Unit 5B Molecular Genetics

1
Unit 5B Molecular Genetics

• Section 1 Mutations

2
Mutations

• Area changes in a nucleotide sequence of the DNA.
• The actual mutation rate is very low as compared
  to the damage rate of DNA due to enzymes which
  specifically repair the DNA.
• Remember Once the DNA sequence has been changed
  / damaged / altered, the DNA molecule will
  continue to reproduce the strand with the
  problem.
• Once the mutation occurs, the chance of another
  mutation occurring in the same strand or at the
  same place does not change, meaning there is
  always an equal chance for mutations.

3
Mutations can be Good

• Mutations can provided for genetic diversity
• Makes gene pool stable with less recessive
  alleles.
• Allows for the study of inheritance.
• Can lead to evolutionary changes in a population.
• If in intron region does not matter.

4
Mutations

• Mutations are changes in the coding of a gene or
  sets of genes
• They can be brought about by mistakes in the
  reading and coding processes
• They can be brought about by chemical treatments
  and pollutants such chemicals are called
  mutagens
• Many mutagens can cause cancer carcinogens
• Classes of mutations
• Chromosomal
• Point mutations
• Frameshift (changes in 1 or 2 bases)

5
Chromosomal Mutations

• Deletion or addition of an additional segment
• Breakage
• Effects 1 to multiple proteins lost very
  serious, often lethal
• Types
• Deletion
• Duplication / Addition
• Inversion
• Insertion
• Translocation

6
1. Chromosome Mutations Cont

• Nondisjunction when chromosomes do not separate
  appropriately in meiosis.
• Typically there is a problem with the kinetochore
  / centromere / spindle fibers.
• Could occur in Meiosis I or II

7
Another Picture of Nondisjunction
8
2. Point Mutations 3 Types

• Base Substitution
• Sometimes no effect, due to redundancy in coding
  for instance, replace AAG (lysine) with AAA (also
  codes for lysine)
• Missense Mutation
• Replace a base so the mRNA does not code for the
  AA correctly.
• Sometimes these are silent nothing happens and
  other times they can be problematic.
• Sometimes the new AA is so alike the other, there
  is no change.
• Nonsense
• The code is changed so that the codon becomes a
  stop codon and no AA is made.
• Destroys the protein being made.

9
Point Mutations With Effect
10
3. Frameshift Mutations

• The starting point is very important for how the
  genetic data are read this because the data are
  read in groups of three bases called codons
• A frameshift mutation occurs when there is a
  shift in the reading frame away from the original
  alignment
• Removal or addition of one or more bases shifts
  the reading frame A change in 1-2 bases
  substantially changes the output

11
3. Frameshift Mutations Cont

• Example using English as an analog system 2
  types possible
• ORIGINAL THEMANCANRUNNOW
• Reads THE MAN CAN RUN NOW
• INSERTION mutation THEMTANCANRUNNOW
• Reads THE MTA NCA NRU NNO W
• DELETION mutation THEMNCANRUNNOW
• Reads THE MNC ANR UNN OW
• red bar indicates the removal of A

12
3. Frameshift Mutations Cont
13
Types of Disorders due to Mutations

• Chromosomal Mutation Disorders
• Deletions
• Wolf-Hirschhorn Syndrom 4p
• Jacobsen Syndrome 11q
• Duplications
• Charcot-Marie-Tooth Disease 17 Peripheral
  Mylein Protein gene
• Translocations (usually on 13, 14, 15, 21, 22)
• Robertsonian Translocation
• Inversions
• ?
• http//en.wikipedia.org/wiki/Chromosome_abnormalit
  ies

14
Types of Disorders due to Mutations

• Nondisjunction Disorders
• Down Syndrome 21
• Down Syndrome 13
• Turner Syndrome XO
• Kleinfelter Syndrome XXY

• Base Pair Disorders
• Cystic Fibrosis
• Sickle Cell Anemia
• Frameshift Disorders
• Abnormal Nephron Development

15
Unit 5B Molecular Genetics

• Section 2 Viruses

16
Viruses

• Noncellular
• Considered to be nonliving because they are not
  capable of replicating with only their own
  mechanisms they are obligate parasites that take
  over cell function intimately associated with
  living organisms.
• Many are causes of disease
• Involved in many ecological processes (disasters)
• Can influence biodiversity through transfer of
  genetic material

17
Virus Organization

• Very tiny
• Small viruses 20 nm larger ones up to 400-500
  nm
• Virion is the name for the infective particle
  that enters other cells
• Nucleic acid core the genome
• Protein capsid used for protection of the
  virion
• Noncellular
• No metabolism
• No respiration, replication or transcription of
  nucleic acid, no translation
• Reproduce only within body of cell are built by
  cells processes

18
DNA and RNA Viruses

• Animal viruses can contain DNA or RNA as a genome
• DNA viral genomes are replicated like the genomes
  of the host cell
• RNA viruses usually replicate their RNA with
  RNA-dependent RNA polymerases
• However, certain RNA viruses must produce a DNA
  intermediate in order to produce more of
  themselves.
• Such viruses are called retroviruses
• They use RNA-dependent DNA polymerase also
  called reverse transcriptase, to make DNA.
• The DNA is then incorporated into the host genome
  by a viral gene product.
• Host RNA polymerases make RNA from the
  incorporated DNA this is then packaged into the
  virions.
• HIV (human immunodeficiency virus) is a
  retrovirus so are some cancer viruses

19
Reversal of the Central Dogma

• Usually information flows from DNA to RNA to
  Protein
• But some tumor viruses use RNA as the genetic
  system (discovered by Temin, 1964)
• Temin proposed a DNA provirus would be made that
  would be an intermediate that would be used to
  make RNA for viral encapsulation
• 1970 Temin and Baltimore discovered the enzyme,
  RNA-dependent DNA polymerase ( reverse
  transcriptase)
• Coded by all tumor viruses
• There are exceptions to this system in tumor
  viruses
• Such viruses are called retroviruses because of
  their backward information flow
• HIV is one such example

20
HIV Infection

• Fusion and uptake of the human immunodeficiency
  virus.

21
HIV Replication
22
Videos of Retroviruses

• http//www.tulane.edu/dmsander/WWW/Video/HIV.mov
• http//highered.mcgraw-hill.com/sites/0072437316/s
  tudent_view0/chapter26/animations.html

23
Unit 5B Molecular Genetics

• Section 3 Biotechnology /
• Genetic Engineering

24
A Revolution in Biological Science

• In mid-1970s, newly developed recombinant DNA
  technology allowed biologists to redirect the
  genetic activity of organisms
• Recombinant DNA Technology DNA from different
  organisms are put together
• Genetic Engineering modification of DNA of an
  organims to produce new genes with specific
  characteristics.
• Biotechnology using biological organisms to
  increased humanity make life better.

25
History and Terminology

• Biotechnology has its roots in microbiology the
  study of micro- organisms (usually bacteria)
• Bacteriophage (viruses of bacteria) were first
  used to try to understand how DNA worked (recall
  Hershey-Chase)
• Scientists learned how to make bacteria competent
  for transformation (recall Griffith) by
  modification of the ionic environment
• Made the cell wall more permeable
• Allowed the cells to take up DNA
• Genetic engineering not possible prior to
  discovery of restriction endonucleases
  (restriction enzymes) by Ham Smith and Daniel
  Nathans (Johns Hopkins Nobel Prize winners
  1978)
• Specifically clip strands of DNA
• Many different types

26
Problem

• A diabetic is rejecting all types of animal
  insulin. The final decision is that he needs
  human insulin. What can we do?

27
Answer

• Create using bacteria
• Splice the required gene from human DNA using a
  restriction enzyme.
• Insert the DNA into the bacteria with a vector.
• Grow the bacteria and allow the bacteria to make
  the insulin.
• Retrieve / harvest the insulin.

28
Overview

• Restriction Enzymes / Splicing / Vectors
• Genomic Libraries
• PCR Polymerase Chain Reaction
• EGP Electrogelphoresis
• DNA Sequencing
• RFLP Restriction Fragment Length Polymorphism
• Relationships
• DNA Fingerprinting

29
Genomic Libraries - cDNA

• The genome is the total DNA in a cell.
• A genomic library is a collection of DNA
  fragments that represent the genome all or
  nearly all the DNA must be present in the library
• The fragments do not have introns only exons.
• A genomic library is made by cutting the total
  DNA with a given restriction enzyme.
• Allows scientists to find various restriction
  enzymes to identify genes.
• The collection of original DNA represented by the
  various bacterial lines constitute the genomic
  library
• Clemson University has a genomic library

30
Genomic Libraries - Negatives

• Genomic libraries have some drawbacks.
• Do not contain introns noncoding regions
• Clip points dont necessarily match open reading
  frames (ORFs) of genes. Several genomic segments
  must be studied to understand the gene and its
  expression
• So, not optimal for study of genes that encode
  proteins
• You have to work backwards
• Expression, or complementary ( cDNA) libraries,
  are derived from the mRNA
• Thus, a cDNA library is complementary to its mRNA
  and does not contain introns
• Comparison of cDNA and genomic DNA allows
  identification of introns and exons but is
  tedious work.

31
Splicing - Restriction Enzymes

• Restriction enzymes are natural, molecular
  scissors normally used to destroy non-host DNA
  (such as bacteriophage)
• Restriction enzymes run along the DNA and cut at
  specific base pair sequences many are
  palindromic
• A nucleic acid palindrome has the same sequence
  on two antiparallel, complementary,
  hydrogen-bonded strands
• e.g. AACGTT will pair with TTGCAA these are
  palindromes or opposites
• The DNA is cut in a staggered fashion and now has
  a sticky end.
• The ends are put together with other strands of
  DNA from other organisms and DNA ligase is used
  to connect them together.
• (When heated, these ends come apart very easily.)

32
Splicing - Restriction Enzyme Specificity

• Restriction enzymes vary in the number of DNA
  bases they recognize from as few as 4 to as
  many as 23
• Restriction enzymes are categorized as n-cutter
  enzymes 4-cutter up to 23-cutter
• Randomly occurring 4-base sequences on average
  are found much more often than 23-base sequences
• Restriction enzymes that recognize large numbers
  of bases tend to cut more accurately because the
  longer the sequence needed for recognition, the
  more specific the recognition a 23-cutter has
  much better specificity than a 4-cutter
• You would choose your restriction enzyme based on
  the sequence you are looking for.

33
Splicing - Restriction Enzyme Specificity Why
Use?

• To look for specific gene sequences to determine
  what is on the chromosome.
• You work backwards from the proteins.
• This is what helped the Human Genome Project

34
Splicing How it works.
35
Splicing - Vector DNA

• A vector is a genome that carries foreign DNA
  into a host cell, usually bacteria.
• Used to transform bacteria that can take up DNA
• Bacteriophage (bacterial viruses (Hershey-Chase
  / Griffith)
• Remember Prokaryote DNA are a circular double
  strand.
• Plasmids are small rings of double-stranded DNA
  with a gene from another organism and are called
  vectors.
• Can carry DNA segments (10kb in size 1Kb1000
  bps)
• Once a plasmid has been injected into bacteria,
  the bacteria utilize the plasmid in its DNA and
  create the protein.
• When mitosis occurs, the gene is replicated and
  more bacteria make the protein. You have cloned
  the recombinant gene.

36
Vector DNA How it works.
37
Why Use?
Genetic Engineering - Vector DNA

• Often carry genes for resistance to antibiotics
• Can test the use of antibiotics
• Example Can be used to provide a selectable
  marker which allows only transformed cells to
  live. Cells containing ampicillin resistance gene
  inserted by transformation can be grown on
  ampicillin-rich media nontransformed cells die
• Can make synthetic drugs
• Example Insulin

38
Vectors - Plasmids and Bacteria
39
(No Transcript)
40
Vectors Limiting Properties

• Size of DNA that can be used is about 15 kb which
  is about 15,000 bps.
• Certain organisms do not accept vectors.

41
Specific DNA Sequences Can Be Detected by
Complementary Probes

• A probe can be a radioactively labeled segment of
  RNA or single-stranded DNA (ssDNA) complementary
  to the target sequence
• To test whether a certain gene is present in any
  particular clone, it is necessary to identify
  colonies whose cells contain DNA that hybridizes
  via hydrogen-bonding with a probe containing a
  sequence complementary to the gene

42
PCR Is Used to Amplify DNA in Vitro

• The Polymerase Chain Reaction (PCR) allows
  amplification of a small amount of targeted DNA
  in a short time. It is very simple but very
  powerful.
• PCR has three steps
• Denaturation. A buffered mixture of primers,
  nucleotides, Taq polymerase and DNA fragments is
  heated to dissociate dsDNA into ssDNA
• Annealing of primers. The solution is cooled and
  the primers bind to complementary sequences of
  the DNA at the ends
• Primer Extension. DNA polymerase then uses the
  nucleotides to extend and make more copies of
  each strand
• The process is repeated over and over to produce
  millions of copies of the original DNA strand

43
PCR
44
PCR Characteristics

• DNA Taq polymerase isolated from the thermophilic
  bacterium Thermus aquaticus is used as it is not
  damaged by the heat
• This polymerase enzyme is very stable and can
  withstand high temperatures
• After 20 cycles a single fragment produces more
  than one million (220) copies
• 30 cycles will produce a billion times the
  original amount (230), enough amplification to
  reveal the presence of a single copy of a
  specific target sequence
• Very sensitive and samples easily contaminated
• http//video.google.com/videoplay?docid4121243690
  121066829qPolymeraseChainReactionVideostotal
  5start0num10so0typesearchplindex0

45
PCR Application

• The use of PCR is virtually limitless
• Criminal investigations (DNA fingerprints) from a
  speck of blood or single hair
• Detection of genetic defects in very early
  embryos by collecting a few sloughed-off cells
  from the amniotic fluid (amniocentesis) and
  amplifying the DNA
• Used to examine historical figures and extinct
  species such as mammoths and dodos

46
PCR Problems

• Easily contaminated since it is so sensitive.
• So if you have any foreign extra DNA it will
  get amplified also and you may get a positive
  match.
• Example Lab Technician

47
Gel Electrophoresis is Widely Used to Separate
DNA and RNA

• DNA and RNA are negatively charged, and move
  through a gel at varying speeds due to different
  molecular lengths (sizes)
• Restriction endonucleases can be used to clip the
  DNA
• DNA fragments are loaded on a gel an electric
  field is applied
• Bigger DNA fragments migrate through the gel more
  slowly than small fragments
• Moves from the negative to positive end because
  the DNA is negative (due to phosphates)

48
EGP What to do now?

• Fragments can be stained and visualized migrating
  as bands under UV light
• DNA is hard to work with on the gel so the
  fragments can be transferred (blotted) to a
  filter, denatured and incubated (grown) with a
  radioactive or fluorescent probe which will
  hybridize to the target sequence to perform a
• Southern Blot
• Northern Blot
• Western Blot

49
Southern Blotting

• Blots for DNA are called Southern blots
• Named after its inventor, E.M. Southern (1975)
• DNA is separated on a gel (EGP)
• Gel is transferred onto nitrocellulose or a nylon
  membrane
• Membrane is incubated with radioactive ssDNA
  probe of the gene of interest
• Probe hybridizes to the blot where there is a
  fragment with a complementary sequence
• The radioactive bands on the blot identify
  fragments of interest such as mutations because
  they will move differently on the gel bed than
  normal

50
Northern Blotting

• Same thing as Southern Blotting but it is used to
  study RNA rather than DNA.
• The chemicals used are slightly different but the
  process is the same.

51
Western Blotting

• Proteins separated in a gel
• Proteins blotted onto a membrane
• Antibodies specific for a particular protein are
  applied
• Antibodies stick to target proteins ONLY
• Revealed by additional antibodies attached to
  enzymes that precipitate a colored product
• Used to detect the proteins made by the HIV virus

52
DNA Sequences Contain Much Information

• Can determine the actual protein encoding
  regions the ORFs
• Regions containing transcriptional signals and
  RNA processing can be recognized
• Amino acid sequences of proteins can be inferred
  from the base sequence much faster and easier
  than from the protein directly
• Introns are missing
• Reveals structure of chromosomes, possibly
  helpful in determining evolution, phylogenies,
  and fighting disease

53
DNA Nucleotide Sequencing

• Radioactive DNA is replicated off the host
  template DNA
• Dideoxynucleotides (ddNTPs lacking OH at 3 and
  2) are incorporated in small quantities in the
  reaction mixture to label sequences which contain
  those deoxybases (the ddNTPs jam DNA polymerase
  because of the lack of OH-)
• Prvents phosphodiester bonds from linking the
  nucleotides.
• Reaction mixtures contain DNA polymerase,
  radioactive primers, single-stranded DNA
  fragment, 4 deoxynucleotides. Four tubes are
  prepared each containing a different
  dideoxynucleotide (ddATP, ddCTP, ddGTP or ddTTP)
• Fragments of varying length are formed in each
  mixture the end points occur at the 4 different
  ddNTP
• Fragments are separated based on length by
  electrophoresis
• Autoradiography reveals the presence of the
  radioactively-labeled DNA fragments
• The DNA sequence is literally read off of the
  gel, using the 4 lanes derived from the 4
  reaction tubes.

54
(No Transcript)
55
(No Transcript)
56
Restriction Fragment LengthPolymorphism (RFLP)
Analysis

• RFLPs can be used to measure the genetic
  relationship between individuals.
• Each individual carries with it a record of the
  variation in genetic organization from its
  previous generations. There is a LOT of variation
  in individuals of a population in the introns
• Except in mitochondria very stable and has not
  changed much.
• Restriction enzymes are used to cut DNA into
  fragments
• The fragments are portions of DNA that contain
  introns and exons you are looking for
  similarities between two samples. The more
  similar they are, the more closely related the
  two organisms are.
• The fragments are of different lengths in
  different individuals since each host DNA is
  unique. When three different individuals DNA are
  cut with a restriction enzyme, 3 different
  fragment sizes are likely to be produced, unless
  they are identical triplets
• Used to
• Determine how closely organisms are related.

57
An RFLP Autoradiogram

• A DNA fingerprint produced by gel
  electrophoresis reveals different banding
  patterns restriction fragment length
  polymorphisms (RFLPs)
• This technology is particularly important in
  determination of paternity and in forensics
• Here, M mother, F father, and C children.
  Note that children have all bands of M and F
  lanes.

58
Genetic Engineering Applications

• Transgenic organisms containing foreign DNA in
  their cells are valuable in research many have
  commercial uses
• Transgenic bacteria now produce insulin for use
  by human diabetics
• Human growth hormone was found only in cadavers
  in small quantities now is made by transgenic
  bacteria, is very helpful for children lacking
  sufficient HGH
• Transgenic animals may be produced by injecting
  the DNA of interest into a fertilized egg cell
• Transgenic sheep have been produced that produce
  useful proteins in their milk

59
Genetic Engineering Examples - Transgenic Mouse

• RGH (Rat Growth Hormone) gene was combined with a
  metalothionein gene, which is stimulated by zinc
  and injected into mouse eggs
• Every time the metalothionein gene was stimulated
  then the RGH was stimulated also.
• Transgenic baby mice were stimulated to grow with
  small dosage of zinc diet became much larger
  than normal
• Potential
• Acondraplasia Humans

60
Mice Knockouts / Gene Therapy

• Genes can be targeted for reduction or
  elimination of function in order to study what is
  missing.
• A nonfunctional gene (knockout allele) is
  inserted into cultured mouse embryonic stem (mES)
  cells
• In some of the cultured stem cells, the gene will
  insert into a chromosome by homologous
  recombination, where the normal allele is
  replaced with the knockout allele this is not
  really understood well.
• Modified mES cells are injected into mouse
  embryos, allowed to develop to maturity. The
  resulting adults are bred for several
  generations, during which researchers attempt to
  find homozygous knockouts for the gene of
  interest
• Can cause the gene targeting to be activated only
  in certain cell types or certain organs, to try
  to understand gene action in those cell types.
• Method is being used to study cardiac, liver,
  cystic fibrosis, and sickle cell.
• Potential to knockout genetic disorders.

61
Knockout Visual
62
Transgenic AnimalsAre Increasingly Important

• Genes can be introduced to animals.
• Human blood clotting factor can be made by the
  sheep and secreted in sheep's milk, which
  hemophiliacs could drink rather than having
  clotting factor transfusions.
• Organism is not harmed
• Get large quantities of products quickley

63
Transgenic PlantsAre Increasingly Important

• Agrobacterium (crown gall bacterium) is often
  used as a vector in plant biotechnology
• The Ti (tumor-inducing) plasmid is used as a
  vector mainly in dicot plants causes elevated
  production of plant growth hormone (cytokinin)
  get bigger plants
• Partially inactivated Ti plasmid (doesnt produce
  galls) is used as a general vector
• Is sometimes hard to get plants to take up DNA
  Genetic shotguns have been developed in which
  gold or other particles coated with DNA are fired
  into the plant cells directly through the cell
  wall modify the plant
• Disease resistance has been transferred to rice,
  also insect resistance in cotton, corn, tobacco
  and other crops.
• Chloroplast engineering is also an active area of
  research

64
Other Videos

• http//highered.mcgraw-hill.com/sites/0072437316/s
  tudent_view0/chapter16/animations.html

65
Safety Guidelinesfor Recombinant Technology

• Initial concerns about the accidental release of
  genetically engineered microbes has not borne
  fruit.
• Initial restrictions have been relaxed somewhat
  with gt20 years experience in this type of work.
• Even so, stringent restrictions do persist in
  biotechnology areas that are poorly understood or
  where there could be potentially hazardous
  repercussions
• Genetic engineering affords great opportunities
  for progress in medicine, food production, and
  forensics. Although many are concerned about the
  possible risks of genetic engineering, the risks
  are in fact slight and the potential benefits,
  such as greatly increased availability of food,
  are great.

66
Unit 5B Molecular Genetics

• Section 4 Genetic Disorders

67
Fragile X
68
Fragile X
69
Turner Syndrome
70
Turner Syndrome
71
Cri-Du-Chat
72
Cri-Du-Chat
73
Down Syndrome
74
Down Syndrome
75
Klinefelter
76
Phenylketonuria (PKU)
77
Cystic Fibrosis
78
Cystic Fibrosis
79
Sickle Cell Anemia
80
Sickle Cell Anemia
81
Tay Sachs
82
Tay Sachs