Northern blot analysis of mRNAs transcribed from bicistronic plasmids. RNA was extracted from untransfected C6 cells and from the same stably transfected C6 pools used in the experiment in Fig. 3. A 20-μg portion of each RNA was electrophoresed in two parallel lanes, blotted, and hybridized with either a LUC-specific probe (A) or a SeAP-specific probe (B). To ensure equal loading, rRNAs were stained with methylene blue prior to hybridization. Lanes: 1, untransfected C6 cells; 2, B/0; 3, B/UTR; 4, B/BiP.

Sequence of the SP163 element. The oligonucleotide primers that were used for amplification of SP163 are underlined. These oligonucleotides correspond to the respective boundaries of the 1,014-nucleotide mouse VEGF 5′UTR. The initiation ATG codon is boxed. Arrowheads point to two possible locations of a presumptive splice junction. Nucleotide 1 of SP163 corresponds to nucleotide 1218 of the mouse VEGF gene (GenBank accession no. U41383), while nucleotide 163 of the SP163 fragment corresponds to nucleotide 2231 of the mouse gene. Substitutions depicted by solid arrows represent mutations introduced into the B/SP163/M5′ mutant, and substitutions depicted by open arrows represent mutations introduced into the B/SP163/M3′ mutant (see Fig. 6).

Translation enhancing activity of VEGF 5′UTR sequences. C6 cells were stably transfected with the monocistronic constructs indicated. SeAP activity was analyzed in pools of transfected clones as described in Materials and Methods, and values were standardized to total protein. For analysis of SeAP activity under hypoxic conditions, cells were shifted to 1% oxygen 24 h before sampling. N, normoxia; H, hypoxia. (Inset) Northern blot analysis of SeAP-containing mRNAs in stably transfected pools. Lanes: 1, M/0-transfected cells; 2, M/UTR-transfected cells; 3, M/SP163-transfected cells; 4, nontransfected cells.

Vectors and constructs used in this study. (A) A bicistronic expression vector in which expression of a bicistronic mRNA is driven by a CMV promoter. A firefly luciferase (LUC) is translated from the first cistron, and a SeAP is translated from the second cistron. Putative IRES elements are inserted into the intercistronic space (ICS). (B) A monocistronic expression vector in which SeAP expression is driven by a CMV promoter. Sequence elements tested for a translation-modulating activity are inserted upstream of the SeAP coding region. (C) Designations of bicistronic and monocistronic constructs used in this study (see Materials and Methods for details). The nucleotide numbers inside the rectangles represent the insert length. ND, not done.

Distribution of VEGF mRNA between subpolysomal and polysomal fractions. (A) Size fractionation of ribosomes by centrifugation through a sucrose density gradient. Cytoplasmic extracts were prepared from cultures of primary astrocytes grown under normoxia (left) or hypoxia (right). Sedimentation was from left to right, and the vertical line indicates the point of division into the subpolysomal (S) and polysomal (P) pooled fractions. OD 260, optical density at 260 nm. (B) Relative abundance of VEGF mRNA in the subpolysomal and polysomal fractions. RNA was extracted from the entire pool, and aliquots representing an equal portion of each pool were analyzed by a quantitative RT-PCR/blot hybridization as described in Materials and Methods. The different splicing variants of VEGF mRNA are indicated by the respective number of amino acids in the encoded protein. The proportion of mRNA in polysomes for the most abundant mRNA species, VEGF₁₆₅ (as determined by densitometric scanning) was 87 and 88% for normoxia and hypoxia, respectively.

IRES activity of SP163. 293 cells were transiently transfected with each of the indicated bicistronic plasmids, and SeAP activity in culture media and LUC activity in cell extracts were determined 36 h posttransfection as described in Materials and Methods. The results are the average of four independent transfections with each plasmid and are expressed as arbitrary LUC activity units and arbitrary SeAP activity units per milliliter. Solid bars, LUC; hatched bars, SeAP. The SeAP/LUC ratios, corrected for differences in levels of the bicistronic mRNAs, are also shown. Plasmid designations are as in Fig. 1. B/ΔSP163 is a deletion mutant of SP163 missing the first 31 nucleotides. B/SP163/M5′ and B/SP163/M3′ are SP163 mutants containing the respective substitutions indicated in Fig. 5. (Inset) Northern blot analysis with a SeAP-specific probe and a LUC-specific probe performed on 20 μg of the total RNA extracted from transfected 293 cells 36 h posttransfection. Lanes: 1, mock-transfected cells; 2, B/0-transfected cells; 3, B/UTR-transfected cells; 4, B/SP163-transfected cells.

Production of LUC and SeAP from a bicistronic mRNA. (A) Production of SeAP from the downstream cistron. Pools of stably transfected C6 clones were grown to 70% confluence, medium was replaced with a fresh medium (t = 0) and aliquots were withdrawn at the indicated time points and analyzed for SeAP activity as described in Materials and Methods. Activity is expressed as cumulative SeAP activity (in arbitrary units of the colorogenic reaction product) per ml of medium and is normalized to total cellular protein and corrected for differences in VEGF mRNA levels (Fig. 4; also see the text). For testing SeAP production during hypoxia, cells were shifted to 1% oxygen at t = 0 and further analyzed as above. N, normoxia; H, hypoxia. (B) Ratio of SeAP to LUC production in the different transfectants in panel A. The SeAP/LUC ratio was calculated from the respective activities determined at the end point of the experiment (t = 24 h). (C) Effect of hypoxia on LUC and SeAP production in transiently transfected 293 cells. At 14 h posttransfection, the medium was changed, half of the culture plates were kept under normoxic conditions, and the other half were placed in a hypoxic chamber (see Materials and Methods). SeAP activity in the culture media and LUC activity in cell extracts were determined 20 h later. Results shown are the average of four independent transfections with each plasmid. The plasmid designations are as shown in Fig. 1. L, LUC activity in arbitrary units; S, SeAP activity in arbitrary units per milliliter of medium; N, normoxia; H, hypoxia.