Andrea Manzi

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.

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.

Inheritance of the rDNA spacer in D.melanogaster

SumamryWe studied the organization of rDNA spacer sequences within several rDNA loci in D. melanogaster. Every locus showed many discrete length classes of rDNA spacer, ranging from 2.4 kb to about 20 kb. Different loci show characteristic distributions of spacers within the various length classes. Using this molecular characteristic as a genetic marker, segregation, recombination and occurrence of unequal crossing-over were studied. The rDNA loci segregated with a Mendelian pattern; interhomologous recombination events occurred at the expected rate; conversely unequal crossing-over within the rDNA locus appears not to be as frequent as expected.

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.

Expression of rDNA insertions during rDNA magnification in D. melanogaster

SummaryIn D. melanogaster, many ribosomal genes contain an intervening sequence that interrupts the 28S rRNA gene. In wild-type flies, type I and type II insertions are rarely transcribed and the transcripts of the interrupted genetic units do not contribute significantly to the production of mature ribosomal RNA.We demonstrate that during rDNA magnification, transcription of short type I sequences and of type II sequences is respectively, about 4- and about 16-fold that observed in unmagnified homozygous bobbed females used as control. In subsequent generation (bbm1) we have observed, for transcription of short type I sequences, that this elevated level is mantained and, for transcription of type II sequences, that the level reverts to one comparable with the control. Transcription of both insertion sequences decreases in subsequent generations (bbm19), reaching a level comparable with the controls.

Changes in abo phenotypic expression without increase in rDNA in Drosophila melanogaster

SummaryFemales of Drosophila melanogaster, homozygous for the abnormal oocyte mutation (abo 2; 44) produce eggs with a greatly reduced probability of developing into adults compared with those of control females. After several generations in abo homozygous stocks, the abo maternal effect is no longer observed. The progressive amelioration of the abo maternal effect in the Canton S background, into which the abo mutation was introduced, was concomitant with an increase in rDNA and variation in the rDNA restriction pattern. To clarify the relationship between the loss of the abo phenotype and the change in rDNA redundancy, we performed genetic and molecular analyses using abo stocks carrying X chromosomes of different origin and carrying different amounts of rDNA. The results we present confirm, in different genetic backgrounds, the previous observations on the behaviour of the abo mutation. However, both the amount and the restriction pattern of rDNA of the different X chromosomes studied remain unchanged after the loss of the abo phenotype. From these observations, it appears that changes in heterochromatic regions other than rDNA are responsible for the loss of the abo maternal effect.

Complete reversion of the abo phenotype in D. melanogaster occurs only when the blood transposon is lost from region 32E

SummaryThe abnormal oocyte phenotype is characterized by instability, as shown by the loss and reappearance of the abo maternal effect under specific genetic conditions. Our previous finding that a correlation exists between the abo phenotype and the presence of a blood transposon in region 32E, led us to perform an extensive genetic and molecular analysis of the most significant aspects of the abo phenotype in different genetic backgrounds. The results of these experiments can be summarized as follows: Complete reversion occurs only when the blood transposon is lost, thus definitively demonstrating that the insertion of the blood transposon in region 32E is the molecular event that causes the pleiotropic abo phenotype. Partial reversion can also occur without loss of the transposon, indicating that different molecular pathways may be involved in the loss of the abo phenotype. Reappearance of the full abo phenotype can occur only in heterozygous lines constructed from partially revertant abo homozygous lines that have not lost the blood transposon.