Nucleotide sequence of adenovirus 2 DNA fragment encoding for the carboxylic region of the fiber protein and the entire E4 region.

The entire nucleotide sequence between coordinates 89.5 and 100% of the Ad 2 DNA genome has been determined using the Maxam and Gilbert method. This sequence of 3766 bp contains information relative to the carboxylic end of the fiber protein and to the entire E4 region. The position within the nucleotide sequence of various open reading frames and of several consensus splicing sequences was correlated with the location by EM and Sl digestion of the E4 mRNA. This correlation allows to suggest an additional splicing event in the maturation process of i or f mRNA and to deduce the structure of most E4 mRNA. The aminoacid sequences of the corresponding proteins are deduced allowing the location of several glycosylation sites. The presence of several open reading frames with a substantial coding capacity permits to postulate on the existence of additional genes located at the 3' end of the fiber gene and the 3' end of the E4 region. The existence of these putative additional genes might explain that termination of transcription is several hundred nucleotides beyond the main known poly A addition sites of the L5 and E4 regions.

Nucleotide sequence of the EcoRI E fragment of adenovirus 2 genome.

The entire nucleotide sequence of the Ad.2 EcoRI E fragment has been determined using the Maxam and Gilbert method. This sequence of 2222 bp, which maps between coordinate 83.4 and 89.7 contains information relative to the early 3 region and to the fiber gene. Altogether with fragment EcoRI D which has been recently sequenced, they cover the entire Early 3 region in which several mRNA were mapped. The aminoacid sequence of the 16K and 14K protein is deduced. The localization of the 14.5K mRNA directing the synthesis of the third E3 known protein is discussed, as well as the hypothetical existence of three other early 3 proteins, which would have a molecular weight of 11K. The initiator ATG triplet of the fiber protein has been found at coordinate 86.1, it is followed up to the end of the fragment by an open reading frame allowing deduction of 80% of the aminoacid sequence of this protein. Sequences known to be frequently present at the border of exon sequence were used to tentatively localize the additional "Z" late leader.

Nucleotide sequence of the EcoRI D fragment of adenovirus 2 genome.

The entire nucleotide sequence of the Ad. 2 EcoRI D fragment has been determined using the Maxam and Gilbert method. This sequence of 2678 bp contains informations relative to late mRNAs ending at position 78 and for which an AATAAA sequence corresponding to their 3' ends is found at residue number 833. Position of the PVIII mRNA is determined thus allowing deduction of the probable amino acid sequence of the PVIII protein. The position and the sequence of the first leader of early 3 mRNAs is determined as well as the sequence and position of the second early leader of region 3 mRNAs, which also correspond to the "y" leader of the fiber mRNA. Following the localization of an open reading frame in which an ATG could initiate protein synthesis it can be predicted that 3a, b, c mRNAs code for the 16K early protein and the probable amino acid sequence of this protein can be deduced. The CAGTTT sequence frequently present at the 5' end of a leader or of a mRNA body as well as the GGTGAG sequence which is found at the 3' end of several leaders were used to postulate the position of various early mRNAs of region 3 and to suggest the existence of an additional splicing event during the processing of mRNAs 3a, b and c. They were also used to predict the position of the additional "x" late leaders. The imbrication of information concerning (i) the family of late mRNAs ending at position 78, (ii) the position of the "x" leader and the "y" leader and (iii) the beginning of early region 3 is also depicted.

Messenger RNA for the Ad2 DNA binding protein: DNA sequences encoding the first leader and heterogenity at the mRNA 5' end.

During the early stage of Ad2 infection of human cells, RNA is transcribed from five separate transcription units. Early region II encodes the mRNA for a 72K single-stranded DNA binding protein (DBP) which functions in DNA replication. This report describes the structure of the first leader of the DBP mRNA and the flanking sequences in the DNA. The leader, labeled in vivo with 32P, was isolated by DNA filter hybridization to the viral restriction fragment Eco RI F, and its RNAase T1 and RNAase A oligonucleotides were analyzed by RNA fingerprinting techniques. Comparison of this RNA sequence information with the DNA sequence of Eco RI F has located a 68 nucleotide region of the Hae III C subfragment at coordinate 75.1 that encodes the leader. This position is near the coordinate to which nascent chain analysis and ultraviolet transcription mapping have mapped an RNA initiation site, or promoter, for the DBP mRNA. The DNA sequence that overlaps the leader on the 3' side contains a donor sequence for splicing this leader to a second downstream leader. The splicing sequence shows a seven base homology with the comparable structure of the Ad2 major late leader, and a mouse globin mRNA splicing sequence. The DNA sequence upstream from the cap, the region oof the potential promoter site does not, however, contain a "TA-TAAA"-type homology of the sort noted by D. Hogness, M. Goldberg and R. Lifton (personal communication) for many cellular transcription units, and by other investigations for the Ad2 major late transcription unit. Also, the leader is found with two distinct capped 5' termini, 7meGpppA and 7meGpppG, which are encoded at adjacent positions in the DNA and thus are from mRNAs which are staggered by one nucleotide in length at the 5' end. The staggering at the 5' terminus and the lack of the upstream homolgy distinguish the DBP mRNA from many viral and cellular messenger. In both these respects, however, the DBP mRNA resembles the late messengers of SV40 and polyoma viruses. In this paper, we discuss the implications of these findings for the mechanism of specifying mRNA 5' ends.

Nucleotide sequence of the EcoRI-F fragment of adenovirus 2 genome.

Using the DNA sequence method of Maxam and Gilbert the entire nucleotide sequence of the adenovirus 2 EcoRI-F fragment was determined. Information contained in that nucleotide sequence, which is 1743 base pairs long, is interpreted with respect to the mapping and processing of the three mRNAs partly encoded by the EcoRI-F fragment. A method to rapidly determine the cleavage site of restriction endonucleases is also reported.

Mapping the DNA fragments produced by cleavage of EcoRI F fragment of adenovirus 2 with HaeIII, HpaII and AluI.

Hydrolysis of the EcoRI F fragment of the adenovirus 2 genome with HaeIII, HpaII and AluI restriction enzymes gives respectively 9, 11, and 11 fragments, the size of which ranges from 20 bp for the smallest HpaII k fragment to 585 for the largest AluI A fragment. The order of fragments was mainly deduced from partial hydrolysis analyses. The relative order of all restriction sites and the distance in nucleotides between them were obtained through secondary analyses of each restriction fragment by the two other enzymes. Position of the KpnI, HindIII, MboI and SmaI sites within the EcoRI F fragment were also reevaluated.

XC-cell fusion induced by murine plasmocytoma cell. III. RNA gene expression correlated to syncytium formation.

The MF2 strain, a mouse myeloma derived cell line, was found to continuously produce C-type viral particles when maintained in tissue-culture. These cells when cultured in an ascitic form by injection to Balb/c mice lost this property. The ability to induce syncytia by cocultivation of the MF2 cell with XC-cells was shown to be related to the viral production. A DNA complementary to viral 70 S RNA was synthesized using the viral reverse-transcriptase endogenous activity. The quality of the probe is discussed and the expression of the viral genome among cellular poly A rich RNA varied concomitently to the syncytium inducing ability as evidenced by molecular hybridization experiments.