Chapter 24, Problem 17DAP

(a)

Summary Introduction

To explain: The way through which nondisjunction of the synthetic chromosome would give rise to a colony that is half red and half white.

Introduction:

Yeast artificial chromosomes (YACs) clone large pieces of deoxyribonucleic acid (DNA) in yeast cells. It can generate unlimited number of DNA copies. Cloning genomic DNA into YAC is useful to analyze very long DNA sequence such as human genes.

The three types of DNA sequence that are necessary to ensure proper propagation and replication of a YAC in a yeast cell are as follows:

1) Centromere for the attachment of mitotic spindle fibers. It attracts one copy of each replicated chromosome into each new daughter cell.

2) Telomere is found at each end of a chromosome. It prevents the linear DNA from degradation.

3) Autonomous replicating sequence (origin of replication) allows assembly of DNA replication machinery and causes the movement of replication fork.

Explanation of Solution

In the given experiment, Heiter and colleagues constructed synthetic chromosomes to find out the components of yeast chromosomes. They constructed plasmids with different parts of chromosomes. They observed proper segregation of synthetic chromosomes during mitosis. According to the assay, wild-type yeast colonies are white and mutated copies of adenine-requiring yeast colonies are red.

During non-disjunction of synthetic chromosomes, one daughter cell and its entire descendants obtain two copies of the synthetic chromosome and give rise to colonies that are white. On the other hand, other daughter cell and all of its descendants obtain no copies of the synthetic chromosome and give rise to colonies that are red. This leads the formation of half-white, half-red colony.

(b)

Summary Introduction

To explain: The way through which loss of the synthetic chromosome would give rise to a colony that is half red and half pink.

Introduction:

Yeast artificial chromosomes (YACs) clone large pieces of DNA in yeast cells. It can generate unlimited number of DNA copies. Cloning genomic DNA into YAC is useful to analyze very long DNA sequence such as human genes.

The three types of DNA sequence that are necessary to ensure proper propagation and replication of a YAC in a yeast cell are as follows:

1) Centromere for the attachment of mitotic spindle fibers. It attracts one copy of each replicated chromosome into each new daughter cell.

2) Telomere is found at each end of a chromosome. It prevents the linear DNA from degradation.

3) Autonomous replicating sequence (origin of replication) allows assembly of DNA replication machinery and causes the movement of replication fork.

Explanation of Solution

If synthetic chromosome loss, one daughter cell and all of its descendants obtain one copy of the synthetic chromosome that give rise to form pink colony. The other daughter cell and all of its descendants obtain no copies of the synthetic chromosome and give rise to form red colony. Thus, a colony formed that is half-pink and half-red.

(c)

Summary Introduction

To determine: The size of the centromere necessary for normal mitotic segregation along with the reason.

Introduction:

Yeast artificial chromosomes (YACs) are necessary to clone large pieces of DNA in yeast cells. It can generate unlimited number of DNA copies. Cloning genomic DNA into YAC is useful to analyze very long DNA sequence such as human genes.

The three types of DNA sequence that are necessary to ensure proper propagation and replication of a YAC in a yeast cell are as follows:

1) Centromere for the attachment of mitotic spindle fibers. It attracts one copy of each replicated chromosome into each new daughter cell.

2) Telomere is found at each end of a chromosome. It prevents the linear DNA from degradation.

3) Autonomous replicating sequence (origin of replication) allows assembly of DNA replication machinery and causes the movement of replication fork.

Explanation of Solution

Heiter and colleagues estimated that the frequency of aberrant mitotic procedures with different types of synthetic chromosomes by counting the frequency of different colony types. They constructed synthetic chromosomes with DNA fragments of various sizes having a known centromere.

Based on the given data, it can be concluded that the centromere size needed for normal mitotic segregation should be less than 0.63 kbp. This is because all the fragments of DNA of 0.63 kbp size contain mitotic stability and cannot show loss of chromosomes.

Thus, the minimum functional centromere should be less than 0.63 kbp, because all fragments of this size or larger determine relative mitotic stability.

Conclusion

Thus, the centromere size required for normal mitotic segregation should be smaller than 0.63 kbp.

(d)

Summary Introduction

To explain: The way through which the synthetic chromosomes can be replicated more-or-less properly.

Introduction:

Yeast artificial chromosomes (YACs) are required to clone large pieces of DNA in yeast cells. It can generate unlimited number of DNA copies. Cloning genomic DNA into YAC is useful to analyze very long DNA sequence such as human genes.

The three types of DNA sequence that are necessary to ensure proper propagation and replication of a YAC in a yeast cell are as follows:

1) Centromere for the attachment of mitotic spindle fibers. It attracts one copy of each replicated chromosome into each new daughter cell.

2) Telomere is found at each end of a chromosome. It prevents the linear DNA from degradation.

3) Autonomous replicating sequence (origin of replication) allows assembly of DNA replication machinery and causes the movement of replication fork.

Explanation of Solution

Telomeres are ribonucleic proteins that are the “protective ends” of “linear chromosomes”. Telomeres generally shorten throughout the lifetime of a person. The synthetic chromosomes are constructed to determine the components of yeast chromosomes.

Telomeres are not required to replicate circular chromosomes beacuse telomeres are important to completely replicate linear DNA. Since, synthetic chromosomes are circular; hence, telomeres are not required for their replication

(e)

Summary Introduction

To explain: The chromosome size required for normal mitotic segregation and the reasons for it.

Introduction:

Yeast artificial chromosomes (YACs) are required to clone large pieces of DNA in yeast cells. It can generate unlimited number of DNA copies. Cloning genomic DNA into YAC is useful to analyze very long DNA sequence such as human genes.

The three types of DNA sequence that are necessary to ensure proper propagation and replication of a YAC in a yeast cell are as follows:

1) Centromere for the attachment of mitotic spindle fibers. It attracts one copy of each replicated chromosome into each new daughter cell.

2) Telomere is found at each end of a chromosome. It prevents the linear DNA from degradation.

3) Autonomous replicating sequence (origin of replication) allows assembly of DNA replication machinery and causes the movement of replication fork.

Explanation of Solution

According to given data, if chromosome size is larger, then there will be less chances of total error rate. Larger is the chromosome, the more closely it is segregated. According to the given data, neither a lesser size below, neither which the synthetic chromosome is fully unstable, nor does a greater size above, which stability wills no longer change.

(f)

Summary Introduction

To determine: Whether the centeromeric and telomeric sequences used in this experiment explain the mitotic stability of normal yeast chromosomes or must other elements be involved.

Introduction:

Yeast artificial chromosomes (YACs) are required to clone large pieces of DNA in yeast cells. It can generate unlimited number of DNA copies. Cloning genomic DNA into YAC is useful to analyze very long DNA sequence such as human genes.

The three types of DNA sequence that are necessary to ensure proper propagation and replication of a YAC in a yeast cell are as follows:

1) Centromere for the attachment of mitotic spindle fibers. It attracts one copy of each replicated chromosome into each new daughter cell.

2) Telomere is found at each end of a chromosome. It prevents the linear DNA from degradation.

3) Autonomous replicating sequence (origin of replication) allows assembly of DNA replication machinery and causes the movement of replication fork.

Explanation of Solution

Graphical representation:

Fig.1 represents the graphical representation of the length of DNA and total percentage of the error rate.

Fig.1: The length of DNA and total percentage of the error rate.

Normal yeast chromosomes are linear in structure and ranges from 250 kbp to 2,000 kbp. They have a mitotic error frequency of 10^-5 per cell division. Based on the given data, a graph can be plotted. This graph is plotted against the length of DNA and total percentage of the error rate.

Hence, it can be concluded that the replication will not be stable even if the synthetic chromosomes are longer in size like normal yeast chromosomes. It is due to the requirement of some additional elements (other, as yet undiscovered, elements) are required for stability that occur in normal chromosomes.