 takes place only at specific sites on the viral RNA templates.
When CTP was added as the sole substrate, it was expected that CMP would be maximally incorporated into the complementary dinucleotide primer AG because C is complementary to G located at the third position from the 3' termini of template RNAs.
The AG-dependent incorporation of [a-3'P]CTP was the highest among all the reactions examined in this study (Table  1 and Fig. 2). The CG-and UG-dependent incorporation of CTP was 30-40% that of AG, both CG and UG being complementary to the 3"terminal UC sequence only at the 3'terminal G residue, suggesting that the primer activities of dinucleotides depend on the complementarity to viral RNA templates. GG exhibited an intermediate activity because of additional UG pairing. Other dinucleotides showing no complementarity to the UC sequence were virtually inactive or showed less than 1%, if any, the activity of AG.
Guanine nucleotide could be transcribed from viral RNAs at positions 2 and 4 of the 3"terminal sequences, and the active primers might be NA (N represents A, G, C, or U) and GC, respectively. The experimental results indicate that GMP incorporation is high for the three species of dinucleotides, GC, AA, and UA (see Table 1 and Fig. 2). Sequence analysis indicated that GC directed the formation of GCG (for details, see Figs. 5 and 6 ), which is fully complementary to viral RNAs between positions 2 and 4. The other three dinucleotides with the C residue at their 3' ends, i.e. AC, CC, and UC, exhibited 10-20% the activity of GC. Again, these low levels of activity might be due to low levels of primer template pairing. All the dinucleotides lacking the complementarity to template RNA  Table 1 at the 3' bases of primers were virtually inactive. Accordingly, AA and UA, both being complementary at their 3' termini with the 3"terminal U residue of all eight viral RNA segments, directed the formation of AAG and UAG by transcribing the C residue at the second position.
Adenine nucleotide is complementary to positions 1 and 4-7 of viral RNA. Among the dinucleotides fully hybridizable to viral RNA at sites prior to each of the template U residues, GC was the only active acceptor for AMP incorporation (see Fig. 2), all the others including CG, which is complementary to the GC sequence located between the third and fourth nucleotides from the 3' termini, being virtually inactive as primers (Table 1). This observation suggests that the primer activities of dinucleotides also depend on the binding positions relative to viral RNA templates and that the transcription initiation takes place only near the extreme ends of viral RNA and prior to the fourth nucleotide from the 3' termini. In agreement with this hypothesis, UMP was not incorporated into any of the dinucleotide primers.
Transcription Initiation Positions on Viral RNA-Among the initiation reactions primed by the best-match dinucleotides, the AG-primed incorporation of CMP (initiation at position 3) was about 13-fold higher than the GC-primed incoqoration of GMP (G initiation at position 4) and about 46-fold higher than the GC-primed incorporation of AMP (A initiation at position 4) (see Table 1 and Fig. 2A). CG was inactive as a primer for AMP incorporation (initiation at position 5). These observations together suggest that among the three positions, i.e. positions 3-5 from the 3' termini of viral RNA segments, the transcription initiation was maximum for position 3 (see Fig. 2 A ). The level was about 10-20% (combined value for GC-dependent incorporation of GMP and AMP) for position 4 and less than 1% for position 5.
In an attempt to determine the transcription initiation activity at position 2, the following two systems were compared AAand UA-dependent incorporation of GMP (initiation at position 2) and CG-and UG-dependent incorporation of CMP (initiation at position 3). These four active dinucleotides hybridize only at their 3' nucleotides with viral RNAs. The level of GMP incorporation was 10-20% the level of CMP incorporation (Fig. 2B), suggesting that the level of transcription initiation at position 2 is 10-20% of the level at position 3. This position effect for efficient initiation of transcription is discussed later.
Priming Activity-Dinucleotide Sequence Relationship-Up to this point, the priming activities of dinucleoside monophosphates with various sequences were compared at a fixed concentration, i.e. 0.24 mM, which is higher than the K,,, value (0.05-0.10 mM) for ApG-primed initiation. It is, however, expected that the priming activities of the dinucleotides with low affinity to the templates might be increased by adding higher concentrations of the respective dinucleotides. To test this possibility, we examined the effect of variation in the primer concentration on the initiation of transcription. Fig. 3 shows the results for AG-, GU-, CC-, and AA-dependent CMP incorporation (initiation at position 3), and both the K,,, and V,,, values are summarized in Table 2. The priming activity of AG was saturated above 1 mM, whereas those of GU and CC continued to increase within the range examined, i.e. up to 2 mM for GU and 4 mM for CC. Although the priming activity of AA at 0.24 mM was negligible, it exhibited low activity at 5 mM. Even with saturated concentrations of these noncomplementary dinucleotides, however, the rate of transcription initiation is not as high as that for AG, and the V,, values are much lower than that for AG (data not shown). Fig. 4 shows the effect of variation in the primer concentration for GC-, AA-, AC-, and GU-dependent incorporation of GMP (initiation at position 2). The rates of all these reactions increased linearly up to 0.5 mM for the GC-and AA-dependent reactions, 2 mM for the AC-dependent reaction, and 4 mM for the GU-dependent reaction. Again, a small but significant level of GMP incorporation was found for GU at 5 mM, of which the priming activity was negligible at 0.24 mM (see Table 1). These observations together indicate that the priming activity of dinucleotides is correlated with the extent of the pairing with viral RNA dinucleotides fully hybridizable to viral RNA exhibit the highest activity; dinucleotides which hybridize only at the 3'-proximal bases exhibit intermediate activity; and dinucleotides which hybridize only at the 5'proximal bases and those showing no complementarity exhibit low activity, if any.
Sequence of Initiated Oligonucleotides-The sequences of the initiated oligonucleotides were determined based on the cleavage patterns with various sequence-specific nucleases. For this purpose, the initiated oligonucleotides were separated from labeled substrates by electrophoresis on polyacrylamide gels and isolated by eluting the gel bands. After nuclease treatment, the cleavage products were identified by electrophoresis on either polyacrylamide gels or DE81 paper. Fig. 5 shows the electrophoretic patterns on a 20% polyacrylamide gel of the major initiated oligonucleotides. Based on the nuclease sensitivity patterns, the products of the AG-and CG-primed CMP polymerization reactions were found to be AGC and CGC, respectively. The products on GC-, AC-, and AA-dependent polymerization of GMP were GCG, ACG, and AAG, respectively. AMP was maximally incorporated in the presence of GC. The main product in this reaction was trinucleotide GCA. The sequences were confirmed by electrophoresis on DE81 paper, as shown in Fig. 6. These results together support the above conclusions as to the position effect of transcription initiation on viral RNA templates and the sequence requirement of dinucleotides for the priming activity.

MATERIALS AND mTHODS
The virus was grown in the allantoic sacs of 10-day-old embryonated eggs for 48 hr at 3 5. 5'C.
Virions were precipitated from the allantoic sacs after stirring for 3 hr at 4'C in the presence of 8% polyethyleneglycol 6000 and then purified by centrifugation through a sucrose step gradient (10). The purified virions were suspended in 10 mM TriS-RC1 (pH 7.8 at 4*C) containing 1 mM DTT and 20% (w/v) glycerol, and stored at -80°C until use. Chemicals and Enz es: Dinucleoside monophosphates were products of pwchased from Amersham, while unlabeled ribonucleoside 5"triphosphates =%macia.
'€I-andy%-labeled  C (2,8). After incubation, 5 111 of an RNA added. The mixture was heated for 2 mi" at 70'C and then directly sample buffer (75% formamide, 20% glycerol, 0.2% SDS and 0.01% BPBJ was carried out in 30% polyacrylamide gel containing 7 M urea. Gel8 were subjected to polyacrylamide gel electrophoresis. Electrophoresis was band was cut out and counted with a liquid scintillation spectrometer. exposed to X-ray films and the radioactivity aesociated with each gel OliqonUCleotide analysis: Labeled oligonucleotides were eluted from the gel bands and lyophilized to dryness. After resoluhilization in H~O, the samples were treated with various nucleases. The products were analyzed by electrophoresis on either 30% polyacrylamide gel (11) or DE81 paper (12). The paper electrophoresis was performed with a pyridine-acetate solution.
Transcription by influenza virus-