A Forward-Genetic Screen and Dynamic Analysis of Lambda Phage Host-Dependencies Reveals an Extensive Interaction Network and a New Anti-Viral Strategy

Latently infecting viruses are an important class of virus that plays a key role in viral evolution and human health. Here we report a genome-scale forward-genetics screen for host-dependencies of the latently-infecting bacteriophage lambda. This screen identified 57 Escherichia coli (E. coli) genes—over half of which have not been previously associated with infection—that when knocked out inhibited lambda phage's ability to replicate. Our results demonstrate a highly integrated network between lambda and its host, in striking contrast to the results from a similar screen using the lytic-only infecting T7 virus. We then measured the growth of E. coli under normal and infected conditions, using wild-type and knockout strains deficient in one of the identified host genes, and found that genes from the same pathway often exhibited similar growth dynamics. This observation, combined with further computational and experimental analysis, led us to identify a previously unannotated gene, yneJ, as a novel regulator of lamB gene expression. A surprising result of this work was the identification of two highly conserved pathways involved in tRNA thiolation—one pathway is required for efficient lambda replication, while the other has anti-viral properties inhibiting lambda replication. Based on our data, it appears that 2-thiouridine modification of tRNAGlu, tRNAGln, and tRNALys is particularly important for the efficient production of infectious lambda phage particles

Highlights of the DNA cutters:a short history of the restriction enzymes

In the early 1950’s, ‘host-controlled variation in bacterial viruses’ was reported as a non-hereditary phenomenon: one cycle of viral growth on certain bacterial hosts affected the ability of progeny virus to grow on other hosts by either restricting or enlarging their host range. Unlike mutation, this change was reversible, and one cycle of growth in the previous host returned the virus to its original form. These simple observations heralded the discovery of the endonuclease and methyltransferase activities of what are now termed Type I, II, III and IV DNA restriction-modification systems. The Type II restriction enzymes (e.g. EcoRI) gave rise to recombinant DNA technology that has transformed molecular biology and medicine. This review traces the discovery of restriction enzymes and their continuing impact on molecular biology and medicine

Origins of Contemporary DNA Tumor Virus Research

The evolution of unicellular organisms into multicellular organisms has demanded the emergence of very precise rules to govern both the spatial siting and the multiplication rates of their respective differentiated cells. A given specialized cell does not grow at any site in its multicellular host, but is restricted to have only certain cell types for its neighbors and to proliferate only upon receipt of a message signifying that more of its kind are needed for the orderly growth or maintenance of the complete organism

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 [African green] monkey [kidney] cells were 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 indicated that Ad2hr400 does not contain SV-40 sequences, which in some adenovirus-SV-40 hybrid viruses allows efficient multiplication in monkey cells. The mutation responsible for the extended host range was 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 5 mutants is located in a region (coordinates 62-70.7; coordinates 62-68 for Ad5hr404) which encodes predominantly the 72K [kilodalton] 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 SV-40 T [tumor] antigen are discussed

Virus-associated RNAs of naturally occurring strains and variants of group C adenoviruses.

We compared the sequences of the virus-associated (VA) RNAs of group C adenoviruses, serotypes 1, 2, 5, and 6, and of three variants of adenovirus type 2 (Ad2) selected for loss of the BamHI restriction site in the VA RNAI gene. In the naturally occurring strains. VA RNAI exists in two forms which differ by two nucleotides: one form is found in Ad2 and Ad6, and the other is found in Ad1 and Ad5. There are three sites of variation in Va RNAII, the Ad1, Ad2, and Ad5 forms each differing from Ad6 VA RNAII at one of the positions. One of the selected variants has a four-base duplication within the BamHI cleavage site, whereas the two others have acquired a VA RNAI sequence indistinguishable from that of Ad5. The findings are interpreted in terms of the secondary structures of the VA RNAs and the interrelationships among the viruses

Control and Regulation of Stem Cells

The roots of stem cell research can be traced back to classical work in embryology and regeneration performed in the 19th century. By the early 1900s, researchers in Europe had come to understand that various types of blood cells were derived from a particular “stem cell,” resulting in physicians’ attempts to administer bone marrow by mouth to patients suffering from anemia and leukemia. Although such therapy was unsuccessful, laboratory experiments eventually demonstrated that mice with defective marrow could be restored to health with infusions into the bloodstream of marrow taken from other mice, stimulating physicians to speculate whether it was feasible to transplant bone marrow from one human to another. By the late 1960s, pioneering work by Till andMcCulloch demonstrated the existence of hematopoietic stemcells. Later, bonemarrow transplantation (a stem cell transplant) was dogged by problems of histocompatability, but as the basis of the HLA system became increasingly understood, successful bone marrow transplantation between unrelated individuals was first demonstrated in 1973. Stem cell research has since exploded, with the derivation of mouse embryonic stem cells in 1981, the culturing of neural stem cells as neurospheres in 1992, and the establishment of the first human embryonic stem cell lines in 1998. A definitive link between stem cells and cancer was established when certain leukemias were shown to originate from hematopoietic stem cells. During the last decade, the concept of adult stem cell plasticity has gained credence, although many findings have proved controversial, and the ethical arguments for and against the use of human embryonic stem cells have been widely debated on national and international levels. Within the last 2 years, it has been reproducibly established that an embryonic stem cell–like state, previously achieved only by somatic cell nuclear transfer into enucleated oocytes (“cloning”) or by fusion with embryonic stem cells, can be induced by reprogramming differentiated adult cells using a simple combination of key transcription factors. Progress in stem cell research is now extremely intense, with more than 5000 research papers on embryonic and adult stem cells published in reputable scientific journals every year. It therefore seemed appropriate to focus the 73rd Symposium on this important and rapidly developing field, providing a unique synthesis of the exciting progress being made in the field of stem cell biology, not only for the Symposia attendees, but for a wider global audience via interviews freely available on the world wide web, and, we anticipate, for readers of these Proceedings