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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
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.
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.
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.
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 (Histag) that serves as a tag for affinity
purification. Mammalian cell expression
systems offer the advantage that posttranslational modifications such as
glycosylation can occur. Glycosylation
reactions do not occur in bacteria.
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.
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).
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 doublestranded 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.