Genes and Proteins

‘Genes’ are DNA sequences (GATC) strung together in linear

strings, ususally somewhere around say 20000 nucleotides in
length. Genes and the DNA sequences between them make up
chromosomes. This genomic information supplies the program
that specifies development into you, me, a frog, a plant, and
everything else.
What do genes do?
They store sequence information that specifies RNA structures
like tRNAs, ribosomal RNAs, etc. Or specify other noncoding
RNA.
They store sequence information for production of amino acid
chains (proteins). This is the classic definition.
Other DNA sequences act as a string to hold genes together, as
attachment points as in centromeres, as telomere caps at
chromosomal ends, etc.
How many genes in humans? 20,000 to 30,000, depending on
what you count as a gene and what you dont.
When we talk about alleles of a gene, we are talking about the genes
on the maternally and paternally inherited chromosomes that are the
same gene (e.g., A) but differ in nucleotide sequence.

The overall number of nucleotide sequence differences between

individual humans has been estimated based on genome sequencing
projects to occur every 1/1000 to 1/2000 nucleotides or so. Some of
these are in genes and affect phenotype, some may be of no impact
whatsoever, but we don’t know for sure.

In genetics we are interested in how sequence changes in genes that

store RNA or protein information will affect the cell or organism. In
some genes, changes in nucleotide sequence will cause clear
phenotypic differences, such as yellow/green, smooth/wrinkled. Or
human diseases. The alleles that cause those phenotypes are
obviously quite severe in their effect.

However, we do not yet understand how the entire collection of

genomic DNA (all the alleles at once), with sequence differences
scattered both in genes and outside genes, collectively determine an
individual phenotypic outcome.
A. Gene with three exons

* *** * * * M

* *
* ** * P
* Represent nucleotide differences
Transcribed/Spliced
Usually the two gene copies contribute equally
to make the final total amount of gene product

B. messenger RNA

polypeptide sequence predicted

Met to STOP

*
Nucleotide changes affects translation, enzymatic activity,
folding, protein-protein interactions, etc?
Garrod’s Inborn Errors of Metabolism

1902. Looked at phenotype

of air-blackened urine, found
in pedigrees especially
involving marriages of first
cousins (so, inborn and
transmissible. Called
Alkaptonuria, results from
defect in gene product
(enzyme) in metabolic
pathway. Patients excrete
HA rather than metabolizing
to MA on down
Other disorders related to previous pathway
Albinism… is trait resulting from change in the tyrosinase
enzyme…this causes an inability to convert tyrosine to DOPA. This
prevents production of the pigmentation compound called melanin.

Phenylketonuria (PKU)… 1/12000 Caucasian births….mutation in

phenylalanine hydroxylase….cannot convert amino acid
phenylalanine to tyrosine…this results in accumulation of
phenylalanine which in turn affects brain development.
Screening for PKU, as well as several other congenital conditions,
is the law. The classic method is called the Guthrie test, which is a
test for bacterial growth that depends on the presence of
phenylalanine. Extra phenylalanine in the blood supports growth
under the conditions of the assay, normal levels do not.
Soft drinks and PKU? Artificial
Sweetener Aspartame used in
Diet Soda is metabolized to
aspartic acid, phenylalanine,
and methanol.
 Beadle and Tatum, Neurospora, and the
One Gene-One Enzyme Hypothesis
Neurosopora crassa. Comes in two mating types, a and α, both
haploid.
Mating types join to form diploid, divide meiotically, and produce
haploid spores.
Wild type organisms can grow on minimal media with salts,
glucose, and biotin. This provides nitrogen, carbon, and an
essential vitamin. Such organisms are called prototrophs.
Mutant Neurosopora require supplements in the media in order to
grow. They are unable to make everything they need from minimal
media alone. These organisms are called auxotrophs. To grow
they need complete media, or minimial media supplemented with
the critical nutrient they cannot make.
Experiments…..Irradiate Neurospora with X-rays. This induces
changes or mutations. Are the mutations genetic or just ‘damage’?
Genetic, because if crossed with normal Neurospora the defects
are inherited.
Background:
Life Cycle of
Neurospora
Summary of Beadle-Tatum Procedure:
Irradiate…this causes genetic damage.
Cross to WT, this allows damaged alleles to be propogated/transmitted.
(Cells now diploid, any defects are complemented (overcome) by normal
genes from WT parent (Heterozygotes))
Allow to sporulate (meiosis). This regenerates haploid organisms.
Select SINGLE spores and grow in complete media (supplemented with all
nutrients). These grow regardless of any underlying metabolic defect caused
by receiving defective genes.
Take samples and grow in minimal media. If they grow they are fine (for that
particular assay). If they don’t grow they have inherited some metabolic
defect. This could be in many possible pathways.
To test specific compound that might restore growth, take those that do NOT
grow in minimal media and inoculate into minimal media plus….specific
nucleotides, specific amino acids, specific vitamins, etc.
If the strain can now grow in one of those tubes, the supplement tells you
what the cell was UNABLE to do. That is, it tells you which metabolic
pathway had been damaged by X-irradiation.
Irradiation, mating, dissection

Growth on Complete Media

Growth on Minimal Media

Growth on supplemented MM

Growth on supplemented MM
 One gene-one enzyme for the
methionine biosynthetic pathway
The one gene-one enzyme idea has dominated genetics
for many years, and it explains many human genetic
diseases.
Example: Cystic Fibrosis …1/2000 Caucasians affected. CF
caused by amino acid change in cystic fibrosis transmembrane
conductance regulator gene (CFTR). Affects mucus viscosity in lungs.
Example: Tay-Sachs disease ...Caused by mutation in a gene
responsible for metabolizing glycolipids (in this case a brain
ganglioside). Higher allele frequency in Ashkenazi Jewish populations
of central Europe, with 1/3600 infants affected.
Example: Sickle Cell Anemia …More prevalent in Middle Eastern
and African Populations. Caused by amino acid change in β-hemaglobin
gene. Heterozygosity thought to confer some resistance to malaria.
Hemoglobin variants may migrate
at different positions by
electrophoresis. This allows them
to be identified based on their
electrical charge. And because
blood is easy to collect, many
samples can be examined. As a
result, we know there are many
variant hemoglobin genes (alleles)
in human populations.

This assay is a kind of

window that lets you ‘see’
the gene itself.
Hemoglobin variants
exist for both the alpha
and beta chains.
Each variety is a unique
allele. The chance of
having a given allele
depends on the
frequency in a given
population. And you
can only have at most
two.
These result in various
hemoglobinopathies,
anemias, or may have
no known consequence.
Molecular techniques
show that other genes
generally show a similar
sort of allelic diversity.
Genomes are massively complex….
First, there are lots and lots of genes (~25,000 in humans). You can say the
word ’25,000’ and it doesn’t mean much, but start counting to 25,000 to see
how many this is. Each of those genes is expressed and regulated at its own
particular level throughout development.
Second, at each gene an individual may have up to two different allelic
copies. Of course at any given gene it is possible that alleles in one
individual may match those of another individual. But its unlikely that there
would also be matches at gene B, gene C, gene Q403, etc up to 25,000.
Third, not only the above, but which allelic copies an individual has will
depend on how many different types of alleles are present in the population to
choose from, and their frequency.
The particular combination of alleles in an individual makes them what/how
they are. Note again that starting with only four genomes (meaning there are
at most four alleles at each gene) [mother (maternal and paternal), and father
(maternal and paternal)], you can still generate offspring that are very
different from one another. How does that work? We don’t know.
And so far genetics has really only considered allelic sequence differences
within ‘genes’ themselves. There is still many other sequence changes in
noncoding/nongenic regions of the genome that we do not yet understand.
Classic genetics proposes a fairly simple one gene,
one enzyme view of biochemical pathways.
A Tougher Look at Biotech

Christophe Vorlet
By DENISE CARUSO
Published: July 1, 2007
THE $73.5 billion global biotech business may soon have to grapple with a discovery
that calls into question the scientific principles on which it was founded.
Last month, a consortium of scientists published findings that challenge the traditional
view of how genes function. The exhaustive four-year effort was organized by the
United States National Human Genome Research Institute and carried out by 35
groups from 80 organizations around the world. To their surprise, researchers found
that the human genome might not be a “tidy collection of independent genes” after all,
with each sequence of DNA linked to a single function, such as a predisposition to
diabetes or heart disease.
Instead, genes appear to operate in a complex network, and interact and overlap with
one another and with other components in ways not yet fully understood. According to
the institute, these findings will challenge scientists “to rethink some long-held views
about what genes are and what they do.”
Compounds on this chart
number maybe a
hundred or so. In a cell
there would be
thousands and
thousands. In addition,
every compound will
have numbers
associated with level,
rate in, rate out, etc.
Enzymes themselves
have rates associated
with transcription,
translation, degradation,
catalytic enhancment by
modifications, etc.
Changes in one place
pulls the whole system
one way or another.
Charts like this
depict cellular
metabolism. They
give a sense of
complexity that
you don’t get
when you
consider one gene
one enzyme.

Biology is moving
to understand the
cell as a system
like this.
Understanding how
thousands of genes
interact during
development to
produce something
unique, like me and
not you, or you and
not me, is a major
challenge. It could be
random, or noisy,
except that genetically
identical twins are
identical. And inbred
mice are identical.
Which means that the
sequences/alleles in
one genome produce
a consistent output.
Another genome will
have a different
output.
New Genomics Approaches Look at Global Gene Expression
Microarrays and High
Throughput DNA
Sequencing approaches
examine the entire output
of all genes at once.