American Society of Hematology

Fig. 5.

$Fig. 5. Comprehensive analysis from patient UPN09. (A) shows the 1.7-kb RT-PCR product obtained using nested PCR, first with RT5out/RT3out and then with RT5Eco/RT3Not. “+” indicates the positive control for this reaction, a clone of HERV-K10. Lanes 1, 2, 3, 4, and 5 are selected fractions from the bottom to top of a sucrose gradient preparation from this patient. The arrow indicates the position of the positive signal. Lane 3 is positive. (B) shows the same fractions analyzed for RT activity. “+” indicates the positive control for this reaction, ie, 0.002 U of MoMLV-RT. Again, lane 3 is positive and the arrow indicates the position of the positive signal. (C) shows SDS-PAGE products detected by fluorography from in vitro translation of several independent RT-PCR clones in pBBV. Lanes 1, 2, 5, and 6 through are independent clones obtained from the RT-PCR product shown in (A), lane 3 above. Lanes 3 and 4 are two different clones of HERV-K10 included for comparison. As can be seen, only lane 2 expresses a protein of apparently full length, which is indicated by the arrow. (D) shows the nucleotide sequence and the deduced amino acid sequence of this clone (pBBV-24) at only those positions that differ from the HERV-K10 prototypic sequence between nucleotides 3950 and 5651 (the RT and RNaseH domains).$

Comprehensive analysis from patient UPN09. (A) shows the 1.7-kb RT-PCR product obtained using nested PCR, first with RT5out/RT3out and then with RT5Eco/RT3Not. “+” indicates the positive control for this reaction, a clone of HERV-K10. Lanes 1, 2, 3, 4, and 5 are selected fractions from the bottom to top of a sucrose gradient preparation from this patient. The arrow indicates the position of the positive signal. Lane 3 is positive. (B) shows the same fractions analyzed for RT activity. “+” indicates the positive control for this reaction, ie, 0.002 U of MoMLV-RT. Again, lane 3 is positive and the arrow indicates the position of the positive signal. (C) shows SDS-PAGE products detected by fluorography from in vitro translation of several independent RT-PCR clones in pBBV. Lanes 1, 2, 5, and 6 through are independent clones obtained from the RT-PCR product shown in (A), lane 3 above. Lanes 3 and 4 are two different clones of HERV-K10 included for comparison. As can be seen, only lane 2 expresses a protein of apparently full length, which is indicated by the arrow. (D) shows the nucleotide sequence and the deduced amino acid sequence of this clone (pBBV-24) at only those positions that differ from the HERV-K10 prototypic sequence between nucleotides 3950 and 5651 (the RT and RNaseH domains).

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