Genomic studies of the Lucké tumor herpesvirus (RaHV-1).

Ranid herpesvirus 1 (RaHV-1) is the etiological agent of the Lucké renal adenocarcinoma of the North American leopard frog Rana pipiens. Construction of cosmid libraries containing RaHV-1 DNA inserts allowed the derivation of a BamHI map for the viral genome. Summation of fragment sizes indicates that the genome is 217 kbp in size, a value in accordance with the most recent published estimate (220 kbp) obtained by field-inversion gel electrophoresis. The DNA sequence of the 39,757-bp insert in 1 cosmid (cos54) was determined and was predicted to contain 21 complete and 3 partial genes. In all, 12 genes have distant counterparts in a fish herpesvirus (ictalurid herpesvirus 1) and are present in 2 blocks, 1 of which is relatively inverted. This indicates that RaHV-1 belongs to the fish virus lineage of the herpesvirus family rather than to the lineage populated by mammalian and avian viruses. The remaining 12 genes in cos54 lack counterparts in any other herpesvirus. One of these encodes a putative DNA (cytosine-5) methyltransferase. This raises the possibility that biological processes induced in the host by RaHV-1 might involve methylation of cellular DNA by the viral enzyme.

Structure and transcription of the gene for translation elongation factor 1 subunit alpha of zebrafish (Danio rerio).

The zebrafish gene for translation elongation factor 1 alpha (EF1 alpha) was isolated from a phage Lambda genomic library and sequence and structure determined. One gene copy of EF1 alpha per haploid set of chromosomes was found and no processed pseudogenes. A highly active promoter region was localized to a 277 bp PstI/PvuII fragment beginning 240 bp upstream from the tsp, but no transcription enhancing, or silencing activity was observed within 1 kbp upstream, or downstream from the promoter. Expression of EF1 alpha appears to be developmentally regulated.

Zebrafish translation elongation factor EF1 alpha mRNA: sequence and secondary structures.

We have determined the complete sequence of the translation elongation factor EF1 subunit alpha (EF1 alpha) mRNA of zebrafish, and the 3'-untranslated sequence of EF1 alpha mRNA of halibut. The 5'-untranslated leader sequence of the EF1 alpha mRNA starts with a polypyrimidine tract. This feature is shared with the mRNAs for ribosomal proteins, where it affects the utilization of mRNA by ribosomes. However, the secondary structures of these leader sequences may differ. 5'-Polypyrimidine tracts of vertebrate EF1 alpha mRNAs participate in the formation of stable stem-loop structures, whereas those of 15 randomly chosen mRNAs for ribosomal proteins do not. This difference may provide a basis for differential control of translation for the two classes of mRNA. The 3'-untranslated sequences of vertebrate EF1 alpha mRNA have diverged little during evolution. Analyses of sequence and putative secondary structures suggest that both sequence-specific interactions and secondary structures may have contributed to sequence conservation.

Fate of herpesvirus DNA in embryos and tadpoles cloned from Lucké renal carcinoma nuclei.

The Lucké renal carcinoma of the northern leopard frog, Rana pipiens, has a herpesvirus aetiology. Lucké tumour nuclei inserted into enucleated frogs' eggs produce development to the swimming tadpole stage. Tissue from these tumour nuclear transplant animals can be induced to survive and differentiate further by allografting to normal tadpoles. We wished to ascertain whether the aetiological agent, the Lucké tumour herpesvirus (LTV), persists in the animals produced by tumour nuclear transplantation. The polymerase chain reaction was used to amplify a 1.2 kbp Hind III restriction fragment of LTV DNA in whole animal homogenates prepared from tumour nuclear transplant tadpoles and normal tadpoles fertilized in vitro. The LTV fragment was not present in the majority (31 of 34) of the cloned animals derived from tumour nuclei, nor was it present in any of five normal tadpoles. Either the 1.2 kbp fragment of LTV DNA was eliminated from most of the cloned animals during the massive reprogramming of the neoplastic genome initiated by insertion of the tumour nuclei into egg cytoplasm, or the nuclei selected for transplantation were primarily those lacking this fragment of LTV DNA. The limited development of the tumour nuclear tadpoles was probably not due to the presence of these viral sequences, but rather reflected the limited plasticity of the tumour cell genome as assayed by nuclear transplantation. Failure to detect the 1.2 kbp fragment of LTV DNA in the majority of mitotic progeny of the Lucké tumour genome does not imply that other parts of the viral genome do not persist.(ABSTRACT TRUNCATED AT 250 WORDS)

Parasexual crosses and hybrid selection with a near haploid variant of the epithelioma papulosum cyprini, cell line EPC: a tissue culture model for the analyses of dominance, recessiveness, and complementation of mutant phenotypes.

We describe techniques for insertional mutagenesis of tissue-cultured piscine cells in which we use transfection with G418 and hygromycin B resistance-conferring plasmids, cell matings by electrofusion, and positive selections of fusion hybrids by dual challenge with the antibiotics G418 and hygromycin B. These techniques are designed to facilitate genetic and molecular analyses of tissue-cultured cells. The experiments were conducted with EPC-1, a new variant of the carp epithelioma cyprini cell line. EPC, with a near haploid number of chromosomes, EPC-1 retains cell morphology and growth characteristics of EPC, including anchorage independence, but shows a higher degree of contact inhibition. The number of metaphase chromosomes of EPC-1 is 53, as opposed to 96 reported for EPC.

The presence of DNA sequences of the Lucké herpesvirus in normal and neoplastic kidney tissue of Rana pipiens.

The Lucké tumour herpesvirus (LTV) is the aetiological agent of the Lucké renal adenocarcinoma of the northern leopard frog, Rana pipiens. LTV virions can be detected by electron microscopy in renal adenocarcinomata after prolonged exposure to low temperature. Tumours maintained at warm temperatures do not contain viral particles. To gain insight into the processes of viral infection, replication and oncogenesis, evidence was sought for the presence of LTV DNA in warm renal tumours and normal renal tissue. The polymerase chain reaction was used to amplify a Hind III restriction fragment of LTV DNA in tissue homogenates. LTV DNA was detected in a significant percentage of normal kidneys as well as in "virus-free" warm tumours and virus-containing cold tumours.

Genomic plasticity of the Lucké renal carcinoma: a review.

The differentiation potential of the Lucké renal carcinoma of the northern leopard frog, Rana pipiens, can be characterized by the nuclear transplantation procedure. Transplantation of tumor nuclei into activated and enucleated ova results, in the best of cases, in swimming larvae which fail to feed. The larvae die in about 10 to 14 days. Rescue of tumor nuclear transplantation tadpole tissue, destined to die, has been accomplished by allografting fragments of that tissue to normal hosts. The allografts persist and differentiate a diversity of tissues which cannot be distinguished by histological analysis from allografted normal control tissue. Allografts are an imperfect mode of assay for histological competence because of the immune response of the host. Lymphocytes and eosinophils invade the grafts in about 40 days. The host immune response occurs in both experimental and control allografts. Consequently, we believe that added histogenetic potential exists in the genome of the Lucké renal carcinoma. We propose that unexpressed differentiative potential of the grafted tissue can be extracted by abrogation of the immune response of the host. A herpesvirus is the etiological agent of the Lucké renal carcinoma. We currently seek to detect viral DNA in tissue derived from tumor nuclear transplant embryos. The presence of the viral genetic material in normal mitotic progeny of Lucké tumor cells, if demonstrated, raises the question of the long-term stability of differentiated cells derived from a virus tumor. Alternatively, absence of viral DNA in the tumor nuclear transplant tissue would suggest that normal differentiation ensues after elimination of the oncogenic DNA from that tissue. Loss of viral DNA may prognosticate stable differentiation.

Structure of transcriptionally active chromatin: radiological evidence for requirement of torsionally constrained DNA.

Synthesis of alpha- and beta-globin RNA in DMSO-induced Friend's erythroleukemia cells and synthesis of immunoglobulin gamma- and kappa-chain RNA, total RNA, 5S RNA, and tRNA in mouse myeloma cells (MPC-11) was inhibited by gamma-irradiation. For all RNA species, synthesis decreased nearly exponentially as a function of radiation dose, whereas RNA size distributions, turnover rates, and specific activities of radioactively labeled RNA were affected only insignificantly. D37 values for the loss of synthesis of various RNA species correspond to target sizes ranging from 21,000 to 53,000 kd, or 30-80 kbp of DNA. These target sizes are several-fold larger than the structural genes in question; however, they correspond well with the size of DNA loops, or "domains" constrained by the nuclear matrix. The data suggest that the eukaryotic transcription unit is the torsionally constrained chromatin loop, transcription of which may be inactivated, or significantly reduced by a DNA single-strand break.

Interactions between structure transitions in a torsionally constrained DNA.

We used S1 nuclease cleavage in conjunction with gel electrophoresis to evaluate torsion-induced cruciform extrusion at two inverted repeat sequences, IRS-B and IRS-C of plasmid pUC12. These structure transitions affect each other through competition for the available torsional free energy according to their relative energies of activation and the magnitude of DNA duplex unwinding associated with each transition. They can be modulated by the level of DNA negative torsion. Interplays between transition sequences occur over long distances and are independent of relative orientation of transition sites. DNA binding factors that enhance or repress structural transitions of specific sequences may, thus, regulate the structural and functional properties of torsionally coupled, distal sequences.

Flanking AT-rich sequences may lower the activation energy of cruciform extrusion in supercoiled DNA.

In the absence of flanking AT-rich segments, cruciform transition energies of DNA palindromic sequences of random base composition are high and mainly dependent upon the base-stacking and -pairing parameters of the palindromic segment. When AT-rich sequences adjoin palindromes, the transition energy of cruciform extrusion is significantly lowered. An inverse relationship exists between the length of the AT-rich stretch and the cruciform transition energy. Long stretches lower the transition energies more than short stretches. At physiological salt and temperature conditions, equilibrium between cruciform extrusion and absorption for the inverted repeat sequences IRS-B and IRS-C of pBR322 derived plasmids is reached in less than five minutes.

Mapping structurally perturbed sites in DNA by replication arrest and run-off replication.

We describe a technique for rapid fine mapping of sites of torsion-induced perturbations of DNA structure. The technique involves strand scission or chemical base modification at structurally perturbed sites, replication arrest in a double-strand DNA sequencing reaction, and size analysis of replication products by electrophoresis on sequencing gels. Besides being less complicated and faster than site identification by conventional end-labeling methods, the technique assures high sequence specificity through the use of oligomeric sequencing primers. This property should be useful for in vivo mapping of DNA structural perturbations with known sequence within complex genomes.

Sequence instability in the long terminal repeats of avian spleen necrosis virus and reticuloendotheliosis virus.

Sequence divergence between the 3' long terminal repeats (LTR) of avian reticuloendotheliosis virus (REV), deletion variant proviral clone 2-20-4, and spleen necrosis virus (SNV)-proviral clones 14-44, 60, and 70-was found to involve two classes of base substitutions: low-frequency interspersed and high-frequency clustered substitutions. Clones 2-20-4 and 14-44 have diverged 4.4% owing to low-frequency substitutions. In contrast, two high-frequency substitution segments have diverged by 30% and 29%, respectively. Clustered substitutions appear to be located either within or next to tandem repeats, suggesting their introduction concomitant with sequence deletions and duplications commonly associated with such repeats. A new 19-bp tandem repeat is found in clone 2-20-4. Its sequence could have evolved from the 26-bp repeats found in the SNV clones.

pUC12-W1: a plasmid vector for the study of cloned DNA conformation.

pUC12-W1 is a new cloning vector for the study of torsion-induced structural transitions of insert DNA. It was derived from pUC12 by deleting three A + T-rich sequences which can undergo structural transitions when torsionally stressed. Transitions at these sites have low energy of activation and undefined structures. They complicate studies on transitions of DNA inserts by diverting torsional force and causing the vector to be undefined in helical and energetic terms. The new vector pUC12-W1, from which these segments have been deleted, will facilitate studies of torsion-induced structural transitions of insert DNA.

Pyrimidine dimer dependent cleavage of single-stranded DNA by T4 UV endonuclease.

T4 UV endonuclease cleaves double- and single-stranded DNA with equal specificity for photo-pyrimidine dimers. Thus, the enzyme can be used for mapping and quantifying pyrimidine dimers in single-stranded DNA as well as in double-stranded DNA. Mapping of pyrimidine dimers shows that rates of UV-dimerization are not only affected by 5', 3' adjacent bases, but also by position within pyrimidine tracts. Di-pyrimidines at 3' ends of tracts are more photoreactive than those at 5' ends.

Size analysis of heterogeneous nuclear RNA in an aqueous gel system.

We have analyzed the size distribution of heterogeneous nuclear RNA (hnRNA) from cultured murine cells in an aqueous gel system consisting of 1.8% polyacrylamide and 0.5% agarose. hnRNA, labeled in vivo with 32P at a low specific activity, was labeled in vitro at its 3'-end with NaB3H4, and fractionated by gel electrophoresis. The ratio of log 32P/3H cpm was determined for each gel fraction and shown to be linearly related to distance migrated. The ratio of 32P/3H cpm was used to predict the molecular weight of 28-S and 18-S RNAs and shown to be within approx. 10% of the known molecular weight. Denaturation of hnRNA with heat or dimethyl sulfoxide gave similar number and weight average molecular weight when analyzed by gel electrophoresis. The same hnRNA analyzed by sucrose gradient velocity sedimentation had a similar number average molecular weight but much higher weight average molecular weight.

Units of transcription for cytoplasmic RNA in mouse myeloma cells.

The size of transcription units for several of the abundant cytoplasmic mRNA species in mouse myeloma cells has been analyzed by the ultraviolet light mapping technique. Inactivation kinetics and target size analyses for production of the predominant RNA species indicate that the mRNAs originate in precursor molecules that are 2--14 times larger than the mature mRNA. This estimate of the size of the transcription unit may be a minimum one since it would not take into account promoter-distal sequences if these were not necessary for processing of the mRNA precursor.

Effects of ultraviolet irradiation and postirradiation incubation on heterogenous nuclear RNA size in murine cells.

We have analyzed the decrease in synthesis of individual size classes of heterogeneous nuclear RNA (hnRNA) in ultraviolet (UV)-irradiated Merwin plasmacytoma (MPC-11) cells at various times of postirradiation incubation. HnRNA from nonirradiated control cells is distributed over a wide range from approximately 60S to 5S, with 42S RNA carrying more label than any other size class. HnRNA from UV-irradiated cells shows a dose-dependent shift in size distribution toward lower molecular weight. The size distribution of hnRNA synthesized after prolonged times of postirradiation incubation is restored toward normal, i.e., synthesis of long RNA molecules increases relative to the synthesis of short ones. Analysis of the total number of hnRNA chains synthesized during a 20-min [(3)H]uridine pulse shows a considerable reduction in their number with increasing UV dose. Murine cell lines are excision-repair-deficient but capable of post replication repair inhibited by caffeine. HnRNA transcripts of cells incubated in its presence were studied. The caffeine, which has no effect on hnRNA size in control cells, inhibits to a considerable extent the restoration of full-length transcripts during postirradiation incubation. The lack of excision repair in MPC-11 was confirmed by the analysis of pyrimidine dimers in trichloracetic acid-insoluble and soluble fractions within 8 h of postirradiation incubation.The size of parental and daughter strand DNA in UV-irradiated cells was correlated with RNA transcript size. The parental DNA in these experiments does not change its size as a consequence of UV exposure and postirradiation incubation. In contrast, daughter DNA strands are short in UV-irradiated cells and they increase in size during postirradiation incubation to reach the size of parental strands after 8 h.

End group of naturally terminated and UV lesion terminated T7 in vitro RNA.

The 3' terminal nucleosides of RNA transcribed in vitro by E. coli RNA polymerase from T7 DNA and UV irradiated TN DNA were determined. The 3' terminal nucleoside of naturally terminated (t1 termination site) RNA cytidine. In the case of RNA terminated at UV lesions, it is cytidine in 0 per cent of the molecules and adenosine in the remaining 30 per cent. Cytidine trialcohols are labile in high concentrations of KOH and at high temperature and appear to convert to uridine.