Mutations that allow human Ad2 and Ad5 to express late genes in monkey cells map in the viral gene encoding the 72K DNA binding protein.

Five host-range mutants (Ad2hr400–hr403, Ad5hr404) of human adenovirus serotype 2 and 5 (Ad2 and Ad5) which overcome the block to growth of wild-type adenovirus in monkey cells have been isolated. They form plaques and multiply efficiently in both monkey and human cells. The alteration in each of these mutants allows the full expression of all viral late genes, in marked contrast to the depressed synthesis of many late proteins in monkey cells infected with the parental Ad2 or Ad5. The altered gene encodes a diffusible product, since the mutation acts in trans to enhance the synthesis of wild-type Ad3 late proteins during co-infections of monkey cells with Ad2hr400 and Ad3. Restriction enzyme analysis of the genomes of all the host-range mutants show that none of them contain major alterations. In addition, an earlier report (Klessig and Hassell, 1978) indicated that Ad2hr400 does not contain SV40 sequences, which in some adenovirus-SV40 hybrid viruses allows efficient multiplication in monkey cells. The mutation responsible for the extended host range has been physically mapped by marker rescue experiments using isolated restriction enzyme fragments of the mutants to transfer the new phenotype to wild-type adenovirus. The alteration in each of the five mutants is located in a region (coordinates 62–70.7; coordinates 62–68 for Ad5hr404) which encodes predominantly the 72K DNA binding protein. More detailed mapping using Ad2hr400 fragments places the mutation (coordinates 62.9–65.6) entirely within the 72K gene. The multifunctional nature of the 72K protein and some of its similarities to SV40 T antigen are discussed.     

Complementation of adenovirus type 5 host range mutants by adenovirus type 12 in coinfected HeLa and BHK-21 cells.

We have studied the ability of adenovirus type 12 (Ad12) to complement the Ad5 transformation-defective host rang (hr) mutants during infection of human cells (HeLa) or hamster cells (BHK-21). The group I mutant hr3 (mapped within 1.3 to 3.7 map units), which is incapable of synthesizing viral DNA, was complemented for both DNA synthesis and infectious virus production in nonpermissive HeLa cells during coinfection with Ad12. Similarly, the group II mutant hr6 (6.1 to 9.4 map units), which does synthesize DNA, was also shown to be complemented for virus production. When the host cells were BHK-21, an established hamster cell line that is permissive for Ad5 but nonpermissive for Ad12 DNA synthesis and virus production, coinfection with Ad5 and Ad12 did not overcome the block to Ad12 DNA synthesis. Coinfection of BHK-21 cells with Ad12 and either hr3 or hr6 leads to the complementation of only the group I mutant (hr3). The inability of Ad12 to complement hr6 in BHK-21 cells may be due to the failure of Ad12 to express an early gene product from the region corresponding to early region 1B (4.5 to 11 map units) Ad5 where hr6 and the other group II mutations are located.     

Isolation of adenovirus type 5 host range deletion mutants defective for transformation of rat embryo cells.

A series of adenovirus type 5 (Ad5) deletion, insertion and substitution mutants, some of which are defective for transformation of rat cells, have been isolated. The mutants were selected as variants which lack the Xba I endonuclease cleavage site at 4 map units on the viral chromosome. The deletions range in size from 150-2300 bp and are located between 1.5 and 10.5 map units. The mutants can be propagated in 293 cells (Ad5-transformed human embryonic kidney cells), but are defective for growth in HeLa or human embryonic kidney cells. No viral DNA synthesis was observed in mutant virus-infected HeLa cells. All but one of the deletion mutants tested were defective for transformation of rat embryo and rat embryo brain cells.     

Control of Adenovirus Gene Expression: Cellular Gene Products Restrict Expression of Adenovirus Host Range Mutants in Nonpermissive Cells

Adenovirus type 5 (Ad5) host range mutants dl312 and hr-1, with lesions in region E1A (0 to 4.5 map units) of the viral genome, fail to accumulate virus-specific early RNA during infection in HeLa cells. In a recent report, we showed that the addition of anisomycin, a stringent inhibitor of protein synthesis, at 1 h after infection of HeLa cells with hr-1 virus resulted in the accumulation of properly spliced and translatable mRNA from all early regions (M. G. Katze, H. Persson, and L. Philipson, Mol. Cell. Biol. 1:807-813, 1981). Based on these results we proposed a model in which expression of early mutant RNA was achieved through inactivation of a cellular protein normally causing a reduction in the amount of viral RNA. These studies have been extended in the present report, which shows that early viral proteins can be detected in Ad5 dl312- and Ad5 hr-1-infected HeLa cells which have been treated for several hours with anisomycin either shortly after infection or before infection. A pulse of drug treatment also resulted in expression of substantial amounts of adenovirus structural proteins after infection with both Ad5 hr-1 and Ad5 dl312, whereas in drug-free controls no late proteins were detected. The Ad5 hr-1 virus previously reported to be DNA replication negative in nonpermissive HeLa cells was found to replicate its DNA, albeit at low levels, when anisomycin was present either from 1 to 5 h postinfection or for 5 h before infection. When infectious virus production was examined in mutant-infected cells the titer of Ad5 dl312 virus was found to increase at least 500-fold in anisomycin-treated HeLa cells. Taken together, these and our previous results suggest that the block in gene expression characteristic for complementation group I Ad5 host range mutants in HeLa cells can be overcome by inactivating cellular gene products serving as negative regulators of viral gene expression.     

Isolation and analysis of adenovirus type 5 mutants containing deletions in the gene encoding the DNA-binding protein.

A genetic system is described which allows the isolation and propagation of adenovirus mutants containing lesions in early region 2A (E2A), the gene encoding the multifunctional adenovirus DNA-binding protein (DBP). A cloned E2A gene was first mutagenized in vitro and then was introduced into the viral genome by in vivo recombination. The E2A mutants were propagated by growth in human cell lines which express an integrated copy of the DBP gene under the control of a dexamethasone-inducible promoter (D. F. Klessig, D. E. Brough, and V. Cleghon, Mol. Cell. Biol. 4:1354-1362, 1984). The protocol was used to construct five adenovirus mutants, Ad5d1801 through Ad5d1805, which contained deletions in E2A. One of the mutants, Ad5d1802, made no detectable DBP and thus represents the first DBP-negative adenovirus mutant, while the four other mutants made truncated DBP-related polypeptides. All five mutants were completely defective for growth and plaque formation on HeLa cell monolayers. Furthermore, the two mutants which were tested, Ad5d1801 and Ad5d1802, did not replicate their DNA in HeLa cells. The mutant Ad5d1804 encoded a truncated DBP-related protein which contained an entire amino-terminal domain derived from the host range mutant Ad5hr404, a variant of Ad5 which multiplies efficiently in monkey cells. While results of a previous study suggest that the amino-terminal domain of DBP could act independently of the carboxyl-terminal domain to enhance late gene expression in monkey cells, the Ad5d1804 polypeptide failed to relieve the block to late viral protein synthesis in monkey cells. The mutant Ad5d1802 was used to study the role of DBP in the regulation of early adenovirus gene expression in infected HeLa cells. These experiments show that E2A mRNA levels are consistently reduced approximately fivefold in Ad5d1802-infected cells, suggesting either a role for DBP in the expression of its own gene or a cis-acting defect caused by the E2A deletion. DBP does not appear to play a significant role in the regulation of adenovirus early regions 1A, 1B, 3, or 4 mRNA levels in infected HeLa cell monolayers since wild-type Ad5- and Ad5d1802-infected cells showed very little difference in the patterns of expression of these genes.     

A specific internal RNA polymerase recognition site of VSV RNA is involved in the generation of DI particles.

The 3′ terminal sequences of four different DI particle RNAs ranging in size from 10S to 30S have been determined directly using rapid RNA sequencing methods or deduced, in the case of the fourth DI RNA, from the complementary sequence of a small RNA transcribed from this part of the genome (Schubert et al., 1978). One DI particle (DI 011) contains covalently linked genomic and antigenomic RNA. The 5′ end of this RNA is identical to that of VSV RNA, as determined by annealing for at least 1 kb, as well as to the other DI particle RNAs used in this study. The 3′ ends of the other three DI particle RNAs are exact copies of the common 5′ terminal sequence for 48 nucleotides in two cases and 45 nucleotides in the third. Beyond these complementary regions the sequences are different for each DI RNA. The fact that these regions differ in length by only three nucleotides, despite the wide differences in the overall size of the DI particle RNAs, indicates that if these DIs were formed by the copy-back mechanisms similar to those proposed by Leppert, Kort and Kolakofsky (1977) and Huang (1977), a specific recognition site for the RNA polymerase must be involved in copying the 5′ terminus. We determined the 5′ terminal sequence from position 43–48 at the end of the complementary region and found it to be 5′-GGUCUU-3′. This hexamer is also part of other highly conserved terminal RNA polymerase initiation sites (Keene et al., 1978; Keene, Schubert and Lazzarini, 1979) and may be a specific internal RNA polymerase recognition site. We conclude that this sequence is one of the elements involved in the genesis of DI particle chromosomes containing short complementary sequences at their termini. The ability of the polymerase to resume synthesis at or near a specific recognition site is discussed.     

An approach to a physico-chemical model of olfactory stimulation in vertebrates by single compounds

During recent years, numerous attempts have been made to correlate both quantitative (Davies &; Taylor, 1959; Engen, 1962; Beck, 1964; Engen, Cain &; Rovee, 1968; Cain, 1969; Dravnieks &; Laffoit, 1970; Laffort, 1969a,b) and qualitative (Davies, 1965; Amoore &; Venstrom, 1965; Döving, 1966a,b; Wright &; Michels, 1964; Leveteau &; MacLeod, 1969) odorous properties of single compounds to their molecular properties. These attempts have been only partially successful.In the present paper we will try to explain the several odorous properties of single compounds on the basis of the non-specific properties of odorants involved in solubility.This model is a first approach, and although it gives statistically highly significant relations, it is not as accurate as those advanced with respect to the physical and sensory dimensions of stimuli in the fields of vision and audition.We will first give the present definitions of the most suitable physicochemical parameters, and then advance quantitative and qualitative models for single compounds. Quantitative odorous properties are: odour threshold, rate of change of odour intensity with odorant concentration in the suprathreshold region, and the somewhat controversial upper odour intensity. Qualitative properties refer to odour character.     

The effect of cycloheximide on the stability of the morphogenetic properties of chick limb-bud mesoderm.

In this study dissociated late stage 20 (stage 20+) chick leg-bud mesoderm cells cultured in cycloheximide (1 μg/ml) were tested to determine the retention of their ability to undergo recognizable limb morphogenesis, according to the technique described by Zwilling (1964). The cycloheximide treatment retards the loss of this morphogenetic property compared with the normal loss that occurs in cultures of untreated cells (Finch and Zwilling, 1971). The exposure to cycloheximide extends the period during which this morphogenetic property can be detected for approximately 15–20 hr. Incorporation experiments show that cycloheximide at 1 μg/ml rapidly inhibits the synthesis of protein. RNA, and DNA in cells exposed to the antibiotic. This inhibitory effect is completely reversible in a relatively short time after removal of the cycloheximide.     

Replication of a mammalian genome: the role of de novo protein biosynthesis during S phase

ts14 is a temperature-sensitive Chinese hamster lung cell mutant that ceases protein biosynthesis within a short time of transfer to nonpermissive temperature (Haralson and Roufa, 1975; Roufa and Haralson, 1975; Roufa and Reed, 1975). This mutant contains a revertible, presumably a point mutation that renders its 60S ribosomal subunit thermolabile (Haralson and Roufa, 1975). In this report, we describe the relationship between the conditional ability of ts14 to synthesize protein during S phase and the replication of its DNA.After transfer to nonpermissive temperature (39°C), where ts14 synthesizes protein at a rate approximately 20 fold less than wild-type cells, synchronous cultures of the mutant performed all the processes required for replication of their DNA. During prolonged incubations at nonpermissive temperature, S phase ts14 completed approximately one round of DNA replication semi-conservatively as judged by density-transfer experiments. Pulse-labeling experiments performed on S phase cells revealed that ts14 synthesized the intermediates of discontinuous DNA replication at nonpermissive and permissive temperatures at similar rates. In these tests, the mutant was not substantially different from wild-type at both culture temperatures. At the nonpermissive temperature, however, ts14 synthesized significantly less nuclear protein (that is, histone) than did wild-type cells, and the mutant's chromatin appeared deficient in histone by virtue of its increased sensitivity to nuclease.     

Transformation of rodent cells by DNA extracted from transformation-defective adenovirus mutants

Complementation group II host range mutants of adenovirus type 5 which map in early region 1B (E1B, 4.5 to 11.0 map units) have been shown to be defective for the synthesis of the E1B 58,000-dalton (58K) antigen in infections of HeLa or KB cells (Lassam et al., Cell 18:781-791, 1979) and unable to transform cultured rodent cells (Graham et al., Virology 86:10-21, 1978). In this report we show that DNA extracted from group II mutants hr6 and hr50 can transform rat cells with the same efficiency as wild-type DNA. Furthermore, group II mutant-transformed hamster cells were shown to contain no detectable E1B 58K tumor antigen but were capable of inducing tumors in newborn hamsters. Hamster cell lines 1019-3 and 1019-C3, transformed by hr50 DNA, produced no detectable quantities of either the E1B 58K or 19K antigen but nonetheless exhibited a fully transformed oncogenic phenotype. Our results show that the E1B 58K antigen is not absolutely required for oncogenic transformation and suggest that even cells lacking the 19K protein can be oncogenic.     

Immortal fibroblasts?

Uncommitted fibroblasts may exist (Kirkwood &; Holliday, 1975; Holliday, Huschtscha, Tarrant &; Kirkwood, 1977). I show that, even if such cells divide significantly faster than committed ones, the uncommitted cells would be overlooked if one uses current culture techniques. Some general guidelines for testing the model are outlined. Uncommitted cells would be useful in studying the biochemical mechanisms of aging and development.     

Warfare and hominid brain evolution.

This paper reviews literature on the evolutionary effects of warfare upon the hominid brain. Alexander &; Tinkle (1968) and Bigelow (1969) are found to be the first to propose that warfare was the principle evolutionary pressure that created the novel substance of the human brain, and that it acted at least from the early Pleistocene. These writers are distinguished from Darwin (1871), Keith (1947) and Wilson (1975) who saw warfare influencing the development of the brain only in historical or near-historical times.The warfare hypothesis of Alexander &; Tinkle is found to be an excellent explanation of the evolution of the human brain, but to be unsatisfactory from a biological viewpoint because they do not explain how warfare evolved in the first place, nor do they attempt to account for the apparent absence of warfare as a behavioral adaptation in species other than some eusocial insects.This author underpins the warfare hypothesis, arguing that it evolved as a necessary consequence of the circumstances of early hominids. Proficient tool use gave domination over predators and opened up new food resources, thereby diminishing two population controls. A population explosion resulted and, at critical densities, when starvation threatened, warfare was the genetically most successful behavioral adaptation. Alternative hypotheses are shown to be inadequate. Finally, the author asks why such an important hypothesis has been ignored for almost a decade.     

The role of migration in the genetic structure of populations in temporally and spatially varying environments II. Island Models

The Island Model introduced by Sewall Wright (1951) has proven to be a useful construction for studying the interaction of genetic drift, population subdivision, and mutation. Interest in the model has recently increased because of its relevance to certain questions involving the rate of differentiation of sub-populations under the neutral allele hypothesis (e.g., Smith, 1970; Latter, 1973). It is perhaps the only realistic population structure in which the test for neutrality proposed by Lewontin and Krakauer (1973) is valid (Lewontin and Krakauer, 1975). If data from natural populations is to be compared to the predictions of the Island Model, it is desirable to have an alternative model with the same migration pattern but with natural selection operating. In this paper one such model will be introduced where the stochastic element comes from random fluctuations in the environment rather than from genetic drift. The model is a direct extension of the one in the previous paper in this series (Gillespie, 1975) which dealt with a population which is subdivided into two patches with restricted migration between them.     

Proteins and messenger RNAs of the transforming region of wild-type and mutant adenoviruses

We have studied the proteins encoded by the transforming region of the closely related human adenovirus serotypes 2 and 5. Messenger RNAs complementary to the two parts of this region, E1A and E1B, were prepared separately by hybridization to cloned DNA fragments encompassing 0.8 to 4.5 map units (for E1A) and 9.8 to 11.1 map units (for E1B). These RNAs were further fractionated by electrophoresis through agarose gels containing methylmercuric hydroxide, and then translated in vitro to identify the proteins encoded by each RNA species. E1A and E1B RNAs isolated at early and at late times after infection were compared. Three size classes of E1A mRNA direct the synthesis of at least five proteins: a28K³ protein encoded by a 0.6 kb mRNA, 42K and 54K proteins encoded by a 0.9 kb mRNA(s), and 48K and 58K proteins encoded by a 1.1 kb mRNA(s). The mRNA for the 28K protein accumulates preferentially at late times. Three size classes of early E1B mRNA direct the synthesis of three proteins: a 15K protein encoded by a 0.9 kb mRNA, an 18K protein encoded by a 1.2 kb mRNA, and a 57K protein encoded by a 2.6 kb mRNA. The mRNA for the 15K protein continues to accumulate at late times, and an additional 22K protein is made, while the 18K and 57K proteins are synthesized poorly, if at all, with late RNA.Substantially different E1A and E1B proteins are encoded by RNA from cells infected with the adenovirus type 5 mutants dl311, dl312, dl313, dl314 and hr1, which are all defective for replication on human cells and, except for dl311, for transformation. dl312, dl314 and hr1 are also defective for early viral gene expression. No viral mRNA could be detected in either dl312 or dl314-infected cells. hr1-infected cells contain a 0.9 kb mRNA encoding E1A 54K and 42K, but instead of 58K and 48K, the 1.1 kb hr1-E1A mRNA is translated into a 26K protein. The E1B mRNAs are present in substantially decreased amounts in hr1-infected cells. dl311-infected cells contain E1A mRNAs of 1.1 and 0.9 kb, encoding 38K and 34K proteins, respectively, and normal E1B mRNAs. The dl313 mRNAs of 1.1 and 0.9 kb contained fused E1A and E1B sequences and were translated into 40K and 36K proteins, respectively. These results are related to the mRNA structures and the biological activity of regions of the individual proteins.