Multiple and Specific Initiation of T4 DNA Replication

Partially replicated T4 DNA molecules (PRM) whose parental or progeny DNA was labeled with bromodeoxyuridine BUdR was analyzed by gradual shearing followed by CsCl banding of the sheared product. Analysis of PRM containing 18-mum replicated DNA showed that each replicated region was 3- to 6-mum long, indicating three to 6 replicative sites per molecule. Analysis of PRM containing 9-mum replicated DNA similarly indicated two to three replicated regions per molecule. DNA from the replicated regions of PRM containing 10-mum replicated DNA ("donor") was hybridized to DNA from mature phage ("recipient"), and the resulting hybrid was subjected to digestion with exonuclease I. The extent of protection of the recipient and more efficient self-annealing of progeny fragments from PRM indicated that the replicated regions represented 8 to 10 nonrandom locations of the genome. Possible significance of multiple sites for initiation of DNA replication is discussed.     

Shutoff on Neuroblastoma Cell Protein Synthesis by Semliki Forest Virus: Loss of Ability of Crude Initiation Factors to Recognize Early Semliki Forest Virus and Host mRNA's

A crude ribosomal wash containing the initiation factors of protein synthesis was isolated from mouse neuroblastoma cells 8 h after infection with Semliki Forest virus (SFV). The activity of this wash was compared with that of a wash from control cells in a cell-free protein-synthesizing “pH5” system, with early SFV mRNA (42S), late SFV mRNA (26S), encephalomyocarditis virus (EMC) mRNA, or neuroblastoma polyadenylated mRNA templates. A pronounced loss of activity (±80%) of the crude ribosomal wash from infected cells was observed with host mRNA (neuroblastoma polyadenylated mRNA) and early SFV mRNA, messengers which contain a cap structure at the 5′ terminus. However, these washes were only slightly less active in systems programmed with (noncapped) EMC mRNA and late SFV mRNA. Although late SFV mRNA (26S) is capped, the synthesis of late (= structural) proteins in infected lysates was insensitive to inhibition by cap analogs. Purified initiation factors eIF-4B (M_r, 80,000) and cap-binding protein (M_r, 24,000) from reticulocytes (but none of the others) were able to restore the activity of infected factors to about 90% of control levels in systems programmed with early SFV mRNA and host mRNA. These observations indicate that infection-exposed crude initiation factors have a decreased level of eIF-4B and cap-binding protein activity. However, after partial purification of these and other initiation factors from infected and control cells, we found no significant difference in activity when model assay systems were used. Furthermore, both eIF-4B and cap-binding protein from infected cells were able to restore the activity of these infection-exposed factors to the same level obtained when these factors isolated from control cells or reticulocytes were added. A possible mechanism for the shutoff of host cell protein synthesis is discussed.     

Early Events in Parvovirus Replication: Lack of Integration by Minute Virus of Mice into Host Cell DNA

Viral DNA sequences were not detected in high-molecular-weight host DNA until well after the onset of viral DNA replication.     

Initiation of heteroduplex-loop repair by T4-encoded endonuclease VII in vitro.

Heteroduplex DNAs with single-stranded loops of 51 nt or 8 nt were constructed in vitro and used in reactions with purified endonuclease VII (endo VII) from phage T4. The enzyme makes double-strand breaks by introducing pairs of staggered nicks flanking the loops. Regardless of loop-size the nicking sites map exclusively at the 3' side of the loop in the looping strand and at the 3' side of the base of the loop in the non-looping strand. The number of potential cleavage sites is small (less than 5) and their distribution depends on DNA sequence. The two closest staggered nicks are 4 bp apart, 2 bp on either side of the loop. Nicking always occurs in the double-stranded part of the molecules; the single-stranded loops are not attacked by endo VII. The nicks are introduced in a stepwise fashion and selection of the strand for the first nick depends on the sequence of 31 base pairs flanking the loops.     

Partial Suppression of Bacteriophage T4 Ligase Mutations by T4 Endonuclease II Deficiency: Role of Host Ligase

Endonuclease II-deficient, ligase-deficient double mutants of phage T4 induce considerably more deoxyribonucleic acid (DNA) synthesis after infection of Escherichia coli B than does the ligase-deficient single mutant. Furthermore, the double mutant can replicate 10 to 15% as well as wild-type T4, whereas the single mutant fails to replicate. When the E. coli host is also deficient in ligase, the double mutant resembles the single mutant. The results indicate that host ligase can substitute for phage ligase when the host DNA is not attacked by the phage-induced endonuclease II.     

Early exposure to IL-4 stabilizes IL-4 mRNA in CD4+ T cells via RNA-binding protein HuR

The mechanisms regulating IL-4 mRNA stability in differentiated T cells are not known. We found that early exposure of CD4+ T cells to endogenous IL-4 increased IL-4 mRNA stability. This effect of IL-4 was mediated by the RNA-binding protein HuR. IL-4 mRNA interacted with HuR and the dominant binding site was shown within the coding region of IL-4 mRNA. Exposure of CD4+ T cells to IL-4 had no effects on HuR expression or subcellular localization, but triggered HuR binding to IL-4 mRNA. Thus, IL-4 plays a positive role in maintaining IL-4 mRNA stability in CD4+ T cells via a HuR-mediated mechanism.     

Inactivation of Bacteriophage T4 by Ethyl Methanesulfonate: Influence of Host and Viral Genotypes

Inactivation of bacteriophage T4 by ethyl methanesulfonate (EMS) is a complex process which depends critically upon the conditions of treatment and upon both the viral and the host genotypes. EMS-inactivated particles are capable of multiplicity and cross-reactivation, indicating the need for caution in using EMS in certain types of mutation studies. The pyrimidine dimer excision systems of the phage and the host do not affect the EMS sensitivity of T4, but the T4x⁺y⁺ system does. Mutational defects in the deoxyribonucleic acid (DNA) ligase and the DNA polymerase systems both of the virus and of its host also affect viral EMS sensitivity.     

Modification of the 5' End of mRNA Leader Sequence Alters the Set of Initiation Factors Essential for Initiation of Translation

Molecular Biology - The abundance of noncanonical mechanisms of eukaryotic initiation of translation indicates their involvement in the regulation of protein synthesis during key events in a cell...     

Initiation of mRNA Translation in Oncogenesis: The Role of eIF4E

The eukaryotic initiation factor 4E (eIF4E) is a key regulator of protein translation whose function is activated by the Akt and Ras proto-oncogenic signal transduction pathways. eIF4E enhances the translation of mRNAs encoding several genes involved in tumorigenesis and acts as a proto-oncogene, in vitro, when overexpressed in immortalized cells. Importantly, eIF4E is frequently found overexpressed in human cancers of multiple histological origins. However, in vivo evidence of the eIF4E neoplastic potential was lacking until now. Here we discuss recent findings that demonstrate eIF4E’s oncogenic role in vivo through direct genetic approaches in the mouse, and identify novel oncogenic functions for this initiation factor in cooperative tumorigenesis and response to therapy.     

Stoichiometry and Change of the mRNA Closed-Loop Factors as Translating Ribosomes Transit from Initiation to Elongation

Protein synthesis is a highly efficient process and is under exacting control. Yet, the actual abundance of translation factors present in translating complexes and how these abundances change during the transit of a ribosome across an mRNA remains unknown. Using analytical ultracentrifugation with fluorescent detection we have determined the stoichiometry of the closed-loop translation factors for translating ribosomes. A variety of pools of translating polysomes and monosomes were identified, each containing different abundances of the closed-loop factors eIF4E, eIF4G, and PAB1 and that of the translational repressor, SBP1. We establish that closed-loop factors eIF4E/eIF4G dissociated both as ribosomes transited polyadenylated mRNA from initiation to elongation and as translation changed from the polysomal to monosomal state prior to cessation of translation. eIF4G was found to particularly dissociate from polyadenylated mRNA as polysomes moved to the monosomal state, suggesting an active role for translational repressors in this process. Consistent with this suggestion, translating complexes generally did not simultaneously contain eIF4E/eIF4G and SBP1, implying mutual exclusivity in such complexes. For substantially deadenylated mRNA, however, a second type of closed-loop structure was identified that contained just eIF4E and eIF4G. More than one eIF4G molecule per polysome appeared to be present in these complexes, supporting the importance of eIF4G interactions with the mRNA independent of PAB1. These latter closed-loop structures, which were particularly stable in polysomes, may be playing specific roles in both normal and disease states for specific mRNA that are deadenylated and/or lacking PAB1. These analyses establish a dynamic snapshot of molecular abundance changes during ribosomal transit across an mRNA in what are likely to be critical targets of regulation.