
B. DNA lying free on flat surface has right number of 10 base pairs per turn & is said to be relaxed
1. Fuse two ends of strands to form circle without twisting and it would still be relaxed
2. Twist opposite to duplex winding direction before fusion into circle —> molecule unwinds (greater number
of bp/turn); DNA is underwound (negatively supercoiled)
3. To resist resultant strain because it ―likes‖ 10 bp/turn & is more stable that way, it becomes twisted upon
itself into supercoiled conformation
4. Circular DNA in nature (mitochondrial, viral, bacterial, etc.) is invariably negatively supercoiled
5. Positively supercoiled DNA is overwound
C. Supercoiling is not restricted to small circular DNAs but also occurs in linear, eukaryotic DNA
1. Linear DNA wrapped around protein cores (nucleosomes) also supercoils
2. Negative supercoiling plays role in allowing chromosomal DNA to be compacted so as to fit into
microscopic cell nucleus
3. Since negatively supercoiled DNA is underwound, it exerts force that helps to separate the 2 strands of the
helix, which is required during both replication & transcription
V. Topoisomerases alter DNA duplex supercoiled state (topology), further supercoil or relax it; seen in both prokaryotic & eukaryotic cells; cells have variety of topoisomerases - 2 classes; different mechanisms
A. Type I topoisomerases - create transient break in one duplex strand, & then allows the intact, complementary
strand to undergo a controlled rotation, which relaxes the supercoiled molecule
1. Essential for processes like DNA replication & transcription to prevent excessive supercoiling from
developing as the complementary strands of the DNA duplex separate & unwind
B. Type II topoisomerases - make transient break in both duplex strands, then pass double stranded DNA segment
(that one or another) through break; reseal severed strands to freeze structure
1. Accompanied by series of dramatic conformational changes
VI. Topoisomerases not only relax & supercoil DNA, but also tie DNA molecule into knots, untie knot, interlink independent circles (catenation) or separate interlinked circles into individual components
A. Topoisomerase II is required to unlink DNA molecules before duplicated chromosomes can be separated
during mitosis
B. Human topoisomerase II is target for a number of drugs (etoposide, doxorubicin) used to kill rapidly dividing
cancer cells; drugs bind to enzyme & keep cleaved DNA strands from resealing
The Structure of the Genome: Denaturation, Renaturation and Complexity
I. Genome - unique content of genetic information (humans - all genetic information in single haploid set of 23
chromosomes); DNA structure probed by X-ray crystallography, genome structure is tougher
A. Sum of genetic information is sum of all DNA segments in fertilized egg at start of life
B. All members of species share same set of genes, even though each individual possesses slightly different
versions (alleles) of many genes
II. DNA denaturation (melting) – heating & separation of the 2 DNA chains held together by weak, noncovalent bonds;
very important property of DNA double helix
A. Dissolve DNA in saline solution, slowly warm it & strands start to separate at specific temperature
1. Within a few degrees, process is complete & solution contains single stranded molecules completely separate
from their original partners
2. Follow denaturation extent by higher single strand absorbance (bases absorb UV light; peaks at ~260 nm);
hydrophobic base interactions drop with denaturation & bases absorb light more easily
3. Melting temperature (T m) is temperature at which shift in absorbance is half completed
B. T m increases if DNA has high G-C content (%G + %C) because of extra H bonds holding it together; even within
single DNA molecule, A-T-rich sections melt before G-C-rich segments
III. DNA renaturation – DNA denaturation not a surprise, but reassociation of single strands into stable double-stranded molecules seemed much less likely
A. J. Marmur et al. (Harvard, 1960) - after denaturation, the separated chains can reassociate into stable double
helices (called renaturation or reannealing)
1. Slowly cooled heat-denatured DNA —> it renatured (OD dropped & it transformed bacteria acting like
genetic material) or
2. Melt DNA at 100°C, then drop temperature quickly to ~25°C below T m & incubate awhile
3. Complementary single-stranded DNAs can reassociate or reanneal
B. Reannealing has served as basis for investigations of DNA complexity & has led to development of nucleic
acid hydridization technology
1. What is genome complexity? - it is the variety & number of DNA sequence copies in the genome
2. In hybridization, complementary nucleic acid strands from different sources can be mixed to form double-
stranded (hybrid) molecules – can be used to answer many questions
C. Factors determining the rate of renaturation of a given DNA preparation
1. Ionic strength of the solution
2. Temperature
3. DNA concentration
4. Period of incubation
5. Size of the interacting molecules
IV. Complexity of viral & bacterial genomes - compare renaturation rates of entire genomes of small virus (SV40; 5.4 x 103 bp), a larger virus (T4; 1.8 x 105 bp) & a bacterial cell (E. coli; 4.5 x 106 bp)
A. Primary difference between these DNAs is their length – to compare their renaturation, it is important that the
reacting molecules have the same length (~1000 bp)
1. Force all DNAs through tiny orifice under high pressure to make them uniform size at ~1000 bp
2. Reanneal at same DNA concentration (mg/ml) & same length —> do so at distinctly different rates
B. The smaller the genome is, the faster is the renaturation of that genome
1. All fragments are same length & DNA concentrations same —> more copies of small genomes
2. Increases chance of collision between complementary fragments of small genomes
C. Get same results in separate or same tubes (rate not affected by presence of unrelated sequences)
1. Viral/bacterial genomes - single, symmetrical curve since all, but a very few sequences in bacterial DNA, are
present at same concentration
2. Every nucleotide sequence in population is as likely to find partner in given time as any other sequence
V. Complexity of eukaryotic genome - DNA fragments in same sample reanneal at very different rates
A. R. Britten & D. Kohne (Caltech) - mammalian genome DNA fragments reannealed at markedly different rates;
not just one gene after another as seems to be the case in bacteria & viruses
B. Various nucleotide sequences in eukaryotic DNA fragments are present at very different concentrations; first
indication that eukaryotic DNA has much more complex organization
1. Curves usually show 3 more-or-less distinct steps representing 3 broad DNA sequence classes
2. Reanneal at different rates since differ in number of times their sequence is repeated within fragment
population
3. The greater the number of copies of a sequence in genome, the greater is its concentration & the faster is its
reannealing rate
C. The classes - highly repeated, moderately repeated & nonrepeated (unique) fractions
The Structure of the Genome: Highly Repeated DNA Sequences
I. Traits – present in at least 105 copies per genome; ~10% of total vertebrate DNA; to follow them, solution must be
very dilute (& their concentration very low) since this fraction reanneals so fast
A. Usually short (a few 100 bp at most); seen in clusters, sequence repeats over & over uninterrupted
B. Sequences arranged in this end-to-end manner are said to be present in tandem (tandem repeats
II. Overlapping categories of highly repeated DNA sequences: satellite, minisatellite & microsatellite DNAs
A. Satellite DNAs - short sequences (~5 – a few 100 bp long) repeated vast number of times in tandem; form very
large clusters of up to several million bp; their function remains a mystery
1. In many species, base composition is often sufficiently different from bulk of genome; that they are easy to
separate during density gradient centrifugation (form satellite band hence the name)
2. Can have >1; Drosophila virilis has 3 (each 7 bp long & very similar; common genetic origin)
3. Longer ones don't form satellites since base makeup differs little from rest of genome
B. Minisatellite DNAs - ~12 - 100 bp long; form clusters of up to 3000 repeats; occupy considerably shorter
stretches of genome than satellite sequences
1. Tend to be unstable & number of copies of particular sequence often increases or decreases from one
generation to next
2. Particular minisatellite locus length is highly variable in population, even among family members
3. Because they are so variable (polymorphic), they are used to identify individuals in criminal or paternity
cases through DNA fingerprinting
4. Changes in their sequences can alter expression (transcription) of nearby genes, which, in turn, can have
serious consequences; some changes implicated in causing cancer & diabetes
C. Microsatellite DNAs – shortest sequences, 1 - 5 bp long; present in small clusters of ~50-100 bp long
1. Scattered quite evenly throughout DNA (at least 30,000 different loci in human genome)
2. DNA replicating enzymes have trouble copying genome regions that contain these small, repetitive
sequences, which causes them to exhibit an extremely high mutation rate
3. Due to their variability within population, they have been used to analyze relationships between ethnic human
populations
a. Many anthropologists argue that modern human species arose in Africa
b. If true, members of different African populations should have greater DNA sequence variation than
humans on other continents, since African populations have had longer to diverge
c. One study analyzed 60 different microsatellite loci & showed that members of African populations had
significantly greater genetic divergence than Asian or European populations。