John F. Pulitzer

Human telomeric position effect is determined by chromosomal context and telomeric chromatin integrity

We investigated the influence of telomere proximity and composition on the expression of an EGFP reporter gene in human cells. In transient transfection assays, telomeric DNA does not repress EGFP but rather slightly increases its expression. In contrast, in stable cell lines, the same reporter construct is repressed when inserted at a subtelomeric location. The telomeric repression is transiently alleviated by increasing the dosage of the TTAGGG repeat factor 1 (TRF1). Upon a prolongated treatment with trichostatin A, the derepression of the subtelomeric reporter gene correlates with the delocalization of HP1α and HP1β. In contrast, treating the cells with 5 azacytidin, a demethylating agent, or with sirtinol, an inhibitor of the Sir2 family of deacetylase, has no apparent effect on telomeric repression. Overall, position effects at human chromosome ends are dependent on a specific higher‐order organization of the telomeric chromatin. The possible involvement of HP1 isoforms is discussed.

Abortive bacteriophage T4 head assembly in mutants of Escherichia coli

Abstract We describe two mutants ( tabB -212 and tabB -127) of Escherichia coli K12 in which T-even phage production is temperature-sensitive. Both mutants are linked to purA and may identify a single new bacterial gene tabB . The uninfected bacterium is indistinguishable from wild type at both 30 °C and 42.4 °C. Sodium dodecyl sulphate—polyacrylamide gel electrophoresis of labelled extracts of tabB mutants infected by T4 wild-type phage shows that the modification of viral head precursors (Laemmli, 1970) does not occur, indicating that capsid formation is blocked. The effect is reversible with at least one of the tabB mutants: a shift to 30 °C leads to the cleavage of a significant fraction of precursors synthesized at 42.4 °C. Two classes of T4 mutants are described: one ( com B ) which grows on tabB even at 42.4 °C, the other ( k B ) which fails to grow on tabB even at the permissive temperature. Both mutants map in T4 gene 31, suggesting an interaction between gene 31 and tabB products. Since gene 31 mutants lead to the random aggregation of head precursors (Laemmli, 1970), we argue that a host product is involved in the ordered polymerization of T4 proteins into capsids or capsid-related structures.

The isolation and characterization of TabR bacteria: Hosts that restrict bacteriophage T4 rII mutants

SummaryWe have isolated mutants of Escherichia coli B (called TabR) that restrict the growth of bacteriophage T4 rII mutants at high temperature. TabR strains lysed very rapidly after infection with rII mutants, and no progeny phage were produced. T4+-infected TabR cells also lysed quickly, but the cells remained intact long enough to give a small burst. We have selected pseudorevertants of rII deletion mutants that grow on TabR at high temperature; tk (thymidine kinase) is a component of one class of these pseudorevertants.T4 strains harboring mutations in genes 12, 16, 25, 34, 36, 45 and 63 were also specifically restricted on TabR strains at high temperature. Bacteriophages T2, T4, T5, T6, and T7 grew normally on TabR, while λ, Φ80, and P1 failed to grow at any temperature. The most restrictive TabR strains were auxotrophic for methionine at high temperature, and most spontaneous Met+ revertants had also lost the ability to restrict rII mutants, suggesting that the TabR phenotype and methionine auxotrophy result from the same mutation.Although the mechanism by which TabR strains exert their restriction has not been determined, one model is described. The potential uses of these and similar strains is discussed.

Design of a system of conditional lethal mutations (tab/k/com) affecting protein-protein interactions in bacteriophage T4-infected Escherichia coli

Abstract The re-direction of host-cell machinery to virus-specific functions, by the physical interaction between viral proteins and pre-existing host proteins, may be a mechanism commonly exploited in virus infection. We argue that the formation of a hybrid complex between an Escherichia coli protein and bacteriophage T4 protein controls the assembly of T4 capsid precursors into ordered structures. This early step in assembly can be blocked either by a mutation in T4 gene 31 (Laemmli et al. , 1970), or by a bacterial mutation ( gro E, tab B) (Georgopoulos et al. , 1972; Coppo et al. , 1973). We show that this step can also be blocked by the interaction of bacterial mutations ( tab B k , tab B com ) and viral mutations k B and com 8 ); com B mutations map in T4 gene 31, while k B mutations map in either gene 31 or 23. Many k 8 mutants are also temperature-sensitive. Phage T4 head assembly is blocked when tab B k (or tab B com ) are infected with T4 k B (or com B ), but not when the bacterial mutant is infected with T4 wild-type, or when tab + cells are infected with k B (or com B ). We interpret this phenomenon as a case of negative complementation between altered host and viral subunits of a hybrid complex and illustrate this idea with the experiments described in the text. We describe a technique by which tab B mutants can be efficiently and specifically selected with k B (or com B ) T4 mutants. Since many k B mutants are temperature-sensitive, temperature-sensitive mutants in other genes also may have latent k properties, and may be used for the isolation of new tab bacterial mutants, identifying other interactions between T4 and E. coli proteins.

Host mutant (tabD)-induced inhibition of bacteriophage T4 late transcription: II. Genetic characterization of mutants

Abstract In this paper we show that the tab D mutants, selected with ts 553 or ts CB53, and described in the accompanying paper (Coppo et al. , 1975): (a) are recessive to tab + ; (b) fail to complement each other, and thus map in the same cistron; (c) by their linkage to rif and their dominance relationships with well characterized amber mutations in the Escherichia coli RNA polymerase operon, probably all map in the gene controlling the synthesis of the β′ subunit of the enzyme. We also describe the isolation of a ts + , k D mutant in phage T4 gene 55, used in the selection of a new tab D mutant ( tab D k292 ). This tab mutant: (a) generates a defective phenotype which differs somewhat from that of the other tab D mutants; (b) complements the other tab D mutants; (c) by its dominance relationship to amber mutants in the RNA polymerase operon, can be assigned to the structural gene coding for the β subunit of the enzyme. A new type of interaction between T4 genes 55 and 45 is also described. The k D properties of ts 553 (gene 55) are suppressed at 30 °C, by a temperature-sensitive mutation in gene 45. This type of interaction between missense mutations in genes 45 and 55 apparently occurs even in tab + strains, since temperature-sensitive mutations in gene 45 accumulate in lysates of two gene 55 mutants ( ts 553 and ts A81).

Host mutant (tabD)-induced of bacteriophage T4 late transcription: I. Isolation and phenotypic characterization of the mutants☆

Abstract A temperature-sensitive mutation (ts553) in bacteriophage T4 gene 55, which codes for a positive control element of viral late transcription, has latent k properties (cf. Takahashi et al., 1975). It can be used to efficiently and specifically select a new class of Escherichia coli mutants (tabD). When a tabD mutant is infected with wild type T4, viral development proceeds almost normally; when tabD) is infected at 30 °C with ts553 late transcription is blocked. The tabD-generated defective phenotype is identical to that observed when tab+ is infected with an amber mutant in gene 55. A temperature-sensitive mutation (tsCB53) in T4 gene 45, which codes for a protein controlling late transcription and replication, also has latent k properties. It selects E. coli mutants, quite similar to those selected with ts553, which grow wild type T4 normally but fail to grow tsCB53 or ts553 at 30 °C; in the latter cases late transcription is blocked but not replication. The tab-generated deficiency is thus in striking contrast to that observed when tab+ is infected with an amber mutant in gene 45, characterized by a block in late transcription and replication. We argue that the products of T4 genes 55 and 45, and the bacterial protein/s identified by tabD mutants form a complex and discuss two alternative modes of interaction which may be relevant to late transcription. Since P55 and P45 bind to RNA polymerase (Ratner, 1974) one or more of the subunits of this enzyme are likely candidates for the tabD protein/s.

Host-virus interactions in the control of T4 prereplicative transcription: II. Interaction between tabC (rho) mutants and T4 mot mutants

Abstract In this paper we describe bacterial mutants ( tab G) that reinforce the effect of T4 mot mutants (Mattson et al. , 1978) ts G1 and am G1. tab G mutants map close to or in the Escherichia coli RNA polymerase rpo B gene. We explore the effect of mot -deficient T4 infections in tab C mutants (Caruso et al. , 1979). We find that: (1) in such infections the synthesis of most T4 proteins of molecular weight greater than 24,000 is inhibited; (2) T4-specific transcription is largely restricted to those species that have been defined as immediate early. We conclude that most tab C bacterial mutants affect transcription of T4 prereplicative genes and confirm that T4 mot mutants also affect transcription of T4 prereplicative genes. We discuss a model where many (most) T4 prereplicative genes are transcribed in two alternative modes: from a rho ( tab C)-sensitive, mot -independent early promoter and from a tab C-insensitive, mot -dependent, middle promoter.

Host--virus interactions in the control of T4 prereplicative transcription. I. tabC (rho) mutants.

Abstract In this paper we describe properties of old ( Takahashi, 1978 ) and new tabCts and tabCcs bacterial mutants. We find that under non-permissive conditions they differently inhibit the synthesis of specific T4 prereplicative gene products. Among such products, that we have been able to identify, are P43 and PrIIA. In contrast, P32 and PrIIB are not affected. Inhibition of P43 (T4 DNA polymerase) synthesis is sufficient to account for depressed DNA synthesis in tabC ( Takahashi, 1978 ). In heterodiploids: (1) all tabC mutants are recessive; (2) all tabC mutants do not complement with each other; (3) at least one, tabCts-5521, becomes dominant at 42.6 °C if rho mutant ts15 (Tab+) ( Das et al., 1976 ) is situated in trans; (4) tabCts-5521 also becomes dominant at 42.6 °C if tabCcs-110 and tabCcs-18 are situated in trans (42.6 °C is non-permissive for T4 development on tabCcs-5521 and permissive for T4 development on tabCcs mutants). We discuss the possibility that in tabC mutants rho protein is altered and insensitive to T4-specific anti-termination functions. We also discuss a model that accounts for the differential effect of tabC mutants on the synthesis of T4 prereplicative proteins.

The HTL1 gene (YCR020W‐b) of Saccharomyces cerevisiae is necessary for growth at 37°C, and for the conservation of chromosome stability and fertility

A small 78 codon ORF, named HTL1 (Chen et al., unpublished results), situated between loci MAK31 and HSP30 on chromosome III of Saccharomyces cerevisiae, is required for growth at 37°C. In this communication, we characterize the ORF and show that disruption of HTL1, besides preventing growth at 37°C, causes genetic and/or epigenetic instability at 26°C: ploidy increases in about 10% of cells grown from individual disruptants and a fraction of disruptant clones are predestined to a rapid and progressive loss of fertility during growth at 26°C. Copyright

New control elements of bacteriophage T4 pre-replicative transcription

Bacteriophage T4 pre-replicative genes are transcribed, by Escherichia coli RNA polymerase, in two alternative modes: an early mode and a middle mode. Middle mode transcription is under the control of at least one viral protein, pmotA. We have identified two additional viral genes, motB and motC, that map in the dispensable region of the T4 genome, between genes 39 and 56. pmotB and pmotC are diffusible factors which provide an alternative to the motA dependent mode of middle transcription of many T4 genes. Deletions of motB and motC are in fact lethal only in combination with a motA mutant. motB controls one of the alternative modes of transcription of the rIIA gene. When motA or motB are missing, transcription of rIIA is quantitatively unaffected; when both are missing the transcription rate drops by about 75%. Control of transcription of the tRNA gene cluster is more complex. Transcription of subcluster 2 is maximally reduced (70%) only by deletions that, besides motB, cut out an adjacent region. We guess that this adjacent region codes for an additional control element, which we call motC. The motB gene is situated in a 750-base region between the left end-points of del(39-56)-1 and -4.