Article Figures & Data
Figures
- Fig. 1.
Kanr cassette insertion mutants. (A) Plasmid constructs. Positions of the Kanr cassette insertions are plotted in relation to landmark features of oriLyt. The name, insertion coordinate, and relative activity for each mutant are noted at the right. Features located on the oriLyt line above the mutants include the UL59 open reading frame and transcript (16), the 29-bp repeats (triangle), the Y block (hollow box), and the two large, imperfect dyad symmetries A and B (3). The core region is shaded and bound by dotted lines, and the flanking regions within which partially inactivated insertions are represented by a gradient. (B) Transient transfection assay of the Kanr cassette insertion constructs. The abilities of the constructs described in panel A to mediate DNA replication were tested as described in Materials and Methods (lanes 1 to 14).DpnI-resistant products of replication were detected by Southern blotting; a replica of the resulting autoradiogram is shown. The tested plasmids are indicated at the top of the panel. The marker (lane 16) contains 0.1 ng each of EcoRI-treated plasmids SP54, SP50, and pGEM7Zf(−). Plasmid SP50 (lane 15), the parent to most of the insertions, and the vector pGEM7Zf(−) (lane 17) were transfected in parallel as wild-type and negative standards, respectively, for comparison. (C) Transient assay of the Kanr cassette insertion constructs from which the insertion was deleted by PstI treatment, leaving a residualPstI linker insertion. Each plasmid was tested in duplicate. The autoradiogram is reproduced here. The deletion constructs, which correspond to the Kanr insertions described in panel A, are indicated at the top of each lane.
- Fig. 2.
Overlapping deletions across HCMV oriLyt. (A) A schematic of the PvuII (nt 89796)-to-KpnI (nt 94860) fragment encompassing oriLyt and deletion constructs. The deleted region of each plasmid is indicated by nucleotide coordinates and by a gap in the line corresponding to the position in the oriLyt core. The open box in the line of pYZ20+1r represents the reiterated dyad sequence (2). Relative replication efficiencies are given at the right; ND, none detected. (B) Quantitative replication assay. The indicated test plasmids were cotransfected with pSP50 or pSP54 and pGEM-7Zf(−) as described in Materials and Methods. An autoradiogram of the resulting Southern blot is reproduced here. Deletion mutants pSP90, pSP72-24, pLH13Δ, pLH34Δ, and pLH50Δ were cotransfected with pSP54 as the positive internal standard. The other deletion mutants were pSP54 derived, and SP50 was used for the internal wild-type comparison. The pSP68 sample was treated with DpnI plus EcoRI andHindIII because pSP68 lacks an EcoRI site, and thus the pSP50 internal standard migrated to the position of pGEM-7Zf(−). (C) Qualitative assay. The indicated test plasmids were assayed for replication competence by transient transfection as described in Materials and Methods. A representative autoradiogram is reproduced here; only the region of the autoradiogram containing replicated (rep’d) signals is shown.
- Fig. 3.
Replicator activities of insertion and deletion mutants relative to those of the wild type. Exterior deletions that impinge upon the oriLyt region (thick grey bar) progressively reduce activity (3, 22). The positions and relative replicator activities of insertions (closed triangles) and deletions (thin grey bars) are plotted. The trough in the activity plot defines the core region (cross-hatched rectangle). Essential regions I and II, defined by deletions that completely abrogated replicator activity (black rectangles I and II), and the intervening deletable segment (open rectangle D) are indicated.
- Fig. 4.
Mutations in essential region I. (A) A schematic of the mutations in essential region I. Essential region I is enlarged directly below a schematic of the oriLyt region highlighting landmark features. For each plasmid, the deleted sequence is indicated by nucleotide coordinates and by a gap in the line. Relative replication efficiencies estimated in the quantitative replication assay are noted at the right; ND, none detected. (B) Quantitative replication assay of the small deletions. For each test plasmid, pSP50 and pGEM-7Zf(−) were used as wild-type and negative internal standards, respectively. The relative replication efficiencies of each plasmid were measured as described in Materials and Methods and are indicated at the right of in panel A. Samples for lanes 14 to 17 were from a transfection experiment and blot separate from those for lanes 1 to 13.
Tables
- Table 1.
Deletion plasmids
Plasmid Central primer(s) Flanking primer(s) Site/vector Coordinates (nt) Reference or source pSP54 XbaI-NheI/pSP51 90504–94860 3 pSP50 KpnI/pSP38, pGEM 89795–94860 3 pYZ1 YUAO3 and -4 YUAO1 and -2 EcoRI-NotI/pSP54 Δ92227–92255 This paper pYZ3a YUAO7 and -8 YUAO1 and 2 EcoRI-NotI/pSP54 Δ92294–92322 This paper pYZ3′ YUAO7 and -8 YUAO1 and -2 NsiI-XcmI/pSP54 Δ92294–92322 This paper pYZ4 YUAO9 and -10 YUAO1 and -2 EcoRI-NotI/pSP54 Δ92338–92360 This paper pYZ5 YUAO11 and -12 YUAO1 and -2 EcoRI-NotI/pSP54 Δ92361–92390 This paper pYZ6 YUAO13 and -14 YUAO1 and -2 EcoRI-NotI/pSP54 Δ92227–92322 This paper pYZ7 YUAO15 and -16 YUAO1 and -2 EcoRI-NotI/pSP54 Δ92400–92420 This paper pYZ8 YUAO17 and -18 YUAO1 and -2 EcoRI-NotI/pSP54 Δ92439–92453 This paper pYZ9 YUAO19 and -20 YUAO1 and -2 NsiI-XcmI/pSP54 Δ92471–92492 17 pYZ9R RP1 and RP2 NsiI-XcmI/pSP54 Δ92471–92492 17 pYZ9R2 9RP1 and 9RP2 NsiI-XcmI/pSP54 Δ92471–92492 17 pYZ10 YUAO21 and -22 YUAO1 and -2 EcoRI-NotI/pSP54 Δ92506–92516 This paper pYZ11 YUAO23 and -24 YUAO1 and -2 EcoRI-NotI/pSP54 Δ92517–92533 This paper pYZ12 YUAO25 and -26 YUAO1 and -2 EcoRI-NotI/pSP54 Δ92542–92573 This paper pYZ13 YUAO31 and -32 YUAO29 and -30 BsmI-EcoRI/pSP54 Δ91498–91697 This paper pYZ14 YUAO33 and -34 YUAO29 and -30 BsmI-EcoRI/pSP54 Δ91598–91797 This paper pYZ15 YUAO35 and -36 YUAO29 and -30 BsmI-EcoRI/pSP54 Δ91698–91897 This paper pYZ15L PstI-XcmI/pYZ14/pYZ15 Δ91598–91897 This paper pYZ15R PstI-XcmI/pYZ15/pYZ16 Δ91698–91997 This paper pYZ15LR PstI-XcmI/pYZ14/pYZ16 Δ91598–91997 This paper pYZ16 YUAO37 and -38 YUAO29 and -30 BsmI-EcoRI/pSP54 Δ91798–91997 This paper pYZ17 YUAO39 and -40 YUAO29 and -30 BsmI-EcoRI/pSP54 Δ91898–92097 This paper pYZ18 YUAO41 YUAO29 BsmI-EcoRI/pSP54 Δ91998–92219 This paper pYZ18L PstI-XcmI/pYZ17/pYZ18 Δ91898–92219 This paper pYZ18LL PstI-XcmI/pYZ16/pYZ18 Δ91798–92219 This paper pSP68 EcoRI-ExoIII/pSP54 Δ92111–92391 3 pYZ19 YUAO43 and -44 YUAO1 and 92654 EcoRI-XcmI/pSP54 Δ92400–92573 This paper pSP90 NotI,ExoIII/pSP61 Δ92454–92979 This paper pSP72-24 SphI,ExoIII/pSP62 Δ92574–92979 This paper pYZ20 NotI-BstXI/pSP54 Δ92887–93145 This paper pYZ22 BstXI-BamHI/pSP54 Δ93142–93361 This paper pLH13Δ PstI/pSP50Kan13 Δ93163–93561 This paper pLH34Δ PstI/pSP50Kan34 Δ93701–94370 This paper pLH50Δ PstI/pSP50Kan50 Δ93561–94370 This paper ↵a pYZ3 contains a single base substitution in the remaining 29-bp repeat as indicated at the bottom of Fig. 4A.
- Table 2.
Oligonucleotides used in mutagenesis
Oligo- nucleo- tide Sequence Coordinates (nt) YUAO1 5′-GGCTTCTCCGTCTACCGG-3′ 91963–91970 YUAO2 5′-CTCGCGCTCCCTAGGTGC-3′ 92892–92909 YUAO3 5′-CACGCATACGCCGTATGTCCGGAATTCC-3′ 92209–92265 YUAO4 5′-GGACATACGGCGTATGCGTGCGTCATCT-3′ 92217–92273 YUAO7 5′-AACGGACTGATCAGAGATGATTTCCGCC-3′ 92277–92332 YUAO8 5′-TCATCTCTGATCAGTCCGTTTTACGTAT-3′ 92284–92340 YUAO9 5′-GCCCCTACGGCGTAAAACGGACTGATGA-3′ 92320–92370 YUAO10 5′-CCGTTTTACGCCGTAGGGGCGGAGCCTA-3′ 92328–92378 YUAO11 5′-TATGCATCCTGGCGTCGCCTAGCATCCG-3′ 92343–92400 YUAO12 5′-AGGCGACGCCAGGATGCATACCCTATAT-3′ 92351–92408 YUAO13 5′-AACGGACTGACCGTATGTCCGGAATTCC-3′ 92209–92332 YUAO14 5′-GGACATACGGTCAGTCCGTTTTACGTAT-3′ 92217–92340 YUAO15 5′-GTTTCGTTAGCTGCAGATGCATCCTAGGGGTGG-3′ 92383–92430 YUAO16 5′-TAGGATGCATCTGCAGCTAACGAAACGTTCTAC-3′ 92390–92437 YUAO17 5′-GGTTCCCGTTCTGCAGCGTAGAACGTTTCGTTA-3′ 92422–92463 YUAO18 5′-ACGTTCTACGCTGCAGAACGGGAACCACCGTAA-3′ 92429–92470 YUAO19 5′-CGGAGGAGAACTGCAGTTACGGTGGTTCCCGTT-3′ 92454–92502 YUAO20 5′-ACCACCGTAACTGCAGTTCTCCTCCGGAACCGG-3′ 92461–92509 YUAO21 5′-GTAAAAATTTCTGCAGTTCCGGAGGAGAAGGGG-3′ 92489–92526 YUAO22 5′-TCCTCCGGAACTGCAGAAATTTTTACCAAATTT-3′ 92496–92533 YUAO23 5′-ATGGTTGCCCCTGCAGGCCCCCCCCGGTTCCGG-3′ 92500–92543 YUAO24 5′-CGGGGGGGGCCTGCAGGGGCAACCATGATTTCC-3′ 92507–92550 YUAO25 5′-GACTGCGCATCTGCAGGGTTGCCCAAATTTGGT-3′ 92525–92583 YUAO26 5′-TTGGGCAACCCTGCAGATGCGCAGTCGGGGCGA-3′ 92532–92590 YUAO29 5′-TGTAACCTGAAACCGCCGTG-3′ 91381–91400 YUAO30 5′-CCGTATGTCCGGAATTCCAC-3′ 92207–92226 YUAO31 5′-GACGTTGGCACTGCAGCGATCGCCACATTCGAT-3′ 91481–91707 YUAO32 5′-GTGGCGATCGCTGCAGTGCCAACGTCATAATCA-3′ 91488–91714 YUAO33 5′-ATATGGCTACCTGCAGTGCCTGTTCTTATGCCG-3′ 91581–91807 YUAO34 5′-AGAACAGGCACTGCAGGTAGCCATATCCGCTTA-3′ 91588–91814 YUAO35 5′-CGTACAAGGGCTGCAGTCTGGCACCGCCTCTTG-3′ 91681–91907 YUAO36 5′-CGGTGCCAGACTGCAGCCCTTGTACGGAAATTT-3′ 91688–91914 YUAO37 5′-AGCGTCTACGCTGCAGACGTAATGGGTGTGGCT-3′ 91781–92007 YUAO38 5′-CCCATTACGTCTGCAGCGTAGACGCTACTCCCG-3′ 91788–92014 YUAO39 5′-ACAGAGGAAGCTGCAGGTGGAGTCTAGGGAGGG-3′ 91881–92107 YUAO40 5′-TAGACTCCACCTGCAGCTTCCTCTGTTTTGGCC-3′ 91888–92114 YUAO41 5′-TCCGGAATTCCTGCAGTCAGCAGGTGTATATTT-3′ 91981–92219 YUAO42 5′-CACCTGCTGACTGCAGGAATTCCGGACATACGG-3′ 91988–92226 YUAO43 5′-GACTGCGCATCTGCAGATGCATCCTAGGGGTGG-3′ 92383–92583 YUAO44 5′-TAGGATGCATCTGCAGATGCGCAGTCGGGGCGA-3′ 92390–92590 92654 5′-CGGCGCATGCGCACTCGAGT-3′ 92635–92654 RP1 5′-TTCTAGAACCGCTGGATGCA-3′ RP2 5′-TCCAGCGGTTCTAGAATGCA-3′ 9RP1 5′-TTCTAGACGCGATGCA-3′ 9RP2 5′-TCGCGTCTAGAATGCA-3′
