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GENES AND ENZYMES
Historical: Garrod
1909: Sir Archibald Garrod, an English physician, published a book entitled
"Inborn Errors of Metabolism"
One of the diseases Garrod descrobed was ALCAPTONURIA (=
ALKAPTONURIA)
Alcaptonuria is inherited as a single gene defect; normals have at least one
dominant allele and those affected are homozygous recessive.
LEGEND:
A_ = normal
aa = affected (alcaptonuric)
Alcaptonuria is a rare disorder, but it is very easy to detect; it causes the urine to
turn black when exposed to air. There are some other symptoms also; as the
affected person ages there tend to be yellow deposits in the eye, and often there is
a darkening and hardening of cartilage tissues, leading to an arthritis-like
condition. Even so, it is not considered a very serious or threatening disease.
When urine from alcaptonurics was analyzed, it was found to contain a chemical
compound that was not present in normals. This compound, homogentisic acid,
(HA) is the substance that turns black when it is oxidized.
Legend:
A_
aa
= normal
= affected (alcaptonuric) (accumulate and excrete HA)
Garrod proposed that the reason that normals do not accumulate homogentisic
acid is that they convert it to another compound called maleylacetoacetic acid,
and furthermore, he suggested that the reason they could do so was that they
have an enzyme that is missing in alcaptonuria.
An enzyme is a protein that catalyzes a specific chemical reaction.
A protein is a chain of amino acids, and a catalyst is a substance that causes a
reaction to proceed without itself being used up or converted in the reaction.
The names of enzymes usually end in ase ie, RNase breaks down RNA,
proteases are useful in digestion because they breakdown proteins etc. The name
of the enzyme that is present in most people but lacking in alcaptonurics is
homogentisic acid oxidase.
Thus we can rewrite the legend:
A_
= normal -- have HA oxidase
aa
= affected (alcaptonuric) -- lack HA oxidase
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In this case we see that the dominant allele corresponds to the presence of one
specific enzyme and that homozygous recessive individuals lack the enzyme,
directly leading to the associated phenotype.
When alcaptonurics are fed high protein diets, they excrete more HA. In
particular, the levels of two of the amino acids that are present in protein,
phenylalanine and tyrosine, seem to correlate to HA production.
This brings us to a much more serious disease called phenylketonuria or PKU
PKU is also inherited as a single gene recessive character. ie
PKU /_ = normal
pku/pku = affected
It is rare, only about 1 in 11,000 live births is affected in the US
In addition to excretion of phenylketones in the urine, there is a much more
serious consequence of this disease. If left untreated, the average IQ of
homozygous recessives will be 17!. 96% of untreated PKU patients have an IQ
less than 50 and 64% have an IQ below 20. Again, the defect can be traced to the
presence or absence of a single enzyme, but in this case, the enzyme is usually
found only in the liver. The enzyme phenylalanine hydrolase (Φ Hase) is
required to convert phenylalanine (phe) to tyrosine (tyr), the first step in getting
rid of any excess that may be present in the blood. When excess phe accumulates,
it can be converted to phenylketones spontaneously, which are then found at
high levels in the urine. Whether the high levels in the blood prevent access of
other essential amino acids into the developing brain cells, or intrerfere with a
pyruvate kinase required for energy in developing brain cells is a point of
contention. In any case, the problem develops very rapidly after birth of an
affected infant. Before birth, the mother, who, at least in the past, has always
been a PKU/pku heterozygote, has the enzyme and her "filtering system" keeps
the level of phe at acceptable levels for normal prenatal development. There is a
much higher than normal level of phe in the infants blood at birth though, and
this has led to the development of a simple, accurate test which is given to all
babies very soon after birth. The test is called the Guthrie test. It consists of a
dried filter paper impregnated with bacteria (B. subtilis) and a toxic analogue of
phe called beta-thienylalanine. When a drop of blood that has lots of phe is
applied, the bacteria can grow; otherwise they cannot. Blood samples taken soon
after birth are collected, and can be tested for a few cents each. Follow up
chemical tests are also needed, since in some cases, especially for small babies,
since they tend to have a high level of phe that does not persist as it will with
PKU patients.
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PROTEIN
DIETARY PROTEIN
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PROTEIN
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PHENYLALANINE
ΦH ase
TYROSINE
Treatment possibilities: 1) you can't feed a patient the enzyme since they will
digest it, 2) you can't inject it since it will probably be recognized as a foreign
protein so will induce an immune response, and 3) it needs to function
specifically in the liver to be effective. 4) Dietary control of the amount of phe
ingested should and does work. Some is needed, since we need Phe to make our
own proteins; but if just enough can be given that little accumulates, then there
will not be a problem. Of course there needs to be close monitoring, but it seems
to be quite effective if the special diet is followed.
It is estimated that 2 potential IQ points are lost for every week an infant goes
untreated! The treatment is usually ended by age 6-8. By then, the brain is well
developed, and high levels of phenylketones do not seem to have a serious
effect. Also, the diet is said to be very bland, and once the child has tasted
"pizza", it is virtually impossible to go back to the diet. Some recent studies
show that performance on math tests etc. probably drops a bit, but not to the
drastic levels seen in untreated cases. There are now special diets for teens and
adults so the levels of phe can be maintained all the way through adulthood if
desired.
This cure has now been available for some time, and there are pku/pku adults
who are of reproductive age. This is especially a problem for females, since they
do not have the enzyme, and even if a fetus they are carrying is heterozygous, its
enzyme will not function until too late. The first several cases led to death or
extreme mental retardation in the infants born to these mothers. I have heard
that some have been able to go back onto the diet in order to have babies in the
normal range, but it does appear to be a risk and few people have the willpower
to succeed.
PKU provides another example where a genetic defect can be absolutely
associated with the lack of a specific enzyme in homozygous recessive
individuals.
The pigment melanin is derived from tyrosine; at least two steps are required in
the conversion. Lets assume that E1 and E2 are two different enzymes needed to
make melanin in the pathway as shown:
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PROTEIN
DIETARY PROTEIN
PHENYLALANINE
TYROSINE
PROTEIN
E1
DOPA
E2
MELANIN
Phenylketones
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A person (or a rat or a rabbit, etc) who is missing E2 is albino, because they cannot
make any of the normal pigment. The absence of the enzyme and thus albinism
in this case is inherited as a homozygous recessive condition. In this case, the
albinos are called DOPA positive, because they do make DOPA, a colorless
compound that is an "intermediate" in the pathway.
A defective E1 also causes albinism, but in this case, they are DOPA negative
albinos. There are indeed at least two genes that are needed to make melanin,
the endproduct of the pathway, from the tyrosine we normally get in our diets.
Again, the defect is inherited as a simple autosomal recessive condition. If we
assume that allele Al1 "codes" for E1, and that Al2, an allele of a different,
independent gene codes for E2 then we can write legends as follows:
Al 1/_ normal
Al 2/_ normal
al1/al 1 albino
al2/al 2 albino
What F2 ratio would be obtained in sib matings of F1 rats that both have the
genotype: Al1/al1, Al2/al2 ?
This is a classical dihybrid cross with two dominant genes. Thus we know that
we expect each gene to segregate 3:1 as far as phenotypes are concerned and that
the F2 ratio should be
9/16 Al1_, Al2_
E1 and E2 present
3/16 Al1/_, al 2/al2
E1 present, E2 absent
3/16al1/al1, Al2/_
E1 absent, E2 present
1/16 al 1/al1, al 2/al2
E1 and E2 missing
In this example, all of the last three classes are albino; they lack either E1 or E2 or
both. Thus the F2 ratio is 9 pigmented to 7 albino. This is an actual case of
epistasis, and this example shows us how epistasis can be explained on a
molecular level.
Another important compound is derived from tyrosine, but in this case the
enzymes are found only in the thyroid gland.
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PROTEIN
DIETARY PROTEIN
PHENYLALANINE
TYROSINE
PROTEIN
E1
DOPA
E2
MELANIN
Phenylketones
I2
IODOTHYROXIN
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There are several metabolic reactions required for making the thyroid hormone
thyroxin, and another for adding the iodine which is required for function.
Again the enzyme required for each step is coded by a different gene. The
consequence of not having one of the enzymes in this pathway is "goiterous
cretinism". Cretinism because without the thyroid hormone, the affected
individual will be stunted in both physical and mental development, and
goiterous, because the normal response of the body for a lack of iodothyroxin is
to try to make more by enlarging the thyroid gland. This is the same effect of
course of a lack of iodine, which would make this an example of a phenocopy-an
environmental mimic of an inherited phenotype.
Fortunately, in this case the treatment is usually very simple; the affected
individual can usually take oral thyroxin, though in some cases injections are
required.
This condition is also readily detected in young children, and so the problem is
essentially gone in developed countries. Newborn screening in Texas turned up
309 cases of PKU and 891 cases of Thyroxin deficiency by 1989, and many more
have likely been detected since.
After the phe and tyr needed for protein syunthesis, melanin, etc. has been
siphoned off, there is still generally an excess over the amounts required for
protein synthesis. The excess is not normally excreted directly, but is instead
converted to smaller breakdown products.
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PROTEIN
1
PROTEIN
DIETARY PROTEIN
2
PHENYLALANINE
3
5
4
TYROSINE
11
PHENYLKETONES
6
DOPA
7
MELANIN
8
p-HPA
9
12
DHPA
10
13
I2
IODOTHYROXIN
HOMOGENTISIC
14
MAA
15-22
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CO + H O
2
2
One person has been discovered with "tyrosinosis" who lacks enzyme 11. He is
(was) normal, so the effects seem to be much less severe than when E5 is missing
(PKU)
Now you see why the amount of homogentisic acid excreted by alkaptonurics is
related to phe and tyr intake!
There are several critical points that you need to take from this figure.
1) Metabolism occurs in a stepwise fashion; each step may use or require a small
amount of energy, but only involves making or breaking one or two bonds at
a time.
2) Each step is catalyzed by a different, unique enzyme, and as we have seen, each
enzyme is coded by a different gene.
3) Metabolic blocks result from a missing enzyme, and in every case we see here,
this results from a homopzygous recessive condition. Thus the blocks are
often referred to also as genetic blocks.
4) Phenotypes can arise in two ways from genetic blocks:
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a)The failure to make an essential endproduct, such as melanin results in
albinism
b) The accumulation of precursors or intermediates that are normally
converted to other compounds. For example, the buildup of Phe leads to
PKU
Beadle and Tatum
Even though it is clear that Garrod understood these concepts in the early 1900s,
they were not truly appreciated until the 1940s. At that time, two men, George
Beadle and Edward Tatum earned a Nobel prize for developing what is now
called the one gene-one enzyme hypothesis.
To me, this work represents the true beginning of the molecular genetics
revolution that has progressed so rapidly in allowing us to understand how
genes work. Let's very briefly look at what Beadle and Tatum did.
First, they realized that they needed to work with a micro-organism, so that
millions of individuals could be screened for mutations. Someone starting today
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would likely chose bacteria, but at the time they were working, no one knew how
to make crosses with bacteria; without crosses heredity could not be studied.
They chose the filamentous fungus Neurospora crassa very deliberately for
several reasons;
1) It produces million of cells that are genetically identical -in a test tube -in a
few days.
2) It is haploid, meaning that any mutants produced will not be masked by a
dominant allele.
3) Crosses can be made between two mating types (either can be the male or
female in a cross) and the products of meiosis can be grown individually.
Progeny are available less than three weeks after a cross is made.
4) Neuospora could be grown on a defined, minimal, medium. The medium
contains only the essential elements (C H O P K I N S Ca Fe Mg, etc) plus a
sugar for a carbon (energy) source and one vitamin, a compound called
biotin. This told them that Neurospora could make "from scratch" all the
other vitamins, all 20 amino acids required for making proteins, the
building blocks for making complex carbohydrates, DNA, RNA and their
building blocks etc.
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Their approach follows: They treated millions of asexually produced conidia
spores with X-rays, the only agent that had been proven to cause mutations by
that time. The survivors were grown on "complete" medium, which is the
opposite of minimal; it contains sources of all vitamins, amino acids, nucleotides
etc. Individuals were then selected, grown on single tubes of complete and tested
for their ability to grow on minimal. Many mutants were found that could not;
these were tested on various media until it would be possible to identify which
biosynthetic pathway was defective.
X-ray survivors that grow on complete medium but not on minimal medium are
next tested on a variety of other test compounds. In the case below, testing conidia
from the culture growing on complete medium shows the mutation has resulted in a
lysine auxotroph.
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Minimal plus
amino acids
Minimal plus
vitamin mixture
Minimal plus
nucleotide bases
Tests on individual amino acids:
ala
leu
arg
lys
asp
met
cys
asn
phe
pro
glu
ser
gln
thr
gly
try
his
tyr
ile
val
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They referred to this type of mutant as an auxotroph, implying that it needs help
in order to grow. They tested each mutant in a cross to verify that it did behave
as a single gene mutation.
They were then often able to pinpoint the defect to one specific metabolic step in
a biosynthetic pathway as follows:
Assume we have the pathway A→B→C→D where D is a compound required for
growth such as an amino acid or vitamin and the arrows are enzymes 1-4 in the
pathway. Some mutants will grow only if given D; others will grow on C or D,
still others on B, C, or D etc. As long as the compound can enter the cell and
occurs after the genetic block, D can still be made. If the last enzyme in the path
is missing, only D will allow growth etc.
Mutant
#1
#2
#3
Lacks
E1
E2
E3
Growth Response on minimal medium plus cpd
A
B
C
D
no
yes
yes
yes
no
no
yes
yes
no
no
no
yes
Thus they were able in several cases to show that a single enzyme was missing in
each mutant, and that each step was inherited as a single gene defect. They stated
that "There are a large number of genes where the function of each gene is to
control the formation of a single enzyme. They found different single gene
mutants that required each of the amino acids, all of the known vitamins, and 4
of the five bases needed to make DNA and RNA. Thus the one gene--one
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enzyme hypothesis was confirmed. Though there has been some evolution in
the concept; we now know for example that sometimes it takes two or more gene
products to make one functional enzyme, the concept that a gene somehow
codes for a specific protein
By the way, almost all the known biochemical pathways were developed from
having mutants such as these; that is, once mutants were available, new
compounds could be tested to see if they could be converted to the compound
required by the auxotroph. Many of the original Neurospora crassa mutant
strains isolated by Beadle and Tatum are maintained at the Fungal genetics Stock
Center and can be obtained free of charge for educational or research purposes at
http://www.fgsc.net/
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