The interaction of Herpes Simplex Virus with murine lymphocytes. I. Mitogenic properties of herpes simplex virus.

Herpes simplex virus (HSV) stimulates DNA synthesis in mouse spleen cultures prepared from normal, macrophage-depleted, and T-cell-depleted spleen cells, but not from thymocytes. In addition, a polyclonal antibody response is observed in HSV-infected spleen cultures. These findings indicate that the cells stimulated to undergo DNA synthesis after HSV infection appear to be the bone marrow-derived lymphocytes. The newly synthesized DNA is host cell and not of viral origin. Heat treatment and ultraviolet irradiation of HSV before addition to spleen cultures prevents the induction of DNA synthesis. We consider the use of this system as assay for the study of cell transformation by HSV and also for the study of host cell control of the expression of the viral genome.

Temperature-sensitive mutants for the replication of plasmids in Escherichia coli. I. Isolation and specificity of host and plasmid mutations.

Temperature-sensitive mutants of Escherichia coli defective in the replication of the plasmid colicinogenic factor E1 (ColE(1)) were isolated following mutagenesis of E. coli K12 strain carrying the ColE(1) factor. Following the mutagenic treatment an enrichment procedure utilizing the replacement of thymine with bromouracil in the ColE(1) DNA duplicated at the restrictive temperature was used. The mutants isolated following this enrichment step were the result of a mutation event either in the host chromosome or in the ColE(1) plasmid. The host mutants fell into three phenotypic classes based on the effect each mutation had on the maintenance of a variety of other extrachromosomal DNA elements. Phenotypic class I mutations affected all E. coli plasmids, both the I and F sex factor types as well as the ColE(1) factor. Phenotypic class II mutations affected the maintenance of the ColE(1) and the F sex factor type plasmids and not the I type, while phenotypic class III mutations affected only ColE(1) replication. None of these mutations was found to have a significant effect on the replication of the E. coli chromosome. The plasmid-linked mutations fell into two phenotypic classes on the basis of the ability of the Flac episome to complement the mutation in the ColE(1) plasmid.

Temperature-sensitive mutants for the replication of plasmids in Escherichia coli. II. Properties of host and plasmid mutations.

Host mutations in Escherichia coli K12 selected for the temperature-sensitive replication of the bacterial plasmid colicinogenic factor E(1) (ColE(1)) exhibit a pleiotropic effect with respect to the effect of the mutation on other extra-chromosomal elements. The mutations also vary with respect to the time of incubation of the cells at 43 degrees C required for complete cessation of ColE(1) DNA synthesis. While the synthesis of the bacterial chromosome appears unaffected, supercoiled ColE(1) DNA replication stops immediately in some mutants and gradually decreases during several generations of cell growth before stopping in others. Mutations isolated in the ColE(1) plasmid resulted in only a gradual cessation of ColE(1) DNA synthesis over several generations of cell growth at 43 degrees C. Conjugal transfer of the ColE(1) and ColV factors occurs normally in the host mutants when the transfer is carried out at the permissive temperature; however, the presence of a group I mutation in the donor cell prohibited conjugal transfer of either plasmid DNA at 43 degrees C to a normal recipient cell. Similarly, the presence of this mutation in the recipient prevented the establishment of ColE(1) or ColV in the mutant recipient cell upon conjugation with a normal donor at 43 degrees C. Various host ColE(1) replication mutants carrying either ColE(1) or ColE(2) were also defective in the mitomycin C-induced production of colicin E(1) or colicin E(2) at 43 degrees C. The majority of the host mutations examined exhibited a temperature sensitivity to growth in deoxycholate in addition to the inhibition of plasmid DNA replication, suggesting a membrane alteration in these mutants when grown at the restrictive temperature.

Balancing regulatory control, scientific knowledge, and public understanding.

In summary, I would like to emphasize the continued need for broad and vigorous basic research, with a balance between the fundamental work that may eventually lead to commercial products and the fundamental work that is necessary for an understanding of the interaction of many types of organisms within the environment. I would like also to reiterate the need for balance in the regulatory approach so that we do not repress innovation in research and development. Over-regulation has many side effects. In addition to repressing innovation and not taking advantage of our research base, over-regulation leads to reluctance by the capital markets to invest in the future of our new industries, thereby halting their development at an early stage. At the same time, under-regulation leads to lack of confidence by the public and paralysis of the industry based on public outcry and legal proceedings. It is my personal belief that the combination of a sound approach to regulatory practice, based on current scientific knowledge, combined with appropriate communication with the public regarding the new products, will lead to an exciting future for all sectors of industry that use the new biotechnology.

Relationship between sulfadiazine resistance and the failure to ferment maltose in Neisseria meningitidis.

The genetic marker for sulfadiazine resistance was transferred by means of purified deoxyribonucleic acid from sulfadiazine-resistant Neisseria meningitidis and N. perflava to a sulfadiazine-sensitive strain of N. meningitidis. Over 80% of the isolates from these experiments, selected on the basis of sulfadiazine resistance, failed to produce acid from maltose. The same proportion of naturally occurring isolates that are sulfadiazine-resistant failed to ferment maltose. The enzymatic block in 17 isolates tested was the loss of maltose permease activity; in two cases, maltose phosphorylase activity was lost also. The permease was present in these cells, however, and could be activated by the addition of sulfadiazine. The results obtained support the hypothesis that these organisms, in becoming resistant to sulfadiazine, have undergone a single mutation.

Deoxyribonucleic acid homologies among species of the genus Neisseria.

Eleven aerobic species of Neisseria, a Mima sp., and a Herellea sp. were tested for deoxyribonucleic acid (DNA) homology in direct hybridization experiments. DNA labeled with either (14)C or (32)P was prepared from five species of Neisseria. Unlabeled DNA from the various microorganisms was immobilized on membrane filters, which, after pretreatment, were incubated with labeled DNA (4,000 counts per min per filter) for 14 hr at 67 C. The measure of relatedness was expressed as the relative percentage of direct binding compared to that obtained with homologous DNA. All serological types of N. meningitidis, including the newly proposed types, were homologous to the standard strain of N. meningitidis with one possible exception, type Z. The genus Neisseria is heterogeneous in nature, forming at least three distinct groups: first, N. meningitidis and N. gonorrhoeae; second, N. perflava, N. subflava, N. sicca, N. flavescens, and N. flava; third, N. catarrhalis and N. caviae. Mima and Herellea species show no significant homology with the Neisseria.

Bacteriocin production by strains of Neisseria meningitidis.

Kingsbury, David T. (Naval Medical Research Institute, Bethesda, Md.). Bacteriocin production by strains of Neisseria meningitidis. J. Bacteriol. 91:1696-1699. 1966.-Strains of Neisseria meningitidis produce substances inhibitory to other strains of meningococcus. These substances are nontransmissible and show a high degree of strain specificity. The properties of one of these substances resemble those of the class of bacterial inhibitors called bacteriocins. Synthesis of this "meningocin" can be increased as much as 200-fold by induction with mitomycin C. It shows a high degree of heat stability and is sensitive to proteolytic enzymes. Six bacteriocins from strains of N. meningitidis have been used to type meningococci. By use of this procedure, strains that were identical serologically were placed into distinct bacteriocin groups.

Estimate of the genome size of various microorganisms.

Bacterial genome sizes, determined by deoxyribonucleic acid reassociation kinetics, vary over a 10-fold range. The smallest studied, Chlamydia trachomatis, had a genome of 6 x 10(5) nucleotide pairs compared to 4.5 x 10(6) for Escherichia coli.

Regulation of biotechnology in the United States: One and a half years of using the 'coordinated framework'.

There are several organizations in the US with responsibilities for regulatory oversight of the planned introduction of recombinant DNA organisms into the environment. Equally, there are many kinds of projects which require assessment. The policies, recommendations and rulings of the various authorities have been integrated into a 'Coordinated Framework' which defines the operation of flexible case-by-case risk assessment. Additions to and revision of the guidelines are being made, a process which will continue in the light of new experience.

Immunological analysis of host and agent effects on Creutzfeldt-Jakob disease and scrapie prion proteins.

Creutzfeldt-Jakob disease (CJD) and scrapie are degenerative neurological diseases caused by unusual infectious pathogens. The term prion has been introduced to underscore the apparent distinctness of these agents from viruses and viroids. The only macromolecule shown to be associated with the infectious agent, the CJD or scrapie prion protein (PrPCJD or PrPSc, respectively), is encoded by the same gene as a normal cellular protein. In several studies biochemical differences have been reported in PrPScs derived from a common host species infected with different putative strains of the scrapie agent, suggesting agent-specific characteristics independent of the host. We analyzed various agent-host combinations by Western blotting of PrPs that were separated by size or charge. The profile of immunoreactive proteins for CJD prions isolated from mice, guinea pigs, and humans appeared distinct. Importantly, PrPCJDS purified from a human brain and from the corresponding first-passage mouse brains were clearly distinguishable. PrPCJDs isolated from CJD prions propagated in NAMRU or B10.Q mice, which are homozygous for a short-incubation-time gene; from the short-incubation-time backcross progeny of (B10.Q x I/LnJ)F1 x B10.Q; or from NAMRU mice inoculated with I/LnJ prions were identical to each other but distinguishable from those of I/LnJ mice, which are homozygous for the long-incubation-time gene. The PrPs from human CJD and ovine scrapie propagated in the same mouse strain appeared the same, but they were distinct from the same isolate of scrapie passaged in hamsters. Lastly, PrPScs purified from five different strains of scrapie propagated in C57BL mice were identical, including strains, ME7 and 139A, which were previously reported to be distinct. This evidence does not support, although it does not exclude, agent-mediated characteristics independent of host-mediated ones for scrapie and CJD.

Integration and transcription of virus DNA in herpes simplex virus transformed cell lines.

The physical state of the HSV I DNA present in two biochemically transformed cell lines, a revertant line and a supertransformed cell line, was determined. These cells all contained fragments of the HSV genome and the transformed and supertransformed cell lines expressed virus thymidine kinase. It was found that the virus DNA in these cells was maintained in a complex state with approximately half of the HSV DNA present in a covalently integrated state and the other half in a non-integrated state. There was no major cell line difference in the distribution of integrated and non-integrated virus DNA. RNA transcripts representing 5% of the HSV I genome are present in each of these lines. This is more than is required to code for the virus thymidine kinase present in the transformed and supertransformed cell lines and suggests the presence of other virus proteins in these cells.

Bacteriophage infecting the myxobacterium Chondrococcus columnaris.

Kingsbury, David T. (University of Washington, Seattle), and Erling J. Ordal. Bacteriophage infecting the myxobacterium Chondrococcus columnaris. J. Bacteriol. 91:1327-1332. 1966.-During a series of screening experiments, seven bacteriophages which infect the pathogenic myxobacterium Chondrococcus columnaris were isolated. Of these, one was chosen for detailed study. This phage has a wide host range among strains of C. columnaris, but does not infect other myxobacterial species tested. Morphologically, this phage resembles coliphage T2, though it is smaller. It has a head diameter of 600 A, a tail length of 1,000 A, and a tail width of 200 A. The head is attached to the tail by a well-defined neck. The turbid plaques produced by this phage are similar in appearance to those produced by coliphage lambda, and average 1 mm in diameter. The phage has a latent period of 100 min, a rise period of an additional 90 min, and a burst size of 23. Calcium ions at a concentration of 0.004 m are required for adsorption. This requirement cannot be met by substitution of magnesium ions. A purified preparation of 2 x 10(12) phage particles was extracted with phenol, and the nucleic acid was identified as deoxyribonucleic acid (DNA). Base ratios of the phage DNA and the DNA of two propagating strains were similar. Streptomycin at a concentration of 70 mug/ml inhibits phage infection at an early stage, probably by inhibiting injection of the phage DNA.

Prions--infectious pathogens causing the spongiform encephalopathies.

The novel properties of the scrapie and Creutzfeldt-Jakob disease (CJD) transmissible agents readily distinguish them from viruses and viroids; thus, they have been labeled "prions". The scrapie prion contains a protein(s) which is required for infectivity; recently a 27,000 to 30,000 MW protein which purifies with the prion has been identified. The similarities between the scrapie and CJD agents suggest that CJD is also caused by a prion. Recent studies show that the time courses of both scrapie and CJD are determined by an autosominal dominant gene denoted PID (prion incubation determinant). In congenic mice infected with CJD, PID appears to be located on chromosome 17 in the major histocompatibility complex (H-2) in the D-subregion. Further studies indicate that replication of the scrapie and CJD prions precedes the development of pathological change. These changes share many similarities with those found in a variety of degenerative neurological disorders of unknown etiology.

Temperature-sensitive mutants for the replication of plasmids in Escherichia coli: requirement for deoxyribonucleic acid polymerase I in the replication of the plasmid ColE 1 .

An Escherichia coli mutant (polA1), defective in deoxyribonucleic acid (DNA) polymerase I, (EC 2.7.7.7) is unable to maintain colicinogenic factor E1 (ColE1), whereas several sex factor plasmids are maintained normally in this strain. polA1 mutant strains containing these sex factor plasmids do not exhibit a readily detectable plasmid-induced polymerase activity. A series of E. coli mutants that are temperature sensitive for ColE1 maintenance, but able to maintain other plasmids, were isolated and shown to fall into two phenotypic groups. Mutants in one group are defective specifically in ColE1 maintenance at 43 C, but exhibit normal DNA polymerase I activity. Mutations in the second group map in the polA gene of E. coli, and bacteria carrying these mutations are sensitive to methylmethanesulfonate (MMS). Revertants that were selected either for MMS resistance or the ability to maintain ColE1 were normal for both properties. The DNA polymerase I enzyme of two of these mutants shows a pronounced temperature sensitivity when compared to the wild-type enzyme. An examination of the role of DNA polymerase I in ColE1 maintenance indicates that it is essential for normal replication of the plasmid. In addition, the presence of a functional DNA polymerase I in both the donor and recipient cell is required for the ColV-promoted conjugal transfer of ColE1 and establishment of the plasmid in the recipient cell.

Encapsulation of lymphocyte DNA by vesicular stomatitis virus.

Vesicular stomatitis virus, grown on the WIL(2)-3A line of continuously growing human lymphocytes, contains DNA in addition to the viral RNA. By contrast, virus grown on BHK 21-C13 fibroblasts has no detectable DNA. The virus-associated DNA is found in both the B and T particles of the virus and is resistant to deoxyribonuclease. The DNA is intimately associated with the virus and appears to be incorporated into the viral ribonucleoprotein core structure produced by treatment of the virus with Nonidet P40 follow edby CsCl isopycnic banding. The virus-associated DNA has an isopycnic density of 1.699 g x cm(-) (-3) in CsCl, identical to that of human DNA. The average molecular weight of the DNA molecules associated with the virus is 9.0 x 10(5), as determined by velocity sedimentation in sucrose density gradients and studies of its contour length in the electron microscope. DNA.DNA reassociation kinetics of this DNA demonstrate that the DNA is of host origin and rules out the possibility that it originates from contaminating microorganisms or mitochondria. The analytical complexity of the virus-associated DNA shows that 50% of the virus-associated DNA sequences are homologous to the repeated DNA sequences of human DNA and the remaining 50% are homologous to human unique sequences. On the average, there is one molecule of DNA for each four virion particles.

Quantitation of the viral DNA present in cells transformed by UV-irradiated herpes simplex virus.

Two cell lines biochemically transformed by UV-irradiated herpes simplex virus (HSV) each contain virus DNA. A comparison of the kinetics of reassociation of 3H-labeled HSV DNA in the presence and absence of either clone 139 (HSV-1 transformed) or clone 207 (HSV-2 transformed) DNA showed that the presence of transformed cell DNA increased the rate of reassociation of approximately 10% of the viral genome while having no effect on the remaining 90%. The Cot1/2 of this reaction was approximately 1,000 in each cell type, as compared to approximately 3,000 for the cellular unique sequences. These results suggest the presence of four to six copies of a 10% fragment of the virus DNA per cell. The DNA from a hamster fibroblast cell line morphologically transformed by UV-irradiated HSV-2 (333-8-9) did not affect the rate of reassociation of HSV-2 DNA, indicating that these cells had less than 3% of a viral genome present.