Antoinette Bolle

Transcription during bacteriophage T4 development: Synthesis and relative stability of early and late RNA☆☆☆

The control of RNA synthesis during infection of Escherichia coli B with bacteriophage T4 has been studied. RNA-DNA hybridization-competition experiments show that between one-third and one-half of the bacteriophage-specific RNA present at 20 minutes after infection at 30 °C consists of RNA species—the socalled “late” species—which are on the average several hundredfold less abundant five minutes after infection. The remainder of the 20-minute RNA consists of species—usually referred to as “early”—that are readily detectable in RNA extracted five minutes after infection. The late RNA species include some that are relatively much more abundant than others. We have investigated the relative stabilities of these two classes of RNA during the late stages of infection by comparing the quantitative partition, between early and late species, of a radioactive precursor incorporated into RNA in widely varying regimes of labelling. The proportion of label in early and late RNA species is constant regardless of the length of the labelling period, leading to the conclusion that the early and late species have comparable average decay times. Measurements of the labelling of late messenger have been made at several times during the first five minutes of infection to test the possibility that all T4 genes might be transcribed for a brief period after the injection of the phage genome into the host cell. Late RNA could not be detected at the beginning of infection. These results support the conclusion that the pattern and evolution of late RNA abundances during T4 development has its origin in changing rates of transcription and not in changing stabilities.

Transcription during bacteriophage T4 development: requirements for late messenger synthesis.

Abstract The prerequisites of late T4 messenger synthesis have been investigated in phageinfected Escherichia coli B, using the methods of DNA-RNA hybridizationcompetition. The absolute requirement of some DNA synthesis for viral late messenger synthesis has been demonstrated in three ways: (1) several DNA synthesis-negative (DO) amber mutants located in 3 different segments of the viral chromosome have been shown to be defective in late messenger synthesis in the non-permissive host; (2) when phage DNA synthesis is made thymine dependent by the proper choice of viral mutant and host, viral late messenger synthesis also becomes thymine-dependent, but early messenger synthesis does not; (3) ultraviolet-irradiated T4+ which do not replicate their DNA do not synthesize viral late messenger. However, DNA synthesis and late messenger synthesis are not linearly related. For example, late messenger synthesis can be sustained in cells infected with T4 gene 30 amber mutants in which DNA synthesis has almost ceased. DNA replication is not a sufficient requirement for late messenger synthesis: the function of at least one, and possibly more, “maturation gene” products is also required. Thus, a maturation-defective mutant, lacking the function of gene 55 ( am BL 292), synthesizes DNA but no late messenger. A mutant in T4 gene 33 ( am N134) is of the same type but “leakier” and eventually makes readily detectable proportions of late viral RNA. A viral dCTPase, the product of T4 gene 56, is synthesized early in normal T4 development. This enzyme helps to prevent the synthesis of cytosine-containing viral DNA. dCTPase-negative T4 amber mutants, even two multiple mutants that synthesize appreciable quantities of viral DNA, are defective in late messenger synthesis. Evidently, two mechanisms must be involved in the synthesis of T4 late messenger: (1) a competent form of DNA must be made available in the infected cell; and (2) one or more “maturation” gene products must act on this competent DNA. T4 gene 55 is likely to specify one such product.

Transcription during bacteriophage T4 development: A demonstration that distinct subclasses of the “early” RNA appear at different times and that some are “turned off” at late times☆

Abstract The appearance of phage-specific RNA synthesized during bacteriophage T4 infection has been examined in detail using RNA-DNA hybridization-competition techniques. The RNA synthesized during the first five minutes of infection includes at least three classes of RNA sequences appearing at different times: the ratio of class A messenger to class B and C RNA decreases about 40-fold between 1.25 and 5 minutes after infection at 30 °C. In the presence of chloramphenicol, class A RNA is synthesized in normal amount but the synthesis of the class C and part of the class B RNA species is blocked. It is suggested that one or more proteins coded for by the class A RNA species may facilitate transcription of class B and C RNA. Contrary to earlier findings, it is shown that the majority of the RNA present at five minutes (classes A, B and C) is in sequences which decrease several-fold in level between 5 and 20 minutes after infection (“true-early” species). Another class of RNA (“quasi-late”) is present at five minutes but increases several-fold by 20 minutes so that it accounts for the majority of the 20-minute RNA which competes with 5-minute RNA in hybridization to phage DNA. The behaviour of the quasi-late RNA is easily distinguished from that of another class which we designate true-late because the former increases about 4- to 15-fold between 5 and 20 minutes while the latter increases more than 100-fold in the same interval. It is suggested that both the true-late and the quasi-late messengers may be active in protein synthesis at 20 minutes and that the “turn-off” of early protein synthesis may be due to control at the level of transcription rather than a mechanism involving translational control.

Regulation of the synthesis of bacteriophage T4 gene 32 protein

Abstract The synthesis of T4 gene 32 product (P32) has been followed by gel electrophoresis of infected cell lysates. In wild-type infections, its synthesis starts soon after infection and begins to diminish about the time late gene expression commences. The absence of functional P32 results in a marked increase in the amount of the non-functional P32 synthesized. For example, infections of T4 mutants which contain a nonsense mutation in gene 32 produce the nonsense fragment at more than ten times the maximum rate of synthesis of the gene product observed in wild-type infections. All of the temperature-sensitive mutants in gene 32 that were tested also overproduce this product at the non-permissive temperature. This increased synthesis of the non-functional product is recessive, since mixed infections (wild-type, gene 32 nonsense mutant) fail to overproduce the nonsense fragment. Mutations in genes required for late gene expression (genes 33 and 53) as well as some genes required for normal DNA synthesis also result in increased production of P32. The overproduction in such infections is dependent on DNA synthesis; in the absence of DNA synthesis no overproduction occurs. This contrasts with the overproduction resulting from the absence of functional P32 which is not dependent on DNA synthesis. These results are compatible with a model for the regulation of expression of gene 32 in which the synthesis of P32 is either directly or indirectly controlled by its own function. Thus, in the absence of P32 function the expression of this gene is increased as is manifest by the high rate of P32 synthesis. It is further suggested that in infections defective in late gene expression and consequently in the maturation of replicated DNA, the increased P32 production is caused by the large expansion of the DNA pool. This DNA is presumed to compete for active P32 by binding it non-specifically to single-stranded regions, thus reducing the amount of P32 free to block gene 32 expression. Similarly, the aberrant DNA synthesized following infections with mutants in genes 41, 56, 58, 60 and 30, although quantitatively less than that produced in the maturation defective infections, can probably bind large quantities of P32 to single-stranded regions resulting in increased P32 synthesis.

Genetic identification of cloned fragments of bacteriophage T4 DNA and complementation by some clones containing early T4 genes

SummaryBacteriophage T4 DNA containing cytosine has been obtained from cells infected with phage mutant in genes 42, 56,denA anddenB. This DNA can be cut by a number of restriction endonucleases. Fragments obtained by digestion of this DNA withEcoRI have been cloned using the vector plasmid pCR1.Clones containing T4 DNA were identified by hybridization with radioactive early and late T4 RNA. A simple marker rescue technique is described for the genetic identification of the cloned T4 fragments. Some of the T4-hybrid plasmids which contain entire T4 genes can complement temperature sensitive and amber mutants of T4.

Controls and polarity of transcription during bacteriophage T4 development

Abstract Hybridization and hybridization-competition experiments, using the separated l and r strands of bacteriophage T4 DNA and 3H-labeled RNAs coded by T4 and its mutants, permit one to study the transcriptional controls for various phagespecific RNA species. In the present study, the T4-coded RNAs have been classified according to: (i) orientation of transcription; (ii) time of appearance; (iii) requirement for protein and DNA synthesis and for expression of the gene 33 and 55 functions; and (iv) true-early or quasi-late behavior, as defined by the respective decrease or increase in the early appearing RNA species during the second half of the viral eclipse period. The “immediate-early” messenger RNAs, produced after phage infection in the absence of protein synthesis, are transcribed counterclockwise from the l strand of T4 DNA. The “delayed-early” RNA is transcribed predominantly from the l strand, whereas the “true-late” RNA, the synthesis of which starts later than five minutes after infection, is mainly transcribed clockwise from the r strand, i.e. with the same orientation as T4 DNA replication. There is only a small difference in the 20-minute l-strand transcription between the wild-type T4 and its mutants blocked in DNA synthesis (D0; genes 42 and 43) or in the late functions (MD, maturation defective) controlled by genes 33 and 55, as revealed by hybridization-competition experiments. Also, 5-minute unlabeled T4-coded RNA competes with almost 90% of the labeled 20-minute RNA complementary to the l strand, indicating that, at most, 10 to 12% of the 20-minute l-specific RNA belongs to the true-late class. Experiments employing r strands show that the 20-minute RNA of the D0 and MD mutants is quite deficient in RNA species coded by wild-type T4 at 20 minutes after infection. Similarly, under non-permissive conditions, mutations in gene rII and in genes 46 and 47, the latter two controlling nuclease activity, depress the r-strand transcription, but mutation in gene 30, which controls T4 ligase, does not have such an effect. The influence of chloramphenicol inhibition at various stages of T4 development on the l and r strand transcription was also evaluated. The activity of the gene 55 product and of DNA replication are continuously required for efficient r strand transcription.

Bacteriophage T4 gene transcription studied by hybridization to cloned restriction fragments.

Cloned restriction fragments of bacteriophage T4 DNA (Mattson et al. , 1977; Selzer et al. , 1978) have been used as hybridization probes to study T4 transcription during infection of Escherichia coli . Eleven early genes and about 35 late genes have been studied. The time at which the genes are active, and their rate of transcription, have been measured by quantitative hybridization to excess DNA immobilized on nitrocellulose filters. The early genes studied include representatives of the three pre-replicative classes previously defined: immediate early (IE), delayed early (DE), and quasi-late (QL). Transcription of genes known or inferred to be in the IE class (genes 30, 39, 52, 40 and/or 41 and 42 ) are transcribed at the highest relative rate during the first four minutes of infection, and are transcribed in the presence of chloramphenicol. Transcription of DE genes rIIA, rIIB and 43 is highest four to eight minutes after infection and does not occur in the presence of chloramphenicol. Transcription of rIIB can be detected prior to transcription of rIIA , confirming previous data (Schmidt et al. , 1970) on rII transcription. The only genes studied which exhibit quasi-late behavior are the transfer RNA genes. They also have IE characteristics. Both l - and r -strand transcripts have been detected from the late region of the chromosome. Some of the l -strand (early) transcription detected from the late region may be anti-late RNA, but it is also possible that there are early genes in one of these regions (between genes 24 and 25 ). Differences in the kinetics of late gene transcription have been observed. A comparison of the earliest times at which late gene transcriptions can be detected shows that initiation of late gene transcription is not entirely synchronous. For example, transcription of genes 50 to 6 is detected by six minutes but transcription of genes 12 to 16 is not detected until 10 minutes. For all of the late genes studied, transcription, once begun, continues until at least 24 minutes. However, some genes ( 50 to 6 ) are transcribed at their maximal relative rate by 12 minutes whereas the relative rate of transcription of other genes ( 12 to 16, 23 ) continues to increase until at least 20 minutes. The relative rates of synthesis of late gene transcripts vary much less than the relative rates of synthesis of late proteins, suggesting that differential synthetic rates of late proteins are not controlled predominantly by messenger RNA synthetic rates.

Construction and properties of recombinant plasmids containing the rII genes of bacteriophage T4

SummaryThe EcoRI digestion products of phage T4 DNA have been examined using a phage DNA transformation assay. A 2.6x106 Dalton fragment was found to contain the rII genes. This fragment was purified and then treated with HindIII endonuclease. The cleavage products were ligated to the vector plasmid pBR313 and viable recombinant plasmids recovered. A genetic assay was employed to demonstrate that the recombinants contained T4 DNA and to localize on the phage genetic map the EcoRI and HindIII sites cleaved during the construction of the plasmids. Preliminary characterization suggests that a fragment covering the beginning of the rIIA gene possibly contains a promotor which is active in uninfected cells.

Fate of cloned bacteriophage T4 DNA after phage T4 infection of clone-bearing cells

Plasmid pBR322 replication is inhibited after bacteriophage T4 infection. If no T4 DNA had been cloned into this plasmid vector, the kinetics of inhibition are similar to those observed for the inhibition of Escherichia coli chromosomal DNA. However, if T4 DNA has been cloned into pBR322, plasmid DNA synthesis is initially inhibited but then resumes approximately at the time that phage DNA replication begins. The T4 insert-dependent synthesis of pBR322 DNA is not observed if the infecting phage are deleted for the T4 DNA cloned in the plasmid. Thus, this T4 homology-dependent synthesis of plasmid DNA probably reflects recombination between plasmids and infecting phage genomes. However, this recombination-dependent synthesis of pBR322 DNA does not require the T4 gene 46 product, which is essential for T4 generalized recombination. The effect of T4 infection on the degradation of plasmid DNA is also examined. Plasmid DNA degradation, like E. coli chromosomal DNA degradation, occurs in wild-type and denB mutant infections. However, neither plasmid or chromosomal degradation can be detected in denA mutant infections by the method of DNA--DNA hybridization on nitrocellulose filters.

Amber mutants of bacteriophage T4D: their isolation and genetic characterization.

We have isolated a large number of mutants of bacteriophage T4D that are unable to form plaques on strain B of Escherichia coli, but are able to grow (nearly) normally on some other strains of E. coli, in particular strain CR63. These mutants, designated amber (am), have been characterized by complementation tests, by genetic crosses, and by their response to chemical mutagens. It is concluded that a particular subclass of base substitution mutations may give rise to amber mutants and that such mutants occur in many genes, which are widely distributed over the T4 genome.