JOURNAL OF BACTERIOLOGY, Oct. 1979, p. 251-260 0021-9193/79/10-0251/10$02.00/0

Vol. 140, No. 1

Physical Characterization of ilv-lac Fusions TIMOTHY D. LEATHERS, JOHN NOTI, AND H. E. UMBARGER* Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907

Received for publication 25 May 1979

Electron microscopic heteroduplex analysis and comparative restriction digests have been used to characterize Xpl(209) and Xpl23(209), the complementary pair of phages used in the Casadaban technique of gene fusion. Derivatives of Xpl (209) constructed to carry fusions of the lac genes to the control regions of the ilvC and ilvEDA operons were also analyzed. These physical maps have provided confirmation of the genetic models for these constructions and physical specifications important in interpreting the behavior of these ilv-lac fusions. The isoleucine and valine biosynthetic genes of Escherichia coli K-12 are organized into at least four transcriptional units (Fig. 1). The ilvEDA genes comprise an operon that is transcribed from ilvE to ilvA (5, 6, 17) and multivalently repressed by the three branched-chain amino acids. ilvG, which lies between ilvE and rbs, specifies valine-resistant acetohydroxy acid synthase activity and is cryptic in the absence of an ilvO mutation in cis. Recent data have shown that the ilvO locus lies between ilvE and ilvG (16). The ilvC gene, specifying the isomeroreductase, lies within 2.5 kilobases (kb) of ilvA and is transcribed in the same direction as the ilvEDA operon, but it represents a separate transcriptional unit, inducible by the substrates of the enzyme (11, 12, 19). The gene fusion technique of Casadaban (4) makes it possible to place the lacY and lacZ genes under the control of any chosen promoter, provided that a bacteriophage Mu insertion can be selected in the vicinity of, and downstream from, that promoter. By this method, a region transcribed from the iluC promoter has been fused to the lac genes (18). A plaque-forming phage that carries this fusion has been isolated, and the DNA from this phage has been used as a template for the in vitro ilvC-directed synthesis of /3-galactosidase (22). Recently, it has been possible to place the lac genes under control of the ilvEDA regulatory region (J. Noti and H. E. Umbarger, manuscript in preparation). A phage derivative has been isolated, and genetic and in vitro studies are under way. Heteroduplex analysis has been employed to characterize these fusions physically by identifying homologous regions among the ilv-lac fusion phages, their parental lambda-lac phages, and previously characterized ilv-containing phages. The resulting physical maps have re-

vealed the approximate location of possible regulatory regions and have provided some physical specifications for the genetic models defining these ilv-lac fusions. Comparative restriction cleavage of the phage DNAs by several sitespecific endonucleases corroborates these data. The dimensions thus established allow predictions to be made about the in vitro expression of these fusions. Since the Casadaban technique has such wide application, data concerning Xpl(209) and Xpl23(209), the complementary pair of phages employed in this method, should be of general interest. MATERIALS AND METHODS Heteroduplex analysis. Heteroduplexes were formed in a formamide procedure originated by Westmoreland et al. (21) and modified by Davis et al. (7). Samples were photographed in a Philips EM300 electron microscope at approximately x 10,000. DNA length measurements were made on enlargements (x3.5 or greater) with the digitizer of a Hewlett-Packard 9800 system, using a fully smoothed program. Single- and double-stranded molecules of phage S13 DNA, the kind gift of E. S. Tessman, were included in each preparation. Heteroduplex studies (8) have shown S13 DNA to be equal in length (within detection) to that of 4X174, recently sequenced as 5,386 base pairs (15). We consistently find a ratio of 9.1 for wild-type lambda lengths to S13 lengths, equivalent to that reported for lambda and OX174 (7). Our estimate of wild-type lambda DNA is therefore 49.0 kb based upon OX174 sequence data. All measurements in this paper reflect this estimate. Preparation of bacteriophage DNA. Bacterial strains and their sources are listed in Table 1. Phage strains and their sources are listed in Table 2. Xpilvlac-3 bearing the ilvD-lac fusion (Noti and Umbarger, manuscript in preparation) was prepared by the general procedure of Casadaban (4). The lac genes carried on Xpl(209) were transposed to a selected site in the ilvEDA operon by virtue of its homology with bacteriophage MuctsKlOlO DNA that had been inserted in iluD in strain CSH26. A temperature-resistant derivative of the resulting lysogen (CU945) was isolated on

251

252

LEATHERS, NOTI, ANI) UMBARGER

,J. BACTFRIOL.

Pyruvate

AHSU1

-aAHS hml-Acetolactate AHSI+il

Pyruvate

IR

0-

TRC~

a-,G-Dihydroxy-DH

a-Ketoisovalerate TRB

~~~~~~isovalerate

Vline

l

AHSI+M AHS u -

cz-Acetohydroxy-IR

OH

a-$-Dihydroxy -

G-methylvalerate

butyrate

TRB

a-Keto-,3-methyl - _lsoleucine valerate

a-Ketobutyrate

tTD Threonine

ilv H

I

9

-TH

H

AHS m

AHS I

C

Y

A

D

E

0

G

i

iR IR

1T 1HT1

HS

TD DH TRB

AHS I

FIG. 1. Biosynthesis of isoleucine and valine. The enzymes catalyzing the indicated steps are abbrev,iated and the corresponding structural genes (where known) are indicated in parentheses as follows: TD (ilv,A), threonine deaminase; AHS I (ilvB) and AHS III (iliuHI) end-product-inhibited acetohydroxy acid synthases; AHS II (ilvG), end-product-noninhibited acetohydroxy acid synthase; IR (iltvC), acetohydroxy acid isomeroreductase; DH (ilzD), dihydroxy acid dehydrase; TRB (ilvE), transaminase B; TRC, transaminase C. ilvG exhibits no activtity in E. coli K-12. In this strain, ilv,O mutations stimulate transcription initiated at the EDA promoter and are absolutely essential for transcription initiated at the ilkG promoter. ilvY specifies a control element for isomeroreductase induction by substrate.

CSH26 CU528

TABLE 1. Bacterial strain list Source or reference Genotype" Cold Spring Harbor ara A (pro-lac) thi galTJ2 iltA454 \cI857Sam7b5l5b5l9xisam6 Smith et al. (17)

CUJ710

ara A

Strain

piltvEDA CUT 11

(pro-lac) thi ilvC2083::Mu-1 cts62KamlO10 ara A,(pro-lac) thi ilC2203::Mu-1 cts62Kam100:: Apl (209) [formerly ilC208.3::Mu- 1 cts62Kam 1O10:

CU71.3

ara A(pro-lac) thi iluC2209::-AMu-:\ApI(209) [for-

Smith and lJmbarger (18) Smith and Umbarger (18)

Api (209)] merly il((1C208&::XpI(209)] ara A (pro-lac) thi ildD2138::Mu-1 cts62Kam 1010 CU944 ara A(pro-lac) thi ilD2139::Mu-1 cts62Kaml100:: ClJ945 Apl (209) ara A(pro-lac) thi il1D2210::-AMu-::Ap1(209) CU946 lacZl3 trpC his-4 tsx-.3 mtl malAl rpsL thi-I M1199 Ail1DACJ15 Xh80cI857St68 Xh8OcI857St68dilu All strains are F

lactose minimal agar plates containing a-ketoisovalerate and a-keto-,8-methylvalerate. When a culture of this strain (CU946) underwent isoleucine limitation, both /8-galactosidase and transaminase B, the ilvE gene product, activities increased coordinately. Induction of this strain with mitomycin C led to the isolation of a plaque-forming phage derivative that complemented our most promoter-proximal ilvE lesions. Frhe heat inducible, lysis-defective phages Xh80dilv and XpilvEDA were prepared by heat induction of lysogens, E. coli strains MII99 and CU528, respectively, by the procedure previously described (19). All other phages were prepared by lytic growth on strain CSH26 as described earlier (22). Extraction of DNA from the phages has been described previously (19). Restriction endonuclease analysis. Bacteriophage DNA was digested with Sall, SmaI, HindIII, EcoRI, KpnI, and PstI obtained from New England BioLabs. Electrophoresis of endonuiclease digests was

Smith and UJmbarger (18) Mucts mutagenesis of CSH26 Lysogenization of CLJ944 with Xpl(209)

Heat-resistant survivor of CU945 Avitabile et al. (3)

performed in horizontal agarose gels (18 by 11 by 0.6 cm). Gels were prepared by autoclaving agarose (SeaKem) in the electrophoretic buffer containing 92 mM Tris, 89 mM boric acid, and 2.4 mM EDTA(Na)4, adjusted to pH 8.0 with boric acid. Depending on the size of the DNA fragments to be separated, 0.2 to 0.3 jug of DNA in glycerol (20%) plus bromophenol blue was layered into a 0.7, 0.8, 1.0, or 1.5% agarose gel and subjected to electrophoresis initially for 5 min at 60 V and thereafter at 30 V for 6 to 8 h. Ethidium bromide (0.1 ,ug/ml) was included in the gels and electrophoresis buffers. The gels were examined by fluorescence with UV light (Transilluminator, Ultraviolet Products, Inc.) and photographed with Tri-X panchromatic film (ASA 400). Eleven PstI-generated fragments and two HindIII-generated fragments of Xh8Odilv DNA were used as molecular length standards. These sizes in kilobases are 10.8, 9.0, 6.0, 4.7, 4.1, 3.6, 1.1, 0.9, 0.6, 0.5, and 0.45 and 1.9 and 1.6, respectively.

llv-lac FUSIONS

VOL. 139, 1979 Phage

ApilvEDA Apl(209) XpI23(209) Apilv-lac-1 Apilv-lac-3

TABLE 2. Phage derivatives used Genotype iAb515 Ab519 A(attP-redB) [ilv'CYADE]

A( b-xis) A(b-xis) A(b-xis) A(b-xis)

[(+'Mu)::( trp'BA' lac'OZY)]" [(-Mu')::(trp'E?DCBA' lac'OZY)1h [(+'Mu)::(ilvC"AYC' lac'OZY)]' [ilv'GOED' trp'BA' lac'OZY]"

253

Source or reference

Smith et al. (17) Casadaban (4) Casadaban (4) Smith and Umbarger (18) J. Noti, Ph.I). thesis, Purdue University, 1979 Avitabile et al. (3)

Xh80dilv (48Oatt8OiAdilv) A(8-att'0P)cI857St68[ ilvGOEDAC] ' Originally described as Aplac'A?YZO'-A209-trp'AB'::(+Mu'). 'Originally described as \plac'A?YZO'-A209-trp'ABCDE'?::(-Mu'). ' The ilvC" fragment would not normally be contiguous with ilvA. It is that part of the iluC gene just beyond the site of the Mu insertion in strain CU711. Therefore, it would be contiguous with ilrC' in wild-type strain K12. "l )erived by excision from the lysogen CU946.

RESULTS Heteroduplexes between DNA of Xpilvlac-1 and DNA of previously characterized phages. XpilvEDA is a bio-type substitution derivative of XcI857Sam7b515b519xisam6 (Xyl99). This phage carries two deletions within the lambda b region that serve as convenient physical markers when heteroduplexes between this phage and b + phages are examined. Analysis of the restriction endonuclease cleavage pattern of XpilvEDA and analysis of the heteroduplexes formed between this and other ilv or lambda phages have allowed the identification of the bacterial genes in this region with some precision (12). Results showed that XpilvEDA carries a 5.3-kb segment of ilv DNA extending from the secondary lambda attachment site in ilvC to a point less than 0.1 kb beyond the beginning of the ilvE structural gene. Heteroduplexes between Xpilv-lac-1 and ApilvEDA (Fig. 2) displayed a double-bubble structure. Measurement of the homologous portions of the left arm revealed a homology of 20.5 ± 0.4 kb. This region extends almost precisely to the point of the b519 deletion. (The absence of the b519 deletion loop indicated that the XDNA of Xpilv-lac-1 does not span this region.) Since the position of the b519 deletion loop has been previously located at 20.3 kb, this length should be considered the maximum for the length of the homologous material in the left arm of the two phages. Homology in the right arm of the heteroduplex is clearly limited by the 16.0 kb of lambda DNA known to be present in XilvEDA. The central double-stranded region of 2.0 kb was bracketed by single-stranded bubbles. The leftmost bubble had one strand, of 3.0 kb, which accounts for the remaining DNA of XpilvEDA extending to the phage attachment site (POA'). Thus, the ilu material carried in common by Xpilh'EDA and Xpilh-lac-1 extends from the secondary lambda attachment site in ilvC to a point

about 2.0 kb away in ilvA, covering approximately two-thirds of the distal portion of this gene (12). This region thus spans the anticipated location of the ilC regulatory apparatus. The remaining ilv'ADE of ApilvEDA is accounted for by the 3.3-kb arm of the rightmost bubble. Examination of heteroduplexes of Xpilv-lac-1 with Xc1857Sam7 (with wild-type lambda physical homology) confirmed the above estimate of left-arm lambda homology and revealed 18.7 kb of right-arm lambda material present in Xpilvlac-1 (Fig. 3a). Therefore, 2.7 kb of lambda DNA is included in the 6.1-kb arm of the rightmost bubble of Xpilv-lac-1/XpilvEDA in Fig. 2. Heteroduplex between DNA of Xpilv-lac1 and that of its parental phage Xpl(209). Xpl(209) is the parental phage from which Apilvlac-1 is derived. Its preparation and behavior indicate that the lacY and lacZ genes are fused to a trp'AB' sequence (deleting the lac control region), which is fused to a segment from the c end of bacteriophage Mu (4, 18). Its structure is discussed more fully below. Examination of heteroduplexes between Xpl(209) DNA and Xpilt, lac- I DNA (Fig. 3b) indicated that the latter has 2.8 kb of ilv, DNA substituted for a short parental sequence of about 0.5 kb, making XpilL-lac-1 a virtual insertion derivative. As anticipated, the left-arm lambda-lac junction and the right-arm lambda-Mu junction are identical in the two phages. The 24.8 ± 0.7 kb of left-arm homology indicates the presence of 4.5 kb of common material beyond the left-arm lambda genes, presumably lac'AYZ. Estimates made by examining heteroduplexes Xpl (209)/Xpl23(209) and Xpl(209)/Xpilv-lac-3 make 4.6 kb a better value for this length (see below). The right-arm homology of 21.2 ± 0.6 kb indicates 2.5 kb of presumed Mu sequence, since 18.7 kb of the Xpilt-lac-1 right arm was shown to be homologous with X. However, 2.8 kb may be a somewhat better estimate on the basis of additional heteroduplexes. In addition, these studies, discussed below, suggested that the 0.5-kb region deleted

254

LEAT'HERS, NOTI. ANI) UMBARG(ER

J. BAC'TERIOL,.

FIG. 2. Heteroduplex between Xpilk-lac-1 and Xpilv EDA DNA. All values represent means of typically 20 to 30 samples and are expressed in kilobases. Standard deviations are indicated. The ilvY gene, recently located between the ilvC and ilvA genes (20), and the promoter-distal ilvC DNA on the 6.1-kb arm of the rightmost bubble have not been indicated on this diagram (see text). The iluY gene has also been omitted from the drawings in Fig. 3. in

Xpilt-lac-1 lies between the lac and Mu reof Xpl (209) and is therefore most probably

gions

trp DNA. The 2.8 kb of ilL substitution indicated is 0.8 kb greater than the 2.0-kb segment held in common by XpiIvEDA. Given the best estimates of the lac and Mu regions, the \pilv-lac-1/ \piltEDA heteroduplex (Fig. 2) suggests that 0.3 kb of additional ilvt material lies to the left of the 2.0-kb common segment and 0.5 kb to the right. (It must be noted that such small lengths are near the resolution limits of these studies.) Since all of the ilvC DNA found in ApilvEDA

estimated to be also present in Xpilv-lac-1, reasonable that this additional 0.3-kb length mav represent contiguous chromosomal ilC sequence. This assumption has been indicated in the interpretative drawing in Fig. 2. It is, however, also possible that all or part of this length may consist of Mu DNA left from the original deletion that formed strain CU713. While the absolute polarity of Mu was selected against in the preparation of the fusion, a transcriptional delay that has been observed in vitro might be explained by a small residual Mu sequence (22). Given the fact that all of the right-hand Mu was it is

region i(ientified in Apl(209) was also found in

Xpilv-lac-1, it is reasonable that the 0.5 kb of ilv DNA estimated to be at the right of the 2.0-kb region represents promoter-distal ilvC DNA (indicated by C" in the interpretative drawing in Fig. 3a and b) excised from the cya side of the prophage insertion in strain CU713. Figure 4 shows diagrammatically the structure proposed for Xpiltv-lac-1. For comparison, Fig. 4 also contains a diagram of the structure proposed earlier for XpiltEDA (12). The structure proposed for XpilL-lac-1 is in agreement with the genetic model proposed earlier (18) and provides the following specifications. 'I'he deletion (Fig. 5) that created strain CU713 removed most or all of the Mu DNA separating lac and ilv and about 0.5 kb of DNA from the original Xpl(209) that was contiguous with lac, presumed to be the trp sequence. An unknown amount of the promoter-distal portion of ilvC may also have been deleted, such that an estimated 0.3 kb of ilvC or Mu, or both, remained between the lac genes and ilvC sequence that was homologous with XpiltvEDA. Excision of \pilt-lac-1 from strain CU713 resulted from recombination be-

VOL. 139, 1979

ilv-lac FUSIONS

x

20.4

± 0.5

I

,~

+ 0.5

18.7 i.

A

a.

9.4

255

* 0.5

N

R

r-

X p ILV-LAG-i

I!'AYZ,1.c

± i0

I'Cm', C., Mu,

10.2

± 0.4

t0.1

0.5

htp 24.0

Xpl (209)

b.

±0.7

21.2 N

I

A

J

.

'AYZ I

Mu

±0.6 R

i

I

Xp/LV-LAC-1

±0.3

2.8

! 0.5

10.1

20.3

x

I,

± 0.6

A

C.

i

18.8

.I

± 0.5

N

.

R

-I

X pl (209)

/ac

I

ftp

',AYZ, AB.Mu

i

± 0.5

8.6

3.1

0.3

+

Mu

d.

X p1 (209)

26.0 t 0.5 A

M

J

.

hZ #P.

19.0 ± 0.6

i

I

'AYZ.'%a,,}

X p /LV-LAC-3

t-

3.4

0.3 4.1 ! 0.3

+

+ 0.4 foc

R

I I

I% IA/,V 5.3

N

trp

IAYZA'

G'.A

I

e.

X p /LV-LAC-3 A

20.4

' 0.4

'

J

16.2

''

! 03

EV N

R

; 2.3 ±0.1

Xp/LV EDA

1l' A .'CAD, 6.0 0.7 .

O

Api (209)

f.

25.1

± 0.6

A

X p123 (209)

± 0.2 Mu

1%

I

J .,AYZ! \

18.8

+ 0.7

N

I

I

',

tCD',* 3.5

t 0.4

FIG. 3. Heteroduplexes between: (a) wild-type lambda (Ac1857Sam7) and Apill-lac-1 DNA; (b) \pl(209) and Xpilv-lac-I DNA; (c) wild-type lambda and ApI(209) DNA; (d) Apl(209) and Apilv-lac-3 DNA; (e) Apilt-lac-.3 and Apilt' EDA DNA; and (f) Apl(209) and ApI23(209) DNA. tween a point about 0.5 kb to the left of the prophage (in ilvC) and a point in ilvA. Therefore, the putative ilvC regulatory region is contained on the phage. The orientation of the ilv

DNA with respect to the lac genes provides confirmation that ilvC is transcribed in the same direction as ilvEDA, as previous studies have indicated (5, 11, 19). A region of about 1 kb

256

LEATHERS, NOTI. ANI) UJMBARGER

J. BACTERIOL.

within the iltD gene. I'he DNA of the phage has been used as a template for in vitro coupled transcription-translation studies (J. Noti, unpublished data). The DNA allowed the synthesis of both transaminase B (the product of the IvtE gene) and B-galactosidase. It did not appear, however, that the synthesis with this DNA as template was under the control of an il/-specific promoter. In contrast, both enzymes in strain CU946, the lysogen from which Apil/-lac-3 was derived, have been shown to be subject to normal, il/-specific control (Noti, unpublished data). Thus, it may be that the excision of Apiltlac-3 does not encompass the ilt-specific control

between i/1C and i/1vA, recently shown to include ilt Y (20), is also contained on the phage. Since it is estimated that 4.1 kb of DNA is required to specify the structural genes for lacY and lacZ (22), it is likely that little lacA' is included in the 4.6-kb lac region found in either Apil/-lac-1 or Apl(209). This observation also increases the likelihood that the 0.5-kb sequence that lies between the lac and Mu regions of Xpl(209) but which is deleted upon formation of Apil/-lac-I is exclusively trp DNA. Structure of Xpl(209). The physical structure of Xpl(209) was largely defined by that derived for Apilt-lac-I above, since the former carries only about 0.5 kb not found on the latter (Fig. 3b). This material was between the lac and Mu segments of Xpl(209) and was probably trp L)NA. Measurements of heteroduplexes between A DNA and Xp1(209) DNA agreed well with these results (Fig. 3c). In particular, the values for the double-stranded regions were equivalent to values derived for Apil/v-/ac-l. Figure 6 summarizes diagrammaticallv the structure of Apl (209). Structure of Xpilv-lac-3. (See Materials and Methods for further details.) Apil/-lac-3 phage contains a fusion of the lacYZ genes to a point

region.

Figure 3d shows a heteroduplex between the DNAs of Apilv-lac-3 and Apl(209). As a Lac' derivative of Apl(209), Apilv-lac-3 would be expected to have retained the parental left-arm homology through these genes. The 26.0 + 0.5 kb mieasured confirmed this expectation and revealed that the 0.5-kb segment presumed to be ttp DNA and deleted in Apil/-lac-1 had been retained bv Api//-lac-3. The homologv between the right arms of Apil/-lac-3 and Apl(209) DNAs appears limited to the lambda genes carried by the latter phage. In other words, the entire Mu region from Apl(209) appears to have been lost in the derivation of Apil/-lac-3. The substituted xp /LV-LAC-1 region of il/v DNA was estimated to be 3.4 + 0.3 /LV /LV LAC kb. Mu A Y Z Cl IC A C'' A A Heteroduplexes between Apil/-lac-3 and 187 28 20 46 203 1051 1031 Api/vEDA are shown in Fig. 3e. The homologies in the left and right arms are as predicted within error. A (2.3 ± 0.1)-kb region of il/v homology is Xp /LVEDA shared by the two molecules. The 6.0-kb strand I/LV of the single-stranded bubble includes 3.2 kb of A 'C ADE ilv DNA extending from the phage attachment 53 160 231 site of ApilvEDA to a point approximately 0.66 FIG. 4. Summary of physical str-ucture dericed for kb from the proposed distal terminus of ilvD Apilv-lac-1 DNA, based upon heteroduplex data. See (12). (The remaining 2.8 kb of DNA in the text for details. Structure ofXpilv, EDA (12) presented bubble would be left-arm A DNA missing in Api/v-lac-3.) Since the 2.3 kb of il/v DNA in the for reference. I/v

Cc

Mu

X

R A

N

bc

Mu

,,p

j Y Z'AB'c

s

M-'C Y -

'I (B

?5:2.81 18.7 -

b) C)

31

38

A

i t .--

D

E

--4--4

2.0

Xp 1(209) -

b)~~~~~~~~~ e !v A

T

4

/

-ff

XpA, Aw

FIG. 5. Physical specifications for events occurring during prepar-ation of Apilv-lac]- from CU710. (a) Structure of DNA in the region of the i/C gene of CU71 1. (b) Deletion (reating CU713. (A) Excision of Apilvlac-1. All values are expressed in kilobases. See text for details.

ilv-lac FUSIONS

VOL. 139, 1979

\pl (209) X 203

'A

LAC Y Z 46

TRP

AB'

1051

Mu 28

187

FIG. 6. Summary ofphysical structure derived for Apl(209) DNA, based upon heteroduplex data. See text for details.

double-stranded region precisely accounts for the remaining bacterial region carried by XpilvEDA, it is clear that Xpilv-lac-3 carries contiguous chromosomal material beyond the beginning of the ilvE structural gene (present in XpilvEDA). Since 2.7 kb is the best estimate for the lambda DNA included in the (4.1 ± 0.3)-kb loop of Xpilv-lac-3/XpilvEDA, it was concluded that Xpilv-lac-3 contains approximately 1.3 kb of ilv DNA beyond UivE. This amount of DNA might specify about 46,000 daltons of protein. The site of at least one ilvG lesion has been shown to be carried by Xpilv-1ac-3 (Noti, unpublished data). The estimate of 3.4 ± 0.3 kb of total ilv DNA present in Xpilv-lac-3 is consistent within experimental error with this model. Figure 7 is a diagrammatic summary of the structure of Xpilv-lac-3. Approximately 1.3 kb (about two-thirds) of the proposed ilvD gene is included, as well as the entire ilvE structural gene. At least part, and perhaps all, of ilvG is present on the phage. Restriction endonuclease digestion of DNA from XcI857 and Xpl(209). Physical mapping of AcI857 by restriction endonuclease digestion has been previously reported (9, 10, 13). Our results are consistent with the earlier studies. Cleavage of the DNA of XcI857 and Xpl(209) was undertaken not only to verify the heteroduplex analysis, but also to define segments of the phage DNA that might be usefully cloned in plasmid vectors. Furthermore, restriction endonuclease analysis of the DNA of similar phages provides a more convenient means of comparing related phages than does heteroduplex analysis. Digestion of AcL857 DNA with the endonucleases SalI, HindIII, SmaI, EcoRI, and KpnI yielded three, seven, four, six, and three fragments, respectively (Fig. 8). Of these, several fragments derived from the right arm of this phage DNA were also found in similar digests of Xpl(209) DNA (Fig. 8). The presence of two SmaI fragments in Xpl(209) corresponding to fragments derived from the right arm of AcL857 DNA indicates that a minimum of 16.1 kb of the right arm of X DNA is carried by Xpl(209). The absence of the characteristic internal 5.3-kb AcI857 EcoRI fragment demands that the lambda homology be less than 21.3 kb, and the probable absence of the internal 9.2-kb Hindlll

257

fragment suggests 20.1 kb as an upper limit. These limits therefore bracket our heteroduplex estimate of 18.7 kb. Owing to the paucity of cleavage sites for the enzymes used in the left arm of X, fewer fragments derived from the left arm of the two phages were identical. The leftmost SmaI fragment of XcI857 was also present in Xpl(209) digests, as were the leftmost KpnI fragments. Thus, at least 18.9 kb of the left arm of X DNA is present in Xpl(209) (the sum of the sizes of the two leftmost KpnI fragments). However, the preserved portion of the X left arm must be less than the length (20.7 kb) of EcoRI fragment 1 from XcI857, since that fragment has been replaced by a 22.0-kb fragment in Xpl(209). These values are consistent with the value of 20.3 kb of common left-arm X DNA found by heteroduplex analysis of the two phage DNAs. The unique EcoRI site in Xpl(209) thus appears to lie about 1.7 kb from the X-lac fusion. This location is consistent with the report of an EcoRI site in lac (1). Two unique HindIII fragments in Xpl(209) replace the four leftmost HindIlI digestion fragments of XcI857. The unique HindIII site in Xpl(209) can be accounted for by the fact that Mu contains a HindIII restriction site 1 kb from the c end (2). Since 2.8 kb of the c end of Mu (estimated by heteroduplex measurements) is present in Xpl(209), this site is 1.8 kb from the X-Mu junction. Restriction endonuclease digestion of Xilv-lac-3 DNA. The DNA of Xpilv-lac-3 was digested with the same restriction endonucleases used to characterize Xpl(209). SalI digestion of Xilv-lac-3 produced two fragments which correspond to the rightmost fragments of Xpl(209) (Fig. 8). Two new fragments unique to Xpilv-lac3 appeared as a result of an additional SalI site not present in Xpl(209). This new site had arisen owing to the insertion of DNA from the operator-proximal portion of ilvEDA. A SalI site was shown to lie just outside of the promoter-proximal portion of the ilvE structural gene (12). Cleavage of Xpilv-lac-3 DNA with HindIII resulted in the formation of six fragments. Three fragments correspond to fragments derived from the right arm of Xpl (209). A Xpilv-lac-3 fragment is the same size (1.4 kb) as an ilv chromosomal HindIII fragment with termini in ilvD and ilvE

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258

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