J. Gel/CI., Vol. 66, No, 2, August 1987. pp. 12-'-132. © Printed in India.

Extragenic suppression of the temperature-sensitivity of a fitA mutation by a fitE mutation in Escherichia coli: Possible interaction between FHA and Fitl] gene products in transcription control'

M HUSSAIN MUNA VAR and R JA YARAMAN

School of Biological Sciences, Madurai Kamaraj University, Madurui 625021. India

MS received J I July 1987

Abstract. Temperature-sensitivity due to the.lil A 76 mutation is suppressed by a mutation in a locus mapping in between the Pps and FitA loci, at approximately -'7·3 min on the E.coli circular genetic map. Genetic evidence suggests that the suppressor mutation might define a distinct genetic locus which has been named FitB. Suppression by the jiil3 mutation is independent of the medium of growth unlike the previously described inrragcnic suppression which is manifested only in rich media. In.f//B/.f//B I mcrudiploids there is a drastic reduction of suppression efficiency in minimal medium. The possible mode of action of the FitA and FitB gene products in transcription control is discussed.

Keywords. FitA; Fit B: suppression; transcription control.

1. Introduction

Earlier work in our laboratory has identified a genetic locus in Escherichia coli, mapping at 37·5 min and defined by a temperature-sensitive mutation (originally named ts76) that leads to transcription abnormalities at 42°C (Jabbar and Jayararnan 1976, 1978; Jayaraman and Jabbar 1980). The map position of this locus precludes its identity as one of the genes coding for the subunits of RNA polymerase, since the genes for all the subunits of the enzyme have been mapped and none of them lies at 37·5 min (for references, see Yura and Ishihama 1(79). It is therefore possible that this locus defines the gene for an accessory transcription factor. Dass and Jayararnan (1085a) isolated an intragenic suppressor mutation (originally called ts24) which could suppress the Ts phenotype of a ts76-bearing strain but coulcl1eacl to temperature-sensitivity and transcription defect at 42°C if present alone. Dass and Jayararnan (198Sa) named the locus 'Fit' (factor involved in transcription). The two mutations therefore have been named fit76 and ./it24. A rifampicin-resistant mutation (rpoB240) which accompanied the fit24 mutation has been shown to cause a medium-and temperature-dependent rifampicin-sensitivity in a fit+ background (Dass and Jayararnan 1985b). Combinations of ./11'-, Iit76,. fit24, fit76~flt24 mutations in rpoB' and rpoB240 genetic backgrounds have been found to affect the expression of different gene groups (Dass and Jayaraman 1987). The present report is the result of our attempts to see whether the Ts phenotype of the /1176 mutation could be extragenically suppressed by mutation(s) in other gene(s). We show that a mutation in a locus mapping very close to the Fit locus

, Dedicated to Professor S Krishnaswarny on his sixty-first birthday.

123

124 M Hussain Munavar and R Jayaraman

could do this. Accordingly the locus of the fit76 and fit24 mutations has been renamed FitA and that of the extragenic suppressor mutation as FitB. We propose that the FitA ancl FitB gene proclucts interact in vivo and that a mutation in FitA could be suppressed by another mutation in FitA itself (intragenic suppression) or in FitB (extragenic suppression).

2. Materials and methods

2.1 Bacterial strains

The Eicoli strains usee! in this work are listed in table 1.

2.2 Genetic techniques

All the genetic techniques employed were conventional ones, as described in Miller (1972). Selection for m'oD+ was made on plates lacking tryptophan, phenylalanine, tyrosine and shikimic acid; pps" was selected on plates containing 0·4% sodium lactate as the sole carbon source. Ts+ /Ts refers to the ability/inability to grow at 42°C. Genetic nomenclature is according to Demerec et al (1966).

3. Results

3.1 Isolation of strain HMJOI

Mutations in the RpoB locus of E.coli have several pleiotropic effects in addition to conferring rifampicin resistance. They are known to suppress the effects of rho mutations (Guarente and Beckwith 1978), dnaA mutations (Bagdasarian et al 1977), rpsi: mutations (Chakrabarti and Gorini 1977), N gene mutations of A. phage (Ghysen and Pironio 1972) etc. Similarly the A and B subunits of DNA gyrase (products of the GyrA and GyrB loci) influence various cellular processes such as maintenance of the supercoiled state of DNA, replication, transcription, repair of DNA damage and activation of expression of silent genes (Gellert et al 1976; Cozzarelli 1980; Gellert 1981; Drilica 1984; Elizur and Chronis-Armer 1984; Wang 1985). Our earlier attempts to isolate Ts+ derivatives from rif" (rpoB) mutants of a

Table 1 List of E. coli strains

Strain

Relevant type

Source/reference

GMS343 GMS343 ts24 BJ501

F- aroD argE IjJsL F- argE fitA24 rpsi:

F- fitA76 pps aroD [JyrD edd pfkA,! rpsi,

F- his fitA76trp purii leu metA rpsL argG fitA76 derivative of CSH57; Dass and Jayaraman (1985a) B J Bachmann

This laboratory

fitA76 derivative of RT500; Dass and Jayaraman (1985a) This work

This work

JAJ572

HMJOI

HM.J02 KL159/F'KLF48 KL159/F'KLF48 jitA76 KL159/F'KLF48 fitA24

Same as .IAJ572; has fitA76 and fitB F-jitA76 fitB aroD pyri) edd pfkA? rpsi: proA his aro Olhis" aroD+

proA his aroivthis" aroD-1- fitA76 proA his aroisthis" aroD-1- fitA24

B J Bachmann

Dass and Jayaraman (1985a) Dass and Jayararnan (1985a)

Suppression of [it A by fit B ill E. coli

125

fitA76 strain led to the isolation of the fltA24 mutation (Dass and Jayaraman 1985a). Using the same strategy we screened several spontaneous nalidixic acid resistant (gyrA) mutants of strain JAJ572 (harbouring the fitA76 mutation), isolated at 30°C, for their ability to grow at 42°C. Out of 330 mutant colonies screened one was Ts+. This isolate was named HMJOl. The experiments described below were carried out with HMJ01 or its derivatives.

3.2 Genetic characterization of HMJOl

3.2a Nalidixic acid resistance and Ts+ phenotype of HMJOI are not related: At first it seemed reasonable to expect that the Ts+ phenotype of HMJOl is due to the nalidixic acid resistance mutation it harbours. However, this did not turn out to be the case. When the nal' mutation was introduced into JAJ572 by PI phage propagated on HMJOl, none of the nal' transductants were Ts-l-. This is reminiscent of our earlier observation (Dass and Jayaraman 1985a) that the rif" mutation which accompanied the fitA24 mutation was not responsible for the Ts-l- phenotype of the fitA 76-fitA24 double mutant. It seemed likely that the Ts+ phenotype of HMJ01 could be due to other reasons and therefore we undertook a more detailed genetic analysis of the same.

3.2b HMJOI harbours the fitA76 mutation: The first possibility to be excluded is that HMJOI is a fitA + revertant. It is known from our earlier work that fitA is cotransducible with the aroD marker at a frequency of approximately 50%. When phage PI propagated on HMJOI was used to transduce the aroD-I- marker into- an araD recipient (GMS 343) and the aroD+ transductants screened for temperature sensitivity, 4·5% of them turned out to be Ts (table 2, cross 1). Although there is a 10-fold difference between the observed and expected cotransduction frequencies (4,5% vs 50%), the significant point to be noticed here is that temperaturesensitive aroD -I- transductants do arise out of a cross between apparently temperature-insensitive parents (the possible reason for the discrepancy is explained below). In order to show that the Ts-aroD-I- transductants from cross 1 do harbour the fitA76 allele, PI was propagated on one of them and used to

Table 2. Genetic characterization of HMJOI
Unsblected
Cross Selected marker/ Cotransduction
number Donor Recipient marker phenotype frequency (o,.{,)
1 HMJ01 GMS343 {[roD+ Ts 4·5 (24/533)
2 GMS343 ({[roD'-Ts GMS343 ([roD + Ts 47 (195/414)
transductant from cross 1)
3 GMS343 (aroD+-Ts BJ50l aroi) + Ts+ o (0/374)
transductant from cross l)
4 HMJOI BJ50l {[roD + Ts+ 75 (702/942)
5 HMJOI BJ501 pps+-aroi)" Ts+ 86 (582/674)
6 HMJOl B.l50l pps+ Ts+ 92 (957/1040)
7 JAJ572 HMJ02 {[roD + Ts 69 (47/68)
8 I-IMJOI BJ50l aroi) + pps? 78 (490/627) Ts": Growth on LB plates at 42°C; Ts: No growth on. LB plates at 42°C

126 M Hussain Munavar and R Jayaraman

transduce the aroD-I- marker into GMS3tI3. Data presented in table 2, cross 2, show that 47'% of the aroD+ transcluctants are indeed Ts. When a fitA76-aroD strain (BJ501) was used as the recipient, none of the aroD+ transductants was Ts+ (table 2, cross 3). These two observations show that HMJOI harbours thetitA 76 mutation but for some reason its cotransduction with aroD is less than expected.

3.2c Identification of a suppressor mutation in J-IMJOI: The low cotransduction of filA76 with the AroD locus from HMJOI could be explained by postulating the presence of a suppressor mutation in the vicinity of the AroD-Fit segment such that the simultaneous inheritance of fit A 76 and exclusion of the suppressor is infrequent. The suppressor mutation could be intragenic (just as fitA24) or extragenic. Our past experience has shown that recornbinational separation of filA 76 and fit A24 mutations is very infrequent, usually less than 1 'Yo. In the present experiments the fit A 76 and suppressor, if any, are separable by approximately 5% .

. It is therefore probable that the suppressor mutation is extragenic, but located very close to the Fit locus. The order of loci in the vicinity of Fit is: Fit-Pps-AroD (counter-clockwise on the circular map). The suppressor mutation may be located to the left of Fit or to the right of AroD, or in between Fit and Pps or Pps and AroD. In order to map the location of the suppressor mutation, afi"tA76-pps-aroD recipient (BJ501) was transduced to aroD+, aroD+ -pps" and pps" using PI propagated on HMJOI as the donor and the respective transductants were screened for Ts+ phenotype. The results presented in table 2 (crosses 4,5 and 6) show that 75% of the aroD+, 86% of aroD+ -pps" and 92% of pps" transductants are Ts+. Since the lowest cotransduction was observed with aroD the location of the suppressor to the right of araD or in between pps and aroD could be ruled out. If the latter (suppressor lying in between pps and aro D) were true, lOO';!" (or ciose to WO%) of the aroD+-pps+ should have become T5+. This was not the case. Moreover, if the suppressor were to lie to the right of aroD, all (or nearly all) the ppS-I- Ts+ transcluctants should also be aroD+. In fact several of them were aroD-. One such pps" -aroD- Ts+ transc!uctant was purified and named I-IMJ02. Therefore the suppressor mutation could lie in between pps anclfit A 76 or to the left of j[t A 76. The high (92%) cotransduction frequency suggests the former as the most probable location of the suppressor mutation. If it were to lie to the left of filA 76, the pps-suppressor cotransduction frequency would be less than the pps-fit A 76 cotransduction frequency which is 70% (Dass and J ayaraman 1985a). The results presented so far show that HMJOI harbours both the [it A 76 mutation and a suppressor mutation which suppresses the Ts phenotype due to the former. The probable genotype of HMJOl with respect to the relevant markers could be fitA 76-suppressor pps" -aroD+.

3.2d Unmasking the latent temperature-sensitivity of H MJ02: If the hypothesis presented above is true, the Ts+ phenotype of HMJOI is due to suppression of the Ts phenotype of filA 76 mutation by the suppressor mutation. It should therefore be possible to unmask its latent temperature-sensitivity by introducing the wild-type allele of the suppressor. Since I-IMJOI does not harbour aroD or pps markers genetic manipulation of this region is not possible. Therefore we used I-IMJ02 which is identical to HMJOI (with respect to relevant markers) except that it is aroD- (see the preceding section). HMJ02 was transduced to aroD+ using PI

Suppression of fit A by fit B in E. coli

127

phage propagated on JAJ572 which is fit A 76-aroO+ but lacks the suppressor mutation, and the aroD+ transductants were screened for Ts phenotype. Data presented in table 2, cross 7, show that a substantial fraction (69°/r») is Ts. This shows that the 1yl phenotype of HMJ02 (and also HMJOl) is indeed due to suppression of the temperature-sensitivity of the fitA 76 mutation by the suppressor mutation.

In the light of the above observations we rename the original Fit locus as Fit A and the 10CllS of the extragenic suppressor mutation as FitB. The cotransduction frequencies between markers in this region have been converted to map distances (min) using WlI'S (1966) formula. Figure 1 presents the most probable order and map positions of markers in this region. It should be pointed out that there is a degree of built-in uncertainty in the absolute map positions since cotransduction frequency of fit A alleles with nearby markers depends upon whether the fit A + or fitA76lfitA24 alleles are involved (see Dass and Jayaraman 1985a). However, it can be said with reasonable confidence that FitA and FitB are flanked by Pps and AroO on the right and the PheS-PheT-InfC gene cluster on the left (see Bachmann 1983).

3.3 Is Fit B a distinct genetic locus?

It should be apparent from what has been presented so far that assigning the suppressor mutation to a distinct genetic locus, different from FitA, is based purely on mapping data. The usual vagaries of genetic analysis, especially while dealing with very closely spaced loci, render such a conclusion open to question. It could be argued that the suppressor mutation described herein could lie within the FitA locus itself. It is therefore necessary to show that the locus of the suppressor mutation is different from FitA. This was attempted as follows. We hac! shown earlier (Oass anel J ayaraman 1985a) that recombinational separation of filA 76 and fit A24 mutations is usually of the order of 1 % or less. If the suppl:essor mutation

0·35

0.207

~------------------

o 055 0·158

I· ~--=----------4

fit A (37·45)

fit 8

( 37·30/')

pps (37·258)

aro D (37·1 )

Figure I. Map positions of /iiA ami FIB. Distances arc in minutes, calculated from cotrunsducuun frequencies using Wu's formula (Wu 19(6). The ./i'tA·aroD ancljitA-pp.l' linkages arc from Dass and Jnyaramaii (llJX5a) (drawn approximately to scale).

128 M Hussain Munavar and R Iayaraman
tit A fit 8
A
'76 24 \
~ I i I I I
\ \ -t- I I
\ , I I
\ \ I I
I
\ \ \ t
\ \ I I
I ~ \ .. 1 f I
+ + aro 0

+

Figure 2. Schematic illustration of a transductional cross fit A 76 filB aroD- (recipient) x filA24 fiIB·'· aroD+ (donor). The first cross-over is shown as a solid line and the second cross-overs as broken lines. R = recipient; D = donor (not drawn to scale).

lies in FitA itself, recornbinational separation between fitA24 and the suppressor could also be of the same order of magnitude. Secondly, filA 76 and filA24 mutations can be distinguished based on the growth patterns of the respective mutants on rich and minimal media at 42°C. While the former is totally Ts on both the media the latter is leaky on rich media. The filA 76-fitA24 double mutant is Ts+ on rich media but Ts on minimal media (Dass and Jayaraman 1987). These differences in growth characteristics could be easily seen on plates. If HMJ02 (fitA76-fitB-aroD) is transduced to araD+ using PI propagated on a fitA24-fitB+ araD+ donor, the aroD+ transductantswill arise as a result of two cross-over events, one to the right and the other to the left of the araD locus. Depending upon the location of the second (left) cross-over the genotypes of the transductants and hence their ability to grow at 42°C, will vary. Figure 2 illustrates such a cross and table 3 presents the predicted genotypes and the phenotypes of the m'oD+ transductants. A cross-over occurring between fitB and fitA24 would yield totally temperature-sensitive aroD+ transductants. Hence, the proportion of such transductants would be a measure of the recombinational separation of fitB and fitA24 mutations. Table 4 presents the results of an experiment in which HMJ02 was transduced to aroD+ using Pl phage propagated on GMS343 ts24 (fitA24 aroD+) and the transductants grouped as indicated. Approximately 54% of the aroD+ transductants are totally Ts (class II) whereas less than 1 % are Ts on minimal medium and Ts+ on LB medium (class III). This shows that recombinational separation of fitB and fitA24 is far more frequent than that of fitA 76 and

Table 3. Predicted correlation between cross-over intervals, genotypes and phenotypes in a cross filA76 jltB aroD (recipient) x jitA24jhB' araD+ (donor)

Interval of the second Genotype of araD+

cross-over transciuctant

Phenotype of aroD' transductant

aroD-jhB jitA76-ji1A24'-jiIB-araD'

jilB-filA24 jitA76-jiIA24+-fiIB+-aroD+

ji1A24-jitA76 fiIA76-jiIA24-fiIB+-aroD'

Ts+ on all media (the parental type but aro D')

Ts on all media

Ts' on rich medium; Ts all minimal medium

jitA76·'-jiIA24-jiIB'-araD'· Ts± on rich medium

Beyond fit A 76

Suppression of [it A by [it B in E. coli

129

Table 4. Transductional cross: GMS343 ts24 (m'oD-' fitA24) (D) x HMJ02 (aroD fit A 76 filB) (R) Selected marker: aroD+

Phenotype of the aroD+ transductant at 42°C

Interval of the secane! Genotype of the araD-'- LB
cross-over transductant medium
aro D-fltB (1) fiIA76-jltA24-'-fiIB-aroD+ +
filB-filA24 (II) fit A 76-}11 A24 + -filB + -aro 0 -t-
f/IA24-fiIA76 (III) filA 76-fiIA24-}itB+ -aroD+ +
Beyond fitA 76 (IV) [it A 76'- -fit A24-}iIB "<aro 0 + ± Minimal

medium Frequency (%)

+ 39-4 (177/449) 53·'] (241/449) 0·89 ( 4/449)

± 6·01 ( 27/449)

fitA24. This would mean that the distance between fitB and fitA24 is greater than that between fitA76 and fitA24. Therefore fitB most probably defines a distinct locus and not a site within FitA itself.

3.4 Negative complementation of [it B by fitB+ allele

The results presented so far have shown that the Ts phenotype due to the fitA 76 mutation could be suppressed by a mutation in the FitB locus. It would be of interest to see the pattern of suppression in a fitB/fitB + merodiploid. In our earlier work we had constructed F' factors harbouring filA 76 and fitA24 mutations (Dass and Jayaraman 1985a). These F's as well as the F' harbouring the fitA + allele were introduced into HMJ01 by crossing with appropriate F'-bearing donors and selecting for his+ transconjugants. (HMJ01 is his- and the F's carry the his+ allele.) The purified transconjugants were scored for their viability (colony formation) in LB anel minimal media at 30°Canel42°C. It should be noted that all the F's carry the fitB + allele irrespective of the fitA alleles. The c1ata presented in table 5 show that introduction of F' fitA + fitB+ into HMJ01 reduces its viability in minimal medium at 42°C three-fold while the viability in LB medium at the same temperature is unaffected. From this observation it would appear that the filB + allele has very little effect, if any, on the suppression potential of the fitB mutation. However, the F' in this case carries the wild type alleles of both FitA and FitB genes. When F' fitA76-fiIB+ or F' fitA24 fitB+ is introduced viability in minimal medium at 42°C is drastically reduced (4000-fold). Thus the fitB+ gene product seems to negatively complement the fitB product with respect to viability in minimal medium at 42°C.

Table 5. Viability of HMJOI and its F' derivatives in rich (LB) and minimal media at 30°C and 42°C

Strain (genotype)

LB medium

Relative colony forming units (42°C/30°C)

Minima! medium

JAJ572 (jiIA76) GMS343 ts24 (jiIA24) HMJOI (fi1A76 filB)

HMJOl.1 (fi1A76 filB/filA' fitB') HMJ01.2 (fIIA76 j1IBlfilA76 j/lB') HMJ01.3 (jilA76 j11BlfilA24 JltB')

10-7 10-" [·15 1·24

1·0

()·98

10-8 10-4 ()·80 0·27

1·8 X lO-4 2·1 X 10-4

[30 J\I/ Hussain Munavar and R l ayaraman

4. Discussion

We have shown in this work that the temperature-sensitivity caused by the fit A 76 mutation could be suppressed by a mutation in another locus. The evidence for this rests on the isolation ofthefitA76 (Ts) mutation from the apparently temperatureinsensitive strain HMJ01. Cotransduction of fit A 76 and a rei 0 -I from HMJO I is neither as high as with the fitA76-aroD"" mutant JAJ572 (Jabbur and Jayaraman 1978; Dass and Jayararnan 1985a) nor as low as with the intragenically suppressed (fit A 76~fi't A24) derivative (Dass and J ayaraman 1985a). A possible reason for this discrepancy could be the closeness of the FitA and the suppressor (FitB) loci as a result of which they tend to be inherited together in transductional crosses. However, in a small number of cases inheritance of fifA76 and ((roO+ and exclusion offitB mutation does occur together, probably by a quadruple cross-over event, two involving the selected ([roO+ marker and two involving the unselected fitA76 marker. From such a transductant fitA76 is cotransducible with aroO+ at normal frequency.

The mapping data presented in this paper suggest that the suppressor mutation is located between the FitA and Pps loci which are themselves fairly close to each other. This makes it difficult to say unequivocally that the suppressor mutation defines a distinct genetic locus. However, three lines of evidence lend support to such a conclusion. They are: 1) the fitE mutation restores the viability of a fit A 76 mutant in LB as well as minimal media at 42°C (see table 5) whereas the intragenic suppressor mutation fit A24 restores viabili ty only in LB (Dass and J ayaraman 1987); 2) recombination in the fitA24-fitB interval is far more frequent than that in the fitA24-fit A 76 interval implying that the distance is more in the former interval; 3) the fitB+ allele negatively complements the suppression potential of the fitB mutation with respect to viability in minimal medium at 42°C. It is difficult to reconcile all the above lines of evidence with the notion that fit B is yet another intragenic suppressor of litA 76. We therefore believe that fitB defines a distinct genetic locus.

The role of Fit A gene in transcription is well documented (our earlier work cited in § 1). Of particular interest are the observations of Dass and J ayaraman (1987) that the efficiency of expression of several physiological functions such as viability in rich and minimal media, gross RNA synthesis, f3-galactosidase synthesis, development of phages T4, T7 and A, sensitivityiresistance to rifampicin etc., could be modulated by combinations offitA+,/i'fA76,fitA24,.f/tA76-fitA24, rJ)oB-I and IpoB240 alleles. These results imply interaction between the Fit A gene product and RNA polymerase and reinforce the earlier conclusion that the former could be an accessory transcription factor. Suppression of the Ts phenotype due to fit A 76 by the fifB mutation suggests that the Fit A and FitB gene products might function in vivo as a complex. The firA 76 mutation could disfavour the formation and/or function of the complex at 42°C. This could be overcome either by a second mutation in FitA itself (intragenic suppression or second site reversion) or a mutation in FitB (extragenic suppression). A possible flaw in this model is the difference in the pattern of suppression by the fit A24 and [i! B mutations. While the former suppresses only in rich media (Dass and Jayararnan 1987) the latter does so in rich as well as minimal media (this work). An alternative model would be to postulate that the fi: A24 mutation overcomes the need for the FitB gene product

Suppression of FIA by [u B itiE, coli

131

but only with respect to the expression of genes needed for growth in rich media. The fitB mutation seems to be crucial for suppression of temperature-sensitivity of the fitA76 mutation in rich as well as in minimal media.

An as yet unanswered question is with regard to the phenotype of the fit A + -fitB allele combination. Since the AroD locus is closer to FitB than FitA, in a transductional cross of the type fit kl- -fitB + -aroD- (recipient) x fitA 76-fitBaroD+ (donor) a larger fraction of the aroD + transductants should be fit A -1--fitBal'oD+. If fltB by itself confers temperature-sensitivity (as does fitA24) there should have been a large fraction of Ts-aroD+ transductants. This has not been observed (see table 2; cross 1). Either fitB by itself does not confer temperaturesensitivity or fit A + -fitB transductants are nonviable in minimal media on which the transductants are selected. These and related questions are currently under investigation in our laboratory.

Acknowledgement

This work was supported by a grant from the Department of Science and Technology, Government of India.

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