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Genetics is a hugely important topic area in biology. From Mendel's
experimentation with pea plants and theories of inheritance, to
Watson and Crick's determination of the physical nature of the
genetic code, right up to the present day in the form of the Human
Genome Project, our understanding of genetics has expanded rapidly
over the last century. There is still so much more to discover.The
field of genomics holds much future promise as it involves
analysing patterns in the full genomes of organisms, something we
know very little about. Progress in terms of medical genetics,
identifying the link between diseases and certain gene variants
remains rapid. These are just some of the interesting branches at
the cutting edge of genetics. Here we provide an elementary
introduction to some core genetic topics.
Inheritance
Gregor Mendel was one of the early pioneers in the field of
genetic inheritance. Mendel used true breeding garden pea plants
(Pisum sativum) to
investigate how seven different phenotypes were represented in the
offspring of parent plants across generations. These experiments
led Mendel to propose that:
- Genes are particulate and there is no blending of
phenotypes
- Each plant contains two genes for each character and the
phenotype defines which of these two alleles is the dominant
one
- Members of each gene pair segregate equally into gametes and
the fusion of gametes does not depend upon which gene it
contains
Mendel gained this insight simply by identifying ratios of
phenotypes in his offspring. He found a pattern of inheritance
independent of the phenotype studied and a pattern where reciprocal
crosses gave the same result.
Results of an experiment similar to Mendel's is given
below:
Now we can quantify the results obtained:
Calculate the ratios of the F$_2$
generations of the plants. Which is the dominant allele in each
crossing?
What is the average ratio in each
case? Using one cross as an example, draw a Punnet square to
illustrate the observed phenotypic ratios.
Take for example the cross between a plant producing yellow peas
and one producing green peas. The F$_1$ generation had plants that
all produced yellow peas. The F$_2$ generation had a 3:1 ratio in
terms of yellow peas: green peas. When breeding the F$_2$ with each
other to produce an F$_3$ generation. Of the 623 yellow pea F$_2$
plants, 199 gave F$_3$ plants all having yellow peas and 424 gave
F$_3$ plants which has yellow peas or green peas. When the green
pea plants of the F$_2$ were selfed the offspring all had green
peas.
Can you work out what is going on?
What is the ratio of true
breeding yellow parents to plants that acted like F$_1$ plants in
the F$_2$ generation?
Draw a Punnet square to explain
the observed ratios.
This above analysis was highly useful in formulating Mendel's Laws
of Inheritance. For scientific theories to be valid, they must of
course be
testable. Mendel
chose to conduct a backcross of a heterozygous F$_1$ with a
homozygous recessive parent.
What ratio would need to be
observed for the hypothesis to be upheld?
Mendel also considered what would occur if two genetic loci were
investigated. For example, consider a cross between plants with
purple flowers and short stems (PPll) with plants with white
flowers and long terms (ppLL). The F$_1$ generation would consist
of plants with purple flowers and long stems.
What would be the genotype of the
F$_1$ generation?
The F$_2$ generation consists of plants with purple flowers and
long stems, purple flowers and short stems, white flowers and long
stems, and white flowers and short stems.
What possible genotypes could
plants having these phenotypes have? Give all the
possibilities.
The F$_1$ plants that give rise to the F$_2$, produce gametes
containing one allele of a gene present at each locus.
What possible combinations of
alleles could be present in a gamete?
A dihybrid cross can be used to calculate the expected ratios of
the phenotypes in the F$_2$ generation. This cross works in a
similar fashion to a Punnet square, except due to it being across 2
loci, the combinations of alleles present in the gametes are listed
for each organism being crossed instead of a single allele. An
example of a dihybrid cross for cat coat colour is given below.
Each genotype is assigned the correct phenotype and the ratio of
the offspring in the generation determined.
Construct a similar dihybrid
cross for the F$_2$ generation of plants (cross of two purple
flowered, long stemmed F$_1$ plants).
Assign a phenotype to each
genotypic possibility. What is the ratio of phenotypes in the F$_2$
generation?
This ratio, if found for the phenotypes in an F$_2$ generation
produced in a breeding experiment, is indicative of the
independent segregation of two
pairs of alleles. This means that each combination of alleles in
the gametes of the F$_1$ plant is equally likely. In other words
there is no interaction between the two genes in terms of
segregating into alleles in the process of meiosis.
Mendel's laws of inheritance provide an interesting overview of
simple genetics. However many of his findings are not held to be
universally true. Situations of
codominance,
genetic linkage and
X-linked inheritance are just
some of those circumstances known to exist as exceptions to
Mendel's rules. Slight modification of Mendel's laws have been
necessary in light of later experimental evidence.
Genetic linkage and recombination
As discussed previously, Mendel's principles have needed slight
modifications to still be valid in light of new experimental
evidence. This is the case for the situation of genetic
linkage.
Mendel stated that genes segregated independently of one another
and this can be seen to be true if individual genes are on
different chromosomes. However, if there is more than one gene per
chromosome then can the inheritance of these two genes be linked?
i.e. is it more likely that certain allelic combinations segregate
together?
Experimental evidence suggested that this could be the case.
Bateson and Punnet for example found that pollen shape and flower
colour showed an inheritance pattern where parental genotypes are
more likely. Thomas Hunt Morgan's Drosphila melanogaster studies
of the inheritance of purple eyes (pr) and vestigial wings (vg) also indicated that linkage
was a real phenomenon. Morgan suggested that the two genes were
carried on the same pair of homologous chromosomes and pairing
during meiosis lead to new genetic combinations, a process known as
recombination. It was
eventually conjectured that chiasmata formation between
homologous chromosomes and crossing over lead to the
exchange of genetic material.
Chromosome mapping
This theory of linkage leads us to a way of mapping the
positions of genes in a genome. Assuming that chiasmata formation
occurs at random along the length of a chromosome, the probability
of a crossover between two genes is proportional to their distance
apart.
If the F$_2$ progeny of a
cross between purple eye, vestigial wing Drosphila crossed with a wild type is conducted,
will the ratios of phenotypes be the same as that observed for the
cross between purple flowered, short stem pea plants with white
flowered, long stem plants? Comment on any
differences.
Thus, if we work out the percentage of recombinants in each
case we can obtain a distance of separation in terms of map units.
This analysis can be extended to the simultaneous mapping of 3
genetic markers. Consider a cross between a wild type Drosphila and a strain
homozygous for recessive mutations causing vestigial wings (vg), vermilion eyes (v) and crossveinless wings
(cv). The phenotypes of
the F$_2$ generation obtained after crossing an F$_1$ of males and
females are shown below:
If the three genes segregated independently, then each of the
progeny classes would be present at equal frequency. This is
clearly not so. It may be seen that the parental phenotypes are far
more likely. A linkage map consistent with all the data offers a
greater insight.
The distance between cv and vg marker genes is expressed as
the percentage of recombinants of this type seen in the
offspring.
$$\frac{38+43+6+4}{1233} \times 100 = 7.3\%$$
The distance between vg and v marker genes is,
similarly:
$$\frac{79+82+6+4}{1233} \times 100 = 13.9\%$$
The distance between cv and v marker genes is:
$$\frac{38+43+79+82}{1233} \times 100 = 19.6\%$$
The sum of the cv-vg
and vg-v map distances is
seen to be larger than the cv-v distance calculated from
the data. This is due to the fact that double crossovers can occur
within an interval. As the probability of crossing over is taken to
be proportional to the distance between the two marker genes, this
can be corrected for by adding double the value of these double
recombinants in the numerator of the fraction expressing the
largest distance.
The distance between cv and v marker genes can now be
calculated as:
$$\frac{38+43+79+82+2(6+4)}{1233} \times 100 = 21.2%$$
Draw the linkage map given by the
data above, showing the distances between marker genes.
Clearly there is a problem when chromosome mapping occurs over
long distances as there is a compression of larger map distances as
recombination frequency tends asymptotically to 50% as marker genes
become spaced further and further apart. This effect can be
minimised by building up the genetic map of a chromosome using
closely linked pairs of markers.
Furthermore, this technique is once again full of many
assumptions. Assuming that chiasmata formation and genetic
recombination occur randomly along the chromosomes is an
oversimplification. Increasing distance away from the centromere
increases the frequency of chiasmata formation. Chiasmata formation
may also differ between sexes and species in terms of frequency and
chiasmata formation may reduce the formation of a 2nd crossover in
adjacent regions. All of these mean that maps established through
restriction mapping methods
often differ from those obtained using these physical
methods.
Gene interactions
Of course the human genome is a highly complex assemblage of
genetic code. Genes can interact with both the external environment
and each other in terms of their expression. The effect of gene
interaction may be seen by considering the relationship between the
genotype of an individual
and its phenotype.
An interesting genetic interaction is epistasy. This is when the effect
of one gene is masked by the effect of another meaning it does not
find expression in the phenotype of an individual. An example of
this is the eyegone gene
in Drosphila which is
clearly epistatic to any eye colour gene as eye formation itself
does not occur! Another interesting genetic phenomenon is the
"viability effect". Certain mutations may actually slow the growth
of certain species in such a way that they become under-represented
in the population. These effects are quite common in mutated genes
in fungi such as Aspergillus
nidulans.
Example
Coat colour in mice is a quality determined by interactions at
several genetic loci. Genes influencing the distribution, type, and
presence of pigment are known to exist. We will consider the case
where the loci are
unlinked
and so are either present on different chromosomes or widely spaced
apart on the same chromosome.
Here are some of the genes involved:
A: A
(
agouti), a, A$^{\text{y}}$
(
lethal yellow), a' (
black and tan)
B: B
(
black), b (
brown)
C: C, c
(
albino), ch (
Himalayan)
D: D, d
(
dilution)
S: S
(
spots), s (
piebald)
So the genotype
aaBB
corresponds to a mouse with a black coat. The genotype
BBcc corresponds to an albino
mouse, demonstrating that the c allele is epistatic to all other
colours. Interestingly these interactions may be more complex. For
example the genotype
AAbb
gives rise to a cinnamon coat colour.
Take for example, the cross between a cinnamon coloured mouse
(
AAbb) and a black mouse
(
aaBB).
The
F$_1$, produces mice of
phenotype AaBb which are all
agouti.
The
F$_2$ are 9 agouti
(A-B-), 3 cinnamon (A-bb), 3 black (aaB-) and 1 brown (aabb)
Using a dihybrid cross, predict
what the ratio of phenotypes in the F$_2$ of a cross between a
mouse with a dilute black coat colour (BBdd)
and a mouse with a brown coat
colour (bbDD). [
Note that
there are no further complexities with phenotype in this
case.]
Restriction mapping
Previously we met a situation where the mapping of a chromosome
could be conducted by considering the recombination frequency of
certain genetic markers in
a cross. Now we consider a method where enzyme catalysed digestion
of DNA can give a cruical insight into its structure.
A wide variety of bacteria produce restriction endonucleases in
order to cleave foreign DNA and so destroy its coding capacity.
This is a useful strategy against infecting bacteriophages. The
cuts that a restriction endonuclease makes to a molecule of DNA may
be flush or staggered, giving protruding sticky ends.
Why do you think that restriction
enzymes don't digest the bacterium's own DNA?
Examples of restriction enzymes include EcoR I, Hind III and
BamHI. Most recognize a specific DNA sequence that is 4-6 bases
long and palindromic. For
example the recognition sequence of EcoR I (produced by the
bacterium E. coli) is 5'
GAATTC 3'.
Purified restriction enzymes are used to cleave DNA molecules into
fragments and separation of fragments by molecular weight can be
achieved through elecrophoresis through an agarose gel that has the
correct pore size for this application. Visualisation of the DNA is
achieved by staining with ethidium bromide and using UV
light.
Depending on the number of sequence recognition sites in the
sample DNA, each restriction enzyme cleaves to produce a different
number of fragments of varying length. It is useful to know that a
plot of
distance migrated
by a fragment against
log
(molecular weight) gives a straight line relationship,
meaning that running fragments of unknown sizes against known
fragments can allow for an estimation of molecular weight.
Restriction sites are used as
physical markers in a similar way
to how genetic markers were used in the
Drosphila cross experiments.
Consider the mapping of a circular bacterial plasmid. Digestion
with a single particular enzyme will produce a single fragment if
there is only one recognition site in the plasmid. If other enzymes
also produce a single fragment, it can be said that there is only
one recognition site for these enzymes in the plasmid as well.
Multiple digests can then
be used to determine where these sites lie in terms of one another.
The sizes of the fragments produced can be ascertained by the
comparative methods outlined earlier: run alongside fragments of a
known length.
Consider the complete and partial restriction of the following
linear molecule of DNA:
Partial digestion using low
concentrations of restriction enzymes or altering experiment time
causes extra bands to appear in a DNA sample after it is run on
agarose gel. These bands correspond to fragments with adjacent
sections attached i.e. digestion has not occured at all possible
restriction sites. These bands are highly useful in classifying the
order of fragments obtained in a complete digest, with the
restriction sites once again acting as markers.
In the example above, extra bands appear in the partial digest at
3.9 kb and 4.3kb as well as at 5.6 kb corresponding to the whole
undigested molecule. A very similar approach may be applied to
circular plasmid molecules.
Consider a circular plasmid pXY1 which is digested using different
combinations of EcoRI, BamHI and TaqI. After running the fragments
on an agarose gel alongside fragments of known size produced by
digesting bacteriophage $\lambda$ with Hind III, the following data
was obtained:
Enzyme(s) Fragment sizes
(kb)
TaqI 3.7, 3.2, 2.1
TaqI + EcoRI 3.7, 3.2, 1.8, 0.3
TaqI + BamHI 3.7, 2.1, 1.6
EcoRI + BamHI 7.1, 1.9
Construct a restriction map of
this circular plasmid pXY1 after reading the following
section.
Take the case of a mystery circular plasmid. The plasmid was
digested with the restriction enzymes PstI, HindIII, and EcoRI and
the size of the fragments produced calculated after electrophoresis
through an agarose gel.
Enzyme(s) Fragment sizes
(kb)
PstI 6.8, 5.9
HindIII 6.4, 6.3
EcoRI 9.2, 3.5
PstI + HindIII 4.9, 4.4, 1.9, 1.5
PstI + EcoRI 5.5, 3.7, 3.1, 0.4
EcoRI + HindIII 6.3, 3.5, 1.8, 1.1
First an outline of the circular plasmid is drawn and the zero
position marked with a vertical line. Now consider a digestion
using one of the restriction enzymes. PstI produces two fragments
of 6.8kb and 5.9kb. Sites of PstI digestion can be marked onto the
diagram to roughly reflect the sizes of the fragments
obtained.
Looking at HindIII, two fragments of 6.4kb and 6.3kb are produced.
These sum to the same value as the PstI fragments indicating that
the total size of the plasmid is 12.7kb. The restriction sites for
HindIII can also be marked onto the diagram either side of the
marked PstI restriction sites. Now look at the PstI + HindIII
digest and label the fragment lengths accordingly.
Considering the EcoRI sites finally, it is clear that they lie
closer together than any of the other sites. Position the
restriction sites such that a 6.3, 3.5, 1.8 and 1.1 kb
fragmentation pattern results when combined with the HindIII sites
already marked. Check the distances with the PstI + EcoRI digest to
ensure they are correct.
Genetic Analysis of Metabolic Pathways
Many fungi and bacteria can be grown on synthetic media of a
known chemical composition. This means single gene mutations
introduced into organisms which affect a certain metabolic pathway
can be characterised. This is because mutation can cause the
requirement for a specific
chemical for growth depending on which step in the pathway if
affected.
Mutants which require a specific supplement are produced and can
be genetically analysed to see how many genes are involved.
Furthermore, the mutants are grown on media containing various
intermediates in the metabolic pathway to investigate where in the
pathway the mutation is acting. This approach is often sufficient
to identify the intermediates and their order of formation.
In an experiment, a 1st year scientist replicates a master plate
containing 26 colonies of the fungus Aspergillus to 5 plates
containing different media. The plates have a nitrogen source that
is an intermediate in the metabolic pathway that degrades
hypoxanthine to NH$_4^+$.
The scientist wants to establish:
1) The growth responses of each of the strains
2) The order of intermediates in the pathway
3) The strains which are blocked in each step giving an idea of
the genetics underlying the growth patterns
The table above shows the process by which the growth of the
fungus is classified, where the "+" indicates growth of the strain
on this medium and a "-" scoring means no growth.
Can you think what the purpose of
the colony number 26 is, given it grows on all the media?
The order of intermediates a $\rightarrow$ can be
determined.
If a mutation occurs affecting a step of a metabolic pathway
close to the initial
susbstrate, then if the species is plated onto media
containing nutrients occuring downstream as intermediates in the
pathway, then growth is still possible.
If mutation occurs affecting a step of the metabolic pathway
close to the final product,
then if a species is plated onto media containing nutrients
occuring upstream as intermediates in the pathway, growth still
won't be possible because of the mutation!
As all of the strains are capable of growth on media containing
e, it must occur closest to NH$_4^+$ in the pathway. Using a
similar analysis the order can be established as:
Hypoxanthine $\rightarrow$ d
$\rightarrow$ a $\rightarrow$ c $\rightarrow$ b $\rightarrow$ e
$\rightarrow$ NH$_4^+$
Can you determine which strains are
blocked in which steps of the pathway? For example the step d
$\rightarrow$ a is blocked by the mutants 1,3,6,9,15,17,18,20 and
25. What does this imply about the species genetically?
Such analysis is highly useful in the
early investigation into the genetics behind metabolic
pathways.
In this article, we've covered
topics ranging from Mendelian inheritance, chromosome mapping,
genetic interactions, the genetic analysis of metabolic pathways
and restriction mapping. Hopefully over the course of this article
you have gained an appreciation of some useful genetic concepts you
will frequently encounter in your future studies.