Proc. Nati. Acad. Sci. USA Vol. 73, No. 10, pp. 3519-3523, October 1976

Biochemistry

Mapping of in vivo messenger RNAs for bacteriophage 4X-174 (agarose gel electrophoresis/restriction endonucleases/DNA-RNA hybridization)

MASAKI HAYASHI, FRANK K. FUJIMURA, AND MARIE HAYASHI Department of Biology, University of California, San Diego, La Jolla, Calif. 92093

Communicated by E. Peter Geiduschek, August 5, 1976

In vivo messenger RNA for bacteriophage ABSTRACT XX-174 was fractionated in agarose gels into a number of discrete species ranging in size from 0.23 X 106 to 2.3 X 108 daltons. These RNA species were eluted from the gels and hybridized to specific fragments derived from XX-174 replicative form DNA by cleavage with restriction enzymes. A map of the orientation of in vivo messenger RNAs with respect to the bacteriophage genetic map was constructed. This map indicated that initiation of messenger RNA occurred before gene B, gene C or D, and probably before gene A and that termination occurred after genes E, F, G, and H. Termination of transcription at any particular site appeared not to be entirely effective such that a number of overlapping transcripts with the same 5'-terminus was observed. Messenger RNA for gene A seemed to be relatively unstable.

Previous studies on the transcription of bacteriophage XX-174 specific messenger RNA (mRNA) indicated that the mRNAs can be fractionated into many species of discrete size by electrophoresis in polyacrylamide gels. For example, Hayashi and Hayashi (1) showed that at least 10 identifiable classes of XX-174 mRNA ranging in size from 0.2 X 106 to 1.8 X 106 daltons (which corresponds to full genome length) can be isolated from pulse-labeled cells infected with wild-type phage. RNA larger than the full genome length frequently can be seen in the gel pattern. By comparison of the molecular weights of XX-174 coded proteins and the sizes of the mRNAs, they concluded that (a) most of the kX-174 mRNAs are polycistronic; (b) some cistrons must be transcribed into more than one size class of RNA, and (c) some RNAs must be the result of more than one round of transcription of the circular, replicative form DNA (RF DNA). These conclusions have been confirmed by Clements and Sinsheimer (2). Furthermore, they claimed that they identified 25 size classes of mRNA. In this paper, we have isolated the different size classes of RNA and hybridized these RNAs to specific DNA fragments derived by cleavage of RF DNA with restriction enzymes to determine the cistrons encoded in each size class of RNA. From these results, a tentative arrangement of XX-174 mRNAs along its genetic map was constructed. Initiation sites of transcription exist before gene B, gene C or D, and probably gene A. RNAs initiated from these sites can terminate after gene E, F, G, or H.

METHODS Electrophoresis of kX-174 RNA. XX-174 mRNA was prepared from phage infected cells as described previously (3). Electrophoresis was performed in a 1.5% agarose gel containing 6 M urea according to the procedure of Rosen et al. (4). Gels were fractionated into 1 mm slices which were soaked overnight in a toluene based scintillation fluid containing 5% Protosol; Abbreviations: R.N.S.C., relative number of sequence copies; RF, *replicative form.

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then radioactivity was assayed in a liquid scintillation counter. Hybridization of Each Size Class of RNA to Specific Fragments of RF DNA Derived by Cleavage of RF DNA with Restriction Enzymes. The RNA sample was prepared and electrophoresed in a 1.5% agarose gel containing 6 M urea. The fractionated gel slices were suspended in 0.125 M K-PO4 buffer (pH 6.5, 0.5 ml/1 mm slice) and shaken overnight at room temperature. Prior to hybridization, the RNA was diluted 50-fold with distilled water and heated at 100° for 10 min. DNA fragments were prepared by digesting XX-174 RF DNA with the appropriate restriction enzymes (Hindll, Hap II, Hpa I or Hin. H-I). Fragments were separated by electrophoresis in 2% agarose gels in E buffer (5). The location of each fragment in the gels was detected by UV light after immersion of the gel in a solution containing 0.5 Ag/ml of ethidium bromide in water. The region of gel containing each fragment was excised and dissolved in saturated KI solution (6). Agarose and KI were eliminated by passage of the solution through a hydroxyapatite column (7). Each heat-denatured fragment equivalent to 0.4 ,ug of RF DNA was mixed with RNA in 0.2 ml of hybridization mixture containing 0.125 M K-PO4 buffer (pH 6.5) and incubated at 650 for 12 hr. After cooling to 370, 30 jig/ml of RNase A (ribonuclease I, ribonucleate 3'-pyrimidino-oligonucleotidohydrolase, EC 3.1.4.22) and 15 units/ml of RNase T1 (guanyloribonuclease, ribonucleate 3'-guanylooligonucleotidohydrolase, EC 3.1.4.8) were added to the mixture which was then incubated for 30 min at 37'. DNA-RNA hybrids were precipitated with ice-cold 6% trichloroacetic acid onto a glass filter and assayed for radioactivity. Conventional membrane filter methods were not used for hybridization because quantitative trapping of small DNA fragments was difficult. Hap II-1 Hpa 1-2 was the second largest fragment derived by digestion of the Hap II-1 fragment with Hpa I enzyme. RESULTS AND DISCUSSION Orientation of Size Classes of RNA on the q5X-174 Genome. Total kX-174 RNA labeled with [3H]uridine and isolated as described in Methods was electrophoresed in an agarose gel. As shown in Fig. 1, the RNA was separated into about 14 peaks. Previously we showed by pulse (30 s with [3H]uridine at 370) and pulse-chase experiments that there was no precursor and product relationship among these RNA size classes (1). Therefore, these RNAs are considered to be primary transcripts, although undetectably fast processing and/or rapid degradation near the termini of the RNAs cannot be completely ruled out. [However, if processing does occur, it is not due to RNase III or RNase I because identical size classes of RNA were isolated from infected cells deficient in these nuclease activities [M. N. Hayashi and M. Hayashi, manuscript in preparation.] Each size class of mRNA was eluted from gels and hybridized

Biochemistry: Hayashi et al.

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CISTRON H HindI.

Proc. Natl. Acad. Sci. USA 73 (1976)

4

CD,

B

A

E

G

F

s

3

i

i

1

IHap 1l-1,Hpa 1-2 I 0N

H Relative No. of Molecules

2 Hin HI-3

e

0.3

I

1-

1.0

m

i *,

22 2.3 3.1 3.0 5.2 9.5

0

III I

m 'I

lx

9.0 O

l

A

FIG. 2. The orientation map of mRNAs on the OX-174 genome. Sizes and the positions of RF DNA fragments on the genome were taken from the results of Lee and Sinsheimer (9). HindUI-1ito 5, Hayashi and Hayashi (5); Hin HI-3 Hap/II-l and F. Fujimura, HapII-1 Hpa I-2, (unpublished results). Direction of transcription is from left (5'-terminus) to right (3'-terminus). The R.N.S.C. ratios of Hap II-1 Hpa I-2 to HindII-5 for peak 0 RNA were 1.85, 1.90, and 1.82 in three independent experiments which imply that the redundancy at the termini of this RNA covered the Hap II-1/Hpa 1-2 region but did not extend into the HindII-5 region. For unknown reasons, the R.N.S.Cs. of peak III to HindII-5, peak IV to Hindll-2 and to Hindll-5, and peak V to HindII-2 (Table 1) were ambiguous. The tentative map positions of these three RNAs were assigned to be consistent with both the hybridization data and the estimated molecular weights. Message A is an unstable functional gene A message (see text). The relative number of molecules of each RNA species was calculated with respect to peak I. promoter site in the gene C to gene D region which is consistent

with published data (12). Fig. 2 also shows that an initiation site exists before gene B. This initiation site was not detected in the in vitro experiments of Chen et al. (12). The relative number of molecules in each size class of RNA was calculated (average of five independent experiments) and is shown in Fig. 2. The ratio of the number of RNA molecules starting before gene B to the number of those starting before gene CID is 1.06 (17.8/16.8) which indicates that in vvo mRNAs are initiated at these two sites with almost equal frequencies. No initiation sites before gene A or gene H can be determined from Fig. 2. It is possible that transcription of peak I RNA is initiated at one of these sites. Another possibility is that the synthesis of functional-unstable gene A mRNA is initiated at a promoter before gene A. The functional and unstable A message also presents a further enigma. It could have been synthesized as a monocistronic message or it could have existed at the5'-end(s) of some message(s). Rapid degradation of the _

10 _

4

U~~~~~~~~~~~ Jo X1

ark Si

E. 0

10

20

30

60 50 40 fraction no.

70

80

FIG. 3. RNAs synthesized in cells infected with a polar F mutant. E. coli HF4704 infected with a gene F amber mutant (H244) and labeled with [3H]uridine as described in Fig. 1 was mixed with E. coli HF4704 infected with wild-type phage and labeled with four [14C] mRNA was 1X-174 1.25 each). ribonucleosides (2.5 -1i/ml, 1g/ml isolated and electrophoresed as in Fig. 1. * * [3H]H244mRNA, 0-- wild-type [24C]mRNA.

polycistronic RNA could have resulted in the formation of some of the messages mapped in Fig. 2 (such as those with their 5'-ends before gene B: II, IV, V, or IX) or perhaps in the formation of some other messages which are unstable and would not be detectable as discrete peaks by gel electrophoresis. Synthesis of functional gene A message from promoters closer to the 5'-end (such as before gene H) is considered unlikely in view of the polarity discussed above, No published information is available concerning termination sites in vitro for XX-174 transcription. Our in doo results would predict that there may be four termination sites located at the ends of genes E, F, G, and H. However, termination at these sites is not stringent. Messages starting before gene B (peaks II, IV, V, IX) could terminate at any of these sites. Similarly, messages starting before gene C or D (peaks 0, III, VI) could terminate at the end of either gene E, F, or H. No mRNA initiating before gene CID and terminating at the end of gene G was observed, but the possibility exists that the transcript was undetected. The in vivo efficiency of termination of a XX-174 transcript appears to depend'upon both the termination site and the initiation site. About 54% of the RNA molecules initiated before gene B or before gene CID terminated after gene E. Sixty-seven percent of the transcripts initiated before gene CID and read through gene F terminated near the end of gene F, whereas only 36% of the corresponding transcripts initiated before gene B did so. No termination was seen after gene G for transcripts initiated before gene CID; the efficiency of termination after gene G for those RNA molecules initiated before gene B and read through gene G were 58%. Termination at the end of gene H seemed most stringent, as 94% of all RNA molecules initiated at gene B and at gene CID and read through gene H terminated there. The existence of a strong termination site at the end of gene H is consistent with the polarity effect of NH2-terminal amber mutations in gene F on genes G and H, but not on gene A (1, 13). The effective gene A message on

termination of transcripts after gene H also suggests that a promoter site must exist before gene A. The correlation of the termini of the 4X-174 mRNAs in Fig. 2 with initiation and termination sites in dvuo depends upon the

Proc. Nati. Acad. Sci. USA 73 (1976)

Biochemistry: Hayashi et al.

3521

to several RF DNA fragments prepared by cleavage with restriction enzymes. The relative number of sequence copies (R.N.S.C.) of RNA complementary to a particular fragment, A, is calculated in the following way:

R.N.S.C.

=

fragment A counts hybridized counts total input to size of total genome size of fragment A

The R.N.S.C. represents the relative frequency of an RNA sequence complementary to the specific region of the genome covered by the DNA fragment. The hybridization results are summarized in Table 1. Peak I RNA can be hybridized to all the DNA fragments used in this experiment. The R.N.S.C. values for all the fragments are identical, indicating that peak I RNA is the result of one round of transcription of RF DNA. The estimated molecular weight of peak 1 (1.8 X 106) agrees with this result. The estimated size of peak 0 suggests that it must contain a length of RNA greater than one genome, as shown by the hybridization data. Peak 0 RNA hybridized to all the DNA fragments but the R.N.S.C. of the Hap II-1 Hpa 1-2 fragment is about twice the R.N.S.Cs. to the other fragments. This result implies that the peak 0 RNA contains two copies of the sequences complementary to the Hap II-1 Hpa 1-2 fragment for every copy of the sequences complementary to the rest of the genome. The R.N.S.Cs. of peaks 0 and I for all fragments indicated that the pulse period (5 min) was sufficient to uniformly label all transcripts with almost equal specific activities. Because the positions of the DNA fragments on the genome are known, it is possible to construct an orientation map of each size class of 4X-174 mRNA from the results in Table 1. This is shown in Fig. 2. The R.N.S.Cs. of minor peaks VII, VIII, X, XI, and XII are ambiguous (Table 1) and the RNAs in these peaks may consist of a mixture of several species. Due to this complication, it is not possible to map the specificities of these RNAs. Our positioning of the ends of transcripts in Fig. 2 is somewhat arbitrary. Exact positions have not been determined. Each RNA species was aligned with the genome to satisfy both the hybridization data and the estimated molecular weight. The respective positions of the 5'- and the 3-ends of each RNA were assumed to be before the NH2-terminal end and after the COOH-terminal end of the cistrons. Because of the relatively small size of cistrons C and D, there is some uncertainty about the positions of the 5'-ends of peaks 0, III, and VI. The location of the ends of peak I RNA is not known. The RNA in peak XIII contains gene D message, because the gene D protein was synthesized in a translational system in dttro with this RNA as the message (Y. Hayashi and M. Hayashi, unpublished results). We tentatively assigned peak XIII to two possible positions as shown in Fig. 2. Functional and Unstable Gene A Message. Previously Vanderbilt et al. (13) showed that amber mutations of S13 (a phage closely related to