Mapping the transcription site of the SV40-specific late 16 S mRNA.

This paper describes the purification of polysomal RNA from SV40-lytically infected CV1 (monkey) cells and separation of the two distinct classes of SV40-specific mRNA sedimenting at 16 S and 19 S. These classes have been hybridized with the whole SV40 DNA genome as well as with the SV40 Hind fragments. The results have permitted the mapping of SV40-specific late 16 S mRNA from approximately 0.945 to 0.175 map units.     

Transcription map for adenovirus type 12 DNA.

The regions of the adenovirus type 12 genome which encode l- and r-strand-specific cytoplasmic RNA were mapped by the following procedure. Radioactive, intact, separated complementary strands of the viral genome were hybridized to saturating amounts of unlabeled late cytoplasmic RNA. The segments of each DNA strand complementary to the RNA were then purified by S1 nuclease digestion of the hybrids. The arrangement of the coding regions of each strand was deduced from the pattern of hybridization of these probes to unlabeled viral DNA fragments produced by digestion with EcoRI, BamHI, and HindIII.. The resulting map is similar, if not identical, to that of adenovirus type 2. The subset of the late cytoplasmic RNA sequences which are expressed at early times were located on the map by hybridizing labeled, early cytoplasmic RNA to both unlabeled DNA fragments and unlabeled complementary strands of specific fragments. Early cytoplasmic RNA hybridized to the r-strand to EcoRI-C and BamHI-B and to the l-strand of BamHI-E. Hybridization to BamHI-C was also observed. The relative rates of accumulation of cytoplasmic RNA complementary to individual restriction fragments was measured at both early and late times. Early during infection, most of the viral RNA appearing in the cytoplasm was derived from the molecular ends of the genome. Later (24 to 26 h postinfection) the majority of the newly labeled cytoplasmic RNA was transcribed from DNA sequences mapping between 25 and 60 map units on the genome.     

Adenovirus type 12-specific RNA sequences during productive infection of KB cells.

The complementary strands of adenovirus type 12 DNA were separated, and virus-specific RNA was analyzed by saturation hybridization in solution. Late during infection whole cell RNA hybridized to 75% of the light (1) strand and 15% of the heavy (H) strand, whereas cytoplasmic RNA hybridized to 65% of the 1 strand and 15% of the h strand. Late nuclear RNA hybridized to about 90% of the 1 strand and at least 36% of the h strand. Double-stranded RNA was isolated from infected cells late after infection, which annealed to greater than 30% of each of the two complementary DNA strands. Early whole cell RNA hybridized to 45 to 50% of the 1 strand and 15% of the h strand, whereas early cytoplasmic RNA hybridized to about 15% of each of the complementary strands. All early cytoplasmic sequences were present in the cytoplasm at late times.     

Expression of Endogenous Retroviral Genes in Leukemic Guinea Pig Cells

The expression of guinea pig retrovirus (5-bromodeoxyuridine[BUdR]-induced GPV) was studied in guinea pig L(2)C leukemic lymphoblasts by use of molecular hybridization of viral complementary DNA (cDNA) to cellular RNA. It was found that L(2)C leukemic lymphoblasts, leukemic spleen, and BUdR-induced virus-producing cells contain virus-specific RNA: 0.05% (800 to 960 copies per cell), 0.02% (360 copies per cell), and 0.3% (5,120 copies per cell), respectively. Adult normal liver and spleen, on the other hand, contain less than 0.2 copy of viral RNA per cell. Both BUdR-induced cells and L(2)C leukemic lymphoblasts contained 14S, 22S, 35S, and 70S RNA species of total and cytoplasmic virus-specific RNA as determined by sucrose velocity gradient analysis and hybridization of sucrose gradient fractions to cDNA. Virus-specific mRNA was identified in both BUdR-induced cells and L(2)C leukemic lymphoblasts by the criterion that it cosedimented with purified polyribosomes in a sucrose gradient and that it changed to a lower sedimentation value if polyribosomes were disaggregated with EDTA prior to centrifugation. Virus-specific mRNA obtained from either the polyribosome region of purified polyribosomes or the released messenger region of EDTA-disaggregated purified polyribosomes consisted of 14S, 20S, and 35S species in both BUdR-induced cells and L(2)C leukemic lymphoblasts. Hybridization of cDNA to the RNA of L(2)C leukemic lymphoblasts and BUdR-induced cells was essentially complete. Additionally, leukemic lymphoblast RNA could displace 95% of the hybridization of BUdR-induced GPV 70S RNA to guinea pig DNA. The midpoints of thermal denaturation of hybrids formed between GPV cDNA and the RNA of either L(2)C leukemic lymphoblasts or the 70S RNA of BUdR-induced GPV were both 89 degrees C in 2x concentrated 0.15 M NaCl plus 0.015 M sodium citrate. These results show that BUdR-induced GPV genes are essentially completely expressed in L(2)C leukemic lymphoblasts and that virus-specific mRNA is present, although fewer copies of RNA are present in L(2)C leukemic lymphoblasts than in BUdR-induced cells.     

Expression of the gene encoding the adenovirus DNA terminal protein precursor in productively infected and transformed cells.

The major product of in vitro translation of early RNA prepared from H5ts125-infected cells and selected by hybridization to adenoviral DNA fragments spanning the region from 14.7 to 31.5 map units had been shown to be identical to the 87-kilodalton terminal protein precursor. A 72- to 75-kilodalton polypeptide whose rRNA can be selected by DNA from this same region and made in the presence of anisomycin was indistinguishable from the 72-kilodalton single-stranded DNA-binding protein encoded by the region from 60.1 to 66.6 map units. The accumulation of cytoplasmic RNA sequences complementary to these l-strand genes under various conditions of infection and in certain lines of transformed cells has been investigated by solution hybridization of cytoplasmic RNA to the separated strands of restriction endonuclease fragments of adenoviral DNA. During the early phase, RNA sequences complementary to the region from 11.6 to 36.7 map units were present at a concentration of 10 to 60 copies per cell, regardless of the nature of the block used to inhibit viral DNA synthesis. By 24 h after infection in the absence of any such block, sequences complementary to the regions from 11.6 to 18.2 map units (IVa2) and from 18.6 to 36.7 map units (E2B) accumulated to concentrations of 4,800 and 280 copies per cell, respectively. The ratio of cytoplasmic E2A RNA sequences to E2B RNA sequences remained close to 10:1 throughout the time period investigated. Of the transformed cell lines which retained E2B DNA sequences that were examined, only the T2C4 line expressed these sequences in cytoplasmic RNA. The implications of these observations for regulation of expression of the adenoviral early l-strand genes are discussed.     

The translational map of the Autographa californica nuclear polyhedrosis virus (AcNPV) genome.

We have mapped early and late viral gene products expressed in Autographa californica nuclear polyhedrosis virus ( AcNPV )-infected Spodoptera frugiperda cells by cell-free translation of virus-specific RNA which was selected by hybridization to cloned restriction endonuclease fragments of AcNPV DNA. Proteins synthesized in vitro were labeled with [35S]methionine and analyzed by SDS-polyacrylamide gel electrophoresis followed by fluorography. At least four early AcNPV -specific polypeptides were found which mapped in two regions of the genome (9-25 and 43-59 map units). These early mRNAs are also synthesized at late times in the infection cycle. Cell-free translation of restriction fragment-selected late AcNPV -specific RNA (24 h post-infection) resulted in the identification and mapping of 24 viral proteins. Curiously, the region between approximately 70 and 80 map units on the viral genome has been found silent with respect to mRNA which is translatable in a cell-free system. However, there may be RNA transcribed from this viral DNA segment.     

Differences in sequence content of nuclear and cytoplasmic polyribosomal RNA from adenovirus-infected cells.

RNA molecules from nuclear and cytoplasmic polyribosomes of adenovirus-infected HeLa cells were compared by hybridization to analyse the sequence content. Nuclear polyribosomes were released by exposure of intact detergent-washed nuclei to poly(U) and purified. Cytoplasmic polyribosomes were also purified from the same cells. To show that nuclear polyribosomes contain ribosomes linked by mRNA, polyribosomes were labelled with methionine and uridine in the presence of actinomycin D in adenovirus-infected cells. Purified nuclear polyribosomes were treated with EDTA under conditions which dissociate polyribosomes into ribosomes and subunits with a simultaneous release of mRNA, and sedimented. The treatment dissociated these polyribosomes, releasing the mRNA from them. Radiolabelled total RNA from each polyribosome population was fractionated in sucrose gradients into several pools or hybridized to intact adenovirus DNA to select virus-specific RNA. Sucrose-gradient-fractionated pool-3 RNA (about 28S) and virus-specific RNA were then hybridized to fragments of adenovirus DNA cleaved by restriction endonucleases SmaI, HindIII and EcoRI by the Southern-blot technique and by filter hybridization. The results showed that nuclear RNA contained sequences, from about 0 to 18 map units, which were essentially absent from cytoplasmic RNA. Furthermore, the amount of virus-specific RNA for a particular sequence was also different in the two populations.     

A very large repeating unit of mouse DNA containing the 18S, 28S and 5.8S rRNA genes

The organization of the 18S, 28S and 5.8S rRNA genes in the mouse has been elucidated by mapping with restriction endonucleases Eco RI, Hind III and Bam HI. Ribosomal DNA fragments were detected in electrophoretically fractionated digests of total nuclear DNA by in situ hybridization with radioiodinated rRNAs or with complementary RNA synthesized directly on rRNA templates. A map of the rDNA which includes 13 restriction sites was constructed from the sizes of rDNA fragments and their labeling by different probes The map indicates that the rRNA genes lie within remarkably large units of reiterated DNA, at least 44,000 base pairs long. At least two, and possibly four, classes of repeating unit can be distinguished, the heterogeneity probably residing in the very large nontranscribed spacer region. The 5.8S rRNA gene lies in the transcribed region between the 18S and 28S genes.