67. A Yeast plasmid vector has an XhoI site in the LEU2 marker gene and a SalI site in the HIS3 marker gene. A
student cloned a gene using the SalI site. How should the student select for the recombinant clone?
1. Plate the transformation mixture on leu- his- medium
2. Plate the

Introduction

Yeast plasmid vectors are commonly used for cloning experiments. In this case, a student has cloned a gene into a yeast plasmid vector that contains XhoI and SalI restriction sites in the LEU2 and HIS3 marker genes, respectively. The question now is: how should the student select for recombinant clones that successfully contain the gene of interest?

Understanding the Markers

1. LEU2 Marker Gene: This gene is used as a selectable marker. When cloned into the vector, it helps to select for yeast cells that have the plasmid. In a leu- strain of yeast, the plasmid will allow the cells to grow because the LEU2 gene complements the defective leucine biosynthesis pathway.

2. HIS3 Marker Gene: Similarly, HIS3 is another selectable marker. Yeast cells that have a functioning HIS3 gene can grow on media lacking histidine, as they can produce histidine by themselves.

Cloning with SalI Site

The student cloned the gene using the SalI restriction site in the HIS3 gene. This means that the HIS3 gene in the recombinant plasmid is disrupted because the gene is cut by the SalI enzyme during the cloning process. The disruption of HIS3 means that the yeast cells with the recombinant plasmid cannot grow on a his- medium unless the HIS3 gene is restored by the cloned insert.

Selection Strategy

To select for recombinant clones, the student must ensure that the LEU2 marker (which is not disrupted by the SalI site) is functional for growth in a leu- strain, while the HIS3 marker is disrupted (so cells will not grow on his- medium unless rescued by the cloned gene). Therefore, the student can use a two-step selection process:

1. First selection: Plate the yeast cells on leu+ (medium containing leucine), so all cells with the plasmid can grow, because the LEU2 marker is intact.

2. Second selection: Replicate the plates onto his- (medium lacking histidine) to ensure that only those cells that have the recombinant plasmid (with a functional HIS3 gene) will grow. This step will help to confirm that the recombinant clones have the desired insert and the HIS3 gene is intact.

Conclusion

The correct selection strategy is to first plate the cells on leu+ medium (which will select for plasmid-bearing cells) and then replica plate them onto his- medium to identify the recombinant clones that have successfully taken up the gene of interest.

Final Answer:

2. Plate the transformation mixture on leu+ his+ medium, followed by replica plating on leu+ his- medium

transformation mixture on leu+ his+ medium, followed by replica plating on leu+ hismedium
3. Plate the transformation mixture on his+ leu-medium, followed by replica plating on leu- hismedium
4. Plate the transformation mixture on leu+ his- medium