The Eukaryotic Genome and Its Expression

1
The Eukaryotic Genome and Its Expression
2
The Structures of Protein-Coding Genes

• Genes have three types of noncoding sequences
• The promoter occurs at the beginning of the gene
  and is the site where RNA polymerase begins
  transcription.
• The terminator occurs at the end of the gene and
  signals the end of transcription.
• Noncoding sequences called introns are
  interspersed with the coding regions, called
  exons.

3
Figure 14.4 The Structure and Transcription of a
Eukaryotic Gene
4
The Structures of Protein-Coding Genes

• The entire sequence, including introns, is
  transcribed. The resulting RNA is the primary
  transcript, or pre-mRNA.
• The transcripts of the introns are removed from
  the pre-RNA and the transcripts of the exons are
  spliced together, resulting in mature mRNA.

5
RNA Processing

• The first two steps of processing pre-mRNA take
  place in the nucleus
• The G cap, a modified GTP, is added to the 5
  end. It facilitates the binding of mRNA to the
  ribosome and protects the mRNA from being
  digested by ribonucleases.
• A poly A tail is added to the 3 end. It is 100
  to 300 residues of adenine (poly A) in length.

6
Figure 14.9 Processing the Ends of Eukaryotic
Pre-mRNA
7
RNA Processing

• RNA splicing removes the introns and splices the
  exons together
• At the boundaries between introns and exons are
  consensus sequences.
• A small ribonucleoprotein particle (snRNP) binds
  to the consensus sequence at the 5 exonintron
  boundary.
• Another snRNP binds near the 3 exonintron
  boundary.
• Then other proteins bind, forming a large
  RNAprotein complex called a spliceosome. This
  complex cuts the RNA, releases the introns, and
  joins the ends of the exons.

8
Figure 14.10 The Spliceosome, an RNA Splicing
Machine (Part 1)
9
Figure 14.10 The Spliceosome, an RNA Splicing
Machine (Part 2)
10
Transcriptional Regulation of Gene Expression

• Each cell in a multicellular organism contains
  all the genes of the organisms genome.
• For normal development, the expression of genes
  must be regulated.
• Regulation of gene expression can occur at many
  points during development.
• Some mechanisms result in the selective
  transcription of specific genes.

11
Transcriptional Regulation of Gene Expression

• With few exceptions, all cells in an organism
  have the same genes, but they express them
  differently.
• For example, both brain and liver cells
  transcribe housekeeping genes that code for
  enzymes and other molecules essential to the
  survival of all cells.
• However, liver cells transcribe some genes for
  liver-specific proteins, and brain cells
  transcribe some genes for brain-specific
  proteins.
• The difference in the production of proteins is
  due to differential transcription.

12
Transcriptional Regulation of Gene Expression

• Most eukaryotic genes have other DNA sequences
  that regulate transcription.

13
Transcriptional Regulation of Gene Expression

• Transcription factors are regulatory proteins
  required for transcription in eukaryotes.
• RNA polymerase does not bind to the promoter
  until several other proteins, until other
  proteins have already bound the proteinDNA
  complex.
• Some DNA sequences, such as the TATA box, are
  common to most promoters others are unique to
  only a few genes.
• Transcription factors play an important role in
  cell differentiation during development.

14
Figure 14.12 The Initiation of Transcription in
Eukaryotes (Part 1)
15
Figure 14.12 The Initiation of Transcription in
Eukaryotes (Part 2)
16
Transcriptional Regulation of Gene Expression

• In addition to the promoter, nearby regulator
  sequences also affect transcription by binding
  regulator proteins that activate RNA polymerase.
• Much farther away are enhancer regions, which
  bind activator proteins and strongly stimulate
  the transcription complex.
• Negative regulatory regions of DNA called
  silencers bind proteins called repressors and
  turn off transcription. Thus they have the
  opposite effect of enhancers.

17
Figure 14.13 The Roles of Transcription Factors,
Regulators, and Activators (Part 1)
18
Figure 14.13 The Roles of Transcription Factors,
Regulators, and Activators (Part 2)
19
Transcriptional Regulation of Gene Expression

• In eukaryotes, genes on different chromosomes may
  require coordination.
• Regulation of various genes can be coordinated if
  all have the same regulatory sequences that bind
  to the same activators and regulators.
• One example is the stress response element in
  plants.
• Stress response elements near each of the
  scattered genes stimulate RNA synthesis.
• RNA then codes for proteins needed for water
  conservation.

20
Figure 14.14 Coordinating Gene Expression
21
Transcriptional Regulation of Gene Expression

• Other mechanisms that regulate transcription act
  on the structure of chromatin and chromosomes.
• The packaging of DNA by the nuclear proteins in
  chromatin can make DNA physically inaccessible to
  RNA polymerase and associated components.

22
Transcriptional Regulation of Gene Expression

• Nucleosomes inhibit initiation and elongation of
  transcription.
• Nucleosomes are inactivated by two protein
  complexes in a process called chromatin
  remodeling.
• Nucleosome disaggregation occurs by acetylation
  of amino groups on the histones, and is
  associated with the activation of genes.
• Nucleosomes reform by deacetylation of the amino
  groups, and is associated with gene deactivation.

23
Figure 14.16 Local Remodeling of Chromatin for
Transcription
24
Transcriptional Regulation of Gene Expression

• Two different kinds of chromatin can be
  distinguished by staining the interphase nucleus.
• Euchromatin stains lightly. It contains DNA that
  is transcribed into mRNA.
• Heterochromatin stains densely and is generally
  not transcribed. Any genes in heterochromatin are
  thus inactivated.

25
Posttranscriptional Regulation

• There are many ways in which gene expression can
  be regulated after transcription.
• Pre-mRNA can be processed in the nucleus by
  cutting and splicing.
• The longevity of mRNA in the cytoplasm can also
  be regulated.

26
Posttranscriptional Regulation

• Alternative splicing of a specific pre-mRNA can
  generate different proteins from a single gene.
• For example, cells in five different tissues
  splice the pre-mRNA for the structural protein
  tropomyosin into five different mRNAs.
• As a result, each of the five tissues in mammals
  (skeletal muscle, smooth muscle, fibroblast,
  liver, and brain) has a different form of
  tropomyosin.

27
Figure 14.20 Alternative Splicing Results in
Different mRNAs and Proteins
28
Translational and Posttranslational Regulation

• Proteins can regulate translation by binding to
  mRNA in the cytoplasm.
• This is important for long-lived mRNAs. It
  prevents the production of unnecessary proteins.
• For example, cyclin, which stimulates the cell
  cycle, must be shut off after it has done its
  job. If not, inappropriate cell division may lead
  to a tumor.

29
Recombinant DNA and Biotechnology
30
Cleaving and Rejoining DNA

• Recombinant DNA technology is the manipulation
  and combination of DNA molecules from different
  sources.
• Recombinant DNA technology uses the techniques of
  sequencing, rejoining, amplifying, and locating
  DNA fragments, making use of complementary base
  pairing.

31
Cleaving and Rejoining DNA

• Bacteria defend themselves against invasion by
  viruses by producing restriction enzymes
• Catalyze the cleavage of DNA into small
  fragments.
• Enzymes cut the bonds between the 3 hydroxyl of
  one nucleotide, and the 5 phosphate of the next.
• There are many such enzymes
• Enzymes recognize and cut a specific sequence of
  bases, called a recognition sequence or
  restriction site (4 to 6 base pairs long).

32
Figure 16.1 Bacteria Fight Invading Viruses with
Restriction Enzymes
33
Cleaving and Rejoining DNA

• Host DNA is not damaged due to methylation of
  certain bases at the restriction sites this is
  performed by enzymes called methylases.
• The enzyme EcoRI cuts DNA with the following
  paired sequence
• 5 ... GAATTC ... 3
• 3 ... CTTAAG ... 5
• Notice that the sequence is palindromic It reads
  the same in the 5-to-3 direction on both
  strands.

34
Cleaving and Rejoining DNA

• Using EcoRI on a long stretch of DNA would create
  fragments with an average length of 4,098 bases.
• Using EcoRI to cut up small viral genomes may
  result in only a few fragments.
• For a eukaryotic genome with tens of millions of
  base pairs, the number of fragments will be very
  large.
• Hundreds of restriction enzymes have been
  purified from various organisms, and these
  enzymes serve as knives for genetic surgery.

35
http//www.scq.ubc.ca/?p249
36
Cleaving and Rejoining DNA

• The fragments of DNA can be separated using gel
  electrophoresis. Because of its phosphate groups,
  DNA is negatively charged at neutral pH.
• When DNA is placed in a semisolid gel and an
  electric field is applied, the DNA molecules
  migrate toward the positive pole.
• Smaller molecules can migrate more quickly
  through the porous gel than larger ones.
• After a fixed time, the separated molecules can
  then be stained with a fluorescent dye and
  examined under ultraviolet light.

37
Figure 16.2 Separating Fragments of DNA by Gel
Electrophoresis (Part 1)
38
Figure 16.2 Separating Fragments of DNA by Gel
Electrophoresis (Part 2)
39
Figure 16.2 Separating Fragments of DNA by Gel
Electrophoresis (Part 3)
40
Cleaving and Rejoining DNA

• Electrophoresis gives two types of information
• Size of the DNA fragments can be determined by
  comparison to DNA fragments of known size added
  to the gel as a reference.
• A specific DNA sequence can be determined by
  using a complementary labeled single-stranded DNA
  probe.
• The specific fragment can be cut out as a lump of
  gel and removed by diffusion into a small volume
  of water.

41
Figure 16.3 Analyzing DNA Fragments
Gel is placed in solution to denature DNA
Known as a Southern blot
Nylon filter pick up DNA from the gel, creating a
blot.
The probe hybridizes with its own unique sequence
on the denatured DNA.
Filter is placed in a solution containing a
radioactively labeled ss DNA probe
42
Cleaving and Rejoining DNA

• Some restriction enzymes cut DNA strands and
  leave staggered ends of single-stranded DNA, or
  sticky ends, that attract complementary
  sequences.
• If two different DNAs are cut so each has sticky
  ends, fragments with complementary sticky ends
  can be recombined and sealed with the enzyme DNA
  ligase.
• These simple techniques, which give scientists
  the power to manipulate genetic material, have
  revolutionized biological science in the past 30
  years.

43
Figure 16.4 Cutting and Splicing DNA
44
Getting New Genes into Cells

• The goal of recombinant DNA work is to produce
  many copies (clones) of a particular gene.
• To make protein, the genes must be introduced, or
  transfected, into a host cell.
• The host cells or organisms, referred to as
  transgenic, are transfected with DNA under
  special conditions.
• The cells that get the DNA are distinguished from
  those that do not by means of genetic markers,
  called reporter genes.

45
Getting New Genes into Cells

• Bacteria have been useful as hosts for
  recombinant DNA.
• Bacteria are easy to manipulate, and they grow
  and divide quickly.
• They have genetic markers that make it easy to
  select or screen for insertion.
• They have been intensely studied and much of
  their molecular biology is known.

46
Getting New Genes into Cells

• Bacteria have some disadvantages as well.
• Bacteria lack splicing machinery to excise
  introns.
• Protein modifications, such as glycosylation and
  phosphorylation, fail to occur as they would in a
  eukaryotic cell.
• In some applications, the expression of the new
  gene in a eukaryote (the creation of a transgenic
  organism) is the desired outcome.

47
Getting New Genes into Cells

• Saccharomyces, bakers and brewers yeast, are
  commonly used eukaryotic hosts for recombinant
  DNA studies.
• In comparison to many other eukaryotic cells,
  yeasts divide quickly, they are easy to grow, and
  have relatively small genomes (about 20 million
  base pairs).

48
Getting New Genes into Cells

• Plants are also used as hosts if the goal is to
  make a transgenic plant.
• It is relatively easy to regenerate an entire
  plant from differentiated plant cells because of
  plant cell totipotency.
• The transgenic plant can then reproduce naturally
  in the field and will carry and express the gene
  on the recombinant DNA.

49
Getting New Genes into Cells

• New DNA can be introduced into the cells genome
  by integration into a chromosome of the host
  cell.
• If the new DNA is to be replicated, it must
  become part of a segment of DNA that contains an
  origin of replication called a replicon, or
  replication unit.

50
Getting New Genes into Cells

• New DNA can be incorporated into the host cell by
  a vector, which should have four characteristics
• The ability to replicate independently in the
  host cell
• A recognition sequence for a restriction enzyme,
  permitting it to form recombinant DNA
• A reporter gene that will announce its presence
  in the host cell
• A small size in comparison to host chromosomes

51
Getting New Genes into Cells

• Plasmids are ideal vectors for the introduction
  of recombinant DNA into bacteria.
• A plasmid is small and can divide separately from
  the hosts chromosome.
• They often have just one restriction site, if
  any, for a given restriction enzyme.
• Cutting the plasmid at one site makes it a linear
  molecule with sticky ends.
• If another DNA is cut with the same enzyme, it is
  possible to insert the DNA into the plasmid.
• Plasmids often contain antibiotic resistance
  genes, which serve as genetic markers.

52
Figure 16.5 (a) Vectors for Carrying DNA into
Cells
53
Getting New Genes into Cells

• Only about 10,000 base pairs can be inserted into
  plasmid DNA, so for most eukaryotic genes a
  vector that accommodates larger DNA inserts is
  needed.
• For inserting larger DNA sequences, viruses are
  often used as vectors.
• If the genes that cause death and lysis in E.
  coli are eliminated, the bacteriophage l can
  still infect the host and inject its DNA.
• The deleted 20,000 base pairs can be replaced by
  DNA from another organism, creating recombinant
  viral DNA.

54
Getting New Genes into Cells

• Plasmid vectors for plants include a plasmid
  found in the Agrobacterium tumefaciens bacterium,
  which causes the tumor-producing disease, crown
  gall, in plants.
• Part of the tumor-inducing (Ti) plasmid of A.
  tumefaciens is T DNA, a transposon, which inserts
  copies of itself into the host chromosomes.
• If T DNA is replaced with the new DNA, the
  plasmid no longer produces tumors, but the
  transposon still can be inserted into the host
  cells chromosomes.
• The plant cells containing the new DNA can be
  used to generate transgenic plants.

55
Getting New Genes into Cells

• When a population of host cells is treated to
  introduce DNA, just a fraction actually
  incorporate and express it.
• In addition, only a few vectors that move into
  cells actually contain the new DNA sequence.
• Therefore, a method for selecting for transfected
  cells and screening for inserts is needed.
• A commonly used approach to this problem is
  illustrated using E. coli as hosts, and a plasmid
  vector with genes for resistance to two
  antibiotics.

56
Figure 16.6 Marking Recombinant DNA by
Inactivating a Gene
57
Biotechnology Applications of DNA Manipulation

• Biotechnology is the use of microbial, plant, and
  animal cells to produce materialssuch as foods,
  medicines, and chemicalsthat are useful to
  people.
• The use of yeast to create beer and wine and
  bacterial cultures to make yogurt and cheese are
  examples of centuries-old biotechnology.
• Gene cloning techniques of modern molecular
  biology have vastly increased the number of these
  products beyond those that are naturally made by
  microbes.

58
Biotechnology Applications of DNA Manipulation

• Many medical products have been made using
  recombinant DNA technology.
• For example, tissue plasminogen activator (TPA),
  is currently being produced in E. coli by
  recombinant DNA techniques.
• TPA is an enzyme that converts blood plasminogen
  into plasmin, a protein that dissolves clots.
• Recombinant DNA technology has made it possible
  to produce the naturally occurring protein in
  quantities large enough to be medically useful.

59
Figure 16.14 Tissue Plasminogen Activator From
Protein to Gene to Drug
60
Table 16.1 Some Medically Useful Products of
Biotechnology
61
Biotechnology Applications of DNA Manipulation

• Selective breeding has been used for centuries to
  improve plant and animal species to meet human
  needs.
• Molecular biology is accelerating progress in
  these applications.
• There are three major advantages over traditional
  techniques
• Specific genes can be affected.
• Genes can be introduced from other organisms.
• Plants can be regenerated much more quickly by
  cloning than by traditional breeding.

62
Biotechnology Applications of DNA Manipulation

• Insecticides tend to be nonspecific, killing both
  pest and beneficial insects. They can also be
  blown or washed away to contaminate and pollute
  non-target sites.
• Bacillus thuringiensis bacteria produce a protein
  toxin that kills insect larvae pests and is
  80,000 times more toxic than the typical chemical
  insecticide.
• Transgenic tomato, corn, potato, and cotton
  plants have been made that produce a toxin from
  B. thuringiensis.

63
Biotechnology Applications of DNA Manipulation

• The process of producing pharmaceuticals using
  agriculture is nicknamed pharming.
• Transgenic sheep are being used to produce human
  a-1-antitrypsin (a-1-AT) in their milk this
  protein inhibits the enzyme elastase, which
  breaks down connective tissue in the lungs.
  Treatment with a-1-AT alleviates symptoms in
  people suffering from emphysema.
• Other products of pharming include blood
  clotting factors and antibodies for treating
  colon cancer.

64
Biotechnology Applications of DNA Manipulation

• Crops that are resistant to herbicides
• Glyphosate (Roundup) is a broad-spectrum
  herbicide that inhibits an enzyme system in
  chloroplasts that is involved in the synthesis of
  amino acids.
• A bacterial gene, which confers resistance to
  glyphosate, is inserted into useful food crops
  (corn, cotton, soybeans) to protect them from the
  herbicide, which otherwise would kill them along
  with the weeds.

65
Biotechnology Applications of DNA Manipulation

• Grains with improved nutritional characteristics
• Genes from bacteria and daffodil plants are
  transferred to rice using the vector
  Agrobacterium tumefaciens.
• Now a genetically modified strain of rice
  produces b-carotene, a molecule that is converted
  to vitamin A in animals.

66
Biotechnology Applications of DNA Manipulation

• There is public concern about biotechnology
• Genetically modified E. coli might share their
  genes with the E. coli bacteria that live
  normally in the human intestines.
• Researchers now take precautions against this.
  For example, the strains of E. coli used in the
  lab have a number of mutations that make their
  survival in the human intestine impossible.

67
Biotechnology Applications of DNA Manipulation

• There are concerns that genetic manipulation
  interferes with nature, that genetically altered
  foods are unsafe, and that genetically altered
  plants might allow transgenes to escape to other
  species and thus threaten the environment.
• Regarding safety for human consumption, advocates
  of genetic engineering note that typically only
  single genes specific for plant function are
  added.
• As plant biotechnology moves from adding genes to
  improve plant growth to adding genes that affect
  human nutrition, such concerns will become more
  pressing.

68
Biotechnology Applications of DNA Manipulation

• The risks to the environment are more difficult
  to assess.
• Transgenic plants undergo extensive field testing
  before they are approved for use, but the
  complexity of the biological world makes it
  impossible to predict all potential environmental
  effects of transgenic organisms.
• Because of the potential benefits of agricultural
  biotechnology, most scientists believe we should
  proceed, but with caution.

69
Biotechnology Applications of DNA Manipulation

• Insecticides tend to be nonspecific, killing both
  pest and beneficial insects. They can also be
  blown or washed away to contaminate and pollute
  non-target sites.
• Bacillus thuringiensis bacteria produce a protein
  toxin that kills insect larvae pests and is
  80,000 times more toxic than the typical chemical
  insecticide.
• Transgenic tomato, corn, potato, and cotton
  plants have been made that produce a toxin from
  B. thuringiensis.

70
Biotechnology Applications of DNA Manipulation

• With the exception of identical twins, each human
  being is genetically distinct from all other
  human beings.
• Characterization of an individual by DNA base
  sequences is called DNA fingerprinting.

71
Biotechnology Applications of DNA Manipulation

• Selective breeding has been used for centuries to
  improve plant and animal species to meet human
  needs.
• Molecular biology is accelerating progress in
  these applications.
• There are three major advantages over traditional
  techniques
• Specific genes can be affected.
• Genes can be introduced from other organisms.
• Plants can be regenerated much more quickly by
  cloning than by traditional breeding.

72
Biotechnology Applications of DNA Manipulation

• Insecticides tend to be nonspecific, killing both
  pest and beneficial insects. They can also be
  blown or washed away to contaminate and pollute
  non-target sites.
• Bacillus thuringiensis bacteria produce a protein
  toxin that kills insect larvae pests and is
  80,000 times more toxic than the typical chemical
  insecticide.
• Transgenic tomato, corn, potato, and cotton
  plants have been made that produce a toxin from
  B. thuringiensis.

73
Biotechnology Applications of DNA Manipulation

• The process of producing pharmaceuticals using
  agriculture is nicknamed pharming.
• Transgenic sheep are being used to produce human
  a-1-antitrypsin (a-1-AT) in their milk this
  protein inhibits the enzyme elastase, which
  breaks down connective tissue in the lungs.
  Treatment with a-1-AT alleviates symptoms in
  people suffering from emphysema.
• Other products of pharming include blood
  clotting factors and antibodies for treating
  colon cancer.

74
Biotechnology Applications of DNA Manipulation

• Crops that are resistant to herbicides
• Glyphosate (Roundup) is a broad-spectrum
  herbicide that inhibits an enzyme system in
  chloroplasts that is involved in the synthesis of
  amino acids.
• A bacterial gene, which confers resistance to
  glyphosate, is inserted into useful food crops
  (corn, cotton, soybeans) to protect them from the
  herbicide, which otherwise would kill them along
  with the weeds.

75
Biotechnology Applications of DNA Manipulation

• Grains with improved nutritional characteristics
• Genes from bacteria and daffodil plants are
  transferred to rice using the vector
  Agrobacterium tumefaciens.
• Now a genetically modified strain of rice
  produces b-carotene, a molecule that is converted
  to vitamin A in animals.

76
Biotechnology Applications of DNA Manipulation

• Crops that adapt to the environment
• A gene was recently discovered in the thale cress
  (Arabidopsis thaliana) that allows it to thrive
  in salty soils.
• When this gene is added to tomato plants, they
  can grow in soils four times as salty as the
  normal lethal level.
• This finding raises the prospect of growing
  useful crops on previously unproductive soils
  with high salt concentration.
• Biotechnology may allow us to adapt plants to
  different environments.

77
Biotechnology Applications of DNA Manipulation

• With the exception of identical twins, each human
  being is genetically distinct from all other
  human beings.
• Characterization of an individual by DNA base
  sequences is called DNA fingerprinting.

78
Biotechnology Applications of DNA Manipulation

• Scientists look for DNA sequences that are highly
  polymorphic.
• Sequences called VNTRs (variable number of tandem
  repeats) are easily detectable if they are
  between two restriction enzyme recognition sites.
• Different individuals have different numbers of
  repeats. Each gets two sequences of repeats, one
  from the mother and one from the father.
• Using PCR and gel electrophoresis, patterns for
  each individual can be determined.

79
Figure 16.17 DNA Fingerprinting
80
Biotechnology Applications of DNA Manipulation

• The many applications of DNA fingerprinting
  include forensics and cases of contested
  paternity.
• DNA from a single cell is sufficient to determine
  the DNA fingerprint because PCR can amplify a
  tiny amount of DNA in a few hours.
• PCR is used in diagnosing infections in which the
  infectious agent is present in small amounts.
• Genetic diseases such as sickle-cell anemia are
  now diagnosable before they manifest themselves.

81
Biotechnology Applications of DNA Manipulation

• There is public concern about biotechnology
• Genetically modified E. coli might share their
  genes with the E. coli bacteria that live
  normally in the human intestines.
• Researchers now take precautions against this.
  For example, the strains of E. coli used in the
  lab have a number of mutations that make their
  survival in the human intestine impossible.

82
Biotechnology Applications of DNA Manipulation

• There are concerns that genetic manipulation
  interferes with nature, that genetically altered
  foods are unsafe, and that genetically altered
  plants might allow transgenes to escape to other
  species and thus threaten the environment.
• Regarding safety for human consumption, advocates
  of genetic engineering note that typically only
  single genes specific for plant function are
  added.
• As plant biotechnology moves from adding genes to
  improve plant growth to adding genes that affect
  human nutrition, such concerns will become more
  pressing.

83
Biotechnology Applications of DNA Manipulation

• The risks to the environment are more difficult
  to assess.
• Transgenic plants undergo extensive field testing
  before they are approved for use, but the
  complexity of the biological world makes it
  impossible to predict all potential environmental
  effects of transgenic organisms.
• Because of the potential benefits of agricultural
  biotechnology, most scientists believe we should
  proceed, but with caution.