Lesson 9 Recombination and Transposition at the Molecular Level Flashcards Preview

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1

Experiment 17A Harlequin Chromosome Staining
Hypothesis

Crossing over may occur between sister chromatids

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Experiment 17A Harlequin Chromosome Staining
Starting materials

A laboratory cell line of Chinese hamster ovary (CHO) cells

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Experiment 17A Harlequin Chromosome Staining
Protocol

1. Expose CHO cells to BrdU fro two cell generations. (2 hours)
2. Near the end of the growth, expose cells to colcemid.
3. Add 0.075 M KCl to spread the chromosomes and then methanol/acetic acid to fix the cells
4. Stain with Hoechst 33258, rinse, and later stain with Giemsa.
5. View under a microscope

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Experiment 17A Harlequin Chromosome Staining
BrdU

5-bromodeoxyuridine. nucleotide analogue. In this experiment, they bind certain types of stain to a different degree compared to normal chromosomes.

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Experiment 17A Harlequin Chromosome Staining
Why was colcemid used?

To prevent the cells from completing mitosis following the second round of DNA replication.

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Experiment 17A Harlequin Chromosome Staining
Interpreting the Data

The microscope visual allowed one to see two different stains of staining, which indicates crossover happened.

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How is Holliday junction formed?

1. Two homologous chromosomes are aligned with each other.
2. A break occurs at identical sites in one strand of both parental chromosomes.
3. The strands then invade the opposite helices and base pair with the complementary strands.
4. This event is followed by a cleavage to form a Holliday junction

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What happens after branch migration?

The cross strands are broken and then are connected to create nonrecombinant chromosomes with a short heteroduplex region. This results in:
nonrecombinant chromosomes with a heteroduplex region

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What happens if the bottom double helix rotates 180 degrees?

This is called isomerization. The uncrossed strands are broken. The strands are then connected to create recombinant chromosomes with a short heteroduplex region. This results in:
Recombinant chromosomes with a heteroduplex region

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Let's look at nonrecombinant chromosomes. Why are they nonrecombinant?

One chromosome is ABAB and the other is abab. In the end, they remain the same. They did not recombine.

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How are recombinant chromosomes different?

Instead of ABAB and abab, they will end up AbAB and aBab.

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What is resolution in the Holliday Model?

The final steps in the recombination process

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How is Holliday Model different from double-stranded break model?

1. The pattern of breakage of one or more DNA strands is different.
2. The newer model proposes that a short region of strand degradation occurs via the action of nucleases that can degrade a DNA strand over a short distance.
3. SInce DNA strand degradation occurs, this model also requires the synthesis of new DNA. This DNA synthesis takes place in relatively short gaps where a DNA strand is missing. The DNA synthesis is called DNA gap-repair synthesis.
4. In the double-stranded break model, gap-repair synthesis occurs in two strands

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DNA gap-repair synthesis

DNA synthesis to replace DNA in double-stranded break model.

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When does gene conversion occur?

When two different alleles become two identical alleles

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What are the two ways that gene conversion can take place?

DNA gap-repair synthesis and DNA mismatch repair

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DNA gap-repair synthesis

1. A chromosome suffers a double-stranded break.
2. Gap is created by digestion of the DNA in the double helix.
3. This digestion eliminates an allele.
4. The two template strands that are used in gap-repair synthesis are from the other double helix. After gap-repair, the damaged chromosome will have the allele of the chromosome used as a template.

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DNA mismatch repair

1. A heteroduplex contains a DNA strand from each of the two original parental chromosomes.
2. The two parental chromosomes may contain an allelic difference within this region. If so, the heteroduplex region that is formed after branch migration will contain an area of base mismatch.
3. Gene conversion occurs when recombinant chromosomes are repaired to produce the same allele.

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Site-specific recombination

A type of recombination in which two DNA segments that are mostly not homologous align themselves at specific sites. Sites are short in sequence. Provide a specific location where recombination will occur. Chromosome breakage and reunion occurs at these specific sites to create a recombinant chromosome.

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How can integration of viral genome happen?

By site-specific recombination

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What is integrate?

An enzyme that recognizes the attachment site sequences and brings them close together.

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What happens when attachment site sequences are brought close together?

The strands are then exchanged and the ends are lighted together so that the viral integrated into the host-cell chromosome.

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Three types of transposition

Simple or conservative, replicative, retroelements

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Simple or conservative transposition

Transposable element is removed from its original site and transferred to a new site

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Replicative transposition

The TE is replicated and the new copy is inserted into a second site

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retroelements (retrotransposons or retroposons)

The element is copied into an RNA intermediate and then into DNA before it is inserted into a new site

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Insertion sequence

Simplest TEs. Commonly found in bacteria. Both ends of the insertion sequence contain inverted repeats (IRs) Insertion sequences may contain a central region that encodes the enzyme transposase, which catalyzes the transposition event.

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Inverted repeats

DNA sequences that are identical but run in opposite directions

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Composite transposons

Contain additional genes that are not necessary for transposition per se. Commonly contain genes that confer a selective advantage to the organism. Prevalent in bacteria. During transposition of a composite transposon, only the inverted repeats at the ends of the transposon are involved in the transpositional event. Whenever insertion sequences are found at both ends of a gene, they create a composite transposon.

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Replicative transposons

Have a sequence organization that is similar to insertion sequences except that replicative transposon have a resolves gene that is found between the inverted repeats.