Example: Two white-flowered plants cross to produce purple flowers, although purple is dominant.
Each contains a mutation in a different gene, encoding a different enzyme needed to make the purple pigment.

Complementation analysis is easiest to do in bacteria, fungi, or C. elegans, where many mutants of a given phenotype can be obtained.

If we isolate a large number of strains with the same defective phenotype,we can cross them in all combinations, and figure out the number of complementation groups.  Any two defective strains that FAIL to complement are in the same complementation group.  Usually each complementation group represents one of the essential enzymes in the pathway.

Problem 1:  Figure out how many complementation groups there are in these examples.

The concept of complementation is extremely important in molecular biology.  For example, the sickle-cell mouse line could only be created because two strains with different defects (lack of mouse or human  globin genes) could be mated to complement each other's defects.  The fact that genes from different species can complement each other was one of the most significant conceptual advances in molecular biology.  Complementation is now used routinely to answer more subtle questions of how genes are regulated.  You absolutely need to understand complementation to understand molecular biology.

Bacterial complementation.
Bacterial genetic systems can show complementation in two important ways--each manipulating a natural process of bacterial genetics.  These two processes have since been modified in biotechnology to provide most of the essential tools of gene cloning.

1.  Specialized Transduction.  A lysogenic bacteriophage can excise itself so as to carry a piece of host DNA by mistake.  The phage will now carry a second copy of an allele (or linked alleles)  into a host cell.  The new bacterium is a partial diploid for the allele(s).

In biotechnology, a phage chromosome can have a piece of foreign DNA ligated into it in the test tube.  Then the phage DNA is packaged into phage, and it can infect a new host where it either (1) produces many copies of the host gene; or (2) lysogenizes the host, to express the cloned DNA.

2.  F' plasmid.The F plasmid can recombine itself into the host chromosome, then recombine itself out again with some host DNA by mistake.  When it enters the next host cell, it carries a second copy of several genes; again, a partial diploid is created.

In biotechnology, a plasmid can have a piece of foreign DNA ligated into it in the test tube; then the plasmid is transformed into E. coli.  Then the plasmid makes many copies, including the cloned gene.

Suppressor mutation analysis.
A variation on complementation is suppressor mutations.   A suppressor mutation corrects a defect in a different gene locus.  A mutant version of gene A makes an altered gene product, which corrects the phenotype of a defective mutation in gene B.

This operon encodes ribosomal proteins, and an elongation factor; in all:

• elongation factor (tuf)
• ribosomal proteins S10 (rpsJ)
• L3 (rplC)
• L4 (rplD)
• L23 (rplW)
• L2 (rplB)
• S19 (rpsS)
• L22 (rplV)
• S3 (rpsC)

Human Growth Hormone Receptor

For other interesting operons, try searching GenBank, the international repository for all known DNA sequences.   (Funded by the U.S. government--your tax dollars at work.)

Control of Gene Expression
What controls how much of a gene product is made?
Several levels:

• DNA sequence--inversion or deletion
• Transcription, sigma factor
• Transcription, promoter sequence, repressor proteins
• Translational repressor
• Posttranslational modification

The Lac Operon
The Lac operon is the classic model for activation and repression of transcription.  Concepts of analysis based on the Lac operon can be applied to other systems including animals and plants.

The following explanation of the Lac operon is modified from MIT Lac Operon.
Jacob and Monod were the first scientists to elucidate a transcriptionally regulated system. They worked on the lactose metabolism system in E. coli. When the bacterium is in an environment that contains lactose:

 It should turn on the enzymes that are required for lactose degradation. These enzymes are:

beta-galactosidase:

This enzyme hydrolyzes the bond between the two sugars, glucose and galactose. It is coded for by the gene LacZ.

Lactose Permease:

This enzyme spans the cell membrane and brings lactose into the cell from the outside environment. The membrane is otherwise essentially impermeable to lactose. It is coded for by the gene LacY.

Thiogalactoside transacetylase:

The function of this enzyme is not known. It is coded for by the gene LacA.

 A bacterium's prime source of food is glucose, since it does not have to be modified to enter the repiratory pathway. So if both glucose and lactose are around, the bacterium wants to turn off lactose metabolism in favour of glucose metabolism. There are regulatory sites upstream of the Lac genes that respond to glucose concentration.

An overall picture of Lac regulation would be this:

• Lactose induces transcription by pulling the LacI repressor off.
• Glucose prevents transcription by pulling the CAP activator off.

Lac Repressor Tutorial

Catabolite Activator Tutorial

When lactose is present, it acts as an inducer of the operon.  It enters the cell, rearranges slightly to form allolactose, then binds to the Lac repressor.  A conformational change causes the repressor to fall off the DNA. Now the RNA polymerase is free to move along the DNA, and RNA can be made from the three structural genes.  The mRNA will be translated to the proteins which transport and metabolize lactose.

 When the inducer (lactose) is removed, the repressor returns to its original conformation and binds to the DNA, so that RNA polymerase can no longer get past the promoter. No RNA and no protein is made.

 Note that RNA polymerase can still bind to the promoter though it is unable to move past it. That means that when the cell is ready to use the operon, RNA polymerase is already there and waiting to begin transcription; the promoter doesn't have to wait for the holoenzyme to bind.

ATP --> ADP + Pi --> AMP + Pi --> cAMP

cAMP binds to a protein called CAP (catabolite activator protein), which is then activated to bind to the CAP binding site. This activates transcription, perhaps by increasing the affinity of the site for RNA polymerase. This phenomenon is called catabolite repression, a misnomer since it involves an activator protein, but understandable since it seemed that the presence of glucose repressed all the other sugar metabolism operons.

This image shows a "close-up" view of CAP regulation:

Corepressor control
Other operons are controlled by their products, rather than their substrates; for example, expression of biosynthetic enzymes to build amino acids.  This is called feedback inhibition.  In the Trp operon, for tryptophan biosynthesis, transcription of mRNA for five enzymes is prevented by binding of the Trp corepressor in the presence of tryptophan.  When tryptophan levels fall, Trp comes off of the corepressor, and the corepressor comes off of the promoter/operator site.  Transcription now occurs, so that the cell has enzymes to make more tryptophan.

Regulatory mutants can have various kinds of mutant phenotypes.  For example:

p-        Promoter fails to bind RNA polymerase.  No transcription occurs.
lacI-    Repressor fails to bind promoter/operator.  Transcription occurs constitutively
            (in the presence or absence of lactose)
o-c      Operator fails to bind repressor.  Transcription is constitutive.
lacZ-    Structural gene is defective.  No enzyme is made.

What will happen?  What kinds of complementation can occur?Does is matter if the two mutant alleles are adjacent on the same chromosome (cis) or separated (trans)?
 
 

Problem 2.  Predict whether the following diploids produce B-galactosidase, in the presence of lactose; in the absence of lactose.  Explain why.Explain in each case whether it matters if the two mutant alleles are located in cis or in trans.

p +    lacZ -      lacI +
-----------------  ----------
p +    lacZ +      lacI -

p +    lacZ -      lacI -
-----------------  ----------
p -    lacZ +      lacI -

p +    lacZ -      lacI +
-----------------   ----------
p +    lacZ +      lacI -

p +    o-c     lacZ +      lacI +    (A constitutive operator NEVER binds repressor,
--------------------------    --------     with or without lactose.)
p +    o +     lacZ -       lacI +

Problem 3. Explain two different genetic processes in bacteria that can create a "partial diploid" for a small part of the genome.  Explain why these processes are useful for bacterial genetic analysis.

Problem 4. State whether  B-galactosidase is expressed by each lac operon diploid, (1) and (2), and briefly state why (one sentence).  Complete possible genotypes for (3) and (4).
 
 

MIT Bacterial Genetics Problems

Quiz on Lac Operon -- Highly Recommended

Bacterial consensus promoters include two regions of six base pairs each, at -10 and -35 bases upstream.  However, no two promoters are exactly alike, and no promoter exactly matches the consensus sequence.  Additional sites for environmental regulators can be found as far as -50 to -300 bases upstream.

When bacteria enter your digestive tract, how do they adjust to your body's defense mechanisms such as acid and antioxidant stress?  When bacteria exit the intestine how do they cope with starvation?

• When bacteria use up their carbon sources, they express RpoS, the starvation sigma factor.  (Review, what is a sigma factor?)
• RpoS joins RNA polymerase to initiate transcription of different environmental stress genes--genes protecting against all the different stresses that the bacteria might encounter before they enter a new human intestine.  This phenomenon is known as cross-protection.
• The stress genes can be used for conditions as unrelated as acid or base resistance.
• The stress genes activated may or may not be part of multi-gene operons.  They may face in opposite directions, from many different promoters, at all different loci around the genomic map.

• Arsenic Resistance Operons. How do bacteria resist arsenic?  An environmental response regulon is turned on by arsenic.  The molecular basis is related to  how cancer cells develop resistance to anti-cancer drugs.

An example of an arsenic resistance operon
from Barry Rosen's Arsenic Research Lab.

• Environmental regulation in Yersinia pestis (bubonic plague bacteria).  Several  complex regulons of genes respond to specific environmental factors, particularly iron and temperature.  At low temperature, the presence of iron tells the bacterium, "I am in blood that has been swallowed by a flea."  The bacterium expresses proteins that upset the flea's digestion, forcing it to regurgitate the bacteria into the blood of its next victim.  (Susan Straley & Robert Perry, Trends in Microbiol., 1995)

• E. coli virulence regulator.  How do virulent E. coli strains kill children?   A regulator protein binds to an operon encoding "pilins," for E. coli to make pili which attach to the intestinal epithelium.

• Tuberculosis model gene expression.  How are genes regulated in a tuberculosis-related pathogen?

M. Donnenberg, U. Maryland
 
• Virulence regulator in "Flesh-eating bacteria."  A gene activator protein in Staphylococcus aureus turns on the virulence regulon that makes the flesh-eating toxins.  This activator can be used as a vaccine against S. aureus.

Balaban & Novick, 1995, PNAS 92:1619.

• DNA Microarrays.  We can now put most of the protein-encoding genes onto a microarray chip, using technology based on the DNA silicon chip industry.  The chip can be used to hybridize to cellular RNA, and measure the expression rates of a large number of genes in a cell.