Unit-2 Genetics Of Prokaryotes And Eukaryotic Organelles And Gene Structure & Expression

UNIT-2 GENETICS OF PROKARYOTES AND EUKARYOTIC ORGANELLES AND GENE STRUCTURE & EXPRESSION

Structure

2.0 Introduction

2.1 Objectives

2.2 Genetics of Prokaryotes and Eukaryotic Organelles:

2.2.1 Phage Phenotypes

2.2.2 Mapping the Bacteriophage Genome

2.2.3 Recombination in Phage

2.2.4 Genetic Transformation, Conjugation and Transduction in Bacteria

2.2.5 Genetics of Mitochondria and Chloroplasts

2.2.6 Cytoplasmic Male Sterility

2.3 Gene Structure and Expression:

2.3.1 Genetic Fine Structure: Fine Structure of Gene

2.3.2 Cis-Trans Test

2.3.3 The Structure Analysis of Eukaryotes Introns and their Significance

2.3.4 RNA Splicing

2.3.5 Regulation of Gene Expression in Prokaryotes and Eukaryotes

2.4 Let Us Sum Up

2.5 Check Your Progress

2.6 Check Your Progress: The Key

2.7 Assignment

2.8 References

2.0 INTRODUCTION

In this world variety of organisms are present from several centuries. They continuous interact with the environment and perform all the necessary activities require for the life. Reproduction is a characteristic living activity found among all the living entities either prokaryotes or eukaryotes. In this chapter we will try to understand the genetics of lower unicellular and higher multicellular organisms.

2.1 OBJECTIVE

This unit is aim to explain the gene related behaviour of viruses (phages), bacteria together with the eukaryotes. One can be learn following facts after thorough movement over to this unit:

• Life Cycle, General & New Phenotype, recombination and genome mapping technique for Bacteriophages,
• Commonly adapted methods of reproduction in bacteria,
• Extrachromosomal genetics of eukaryotic organelles like mitochondria and chloroplast including the role of cytoplasm in some features of heredity,
• Physical status of gene and its single or unit behaviour (cis-trans test),
• Non-coding region(Intron) of mRNA but having importance,
• Processing of RNA by splicing mechanism and
• Genes having different units or factors for controlling its working.

2.2 GENETICS OF PROKARYOTES AND EUKARYOTIC ORGANELLES

2.2.1 Phage Phenotypes

Bacteriophage - A bacteriophage (from 'bacteria' and Greek phagein, 'to eat') is any one of a number of viruses that infect bacteria. The term is commonly used in its shortened form, phage.

Typically, bacteriophages consist of an outer protein hull enclosing genetic material. The genetic material can be ssRNA (single stranded RNA), dsRNA, ssDNA, or dsDNA between 5 and 500 kilo base pairs long with either circular or linear arrangement. Bacteriophages are much smaller than the bacteria they destroy - usually between 20 and 200 nm in size.

T2 and its close relative T4 are viruses that infect the bacterium E. coli. The infection ends with destruction (lysis) of the bacterial cell so these viruses are examples of bacteriophages ("bacteria eaters").

General Phenotype - Generally each virus particle (virion) consists of:

·  a protein head (~0.1 µm) inside of which is a single, circular molecule of double-stranded DNA containing 166,000 base pairs. (Figure 2.1)

·  a protein tail from which extend thin protein fibers

Life Cycle - The virus attaches to the E. coli cell. This requires a precise molecular interaction between the fibers and the cell wall of the host. The DNA molecule is injected into the cell. Within 1 minute, the viral DNA begins to be transcribed and translated into some of the viral proteins, and synthesis of host proteins is stopped. At 5 minutes, viral enzymes needed for synthesis of new viral DNA molecules are produced. At 8 minutes, some 40 different structural proteins for the viral head and tail are synthesized. At 13 minutes, assembly of new viral particles begins. At 25 minutes, the viral lysozyme destroys the bacterial cell wall and the viruses burst out — ready to infect new hosts.

o  If the bacterial cells are growing in liquid culture, it turns clear.

o  If the bacterial cells are growing in a "lawn" on the surface of an agar plate, then holes, called plaques (Figure 2.2), appear in the lawn.

New Phenotypes - Occasionally, new phenotypes appear such as a change in the appearance of the plaques or even a loss in the ability to infect the host.

Examples:

·  h

o  Some strains of E. coli, e.g. one designated B/2, gain the ability to resist infection by normal ("wild-type") T2. The mutation has caused a change in the structure of their cell wall so that the tail fibers of T2 can no longer bind to it. However, T2 can strike back. Occasional T2 mutants appear that overcome this resistance. The mutated gene, designated h (for "host range"), encodes a change in the tail fibers so they can once again bind to the cell wall of strain B/2. The normal of "wild-type" gene is designated h+ .

o  When plated on a lawn containing both E. coli B and E. coli B/2,

§  the mutant (h) viruses can lyze both strains of E. coli, producing clear plaques, while

§  the wild-type (h+) viruses can only lyze E. coli B producing mottled or turbid plaques.

·  r

o  Occasional T2 mutants appear that break out of their host cell earlier than normal.

o  The mutation occurs in a gene designated r (for "rapid lysis"). It reveals itself by the extra-large plaques that it forms.

o  The wild-type gene, producing a normal time of lysis, is designated r+. It forms normal-size plaques.

As with so many organisms, the occurrence of mutations provides the tools to learn about such things as

·  The function of the gene;

·  Its location in the DNA molecule (mapping).

2.2.2 Mapping the Bacteriophage Genome

A bacteriophage (from 'bacteria' and Greek ‘phagein= to eat') is any one of a number of viruses that infect bacteria. The term is commonly used in its shortened form, phage. Typically, bacteriophages consist of an outer protein hull enclosing genetic material. The genetic material can be ssRNA (single stranded RNA), dsRNA, ssDNA, or dsDNA between 5 and 500 kilo base pairs long with either circular or linear arrangement. Bacteriophages are much smaller than the bacteria they destroy - usually between 20 and 200 nm in size.

T2 and its close relative T4 are viruses that infect the bacterium E. coli. The infection ends with destruction (lysis) of the bacterial cell so these viruses are examples of bacteriophages ("bacteria eaters") Bacteriophage genome can be mapped by following method.

Techniques for the Study of Bacteriophage’s genome

Viruses reproduce only within host cells; so bacteriophages must be cultured in bacterial cells. To do so, phages and bacteria are mixed together and plated on solid medium in a Petri plate. A high concentration of bacteria is used so that the colonies grow into one another and produce a continuous layer of bacteria, or “lawn,” on the agar. An individual phage infects a single bacterial cell and goes through its lytic cycle. Many new phages are released from the lysed cell and infect additional cells; the cycle is then repeated. The bacteria grow on solid medium; so the diffusion of the phages is restricted and only nearby cells are infected. After several rounds of phage reproduction, a clear patch of lysed cells (a plaque) appears on the plate (Figure 2.2). Each plaque represents a single phage that multiplied and lysed many cells. Plating a known volume of a dilute solution of phages on a bacterial lawn and counting the number of plaques that appear can be used to determine the original concentration of phage in the solution.

(1) Mapping by Recombination Frequencies - The strain B of E. coli can be infected by both h+ and h strains of T2 bacteriophage. In fact, a single bacterial cell can be infected simultaneously by both.

Let us infect a liquid culture of E. coli B with two different mutant T2 viruses

·  h r+ and

·  h+ r (Figure 2.4)

When this is done in liquid culture, and then plated on a mixed lawn of E. coli B and B/2, four different kinds of plaques appear.

Genotype / Phenotype / Number of Plaques
hr+ / clear, small / 460
h+r / turbid, large / 460
h+r+ / turbid, small / 40
hr / clear, large / 40
Total = / 1000
hm+ / 470
h+m / 470
h+m+ / 30
hm / 30
Total = / 1000

The most abundant (460 each) are those representing the parental types; that is, the phenotypes are those expected from the two infecting strains. However, small numbers (40 each) of two new phenotypes appear. These can be explained by genetic recombination having occasionally occurred between the DNA of each parental type within the bacterial cell.

Just as in higher organisms, one assumes that the frequency of recombinants is proportional to the distance between the gene loci. In this case, 80 out of 1000 plaques were recombinant, so the distance between the h and r loci is assigned a value of 8 map units or centimorgans.

Now repeat coinfecting E. coli B with two other strains of T2:

·  hm+ and

·  h+m

Again, 4 kinds of plaques are produced: parental (470 each) and recombinant (30 each).

The smaller number of recombinants indicates that these two gene loci (h and m) are closer together (6 cM) than h and r (8 cM). But the order of the three loci could be either

mr+ / 440
m+r / 440
m+r+ / 60
Mr / 60
Total = / 1000

·  m–6–h—8—r or

·  h–6–m-2-r

To find out which is the correct order, perform a third mating using

·  mr+ and

·  m+r

This makes it clear that the

order is m—h—r, not h—m—r. But why only 12cM between the outside loci (m and r) instead of the 14cM produced by adding the map distances found in the first two matings?

(2) Mapping by A Three-Point Cross - The answer comes from performing a mating between T2 viruses differing at all three loci:

·  hmr and

·  h+m+r+

(Note: this time one parent has all mutant; the other all wild-type alleles — don't be confused!)

Group 1 / hmr / 435
Group 2 / h+m+r+ / 435
Group 3 / h+mr+ / 25
Group 4 / hm+r / 25
Group 5 / hmr+ / 35
Group 6 / h+m+r / 35
Group 7 / hm+r+ / 5
Group 8 / h+mr / 5
Total = / 1000

The result: 8 different types of plaques are formed.

·  parentals; that is, nonrecombinants in Groups 1 and 2;

·  recombinants - all the others

Analyzing these data shows how the two-point cross between m and r understated the true distance between them.

Let's first look at single pairs of recombinants as we did before (thus ignoring the third locus).

·  If we look at all the recombinants between h and r but ignore m (as in the first experiment), we find that they are contained in Groups 5, 6, 7, and 8 -7 giving the total of 80 that we found originally.

·  If we look at recombinants between h and m but ignore r (as in the second experiment), we find that they are contained in Groups 3, 4,7, and 8 - giving the same total of 60 that we found before.

·  But if we focus only on m and r (as we did in the third experiment), we find that the recombinants are contained in Groups 3, 4, 5, and 6 - giving the same total of 120 as before while the non-recombinants are not only in Groups 1 and 2 but also in Groups 7 and 8. The reason: a double-crossover occurred in these cases, restoring the parental configuration of the m and r alleles.

·  Because these double crossovers were hidden in the third experiment, the map distance (12 cM) was understated. To get the true map distance, we add their number to each of the other recombinant groups (Groups 3,4,5, and 6) so 25 + 5 +25 +5 +35 + 5 + 35 + 5 = 140, and the true map distance between m and r is the 14 cM that we found by adding the map distances between h and r (8 cM) and h and m (6 cM).

The three-point cross is also useful because it gives the gene order simply by inspection:

·  Find the rarest genotypes (here Groups 7 and 8), and

·  the gene NOT in the parental configuration (here h) is always the middle one.

2.2.3 Genetic Recombination in Phage

Site-specific genetic recombination is very common method in phage for exchanging the genetic material. Unlike general recombination it is guided by a recombination enzyme that recognizes specific nucleotide sequences present on one or both of the recombining DNA molecules. Base-pairing between the recombining DNA molecules need not be involved, and even when it is, the heteroduplex joint that is formed is only a few base pairs long. By separating and joining double-stranded DNA molecules at specific sites, this type of recombination enables various types of mobile DNA sequences to move about within and between chromosomes.

Site-specific recombination was first discovered as the means by which a bacterial virus, bacteriophage lambda, moves its genome into and out of the E. coli chromosome. In its integrated state the virus is hidden in the bacterial chromosome and replicated as part of the host's DNA (Figure 2.8). When the virus enters a cell, a virus-encoded enzyme called lambda integrase is synthesized. This enzyme catalyzes a recombination process that begins when several molecules of the integrase protein bind tightly to a specific DNA sequence on the circular bacteriophage chromosome. The resulting DNA-protein complex can now bind to a related but different specific DNA sequence on the bacterial chromosome, bringing the bacterial and bacteriophage chromosomes close together. The integrase then catalyzes the required DNA cutting and resealing reactions, using a short region of sequence homology to form a tiny heteroduplex joint at the point of union (Figure 2.5). The integrase resembles a DNA topoisomerase in that it forms a reversible covalent linkage to DNA wherever it breaks a DNA chain. The same type of site-specific recombination mechanism can also be carried out in reverse by the lambda bacteriophage, enabling it to exit from its integration site in the E. coli chromosome in order to multiply rapidly within the bacterial cell. This excision reaction is catalyzed by a complex of the integrase enzyme (Figure 2.6) with a second bacteriophage protein, which is produced by the virus only when its host cell is stressed. If the sites recognized by such a recombination enzyme are flipped, the DNA between them will be inverted rather than excised (Figure 2.7). Many other enzymes that catalyze site-specific recombination resemble lambda integrase in requiring a short region of identical DNA sequence on the two regions of DNA helix to be joined.