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The early genes of the human adenotirus type 2 (Ad 2) expressed prior to the onset of DNA replication at 6 h post-iifection, are involved in regulatory functions necessary for the virus growth cycle and in the oncogenic transformation of mammalian cells. After initial transcription in the nucleus by host cell mechanisms, the early viral RNA species are modified by polyadenylation on the 3'-end, addition of a S'methyl cap (m'Gppp) and excision of intervening sequences (sp~c~g) to yield mature polysomal mRNA species. The physical map of the early Ad 2 genes reveals 4 separate gene regions, El -E4, located at the map coordinates 1.5-I 1 (El) and 76-86 (E3) on the r-strand, and 68-62 (E2) and 99-91.5 (E4) on the I-strand (r and 1 indicate rightward and leftward transcription of the Ad 2 genome, presented in 100 map units). Each early gene block codes for a family of spliced partially overlapping mRNA species, usually with common 3'end 5'-termini [l-6]. (Two additional sets of early viral RNA sequences may be present in low copy number, complementary to the map regions 11-14.5 and 20-23.5 on the f-strand 171.) The gene product of the region E2 is a DNA-bin~mg protein (M, 73 000) involved in Ad 2 DNA replication [8,9] and perhaps late gene expression [IO]. The characterization of the mRNA of this DNA-binding protein (DBP) is therefore of particular interest. We had calculated the coordinates of the main body of this mRNA by determining the % of radioactivity of DBP mRNA which hybridized to particular subfrag ments of the EcoRI B fragment (map position 58.5-70.7) ill ]. Our findings are in good agreement with the data obtained by an Sr nuclease technique [ 121 and by electron microscopy [6]. We showed that the Elsevier/North-Holland Biomedical Press 3'-terminus of the DBP mRNA lies within the restriction fragment BgiII J (60. 2-63.6) at the map position 61 .I C 0.4. Here, we prepared A&I and N&r11 subfragments of BglII J, and by hybridization to small poly(A)-containing fragments of the DBP mRNA isolated an -300 basepair subfragment which is homologous to the 3'-terminal region of this mRNA. Analysis of the primary structure of this subfragment identified the DNA sequence which codes for the 3'-terminus of the DBP mRNA. Materials and methods Restriction endonucleasesRestriction endonucleases EcoRt, BglII, HpaII, AU, KpnI, HaeIII, BaZI and ma1 [ 131 were isolated by standard biochemical procedures including ammonium sulfate fractionation, phosphocellulose, DEAEcellulose and hydroxylapatite chromatography, gel filtration, heparin-agarose and aminoalkyl-agarose affinity chromato~aphy.AvaI and HphI were purchased from New England BioLabs. DNA restriction fragmentsThe preparation of Ad 2 DNA,cleavage with restriction endonucleases and the isolation of the viral DNA fragments from 1.4% agarose gels has been described [3]. Purification of small restriction fragments from polyacrylamide gels was done as in [ 143.2.3.~xI75RFIDMA RF I DNA of #xl 74~723 was isolated as suggested by W. Riiger, Boch~.~aeIII fra~ents of@xl74am3[ ...
The early genes of the human adenotirus type 2 (Ad 2) expressed prior to the onset of DNA replication at 6 h post-iifection, are involved in regulatory functions necessary for the virus growth cycle and in the oncogenic transformation of mammalian cells. After initial transcription in the nucleus by host cell mechanisms, the early viral RNA species are modified by polyadenylation on the 3'-end, addition of a S'methyl cap (m'Gppp) and excision of intervening sequences (sp~c~g) to yield mature polysomal mRNA species. The physical map of the early Ad 2 genes reveals 4 separate gene regions, El -E4, located at the map coordinates 1.5-I 1 (El) and 76-86 (E3) on the r-strand, and 68-62 (E2) and 99-91.5 (E4) on the I-strand (r and 1 indicate rightward and leftward transcription of the Ad 2 genome, presented in 100 map units). Each early gene block codes for a family of spliced partially overlapping mRNA species, usually with common 3'end 5'-termini [l-6]. (Two additional sets of early viral RNA sequences may be present in low copy number, complementary to the map regions 11-14.5 and 20-23.5 on the f-strand 171.) The gene product of the region E2 is a DNA-bin~mg protein (M, 73 000) involved in Ad 2 DNA replication [8,9] and perhaps late gene expression [IO]. The characterization of the mRNA of this DNA-binding protein (DBP) is therefore of particular interest. We had calculated the coordinates of the main body of this mRNA by determining the % of radioactivity of DBP mRNA which hybridized to particular subfrag ments of the EcoRI B fragment (map position 58.5-70.7) ill ]. Our findings are in good agreement with the data obtained by an Sr nuclease technique [ 121 and by electron microscopy [6]. We showed that the Elsevier/North-Holland Biomedical Press 3'-terminus of the DBP mRNA lies within the restriction fragment BgiII J (60. 2-63.6) at the map position 61 .I C 0.4. Here, we prepared A&I and N&r11 subfragments of BglII J, and by hybridization to small poly(A)-containing fragments of the DBP mRNA isolated an -300 basepair subfragment which is homologous to the 3'-terminal region of this mRNA. Analysis of the primary structure of this subfragment identified the DNA sequence which codes for the 3'-terminus of the DBP mRNA. Materials and methods Restriction endonucleasesRestriction endonucleases EcoRt, BglII, HpaII, AU, KpnI, HaeIII, BaZI and ma1 [ 131 were isolated by standard biochemical procedures including ammonium sulfate fractionation, phosphocellulose, DEAEcellulose and hydroxylapatite chromatography, gel filtration, heparin-agarose and aminoalkyl-agarose affinity chromato~aphy.AvaI and HphI were purchased from New England BioLabs. DNA restriction fragmentsThe preparation of Ad 2 DNA,cleavage with restriction endonucleases and the isolation of the viral DNA fragments from 1.4% agarose gels has been described [3]. Purification of small restriction fragments from polyacrylamide gels was done as in [ 143.2.3.~xI75RFIDMA RF I DNA of #xl 74~723 was isolated as suggested by W. Riiger, Boch~.~aeIII fra~ents of@xl74am3[ ...
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