Chap. 5 Problem 1

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Chap. 5 Problem 1
Recessive mutations must be present in two copies
(homozygous) in diploid organisms to show a
phenotype (Fig. 5.2). These mutations show a
loss-of-function phenotype. Lethal recessive
mutations can be maintained in diploid organisms
in heterozygous form. In contrast, dominant
mutations must be present in only one copy
(heterozygous) in diploid organisms to show a
phenotype. Dominant mutations often cause a
gain-of-function phenotype and can be lethal in
heterozygotes.
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Chap. 5 Problem 2
A temperature-sensitive (ts) mutation is a
mutation that causes a phenotype only under the
condition of high temperature (is conditional).
ts mutations often are caused by amino acid
substitutions that make a protein incorrectly
folded and therefore inactive at high
(nonpermissive), but not low (permissive)
temperatures. ts mutations are useful for
analysis of the functions of essential genes in
haploid organisms (Fig. 5.6a). It is not possible
to create a viable cell that lacks an essential
gene.
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Chap. 5 Problem 3
Complementation analysis can be used to determine
if two recessive mutations reside in the same or
in different genes. The wild type phenotype will
be observed in a heterozygous diploid organism if
the mutations reside in different genes (Fig.
5.7). The mutant phenotype will be observed in a
heterozygous diploid organism if the mutations
reside in the same gene since neither allele is
functional. Dominant mutations cannot be analyzed
by complementation analysis as the mutant
phenotype will be observed in the presence of the
wild type allele.
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Chap. 5 Problem 7
A cDNA library is a collection of cloned DNA
fragments corresponding to all mRNAs transcribed
in a certain tissue or organism. The DNA
fragments are derived by reverse transcription of
mRNA. A genomic DNA library is a collection of
cloned DNA fragments representing all of the DNA
of an organism. This includes both protein-coding
and non-protein-coding segments of DNA. To clone
a gene expressed only in neurons, you could start
with either of the genomic DNA libraries, or you
could use the cDNA library produced from neurons.
You should not use the skin cell cDNA library, as
the cDNA for the neuronal gene will not be
present in the library.
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Chap. 5 Problem 10
To express a foreign gene, the plasmid must have
a promoter that will drive expression of the gene
(Fig. 5.31). One common method by which expressed
proteins are purified is via the attachment of an
amino acid sequence such as a polyhistidine
sequence (His-tag) that serves as a tag for
affinity purification. Mammalian cell expression
systems offer the advantage that
post-translational modifications such as
glycosylation can occur. Glycosylation reactions
do not occur in bacteria.
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Chap. 5 Problem 13
A number of DNA polymorphisms exist in DNA. SNPs
are single nucleotide changes between
individuals. SSRs are repeating one-, two-, or
three-base sequence duplications that vary in
length between individuals. Both of these
polymorphisms can be linked to a disease gene and
used to map its location in the genome. The
tighter the linkage of a marker to a disease gene
the closer its location to gene.
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Chap. 5 Problem 15
In expression analysis, the expression of a
candidate disease gene is is compared in tissues
from normal and affected individuals by Northern
blotting. Northern blotting allows a comparison
of both the level and length of transcripts from
a gene, which may be changed in the tissue from
the diseased individual. If the disease is caused
by a point mutation that changes an amino acid in
the encoded protein, then expression analysis may
be inconclusive. In this case, DNA sequencing
will be required to identify the SNP that causes
the disease (Fig. 5.38).
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Chap. 5 Problem 17
Dominant negative alleles inactivate the function
of a wild type allele in the heterozygous state
(Fig. 5.44). Thus both copies of the gene of
interest need not be modified to observe a
phenotype.
In RNA interference, gene expression is
inactivated by selective destruction of the mRNA
transcribed from that gene. To accomplish this, a
short double-stranded RNA is synthesized in vitro
(Fig. 5.45 a b) and then introduced into cells.
The base pairing of the introduced RNA with the
target mRNA leads to nuclease destruction of the
mRNA and silencing of the gene. Alternatively,
the RNA can be synthesized in vivo.