I. Examples of spatial and temporal regulation
of gene expression in eukaryotes
A. Spatial regulation
of genes - differential expression in different tissues
1. Tubulin genes in plants
a. Used in making microtubules
b. Cytoskeleton
2. Alpha and beta subunits; one of each to form tubulin
a. Different alpha and beta subunits expressed in different tissues
b. Use of specific antibodies to determine which tissues express which
subtype
B. Temporal regulation
of genes - differential expression at different times
1. Hemoglobin genes in humans
a. Multigene family
b. Due to duplication of genes
c. Some are pseudogenes
(1) Frameshift mutations
(2) Nonsense mutations
d. Organized into cluster (chromosome 16) and cluster
(chromosome 11)
2. Expression of different forms at different times in development
a. Embryonic: (zeta) (alpha type); epsilon (beta type)
b. Fetal: Ggamma and Agamma
c. Adult: alpha2, alpha1, delta, beta
d. Embryonic hemoglobin has greater affinity for O2, lower affinity
for CO2
(1) Allows exchange of blood gases with mother
II. Regulation of gene expression
A. Overview
1. Much more complex than prokaryotes
a. Transcription and translation in different locations
b. RNA processing
2. Compartmentalization in cell
a. Regulation in nucleus at level of DNA or RNA
b. Regulation in cytoplasm at level of RNA or protein
3. Need for internal signaling systems to turn on and off genes
a. From exterior of organism to internal cells
b. From exterior of cell to nucleus
B. Transcriptional
factors regulate transcription by interacting with DNA
C. Spliceosomes
remove introns
1. Multiple introns can be removed sequentially
a. may not be one after other (1, 2, 3, 4)
b. but order of removal follows pattern (2, 6, 3, 12, etc.)
2. Can also remove two adjoining introns and exon between
a. alternative splicing
b. frequently affects the function of the protein
c. one gene - multiple polypeptides
d. economy of DNA to produce multiple proteins
3. Antibody genes
a. arranged by domains
b. each domain has a specific function
c. can select one of multiple variations on domain
d. like mixing and matching coordinated outfits
D. mRNA stability
regulated in cytoplasm
1. Length of poly A tail
a. Histones have no poly A tail, short lived
2. Structure of 3' untranslated region (3' UTR)
a. Short lived if AUUUA repeated several times
3. Metabolic state of cell
a. Hormonal influence
4. Mechanisms unclear; may be other factors involved as well
a. Just getting a glimpse of what is occurring here
E. Induction
of transcription by environmental and biological factors
1. Heat shock genes
a. Expression of ‘emergency' genes - for dire environmental conditions
b. Protein present in nucleus
(1) heat-shock transcription factor
(2) when temperature rises, HSTF is phosphorylated
(3) phosphorylated form binds to DNA at specific sequences
(4) called heat shock response elements HSE
(5) stimulates transcription of genes nearby (heat shock proteins)
2. Light
a. Mechanism sketchy
b. Enzyme involved in photosynthesis synthesized when light present
c. Involves absorption of light by phytochrome
d. Biochemical cascade leading to binding of proteins to DNA and transcription
of enzyme
3. Steroid hormones
a. Lipid-soluble, pass through cell membrane
b. Bind to receptors inside cell
c. Receptor enters nucleus and binds to specific sites on DNA
4. Peptide hormones
a. Can't enter cell, bind to membrane-bound receptors
b. conformational change in receptor
c. Cascade of events leading to signals in nucleus
d. Frequently involves phosphorylation, cAMP production
e. Multiple hormones, multiple receptors, but overlapping (or opposite)
triggering responses
(1) result is fine tuning of turning on and off genes based on different
events outside the cell
III. Molecular control of transcription
in eukaryotes
A. Overview
1. Basal transcription factors - bind to sequence within promoter
a. Turn on a specific gene
2. Special transcription factors - bind to response elements
a. Turn on or off a region of the chromosome
b. Enhancers - turn on
c. Silencers - turn off
B. Enhancers
1. Act over large distances (several thousand base pairs)
2. Influence on gene expression is independent of orientation
3. Effects are independent of position (upstream, downstream, or within
intron of gene)
4. Unlike promoters, which are 5' to gene, function in one orientation
5. Most function in specific tissues; ignored in other tissues
6. Yellow gene in Drosophila has 4 tissue specific enhancers (p. 581)
7. SV40 enhancer is tandem repeat; no nucleosomes bind in this region
a. Lack of nucleosomes may expose DNA to polymerase,
b. Can then transcribe regions surrounding (Bumping off nucleosomes as
it goes)
8. Also suggested that proteins that bind to enhancer encourage the binding
of transcription factors
a. like needle threader helps to thread needle
b. See fig. 22.12, p. 583
C. Transcription
factors
1. Many identified, most have DNA-binding domain and transcriptional activation
domain
2. Not completely understood
3. Involves interaction between proteins
4. Characteristic structural motifs important in binding DNA
a. Zinc fingers
b. Helix-turn-helix
(1) one helix binds DNA
(2) others form protein dimers
(3) similar to homeodomain
(a) Drosophila genes
(b) homeotic transformation - substituting one body part for another
c. Leucine zipper
(1) leucine every 7th a.a.
(2) form dimers
(3) usually adjacent to positively charged stretch of a.a.
d. Helix-loop-helix
(1) helical regions permit dimerization between two peptides
e. May turn on or off transcription by formation of heterodimers
IV. Gene Expression and Chromosome Organization
A. Overview
1. Presence of nucleosomes blocks transcription
2. Transposition of genes near or far from enhancers affects expression
3. May be additional aspects of chromosome structure which affect expression
B. Transcription
in lampbrush chromosome loops
1. Highly condensed amphibian oocyte chromosomes
2. But lateral loops where uncoiled for transcription
C. Polytene Chromosome
puffs
1. Hundreds of sister chromatids lined side by side
2. Dark banding
3. Highly transcribed regions more disperse, loss of banding, puff out
4. Ecdysone or heat shock cause puffing to occur
D. Molecular
organization of transcriptionally active DNA
1. Transcribed DNA remains packaged in nucleosomes
2. But more sensitive to DNAse
3. DNAse I hypersensitivity sites
a. Use low levels of DNAse I
b. Nicks preferentially at sequences upstream of promoter or enhancer regions
4. Specialized chromatin structures
a. If flank a site, insulate from DNAse
b. Gene(s) which are flanked expressed uniformly regardless of where located
in chromosome
5. Euchromatin and heterochromatin
a. Most genes in euchromatin
b. If moved to heterochromatin, don't function properly
(1) position effect variegation
(2) variability in phenotype caused by changing position of gene
6. Gene amplification
a. Ribosomal RNA genes in amphibian oocytes
b. Extrachromosomal copies of specific genes
c. In humans, homogeneously staining regions and double minutes
V. Activation and inactivation of whole
chromosomes
A. Sex chromosomes
1. Different number of copies of chromosomes
2. Differing dosages
3. Dosage compensation by inactivation, hyperactivation or hypoactivation
4. In all, coordinate regulation of entire chromosome
B. Mammals
- bar bodies
1. Inactivation
2. X inactivation center (XIC)
3. Spreads in opposite directions toward end of chromosome
4. Not all genes are transcriptionally silent (XIST)
a. No open reading frame
b. Believe that transcript binds to X chromosome to inactivate
C. Drosophila
- hyperactivation of male X chromosome transcription
1. Four genes expressed to permit hyperactivation
2. If null genes, male-specific lethality
3. One gene similar to helicase; may keep chromosome opened for transcription
D. Nematode -
XO males and XX hermaphrodites
1. Partial repression of genes in both Xs of hermaphrodites
2. Hypoactivation
3. Protein which binds to X chromosome, but only if two copies of chromosome
are present
4. When binds, appears to repress transcription
VI. Gene expression and cancer
A. Tumors
1. Benign
2. Malignant
B. Oncogenes
1. Kinases
a. Turn on and off collection of proteins by phosphorylation
2. Altered growth factor genes
a. loss of control of growth
3. Receptors for growth factors and hormones
a. Loss of control
b. Cascade effects, but turn on or off the wrong things, or due to wrong
signal
4. Transcription factors
a. Stimulate gene expression
b. Typically developmental genes; cancer cells dedifferentiate
C. Proto-oncogenes
1. Viral oncogenes often similar to cellular proteins with important regulatory
functions
2. Viral genes lack introns (from packaging of processed mRNA molecules)
3. Protooncogenes have introns
4. Oncoviruses cannot infect without presence of helper virus
a. loss of own genome to carry oncogene
5. In virus, oncogenes disrupt possibly because produce more product
a. Enhancers in genome cause greater transcription
b. Expression at inappropriate times
c. Mutation in viral gene causes a new function
VII. Genetic basis of human cancer
A. Mutant cellular
oncogenes
1. Typically point mutations
2. For c-ras, three different mutations, become dominant activators of
uncontrolled cell growth
B. Chromosome
rearrangements and cancer
1. Philadelphia chromosome
a. Reciprocal translocation
b. Creation of fusion gene
2. Burkitt's lymphoma
a. Chromosome 8 with one of three chromosomes containing immunoglobulins
b. Overexpression of c-myc gene when near immunoglobulins
3. Tumor-suppressor genes
a. Sometimes loss of gene function leads to cancer
b. Retinoblastoma - if one good gene present, no cancer
c. Gene functions to suppress tumor formation
C. If inherit
one copy of bad gene, higher probability of getting mutation in second
gene
1. Leading
to cancer
2. Predisposition
3. Two-hit
hypothesis - both genes must go bad before cancer
4. Colon
cancer - gene involved in DNA repair
5. Breast
cancer - gene that binds to DNA
6. Cancer
is stepwise process of accumulating mutations in genes
For questions, comments
and additional information, contact mfhicks@pstcc.edu
Last Updated: June 24
2001
Site map: Margaret
F. Hicks Home - Biology 2120 -
Notes
- Eukaryotic Gene Regulation
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