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Disse sgetermer er blevet fremhvet: shuster sporulation 
Optimizing Sporulation for Different S

Optimizing Sporulation for Different S. cerevisiae Strain Backgrounds.
E. Brooks, S. Tom, E. Schwartz, S.L. Elrod and E.O. Shuster
University of California, Davis

Major Conclusions:

  1. No single "magic" sporulation protocol supports optimal sporulation for all strains.
  2. Media supporting high levels of sporulation on plates do not always support high levels in liquid (& vise versa!).
  3. Sporulation efficiency is affected by both pre-sporulation and sporulation conditions.
  4. We have defined sets of "consensus" media  conditions for plate and liquid sporulation, at least one of which supports good sporulation for each strain tested.
    a)    In general, sporulation media containing dextrose and yeast extract do NOT support high levels of sporulation (>70% of max.) on plates, although they may in liquid.
    b)    In general, acetate-based pre-sporulation media support higher levels of sporulation in liquid than do those containing glucose.
  5. In many strains, the proportion of asci containing 3 or 4 spores is higher at 18C than at 30C.

Standardized test protocols (temperature can be altered to suit experiment):
Sporulation on plates:

  1. Grow fresh patches on medium of choice.
  2. Replica-plate to pre-sporulation medium; incubate at 30C for 18-24 hrs.
  3. Replica-plate to sporulation plates; incubate at 30C (or lower).  Check for spores after 2, 5 or 10 days (strains vary; lower temperatures may require longer times).  Older asci may come apart more easily during dissection, if they have not prematurely dissociated.

Sporulation in liquid:

  1. Grow fresh overnight in standard medium.
  2. Inoculate pre-sporulation medium to allow growth in 18-24 hrs to late log (1-2 x 107cells/ml) for acetate media or early stationary (1-2 x 108) for dextrose media.
  3. Wash cells 1-2x with H2O, resuspend in sporulation medium at (1-2 x 107 cells/ml).
  4. Incubate in shaking H2O bath at 30C (or lower) for 30-72 hours (48 hrs usually fine).

 

Strains Name

BR2171-3B

BR2495

EB1501

NH39

NKY278

SE1200

SE1202

YPH274

Y55

Background

Unknown

Unknown

S288c

A364a

SK1

AP1

W303

S288c

Y55

Source

Roeder lab

Roeder lab

Holm lab

Hartwell lab

Kleckner lab

Byers lab

Rothstein lab

Heiter lab

Haber lab

 

Effect of temperature on sporulation

little/no effect

cs (18 &/or 15C)

ts (34.5C)

cs & ts

NKY278 (SK1)
Y55

BR2171-3B
SE1200 (AP1)

EB1501 (S288c)

BR2495
NH39 (A364a)
YPH274
SE1202 (W303)

 

Pre-sporulation conditions

Mediac

KAC(%)

dextrose(%)

Yeast ext.(%)

Peptone(%)

YNB w/o aa(%)

amino acids

10% YEPD

0.00

10.00

0.80

0.30

0.00

-

1% YPA

1.00

0.00

1.00

2.00

0.00

-

2% YPA

2.00

0.00

1.00

2.00

0.00

-

MSAc

2.00

0.00

0.00

0.00

0.67

a

2% YEPD

0.00

2.00

1.00

2.00

0.00

-

PSP2

1.00

0.00

0.10

0.00

0.67

a

SAcCa

2.00

0.00

0.00

0.00

0.67

b

6% YEPD

0.00

6.00

1.00

2.00

0.00

-

a- Media supplemented with 20mg/ml ade, ura, his, arg, met; 60mg/ml tyr, lys,; 80mg/ml trp, leu, iso.
b- Media supplemented with 2% caseamino acids and 20mg/ml ade, his, trp, ura.
c- 2% agar added for plates.

 

Sporulation conditions

Mediab

KAC(%)

dextrose(%)

raffinose(%)

Yeast ext. (%)

amino acids

SPM

0.30

0.00

0.02

0.00

a

2% KAc

2.00

0.00

0.00

0.00

a

1% KAc

1.00

0.00

0.00

0.00

a

CSH spo

1.00

0.05

0.00

0.10

a

SPO

1.50

0.05

0.00

0.25

a

0.3% KAc

0.30

0.00

0.00

0.00

a

SPO7

1.50

0.10

0.00

0.25

a

SPO8

0.30

0.00

0.02

1.00

-

a- Media supplemented with 10mg/ml of ade, arg, his, iso, leu, lys, met, phe, thr, trp, ura, val.
b- 2% agar added for plates

 

Plates

 

"Best"

"Consensus"

Strain

Media

%spo +/- SD

6% YEPD/SPM

2% YPA/1% KAc

PSP/1% KAc

BR2171-3B

MSAc/2%KAc

66.2 +/- 0.6

57.2 +/- 6.5

37.5 +/- 2.6

50.5 +/- 9.5

BR2495

MSAc/2%KAc

71.5 +/- 2.2

50.8 +/- 5.0

26.8 +/- 15.4

68.2 +/- 3.4

EB1501

6% YEPD/1%KAc

29.3 +/- 12.1

21.2 +/- 12.3

1.8 +/- 1.4

3.5 +/- 1.7

NH39

2% YPA/1%KAc

23.4 +/- 7.3

8.5 +/- 5.6

23.4 +/-7.3

1.4 +/- 1.5

NKY278

6%YEPD/1%KAc

95.8 +/- 1.4

83.0 +/- 3.8

54.6 +/- 2.6

65.8 +/- 7.5

SE1200

6%YEPD/SPM

67.6 +/- 6.6

67.6 +/- 6.6

9.2 +/- 4.6

21.0 +/- 9.4

SE1202

2%YEPD/0.3%KAc

58.2 +/- 2.2

56.6 +/-3.4

34.2  +/- 6.4

23.8 +/- 7.3

YPH274

1%YPA/SPM

28.5 +/- 1.5

15.5 +/- 4.2

24.5 +/-13.1

12.0 +/- 1.4

Y55

6%YEPD/SPM

98.3 +/- 1.8

98.3 +/- 1.8

91.0 +/- 5.3

97.2 +/- 0.6

 

Liquid

 

"Best"

"Consensus"

Strain

Media

%spo +/- SD

1%YPA/1%KAc

6%YEPD/CSH SPO

PSP2/1%KAc

BR2171-3B

1%YPA/CSH SPO

72.5 +/- 2.8

55.8 +/- 7.4

62.3 +/- 9.5

15.0

BR2495

PSP2/1% KAc

39.0

30.0 +/- 5.7

19.5 +/- 13.4

39.0

EB1501

1%YPA/1%KAc

52.0 +/- 7.8

52.0 +/- 7.8

32.3 +/- 8.8

27

NH39

6%YEPD/CSH SPO

28.0 +/- 3.5

12.0 +/- 2.1

28.0 +/- 3.5

0

NKY278

1%YPA/CSH SPO

97.8 +/- 1.1

97.5 +/- 1.4

92.8 +/- 2.5

91

SE1200

1%YPA/1%KAc

77.3 +/- 6.7

77.3 +/- 6.7

51.5 +/- 4.9

60.5 +/- 4.9

SE1202

1%YPA/1%KAc

51.8 +/- 0.4

51.8 +/- 0.4

37.3 +/- 7.4

39.5

YPH274

PSP2/1%KAc

44.5

32.8 +/- 3.9

23.8 +/- 1.8

44.5

Y55

1%YPA/1%KAc

83.8 +/- 0.4

83.8 +/- 0.4

82.0 +/- 4.2

60.5