The T4-related bacteriophages are a group of bacterial viruses that share morphological similarities and genetic homologies with the well-studied Escherichia coli phage T4, but that diverge from T4 and each other by a number of genetically determined characteristics including the bacterial hosts they infect, the sizes of their linear double-stranded (ds) DNA genomes and the predicted compositions of their proteomes. The genomes of about 40 of these phages have been sequenced and annotated over the last several years and are compared here in the context of the factors that have determined their diversity and the diversity of other microbial genomes in evolution. The genomes of the T4 relatives analyzed so far range in size between ~160,000 and ~250,000 base pairs (bp) and are mosaics of one another, consisting of clusters of homology between them that are interspersed with segments that vary considerably in genetic composition between the different phage lineages. Based on the known biological and biochemical properties of phage T4 and the proteins encoded by the T4 genome, the T4 relatives reviewed here are predicted to share a genetic core, or "Core Genome" that determines the structural design of their dsDNA chromosomes, their distinctive morphology and the process of their assembly into infectious agents (phage morphogenesis). The Core Genome appears to be the most ancient genetic component of this phage group and constitutes a mere 12-15% of the total protein encoding potential of the typical T4-related phage genome. The high degree of genetic heterogeneity that exists outside of this shared core suggests that horizontal DNA transfer involving many genetic sources has played a major role in diversification of the T4-related phages and their spread to a wide spectrum of bacterial species domains in evolution. We discuss some of the factors and pathways that might have shaped the evolution of these phages and point out several parallels between their diversity and the diversity generally observed within all groups of interrelated dsDNA microbial genomes in nature.

The DNA polymerases (gp43s) of the related bacteriophages T4 and RB69 are B family (polymerase ␣ class) enzymes that determine the fidelity of phage DNA replication. A T4 whose gene 43 has been mutationally inactivated can be replicated by a cognate RB69 gp43 encoded by a recombinant plasmid in T4-infected Escherichia coli. We used this phage-plasmid complementation assay to obtain rapid and sensitive measurements of the mutational specificities of mutator derivatives of the RB69 enzyme. RB69 gp43s lacking proofreading function (Exo ؊ enzymes) and/or substituted with alanine, serine, or threonine at the conserved polymerase function residue Tyr 567 (Pol Y567(A/S/T) enzymes) were examined for their effects on the reversion of specific mutations in the T4 rII gene and on forward mutation in the T4 rI gene. The results reveal that Tyr 567 is a key determinant of the fidelity of base selection and that the Pol and Exo functions are strongly coupled in this B family enzyme. In vitro assays show that the Pol Y567A Exo ؊ enzyme generates mispairs more frequently but extends them less efficiently than does a Pol ؉ Exo ؊ enzyme. Other replicative DNA polymerases may control fidelity by strategies similar to those used by RB69 gp43.Bacteriophage RB69 is a relative of phage T4, with which it shares many similarities in genetic organization (1, 2) and structures and functions of the phage-encoded DNA replication proteins (3,4). Replication fidelity in T4 and presumably also in RB69 is determined almost exclusively by the fidelities of the phage-encoded DNA polymerase and its associated proofreading 3Ј-5Ј exonuclease (5). This useful simplicity reflects the fact that T4 DNA replication appears to be devoid of DNA mismatch repair; phage T4 is not subject to the action of the several Escherichia coli mismatch repair systems (6) and seems unable to repair mutational heteroduplexes on its own. Screens for T4 mutator mutations have failed to uncover evidence for the involvement of mismatch repair in mutagenesis, and the mutational dose response to base analogues does not display the mismatch repair-dependent lag seen in E. coli (5).The DNA polymerases of phages T4 and RB69 (gp43, product of phage gene 43) are members of the polymerase ␣ class or B family of DNA polymerases, which includes the replicative polymerases ␣, ␦, and ⑀ of eukaryotic cells and the polymerases of several of their DNA viruses (7). Some archaeons also encode gp43-like B family enzymes (8 -10). As such, T4 gp43 and RB69 gp43 are attractive subjects for studies of mechanisms of replication by this class of enzymes, particularly because of the amenability of the phage system to combined genetic and biochemical analyses (11)(12)(13)(14). A recently determined crystal structure of RB69 gp43 reveals five discrete domains termed N, Exo, Palm, Fingers, and Thumb (15). This structure is in the "open" configuration and provides a preliminary framework for understanding the dynamics of DNA polymerase interactions with the DNA primer template, with incoming dNTPs, and ...

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Background: Bacteriophages are an important repository of genetic diversity. As one of the major constituents of terrestrial biomass, they exert profound effects on the earth's ecology and microbial evolution by mediating horizontal gene transfer between bacteria and controlling their growth. Only limited genomic sequence data are currently available for phages but even this reveals an overwhelming diversity in their gene sequences and genomes. The contribution of the T4-like phages to this overall phage diversity is difficult to assess, since only a few examples of complete genome sequence exist for these phages. Our analysis of five T4-like genomes represents half of the known T4-like genomes in GenBank.

ObjectiveResults of the screening of disease causative mutations in congenital hypothyroidism (CH) vary significantly, depending on the sequence strategy, patients’ inclusion criteria and bioinformatics. The objective was to study the molecular basis of severe congenital hypothyroidism, using the next generation sequencing (NGS) and the recent guidelines for assessment of sequence variants.Design243 patients with CH (TSH levels at neonatal screening or retesting greater than 90 mU/l) and 56 control subjects were included in the study.MethodsA custom NGS panel targeting 12 CH causative genes was used for sequencing. The sequence variants were rated according to American College of Medical Genetics and Genomics (ACMG) guidelines.ResultsIn total, 48 pathogenic, 7 likely pathogenic and 57 variants of uncertain significance were identified in 92/243 patients (37.9%), while 4 variants of uncertain significance were found in 4/56 control subjects (7.1%). 13.1% (12/92) of the cases showed variants in ‘thyroid dysgenesis’ (TD) genes: TSHR, n = 6; NKX2-1, n = 2; NKX2-5, n = 1; PAX8, n = 3. The variants in ‘dyshormonogenesis’ (DH) genes were found in 84.8% (78/92) of cases: TPO, n = 30; DUOX2, n = 24; TG, n = 8; SLC5A5, n = 3; SLC26A4, n = 6; IYD, n = 1. 8 patients showed oligonenic variants. The majority of variants identified in DH genes were monoallelic.ConclusionsIn contrast to earlier studies demonstrating the predominance of TD in severe CH, the majority of variants identified in our study were in DH genes. A large proportion of monoallelic variants detected among DH genes suggests that non-mendelian mechanisms may play a role in the development of CH.

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BackgroundPrimary hyperparathyroidism (PHPT) is a relatively rare disorder among children, adolescents and young adults. Its development at an early age is suspicious for hereditary causes, though the need for routine genetic testing remains controversial.ObjectiveTo identify and describe hereditary forms of PHPT in patients with manifestation of the disease under 40 years of age.DesignWe enrolled 65 patients with PHPT diagnosed before 40 years of age. Ten of them had MEN1 mutation, and PHPT in them was the first manifestation of multiple endocrine neoplasia type 1 syndrome.MethodsThe other fifty-five patients underwent next-generation sequencing (NGS) of a custom-designed panel of genes, associated with PHPT (MEN1, CASR, CDC73, CDKN1A, CDKN1B, CDKN1C, CDKN2A, CDKN2C, CDKN2D). In cases suspicious for gross CDC73 deletions multiplex ligation-dependent probe amplification was performed.ResultsNGS revealed six pathogenic or likely pathogenic germline sequence variants: four in CDC73 c.271C>T (p.Arg91*), c.496C>T (p.Gln166*), c.685A>T (p.Arg229*) and c.787C>T (p.Arg263Cys); one in CASR c.3145G>T (p.Glu1049*) and one in MEN1 c.784-9G>A. In two patients, MLPA confirmed gross CDC73 deletions. In total, 44 sporadic and 21 hereditary PHPT cases were identified. Parathyroid carcinomas and atypical parathyroid adenomas were present in 8/65 of young patients, in whom CDC73 mutations were found in 5/8.ConclusionsHereditary forms of PHPT can be identified in up to 1/3 of young patients with manifestation of the disease at <40 years of age. Parathyroid carcinomas or atypical parathyroid adenomas in young patients are frequently associated with CDC73 mutations.

DNA polymerase (gp43) of phage T4 plays two biological roles, one as an essential DNA binding replication enzyme and the other as an mRNA-specific autogenous translational repressor. Binding of T4 gp43 to its mRNA target (translational operator RNA) interferes with gp43-DNA interactions, but it is unclear how the protein determinants for binding DNA are affected by the dynamics of gp43-mRNA interactions. We have used RB69 gp43, a natural variant of the T4 enzyme whose crystal structure has been determined to identify protein sites that respond to the interaction with specific RNA. We used protein phosphorylation markers, photocross-linking studies, protease sensitivity assays, and mutational analyses to examine the effects of operator RNA on the enzyme's five structural domains (N, exo, palm, fingers, and thumb). Our studies suggest that this RNA affects gp43-DNA interactions through global effects on protein structure that occlude DNA-binding sites but leave the enzyme accessible to interactions with the sliding clamp (RB69 gp45) and possibly other polymerase accessory proteins. We discuss the possible biological significance of putative RNA-binding motifs in the N and palm domains of RB69 gp43.