HYPERPRODUCTION OF DIHYDROFOLATE REDUCTASE IN DZPLOCOCCUS PNEUMONZAE AFTER MUTATION IN THE

STRUCTURAL GENE. EVIDENCE FOR AN EFFECT AT THE LEVEL OF TRANSCRIPTION

F. M. SIROTNAK AND R. W. McCUEN

Sloan-Kettering Institute for Cancer Research, New York, N . Y. 10021

Manuscript received February 12, 1973 Revised copy received April 20, 1973 Transmitted by PHILIP E. HARTMAN

ABSTRACT

Dihydrofolate reductase is markedly hyperproduced in strains of Diplococ- cus pneumoniae which bear any one of a unique group of sense to sense mu- tations (amer) in the corresponding structural gene. Increased enzyme levels mediated by the amer mutations are apparently the result of increased rates of de novo synthesis. The basis for these effects could be transcriptional, trans- lational or could involve an increase in messenger RNA stability. Data reveal- ing no difference in stability of the related mRNA in a variety of mutants and the wild-type strain appear to eliminate the last possibility. Other data sup- port the idea of an effect on transcription. This includes the extreme sensitivity of amer mutation expression following genetic transformation, to inhibition by actinomycin D and rifampacin, and the presence in one extremely high level mutant (amer-3 with 120 times the wild-type enzyme content) of increased amounts of mRNA. The data are most compatible with the idea of a regulatory function for the dihydrofolate reductase protein in this organism.

HE exceptionally large increase in the production of dihydrofolate reductase in DipZococcus pneumoniae following mutational alteration of the corre-

sponding structural gene has been the subject of a number of reports from this laboratory ( SIROTNAK and HACHTEL 1969; SIROTNAK, HACHTEL and WILLIAMS 1969; SIROTNAK 1969, 1970, 1971). In view of the evidence presented, it seems clear that high levels of this enzyme protein in mutant strains occur by an in- crease in the rate of de novo synthesis. The mutations (ame’) involved are dis- tributed over a number of widely separated sites in the gene. More significantly, these mutations appear to be of the sense to sense type, since they only modify the enzyme with no deleterious effect on catalytic function. The idea that a struc- tural gene mutation can quantitatively modify, in an upward manner, the ex- pression of the gene is relatively unique in respect to general notions of struc- tural gene function. Other examples of this type of quantitative genetic effect in microorganisms, which are possibly related, have also been recently reported (KOVACH et al. 1969; COVE and PATMAN 1969; KOVACH et al. 1970; DORFMAN et al. 1971; LOMAX and WOODS 1971; ROTHMAN-DENES and MARTIN 1971; KOVACH et al. 1972). In the present case, the mutational effects on enzyme pro- Genetics 74: 543-556 August, 1973

544 F. M. SIROTNAK A N D R. W. MC CUEN

duction could conceivably result via an alteration at the transcriptional or trans- lational level of protein synthesis, or even involve a change in the stability of the corresponding messenger RNA. Data are presented here which would appear to eliminate this last possibility. Other data, also presented, support the idea of an effect on transcription by at least one specific group of these mutations. Barring the possibility of a trivial (indirect) effect on regulation (unlikely in the absence of any severe catalytic impairment of dihydrofolate reductase), these results are suggestive of a bifunctiopal role, i.e., both catalytic and regulatory, for the dihy- clrofolate reductase protein.

MATERIALS A N D METHODS

Source of strains: The wild-type strain used in these studies was the R6 variant obtained from DR. R. D. HOTCHKISS, Rockefeller University. The mutant strains used bear individual cme? mutations in the dihydrofolate reductase gene and were prepared by DNA-mediated trans- formation (SIROTNAK and HACHTEL 1969) of the wild-type strain. This procedure provides some assurance that each transferred amer mutation exists in an identical genetic background. One recombinant strain bearing two amer mutations was also used. Mutant donor strains for DNA were selected for resistance to amethopterin. The procedures for strain isolation, genetic and biochemical characterization, and recombination analysis have been described ( SIROTNAK, LUNT and HUTCHISON 1964; SIROTNAK and HACHTEL 1969; SIROTNAK, HACHTEL and WILLIAMS 1969; SIROTNAK 1970; 1971). The mal- strain, a derivative of the original R6 wild-type strain bearing the MK2 mutation in the amylomaltase gene, was generously provided by DR. S. LACKS, Brook- haven National Laboratories.

Enzyme assays: Dihydrofolate reductase activity in crude extracts of sonically disrupted washed cells was determined by a modified procedure (SIROTNAK, DONATI and HUTCHISON 1964) of OSBORN and HUENNEKENS (1959). Amylomaltase activity was measured by the procedure of LACKS and HOTCHKISS (1960). The production of glucose during the reaction of recrystallized maltose with cell lysate was determined by the glucostat reaction (Worthington Biochemicals).

Fractionation of C'4 labelled bulk RNA: Aliquots of cell culture exposed to C14-uridine were centrifuged and resuspended in 1.0 ml of 0.05 1LI tris HC1 and 0.1 M NaC1. Cells were lysed with 0.1% sodium deoxycholate, and nucleic acids precipitated with 5 volumes of cold 10% trichloroacetic acid (TCA). Precipitate was washed three tmes by resuspension in 10% TCA and then dissolved in 2 ml of 0.5 N NaOH and incubated for 16 hours a t 37°C. After adjusting the pH to 7.0 with 6 N HC1, RNA was extracted by adding 2 ml of cold 10% TCA and centrifug- ing. The pellet was resuspended in another 2 ml TCA and centrifuged again. Supernatants from both extractions were combined and radioactivity counted by scintillation spectrophotometry. Inhibition of RNA synthesis was obtained by the addition of actinomycin D (generously SUP-

plied by DR. F. W. HOLLY, Merck, Sharp and Dohme Inc.) or rifampacin (obtained as a gift from Lapetit, Inc.).

Pulse-labelling and fractionation of messenger RNA for "double-label" experiments: Loga- rithmic phase cultures (100 ml at O.D. 0.3) in partially defined medium (SIROTNAK, LUNT and HUTCHISON 1960) were exposed for periods of 1 or 2 min to either C14-uridine (10 pc in 100 pg) or H3-uridine (25pc in 100 p g ) . The cultures were then chilled by pouring immediately over frozen buffer (0.01 M Tris HC1 and 0.005 M MgCl,, pH 7.3), combined and centrifuged. The cell pellet was frozen with the aid of an alcohol-dry ice bath and the cells disrupted by grinding in a pre-chilled mortar. After adding 40 pg of DNAase, the paste was resuspended in 10 ml of Tris-MnC1, buffer and one-tenth volume of 10% sodium lauryl sulfate was added. After stand- ing for 15 min at room temperature, the RNA was purified by phenol extraction (GIERER and SCHRAMM 1956).

CO-chromatography of pulse-labelled RNA: The mixture of H3- and C14-labelled RNA was chromatographed on a methylated albumin-kiselguhr column (MANDEL and HERSHEY 1960).

G E N E REGULATION IN DIPLOCOCCUS 545

Usually 1 mg of RNA was added to the column. Elution was obtained with a NaCl gradient. Fractions were examined for absorbance at 260 mp and radioactivity was measured by scintil- lation spectrophotometry after precipitation with trichloroacetic acid and filtration on nitrocel- lulose membranes. Counting was done in a Packard Tricarb Spectrophotometer for two or three ten-min periods. H3-counts were corrected for pick-up of C14 counts in the tritium channel.

Large-scale transformation experiments: Recipient cultures of the wild-type R6 strain or the amylomaltase negative strain (Mk2) were rendered competent for transformation by a period of growth in competence medium (SIROTNAK 1971). A volume of 100 ml of culture was harvested during the period of peak competence (cell conc. = 1.0-1.5 x lOQ/ml), chilled and diluted ten- fold in 0.05 M potassium phosphate buffer (pH 7.9) containing 2.5 mM MgCl,, 0.45 mM CaCI,, 11 mM glucose and 0.167 mM cystine. A saturating concentration of DNA (10-50 pg/ml) from an amer mutant or wild-type donor strain was added and the suspension incubated for 25 min a t 30°C to allow maximum transformation. After the addition of 1.0 mg of deoxyribonuclease (Worthington), a mixture of 3 gm heart infusion (DiEco) and 3 gm yeast extract (Difco) was added and the culture incubated at 37°C. Aliquots (40-80 ml) were removed at zero time, and at various times thereafter, and the cells were examined for dihydrofolate reductase or amylomaltase activity. The procedure for preparing DNA has been described (SIROTNAK, LUNT and HUTCH- ISON 1964).

All strains used as donors of DNA also bear the strr-41 mutation (derived from a strain sup- plied by DR. R. D. HOTCHKISS) for streptomycin resistance. The transformation of this character is used as an indicator of transformation efficiency. After the competent culture is exposed to DNA, a small aliquot is diluted five-fold in a semisynthetic casein hydrolysate medium ( SIROT- NAK, LUNT and HUTCHISON 1960) and incubated for 75 min at 37°C to allow for maximum expression of this genetic property. Streptomycin-resistant transformants are counted as colonies after overnight growth following dilution in drug medium. The level of transformation usually achieved with the described procedure is in the range of 10-15%.

RESULTS

Amer mutant strains: Three genetically distinguishable classes of ame' muta- tions have been identified in the dihydrofolate reductase structural gene of D. pneumoniae (SIROTNAK 1971). Each genetic class is represented by at least one mutation in the group of strains selected for the present study. Five of the six mutant strains bear individual mutations, namely amer-3, -30, -37, -51 and -89. @ne strain, prepared by recombination, bears two mutations, amer-20 and -51. The relative distribution of these mutations at sites within the dihydrofolate re- ductase gene has already been given (SIROTNAK and HACHTEL 1969; SIROTNAK 1970). The level of enzyme synthesis determined by mutation in each of these strains varies from as little as 3- to as much as 120-fold over that characteristic of the wild type.

Relative stabilities of amer mutant and wild-type specific mRNA: In light of current knowledge of protein synthesis, it is difficult to imagine how any struc- tural gene mutation, particularly one which generates a fully translatable nu- cleotide triplet, could drastically alter the properties of the related messenger RNA. Nevertheless, because of the unusual quantitative effects of the ame' muta- tions under discussion here, an examination was made for a possible difference in degradation of the dihydrofolate-reductase-specific mRNA from mutant and wild-type strains.

Mutant and wild-type cultures of D. pneumoniae were treated with actino- mycin D or rifampacin to stop RNA synthesis and dihydrofolate reductase ac-

546

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F. M. SIROTNAK A N D R. W. MC CUEN

control

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( 0 1 actinomycin D (pglml) ( x ) rifampicin (pg1ml 1

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FIGURE 1 .-The inhibition of RNA synthesis in wild-type D. pneumoniae by actinomycin D and rifampacin.

l - 1 1.1

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t (min.) FIGURE 2.-The effect of actinomycin D on the accumulation of dihydrofolate

wild-type D. pneumoniae. reductase in

GENE REGULATION IN DIPLOCOCCUS 547

tivity was measured at varying intervals of time. The decreasing rate of enzyme accumulation observed after cessation of RNA synthesis reflects the changing capacity for synthesis and is a functional measure of specific mRNA concentra- tion. Similar measurements of relative RNA concentration by synthetic capacity have been made by others (HARTWELL and MAGASANIK 1963; KAEMPFER and MAGASANIK 1967; LEIVE and KOLLIN 1967). The data in Figure 1 show the effect of each drug, at the level (1 pg/ml) used in these experiments, on CI4-uridine incorporation into RNA of the wild-type strain. Inhibition of RNA synthesis was immediate and essentially complete at this concentration of drug. The same re- sult was obtained with the amer mutant strains.

The synthesis of dihydrofolate reductase in wild-type cultures following the addition of actinomycin D is shown in Figure 2. Compared to the control culture, there is a nearly immediate effect on the rate of synthesis in the drug-treated culture, with a complete cessation of synthesis in about 15 min. By comparing the level of enzyme accumulated following cessation of synthesis to those levels accumulated at varying time intervals after the addition of drug, it was possible to obtain a measure of the rate of specific mRNA decay. From the data shown in Figure 3, it can be seen that the dihydrofolate reductase specific mRNA in the wild-type strain decays at a logarithmic rate after the first two or three minutes following the addition of drug. The half-life for decay determined from the graph is 2.9 min.

The decay rates for dihydrofolate reductase specific mRNA were also deter- mined in six mutant strains. The results are summarized in Table 1. The average half-life for the same mRNA in these mutants was essentially identical (2.7 to

- 9

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= 2.9 min.

2 I 1- 0 8 16 26

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FIGURE 3.-The decay of synthetic capacity for dihydrofolate reductase in wild-type D. pneumoniue after the addition of actinomycin D.

548 F. M. SIROTNAK A N D R. W. MC CUEN

TABLE 1

The decay of synthetic capacity for dihydrofolate reductase in mutant and wild-type D. pneumoniae after the addition of actinomycin D

Dihydrofolate reductase Specific mRNA Strain specific activity T t/2* (min.)

Wild-type 1 .o 2.9 amer-3 120.0 2.7 amer-20 3.0 2.9 amer-27 8.0 2.8 amer-51 10.0 3.0 amer-89 60.0 3.1 amer-20-51 60.0 2.9

* The average of two values obtained in separate experiments. A comparison of mutant and wild-type cells was made in each experiment.

3.1 min) to the value (2.9 min) obtained for the wild-type mRNA. The relative levels of dihydrofolate reductase in these strains are also given in Table 1.

Kinetics of mutant enzyme formation following genetic transfer of amer mu- tations to wild-type cells: With the development of a high efficiency D. pneu- moniae transformation procedure ( SIROTNAK 1971 ), it became possible to study the expression of an ame' mutational alteration following integration of the mu- tant genetic material into the wild-type dihydrofolate reductase structural gene. We compared the kinetics of expression of this quantitative mutant enzymic property to those observed following the transformation of a mutation in the amylomaltase gene which determines a simple qualitative enzymic effect (LACKS 1966; LACKS and HOTCHKISS 1960).

It was only feasible to examine the kinetics of expression following transforma- tion of a few amer mutations, since a large quantitative difference between steady-state enzyme synthesis in mutant and wild-type cells is necessary. Also, a single cross involving both amer and mal- mutations (amesmal- recipient x ame'mal+ donor) could not be utilized in a comparison between both transforma- tions because the mal- recipient strain transformed with a relatively low fre- quency. The data shown in Figure 4 were obtained following the transfer of the amer-3 mutation, which determines a 120-fold increase in dihydrofolate re- ductase level, and two closely linked mutations (ame'-20 and -51), from a re- combinant DNA donor, which jointly determine a 60-fold increase in enzyme level. The data are plotted as a difference between enzyme levels seen in parallel cultures of wild-type recipient cells exposed to mutant DNA or wild-type DNA. The relative amount of mutant enzyme, at each interval after expression is ini- tiated, was calculated as the fraction of the total enzyme observed at the steady- state level of synthesis in cultures treated with the amer DNA. Absolute steady- state levels achieved in the experiments shown were six-fold over the wild-type level with amer-3 DNA and three-fold greater with amer-20-5 1 DNA.

The period of DNA exposure used in these experiments permits the transfor- mation of all of the competent cells present (5-10% take up the amer mutation).

GENE REGULATION I N DIPLOCOCCUS

0.M (a)growth I

549

ik (b) dihydrofolate I G- 3 DNA (.)amer- 20- 51 DNA F

I , I , I I I I I

t (min.) 0 20 40 60 m

FIGURE 4.-The kinetics of expression of dihydrofolate reductase in amer transformants of wild-type D. pneumoniae.

Although the appropriate donor DNA segments are integrated into the recipient genome within a few minutes after transformation (Fox and HOTCHKISS 1960; LACKS 1962), no detectable synthesis of mutant dihydrofolate reductase occurred during this period. The amount of growth observed was also negligible. After add- ing nutrients and increasing the temperature to 37°C (zero time in Figure 4), both growth and new enzyme synthesis began immediately. Synthesis gradually increased for about 20 min, then exhibited a sharp rise until the steady-state level was approached. Although the achievement of the steady-state level of synthesis requires about 50 min, 80% of mutant enzyme synthesis occurs within a period of 15 min, i.e., from 20 to 35 min after the addition of nutrients. In addition, the steady-state level of synthesis was reached within one doubling in cell number. A nearly identical result is shown in Figure 4 for the expression following trans- formation with amer-20-51 DNA. Other results obtained with amer-89 DNA were also similar.

The kinetics of expression in the same system after the transformation of the mal+ qualitative property are shown in Fiigure 5. This was a parallel experiment done on the same day as the amer transformation. The recipient strain bears a mal- mutation (Mk2) in the amylomaltase gene, which inactivates the corre- sponding enzyme protein (LACKS 1966). Transformants obtained with the mal+ wild-type DNA synthesize a normal amylomaltase. Expression of the trans- formed property does not require an increase in the steady-state level of synthesis relative to that exhibited by the recipient mal- strain for the defective enzyme. The data plotted in Figure 5 represent the fraction at various times of the total

550

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F. M. SIROTNAK A N D R. W. MC CUEN

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FIGURE 5.-The kinetics of expression of amylomaltase in mal+ transformants of mal- D. pneumoniae.

G E N E REGULATION IN DIPLOCOCCUS 551

amylomaltase activity observed at steady-state level of synthesis. The pattern of expression is unlike that observed for dihydrofolate reductase in the amer trans- formants. Expression of the mal+ property is already underway when growth of the transformed culture is initiated and continues in a nearly linear fashion until the steady-state level is reached in about 25 min. This result is similar to that reported earlier for the same transformation (LACKS and HOTCHKISS 1960).

The expression of mutant levels of dihydrofolate reductase synthesis following the amer transformation is extremely sensitive to inhibition by actinomycin D or rifampacin. A concentration of 1 yg/ml of drug was used in these experiments. This amount of either drug has already been shown (Figure 1) to cause an im- mediate and almost complete inhibition of RNA synthesis in this organism. The plot of the difference in expression following treatment of the culture with wild- type and amer-3 DNA, in the presence and absence of actinomycin D, is shown in Figure 6. The addition of drug to parallel cultures in various stages of expres- sion resulted in an immediate cessation of expression. The same result was ob-

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FIGURE 7.-Co-~hromatography of H3 labelled amer-3 RNA and C14 labelled wild-type RNA. The ratio of H3 to C14 counts in the RNA sample was 1.87. The upper portion of the figure repre- sents the difference in the proportion of each label in the various fractions after values were normalized to 1.87.

552 F. M. S I R O T N A K A N D R . W. MC CUEN

tained with rifampacin, and with actinomycin D and rifampacin during expres- sion following transformation with amer-20-5 1 DNA.

CO-chromatography of pulse-labelled RNA from amer mutant and wild-type strains: If the effects of the amer structural gene mutations on dihydrofolate reductase synthesis are mediated at the level of transcription, i t should be possible, in the case of a very large quantitative difference, to demonstrate by physical means a difference in the amount of specific mRNA present in mutant cells.

A culture of the ame'3 strain, exhibiting a 120-fold increase in enzyme level, was pulse labelled with H3-uridine, chilled, and mixed with a chilled wild-type culture pulsed with C14-uridine. Following extraction, the RNA was chromato- graphed on a methylated albumin-kieseguhr column. The elution pattern ob- tained with a NaCl gradient for O.D. 260 and H3 and C'" labels is shown in Figure 7. The three O.D. 260 peaks correspond to 4s transfer RNA, and 16s and 23s ribosomal RNA. The radioactivity profiles for each label correspond very closely. However, as the upper portion of the figure illustrates, a difference exists in the fractionation range which corresponds to rapidly labelled mRNA ( HAYASHI et al. 1963; MARTIN 1963). Higher levels of the tritium label, corresponding to RNA from amer-3 cells, occur in this range. A similar result was obtained in three replicate experiments with individually labelled cultures from different

1.2

1.0

0 *= 3

v u

fraction number FIGURE 8.-Co-&romatogrsphy of wild-type RNA labelled separately with HS and 0 4 . The

ratio of H3 to C14 counts in the RNA sample was 1.55. The upper portion of the figure represents the difference in the proportion of each label in the various fractions after values were normalized to 1.55.

GENE REGULATION IN DIPLOCOCCUS 553

days. In three control experiments, separate cultures of wild-type cells were pulse-labeled with H3- and Q4-uridine. The chromatography of RNA from one of the pooled cultures is shown in Figure 8. No difference in the elution of each label was ever observed. These results appear to suggest a quantitative difference in the composition of the mRNA fraction from the amer-3 and wild-type cells.

DISC US S I O N

It seems clear that the amer mutations in the dihydrofolate reductase structural gene of D. pneumoniae, or at least the group examined here, do not alter the stability of the related mRNA. Previous data (SIROTNAK 1971) have also shown a lack of effect of these and related amer mutations on the turnover of the dihy- drofolate reductase protein itself. The effect of these mutations on the synthesis of dihydrofolate reductase, therefore, must be mediated at either the transcrip- tional or translational level of protein synthesis.

A direct effect on translation could conceivably occur, if the coding alteration, resulting from an ame' mutation, eliminated the requirement for a minor species of transfer RNA and increased the rate of translation of the entire cistron. Although this explanation is theoretically possible, it is difficult to evoke, exclu- sively, in the case of the amer group of mutations. First, in a structural gene hav- ing only about 150 translatable triplets ( the protein product is a single polypep- tide with an MW of 20,000, SIROTNAK and SALSER 1971), it is unlikely that the large number of ame' sites identified all affect the coding for a minor species of transfer RNA. Moreover, since many of the quantitative effects of the ame' mu- tations are enormous, minor species of tRNA elzcoded within the wild-type struc- tural gene would have to exist in exceptionally minute amounts. Differences in the relative amounts of major and minor tRNA species actually found in bacteria are apparently nowhere near those differences needed to satisfy this requirement (KELMERS, NOVELLI and STULBERG 1965; KELLOG et al. 1966; SOLL et al. 1966; SOLL, CHERAYEL and BOCK 1967).

A considerable amount of support for an effect on transcription by at least one mutation (ame'-3), and to a lesser extent, ame'-20 and -51 from a different genetic group (SIROTNAK 1970, 1971; SIROTNAK and HACHTEL 1969) was ob- tained in the present study. The expression of the amer-3 and amer-20-51 en- zymic properties following transformation of the wild-type was very effectively blocked by the addition of actinomycin D or rifampacin. The cessation in expres- sioE of the ame' property after the addition of drug was immediate and complete regardless of when the drug was added prior to achieving a steady-state level of synthesis. If the increase toward a new steady-state level did not require a con- tinuing increase in specific mRNA synthesis, then at some point followillg ini- tiation of expression, the increase in dihydrofolate reductase level would be at least partially insensitive to drug. The greater time required for the expression of the mutact dihydrofolate reductase levels in the amer transformants as compared to the mal+ property could be attributed. at least in part, to the necessity for in- creased specific mRNA synthesis.

554 F. M. SIROTNAK A N D R. W. M C C U E N

As more direct evidence for a transcriptional effect, strains bearing the ame’-3 mutation were found by co-chromatography of double-labeled RNA to synthesize measurably higher levels of mRNA. The increase in the ame’3 strain of RNA corresponding to mRNA is commensurate with the extremely high level (120- fold over the wild-type) of dihydrofolate reductase synthesized in this strain. This enzyme protein represents close to 1 % of the total protein synthesized in this strain ( SIROTNAK, HACHTEL and WILLIAMS 1969). Assuming this protein is only one of a number encoded in what is likely a polycistronic mRNA mole- cule, the difference in the amount of mRNA found in this strain is not surprising.

An explanation of these results in terms of an effect at the level of messenger RNA transcription is difficult to provide unless one presumes some type of regula- tory role for the dihydrofolate reductase gene. The idea that the gene itself may contain some sort of recognition site (operator-promoter?) seems untenable, since sites for amer mutation are involved in coding for the dihydrofolate reductase protein. The lack of any catalytic inactivation of dihydrofolate reductase by the amer mutation renders unlikely the more trivial possibility of an indirect effect, at least on a conventional form of regulatioc, due to the disruption of end-product (folate coenzyme) synthesis. An indirect effect on regulation could also occur if the ame‘ mutations rendered the dihydrofolate reductase protein more sensitive to a feedback inhibitor or some other ligand continually present internally. How- ever, one would expect the same effect when this enzymatic activity is shut down by the presence of a specific inhibitor like amethopterin. Theoretically, then, as more and more drug is added to a wild-type culture, the enzyme level in response to the consequent depletion of folate coenzyme should rise to the fully “dere- pressed” level. Clearly, all of the evidence presented, so far, does not support this idea. Moreover, actual measurements of dihydrofolate reductase have been made (MCCUEN and SIROTNAK, unpublished results) prior to and following the addi- tion of various amounts of amethopterin, which gradually reduce folate coenzyme pools and eventually growth (one to two hours). When the wild-type strain and mutants bearing enzyme with reduced drug binding affinity were examined in this way, enzyme levels were always unaltered in the presence of drug. Finally, the available genetic evidence suggests (SIROTNAK 1970) the lack of any regula- tory control on dihydrofolate reductase synthesis in the first place.

A possibility, still compatible with the available evidence, is that the dihydro- folate reductase protein has a regulatory function in addition to a catalytic func- tion. The amer mutations, then, are able to alter selectively the regulatory function. This interpretation could account for the sigmoidal expression kinetics obtained for the amer property following transformation (Figure 4). Although synthesis of the mutant enzyme occurs early following initiation of expression, the rate is low in transformants because the majority of the enzyme is still wild type. As sufficient mutant enzyme is synthesized, the regulation shifts, allowing the high level of synthesis of the amer donor strain. The existence of “bifunctional proteins” of this sort have been recently proposed by other workers (see refer- ences listed in the introduction). Moreover, mutations with effects similar to the amer mutations have also been described (FRAENKEL and BANERJEE 1970;

GENE REGULATION IN DIPLOCOCCUS 555

PARDEE et al. 1971; BANERJEE and FRAENKEL 1972). The manner by which the dihydrofolate reductase protein could function in this “self-regulatory” fashion is not readily evident at present.

The authors gratefully acknowledge the interest and support of DR. DORRIS J. HUTCHISON and the excellent technical assistance of MISS JOANNE M. GALLO. This work was supported in part by Grant CA 08748 from the National Cancer Institute.

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