Biomedical subjects
Publications and source records attributed to H Westphal.
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One measurable biological activity of early adenovirus genes is their ability to promote growth of the defective adeno-associated virus, AAV. We have identified an ordered sequence of communications among the early genes of adenovirus type 2 (Ad2) that results in expression of the helper activity. We purified DNA fragments and mRNAs corresponding to early Ad2 regions E1, E2A, E3 and E4 and injected them via glass capillaries into AAV-infected cells. DNAs were placed in the nucleus, mRNAs in the cytoplasm. AAV DNA and proteins synthesized in response to the injected Ad2 nucleic acids were extracted from as few as 100 cells and identified by gel electrophoresis. Our results reveal a cascade of early Ad2 gene regulation, E1 leads to E2A leads to E4, with E4 providing the helper effect for AAV.
Ad2+ND4del is an adenovirus type 2-simian virus 40 hybrid virus nondefective for growth in human cells. The virus was first observed when stocks of Ad2+ND4, a hybrid isolated from primary monkey kidney cells, were propagated in human cells. This paper describes the DNA sequence at two sites of DNA recombination, the site of the left adenovirus type 2-simian virus 40 junction and the site of a deletion of internal simian virus 40 sequences. Since the deletion was observed when the virus was switched from monkey to human cells, an analysis of gene expression in the region of DNA rearrangement may prove useful for the elucidation of molecular events that accompany virus growth in different hosts.
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Adenovirus type 2 mRNAs were injected via glass capillaries into Vero cells, a line of African green monkey kidney cells permissive for adenovirus growth. Polypeptides synthesized after injection were labeled with 35S-labeled amino acids, precipitated with antiviral sera, and analyzed by polyacrylamide gel electrophoresis. Both early and late viral mRNAs give rise to authentic polypeptides. The in vivo translation of mRNAs can be measured as late as 24 hr after injection. The ability to analyze the protein products of microinjected mRNAs directly should greatly extend the applications of the procedure. Vero cells injected with early mRNA from adenovirus type 2 support the growth of adenovirus-associated virus, a defective virus that is dependent on adenovirus helper functions. This result demonstrates that a measurable biological activity, the ability to overcome the defectiveness of adenovirus-associated virus, resides in early adenovirus mRNA.
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We describe the structure of cytoplasmic RNA species transcribed from the DNA of adenovirus-associated virus, a defective parvovirus. The RNA was hybridized with minus strand template DNA and visualized in the electron microscope. Alternatively, the DNA.RNA duplex molecules were digested with nuclease S1 or Escherichia coli exonuclease VII and analyzed by agarose gel electrophoresis. A set of RNA species was observed with 5' terminal at map positions 5, 13, 19, or 39 and a 3' terminus and poly(A) tail at position 96 (one map unit is equivalent to 1% of genome length). Most of these RNAs are spliced and lack sequences approximately between positions 40 and 49. Some RNA preparations also contained unspliced molecules with 5' and 3' terminal at positions similar to those in the spliced RNA.
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Purified and partially resolved vesicular stomatitis virus (VSV) messenger RNA has been annealed to the VSV genomic RNA and visualized in the electron microscope under conditions in which duplex regions have a wider image width than single-stranded RNA. The locations of the intercistronic boundaries between the messages have been mapped on the VSV genome. The contour of the double-stranded regions is occasionally interrupted by looped-out single-stranded RNA. The loops are comprised of post transcriptionally synthesized polyadenylate. Most of these structures are found at the intercistronic boundaries and covalently bridge adjacent message sequences. In this paper, we discuss the possible significance of these loops.
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Nuclei of KB cells harvested at late stages of productive infection with adenovirus type 2 (Ad2) harbor RNA molecules which measure up to 13 mum in length, as determined by electron microscopy of denatured RNA. While some of the molecules display features of secondary structure that are characteristic for precursor rRNA, our interest was in those showing almost no intramolecular folding. When hybridized to double stranded viral DNA under conditions which favor RNA:DNA duplex formation, nuclear AD2 RNA displaces the homologous DNA region and generates R loop structures whose size is proportional to the length of the hybridizing RNA. Slowly sedimenting RNA forms small R loops, whereas RNA of high sedimentation velocity generates loops that span a large proportion of the DNA length. Using SV40 sequences within Ad2+ND4 hybrid DNA as a position marker, we oriented many of the R loops on the conventional Ad2 map. Our analysis was restricted to the most abundant sequences of late Ad2 nuclear RNA participating in R loop formation. A small but significant proportion of large RNA generates loops between map positions 0.3 and 0.9. The much more frequent RNA of intermediate size (although larger than mRNA) hybridizes with midpoints near map positions 0.55 and 0.88--that is, near the gene locations for hexon and fiber. Our findings are compatible with the idea that the nuclear RNAs visualized in this study are intermediates in a processing pathway leading to mature forms of late Ad2 mRNA.
Separated strands of adenovirus DNA were annealed with early cytoplasmic RNA and visualized in the electron microscope. DNA-RNA duplex regions within the DNA filaments could be recognized by their heavy contour. This contour was often interrupted at distinct locations by loops of displaced, single-stranded DNA. Loops have been observed and mapped in all four early regions of the genome. The structures appear to signal hitherto unknown mechanisms of eukaryotic gene expression.
R loops were generated with late adenovirus type 2 (Ad2) mRNA in double-stranded viral DNA, and visualized by electron microscopy. Unpaired DNA sequences in Ad2:Ad2+ND4 heteroduplex DNA served as a visual marker for the orientation of R loops with respect to the conventional DNA map. The most abundant classes of late Ad2 mRNA observed by this technique hybridized, in order of R-loop frequency, with midpoints near posit1ons 0.57, 0.88, 0.77, and 0.40 to 0.50 of the DNA map. The R loop at position 0.57, 0.88, 0.77, and 0.40 containing the hexon gene; the one at position 0.88 corresponded to a region containing the fiber gene. The relative frequencies of these two R loops paralleled those of the encoded gene products. The mRNA sizes, calculated from those of the respective R loops, were slightly larger than needed to code for these polypeptides. Using the R-loop technique, two locations at which adjacent mRNA's hybridized to different strands were accurately mapped at positions 0.61 and 0.91 of the DNA. The map positions of late Ad2 mRNA correlated well to published RNA and protein maps.
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Late adenovirus messenger RNA was annealed to complementary regions of partially melted viral double-stranded DNA. The RNA. DNA hybrid regions within the DNA molecules were visualized as loops in the electron microscope. Loops occurred at several regions of the DNA, most frequently, however, at a location near the center of the molecule. This hybridization technique appears well suited for an accurate mapping of messenger RNA, as well as for studies of RNA processing.
Escherichia coli ribonuclease III cleaves adenovirus messenger RNA and mammalian 28S and 18S ribosomal RNA. Fragmentation is not random, but in each case a specific collection of products is generated. This points to the potential use of the enzyme as a tool for specific fragmentation of RNA. Cleavage by RNase III abolishes the capability of adenovirus messenger RNA to direct cell-free synthesis of virus polypeptides.
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