The gene for polypeptide IX of human adenovirus type 7

This paper describes the nucleotide sequence of subgroup B human adenovirus type 7 (Ad7) between positions 3351-4010, and of cloned cDNA derived from mRNAs encoded by this segment. One of these (mRNA VII) is shown to be unspliced, and has its 5'- and 3'-ends encoded by positions 3460 (determined by the nuclease S1 technique) and 3939, respectively. The mRNA sequence contains a single open reading frame for protein biosynthesis between the first available initiation triplet AUG at position 3481 to the stop codon UAA at position 3895. It can specify a polypeptide of 138 amino acids (14 098 daltons) which must be polypeptide IX of Ad7, as is evident from its mapping position, and from a comparison with the sequence of the protein IX gene of subgroup C adenovirus type 5.     

The nucleotide sequence of the polypeptide IX gene of human adenovirus type 3

The nucleotide sequence of the DNA segment encompassing the polypeptide IX gene of class B human adeno-virus serotype 3 (Ad3) has been determined using cloned restriction fragments. There is only a single, open translational reading frame capable of specifying a protein of 138 amino acids, comparable to the M_r 12000–13000 of protein IX detected in virions (Wadell, 1980). The corresponding region of a closely related class B virus, Ad7, is virtually identical (Dijkema et al., 1981), but the comparable segments of class C viruses Ad2 or Ad5 are much less homologous (Aleström et al., 1980; Maat et al., 1980). There are 150 single bp changes and 19 deletion-insertions, at least one frameshift, together affecting 210 nucleotides within the 455 bp comparison positions of the protein-coding regions of Ad2 (423 bp) and Ad3 (417 bp). Each of the 19 deletion-insertions involves an integral multiple of 3 bp in phase with the open translation frame. There is no “TATA” promoter box in Ad3 DNA at the position comparable to that of Ad2. The deduced protein sequences near the amino-terminus are extensively conserved between the two classes of viruses, but the carboxy-terminal portion and the nucleotide sequences flanking the gene are much more diverged. In both classes, these N- and C-terminal regions of the inferred proteins are linked by an alanine-rich chain, an arrangement suggestive of two functional domains.     

Methylation of late adenovirus 2 nuclear and messenger RNA.

Methylation of adenovirus 2 (Ad 2) late RNA was studied. RNA was double-labeled with [³H-methyl]-methionine and [¹⁴C]-uridine 15–20 h postinfection. Nuclear RNA (rRNA) and cytoplasmic RNA (mRNA) was extracted, and fractionated into polyA(+) and (?) molecules using poly(U)-Sepharose. Ad 2 specific RNA was purified by 2 cycles of hybridization to and elution from Ad 2 DNA immobilized on filters. The Ad 2 polyA(+) and (?) nRNA and mRNA fractions had the same ${}^3\text{H}{}^{14}\text{C}$ ratios, and were estimated to contain a minimum of 1.4 methylated nucleotides per 1000 bases. Viral RNA was digested with RNase T₂ and chromatographed on DEAE-Sephadex in 7 M urea at pH 7.6. All four Ad RNA fractions contained methylated constituents consistent with: (1) two classes of methylated “capped” 5′-termini with general structures m⁷ GpppN^mpNp and m⁷ GpppN^mpN^mpNp; (2) internal base methylations; (3) minor amounts of internal ribose 2′-0-methylations. Two classes of 5′-termini have previously been reported for animal cell mRNA, but not for mRNA from a variety of viruses. Internal methylations may be unique to RNA molecules transcribed in the nucleus, since they have not been found in RNA from cytoplasmic viruses. No gross differences were observed in the DEAE-Sephadex elution profiles of the methylated constituents of the four types of Ad 2 RNA. These results suggest that the majority of methylation events occur in the nucleus, and raise the possibility that Ad 2 methylated late nRNA may differ significantly from SV40 late nRNA (Lavi, S., and Shatkin, A.J. (1975) Proc. Natl. Acad. Sci. USA $\text{72}$, 2012–2016).     

N-6-methyl-adenosine in adenovirus type 2 nuclear RNA is conserved in the formation of messenger RNA

In the biogenesis of adenovirus type 2 messenger RNAs, methylation occurs at the 5′ end (cap) and to internal adenosine residues to yield N⁶-methyl-adenosine (m⁶A) (Sommer et al., 1976; Moss &; Koczot, 1976; Wold et al., 1976). The kinetics of accumulation of ³H from methyl-labeled methionine and ¹⁴C from uridine into Ad-2²-specific RNA was measured late in Ad-2 infection. As reported previously (Nevins &; Darnell, 1978a), the rate of accumulation of [¹⁴C]uridine label in nuclear RNA was approximately four- to fivefold faster than in the cytoplasmic RNA, indicating a conservation of about 20% for the total RNA. The initial rates of [³H]methyl label in m⁶A in nuclear RNA and in the cytoplasmic RNA were approximately equal, suggesting a complete (or nearly complete) conservation of m⁶A.In accord with the accumulation kinetics, the ratio of ³H to ¹⁴C was higher in cytoplasmic RNA than in nuclear RNA that hybridized to equivalent regions of the Ad-2 DNA.A mathematical model was designed to evaluate the accumulation of methyl label in m⁶A, taking into consideration the three major parameters that affect the accumulation curves: equilibration of the S-adenosyl-methionine pool, the nuclear dwell time of sequences destined to be mRNA, and the cytoplasmic stability of mRNA. The half-time ($\text{t}\text{1}\text{2}$) for pool equilibration was determined experimentally to be 22 minutes and the nuclear dwell time and the mean life-time of cytoplasmic mRNA were estimated from ¹⁴C label to be about 30 and 70 minutes, respectively.The model gave an excellent fit to the data when the $\text{t}\text{1}\text{2}$ for pool equilibration time of 24 ± 2 minutes, a nuclear dwell time of 25 ± 10 minutes, and a mean cytoplasmic mRNA life-time of 75 ± 30 minutes were used to evaluate accumulation curves. Even when data from a restricted region of the genome, $\text{40.5–52.6}$, which encodes the main portion of at least five 3′ co-terminal mRNAs whose spliced junction with the tripartite leader sequence varies from $\text{38, 40, 43, 45}$, and $\text{48}$ was analyzed, it appeared that m⁶A was conserved.Finally, m⁶A was found to be added in a brief label (3.5 min) mainly to nuclear molecules that were longer than any cytoplasmic RNA. The conservation of m⁶A and its addition prior to splicing raise the possibility that internal methylations are involved, in the formation of mRNA.     

Phosphorylation of a 60 kDa polypeptide from Xenopus oocytes blocks messenger RNA translation.

The stored mRNP particles of Xenopus oocytes contain protein kinase activity and two major phosphoproteins of 60 kDa (pp60) and 56 kDa (pp56). These proteins can be phospholabelled in the particles either in vivo or in vitro and then isolated by SDS-PAGE. On renaturing pp60 in the presence of globin mRNA, a stable RNA-protein complex is formed. The complex has a uniform density in Cs salt gradients, corresponding to the binding of about 10 protein molecules to each mRNA, probably at the poly(A) sequence. Compared with uncomplexed mRNA, the RNP complex is translated poorly both in vitro and in vivo. Translation of the complex can be regained after treatment with protein phosphatase. It is shown that dephosphorylation destabilizes the binding of protein to RNA, making the mRNA accessible for translation. Studies with native mRNP particles show that their translation also can be enhanced by dephosphorylation.     

Effect of different conformations of galactose messenger RNA on gene expression and messenger half-life in vitro

From a DNA-directed cell-free system, functional gal mRNA is obtained which directs the cell-free synthesis of the three galactose enzymes of Escherichia coli. A substantial fraction of this gal mRNA has the properties of a polycistronic messenger. Exposure to elevated temperatures in the presence or absence of magnesium ion results in pronounced changes of the capacity of this mRNA to give rise to the synthesis of the three enzymes. Depending on the conditions of the pre-treatment, the absolute amounts as well as the ratio of the three gene products synthesized can be changed. The different forms of gal messenger so obtained also exhibit different susceptibilities towards functional inactivation during the enzyme synthesis reaction. As the changes in template activity are reversible, it is concluded that the different treatments cause reversible transitions between different conformations of the gal mRNA.     

Hybridization maps of early and late messenger RNA sequences on the adenovirus type 2 genome.

Specific fragments of adenovirus type 2 DNA, generated by cleavage with restriction endonucleases endoR.EcoRI, endoR.HpaI and endoR.HindIII were used in hybridization-mapping experiments. The complementary strands of individual cleavage fragments were separated by the method of Tibbetts &; Pettersson (1974). Liquid hybridizations were performed with ³²P-labeled separated strands of cleavage fragments and messenger RNA extracted from cells early and late after adenovirus infection. The fraction of each fragment strand which was represented in “early” and “late” messenger RNA was determined by chromatography on hydroxylapatite. Early messenger RNA was found to be derived from four widely separated regions, two on the 1- and two on the h-strand (h- and l- refer to the strand with heavy and light buoyant density in CsCl when complexed with poly(U, G)). Messenger RNA, present exclusively late after infection, is derived from several locations, predominantly from the l-strand with a major block of continuous sequences extending between positions 0.25 and 0.65 on the unit map of the adenovirus type 2 genome.     

The structure of pre-messenger RNA and messenger RNA from erythroid cells

Pre-mRNA fractions (greater than 45 S) were characterized by electron microscopy. High salt concentrations (0.2 M ammonium acetate, pH 8) yield linear molecules of different length (0.5--17 micrometer). In 10% of the molecules a compact-nonlinear contour (cn-contour) is detectable at one end. A significant enhancement of the number of cn-contour carrying molecules is observed after binding pre-mRNA to poly(U)-sepharose. The terminal cn-contour could be the depiction of a secondary and/or tertiary structure including the poly(A)-tail. 9 S globin mRNA appear in 80% with virtually the same cn-contour as detected in pre-mRNA molecules. After denaturing the mRNA in 80% formamide--4M urea in connection with heating to 90 degrees C from 10 min, a percentage of 77% of stretched, linear molecules results. This structural transformation is reversible when the denatured RNA is precipitated and redissolved in 0.2 M ammonium acetate. 73% of the stretched molecules are characterized by a mean length of 0.44 micrometer. This value is twice as high as commonly assumed for a globin mRNA chain.     

Pre-early adenovirus 5 gene product regulates synthesis of early viral messenger RNAs.

The studies described here demonstrate that the expression of many early adenovirus mRNAs is dependent upon the activity of a pre-early viral product. This viral gene product is defective in adenovirus 5 host range (Ad hr) group I mutants. Adenovirus 5 host range mutants were previously isolated by their ability to replicate in the adenovirus 5-transformed human embryonic cell line 293 and by their inability to replicate efficiently in HeLa cells (Harrison, Graham and Williams, 1977). The group I complementation class of host range mutants has been mapped by marker rescue between 0 and 4.4 units (Frost and Williams, 1978). We have used the S1 nuclease gel technique to examine the expression of early mRNA after infection of HeLa cells with Ad5 hr group I and II mutants. The Ad5 hr group II mutants stimulate the synthesis of a wild-type pattern of early mRNAs. In contrast, infection of HeLa cells with Ad5 hr group I mutants gives rise to only two early mRNAs. These mRNAs map from 1.5–4.4 units, or in the same region as the Ad5 hr group I mutations. Since infection of HeLa cells with Ad5 hr group I mutants was defective for synthesis of cytoplasmic mRNAs complementary to three early regions in the right half of the genome and to the early region 4.5–11.0 units, we also analyzed nuclear RNA from these cells by the S1 nuclease gel technique for the presence of precursor RNA chains. Nuclear precursors were not detected in Ad5 hr group I-infected HeLa cells, suggesting that the gene product defective in these mutants is required for synthesis of stable nuclear RNA from the three early regions in the right half of the genome and from the early region 4.5–11.0 units.