Transiently transfected MEL cells do not reveal transcriptional interference. Shown are dot plots; green fluorescent intensity is on the x axis on a 4-decade log scale, and the yellow fluorescent intensity is on the y axis, also on a 4-decade log scale. Each dot represents one of 20,000 cells assayed; >95% remain untransfected, a percentage confirmed by cotransfection with pCMV-βgal (data not shown). Quadrants divide expressing and nonexpressing cells; quadrant lines are drawn to be above the level of 99% of cells with no construct. G, single-gene construct containing a single GFP-containing transcription unit under the same promoter and polyadenylation signal as diagrammed in Fig. 1; Y, construct with single YFP-containing transcription unit; D, two-gene divergent construct; C, two-gene convergent construct; YG, two-gene tandem construct; GY, two-gene tandem construct.

RMCE introduces constructs into the genome in two orientations. (A) Sample autoradiogram of Southern analyses used to determine the orientation of two-gene constructs integrated into the RL5 genomic location. Shown are the PstI-digested genomic DNAs of eight tandem YG clones that were probed with a radiolabeled PCR product recognizing both GFP and YFP equivalently. M denotes the lane containing a radiolabeled 1-kb ladder. Lanes A show clones assigned to orientation A based on the pattern of the fragments (1.1 and 6.1 kb, as indicated on the left). Lanes B show clones assigned to orientation B (with 2.5- and 4.7-kb fragments). A/B are mixed clones that appear to have cells with both orientations; only approximately 11% of the clones were found to be either aberrant or mixed, and these were excluded from further analyses. (B) Diagrammatic representation of the derivation of the PstI restriction fragments that are diagnostic for either orientation A or orientation B with tandem YG integrated into the RL5 genome (thick line). Other diagram conventions are the same as in Fig. 1.

Genomic orientation modulates transcriptional interference in integrated tandem constructs. (A) A pair of bar graphs show the relative fluorescence for the single gene and the tandem two-gene constructs integrated into RL5 in either orientation A or B. Dark gray bars are average green fluorescence, and light bars are average yellow fluorescence, expressed as a percentage of the mean fluorescence of single genes in the same orientation. G and Y are the single genes, YG and GY are the tandem two-gene constructs, ΔGY is the tandem GY construct with the deletion of the upstream CMV basal promoter, and GΔY is the tandem GY construct with the downstream CMV basal promoter deleted. Error bars represent the standard deviation of the mean fluorescence for six or more independent clones analyzed for each construct and orientation of that construct. All levels of expression are significantly different (P < 0.001) from the expression of the single gene, except the ones labeled with an asterisk (P > 0.1). Shown between the two bar graph panels are contour FACS plots of representative clones for each construct in each orientation, as in Fig. 4. (B) Average of the fluorescence means for the same constructs integrated into RL6, normalized to the mean fluorescence of the single gene. All levels of expression are significantly different (P < 0.01) from the expression of the single gene, except the ones labeled with # (P > 0.2).

Expression levels from integrated divergent and convergent constructs are suppressed. (A) Shown as two panels of bar graphs are the relative fluorescence for the single-gene (G and Y) and the divergent (D) and convergent (C) two-gene constructs integrated into RL5 in either orientation A or B. Dark gray bars are average green fluorescence, and light bars are average yellow fluorescence, expressed as a percentage of the mean fluorescence of single genes in the same orientation. Error bars represent the standard deviation of the mean fluorescence for three or more independent clones analyzed for each construct and orientation of that construct. All levels of expression are significantly different (P < 0.001) from the expression of the single gene. Shown between the two bar graph panels are contour FACS plots of representative clones for each construct in each orientation. The x axis is green fluorescent intensity on a 4-decade log scale, and the y axis is yellow fluorescent intensity also on the same log scale. Outliers are shown as small dots. Quadrants divide expressing and nonexpressing cells; quadrant lines are drawn to be above the level of 99% of cells with no construct. (B) Average of the fluorescence means for the same constructs integrated into RL6, again normalized to the mean fluorescence of the single genes. All levels of expression are significantly different (P < 0.01) from the expression of the single gene.

Diagrammatic representations of the constructs developed for this study. (A) The arrangement of the two reporter transcription units is depicted by arrows, which indicate the direction of transcription. Each transcription unit contains the CMV promoter or enhancer driving GFP or YFP and the SV40 large T-antigen polyadenylation signal (pA). The dark gray arrow is the GFP-containing transcription unit, and the light arrow is the YFP-containing transcription unit; they are shown in the four possible orientations: divergent (D), convergent (C), and tandem (YG and GY). Thin lines represent vector sequences derived from pGEM. The two dark squares bearing triangles denote the two inverted 34-bp loxP sites, which are recognized by the CRE recombinase. (B) Tandem GY construct. The construct has been drawn to scale with the CMV promoters (stippled arrows), TATA boxes (TATAA), the starts of transcription (+1), translation initiation sites (ATG) of the fluorescent proteins (gene bodies are dark and light rectangles, GFP and YFP, respectively), and polyadenylation signals (pA; striped blocks) indicated. S, SnaB1; N, NcoI; and P, PstI. The brackets delimit the basal promoter deletions for the two constructs, ΔGY and GΔY, and the dotted lines show the regions of hybridization to the radiolabeled probe used for Southern analyses.

Fluorescence of clones correlates with steady-state levels of RNA. (A) Northern blot analyses of total RNA isolated from 14 independent RL5 clones hybridized to a radiolabeled probe that recognizes both GFP and YFP (in the upper and middle panels, showing long and short exposures, respectively), and to a 1.2-kb fragment of GAPDH, as a loading control (in the lower panel). Lanes: 1, RNA isolated from a clone carrying a single GFP in orientation A; 2, GFP alone in orientation B; 3 and 4, YFP alone in orientations B and A, respectively; 5 and 6, convergent in orientations A and B, respectively; 7 and 8, divergent in orientations B and A, respectively; 9 and 10, tandem YG in orientations B and A, respectively; lanes 11, 12, 13, and 14, tandem GY in orientations B, A, B, and B, respectively; 15, RNA isolated from untransfected RL-5. (B) Total fluorescence (sum of mean GFP and YFP fluorescences, determined by FACS as in Fig. 4) of each of the clones shown above as a function of the normalized content of fluorescent protein transcripts detected in the Northern blot shown in panel A. The line is the trend line determined by regression analysis with a correlation coefficient (R²) of 0.830.

Fraction of total fluorescence due to YFP correlates with the fraction of mRNA from YFP. (A) Diagrammatic representation of the RT-PCR and restriction fragment length polymorphism analysis used to distinguish GFP and YFP transcripts. The oligomers used amplify YFP and GFP with equal efficiency. The PCR products for both GFP and YFP are identical, with the exception of a PstI site present in products derived from YFP transcripts that is lacking in GFP. (B) Sample ethidium bromide-stained agarose gel illustrating RT-PCR products digested with PstI to differentiate GFP (uncut at 595 bp) and YFP (cleaved to give 459- and 136-bp products). Poly(A) RNAs from 11 independent clones were subjected to RT-PCR followed by restriction fragment length polymorphism analysis. Lanes 1 to 4 are derived from the single-gene clones G and Y as in Fig. 6. Lanes 5 to 8 are derived from two-gene convergent and divergent clones as in Fig. 6. Lane 9 is from a YG orientation B clone. Lanes 10 and 11 are GY orientations B and A, respectively. Lane 12 is the same as lane 1, but without the reverse transcriptase step, and lane 13 represents a mixture of poly(A) RNAs in a GFP/YFP transcript ratio of 4:1. Lane M contains the 1-kb ladder from Life Sciences. (C) The proportion of yellow fluorescence is plotted against the proportion of YFP transcripts. The proportion of yellow transcripts expressed as a percentage of total (GFP plus YFP) transcripts in that clone was determined radiometrically (in duplicate) on polyacrylamide gels by the RT-PCR and restriction fragment length polymorphism technique illustrated in panels A and B. The line is the trend line determined by regression analysis with a correlation coefficient (R²) of 0.991.