Transcript Wednesday, September 5

20.1 – 1
Look at the illustration of “Cloning a
Human Gene in a Bacterial Plasmid”
(Figure 20.4 in the orange book). If the
medium used for plating cells in step 5
did not contain ampicillin, cells
containing no plasmid would be allowed
to grow into colonies. What color would
those colonies be, and why?
Cloning a human gene
in a bacterial plasmid
Cloning a human gene
in a bacterial plasmid
Bacterial
cell
Human
cell
Isolate DNA
ampr
gene
Bacterial
plasmid
lacZ
gene
Human
chromosomes
Restriction enzymes
ampr
gene
Bacterial
plasmid
gene of
interest
Mix together
Mix together
Add bacteria
Add bacteria
Grow on ampicillin
Grow on ampicillin
Add lactose mimic
Add lactose mimic
20.1 – 1
Look at the illustration of “Cloning a
Human Gene in a Bacterial Plasmid”
(Figure 20.4 in the orange book). If the
medium used for plating cells in step 5
did not contain ampicillin, cells
containing no plasmid would be allowed
to grow into colonies. What color would
those colonies be, and why?
20.1 – 1
White (No functional lacZ gene is
present.)
20.1 – 2
Imagine you want to study human
β-globin, a protein found in red
blood cells. To obtain sufficient
amounts of the protein, you decide
to clone the β-globin gene. Would
you construct a genomic library or a
cDNA library? What material would
you use as a source of DNA or
RNA?
20.1 – 2
A cDNA library, made using
mRNA from developing red blood
cells, which would be expected to
contain many copies of β-globin
mRNA’s.
20.1 – 3
What are two potential difficulties in
using plasmid vectors of human
proteins from cloned genes?
20.1 – 3
Some human genes are too large to
be incorporated into bacterial
plasmids. Bacterial cells lack the
means to process RNA transcripts,
and even if the need for RNA
processing is avoided by using cDNA,
bacteria lack enzymes to catalyze
the post-translational processing
that many human proteins undergo.
20.2 – 1
Suppose you carry out electrophoresis
on a sample of genomic DNA isolated
from an individual and treated with a
restriction enzyme. After staining the
gel with a DNA-binding dye, what would
you see? Explain.
20.2 – 1
Any restriction enzyme will cut DNA
in many places, generating such a
large number of fragments that they
would appear as a smear rather than
distinct bands when the gel is stained
after electrophoresis.
20.2 – 2
Explain why restriction fragment length
polymorphisms (RFLPs) can serve as
genetic markers even though they
produce no visible phenotypic
differences.
20.2 – 2
RFLPs are inherited in a Mendelian
fashion, and variations in RFLPs
among individuals can be detected by
Southern blotting.
20.3 – 1
What is a major difference between a
genetic (linkage) map and a physical
map of a chromosome?
20.3 – 1
In a genetic linkage map, genes and
other markers are ordered with
respect to each other, but only the
relative distances between them are
known. In a physical map, the actual
distances between markers,
expressed in base pairs, are known.
20.3 – 2
In general, how does the approach to
genome mapping used in the Human
Genomic Project differ from the
shotgun approach?
20.3 – 2
The three-stage approach employed
in the Human Genome Project
involves genetic mapping, physical
mapping, and then sequencing of
short, overlapping fragments that
previously have been ordered relative
to each other.
continued…
20.3 – 2
The shotgun approach eliminates the
genetic mapping and physical mapping
stages; instead, short fragments
generated by multiple restriction
enzymes are sequenced and then
subsequently ordered by computer
programs that identify overlapping
regions.
20.4 – 1
Current estimates are that the human
genome contains about 25,000 genes,
but there is evidence for many more
different polypeptides. What process
might explain this discrepancy?
20.4 – 1
Alternative splicing of RNA
transcripts from a gene and posttranslational processing of
polypeptides.
20.4 – 2
What is the major value of DNA
microarray analysis for studying gene
expression?
20.4 – 2
It allows the expression of thousands
of genes to be expressed
simultaneously, thus providing a
genome-wide view of which genes are
expressed in different tissues, under
particular conditions, or at different
stages of development.
20.4 – 3
Why is the genetic variation among
people so much less than it is among
individuals of many other species?
20.4 – 3
Because the human species arose
more recently than many other
species, there has been less time for
genetic variations in coding and
noncoding DNA to accumulate.
20.5 – 1
What is the advantage of using stem
cells for gene therapy?
20.5 – 1
Stem cells continue to reproduce
themselves.
20.5 – 2
List at least three different properties
that have been acquired by crop plants
via genetic engineering.
20.5 – 2
•Herbicide resistance
•Pest resistance
•Disease resistance
•Delayed ripening
•Improved nutritional value