can the yeast s.cerevesiae lost the pRS426 ura plasmid?

[Autophagator]

Hi all¡
I am performing my doctorate with yeast S.cerevisiae. Its a completely new organism for me, and now I am having troubles with him/her/she/he/it whatever.. Recently, I did transform a simple mutant (ura+) to create a double mutant. The simple mutant was ura+ ( with pRS426 plasmid) and grow normally in SC-ura plaques. However, after transformation (with other DNA and selection non relationated), the double mutant don’t grow in liquid media SC-ura. Why?? I don’t understand¡¡ I did repeat the test several time, and still happens. Can anybody suggest me an idea, test, paper, link o something to know what happens here????
Thank a lot in advance.

[hairongxu88]

I don't know

[mkc3]

Hi, I think you need to provide more details. The strain you are transforming carries the pRS426 plasmid but after transforming with a second plasmid, did you use double selection to maintain the pRS426 plasmid? It may very well be lost if you did not as the pRS426 plasmid is a 2 micron plasmid with no centromere sequence. As a result, the partitioning of the 2 micron plasmid between mother and daughter cell is unequal, unlike the partitioning of a chromosome, and if selection pressure isn't applied, the daughter cell may not receive any of the pRS426 plasmid at all.

On the other hand, a CEN plasmid like pRS416 would be more stable as it is attached to the spindle through its centromere sequence. However, I would still maintain -Ura selection as it is still less stable than a chromosome.

You might want to look up the original pRS426 paper (Christianson et al, 1992, Multifunctional yeast high-copy-number shuttle vectors, <I>Gene</I>) and the pRS300 series paper (Sikorski & Hieter, 1989, <I>Genetics</I>)

Good luck.

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Originally Posted by Autophagator

Hi all¡
I am performing my doctorate with yeast S.cerevisiae. Its a completely new organism for me, and now I am having troubles with him/her/she/he/it whatever.. Recently, I did transform a simple mutant (ura+) to create a double mutant. The simple mutant was ura+ ( with pRS426 plasmid) and grow normally in SC-ura plaques. However, after transformation (with other DNA and selection non relationated), the double mutant don’t grow in liquid media SC-ura. Why?? I don’t understand¡¡ I did repeat the test several time, and still happens. Can anybody suggest me an idea, test, paper, link o something to know what happens here????
Thank a lot in advance.

[mkc3]

Hi, pRS426 is unstable in the absence of selective pressure so you need to maintain an absence of uracil even when you are doing the second transformation. It only has a 2 micron origin, hence unlike chromosomes that attach to the mitotic spindle and are partitioned equally, its partitioning between mother and daughter cell is random, with a tendency to partition to the mother cell. Hence, in the absence of selective pressure, you are probably going to get a significant number of daughter cells that are free of pRS426. When you later try and grow these pRS426-free transformants in the absence of uracil again, they fail to grow.

The CEN plasmid pRS416 would be more stable given that it has a centromere sequence which allows it to be partitioned by the mitotic spindle during cell division. However, it is still less stable than a chromosome and selective pressure should be maintained.

You can read more in Christianson et al. (1992), Gene and Sikorski & Hieter (1989), Genetics.

I hope that helps you.

[mkc3]

Hi,

pRS426 is a 2 micron episomal plasmid. Its distribution during mitosis between mother and daughter cell is random as it does not attach to the spindle, and tends to be biased towards staying with the mother cell. As a result, you will have to maintain selection for pRS426 with Ura dropout medium, even when you transform it with the second plasmid (so use double dropout medium to select for transformants harbouring both plasmids).

A good reference to start with might be the Christianson et al. paper in Gene, 1992, I believe, which is the paper that first published pRS426. You can also check papers in the 1970s and 1980s that studied the stability of 2 micron episomal plasmids (YEp) in yeast.

I hope that helps!

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Originally Posted by Autophagator

Hi all¡
I am performing my doctorate with yeast S.cerevisiae. Its a completely new organism for me, and now I am having troubles with him/her/she/he/it whatever.. Recently, I did transform a simple mutant (ura+) to create a double mutant. The simple mutant was ura+ ( with pRS426 plasmid) and grow normally in SC-ura plaques. However, after transformation (with other DNA and selection non relationated), the double mutant don’t grow in liquid media SC-ura. Why?? I don’t understand¡¡ I did repeat the test several time, and still happens. Can anybody suggest me an idea, test, paper, link o something to know what happens here????
Thank a lot in advance.