BIO 308 - Genes and Development - Dr.
Daley
Nucleus
or Cytoplasm: Which Controls Heredity?
•
Thomas Hunt Morgan
allied himself with embryologist that believed the control of development lay
within the cytoplasm
•
Edmund Beecher Wilson -
allied himself with Theordor Boveri - nucleus contained the instructions for
development
Split
Between Embryology and Genetics
•
EB Wilson and Nettie
Stevens demonstrated a critical correlation between nuclear chromosomes and
organismal development - XO and XY became male and XX became female
•
Then Morgan began to
find mutations correlated with sex and the X chromosome
–
He now viewed genes as
being physically linked to one another on the chromosomes
–
Thus Morgan had shown that
nuclear chromosomes are responsible for the development of inherited characters
Split
Between Embryology and Genetics
•
By the 1930’s -
this type of genetics began to be considered to be separate from embryology
•
Geneticists needed to
demonstrate the existence of inherited variants during early development
–
They also needed to show
how the same chromosomes could produce different cell types
•
So early embryologist
did not want to ground their science in gene action
Developmental
Genetics
•
Early - Salome
Gluecksohn-Schoenheimer & Conrad Hal Waddington contributed to the reunion
of development and genetics
•
Genomic equivalence -
–
salamander regeneration
- lens removed then a new lens can form from the cells of the dorsal iris
•
A common example of metaplasia - Respiratory epith. -
undergo metaplasia in response to inhaled irritants in tobacco smoke
Wolffian
Regeneration
Amphibian
Cloning - Restriction of Nuclear Potency
•
Totipotent - cells
nucleus is capable of directing the entire development of the organism when
transplanted into an enucleated egg
•
King and Briggs in 1952
- transplanted a blastula stage nucleus into an enucleated egg and got the
embryo to the tadpole stage
Amphibian
Cloning - Restriction of Nuclear Potency
•
Later stages were less
able to direct development -
nuclei of somatic cells of tail bud stage - no develop
•
Thus most somatic cells
lose their ability to direct development as they become determined and
differentiated
Success
of Nuclear Transplants
Amphibian
Cloning
The Pluripotency of Somatic Cells
•
Gurdon and colleagues -
worked with Xenopus
•
Similar results to King
and Briggs
•
However intestinal
endodermal cells were generally more capable of directing development
Clone
of Xenopus
Intestinal
Nuclei and Development
Cloning
in Mammals
•
Ian Wilmut in 1997 -
announced cloning of a somatic cell into an adult sheep - Dolly
•
Cells from mammary gland
of adult pregnant ewe - into culture
–
Maintained in G0
of cell cycle
•
Next oocytes from
different strain - removed nuclei
•
Next the fusion of donor
cells and enucleated oocyte via electrical pulses
–
Fused the cells and
activated development
Cloning
in Mammals
•
Next the embryos
transferred to uteri of pregnant sheep
•
Of 434 sheep oocytes -
only one survives - Dolly
•
DNA analysis confirmed
that the nuclei of Dolly’s cells
were derived from the donor nucleus
•
Similar results with
cows - Kato et al. 1998 and mice (Wakayama et al. 1998)
Cloned
Mammals
Cloning
in Mice
Differential
Gene Expression
•
Only a small percentage
of the genome is expressed in each cell & a portion of the RNA synthesized
in the cell is specific for that cell type
•
Polytene chromosomes
(DNA that replicates without mitosis and is - 512 (29), 1024 (210)
or more parallel DNA strands held together)
•
The puffed out regions
are transcribing RNA
DNA-RNA
Hybridization
•
Used to confirm
differential gene expression
•
Accomplished by
annealing single-stranded pieces of RNA and DNA - to allow complementary
strands to form double stranded hybrids
•
Some mRNA in all cells -
encode metabolic enzymes
•
Other mRNA’s only
in certain cell types and not in others
–
How this occurs - next
lecture
RNA
Localization Techniques
•
Northern blotting
–
First extract RNA from
different organs of the same embryo
•
Developmental Northern blots
–
Place samples on gel and
separate by applying electrical current - electrophoresis
•
Separated by size - smallest move the fastest
–
Place separated
RNA’s on nitrocellulose paper
–
Now incubate papers in
solution of radioactive single-stranded DNA fragment from a particular gene
•
The DNA only binds to regions of the filter paper where
complementary RNA is located
RNA
Localization Techniques
•
Northern blotting cont.
–
X-ray film is placed
above the filter and incubated in the dark
–
Dark spots on film -
indicate where the radioactive DNA has bound
Northern
Blot
Northern
Blot
RNA Localization
Techniques
•
In situ Hybridization
–
The Embryos or organs
are fixed to preserve structure & prevent RNA from being degraded
–
Section for microscopy - placed on slide
–
Radioactive DNA probe
added - binds only where complementary mRNA is present
–
Slide cover with
photographic emulsion for autoradiography
In situ Hybridization
RNA
Localization Techniques
•
Polymerase chain
reaction (PCR)
–
Method of in vitro gene
cloning - making numerous copies of the same DNA sequence
–
Method provides a means
for finding mRNA for specific proteins in embryos - especially helpful because
only a small amount would be present
–
mRNA via reverse
transcriptase into complementary DNA
–
DNA polymerase & S1
nuclease - now make double stranded cDNAs
–
Next specific cDNA is
targeted for amplification
Transgenic
Cells and Organisms
•
Inserting new DNA
–
Cloned DNA can be
injected into nucleus - in transfection, DNA is incubated so that the host
cells takes it up - incorporation of the donor DNA is rare
–
If however the new gene
is mixed with a gene giving resistance to an antibiotic, then only those cells
with the new genes survive in a antibiotic culture
–
Electroporation -
high-voltage pulse pushes DNA into host cell
Chimeric
Mice
Embryonic
stem cells (ES cells) with new gene microinjected into host embryo
•
Transgenic ES cells
integrate into embryo
•
Chimeric mouse (two
different genomes)
Transgenic
Mice
Transgenic
Mice
Gene
Targeting Experiments
•
“Knockout
experiments”
•
ES cells are cultured -
target gene cut with a restriction enzyme - cut gene replace with neomycin
resistant gene - ES cells selected by neomycin resistance
•
Next ES cells injected
into blastocyst - then into uterus of pregnant female rat
Gene
Targeting
Gene
Targeting
Antisense
RNA - Determining message function
•
Use antisense copies of
a mRNA to block the function of the message - reveals the function of the
message
•
Clone DNA into vectors
that have promoters at both ends of the inserted gene
Antisense
RNA - Determining message function
•
RNA polymerase - promoter
will initiate transcription in the wrong direction
–
The transcript is
complementary to the natural one - called antisense RNA
•
Injection of antisense
RNA into cells with normal mRNA - the two bind - then degraded - functional
deletion of the message
Embryonic
Stem Cells (ES)
Embryonic
Stem Cells (ES)