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Results: 5

1.
Figure 2

Figure 2. From: ?-Globin mRNA decay in erythroid cells: UG site-preferred endonucleolytic cleavage that is augmented by a premature termination codon.

Sequence of human β-globin mRNA, where nucleotides in bold are complementary to DNA primers used in primer extensions.

Audrey Stevens, et al. Proc Natl Acad Sci U S A. 2002 October 1;99(20):12741-12746.
2.
Figure 3

Figure 3. From: ?-Globin mRNA decay in erythroid cells: UG site-preferred endonucleolytic cleavage that is augmented by a premature termination codon.

S1 nuclease mapping of 5′ decay products of normal and nonsense-containing human β-globin transcripts. S1 nuclease protection was performed as in Fig. 1 by using 0.74 ng of synthetic β-globin mRNA (lane 2) (21), 15 μg of poly(A) MEL cell RNA, or 15 μg of yeast RNA and a 3′-labeled BfaI–BamHI probe that spans nucleotides 31–351 of human β-globin mRNA. Decay intermediates are identified with circles.

Audrey Stevens, et al. Proc Natl Acad Sci U S A. 2002 October 1;99(20):12741-12746.
3.
Figure 5

Figure 5. From: ?-Globin mRNA decay in erythroid cells: UG site-preferred endonucleolytic cleavage that is augmented by a premature termination codon.

β-globin mRNA endonuclease activity associated with MEL-cell polysomes. (Left) An in vitro-synthesized, 5′-32P-labeled transcript consisting of the 5′-most 347 nt of human β-globin mRNA (plus 20 nt of vector) was incubated for 30 min at 37°C with no added protein (lane 2, input), polysome extract from uninduced (MEL−, lane 3) or 48-hr DMSO-induced (MEL+, lane 4) MEL cells, or two fractions containing β-globin mRNA endonuclease activity recovered from Mono S fractionation of the polysome extract (lanes 5 and 6). Lane 7 shows a partial T1 digest of the β-globin transcript; an open circle (○) shows products with counterparts in vivo. Right is a longer exposure of Left.

Audrey Stevens, et al. Proc Natl Acad Sci U S A. 2002 October 1;99(20):12741-12746.
4.
Figure 4

Figure 4. From: ?-Globin mRNA decay in erythroid cells: UG site-preferred endonucleolytic cleavage that is augmented by a premature termination codon.

Primer extension mapping the 5′ ends of the human β-globin mRNA decay products. Total RNA (10 μg), or synthetic human β-globin mRNA (7.2 ng) (21) was annealed to oligonucleotide DNAs (see Fig. 2 and Table 1) complementary to mRNA nucleotides 112–136 (A), 226–250 (B), or 341–365 (C) and assayed by primer extension. Sequencing ladders (T or G ladders) provided molecular size markers, and a control primer extension (synthetic globin RNA) identified sites of reverse transcriptase stalling. Numbers to the left map potential cleavage sites relative to the full-length 5′ end, which is defined as 1. Notably, because Thal 10 mRNA contains one less nucleotide at codon 44 relative to Norm 2 mRNA, the extension band resulting from cleavage at nucleotide 167 is shorter by one nucleotide. Results shown are representative of at least two independently performed experiments that used RNA from transfectants grown and harvested on different days. Full-length products are identified with an asterisk (✶).

Audrey Stevens, et al. Proc Natl Acad Sci U S A. 2002 October 1;99(20):12741-12746.
5.
Figure 1

Figure 1. From: ?-Globin mRNA decay in erythroid cells: UG site-preferred endonucleolytic cleavage that is augmented by a premature termination codon.

S1 nuclease mapping of 3′ decay products of normal and nonsense-containing human β-globin transcripts. (A) Mice transgenic for a nonsense-free (Norm) or nonsense-containing (Thal) human β-globin allele were made anemic, and RNA was isolated from peripheral blood (13). Additionally, MEL cells stably expressing the nonsense-free (Norm 2) or nonsense-containing (Thal 10) human β-globin allele were propagated in the absence (uninduced) or presence (induced) of 2% DMSO, and RNA was isolated from total (T), nuclear (N), and cytoplasmic (C) fractions (19). A 5′-32P-labeled 590-bp MseI–MseI fragment of human β-globin cDNA and a 5′-32P-labeled 183-bp BamHI–BamHI fragment of mouse α-globin cDNA were used as probes in S1 nuclease mapping (13). Full-length human β-globin mRNA protected 524 nt (hβ-Gl mRNA), mouse α-globin mRNA protected 183 nt (mα-Gl mRNA), and mRNA decay products are identified by arrows. The 159-nt protected fragment (□) derives from intron 2-containing human β-globin pre-mRNA (data not shown). Total (T), nuclear (N), and cytoplasmic (C) RNA from each transfectant was assayed by using the amounts specified above each lane (2, 15, or 30 μg). −RNA, no RNA; −S1, no S1 nuclease. The locations of the premature termination codon (PTC) and 5′ decay products are diagrammed. (B and C) A 5′-32P-labeled NaeI–BamHI fragment of a human β-globin cDNA was used as probe for S1 nuclease mapping. Full-length mRNA protected 351 nt (✶). In B, 10 μg of total RNA was used except for lane 2, where 1.5 μg was used. In C, 10 μg of total, 10 μg of poly(A), and 0.5 μg of poly(A)+ RNA were used. The sizes of the protected fragments are indicated according to the distance from the 5′ end of full-length β-globin mRNA to the left in B and right in C. M, molecular weight standards.

Audrey Stevens, et al. Proc Natl Acad Sci U S A. 2002 October 1;99(20):12741-12746.

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