Gene expression

What is a gene?

1941 George Wells Beadle & Edward Lawrie Tatum proposed the one gene - one enzyme (polypeptide) concept

Genes act by regulating distinct chemical events

First clear understanding of how genes work to produce phenotype

Neurospora crassa life cycle

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/N/Neurospora.html

Advantages of Neurospora for studying genetics

Can be grown quickly on simple culture medium.

Sugar, inorganic salts, one vitamin (biotin)

Spends most of life cycle in the haploid condition

So recessive mutations will show up in its phenotype

No need for test crosses

Can be asexually propagated in large numbers by conidia (asexual spores)

Can be mated to produce ordered tetrads

Life cycle of Neurospora crassa

Have two mating types (like sexes)

A and a

Matings occur only between two mycelia of differing mating types

Can be crossed by placing two types of mycelia on the same plate

Mycelia fuse, then nuclei fuse to form diploid zygote

Diploid zygote undergoes two stages of meiosis, followed by one mitotic division

These cell divisions occur in a narrow tube called the ascus (spore sac)

Which causes the meiotic and mitotic products to remain in order (ordered tetrads)

Thus, if the zygote nucleus is heterozygous for a gene (shown here as a and A) and there is no crossing over near that locus during meiosis I

The ascus will end up with four spores at one end containing one allele (a) and four spores at the other end containing the other allele (A)

Experimental protocol

Grew normal (“wild-type”) Neurospora on minimal medium (sugar, inorganic salts, one vitamin (biotin)

Induced mutations in some conidia by exposing them to ultraviolet rays

Germinated and grew individual irradiated spores on a "complete" medium (enriched with various vitamins and amino acids)

Did crosses

Mated mycelium with spores of the other mating type

Dissected out ascospores individually and plated each one on complete medium.

Tested progeny on minimal medium

Subcultured clones of each of these genotypes on minimual medium (1^st in figure), observed outcome

Sometimes grew on minimal medium; sometimes didn't.

Evaluated which functions were missing

When a genotype could not grow on minimal medium

Subcultured clones of genotype onto various media (2^nd culture tube in figure) that differed in the composition of nutritional supplements (sugars, amino acids, vitamins, salts)

Eventually, each mutated strain was found to have acquired a need for one nutrient

For example, in 3^rd culture tube in figure, fungus needs added vitamin thiamine

Interpretation

Beadle and Tatum reasoned that radiation had caused an allele at a gene that permits the synthesis of thiamine from the simple ingredients in minimal medium to mutate to an allele that does not.

The mutation altered a gene so it could no longer produce the normal enzyme, resulting in a physical symptom (phenotype), like the need for nutritional supplements.

One gene à one enzyme

Further experimentation

Eventually deduced the thiamine synthesis pathway

By adding different precursors of thiamine, one at a time, to the culture medium, they narrowed down the defect to the absence of a single enzyme.

If they added to the minimal medium any precursor past that enzyme in the pathway, growth occurred.

If they added a precursor before that blocked step, growth did not occur

For their work, Beadle and Tatum shared, with J. Lederberg, the 1958 Nobel Prize in Physiology or Medicine.

Class activity

Here’s a table of an experiment with Neurospora that is like Beadle’s and Tatum’s

Nutritional mutants were isolated from “wild-type” Neurospora

These responded to additions to minimal media be growth (+) or lack of growth (-)

Given the following responses of single gene mutations

Diagram a metabolic pathway consistent with the data

Indicate where the pathway is blocked in each mutant strain

	Supplements added to minimal medium
Mutant strain	citrulline	GSA	arginine	ornithine	glutamic acid
1	+	-	+	-	-
2	+	+	+	+	-
3	+	-	+	+	-
4	-	-	+	-	-

Analysis

All strains rescued by arginine

Arginine is “end product”

Which strain rescued by only arginine?

Strain 4

Strain 4 mutated at gene converting precursor D to arginine

Gene d

Which strain rescued by arginine or another compound?

Strain 1 – rescued by arginine or citrulline

Mutated at gene converting Precursor C to citrulline

Gene c

Which strain rescued by arginine, citrulline, or another compound?

Strain 3 – rescued by arginine, citrulline, or ornithine

Mutated at gene converting Precursor B to ornithine

Gene b

Which strain unaccounted for?

Strain 2 – requires only GSA for rescue

Mutated at gene converting Precursor A to GSA

Gene a

What is precursor A?

Could be glutamate

No strains rescued by glutamate

Wildtype pathway

Today’s view of genes

Beadle and Tatum thought of a gene as an physical entity that produced an enzyme

Or, more broadly, a protein

However, we now know that some proteins have multiple subunits, and consist of two or more polypeptide chains

Polypeptide chain is chain of amino acids

One polypeptide can mutate while others remain normal

e.g. human hemoglobin molecule contains two types of polypeptides, alpha and beta, each produced by different genes

Here are the gene clusters, located on two chromosomes

Alpha locus

Two alpha globin genes, essentially identical

Zeta globin genes are expressed early in development and are part of fetal hemoglobin

Beta locus

Two beta globin genes that are part of adult hemoglobin

Plus other genes expressed earlier in development, as shown in table

Embryonic hemoglobins	Chain composition
Embryonic gower 1	zeta(2), epsilon(2)
Embryonic gower 2	alpha(2), epsilon (2)
Embryonic Portland	zeta(2), gamma (2)
Fetal hemoglobin (HbF)	alpha(2), gamma(2)
Adult hemoglobins
hemoglobin A (HbA)	alpha(2), beta(2)
Minor hemoglobin A2 (HbA₂)	alpha(2), delta(2)

One allele of the polypeptide HbA produces HbS hemoglobin

Sickling hemoglobin

A person homozygous for HbS has sickle-cell trait

Causes rbcs to collapse and become angular

Sickled rbc phenotype: HbSHbS genotype

Normal rbc phenotype: HbA__ genotype

Sickled cells don’t flow smoothely and create clots, restricting blood flow in organs throughout the body

But, a person heterozygous for HbS is more resistant to malaria

Heterozygote advantage