Frameshift suppressors

J. Mol. Biol. (1972) 66, 483493

Frameshift Suppressors II.? Genetic Mapping and Dominance Studies DONALD L. RIDDLES

AND JOHN R. ROTH

Department of Moleculur Biology, University of California Berkeley, Calij. 94720, U.S.A. (Received 13 September 1971) Suppressors speoiflo for frameshift mutations have been studied. Six distinct suppressor looi have been identified on the genetic map of Salmonella typhimurium. These suppressor genes have been designated SufA through mfF. Suppressor mutations mfA, B, D and E are dominant, while sufF mutations are recessive to the wild-type allele. All dominant suppressor types are induced by the mutagen ICR-191. The recessive aufF mutations are induced by nitrosoguanidine. One dominant suppressor, .sufD, coincides iu map position to a glyoyl-transfer RNA gene in Escherichia co.%.

1. Introduction Frameshift mutations are additions or deletions of nuoleotides in DNA which result in out-of-phase translation of the messenger RNA. Since the genetic code is read as unpunctuated base triplets, additions or deletions in the message which are not multiples of three bases result in a shift in the reading frame. These mutations are induced by acridine-like compounds, which are believed to intercalate between adjacent base pairs in the DNA (Orgel, 1965 ; Ames & Whitfield, 1966). We have previously described the isolation of bacterial strains which carry external suppressors specific for frameshift mutations (Riddle t Roth, 1970). Suppressorcarrying strains were isolated as revertants of frameshift mutations in the histidine operon of Salmonella typhimurium. We found that 13 of 21 frameshift mutations tested gave rise to external suppressors among their revertants. Most suppressors were produced by the frameshift-inducing mutagen, ICR-1915; however, nitrosoguanidine induced suppressors of several frameshift mutations in addition to the suppressors induced by ICR-191. Independent suppressor mutations (48) were placed in nine phenotypic classes on the basis of their specificity of suppression. That is, nine distinct patterns of cross-suppression were observed when the suppressors were tested for their ability to suppress each of the frameshift mutations in our sample. Work described here was initiated in order to determine: (1) if the nine suppressor classes represent distinct suppressor loci; (2) if there is any correspondence between the map position of frameshift suppressors and known nonsense or missense suppressors; and (3) if the suppressors are dominant or recessive. i Paper I in this series is Riddle & Roth, 1970. $ Present sddress: Bepertinent of Biologic81 Sciences, University of Californie, Sent8 Barb8r8, Calif. 93106, U.S.A. 5 Abbreviations used: ICR-191, 2-ohloro-6-methoxy-9-(3-(2-chloroethyl) aminopropylsmino) a&dine dihydrochloride; NC, N-methyl-N’-nitro-N-nitrosoguanidine. 483

484

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AND

J. R.

ROTH

2. Materials and Methods (a) Bacterial

&aims

The strains used in this study are listed in Table 1; all are derivatives of S. typhimurium strain LT2. The Hfr strains used in preliminary mapping experiments, and the S. typhimurium episome, F’T77, were kindly provided by Dr K. E. Sanderson. An Exhetichia co&i strain carrying the F-prime episome KLFlO was obtained from Dr C. W. Hill. The F-prime episomes, F’32, KLPl6, KLFlO and F’13, are of E. coli origin, but are harbored by S. typhimurium strains (Table 1). TABLE

1

Bacterial strains Hfr strains SA534 SA536 SA549 TR1146

Hfr Hfr Hfr Hfr

F-prime donors TR116 TR1624

Strains used in co-transduction TR226 TR1435 TR1525, TR16267, TR1654$ TR1534, TR1635t, TR1668$ TR1601, TRlGlOt, TR1665$ TR1609 TR1620 TR1634, TR1635t, TR1666$ TR1697 TR1769, TR1770t

TR1769,

TR17515

TR1760,

TR17525

serAl3 serAl3 serAl3 serAl3

leu-1119

purE66 pro-635/F/13 lac+ purE + hi801242 hiaC3072 serAY90 1y8-554 ilv-563jKLF16 serA+ lya + his01242 hisD3749 tyr-545 ~~1-540 Eeu-1130/F’T77 xyl+ his01242 hkF3704 thi-502 &+565/KLFlO thi+ his01242 hid3737 aroD purF145 glpT201 leu-1131/F’32 aroD + purF + glpT +

TR1724 TR1731 TR1801

Strains used in dominance TR1746, TR1674$ TR1756, TR1749$ TR1758, TR17603

K4 K6 K2 K5

tests hisG200 aroD s~pK584~O~ his01242 hid3072 sufE35 hi801242 hid3737 aerA 1~8-554 hi801242 hid3737 purE801 trpA8 his01242 hiaD tyr-545 xyl-540 his01242 hisD3749 tyr-545 2~1-540 mtl-251 hi801242 hid3072 1~5-554 sufD42s~pK584~~* his01242 hi&3737 thi-502 his01242 hisF3704 thi-502 rif-201 his01242 hid3737 htiW7834

tests

TR1762, TR17535 TR1763, TR1754$ TR1786, TR1783$

A complete list of the frameshift & Roth, 1970).

hi801242 hisC3072 purEI trpA8 sujF44/F’13 purE + lac+ his01242 hisD3749 tyr-545 2~1-540 8ufA6/F’T77 xyl+ hi801242 hisC3072 serA790 1~8-554 sufDdBIKLF16 serA+ 1ys+ his01242 hid3072 aerA 1~9-554 sufD28lKLF16 serA+ lys + his01242 hid’3072 serA790 1~8-554 sufD30/KLF16 serA+ lys + thi+ his01242 hisF3704 thi-502 aufE35/KLFlO hi801242 hieF3704 thi-502 aufE33/KLFlO thi+ hi801242 hid3737 aroD purF145 glpT201 sufB2/F’32 aroD+ purF+ glpT+ suppressors studied in this report hes been published

(Riddle

t This strain carries hi&3072 instead of hi&3737 or hisD3749, but is otherwise isogenic. Since no single fremeshift mutation is suppressed by all suppressors, it was necessary to construct these isogenic strains so that ell suppressors could be tested for co-transduction with each marker. $ This strain carries h&F3704 instead of hid3737 or hisD3749 but is otherwise isogenic. 3 This strain has the chromosomal genotype given, but lacks the F-prime episome.

MAPPING

FRAMESHIFT

SUPPRESSORS

485

The histidine regulatory mutant !&W7834 was obtained from Dr B. N. Ames; due to the h&W mutation, this strain is unable to grow at 25°C. A derivative of this strain, TR1’769, was used in mapping the .sufB locus. The presence of the hisW mutation in these strains can be detected by the absence of growth at 25°C on nutrient broth plates, while growth at 37°C is normal. (b) Nomenclatzlre We have used the prefix Buf with the isolation numbers of our frameshift suppressor mutants in a fashion analogous to the SUP designations for nonsense and missense suppressors. In accordance with the nomenclature of Demerec, Adelberg, Clark & Hartman (1966), we have used lettered 8uf designations for the suppressor genes 83 shown in Fig. 1. Throughout the text of this paper, wild-type alleles of suppressor genes are designated sufWT,and mutant alleles (those with suppressor activity) are expressed as suf. (c) Media and transduction Complete medium is O*S% Difco nutrient broth with 0.5% N&l. The E medium of Vogel & Bonner (1956) supplemented with 2% glucose was used a~3minimal medium. Solid media contained 1.5% Difco agar. Auxotrophic mutants were grown on E medium supplemented with amino acids at a concentration of 0.1 mM, except serine which was used at a concentration of 4 mM. For counter-selection of strains carrying xyl mutations, E medium lacking citrate (Berkowitz, Hushon, Whitfield, Roth & Ames, 1968) was supplemented with 0.2% xylose. When mtl (mannitol non-fermentation) was used as a genetic marker, the presence of the mutation was scored on McConkey’s indicator plates containing 0.2% mannitol. The sensitivity of bacterial strains to rifampicin wm determined on solid minimal medium by streaking cultures radially from a filter disc saturated with 10 ~1. of 1 mg/ml. solution of rifampicin. Transductional crosses were performed as previously described (Riddle & Roth, 1970). (d) P-prime transfers and segregation Episome transfer was performed by mixing drops (about lo8 cells) of stationary phase cultures of the donor and recipient strains on solid selective medium. Episomes were removed by growth of merodiploid strains in nutrient broth overnight. Growth under these non-selective conditions results in loss of the episome from O-1 to 10% of the cells in the population. Such spontaneous segregants can then be isolated. 3.

Results

(a) Six frameshift

suppressor

loci

We have genetically mapped all of the frameshift suppressors included in our initial study (Riddle & Roth, 1970). Six distinct loci for frameshift suppressors are shown in Figure 1. We have assigned the letters A through P as designations for

these suppressor genes. The approximate map positions of the suppressor mutations were determined in conjugation crosses using the Hfr strains indicated in Figure 1. More exact locations were subsequently determined by testing the suppressors for co-transduction with various mutations known to map in the same region. Once a selective marker was found which was co-transduced with one suppressor in a given phenotypic class, all the independent suppressor mutations in that class were screened for similar co-transduction. Without exception, all suppressors of a given phenotypic class map at the same locus. The suppressors sujA, B, D, E and P are co-transduced with at least one of the selective markers listed in Table 2. Strains carrying these selective nutritional markers and a suppressible his frameshift mutation, were crossed with P22 transducing phage grown on strains carrying suppressors from each of the nine suppressor classes

488

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J. R.

ROTH

FIG. 1. The location of frameshift suppressor loci on the genetic map of 8. @@&~riunt. The arrowheads represent the origins of chromosome transfer of the Hfr strains used in preliminary mapping experiments. Frameshift suppressor (c@) loci, and markers which can be co-transduoed with them, are enclosed in brackets. The .x&? locus, in parenthesis, has been mapped only by conjugation crosses.

(Table 2). Recombinants were selected which had received the wild-type allele of the marker being tested. These recombinants were then tested for co-inheritance of the frameshift suppressor, by testing suppression of the his frameshift mutation carried in the recipient. The percentage figures given in Table 2 represent the percentage of His+ transductants among the selected recombinants. The number of recombinants tested is given in parentheses. These crosses demonstrate that each of the suppressors tested maps at one of six distinct genetic loci. Conjugational crosses established that the sufC locus is between gal and trp, close to pyrC (see Fig. 1). No selective nutritional marker was found which could be cotransduced with su.C. The sujC locus is clearly distinct from the other suf loci since it is not co-transducible with any of the markers to which the other suf loci are linked (Table 2). The co-transduction frequencies shown in Table 2 were confirmed in each case by performing the crosses another way. Strains carrying both the auxotrophic mutation and the frameshift suppressor were crossed with P22 grown on a wild-type strain. Again, recombinants were selected which had received the wild-type allele of the marker being tested. These recombinants were then tested for loss of the suppressor gene originally carried in the recipient. In these crosses, the results shown in Table 2 were confirmed. One interesting aspect of the data in Table 2 is that suppressors of phenotypic class V show unusually low co-transduction frequencies with both outside markers, serA and lys (in comparison with classes IV and VI). This may reflect poor viability of transductants inheriting these suppressors, or it may result from an effect of these mutations on the frequency of recombination in the adjoining region of the chromosome. (b) The order of markers on the genetic map A gene order was established for several suppressors with respect to neighboring markers. The sufA locus is 11 o/0co-transducible with xyl but is not co-transduced with

(1100) (880) (183) (110) (280) (204)

32 33 0 (680) 0 (720)

0 (137) 0 (445)

(129)

0

36 44

0 (89) 0 (90) 0 (154) 0 (176)

0 (210) 0 (202)

12 19 0 0 0 0

35

10 13 41 45 27 28 29 30

t3 8 2 11

@Kf

number

0 (12) 0 (20) 0 (18) 0 (18) 0 (16) 0 (1’3) 0 (18) 0 (18) 0 (1’3) 0 (20) 0 (12) ---

60 (70) 60 (76)

0 (14) 0 (1‘3)

0 (465) 0 (600)

0 (296) 0 (410)

0 (436)

9 (262) 11 (393)

6 (176) 4 (218)

13 (307) 11 (397)

0 (121) 0 (160)

0 (368) 0 (385)

0 (655) 0 (185)

7 (196) 9 (244) 0 (211) 0 (198) 0 (430) 0 (721) 0 (646)

0 (526) 0 (274) 0 (370) 0 (614) 0 (688) 0 (160) 10 (134) 7 (179) 2 (190) 1 (239)

serA

Percentage co-inheritance of suppressor mutations with the indicated marker by P22 transductiont$

(143) (189)

PW

(726) (168) (212)

40 (784) 26 (141)

0 (198) 0 (460)

0 (600) 0 (770) 40 (1140)

0 (430) 0 (261)

0 (386) 0 (289)

0 0 0 0 0 0

(203) (162) (678) (142) (178) (310)

(84) (102) (111) (306) (713)

0 (361) 0 (288)

26 (297) 27 (661)

0 0 0 0 0

0 (330) 0 (176)

0 0 0 0 0 0

t Recombinants were selected which had received the wild-type allele of the indicated marker. Co-inheritsnce of the frameshift suppressor carried in the donor was determined by testing for suppression of the kis frameshift mutation carried in the reoipient strain (Table 1). $ The number of transductants tested is given in parentheses. 8 The presenoe of the &W mutation can be detected by its cold-sensitivity phenotype. Because of the instability of this J&l+’ marker, coldresistance was not selected in these crosses. Strains carrying the htiW mutation and a his frameshift mutation were transduced to His+ at 37°C with phage grown on the suppressor-oarrying strains. Transductants carrying suppressors were single-oolony isolated and their coldsensitivity was scored at 25°C on nutrient broth plates. (1No marker has been found which can be co-transduced with the sufC locus.

IX

VII VIII

VI

V

IV

III

II

I

Genetic locus

suppressors

Phenotypic Cl&IS

2

Co-transduction frequencies of frameshift suppressor mutations with markers of known location

TABLE

D.

488

L.

RIDDLE

AND

J.

R.

ROTH

rntl. Since xyl and mtl are themselves 10% co-transducible, the inferred gene order is &A-xyl-mtl. The sufB locus is co-transducible with the his regulatory gene, his W, but not with glpT. The gene orders aroD-purli’-sufB and purP-glpT-sufB were determined by three-point conjugation crosses (see Table 3). TABLE

3

Mapping of sufBt with respect to aroD, purl? and glpT by conjugation purF +

aroD + Donor:

1 Recipient

,uf”T

g&T +

TR1145 (Hfr K5) :

2 aroD-

Unselected markers

aroD +

purF+ sufB pwF + auf”’ purF- sufB purF - 8UfWT

glpT+ glpT + glpTglpT -

PUTF -

sW’ -

Regions of required orossover

Selected markers

aroD + purF +

4

3

5

TR1783

No. of recombinants

1 83 (3 or 4) l&5 l&2 1, 2, & (3 or 4), & 5

sufB suf “’ BufB suf WT

l&4 l&5 l&3 1, 3, 4 t 5

@ufB

196 (28%) 466 (68%) 24 ( 3.4%) 4 ( 6.6%) total s90

20 ( 8%) 195 (78%) 31 (12.4%) 4 ( 1.6%) total 250

t The aufB locus is co-transducible with hiaW, but the gene order with respect to 7&W was not determined. No co-transduction of gZpT with either 8ufB or h&W could be demoted. TABLE

4

Mapping of sufE with respect to thi and rif by P22 transduction rif Donor :

TR1435

Recipient

: TR1697

1

thi + 2

rif” Selected marker &fE

Unselected marker thi+ thi+ thithi-

rif= rif” rifR rif”

Regions of required crossover 28~4 l&4 3&4 1, 2, 3 & 4

=fE 3

thi -

4 8ufW’

No. of transductants 42 (18%) 8 ( 3%) 190 (79%) 0 total 240

The donor, TR1435, carries SufE, normal thi genes and is sensitive to rifampicin, The recipient, TR1697, does not carry the suppressor, but requires thiamine and is resistant to rifampicin. Transductants carrying sufE were detected by suppression of the hia frameshift mutation, hkF37’04, carried in the recipient.

MAPPING

FRAMESHIFT

SUPPRESSORS

489

The sufD lOCU8is co-transducible with both serA and lys, but these two marker8 are not themselves co-transducible. The inferred gene order is lys-sufD-serA. The sufE locus was ordered with respect to rif (resistance to rifampioin) and thi (thiamine requirement) by a three-point transductional cross (Table 4). The inferred gene order is rif-thi-sufE. In two-point transduction tests, sufE is 40% co-transducible with thi and 4% co-transducible with rif. (c) Nine phenotypic classes: six genetic loci The suppressor mutations mapping at sufD (between serA and Zys) include three phenotypic classes (IV, V and VI), which exhibit overlapping but distinguishable patterns of cross-suppression (Riddle & Roth, 1970). Similarly, the sufE locus (near thi) include8 suppressor mutations of two phenotypic classes (VII and IX). The fact that more than one 8uppres8or class map8 at one locus suggests that distinct allele8 of one gene exist which have slightly different suppression properties. However, the possibility that there are separate, but closely linked, suppressor genes cannot be ruled out on the basis of the mapping data presented here. Each of the other suppressor loci includes suppressors of a single phenotypic class. (d) Proximity

of sufD to a nonsense suppressor gene

The frameshift suppressor locus, sufD, is very close to a UGA nonsenSe suppressor gene which is also located between serA and lys (Reeve8 & Roth, 1971). Three-point crosses were performed, which demonstrate that these loci are distinct and the frameshift and nonsen8e suppressor genes were ordered with respect to the nearby serA and TABLE 5 Mapping of 8ufD and a UGA nonseme suppressor by P22 transduction SUpUGA

serA + Donor:

1 Recipient

Seleoted marker

2

3

4

@zkpWT

Unselected

marker

sufD

5 lY8

Regions of required crossover

.supWT sufD supWTaufWT supucAsufD supUGAsuf”’

l&2 1, 2, 4 t 4 l&3 l&4

supWT sufD

4&5 3&5 2, 3, 4 33 5 2&5

No. of trsnsductants 1463 (93%)

total

lys +

lye?+

: TR1760 serA

serA +

suf WT

TR226

8upWT8ufW’ supUG”sufD supUGA.wfW’

tot81

8: (5%) 37 (2%) 1583 843 (95%) 30 (4%) 0 8 (1%) 881

The donor, TR226, carries a UGA suppressor mutation but is otherwise wild type. The recipient, TR1750, oarries the frameshift suppressor, sufD, and the serA and Zy8 msrkers. The suppressor genotype of Ser+ or Lys+ transductsnts was determined by: (1) suppression of the hia frsmeshift mutation, hkC3072, carried in the recipient; snd (2) suppression of the F’lac UGA mutation, lat.2219 (Riddle BERoth, 1970).

490

D. ser A

L.

RIDDLE

AND

J. R.

ROTH

sup “GA

Estimated distance

FIU. 2. Suppressor loci between 8erA and 44s. The values given below the heavy line are cotransduction frequencies of the indicated mcrrkers. The estimated number of genes between these loci is given on the bottom line. Co-transduction frequencies of the TJGA suppressor with serA and lys are from Reeves & Roth (1971).

lys loci. These co-transduction data (Table 5) are consistent only with the gene order shown in Figure 2. In order to test directly co-transduction of the suppressors, the following cross was performed. Phage prepared on TR1620, a strain csrrying both the frameshift suppressor and the UGA suppressor, was used to transduce a his frameshift mutant, TR1526, to prototrophy. Transductants csrrying the frameshift suppressor were then scored for co-inheritance of the UGA suppressor. The presence of the UGA suppressor in transductants was determined by: (1) ability to support growth of UGA mutants of P22 (Lew & Roth, 1970) ; and (2) the ability to suppress lac UGA mutations (Berkowitz et al., 1968). The frameshift suppressor, 8u.D and the UGA suppressor are 75% co-transducible (Figure 2). Based on the method of Wu (1966), we estimate that these suppressor genes are situated four to five genes apart, assuming 50 genes per transducing fragment. We conclude that the frameshift suppressor and UGA suppressor are mutations of distinct genes. These suppressors &o differ in that the UGA suppressor is recessive (Reeves t Roth, 1971) while the frameshift suppressor is dominant (see below). (e) Dominant frameshift suppessors As shown in Table 6, mfA, B, D and E were found to be dominant. Dominance tests were performed by infecting suppressor-carrying strains with 8n F-prime episome carrying wild-type copy of the suppressor gene. If suppressor activity is maintained in such a merodiploid (sufWT/suf), we conclude that the suppressor is dominant. If suppressor activity is lost, the suppressor is judged to be recessive to the wild-type allele. At least one suppressor mutation from each of eight phenotypic classes was tested for dominance. Strains were constructed which carry both a frameshift suppressor and an auxotrophic mutation known to be wmplemented by the episome. Transfer of the episome WLISselected by means of this auxotrophic mutation. Suppressor activity was then detected as a His+ phenotype resulting from suppression of tb his frameshift mutation carried in the recipient strain. In Table 6 the genotype of the haploid parent

ICR-191 ICR-191

ICR-191

ICR-191

ICR-191

IV

V

VI

VII

IX

NG

ICR-191

VIII

ICR-191

I

Suppressor induced by

II

Phenotypio cless

h&F3704 hi&3072

hid3072

hid3072

hid3072

hiaC3072

hid’3737

hiaD

auf-art

auf-33

mlj-35

suf-30

sqj-42 auf-23

suj-2

a~j-6

Suppressor

of strain tested~

Suppressed frameshift

Genotype

F’13lac+purE+

pwE-

Lac-Pur-His+

Phi-His+

Ser-Lys-His+ Thi-Hia+

Ser-Lys-His+

Ser-Lys-His+

Xyl-His+ Are-Pur-His+

Phenotype haploid =e%wt

of

Recessive

Dominant

Domiimnt

Dominant

Dominent

Domirmnt

Dominmrt Dominsnt

Domirmnoe

All of them (euj36, 37, 43, and 44) were found to be recessive.

Laa+Pur+His-

!Chi+His+

KLF lOthi +

thi thi -

Ser+Lys+His+

Ser+Lys+His+

Aro+Pur+His+

Xyl+His+

Phenotype of merodiploid

Ser+Lys+His* Thi+His+

KLFl&erA+Zys”

KLFl6.serA+lye’

FT77xyl+ F’32aroD+prF+

F-prime episome

KLF16serA+Zyet KLFlOthi+

8erA-lys-

stwA - @J88erA - 1yS-

aroD-pwF-

ZrJI-

Additional markers

t The aomplete genotypes of these strains ere listed in Table 1. 3 All suppressor mutations mapping 8t s-yjF were tested for dominance.

Suppressor locus

6

Dominance tests

TABLE

492

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J. R.

ROTH

strain is presented in columns 4,5, and 6. These strains carry the his frameshift mutation listed in column 4, which is suppressed by the suppressor listed in column 5. The wild-type F-prime episomes (shown in column 7 and in Fig. 1) were then transferred into these strains and suppressor activity was tested (column 8). If a His + phenotype was observed in the merodiploid (sufwT/suf) the suppressor was judged to be dominant. In the absence of suppressor mutations, the presence of the F-prime episomes did not make his frameshift mutants phenotypically His + . It was therefore concluded that the F-prime episomes themselves did not carry frameshift suppressor mutations. The F-prime strains were shown to be merodiploids, and not recombinants, by spontaneous loss of episomal genes to produce F- haploid strains (see Materials and Methods). As shown in column 9 of Table 6, these F- haploid strains exhibited the same suppressor phenotypes as the original haploid strains represented in columns 4, 5, and 6. Since the F-prime episomes, KLFlO, KLFIG, F’13 and F’32, are of E. coli origin and there is little or no recombination with the Salmonella chromosome, it was not necessary to use ret- strains in order to maintain the merodiploids. E. coli F-prime episomes can be used for dominance tests because homologous genes almost always occupy the same position on the genetic map of E. coli and 8. typhimurium (Sanderson, 1970; Taylor, 1970). In those cases where suppressor mutations located in the chromosome appeared to be dominant, suppressor mutations were induced in episomal genes in order to demonstrate unequivocally that the F-prime episomes carry the wild-type allele of the suppressor gene. For each dominant suppressor type, a strain was constructed which carries a his frameshift mutation and is diploid for the suppressor locus (~uf~‘/suf”~). His+ revertants were induced with ICR-191 as previously described (Riddle & Roth, 1970). The desired suppressor-carrying revertants were identified by : (1) the ability to transfer suppressor function along with other episomal markers; and (2) spontaneous loss of the suppressor gene with associated episomal genes. Episomal suppressor mutations were obtained for sufA, B, D and E. The ability of suppressors to arise in the episome of merodiploid strains demonstrates that the F-prime episomes carry a wild-type suppressor allele and that these suppressors are dominant. (f) A recessiveframeshift suppressor Suppressor mutations mapping at sufF are recessive to the wild-type allele. Suppressor function was absent in sufF merodiploid strains (His- phenotype), but returned in haploid segregants resulting from loss of the F-prime episome (Table 5). The presence of F’13 in his+ strains does not result in a His- phenotype, indicating that the episome does not prevent function of the histidine biosynthetic pathway. All four suppressor mutations mapping at sufF were found to be recessive. The presence of one of these suppressors, sufF44, results in temperature-sensitive growth of strains even in histidine-containing medium. This indicates that proper functioning of the sufF product may be essential for cell viability. Suppression may result from a partial loss of the sufF function.

4. Discussion Our working hypothesis has been that frameshift suppression involves altered transfer RNA as does nonsense and missense suppression (Gorini, 1970). If this is the case, we would expect : (1) the suppressor mutations to be widely distributed over the

MAPPING

FRAMESHIFT

SUPPRESSORS

493

genetic map; (2) the suppressors to be dominant; and (3) that some suppressor loci might correspond to known tRNA genes. We have identified six distinct suppressor loci. The four suppressor types induced by ICR-191 (sufA, B, D and E) are dominant. The sufD gene maps at a position corresponding to the location of a glycine tRNA struotural gene on the E. coli chromosome described by H.ill, Squires t Carbon (1970). Direct evidence is presented in the accompanying paper (Riddle & Roth, 1972) that strains carrying sufD contain an altered glycine tRNA, and that two other dominant suppressor mutations, sujA and sufB, affect the structure of proline tRNA. Since sufF suppressors are recessive, it is unlikely that this gene is a tRNA structural gene. While suppressor mutations in tRNA structural genes would be expected to be dominant, a recessive mutation is generally associated with loss of function rather than a qualitative change in function. The s@F gene ‘may be involved in enzymic modification of tRNA. Strains carrying a sufF mutation may accumulate a partially unmodified tRNA, which occasionally fails to maintain the reading frame during translation and thus may suppress frameshift mutations. The sufC locus has not been mapped with sufficient accuracy to permit dominance tests. Like sujF mutations, su@ mutations are induced by nitrosoguanidine but are not inducible by ICR-191. Based on this similarity, we suspect that sufC suppressors also may be of the recessive type. This investigation was supported by U.S. Public Health Service research grant AM121 15 from the Institute of Arthritis and Metabolic Diseases, and training grant GM367 from the Institute of General Medical Sciences. REFERENCES Ames, B. N. & Whitfield, H. J., Jr. (1966). Cold Spr. Harb. Symp. Quant. Biol. 31, 221. Berkowitz, D., Hushon, J. M., Whitfield, H. J., Jr., Roth, J. & Ames, B. N. (1968). J. Bact. 96, 215. Demerec, M., Adelberg, E. A., Clark, A. J. & Hartman, P. E. (1966). Genetics, 54, 61. Gorini, L. (1970). Ann. Rev. Genetics, 4, 107. Hill, C. W., Squires, C. & Carbon, J. (1970). J. Mol. Biol. 52, 557. Lew, K. K. & Roth, J. R. (1970). I’&-ology, 40, 1059. Orgel, L. E. (1965). Advunc. Enzymol. 27, 289. Reeves, R. H. & Roth, J. R. (1971). J. MOE. Biol. 56, 523. Riddle, D. L. t Roth, J. R. (1970). J. Mol. Biol. 54, 131. Riddle, D. L. & Roth, J. R. (1972). J. Mol. Biol. 66, 495. Sander-son, K. E. (1970). Bact. Rev. 34, 176. Taylor, A. L. (1970). Bact. Rev. 34, 155. Vogel, H. J. & Bonner, D. M. (1956). J. Biol. Chem. 218, 97. Wu, T. T. (1966). Geneth, 54, 405.