Genetic Transfer & Recombination In Bacteria

• Genetic recombination - transfer of DNA from one organism (donor) to another recipient. The transferred donor DNA may then be integrated into the recipient's nucleoid by various mechanisms (homologous, non-homologous).

• Homologous recombination- homologous DNA sequences having nearly the same nucleotide sequences are exchanged by means of Rec A proteins. This involves breakage and reunion of paired DNA segments as seen in (Natural mechanisms of genetic recombination in bacteria include:

a. transformationb. transductionc. conjungation

Transformation

• Genetic recombination in which a DNA fragment from a dead, degraded bacterium enters a competent recipient bacterium and it is exchanged for a piece of the recipient's DNA.

• Involves 4 steps

1. A donor bacterium dies and is degraded 2. A fragment of DNA from the dead donor bacterium binds to DNA binding proteins on the cell wall of a competent, living recipient bacterium

3. The Rec A protein promotes genetic exchange between a fragment of the donor's DNA and the recipient's DNA

4. Exchange is complete

The 4 steps in Transformation

http://www.cat.cc.md.us/courses/bio141/lecguide/unit4/genetics/recombination/transformation/transformation.html

Transduction

• Genetic recombination in which a DNA fragment is transferred from one bacterium to another by a bacteriophage

Structure of T4 bacteriophage Contraction of the tail sheath of T4

What are Bacteriophages?

Bacteriophage (phage) are obligate intracellular parasites that multiply inside bacteria by making use of some or all of the host biosynthetic machinery (i.e., viruses that infect bacteria

Transduction (cont’d)• There are two types of transduction:

– generalized transduction: A DNA fragment is transferred from one bacterium to another by a lytic bacteriophage that is now carrying donor bacterial DNA due to an error in maturation during the lytic life cycle.

– specialized transduction: A DNA fragment is transferred from one bacterium to another by a temperate bacteriophage that is now carrying donor bacterial DNA due to an error in spontaneous induction during the lysogenic life cycle

Seven steps in Generalised Transduction

1. A lytic bacteriophage adsorbs to a susceptible bacterium.

2. The bacteriophage genome enters the bacterium. The genome directs the bacterium's metabolic machinery to manufacture bacteriophage components and enzymes

3. Occasionally, a bacteriophage head or capsid assembles around a fragment of donor bacterium's nucleoid or around a plasmid instead of a phage genome by mistake.

Seven steps in Generalised Transduction (cont’d)

4. The bacteriophages are released.

5. The bacteriophage carrying the donor bacterium's DNA adsorbs to a recipient bacterium

Seven steps in Generalised Transduction (contd)

http://www.cat.cc.md.us/courses/bio141/lecguide/unit4/genetics/recombination/transduction/transduction.html

6. The bacteriophage inserts the donor bacterium's DNA it is carrying into the recipient bacterium .

7. The donor bacterium's DNA is exchanged for some of the recipient's DNA.

Six steps in Specialised Transduction

1. A temperate bacteriophage adsorbs to a susceptible bacterium and injects its genome .

2. The bacteriophage inserts its genome into the bacterium's nucleoid to become a prophage.

Six steps in Specialised Transduction (cont’d)

3. Occasionally during spontaneous induction, a small piece of the donor bacterium's DNA is picked up as part of the phage's genome in place of some of the phage DNA which remains in the bacterium's nucleoid.

4. As the bacteriophage replicates, the segment of bacterial DNA replicates as part of the phage's genome. Every phage now carries that segment of bacterial DNA.

Six steps in Specialised Transduction (cont’d)

5. The bacteriophage adsorbs to a recipient bacterium and injects its genome.

6. The bacteriophage genome carrying the donor bacterial DNA inserts into the recipient bacterium's nucleoid.

http://www.cat.cc.md.us/courses/bio141/lecguide/unit4/genetics/recombination/transduction/spectran.html

Bacterial Conjugation

Bacterial Conjugation is genetic recombination in which there is a transfer of DNA from a living donor bacterium to a recipient bacterium. Often involves a sex pilus.

• The 3 conjugative processes

I. F+ conjugation

II. Hfr conjugationIII. Resistance plasmid conjugation

F+ Conjugation- Genetic recombination in which there is a transfer of an F+ plasmid (coding only for a sex pilus) but not chromosomal DNA from a male donor bacterium to a female recipient bacterium. Involves a sex (conjugation) pilus. Other plasmids present in the cytoplasm of the bacterium, such as those coding for antibiotic resistance, may also be transferred during this process.

I. F+ Conjugation Process

The 4 stepped F+ Conjugation

1. The F+ male has an F+ plasmid coding for a sex pilus and can serve as a genetic donor

2. The sex pilus adheres to an F- female (recipient). One strand of the F+ plasmid breaks

The 4 stepped F+ Conjugation (cont’d)

3. The sex pilus retracts and a bridge is created between the two bacteria. One strand of the F+ plasmid enters the recipient bacterium

4. Both bacteria make a complementary strand of the F+ plasmid and both are now F+ males capable of producing a sex pilus. There was no transfer of donor chromosomal DNA although other plasmids the donor bacterium carries may also be transferred during F+ conjugation.

http://www.cat.cc.md.us/courses/bio141/lecguide/unit4/genetics/recombination/conjugation/f.htm l

II. Hfr Conjugation

Genetic recombination in which fragments of chromosomal DNA from a male donor bacterium are transferred to a female recipient bacterium following insertion of an F+ plasmid into the nucleoid of the donor bacterium. Involves a sex (conjugation)pilus.

5 stepped Hfr Conjugation

1. An F+ plasmid inserts into the donor bacterium's nucleoid to form an Hfr male.

2. The sex pilus adheres to an F- female (recipient). One donor DNA strand breaks in the middle of the inserted F+ plasmid.

5 stepped Hfr Conjugation (cont’d)3. The sex pilus retracts and a bridge forms between the two bacteria. One donor DNA strand begins to enter the recipient bacterium. The two cells break apart easily so the only a portion of the donor's DNA strand is usually transferred to the recipient bacterium.

4. The donor bacterium makes a complementary copy of the remaining DNA strand and remains an Hfr male. The recipient bacterium makes a complementary strand of the transferred donor DNA.

5 stepped Hfr Conjugation (cont’d)

5. The donor DNA fragment undergoes genetic exchange with the recipient bacterium's DNA. Since there was transfer of some donor chromosomal DNA but usually not a complete F+ plasmid, the recipient bacterium usually remains F-

http://www.cat.cc.md.us/courses/bio141/lecguide/unit4/genetics/recombination/conjugation/hfr.htm l

III. Resistant Plasmid Conjugation

Genetic recombination in which there is a transfer of an R plasmid (a plasmid coding for multiple antibiotic resistance and often a sex pilus) from a male donor bacterium to a female recipient bacterium. Involves a sex (conjugation) pilus

4 steped Resistant Plasmid Conjugation

1. The bacterium with an R-plasmid is multiple antibiotic resistant and can produce a sex pilus (serve as a genetic donor).

2. The sex pilus adheres to an F- female (recipient). One strand of the R-plasmid breaks.

4 stepped Resistant Plasmid Conjugation (cont’d)

3. The sex pilus retracts and a bridge is created between the two bacteria. One strand of the R-plasmid enters the recipient bacterium.

4. Both bacteria make a complementary strand of the R-plasmid and both are now multiple antibiotic resistant and capable of producing a sex pilus.

http://www.cat.cc.md.us/courses/bio141/lecguide/unit4/genetics/recombination/conjugation/r.html

Organization of Eukaryotic Chromosomes

• A eukaryotic chromosome contains a long, linear DNA molecule

• Three types of DNA sequences are required for chromosomal replication and segregation– Origins of replication– Centromeres– Telomeres

• Genes are located between the centromeric and telomeric regions along the entire chromosome– A single chromosome usually has a few hundred to

several thousand genes

• In lower eukaryotes (such as yeast)– Genes are relatively small

• They contain primarily the sequences encoding the polypeptides

• ie: Very few introns are present

• In higher eukaryotes (such as mammals)– Genes are long

• They tend to have many introns

• Sequence complexity refers to the number of times a particular base sequence appears in the genome

• There are three main types of repetitive sequences– Unique or non-repetitive– Moderately repetitive– Highly repetitive

• Unique or non-repetitive sequences– Found once or a few times in the genome– Includes structural genes as well as intergenic areas

• Moderately repetitive– Found a few hundred to a few thousand times – Includes

• Genes for rRNA and histones• Origins of replication• Transposable elements

• Highly repetitive– Found tens of thousands to millions of times– Each copy is relatively short (a few nucleotides to several

hundred in length)

– Some sequences are interspersed throughout the genome

• Example: Alu family in humans

– Other sequences are clustered together in tandem arrays• Example: AATAT and AATATAT sequences in Drosophila• These are commonly found in the centromeric regions

Renaturation Experiments

The rate of renaturation of complementary DNA strands provides a way to distinguish the three different types of repetitive sequences

The renaturation rate of a particular DNA sequence depends on the concentration of its complementary partner Highly repetitive DNA will be the fastest to renature

Because there are many copies of complementary sequences

Unique sequences will be the slowest to renature It takes added time for these sequences to find each

other

60-70% of human DNA are unique

sequences

Eukaryotic Chromatin Compaction

If stretched end to end, a single set of human chromosomes will be over 1 meter long! Yet the cell’s nucleus is only 2 to 4 m in diameter

Therefore, the DNA must be tightly compacted to fit

The compaction of linear DNA in eukaryotic chromosomes involves interactions between DNA and various proteins Proteins bound to DNA are subject to change during the

life of the cell These changes affect the degree of chromatin

compaction

Nucleosomes

The repeating structural unit within eukaryotic chromatin is the nucleosome

It is composed of double-stranded DNA wrapped around an octamer of histone proteins An octamer is composed two copies each of four

different histones 146 bp of DNA make 1.65 negative superhelical turns

around the octamer

Overall structure of connected nucleosomes resembles “beads on a string”

This structure shortens the DNA length about seven-fold

Vary in length between 20 to 100 bp, depending on species and cell type

Diameter of the nucleosome

Histone proteins are basic They contain many positively-charged amino acids

Lysine and arginine These bind with the phosphates along the DNA

backbone

There are five types of histones H2A, H2B, H3 and H4 are the core histones

Two of each make up the octamer

H1 is the linker histone Binds to linker DNA Also binds to nucleosomes

Play a role in the organization and compaction

of the chromosome

Nucleosomes Join to form a 30 nm Fiber

Nucleosomes associate with each other to form a more compact structure termed the 30 nm fiber

Histone H1 plays a role in this compaction At moderate salt concentrations, H1 is removed

The result is the classic beads-on-a-string morphology

At low salt concentrations, H1 remains bound Beads associate together into a more compact

morphology

The 30 nm fiber shortens the total length of DNA another seven-fold

Its structure has proven difficult to determine The DNA conformation may be substantially altered

when extracted from living cells

Two models have been proposed Solenoid model Three-dimensional zigzag model

Regular, spiral configuration containing six

nucleosomes per turn

Irregular configuration where nucleosomes have little face-to-face

contact

Further Compaction of the Chromosome

The two events we have discussed so far have shortened the DNA about 50-fold

A third level of compaction involves interaction between the 30 nm fiber and the nuclear matrix

The nuclear matrix is composed of two parts Nuclear lamina Internal matrix proteins

10 nm fiber and associated proteins

The third mechanism of DNA compaction involves the formation of radial loop domains

Matrix-attachment regions

Scaffold-attachment regions (SARs)

or

MARs are anchored to the nuclear

matrix, thus creating radial loops

25,000 to 200,000 bp

Further Compaction of the Chromosome

The attachment of radial loops to the nuclear matrix is important in two ways 1. It plays a role in gene regulation

2. It serves to organize the chromosomes within the nucleus

Each chromosome in the nucleus is located in a discrete and nonoverlapping chromosome territory

Heterochromatin vs Euchromatin

The compaction level of interphase chromosomes is not completely uniform Euchromatin

Less condensed regions of chromosomes Transcriptionally active Regions where 30 nm fiber forms radial loop domains

Heterochromatin Tightly compacted regions of chromosomes Transcriptionally inactive (in general) Radial loop domains compacted even further

There are two types of heterochromatin Constitutive heterochromatin

Regions that are always heterochromatic Permanently inactive with regard to transcription

Facultative heterochromatin Regions that can interconvert between euchromatin and

heterochromatin Example: Barr body

Compaction level in euchromatin

Compaction level in heterochromatin

During interphase most chromosomal

regions are euchromatic

C – VALUE PARADOX

C – value: the haploid DNA content in an individual.

G – value: the number of genes present in haploid genome.

I – value: the amount of information embedded in haploid individual.

C – value paradox: lack of association between c – value and degree of complexity of various multi cellular organisms. This phenomenon was named by THOMAS in 1971.

Organism Estimated size Estimated gene number

Average gene density

Chromosome number

Homo sapiens(humans)

2,900 million bases

~30,000 1 gene per 1,00,000 bases

46

Rattus norvegicus (rat)

2,750 million bases

~30,000 1 gene per 1,00,000 bases

42

Mus musculus (mouse)

2,500 million bases

~30,000 1 gene per 1,00,000 bases

40

Drosophila melanogaster (fruit fly)

180 million bases 13,600 1 gene per 9,000 bases

8

Arabidopsis thaliana (plant)

125 million bases 25,500 1 gene per 4000 bases

5

Saccharomyces cerevisiae (yeast)

12 million bases 6,300 1 gene per 2000 bases

16

Escherichia coli (bacteria)

4.7 million bases 3,200 1 gene per 1400 bases

1