Download TB1 - BIOCHEM, Broyles

Broyles’ Specific Learning Objectives
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Molecular Basis for Sickle Cell Disease
o DNA mutation – a single nucleotide change in the #6 codon of the β globin gene
o Amino acid change – glutamate is changed to valine (due to nucleotide change)
o Prenatal molecular diagnosis –
 Hemoglobin (Hb) electrophoresis – the change in nucleotide (valine for glutamic acid)
causes a less negative charge to be formed; therefore lessening the run on the gel when a
positive charge is applied
 RFLP – a mutation in the DNA results in a net gain or loss of the cutting site for the
restriction enzyme; therefore mutant and normal DNA are cut into different sized
fragements
o Fetal hemoglobin – alleviates both sickle cell and β-thalassemia (known to have worked on
population in Saudi Arabia; homozygous carriers for sickle cell; showed hereditary persistence in
fetal hemoglobin (HPHF); no clinical side effects; must be held in a range of 20%-25%;
increases oxygen uptake and decreases sickling
Pathway of gene expression – genotype to molecular phenotype
o I: Replication – DNA in chromatin
o II: Chromatin structure in nucleosomes
o III: transcription
o IV: post-transcript modifications (processing control)
o V: mRNA (nuclear); degradation within the nucleus
o VI: transport our of nuclear “pore”
o VII: masked mRNA
o VIII: mRNA forms polysome
o IX: translation into polypeptide
o X: post-translational modifications and assemblies into active protein
Chromatin structure
o Chromosomes (DNA + protein)
 Basic structural unit is the nucleosome – DNA wraps around protein core 2 times/core;
nucleosome is a histone octomer with a net positive charge, attracts negative charge of
DNA; degree of condensation of chromatin is inversely proportional to gene expression –
form beads on a string
 Chromatin fibers develop of packed nucleosomes
 Series of loop domains form on a chromosome
 Loops fold further
 Final form is tight clusters of multi folded chromatins
o Nucleosomes
 Core is formed by 8 histones (2 of each) in the octomer
 H2A, H2B, H3, H4 – H3 and H4 form dimer; H2A and H2B form dimer – dimers
get together and form an octomer
 H1 is outside of nucleosomes and links nucleosomes together by interacting with
DNA and other proteins
o DNase I – used to determine whether a gene is “active” and in the open conformation and
available for transcription
 Endonuclease isolated from Bovine pancreas, helps determine the 2 steps in gene
activation
 Decondensation of chromatin to uncover a gene making it available for
transcription - Opens up the chromatin structure, creates a potential for gene
activation - genes in open configuration will be digested
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Activation and transcription of the gene – binding of RNA polymerase and gene
regulatory proteins (trans-acting factors)
o 4 characteristics of an “active” gene
 Can be digested with DNase I
 Tends to have hypomethylated DNA
 Hypermethylated genes tend to be inactive (methylation occurs on cytosine
residues and turns gene off)
 Active methyl genes have fewer methyl groups on the DNA
 Has more loosely bound histones due to acetyl groups on the N-terminus of histone
proteins which lessens the histones positive charge, especially occurs on H3 and H4
 Tends to have TA (trans-acting) factors called nonhistone chromosomal proteins involved
in gene regulation
Transcription and its regulation
o 3 classes of RNA polymerases –
 RNA polymerase I (pol I) – in nucleolus; transcribes rRNA; insensitive to α-amanitin
 Pol II – in nucleoplasm; transcribes mRNAs; strongly inhibited by α-amanitin; most
interested in pol II
 Pol III – in nucleoplasm; transcribes a small rRNA and tRNAs; inhibited by high
concentrations of α-amanitin
o Order of assembly of the pol II transcription preinitiation complex
 RNA pol II complex assembles in an orderly fashion
 TATAAA box on DNA strand is recognition sequence (located 30 bp upstream from the
start of transcription)
 At 30 bp is TFIID (transcription facto II b/c of association with RNA pol II and D is in a
lettered series)
 Called the TATA binding protein (TBP), first to recognize the TATA sequence
 TFIIA then binds to TFIID
 TFIIB binds
 Pol II and TFIIF then bind
 TFIIE and TFIIH then bind the to the complex
 Need a boost from a gene regulatory protein and some ATP energy to start the process of
transcription
 All together is called the pre-initiation complex
 TBP is special – causes DNA to bend at site of recognition to mark the start of assembly
of the pre-initiation complex
o Definitions of terms pertaining to regulation of transcription
 Cis – acting sequences are DNA regions involved in control of gene expression, to which
trans-acting factors bind…i.e.
 Proximal promoter – DNA control sites between 100 base pairs upstream and the
site of transcription…the TATA box is an example
 Distal promoter - DNA sites between 100 and 200 base pairs upstream, known to
bind to a variety of trans-acting factors
 Enhancers – DNA regions involved in positive regulation distances away,
upstream or downstream from the gene and oriented in either direction with
respect to the gene (5’ to 3’ or 3’ to 5’)
 Silencers – DNA regions involved in negative control (repression) opposite
enhancers
 Trans-acting factors are gene regulatory proteins that either activate or repress genes by
binding to cis-acting DNA sequences and/or to other DNA binding proteins
 Transcription factors (TFs) – general transcription factors (GTF) – proteins other than
RNA polymerase that are required for the formation of a transcription initiation complex
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TBP (TATA binding protein) – a subunit of TFIID that specifically binds to the TATA
box which is a DNA sequence found in many pol II promoters
 TAFs (TBP – associated factors) – a class of trans-acting factors that bind TBP and
bridge by also binding another DNA-binding protein
o Anatomy of a protein encoding eukaryotic gene
 Page D-34 in syllabus – visual representation of the definitions pertaining to the
regulation of transcription
o Structural motifs of trans-acting gene regulatory proteins
 HTH – helix-turn-helix
 Zinc finger (Cys-Cys and Cys-His), e.g. TFIIIA or GATA-1 (a protein involved in
regulation of human globin genes)
 Leucine zipper – a class of trans-acting factor with a special motif for interacting with
other proteins as well as DNA
 Helix-loop-helix (HLH) or basic helix-loop-helix (bHLH)
o Cis-acting DNA sequences to which transcription factor TBP (of TFIID) and trans-acting
factors GATA-1, SP-1 and CBP bind
 TATA box: recognized by TFIID, specifically TBP – functions in pol II transcription
initiation
 GGGCGG: recognized by Sp-1 (trans-acting factor) – functions in regulation of many pol
II genes
 CCAAT: recognized by CBP (cat-binding protein) – functions in regulation of many pol
II genes
 GATA: recognized by GATA-1 protein – functions in the regulation of erythroid-specific
genes
Human globin gene clusters
o α- like globin genes are on chromosome 16
o β- like globin genes are on chromosome 11
 each globin gene has 3 exons and 2 introns (structurally related)
 both gene clusters are developmentally regulated and coordinated
 coordination results in a set of embryonic hemoglobins (Hbs) that are developmentally
replaced with a set of fetal Hbs and later replaces with adult Hbs
o α- thalassemia – relative or absolute deficiency of α chains
o β- thalassemia – relative or absolute deficiency of β chains
o Switching of globin genes during development
 Embryonic hemoglobins are produced during the 1st trimester of development; large,
nucleated cells formed in the yolk sac; leads to an activation and expression of alpha-like
zeta globin and beta like epsilon globin (bind oxygen most efficiently)
 Fetal hemoglobins are produced in the 2nd and 3rd trimesters; cells are enucleated and
formed primarily in the liver and spleen; 2 alpha and 2 gamma chains dominate (bind
oxygen better than adult Hb)
 Adult hemoglobins are produced after birth; cells are enucleated formed primarily in the
bone marrow; 2 alpha and 2 beta chains dominate (least efficient binding of oxygen, but
most efficient in delivering oxygen to needing cells)
o 3 drugs stimulate expression of fetal hemoglobin
 5-azacytidine – adult cytosines in the promoter region get methylated and the gene is
turned off (effectively halting production of fetal hemoglobin); azacytidine prevents the
methylation of cytosine, therefore allowing the continuation of expression of fetal gamma
hemoglobin genes; may be mutagenic and cause cancer
 A derivative, 5-aza-2’-deoxycytidine works in the same way, but is more efficient
and appears to be less toxic
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Hydroxyurea – used in sickle cell disease patients (SCD); increases fetal hemoglobin
production, exact treatment mechanism is unknown; might be mutagenic and decreases
pain associated with bone marrow transfusions; is more effective in children with SCD
 Butyric Acid – increases fetal Hb production by inhibiting the histone deacatylases; less
toxic and causes more fetal hemoglobins to be produced; discovered by accident and
utilizing both basic and clinical science; stimulates gamma globin gene expression
Stem cells, cellular differentiation, development, cancer and gene therapy
o Steps in cellular differentiation – (2 stage process)
 Determination – stage 1 of gene expression (chromatin condensation); uncovering a gene
within chromatin
 Maturation – stage 2 of gene expression building an active transcription complex on the
uncovered, available gene (assembly of a pol II complex with TFs and specific TAs)
o Pluripotent hematopoietic stem cells (PHSC)
 Able to form daughter cells that become all blood elements (T-cells, plasma cells,
erythrocytes, megakaryocytes, leukocytes, macrophages, eosinophils, natural killer cells)
 Assayed in mice by destroying blood cell production through gamma ray attack,
hematopoietic stem cells from another animal are then injected, later: circulating blood
cells have a donor cell marker, nodules are found on the spleen and contains precursors to
red blood cells, white blood cells and megakaryocytes
o Scheme of blood cell lineages in humans
 PHSC (pluripotent stem cell) → lymphoid stem cells and myeloid stem cells →
differentiates from multipotent cells to unipotent cells (no histological differences,
change occurs in the nucleaus)→ eventually form: T-cells, plasma cells, erythrocytes,
megakaryocytes, leukocytes, macrophages, eosinophils, natural killer cells
o Role of stem cells in development and tissue renewal cancers often arise from stem cells or
progenitor cells
 Cancers often arise from stem cells or progenitor cells
 Many tissues of the body have stem cells, any tissue that regenerates frequently, skin, gut,
lungs…etc
 Most often, cancers are found in tissues that contain stem cells, the earlier in the life of
the stem cell, the more malignant the tumor
o Stem cells as a vehicle for gene therapy in humans
 Search for a cell in adults that possesses the properties of an embryonic stem cell and
capable of differentiating into many other types of cells (need to kill embryos to obtain
ES cells would be decreased)
 This would undoubtedly benefit gene therapy in diseased patients
o Gene transfer of cells in culture, in transgenic animals and in animals cloned by nuclear
transfer
 Allowed new animal models of human cancers and other diseases with reasonable hopes
of increased understanding and eventually deriving cures
Medical importance of gene regulation (D-66)
o Seeking to find a cure in molecular diseases is based in understanding gene regulation
o Seek to cure a regulatory disease through a better understanding and application of the
biochemistry and molecular biology of regulation itself
o Most major diseases present problems with gene regulation
 AIDS, cancer, alzheimer’s, birth defects, arthritis and atherosclerosis
o Central problem of biomedicine in the 21st century is to understand and be able to
manipulate cellular differentiation (process of how cells become different from one
another)
 Central problem in cellular differentiation is gene regulation