104

Appendix

A physical map of the bioAB region in the X bio transducing phage

(E. coli bio operon; regulatory region: heteroduplex mapping; electron

tions)

microscopy; IS1 insertion; dele-

Graiyna Konopa , *a Waclaw Szybalski **a, Jeroo Kotval *b and Allan Campbell b

0 McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, WI 53706, und ’ Deportment oj Biologicul

Sciences, Stanford University, Stanford, CA 94305 (U.S.A.)

(Received April 2nd, 1982)

(Accepted May 5th. 1982)

SUMMARY

The center of the p’,opC, promoter-operator region for the bioABFCD operon is located 1.7 1 kb clockwise

from the attX site on the Escherichia coli genome, as determined by the position of the ~13 1 (ISI ) insertion.

The order of several bio endpoints to the right of p131 is Xbio267, 122, 169, 74, 1, and 69. The endpoints of

the two bio deletions, A61 in bioA and A3h in bioB, were also determined.

In the preceding communication the physical

structure of the entire bioABFCD operon and

neighboring regions was outlined, and the position

of the controlling p’,o& region indicated on the

basis of the restriction mapping and sequencing

data of Otsuka and Abelson (1978) and Barker et

al. (198 1). Here we present our electron micro-

scopic heteroduplex data which locate the ~131

(ISI ) insertion (Ketner and Campbell, 1975) in

relation to attB and specify the position of several

nearby bio endpoints and deletions in relation to

* Present addresses: (G.K.): Department of Biochemistry, Uni-

versity of Gdadsk, “1. KIadki 24, 80-822 Gdarisk (Poland);

(J.K.): Department of Microbiology and Immunology, Albany

Medical College, Albany, NY 12208 (U.S.A.).

** To whom reprint requests should be directed.

Abbreviations: bp, base pairs; IS, insertion sequence; kb, kilo-

base pairs; o, operator; p, promoter; A, deletion; %X unit. 485

bp; Xarr2, XarrLarrR or XattBOP’atrPOB’ (Fiandt et al., 1976).

0378-I 119/82/0000-0000/$02.75 ‘Q 1982 Elsevier Biomedical Press

this ISI marker (see Figs. 1 and 2, and Table I).

Deletion A61 removes part of the bioA gene and

fuses it to the gal region. This deletion was crossed

into the phage XgalSbio69cI857 (Ketner and

Campbell, 1974; Barker and Campbell, 1980) re-

sulting in Xgaf8(A61)bio69cI857. Phage Xbio3h-1

of Kayajanian (1968) carries within the bio DNA

the A3h deletion extending from bioB to uvrB

(Szybalski and Szybalski, 1982). Other bio end-

points shown in Fig. 1 are within gene bioB with

the exception of hbiol, which contains the entire

gene bioB, and hbio69, which also contains most

of bioC. Phage hbio267, 122, 169, and 74 were

isolated by J.K., and some of them were used for

testing the effect of rightward function on leftward

bioA transcription (Kotval et al., 1982).

The present data complement those of Szybal-

ski and Szybalski (1982) and suggest that the

center of the p,op, region is located about 1.71 kb

from the attR site, i.e., 0.23 kb closer to the att site

than the data of Otsuka and Abelson (1978) would

105

0 1 2 3 4 kb -- I

attlR V

1 I I II II

bio 267 I

I --

1 I

----A3h---_-_-_-_-_---

Fig. 1. Physical and genetic map of the regulatory and neighboring regions of the bio operon, showing the endpoints of the bio DNA in

several Xbro phages. The top scale shows the length in kb of bio DNA starting from the aft R site. The open rectangles represent the bio

proteins, positioned as described in the preceding communication of Szybalski and Szybalski (1982). pa is the rightward and j?A is the

leftward bio promoter, and they flank the o operator in the ,?a@*. Symbol ~131 specifies the site of the 1% insertion in Xbiolbio

p98bio ~131, positioned according to the electron microscopic analysis of the heteroduplex Xbiolbio p98bio p131/Xbio+att’imm434

(see Fig. 2). The positions of 5’-ends of the pA and pa-promoted bio transcripts (open arrows) in relation to the ~13 1 insertion site are

taken from Barker et al. (1981). The heavy lines represent bio DNA and the thinner lines (left of artR) correspond to X DNA. The

right endpoint of the A61 deletion in Xgul8(A6l)bio69 and the left endpoint of the A3h deletion in Xbio3h-1 (see also Fig. 2 in

Szybalski and Szybalski, 1982) are positioned in respect to ~131 and all other bio’s. These positions and that of the bio endpoints are

based on the data in column 4(A) of Table I (pl31-bio measurements) and are drawn to scale. For the structure of harr2ir7nn434

(b519b515arrRbiot uorB+pg/-atrLXimm434) see Fiandt et al. (1976) and Szybalski and Szybalski (1982).

seem to indicate. The sequence data of Barker et

al. (1981) and Otsuka and Abelson (1978) specify

the distances between the ~131 insertion site and

the start points of the pA and p,-promoted tran-

scripts, but they do not extend to the att site or all

termini of the bio genes.

ACKNOWLEDGEMENTS

This report was supported by grants 5-POl-CA-

23076 and 5-P30-CA-07175 from the National

Cancer Institute (to W.S.) and by grant AI08573

from the NIAID (to A.C.).

Fig. 2. Electron photomicrograph of the heteroduplcx, Xhiolhw p98bio pl31/b5 196.5 15bio’ (rtr2mnt434. The single-stranded loops A

and B correspond to the b519 and h515 deletions, respectively. loop C to the ~131 (ISf) insertion. and loop D to the combined

&I’- -uurB’ -pgf region of katt’ (see legend to Fig. 1). The I~~nh~mology E is between the immh and wrww434 regions.

TA

BL

E

I

Phys

ical

en

dpoi

nts

of

the

bio

DN

A

inse

rtio

ns

and

dele

tions

h&i0

st

rain

B

(1)

Phys

ical

di

stan

ces

b515

to

bio

end

poin

t b

(%A

*SD

.)

(n)

(2)

pl3

1 to

bi

o en

dpoi

nt

c

(%X

2S.

D.)

(n

)

(3)

Phys

ical

di

stan

ces

Cal

cula

ted

leng

th

of

bio

DN

A

d (k

b)

(A)

(B)

(4)

(5)

h en

dpoi

nt

to

Cal

cula

ted

’ im

m 4

34 e

h

endp

oint

(%;X

*S

D.)

(n

) (%

A)

(6)

(7)

Xbi

o267

nin5

Xbi

ol22

imm

434n

in5

Xbi

o169

imm

434

Xbi

o74i

mm

434

Xbi

olbi

op98

p131

Xga

/( A

61)b

io69

hbio

3h-I

(A3h

)

7.77

~0.5

7 (2

3)

0.26

-cO

.06

(12)

1.

83

1.90

6.

3020

.50

(27)

67

.20

8.27

r0.7

1.

(25)

0.

83*0

.09

(17)

2.

11

2.14

__

_ __

8.58

-to.

78

(44)

1.

07-c

0.08

(27

) 2.

23

2.29

.-

- __

8.52

20.5

3 (4

4)

1.20

20.1

0 (2

2)

2.29

2.

26

__^

_-

9.77

r0.6

6 (4

5) g

2.

3920

.19

(44)

2.

87

2.87

5.

1620

.29

(46)

68

.34

___

1.48

~0.2

1 (1

9)

h 3.

50 i

__

7.14

-tO

.57

(25)

66

.36

-__

0.50

~0.1

0 (2

0) h

-_

__

__

_ __

a Se

e Fi

g.

1 (a

nd

Tab

le

III

of

Szyb

alsk

i an

d Sz

ybal

ski,

1982

; pr

eced

ing

pape

r).

b M

easu

red

in h

eter

odup

lexe

s of

th

e ty

pe

hbio

/Xb5

19b5

15bi

o’ar

t’im

m43

4 as

the

di

stan

ce

from

th

e b5

15 d

elet

ion

loop

to

th

e “a

rt2

loop

”,

the

nonh

omol

ogy

mar

king

th

e bi

o-A

junc

tion

in

the

Xbi

o (s

ee S

zyba

lski

an

d Sz

ybal

ski,

1982

).

S.D

., st

anda

rd

devi

atio

n;

n,

num

ber

of

mol

ecul

es

mea

sure

d (i

n pa

rent

hese

s).

’ M

easu

red

in

hete

rodu

piex

es

of

the

type

X

bio/

Xbi

oibi

o ~

98~

131

as

the

dist

ance

fr

om

the

~131

(I

SI)

in

sert

ion

loop

to

th

e bi

o en

dpoi

nt.

d(A

) C

alcu

late

d in

X

h un

its

as

the

sum

of

th

e di

stan

ce

from

at

f to

~13

1 (3

.52%

&h,

as

mea

sure

d in

th

e hb

rolb

io

p98p

131/

harr

2 he

tero

dupl

ex)

and

the

dist

ance

fr

om

~131

to

th

e bi

o

endp

oint

, an

d co

nver

ted

to

kb.

(B)

Cal

cula

ted

as

the

dist

ance

fr

om

b51S

to

th

e bi

o en

dpoi

nt

min

us

the

bS15

-nrt

R

dist

ance

(i

n SX

),

and

conv

erte

d to

kb

. T

he

b515

-attR

di

stan

ce

is

3.86

kO.4

0 %

X (

Fian

dt

et a

l.,

1976

).

’ D

ista

nce

from

th

e “a

ft*

loop

” to

th

e im

m43

4/im

mX

no

nhom

olog

y in

het

erod

uple

xes

hbio

/XbS

19b5

15bi

o+at

r’im

m43

4.

fCal

cuIa

ted

as

the

diff

eren

ce

betw

een

the

left

im

m43

4 en

dpoi

nt,

take

n as

73

S%h

(Szy

bals

ki

and

Szyb

alsk

i, 19

79)

and

the

h en

dpoi

nt-t

o-im

m43

4 di

stan

ce.

s The

mea

sure

d di

stan

ce

from

ar

rR

to ~

131

(IS

I)

is 3

.522

0.47

8X

(see

fo

otno

te

d).

The

IS

f le

ngth

is

not

in

clud

ed

in

this

b5

1S-L

:o

endp

oint

di

stan

ce.

h D

ista

nce

from

~1

31

left

war

d to

th

e ri

ght

endp

oint

of

de

letio

n A

61

(lin

e 6)

or

ri

ghtw

ard

to

the

left

en

dpoi

nt

of

dele

tion

A3h

(l

ine

7) (

see

Fig.

1)

i Dis

tanc

e fr

om

dele

tion

A61

to

th

e bi

o en

dpoi

nt

in

the

Xga

18(A

61)b

io69

/Xar

r’im

m43

4 he

tero

dupl

ex.

108

REFERENCES

Barker. D.F. and Campbell, A.M.: Use of hro-lac fusion strains

to study regulation of biotin biosynthesis in Escherrchio cob.

J. Bacterial. 143 (1980) 7899800.

Barker, D.F.. Kuhn, J. and Campbell, A.M.: Sequence and

properties of operator mutations in the hro operon of

Escherrchia co/i. Gene I3 (1981) 89- 102.

Fiandt, M.. Gottesman, M.E., Shulman. M.J.. Szybalski, E.H..

Szybalski. W. and Weisberg, R.A.: Physical mapping of

coliphage hart’. Virology 72 (1976) 6- 12.

Kayajanian, G.: Studies on the biotin-transducing, defective

variants of bacteriophage X. Virology 36 (1968) 30-41.

Ketner, G. and Campbell, A.: A deletion placing the galac-

tokinase gene of Eschmchia co/i under control of the biotin

promoter. Proc. Nat]. Acad. Sci. USA 7 I (1974) 2698~~2702.

Ketner. G. and Campbell, A.: Operator and promoter mu-

tations affecting divergent transcription in the hrr~ gene

cluster of Escherrrhrcr CO/I. J. Mol. Biol. I96 (1975) 13-27.

Kotval. J., Campbell, A.. Konopa. G. and Szybalski. W.:

Leftward transcription in the Esc,hrrrchra dt operon does

not require the products of the rightward transcript. Gene

17 (1982) 219-222.

Otsuka, A. and Abelson, J.: The regulatory region of the biottn

operon in Escherichru co/r. Nature 276 (1978) 689-694.

Szybalski. E.H. and Szybalski, W.: A physical map of the

Escherrchm w/i bra operon. Gene I8 ( 1982) 93- 103.

Communicated by M. Lieb.