CH 14 Applications of Recombinant DNA Technology

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CH 14Applications of Recombinant DNA Technology
Analysis of Biological Processes 1. Developments
in DNA technology have allowed advances in
research. Useful new techniques include a.
Restriction mapping. b. Southern blotting to
identify genomic regions. c. Northern blotting to
study RNA. d. DNA sequencing. 2. Applications
of these technologies have been applied to the
study of a. Functional organization of genes and
regulation of gene expression. b. Key regulatory
and target genes in development. c. Genetic
influences on cancer and aging. d. Evolutionary
relationships between organisms.

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Using Northern Blots to study transcriptionGluco
se Repression of Transcription of the Yeast GAL1
Gene

• GAL (galactose) genes in the yeast Saccharomyces
  cerevisiae encode enzymes for break down of
  galactose, which serves as their inducer.
• Glucose represses GAL gene expression, and
  existing GAL mRNAs are degraded, as shown by
  Northern blot analysis of yeast samples taken at
  time points and probed with the GAL genes.
• Glucose added at time zero and amount of GAL1
  transcripts analyzed at various times thereafter
  by Northern blotting and probing

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DNA Molecular Testing for Genetic Disease
Mutations

• 1. Many human diseases result from protein
  defects caused by DNA mutations. DNA testing is
  increasingly available for genetic diseases,
  including
• a. Huntington disease.
• b. Hemophilia.
• c. Cystic fibrosis.
• d. Tay-Sachs disease.
• Sickle-cell anemia.

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Concept of DNA Molecular Testing

• 1. Designing DNA molecular tests requires
  knowledge of gene mutations that cause a disease,
  derived from sequencing the gene involved.
• 2. Often many different mutations of a gene can
  cause loss of function and lead to disease. The
  breast cancer genes BRCA1 and BRCA2 are examples
• a. Normal BRCA1 and BRCA2 genes control cell
  growth in breast and ovarian tissue.
• b. Mutations in the BRCA1 and BRCA2 genes can
  lead to cancer. Hundreds of mutations in these
  genes have been identified.
• Each BRCA1 or BRCA2 mutation confers a different
  risk of developing cancer, ruling out a single
  DNA molecular test to assess an individuals
  breast cancer risk associated with these genes.
• Genetic testing reveals the presence of a
  mutation associated with a genetic disease.
  Genetic testing is usually done on a targeted
  population of people with symptoms or a family
  history of the disease.

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Purposes of Human Genetic Testing

• 1. Human genetic testing serves three main
  purposes
• a. Prenatal diagnosis (fetal analysis).
• b.
• c.

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Fetal Analysis

• Fetal Analysis is concerned with whether a fetus
  is normal. A sample of fetal cells is needed for
  the analysis. There are currently two methods of
  obtaining the necessary sample

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Fetal Analysis
Fig. 10.11 Amniocentesis, a procedure used for
prenatal diagnosis of genetic defectsAmniocentes
is is -Fluid contains fetal skin
cells -Seldom done before the 12th week of
pregnancy, due to small amounts of amniotic fluid
and risk to the fetus. -Cells cultured and
examined -Complicated and costly, so used
primarily in high risk cases
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Fetal Analysis Fig. 10.12 Chorionic villus
sampling, a procedure used for early prenatal
diagnosis of genetic defects

• Chorionic villus sampling can be done earlier, in
  the 8th12th weeks of pregnancy, by removal of
  chorionic villus tissue either through the
  abdomen as in amniocentesis, or via the vagina
• Chorion is a

Fig 10.12
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Fetal Analysis

• Once fetal cells are obtained they are usually
  cultured in the laboratory, although chorionic
  villus sampling may provide enough tissue to
  assay directly.
• Cells are examined for protein or enzyme
  alterations or deficiencies, DNA mutations and
  chromosomal abnormalities.
• Amniocentesis is costly and cannot be performed
  until the second trimester, removing early
  abortion as an option in cases of severe genetic
  defects.
• Chorionic villus sampling can be done earlier,
  but carries a higher risk of fetal death and
  inaccurate diagnosis due to the presence of
  maternal cells.

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Fetal Analysis

• If both parents are carriers (heterozygotes) for
  the mutant allele, the probability is 1/4 that
  the fetus is an affected homozygote, 1/2 that it
  is a carrier, and 1/4 that it is homozygous for
  the normal allele. Genetic testing can determine
  the result of a particular conception.
• b. Genetic testing may be used during in vitro
  fertilization to eliminate before implantation
  embryos with mutated genes that could result in
  serious disease... Ethical concerns?

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Newborn screening

• Examples of tests for specific mutations using
  blood from newborns include
• a. Phenylketonuria (PKU)
• b. Sickle-cell anemia
• c. Tay-Sachs disease

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Phenylketonuria (PKU)

• Phenylketonuria (PKU) is commonly caused by a
  mutation on chromosome 12 in the phenylalanine
  hydroxylase (PAH) gene (12q24.1)
• 2. Phenylalanine is an essential amino acid, but
  excess is harmful, and so is normally converted
  to tyrosine. Excess phenylalanine affects the
  CNS, causing mental retardation, slow growth and
  early death.

Tyrosinase
PAH
Fig. 10.1 Phenylalanine-tyrosine metabolic
pathways
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Phenylketonuria (PKU)

• PKUs effect is pleiotropic (wide reaching). Some
  symptoms result from excess phenylalanine. Others
  result from inability to make tyrosine these
  include fair skin and blue eyes (even with
  brown-eye genes) and low adrenaline levels.
• Diet is used to manage PKU by providing just
  enough phenylalanine for protein synthesis, but
  not enough that it accumulates. To be effective,
  the special diet must commence in the first two
  months after birth, continue at least throughout
  childhood, and be resumed before pregnancy in PKU
  women to avoid phenylalanine levels that would
  affect the fetus.
• 5. All U.S. newborns are screened for PKU using
  the Guthrie test
• a. A drop of blood on filter paper is placed on
  solid media containing b-2-thienylalanine and the
  bacterium Bacillus subtilis.
• b. Normally, b-2-thienylalanine inhibits growth
  of Bacillus subtilis.
• Phenylalanine allows Bacillus subtilis to grow in
  the presence of b-2-thienylalanine, so bacterial
  growth indicates high phenylalanine levels in the
  blood, and the possibility that the infant has
  PKU.
• 6. NutraSweet ...

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Sickle Cell Anemia

• J. Herrick (1910) first described sickle-cell
  anemia, finding that red blood cells (RBCs)
  change shape (form a sickle) under low O2
  tension.
• Sickled RBCs are fragile, hence the anemia.
• They are less flexible than normal RBCs, and form
  blocks in capillaries, resulting in tissue damage
  downstream.
• Effects are pleiotropic, including damage to
  extremities, heart, lungs, brain, kidneys, GI
  tract, muscles and joints. Results include heart
  failure, pneumonia, paralysis, kidney failure,
  abdominal pain and rheumatism.
• d. Heterozygous individuals have

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Sickle Cell Anemia

• 2. Linus Pauling and coworkers (1949) used
  electrophoresis and showed
• Hemoglobin from individuals with sickle-cell
  anemia (Hb-S) has altered mobility compared with
  normal hemoglobin (Hb-A).
• Hemoglobin from individuals with the sickle-cell
  trait shows equal amounts of Hb-A and Hb-S,
  indicating that
• Therefore, the sickle-cell mutation changes the
  form of its corresponding protein, and protein
  structure is controlled by genes.

Sample loaded
-
Fig. 10.6 Electrophoresis of hemoglobin variants
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Human ?-globin

• ?-globin subunit of hemoglobin
• Hemoglobin
• Binds O2 in lungs
• Delivers O2 to tissues in exchange for CO2
• Binds CO2 in tissues releases it in the lungs
  (although most CO2 carried by bicarbonate)

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Human ?-globin
Most aa coil into ?-helices fold around heme
group
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Human ?-globin
Heme groups iron atom surrounded by
protoporphyrin ring Heme groups are examples of
nonprotein substances added to proteins after
translation
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Human Hemoglobin
Hemoglobin is formed by four polypeptide chains,
two molecules of the a polypeptide and 2 of the b
polypeptide, each associated with a heme group
Quaternary
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Human Hemoglobin

• How does hemoglobin bind O2 and CO2?
• Hydrophobic heme groups in pocket lined with
  hydrophobic amino acids (both ? ? subunits)
• O2 binds to hydrophobic heme pocket
• Surface amino acids of hemoglobin are
  hydrophilic
• Allows hemoglobin to function in erythrocytes
• Arginine at carboxyl end of ?-globin bind CO2
• Cant bind O2 and CO2 simultaneously
• CO2 binding
• Release of CO2

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Sickle Cell Anemia

• V.M. Ingram (1956) found that the 6th amino acid
  of the b chain in sickle-cell hemoglobin is
  valine (no electrical charge) rather than the
  negatively charged glutamic acid in the b chain
  of normal hemoglobin
• Outline of the genetics and gene products
  involved in sickle-cell anemia and trait
• a. Wild-type b chain allele is bA, which is
  codominant with bS

Fig. 10.8 The first seven N-terminal amino acids
in normal and sickled hemoglobin ? polypeptides
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Sickle-Cell Anemia
Hydrophobic valine at aa 6 embeds itself into the
hydrophobic heme pocket in a ? subunit of another
deoxygenated hemoglobin
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Sickle-Cell Anemia
Several Hb S molecules attach to each other to
form a long double- stranded polymer
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Sickle-Cell Anemia
Seven double-stranded polymers wind around each
other to form a sickle hemoglobin fiber Sickle
hemoglobin fibers cause erythrocytes to assume a
sickle shape Sickle cells clog capilaries -gt
tissue damage
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Fig. 14.2 The beginning of the ?-globin gene,
mRNA, and polypeptide showing the normal Hb-A
sequences and the mutant Hb-S sequences

• RFLPs are associated with many genetic disorders.
  Sickle-cell anemia is an example
• A single base-pair change in the b-globin gene
  results in abnormal hemoglobin, Hb-S, rather than
  the normal Hb-A. Hb-S molecules cause sickling of
  red blood cells.
• b. The Hb-S mutation is an AT-to-TA base pair
  change in the 6th codon of b-globin, resulting in
  a valine rather than a glutamic acid, and also
  eliminating a

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probe
Fig. 14.3 Detection of sickle-cell gene by the
DdeI restriction fragment length polymorphism

• In the normal b-globin (Hb-A) gene there are
  three DdeI sites, while the sickling form, Hb-S,
  has only two DdeI sites. This difference can be
  detected using Southern blot hybridization of

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Examples of amino acid substitutions found in ?
polypeptides of various human hemoglobin variants
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Fig. 4.5 The biochemical step for the conversion
of the brain ganglioside GM2 to the ganglioside
GM3, catalyzed by the enzyme N-acetylhexosaminidas
e A (hex A)

• 1. Caused by recessive mutation at the gene hexA
  (15q23-q24), which encodes the lysosomal enzyme
  N-acetylhexosaminidase A( HexA). The HexA enzyme
  cleaves a terminal N-acetylgalactosamine group
  from a brain ganglioside, a complex nerve
  membrane glycolipid

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Tay-Sachs disease

• 2. Infants homozygous recessive for this gene
  will have nonfunctional HexA enzyme. Unprocessed
  ganglioside accumulates in brain cells, and
  causes various clinical symptoms
• a. Infants have enhanced reaction to sharp
  sounds.
• b. A cherry-colored spot surrounded by a white
  halo may be visible on the retina.
• c. Rapid neurological degeneration begins about
  one year of age, as brain loses control of normal
  functions due to accumulation of unprocessed
  ganglioside.
• d. Progress is rapid, with blindness, hearing
  loss and serious feeding problems leading to
  immobility by age 2.
• e. Death often occurs at 34 years of age, often
  from respiratory infection.
• 3. The disease is incurable. Carriers and
  affected individuals can be detected by genetic
  testing.