The nucleotide sequence of the transforming HindIII-G fragment of adenovirus type 5 DNA. The region between map positions 4.5 (HpaI site) and 8.0 (HindIII site).

The nucleotide sequence of the region between map positions 4.5 (HpaI-site) and 8.0 (HindIII-site) of adenovirus type 5 (Ad5) DNA has been determined. This stretch of DNA is part of the transforming HindIII-G fragment, which is 2809 nucleotides long. The sequenced segment was found to have a long open reading frame for protein biosynthesis, starting 23 nucleotides from the HpaI site and extending all the way to the HindIII-G site, which could code for a protein of at least 44 000 daltons. The possible correlation beteen the coding capacity of the HindIII-G fragment and the "transforming" proteins specified by it will be discussed in the light of the recent data on the splicing of early mRNAs.     

The nucleotide sequence of adenovirus type 5 early region E1: the region between map positions 8.0 (HindIII site) and 11.8 (SmaI site)

The nucleotide sequence of the region between map positions 8.0 (HindIII site) and 11.8 (SmaI site) of adenovirus type 5 (Ad5) has been determined. Together with the sequences reported earlier (Van Ormondt et al., 1978; Maat and Van Ormondt, 1979) it encompasses the entire leftmost early region E1 of Ad5 DNA (4126 base pairs). The total sequence revealed a number of potential regulatory signals (promoter sites, ribosome binding sites, 3'-poly(A)-associated sequences), which confirm that region E1 is divided into subregions, E1a and E1b, and a region coding for semi-late viral protein IX. By taking into account the adenovirus 2 (Ad2) RNA-splicing data of Perricaudet et al. (1979; 1980) and the Ad2 RNA mapping data of Chow et al. (1979) we predict that E1a codes for polypeptides of 32, 26 and ca. 13 kd, and subregion E1b for polypeptides of 67 kd and 20 kd; the expected molecular weight of protein IX is 14.4 kd.     

The genes of influenza virus

The 5′ terminal sequences of several adenovirus 2 (Ad2) mRNAs, isolated late in infection, are complementary to sequences within the Ad2 genome which are remote from the DNA from which the main coding sequence of each mRNA is transcribed. This has been observed by forming RNA displacement loops (R loops) between Ad2 DNA and unfractionated polysomal RNA from infected cells. The 5′ terminal sequences of mRNAs in R loops, variously located between positions 36 and 92, form complex secondary hybrids with single-stranded DNA from restriction endonuclease fragments containing sequences to the left of position 36 on the Ad2 genome. The structures visualized in the electron microscope show that short sequences coded at map positions 16.6, 19.6 and 26.6 on the R strand are joined to form a leader sequence of 150–200 nucleotides at the 5′ end of many late mRNAs. A late mRNA which maps to the left of position 16.6 shows a different pattern of second site hybridization. It contains sequences from 4.9?6.0 linked directly to those from 9.6?10.9. These findings imply a new mechanism for the biosynthesis of Ad2 mRNA in mammalian cells.     

Proof of recombination between viral and cellular genomes in human KB cells productively infected by adenovirus type 12: structure of the junction site in a symmetric recombinant (SYREC)

In previous work we have described a symmetric recombinant (SYREC1) between Ad12 DNA and human KB cell DNA. This recombinant DNA molecule has been generated during productive infection and is encapsidated into virions. From the DNA of a similar symmetric recombinant (termed SYREC2) between the left terminus of Ad12 DNA and human KB cellular DNA, the site of linkage between the two DNAs was cloned and sequenced. It was demonstrated that the first 2081 Ad12 nucleotides counting from the left viral terminus are conserved and linked to a sequence of GC-rich (70.4% G + C) KB cell DNA which occurs about 20 times per cellular genome. Except for a common 5'-CTGGC-3' pentanucleotide between the Ad12 DNA and KB cell DNA sequences, extensive patch homologies were not apparent at the site of junction. Similarly, comparisons of the deleted Ad12 DNA sequence and the cellular sequence replacing it did not reveal patch homologies. The 304 bp abutting the Ad12 terminus were shown to hybridize to KB cell DNA. These results provided definitive proof for the occurrence of recombinants between viral and cellular DNAs in human cells productively infected by Ad12 as previously shown by less direct experiments (Burger and Doerfler, 1974; Schick et al., 1976). Across the site of junction, an open reading frame exists which extends the truncated 54-kDal protein of the E1b region of Ad12 DNA for another 66 amino acids encoded by KB cellular DNA. This sequence is terminated by two UGA translational termination signals. The hypothetical protein has not yet been isolated.     

Transforming Region of Group A, B, and C Adenoviruses: DNA Homology Studies with Twenty-Nine Human Adenovirus Serotypes

The 31 human adenovirus (Ad) serotypes form five groups based upon DNA genome homologies: group A (Ad12, 18, 31), group B (Ad3, 7, 11, 14, 16, 21), group C (Ad1, 2, 5, 6), group D (Ad8, 9, 10, 13, 15, 17, 19, 20, 22-30), and group E (Ad4) (M. Green, J. Mackey, W. Wold, and P. Rigden, Virology, in press). Group A Ads are highly oncogenic in newborn hamsters, group B Ads are weakly oncogenic, and other Ads are nononcogenic. However, most or all Ads transform cultured cells. We have studied the homology of Ad5, Ad7, and Ad12 transforming restriction endonuclease DNA fragments with DNAs of 29 Ad types. Ad5 HindIII-G (map position 0-7.3), Ad7 XhoI-C (map position 0-10.8), and Ad12 (strain Huie) EcoRI-C (map position 0-16) and SalI-C (map position 0-10.6) fragments were purified, labeled in vitro (nick translation), and annealed with DNAs of Ad1 to Ad16, Ad18 to Ad24, and Ad26 to Ad31. Hybrids were assayed by using hydroxylapatite. Ad5 HindIII-G hybridized 98 to 100% with DNAs of group C Ads, but only 1 to 15% with DNAs of other types. Ad7 XhoI-C fragment hybridized 85 to 99% with DNAs of group B Ads, but only 6 to 21% with DNAs of other types. Ad12 (Huie) EcoRI-C hybridized 53 to 68% with DNAs of five other Ad12 strains, 53% with Ad18 DNA, 56% with Ad31 DNA, but only 3 to 13% with DNAs of other types. In vitro-labeled Ad12 (Huie) SalI-C hybridized 35 to 71% with DNAs of 6 other Ad12 strains, 44% with Ad18 DNA, 52% with Ad31 DNA, but only 2 to 7% with DNAs Ad7, Ad2, Ad26, or Ad4. When assayed using S-1 nuclease, SalI-C annealed 17 to 44% with DNAs of group A Ads. The melting temperatures of the hybrids of Ad5 HindIII-G with all group C Ad DNAs were 84 degrees C in 0.12 M sodium phosphate (pH 6.8). The melting temperature of the Ad12 (Huie) EcoRI-C hybrid with Ad12 (Huie) DNA was 83 degrees C, but was only 71 to 77 degrees C with DNAs of other group A Ads. Thus, group C and group B Ads both have very homologous transforming regions that are not represented in DNAs of non-group C Ads or non-group B Ads, respectively. Similarily, group A Ads have unique but less homologous transforming regions. These different transforming nucleotide sequences may be reflected in the different oncogenic properties of group A, B, and C Ads.     

Structure of viral DNA in a rat cell line transformed by the cloned EcoRI-C fragment of adenovirus 12.

A DNA segment carrying viral DNA was cloned from a rat cell line transformed by the cloned EcoRI-C fragment (0 to 16.4 map units) of human adenovirus type 12(Ad12), and the viral sequence in the clone was analysed. The cloned segment contained the region from nucleotide positions 118 to 3520 of the Ad12 genome in the middle. No unique structure was found at the viral and non-viral DNA junctions. When examined the transforming activity, the conserved viral sequence was able to transform rat 3Y1 cells efficiently. Southern blotting analysis of the viral sequence in five re-transformed cell lines showed that the viral sequence was inserted at different sites of cellular DNA. These results indicate that (I) the Ad12 DNA moiety from the enhancer-promoter region of the E1A gene to the end of the E1B gene contains enough information for efficient transformation of the rat cell, and (II) integration of the viral sequence at unique cellular sites is not prerequisite for transformation.     

The nucleotide sequence of the genes encoded in early region 2b of human adenovirus type 7

The nucleotide sequence of a cloned DNA segment encoding the early region 2b from the group B human adenovirus Ad7 has been determined. When compared to Ad2, a group C adenovirus, these sequences were found to be approx. 80% homologous within the l-strand gene-coding regions. Most changes are transitions or transversions, although several deletions/insertions also occur within the N-terminal domain of one of the coding regions. The substantial nucleotide homology results in a high degree of amino acid conservation in the predicted polypeptides encoded by the early region 2b genes. Two major open reading frames, corresponding to the Mr 87000 and Mr 140000 polypeptides of Ad2, are found in the l strand of Ad7 between genome coordinates 28.5 to 23.1 and 13.8, respectively. The r strand of the DNA in this region encodes the three leader segments joined to the 5' end of the most late viral mRNAs, and also encodes the i-leader segment found between the second and third leaders on some mRNAs. The positions of the donor and acceptor splice sites of the three leaders are conserved and can be identified by homology to Ad2. Only two of the unidentified open reading frames (URF) in Ad2 (Gingeras et al., J. Biol. Chem., in press) can be found in Ad7. URF1, encoding an Mr 13500 polypeptide at genome coordinate 17, is predominantly conserved in nucleotide and amino acid sequence, but contains one half as many arginine amino acids as does URF1 of Ad2. URF2, encoding an Mr 13600 protein which lies within the i-leader region, is not well conserved in either nucleotide or amino acid sequence.