Genetic Engineering Exam I

Take home Exam I (25 points)

The following questions are based on
(a) Zuobenko et al. Nucleic Acids Research, 1994, Vol. 22, No. 19 3819-3824
(b) Lecture 5

1. What is the recombination mechanism underlying the integration process of foreign DNA into plastid genome? (1)

(i) introduction of the transforming DNA on the surface of microscopic tungsten particles using the biolistic process or polyethylene glycol treatment (ii) integration of the transforming DNA into the plastid genome
by homologous recombination and (iii) selective elimination of wild-type plastid genome copies during the course of repeated cell divisions

2. How is DNA vector design used for plastid transformation different from that of the nuclear transformation? (2)

In plastid transformation, pPRV1 based on a pUC119 plasmid derived from E.coli is used as a plastid transformation vector. These vectors replicate in E.coli but not in the plastids. A selectable spectinomycin resistance gene (aadA) for highly efficient recovery of plastid transformants and a multiple cloning site (MCS) are used. There is no readthrough transcription from outside promoters when these genes are inserted into the plastid, which makes this vectors an ideal choice. The transplastomes are stable and maternally inherited. Since the number of chloroplast DNA copies are up to 10,000, the development of selectable marker genes in the vector design process is critical for the recovery of a pure population. An additional marker for kanamycin resistance (kan gene) can be used too.
In nuclear transformation, T-DNA from Agrobacterium is used.

3. Can you use Agrobacterium for delivering T-DNA into plastids? Give reason (2)

No. Agrobacterium T-DNA only targets the nuclear genome.


4. Give 2 (not the whole list) most compelling (in your opinion) facts that support the endosymbiont theory, also state what this theory postulates. (2)


1- Plastid ribosomes are more similar to prokaryotic ribosomes than to their cytoplasmic counterparts: cytoplasmic ribosomes- 80S (40S + 60S subunits) Plastid and prokaryotic ribosomes- 70S (30S + 50S subunits) Antibodies raised against 70S and 30S subunits of plastid ribosomes are active against E. coli.
2- Promoters of most chloroplast genes contain DNA sequences similar to the E. coli ‘-10’ and ‘-35’ promoter motifs.
The endosymbiotic theory postulates that the origin of mitochondira is from proteobacteria, and the origin of chloroplast is from cyanobacteria. According to this theory, these organelles originated as separate prokaryotic organisms taken inside the cell as endosymbionts to form organelles of eukaryotic cells.


5. Could you use a plastid transformation vector used for transformation of tobacco plastid to work on carrot plastids? Why? (2)

6. Why tobacco plants expressing Cry2Aa2 gene from plastid produce 100 fold higher expression than those expressing it from nucleus (see lecture 5). (2)

In nuclear transgenic plants, expression of multiple genes requires introduction of individual genes and time-consuming subsequent backcrosses to reconstitute multi-subunit proteins or pathways. However, multiple genes can be inserted in a single transformation event when used in chloroplasts. Also the number of chloroplast DNA copy is much higher than the nuclear genome.

7. The most challenging aspect of plastid transformation is achieving homoplasmy. Describe what is homoplasmy and how is it promoted in a plastid transformation experiment. (2)

Homoplasmy is the presence of mutation, or insertion of a foreign gene, into all of the plant plastid organelles DNA, all of the chloroplasts. Since there are over ten thousands of plastid genomes per every eukaryotic cell, it is challenging to have the insertion present in all of the copies. It can be promoted by several cycles of antibiotic selection, sorting and biogenesis. When transgenic lines are subjected to several additional rounds of regeneration and selection, the creation of homoplasmic lines are promoted.

8. What are the commonly used drugs for plastid transformation? (2)
spectinomycin, streptomycin, and kanamycin.


9. If you took a plasmid vector designed for nuclear transformation, and can you use it for recovering transplastomic plants? Why or why not? (2)

No.

10. Consider these crosses between a transplastomic plant (Tp) resistant to spectinomycin (spec.) and a WT plant: (2)

(i) Tp (♀) x WT (♂)
(ii) WT (♀) x Tp (♂)
(iii) Tp (♀) x Tp (♂)

(a) Seeds of cross (i) will be resistant to Spec. YES
(b) Seeds of cross (ii) will be resistant to Spec. NO
(c) Progeny of cross (iii) will be resistant to Spec. YES
(d) If resistance is transmitted to next generation, what percentage of progeny will carry resistance? 100%


11. Give two features of plastid translation machinery that are different from that of nucleus. (2)
The mRNAs are not capped.
The mRNAs are not poly-adenylated.

12. Structure of plastid genome is linear ? NO

13. Plastid genomes are highly conserved between dicot and monocot plants? YES

14. Give 4 distinct advantages of plastid transformation (2)

No gene slicing
No vector sequences
Hyper expression
No position effect