The arrangement of genes 7 and 2 is consistant with a polar arrangement but gene 2 does seem to have a proper Shine-Dalgarno sequence [12]. In these cases the orfs for genes 12 and 5 do not have proper Shine-Dalgarno sequences associated with them (Table 2). Table 2 Base sequences at orfs of Φ2954 gene sequence 1 UAGGAAGUUUGAACC AUG GCUAGAAGAAUC 2 GCCGAGUGGCUCCGA AUG AAGGAUGACACU 3 UGCCAAGGGGUUAAU AUG UCAACCGCUCU 4 UCAAGGAAACCUUGU AUG AAGAUGUUACCG 5 GCCGGUUAAUCCGCG GUG AGCAAACAAGGC 6 CGACGACUCGGGAGU AUG CAACAGUAUCUG 7 GUAUGGGAGUGUAAA AUG GAUCUUAUUAAA 8 AACAAGGAGCAAGAA AUG GCUAAGCCACCC 9 UGGCAGGAGAUUCAU AUG UUCGCTAAAAGC 10 CGUAGUAGUGAAACC AUG AAUAAAGTTCTG 16 CUUCGGGUUGAGCAC AUG GCCCAUGCCAGA 12 AACAUCGCCGCUCUG

AUG GGUGCUGUAAAC 14 AGAGGUGUUUUCGAU AUG UUGAAAGUUCAG 15 CAUGAGGUCUUGCGA AUG AACACUUAUCAA Reverse genetics The cDNA copies of the genomic segments were inserted into a derivative

of plasmid pT7T319U (GenBank: U13870.1) that had the T7 RNA polymerase promoter selleck chemicals replaced by the promoter of SP6 RNA polymerase so that transcription would start efficiently at the terminal G of segments S and M. The fidelity of the cDNA constructs was tested by their activity in the production of live virus resulting from their electroporation into strain LM3313. LM3313 is a derivative of LM2489 carrying plasmid pLM2790 that expresses the SP6 RNA polymerase. Three different constructs of the L segment were utilized; they contained 5′ sequences beginning with the normal ACAA start, or with GACAA Z-IETD-FMK chemical structure or GCAA. Segments M and S were normal (Figs. 2 and 3). The cDNA plasmids are ColE1 derivatives and unable to replicate in pseudomonads. The constructs with ACAA or GACAA produced about six CUDC-907 purchase thousand plaques from 1010 cells while the construct of GCAA produced about one hundred plaques. The amount of transcript with ACAA would be expected to be much lower than that for GACAA, suggesting that its efficiency in plaque production would be greater than that for GACAA on a per molecule basis. While the phage resulting from the L segment with the normal 5′ sequence of ACAA behaved identically to that of wild type Φ2954, the phage with the GCAA sequence showed novel transcription behavior.

Whereas wild type Φ2954 nucleocapsids transcribe only genomic segments S and M in vitro; the mutant with GCAA instead of ACAA at the 5′ terminus transcribes all three genomic segments in vitro (Fig. Nitroxoline 5). The differences in template activity for genomic segment L as opposed to S and M is used by most members of the Cystoviridae to effect temporal regulation of transcription. In the case of Φ6 the L segment has the sequence GU at the 5′ end of the plus strand while S and M have GG. The polymerase favors G over U as the second nucleotide and it is proposed that the second base is the first to be paired during transcription [13]. A host protein, YajQ, is able to alter this selection so as to enable active transcription of the L segment upon entry into the host cell [4].