Kit 1D

The MudP and MudQ (Mud-22) Mapping Set

 

Hybrid genetic elements have been constructed that carry two-thirds of the temperate phage P22 between the ends of transposon Mu; these are called MudP and MudQ or Mud-22 (Youderian et al., 1988. Genetics 118:581-592). Insertions of these elements in the Salmonella chromosome produce locked-in P22 prophages that cannot excise. Upon induction the phage replicates, in situ, amplifying the neighbouring regions of the chromosome and packaging three contiguous headsful of adjacent DNA in one direction from the P22 packaging site, pac.

Workers in the Roth lab and in the Youderian lab have constructed a set of lysogens. Each lysogen, on induction, packages ca. 3-4 mins of the chromosome starting from the point of insertion and going unidirectionlly, clockwise or counter-clockwise. The construction and use of this set is detailed in Benson and Goldman, 1992, J. Bacteriol. 174:1673-1681.

This set was kindly provided by Nick Benson, University of Utah, to the SGSC.

The set can be used for mapping in either of two ways:

A. Genetic methods - Described by Benson and Goldman (above).

B. Molecular methods - The strains of the set are induced for P22 phage production, P22 phage is harvested and the DNA isolated. This DNA can be bound to nitrocellulose filters and probed with Salmonella DNA of unknown location on the map. The DNA was isolated by the following method (also described in Liu and Sanderson, 1992. J. Bacteriol. 174:1662-1672):

I. Induction of the P22 phage and Isolation of DNA:

1. Grow 5 mls of overnight cells in LB broth plus chloramphenicol (Cam) 40 ug/ml.

2. Add 25 mls of LB plus mitomycin C to a final concentration of 2.0 ug/ml.

3. Grow with shaking overnight at 37 degrees C.

4. Add 1.0 ml of chloroform and shake for 30 seconds. Spin for 10 minutes at 8K.

5. Move the supernatant to a fresh tube, spin at 16K for 1 hour.

6. Remove the supernatant and dissolve the pellet in 1.5 ml of TE (10/1) buffer (pH 8.0). This pellet is composed primarily of phage.

(Steps 7-12 are methods used at the SGSC for isolation of DNA from the P22 phage; other methods could be used in its place.)

7. Move 100 ul of the solution to a fresh Eppendorf tube; freeze the rest at -80 degrees C.

8. Add 400 ul of water, extract once with equal volume of phenol: chloroform: isoamyl alcohol (25:24:1).

9. Move the supernatant to a fresh tube, add NaCl to a final concentration of 0.25M. Precipitate the DNA with two volumes of 100% ethanol.

10. Move the precipitated DNA to 0.5 ml of water, extract twice with phenol: chloroform: isoamyl alcohol.

11. Repeat step 9.

12. Remove the supernatant, air dry, and rehydrate the pellet in 100 ul of TE (10/1) buffer.

II. The DNA is then bound to nitrocellulose filters and probed with DNA of unknown location.

 

Mud-P22 Mapping Strains Provided in Kit 1D:

from Benson and Goldman, 1992, J. Bacteriol. 174:1673-1681.

Strain No.

SGSC No.

Minute(a)

Genotype of Mud insertion(b)

Packaging direction(c)

TT15223

1979

0.00

thr-469::MudP

A

TT15224

1980

0.00

thr-469::MudQ

B

TT15225

1981

1.5

pyrA2413::MudP

-

TT15226

1982

3.5

nadC218::MudP

A

TT15227

1983

3.5

nadC218::MudQ

B

TT15229

1985

7

proA692::MudQ

A

TT15231

1987

7

proA692::MudP

B

TT15230

1986

8.5

proC963::MudP

A

TT15232

1988

12

purE2154::MudQ

A

TT15235

1990

12

purE2154::MudP

B

TT15237

1992

16

cobD498::MudP

A

TT15236

1991

16

cobD498::MudQ

B

TT15238

1993

17

nadA219::MudP

A

TT15629

2037

17

nadA219::MudQ

B

TT15239

1994

25

putA1019::MudQ

A

TT15240

1995

25

putA1019::MudP

B

TT15241

1996

27

purB1879::MudQ

-

TT15242

1997

27

purB1879::MudP

-

TT15244

1999

30

aroD561::MudP

A

TT15243

1998

30

aroD561::MudQ

B

TT17397

2397

35

zde-3634::MudP

-

TT17398

2396

35

zde-3634::MudQ

-

TT15245

2000

37

pyrF2690::MudQ

A

TT15246

2001

37

pyrF2690::MudP

B

TT15630

2038

40

tre-152::MudP

A

TT15625

2033

40

tre-152::MudQ

B

TT15251

2006

44

hisD9950::MudP

-

TT15249

2004

45

zea-3666::MudQ

A

TT15250

2005

45

zea-3666::MudP

B

TT15252

2007

52

aroC566::MudP

-

TT15254

2009

53

cysA1586::MudP

A

TT15258

2013

53

cysA1586::MudQ

B

TT15632

2040

54

guaAB5641::MudQ

A

TT15255

2010

54

guaAB5641::MudP

B

TT15256

2011

56

purG2149::MudP

A

TT15257

2012

56

purG2149::MudQ

B

TT15260

2015

58

nadB226::MudQ

-

TT15261

2016

61

proU1884::MudP

A

TT15628

2036

61

proU1884::MudQ

B

TT15263

2018

64

cysHIJ1574::MudP

B

TT15262

2017

64

cysHIJ1574::MudQ

-

TT16706

2073

65

zgc-1715::MudP

A

TT16707

2074

65

zgc-1715::MudQ

B

TT16708

2075

69

zgf-1716::MudP

A

TT16709

2076

69

zgf-1716::MudQ

B

TT17190

2330

72

zgi-3717::MudP

A

TT17191

2331

72

zgi-3717::MudQ

B

TT15631

2039

75

cysG1573::MudQ

-

TT15264

2019

75

cysG1573::MudP

B

TT17165

2329

76

envZ1005::MudQ

A

TT15265

2020

76

envZ1005::MudP

B

TT15267

2022

81

pyrE2419::MudP

A

TT15266

2021

81

pyrE2419::MudQ

B

TT15269

2024

85

ilvA2648::MudQ

A

TT15268

2023

85

ilvA2648::MudP

B

TT15270

2025

86

metE2131::MudQ

A

TT15271

2026

86

metE2131::MudP

B

TT15273

2028

88

pnuE41::MudQ

A

TT15272

2027

88

pnuE41::MudP

B

TT15635

2043

90

purD1874::MudQ

-

TT15276

2031

93

melAB396::MudP

A

TT15275

2030

93

melAB396::MudQ

B

TT15277

2032

95

purA1881::MudP

A

TT15638

2046

97

zjh-3725::MudP

A

TT15633

2041

97

zjh-3725::MudQ

B

(a) All map positions are from Sanderson and Roth, 1988. Microbiol. Rev. 52:485-532.

(b)All strains contain the background genotype fels2 leuA414(am)r(-).

(c) A and B, clockwise and counterclockwise packaging, respectively.

 

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