1. A B D C E 5 kb 2 kb 1 2 3 4 Copies per 1,000 GAPDH copies. UL US TRL
IRL
IRS
TRS
UL49.5
UL46 (VP11/12)
UL50
UL (VP13/14)
UL49 (VP22)
UL (VP16)
UL45
156
155
154
152
151
153 A
UL44 (gC)
144
143
157
158
196
195
198
197
200
199
150
149 B
5 kb
2 kb
UL49
UL47
UL46
UL48 1 2 3 4 C D E
Copies per 1,000 GAPDH copies.
Primer set
Gene
UL46 (VP11/12)
UL47 (VP13/14)
UL48 (VP16)
UL49 (VP22)
UL44 (gC)
ICP4
UL46-47
UL47-48
UL48-49
28 h
0.44
1.91
5.95
5.07
0.10
0.16
0.00
53 h
1.28
2.65
13.09
12.85
0.26
0.37
0.17
0.02
0.01
74 h
3.11
6.07
32.56
30.33
1.42
1.74
0.55
0.11
0.04
100 h
9.91
19.93
88.36
87.10
5.21
5.41
2.95
0.28
1.17
124 h
17.00
37.97
173.15
182.10
24.95
14.37
2.38
0.32
1.16
148 h
22.09
60.01
247.73
222.54
30.20
21.50
2.70
0.36
0.99

2. Fig. S3. Northern blot and RT-qPCR analysis of polycistronic transcripts within the MDV UL46-UL49 locus in vitro. (A) Schematic representation of the genomic structure of MDV showing the locations of the TRL, TRS, UL, US, IRL, and IRS regions. It has been previously suggested that multiple transcripts are produced from this locus during in vitro replication using northern blotting analysis (Yanagida et al., 1993) and these transcripts are shown here, including locations of primers used in this study. (B) For generation of DIG-labeled probes specific for UL49, UL48, UL47eGFP, or UL46, primer combinations of , , , and were used, respectively. (C) Northern blot analyses using specific probes in 1) control (CEC) cells or 2) vUL17eGFP-, 3) vPCMVUL47eGFP-, or 4) vPCMVUL48-infected CECs at 7 days pi. Location of ribosomal RNAs (~2 and ~5 kb) are indicated. (D) Ethidium bromide stain of total RNA prior to transfer to membrane and northern blotting analyses (representative image). (E) Transcripts for UL46, UL47, UL48, UL49, and polycistronic transcripts encoding UL46-UL47, UL47-UL48, and UL48-UL49 from vUL47eGFP-infected CEC cultures over 6 days. The number of transcripts per 103 GAPDH transcripts is shown for each primer combination and shows that very little of the transcripts amplified from transcript specific primers are due to contaminating polycistronic transcripts.