Method

Experiment 1

Experiment 2

One Method Preserving Organism Suger

Growth means that gene A is not essential.

No growth means that gene B is essential.

Growth means that gene A is not essential.

No growth means that gene B is essential.

Conclusion: If each gene is inactivated in turn, a "minimal essential genome" can be determined.

13.22 Using Transposon Mutagenesis to Determine the Minimal Genome By inactivating genes one by one, scientists can determine which genes are essential for the cell's survival.

Another way to define the minimal genome is to take the organism with the simplest genome, deliberately mutate one gene at a time, and see what happens. Mycoplasma genitalium has the smallest known genome—only 470 genes. Even so, some of its genes are dispensable under some circumstances. It has genes for metabolizing both glucose and fructose. In the laboratory, the organism can survive on a medium supplying only one of those sugars, making the genes for metabolizing the other sugar unnecessary. But what about other genes? Experiments using transposons as mutagens have addressed this question. When the bacterium is exposed to transposons, they insert themselves into a gene at random, mutating and inactivating it (Figure 13.22). The mutated cells are sequenced to determine which gene was mutated, and then examined for growth and survival.

The astonishing result of these studies is that M. genital-ium can survive in the laboratory without the services of 133 of its genes, leaving a minimum genome of 337 genes! This "genomic downsizing" has also been found in other prokaryotes. The bacterium that causes leprosy, Mycobac-terium leprae, is a cousin of Mycobacterium tuberculosis, mentioned above. But M. leprae has "discarded" 2,000 of the genes present in its cousin. For example, it lacks genes for the proteins of the electron transport chain (see Chapter 7), and is therefore slow-growing. But it retains the anabolic pathways it needs to survive when external nutrients are scarce.

Chapter Summary

Probing the Nature of Genes

► Prokaryotes and viruses are useful for the study of genetics and molecular biology because they contain much less DNA than eukaryotes, grow and reproduce rapidly, and are haploid.

Viruses: Reproduction and Recombination

► Viruses were discovered as disease-causing agents small enough to pass through a filter that retains bacteria. The basic viral unit, called a virion, consists of a nucleic acid genome, which codes for a few proteins, and a protein coat called a capsid.

► Viruses are obligate intracellular parasites: they need the biochemical machinery of a living cell in order to reproduce.

► There are many types of viruses, classified by their size and shape, by their genetic material (RNA or DNA), or by their host organism. Review Figure 13.1

► Bacteriophage are viruses that infect bacteria. In the lytic cycle, the host cell bursts, releasing new phage particles. Some phage can also undergo a lysogenic cycle, in which their DNA is inserted into the host chromosome, where it replicates for generations. When conditions are appropriate, the phage DNA exits the host chromosome and enters a lytic cycle. Review Figure 13.2

► Some viruses have promoters for host RNA polymerase, which they use to transcribe their own genes. Review Figure 13.3

► Most of the many types of RNA and DNA viruses that infect animals cause diseases. Some animal viruses have an envelope derived from the host's plasma membrane.

► Retroviruses, such as HIV, have RNA genomes that they reproduce through a complementary DNA intermediate. Other RNA viruses use their RNA to make mRNA to code for enzymes and replicate their genomes without using DNA. Review Figures 13.4, 13.5

► Many viruses are spread by vectors, such as insects.

Prokaryotes: Reproduction and Recombination

► When bacteria divide, they form clones of identical cells that can be observed as colonies when grown on solid media. Review Figure 13.6

► A bacterium can transfer its genes to another bacterium by conjugation, transformation, or transduction.

► In conjugation, a bacterium attaches to another bacterium and passes a fragment of its DNA to the recipient cell. Review Figures 13.7, 13.8, 13.9

► In transformation, fragments of bacterial DNA are taken up by a cell from the environment. These genetic fragments may recombine with the host chromosome, thereby permanently adding new genes. Review Figure 13.10a

► In transduction, phage capsids carry bacterial DNA from one bacterium to another. Review Figure 13.10b

► Plasmids are small bacterial chromosomes that are independent of the main chromosome. R factors, which are plasmids that carry genes for antibiotic resistance, are a serious public health threat. Review Figure 13.11

► Transposable elements are stretches of DNA that can move from one place to another on the bacterial chromosome—either by actually moving or by making a new copy, which is inserted at a new location. Review Figure 13.12

Regulation of Gene Expression in Prokaryotes

► In prokaryotes, the synthesis of some proteins is regulated so that they are made only when they are needed.

► Constitutive enzymes whose products are essential to the cell at all times, are synthesized constantly. A compound that stimulates the synthesis of an enzyme needed to process it is called an inducer, and the enzyme is called an inducible enzyme. Review Figures 13.13, 13.14

► An operon consists of a promoter, an operator, and two or more structural genes. Promoters and operators do not code for proteins, but serve as binding sites for regulatory proteins. When a repressor protein binds to the operator, transcription of the structural genes is inhibited. Review Figures 13.15, 13.16

► The mechanisms that regulate the expression of prokaryotic genes include inducible operator-repressor systems, repressible operator-repressor systems, and systems that increase the efficiency of a promoter. Review Table 13.2

► The lac operon is an example of an inducible system. When lactose is absent, a repressor protein binds tightly to the operator. The repressor prevents RNA polymerase from binding to the promoter, turning transcription off. Lactose acts as an induc-er by binding to the repressor. This binding changes the repres-sor's shape so that it can no longer bind to the operator. With the operator unbound, RNA polymerase binds to the promoter, and transcription is turned on. Review Figure 13.17. See Web/CD Tutorial 13.1

► Repressor proteins are coded by constitutive regulatory genes.

► The trp operon is an example of a repressible system. The presence of tryptophan, the end product of a metabolic pathway, represses synthesis of the enzymes involved in that pathway. Tryptophan acts as a corepressor by binding to an inactive repressor protein and making it active. When the activated repressor binds to the operator, transcription is turned off.

Review Figure 13.18. See Web/CD Tutorial 13.2

► The efficiency of a promoter can be increased by regulation of the level of cAMP, which binds to a protein called CRP. The CRP-cAMP complex then binds to a site near the promoter, enhancing the effectiveness of RNA polymerase binding and hence transcription. Review Figure 13.19

Control of Transcription in Viruses

► In bacteriophage that can undergo a lytic or a lysogenic cycle, the decision as to which pathway to take is made by operator-regulatory protein interactions. Review Figure 13.20

Prokaryotic Genomes

► Functional genomics relates gene sequences to protein functions. Review Figure 13.21

► By mutating individual genes in a small genome, scientists can determine the minimal genome required for cellular life.

Review Figure 13.22

See Web/CD Activity 13.1 for a concept review of this chapter.

Self-Quiz

1. Which of the following is not true with regard to the lac operon?

a. When lactose binds to the repressor, the latter can no longer bind to the operator.

b. When lactose binds to the operator, transcription is stimulated.

c. When the repressor binds to the operator, transcription is inhibited.

d. When lactose binds to the repressor, the shape of the repressor is changed.

e. When the repressor is mutated, one possibility is that it does not bind to the operator.

2. Which of the following is not a type of virus reproduction?

a. DNA virus in a lytic cycle b. DNA virus in a lysogenic cycle c. RNA virus by a double stranded RNA intermediate d. RNA virus by reverse transcription to make cDNA

e. RNA virus by acting as tRNA

3. In the lysogenic cycle of a bacteriophage, a. a repressor, cI, blocks the lytic cycle.

b. a bacteriophage carries DNA between bacterial cells.

c. both early and late phage genes are transcribed.

d. the viral genome is made into RNA which stays in the host cell.

e. many new viruses are made immediately, regardless of host health.

4. An operon is a. a molecule that can turn genes on and off.

b. an inducer bound to a repressor.

c. regulatory sequences controlling protein-coding genes.

d. any long sequence of DNA.

e. a group of linked genes.

5. Which statement about both transformation and trans-duction is true?

a. DNA is transferred between viruses and bacteria.

b. Neither occurs in nature.

c. Small fragments of DNA move from one cell to another.

d. Recombination between the incoming DNA and host cell DNA does not occur.

e. A conjugation tube is used to transfer DNA between cells.

6. Plasmids a. are circular protein molecules.

b. are required by bacteria.

c. are tiny bacteria.

d. may confer resistance to antibiotics.

e. are a form of transposable element.

7. The minimal genome can be estimated for a prokaryote a. by counting the total number of genes.

b. by comparative genomics.

c. as about 5,000 genes.

d. by transposon mutagenesis, one gene at a time.

e. does not include any genes coding for tRNA.

8. When tryptophan accumulates in a bacterial cell, a. it binds to the operator, preventing transcription of adjacent genes.

b. it binds to the promoter, allowing transcription of adjacent genes.

c. it binds to the repressor, causing it to bind to the operator.

d. it binds to the genes that code for enzymes.

e. it binds to RNA and initiates a negative feedback loop to reduce transcription.

9. The promoter in the lac operon is a. the region that binds the repressor.

b. the region that binds RNA polymerase.

c. the gene that codes for the repressor.

d. a structural gene.

10. The CRP-cAMP system a. produces many catabolites.

b. requires ribosomes.

c. operates by an operator-repressor mechanism.

d. is an example of positive control of transcription.

e. relies on operators.

For Discussion

1. Viruses sometimes carry DNA from one cell to another by transduction. Sometimes a segment of bacterial DNA is incorporated into a phage protein coat without any phage DNA. These particles can infect a new host. Would the new host become lysogenic if the phage originally came from a lyso-genic host? Why or why not?

2. Compare the life cycles of the viruses that cause influenza and AIDS (Figures 13.4 and 13.5) with respect to:

• How the virus enters the cell

• How the virion is released in the cell

• How the viral genome is replicated

• How new viruses are produced

3. Compare promoters adjacent to "early" and "late" genes in the bacteriophage lytic cycle.

4. In the lactose (lac) operon of E. coli, repressor molecules are encoded by the regulatory gene. The repressor molecules are made in very small quantities and at a constant rate per cell. Would you surmise that the promoter for these repressor molecules is efficient or inefficient? Is synthesis of the repressor constitutive, or is it under environmental control?

5. A key characteristic of a repressible enzyme system is that the repressor molecule must react with a corepressor (typically, the end product of a pathway) before it can combine with the operator of an operon to shut the operon off. How is this different from an inducible enzyme?

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  • Dominik
    When tryptophan accumulates in a bacterial cell?
    8 years ago

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