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. 2018 May 15;1(1):41–53.

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© 1991 by the University of Health Sciences/The Chicago Medical School. All rights reserved.

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Expression of the UHU genes. A. 10 μg RNA from CHO cells transfected with the UHU₁₅₆ genes (lanes 1–3) or the UHU₁₁₇ genes (lanes 4 and 5) was analyzed by S1 nuclease mapping using the appropriate homologous gene labeled at the 3′ end of the Narl site at codon 43 as a probe. The protected fragments are: H2a_H- protection to the TAA codon by the endogenous hamster H2a mRNAs; U1–protection by transcripts from the UHU genes extending to the normal U1 3′ end. P is the undigested probe band. The positions of the marker fragments, pUC18 digested with HpaII, are indicated. B. 10 μg total cell RNA from CHO cells transfected with the UHU₁₅₆ genes (lane 2) or the UHU₁₁₇ genes (lanes 3 and 4) was analyzed by S1 nuclease mapping using the UHU₁₅₆PL (lanes 1 and 2) or UHU₁₁₇PL (lanes 3 and 4) probes described in Fig. 4C labeled at the 3′ end of the Narl site at codon 43. These probes diverge from the transfected gene after the U1 3′ end as in Figure 4B, allowing detection of all longer transcripts as a single protected fragment (RT). The protected fragments are: H2a_H–protection to the TAA codon by the endogenous hamster H2a mRNAs; U1–protection by transcripts from the UHU genes extending to the normal U1 3′ end; RT–protection by transcripts which extend past the U1 3′ end. Lane 1 is 1 μg RNA from untransfected CHO cells. C. 5 μg total cell RNA, cells transfected with the UHU₁₁₇ gene (lane 1), the UHU₁₅₆ gene (lane 2), or untransfected CHO cells (lane 3) were analyzed by S1 nuclease mapping using the U5H gene labeled at the 5′ end of the NarI site (codon 43) as a probe. The protected fragments are: H2a_H–protection to the ATG codon by the endogenous hamster H2a mRNAs; U1–protection by transcripts from the UHU genes extending to the normal U1 5′ end. Lane M is marker pUC18 digested with HpaII. D. 10 μg total cell RNA from cells transfected with the UHU₁₁₇ genes (lanes 1 and 2) or the CHO-156C cells transfected with the UHU₁₅₆ gene (lanes 3 and 4) were analyzed by S1 nuclease mapping using the UHU₁₁₇ gene (lanes 1–3) or the UHU₁₅₆ gene (lane 5) labeled at the 3′ end of the NarI site (codon 43) as a probe. The protected fragments are labeled as in Figure 5B. The fragment labeled T is due to protection of all transcripts from the UHU₁₅₆ gene to the point of divergence of the UHU₁₅₆ and the UHU₁₁₇ genes. Band P is the undigested probe. Lane 4 is marker pUC18 digested with HpaII. The same CHO-156C [UHU156] (lane 6) and UHU₁₁₇ RNAs (lane 7) were analyzed by S1 nuclease mapping using the U₅H gene labeled at the 5′ end of the NarI site as a probe. The protected fragments are labeled as in Figure 5C. The fragment labeled U1_RT results from protection by transcripts which initiated upstream from the normal U1 start site. Band P is undigested probe DNA. The position of the marker fragments is shown. The hamster histone RNAs were not detected efficiently in lane 7 for unknown reasons.

Expression of the UHU genes. A. 10 μg RNA from CHO cells transfected with the UHU₁₅₆ genes (lanes 1–3) or the UHU₁₁₇ genes (lanes 4 and 5) was analyzed by S1 nuclease mapping using the appropriate homologous gene labeled at the 3′ end of the Narl site at codon 43 as a probe. The protected fragments are: H2a_H- protection to the TAA codon by the endogenous hamster H2a mRNAs; U1–protection by transcripts from the UHU genes extending to the normal U1 3′ end. P is the undigested probe band. The positions of the marker fragments, pUC18 digested with HpaII, are indicated. B. 10 μg total cell RNA from CHO cells transfected with the UHU₁₅₆ genes (lane 2) or the UHU₁₁₇ genes (lanes 3 and 4) was analyzed by S1 nuclease mapping using the UHU₁₅₆PL (lanes 1 and 2) or UHU₁₁₇PL (lanes 3 and 4) probes described in Fig. 4C labeled at the 3′ end of the Narl site at codon 43. These probes diverge from the transfected gene after the U1 3′ end as in Figure 4B, allowing detection of all longer transcripts as a single protected fragment (RT). The protected fragments are: H2a_H–protection to the TAA codon by the endogenous hamster H2a mRNAs; U1–protection by transcripts from the UHU genes extending to the normal U1 3′ end; RT–protection by transcripts which extend past the U1 3′ end. Lane 1 is 1 μg RNA from untransfected CHO cells. C. 5 μg total cell RNA, cells transfected with the UHU₁₁₇ gene (lane 1), the UHU₁₅₆ gene (lane 2), or untransfected CHO cells (lane 3) were analyzed by S1 nuclease mapping using the U5H gene labeled at the 5′ end of the NarI site (codon 43) as a probe. The protected fragments are: H2a_H–protection to the ATG codon by the endogenous hamster H2a mRNAs; U1–protection by transcripts from the UHU genes extending to the normal U1 5′ end. Lane M is marker pUC18 digested with HpaII. D. 10 μg total cell RNA from cells transfected with the UHU₁₁₇ genes (lanes 1 and 2) or the CHO-156C cells transfected with the UHU₁₅₆ gene (lanes 3 and 4) were analyzed by S1 nuclease mapping using the UHU₁₁₇ gene (lanes 1–3) or the UHU₁₅₆ gene (lane 5) labeled at the 3′ end of the NarI site (codon 43) as a probe. The protected fragments are labeled as in Figure 5B. The fragment labeled T is due to protection of all transcripts from the UHU₁₅₆ gene to the point of divergence of the UHU₁₅₆ and the UHU₁₁₇ genes. Band P is the undigested probe. Lane 4 is marker pUC18 digested with HpaII. The same CHO-156C [UHU156] (lane 6) and UHU₁₁₇ RNAs (lane 7) were analyzed by S1 nuclease mapping using the U₅H gene labeled at the 5′ end of the NarI site as a probe. The protected fragments are labeled as in Figure 5C. The fragment labeled U1_RT results from protection by transcripts which initiated upstream from the normal U1 start site. Band P is undigested probe DNA. The position of the marker fragments is shown. The hamster histone RNAs were not detected efficiently in lane 7 for unknown reasons.