Lysine tRNA is the predominant tRNA in murine mammary tumor virus

The method of aminoacylation and subsequent identification of the esterified amino acids was used to characterize the transfer RNAs in murine mammary tumor virus. Lysine tRNA was the major tRNA in both “free” 4S RNA and “7OS-associated” 4S RNA in virus derived from either tissue culture or mouse milk.     

Effects of aphidicolin on retrovirus DNA synthesis in vivo

Renaturation of $\text{Aequorea}$ green-fluorescent protein (A-GFP) was achieved for the first time following denaturation in guanidine-HCl or acid. Denaturation was accompanied by the concerted loss of visible fluorescence, alteration of absorption characteristics, and large negative deflection of CD signal in the far UV. Dialysis of a guanidine-denatured sample at pH 8 resulted in 64% renaturation (return to native absorption) and neutralization of an acid-denatured sample restored 90% of the native absorption. Renatured GFP is highly fluorescent and indistinguishable from native GFP with respect to the shape of excitation and emission spectra. Both native and denatured proteins exhibit resistance to trypsin hydrolysis and have identically broad pH and heat stability profiles, all of which suggest full renaturation.     

Transfer ribonucleic acid nucleotidyltransferase activity in virions of type-C viruses.

A nucleotidyltransferase activity has been found associated with a number of mammalian and avian oncornaviruses. This activity catalyzes the incorporation of adenosine monophosphate and cytosine monophosphate into acid insoluble forms. The transferase activity from Rauscher murine leukemia virus has been characterized. The endogenous reaction is stimulated by various tRNAs particularly the 4S RNA isolated from Rauscher leukemia virus, whereas other RNAs have no effect. The product of the reaction is alkali and RNase sensitive, insensitive to DNase, and its size is similar to tRNA. Finally, the terminal nucleotide analysis of the product of the reaction indicates the presence of a CCA terminus. The properties of the activity found in the type-C viruses are in accord with those of known tRNA nucleotidyltransferases from other sources.     

Separation procedure and sugar composition of oligosaccharides in the surface glycoprotein of friend murine leukemia virus

The sugar composition of the surface glycoprotein from Friend murine leukemia virus was determined by gas-liquid chromatography of the alditol acetates and by the thiobarbituric acid method, respectively. N-Acetylglucosamine, mannose, galactose, sialic acid and fucose were found in a molar ratio around 15.2:11.6:7.4:3.3:1.0. Ten ogligosaccharide fractions were obtained from glycoprotein preparations by a suitable sequence of degradation (with pronase, endo-β-N-acetylglucosaminidase H, neuraminidase, and by hydrazinolysis) and separation procedures (concanavalin A-affinity chromatography and gel filtration). The qualitative sugar composition of these fractions was analyzed by in vivo labelling with D-[6-³H]glucosamine, D-[2-³H]mannose, D-[6-³H]galactose, or L-[6-³H]fucose, and their molecular weights were estimated from the gel elution volumina. Four fractions of N-glycosidically linked oligosaccharides of the oligomannosidic (‘high mannose’) type oligomannosidic₇-oligomannosidic₁₀, about seven to ten sugar residues), two of the mixed (M₁₁ and M₁₂), and four of the N-acethyllactosaminic (‘complex’) type (N-acetyllactosaminic₉, probably nine sugar residues; (N-acetyllactosaminic_a-N-acetyllactosaminic_c, size unknown) were thus identified.     

A comparison of the enzymatic responses of the DNA polymerases from four RNA tumor viruses.

DNA polymerases from avian, feline, murine and simian RNA tumor viruses exhibit substantial differences in optimal assay conditions and vary widely in their template-primer preferences. Avian DNA polymerase utilizes both natural and synthetic template-primers efficiently in the presence of Mg⁺⁺ as well as Mn⁺⁺. By contrast, the mammalian viral DNA polymerases are much more responsive to poly(A)·oligo(dT) than to other template-primers, and exhibit up to 20-fold greater activity with Mn⁺⁺ than with Mg⁺⁺. In addition, simian sarcoma virus DNA polymerase shows no detectable response to poly(C)·oligo(dG) over a wide variety of conditions stimulatory to the other viral enzymes.     

RNA metabolism of murine leukemia virus: detection of virus-specific RNA sequences in infected and uninfected cells and identification of virus-specific messenger RNA

Virus-specific RNA sequences were detected in mouse cells infected with murine leukemia virus by hybridization with radioactively labeled DNA complementary to Moloney murine leukemia virus RNA. The DNA was synthesized in vitro using the endogenous virion RNA-dependent DNA polymerase and the DNA product was characterized by size and its ability to protect radioactive viral RNA. Virus-specific RNA sequences were found in two lines of leukemia virus-infected cells (JLS-V11 and SCRF 60A) and also in an uninfected line (JLS-V9). Approximately 0.3% of the cytoplasmic RNA in JLS-VII cells was virus-specific and 0.9% of SCRF 60A cell RNA was virus-specific. JLS-V9 cells contained approximately tenfold less virus-specific RNA than infected JLS-VII cells. Moloney leukemia virus DNA completely annealed to JLS-VII or SCRF 60A RNA but only partial annealing was observed with JLS-V9 RNA. This difference is ascribed to non-homologies between the RNA sequences of Moloney virus and the endogenous virus of JLS-V9 cells.Virus-specific RNA was found to exist in infected cells in three major size classes: 60–70 S RNA, 35 S RNA and 20–30 S RNA. The 60–70 S RNA was apparently primarily at the cell surface, since agents which remove material from the cell surface were effective in removing a majority of the 60–70 S RNA. The 35 S and 20–30 S RNA is relatively unaffected by these procedures. Sub-fractionation of the cytoplasm indicated that approximately 35% of the cytoplasmic virus-specific RNA in infected cells is contained in the membrane-bound material. The membrane-bound virus-specific RNA consists of some residual 60–70 S RNA and 35 S RNA, but very little 20–30 S RNA. Virus-specific messenger RNA was identified in polyribosome gradients of infected cell cytoplasm. Messenger RNA was differentiated from other virus-specific RNAs by the criterion that virus-specific messenger RNA must change in sedimentation rate following polyribosome disaggregation. Two procedures for polyribosome disaggregation were used: treatment with EDTA and in vitro incubation of polyribosomes with puromycin in conditions of high ionic strength. As identified by this criterion, the virus-specific messenger RNA appeared to be mostly 35 S RNA. No function for the 20–30 S was determined.     

Triplication of a unique genetic segment in a simian virus 40-like virus of human origin and evolution of new viral genomes

The non-defective (heavy) virions from a simian virus 40-like virus (DAR virus) isolated from human brain have been serially passaged at high input multi-plicities in primary monkey kidney cells. The ³²P-labeled, progeny DAR-viral genomes have been purified and tested for sensitivity to the R_I restriction endouclease from Escherichia coli (Eco RI3 restriction nuclease). The parental DAR-viral genomes share many physical properties with “standard” simian virus 40 DNA and are cleaved once by the Eco RI restriction nuclease. After the fourth serial passage, three populations of genomes could be distinguished: Eco RI resistant, Eco RI sensitive (one cleavage site) and Eco RI “supersensitive” (three, symmetrically-located, cleavage sites). The Eco RI cleavage product of the “supersensitive” form is one-third the physical size (10.4 S) of simian virus 40 DNA and reassociates about three times more rapidly than sheared, denatured simian virus 40 DNA. From the fourth to the eighth serial passages, the genomes containing this specific triplication of viral DNA sequences were selected for and became the predominant viral DNA species.     

Characterization of a rearrangement in viral DNA: mapping of the circular simian virus 40-like DNA containing a triplication of a specific one-third of the viral genome

Serial passage of the non-defective form of a simian virus 40-like virus (DAR) isolated from human brain results in the appearance of three distinct classes of supercoiled DNAs: R_I resistant, R_I sensitive (one cleavage site) and R_I “supersensitive” (three cleavage sites). The R_I cleavage product of the “super sensitive” form is one-third the physical size of simian virus 40 DNA (10.4 S) and reassociates about three times more rapidly than “standard” viral DNA. To identify the portions of the DAR genome present in the 10.4 S segment, the plus strand of each of the 11 fragments of ³²P-labeled simian virus 40 DNA, produced by cleavage with the Hemophilus influenzae restriction endonuclease, was hybridized in solution with the sheared R_I cleavage product of the “supersensitive” class of viral DNA. Reaction was observed with fragments located in two distinct regions of the simian virus 40 genome: (1) Hin-A and C; (2) Hin-G, J, F and K.Further studies indicated that simian virus 40 complementary RNA transcribed in vitro with Escherichia coli RNA polymerase from one strand of simian virus 40 DNA reacts with both strands of the denatured 10.4 S cleavage product when hybridization is monitored with hydroxyapatite. Treatment of the 10.4 S DNA-simian virus 40 cRNA hybrid with the single-strand spcific nuclease, S₁, converted approximately 50% of the radioactive counts to an acid-soluble product. These results indicate that the 10.4 S product contains a transposition of sequences originally present on one of the DAR DNA strands to the other strand. Examination of heteroduplexes formed between the 10.4 S segment and unique linear forms of DAR DNA produced with the R · Eco RI restriction endonuclease have confirmed these observations. Thus it appears that a molecular rearrangement(s) has resulted in the recombination and inversion of viral DNA sequences from two separate loci on the parental DAR genome. This 1.1 × 10⁶ dalton segment is reiterated three times in a supercoiled molecule equivalent in physical size to parental DAR DNA.     

Relationship between expression of epidermal growth factor and simian virus 40 T antigen in a line of transgenic mice

The pattern of expression of the simian virus 40 (SV40) T antigen gene and resultant dysplasia were re-examined in a line of transgenic mice in which the T antigen gene was under the control of the SV40 early promoter. We found that T antigen expression in the kidney, and resulting dysplastic lesions, occurred exclusively in the distal convoluted tubules and the ascending limbs of Henle. Epidermal growth factor (EGF) expression in the kidney of normal mice was similarly immunolocalized. The correlation between high EGF immunoreactivity in normal mouse tissues and T antigen expression in the transgenic counterpart was also seen in the choroid plexus epithelium and in the submandibular glands of male mice. T antigen was not found in the submandibular gland of transgenic females. Similarly, EGF was only rarely detected in the normal female submandibular gland. In contrast to the correlation between T antigen expression in the transgenic mice and EGF expression in the corresponding tissues of the normal mice, within the dysplastic lesions of the transgenic mice EGF expression was severely diminished. Adenocarcinomas of the male submandibular gland from another line of transgenic mice that expresses theInt-1 transgene, showed similarly reduced levels of immunostaining for EGF. Thus, reduced expression of EGF might be a general feature of dysplasia and tumorigenesis in those tissues that normally express EGF.     

Studies of simian virus 40 DNA. VII. A cleavage map of the SV40 genome

A physical map of the Simian virus 40 genome has been constructed on the basis of specific cleavage of Simian virus 40 DNA by bacterial restriction endonucleases. The 11 fragments produced by enzyme from Hemophilus influenzae have been ordered by analysis of partial digest products and by analysis of an overlapping set of fragments produced by enzyme from Hemophilus parainfluenzae. In addition, the single site in SV40 DNA cleaved by the Escherichia coli R_I restriction endonuclease has been located. With this site as a reference point, the H. influenzae cleavage sites and the H. parainfluenzae cleavage sites have been localized on the map.