DNA intermediates of meiotic recombination in synchronous S. pombe at optimal temperature

Crossovers formed by recombination between homologous chromosomes are important for proper homolog segregation during meiosis and for generation of genetic diversity. Optimal molecular analysis of DNA intermediates of recombination requires synchronous cultures. We previously described a mutant, pat1-as2, of the fission yeast Schizosaccharomyces pombe that undergoes synchronous meiosis at 25°C when an ATP analog is added to the culture. Here, we compare recombination intermediates in pat1-as2 at 25°C with those in the widely used pat1-114 temperature-sensitive mutant at 34°C, a temperature higher than optimal. DNA double-strand breaks at most hotspots are similarly abundant in the two conditions but, remarkably, a few hotspots are distinctly deficient at 25°C. In both conditions, Holliday junctions at DNA break hotspots form more frequently between sister chromatids than between homologs, but a novel species, perhaps arising from invasion by only one end of broken DNA, is more readily observed at 25°C. Our results confirm the validity of previous assays of recombination intermediates in S. pombe and provide new information on the mechanism of meiotic recombination.     

Correlation of Phenotype with the Genotype of Egg-Contaminating Salmonella enterica Serovar Enteritidis

The genotype of Salmonella enterica serovar Enteritidis was correlated with the phenotype using DNA-DNA microarray hybridization, ribotyping, and Phenotype MicroArray analysis to compare three strains that differed in colony morphology and phage type. No DNA hybridization differences were found between two phage type 13A (PT13A) strains that varied in biofilm formation; however, the ribotype patterns were different. Both PT13A strains had DNA sequences similar to that of bacteriophage Fels2, whereas the PT4 genome to which they were compared, as well as a PT4 field isolate, had a DNA sequence with some similarity to the bacteriophage ST64b sequence. Phenotype MicroArray analysis indicated that the two PT13A strains and the PT4 field isolate had similar respiratory activity profiles at 37°C. However, the wild-type S. enterica serovar Enteritidis PT13A strain grew significantly better in 20% more of the 1,920 conditions tested when it was assayed at 25°C than the biofilm-forming PT13A strain grew. Statistical analysis of the respiratory activity suggested that S. enterica serovar Enteritidis PT4 had a temperature-influenced dimorphic metabolism which at 25°C somewhat resembled the profile of the biofilm-forming PT13A strain and that at 37°C the metabolism was nearly identical to that of the wild-type PT13A strain. Although it is possible that lysogenic bacteriophage alter the balance of phage types on a farm either by lytic competition or by altering the metabolic processes of the host cell in subtle ways, the different physiologies of the S. enterica serovar Enteritidis strains correlated most closely with minor, rather than major, genomic changes. These results strongly suggest that the pandemic of egg-associated human salmonellosis that came into prominence in the 1980s is primarily an example of bacterial adaptive radiation that affects the safety of the food supply.     

Phage Resistance in a Phage-Insensitive Strain of Streptococcus lactis: Temperature-Dependent Phage Development and Host-Controlled Phage Replication

Streptococcus lactis ME2 is a dairy starter strain that is insensitive to a variety of phage, including 18. The efficiency of plating of 18 on ME2 and N1 could be increased from <1 × 10⁻⁹ to 5.0 × 10⁻² and from 7.6 × 10⁻⁷ to 2.1 × 10⁻², respectively, when the host strains were subcultured at 40°C before plating the phage and the phage assay plates were incubated at 40°C. Host-dependent replication was demonstrated in N1 at 30°C and in N1 and ME2 at 40°C, suggesting the operation of a temperature-sensitive restriction and modification system in ME2 and N1. The increased sensitivity of ME2 and N1 to 18 at 40°C was also demonstrated by lysis of broth cultures and increased plaque size. ME2 grown at 40°C showed an increased ability to adsorb 18, indicating a second target for temperature-dependent phage sensitivity in ME2. Challenge of N1 with a 18 preparation that had been previously modified for growth on N1 indicated that at 40°C phage development was characterized by a shorter latent period and larger burst size than at 30°C. The evidence presented suggests that the high degree of phage insensitivity expressed by ME2 consists of a variety of temperature-sensitive mechanisms, including (i) the prevention of phage adsorption, (ii) host-controlled restriction of phage, and (iii) suppression of phage development. At 30°C these factors appear to act cooperatively to prevent the successful emergence of lytic phage active against S. lactis ME2.     

Decay of incorporated radioactive phosphorus during reproduction of bacteriophage T2

The multiplication of vegetative T2 bacteriophage in B/r bacteria has been followed by studying the lethal effects of decay of incorporated radiophosphorus P³² at various stages of the eclipse period. Experiment I. Non-radioactive B/r bacteria were infected with highly radioactive (i.e. P³²-unstable) T2 and infection allowed to proceed at 37°C. for various numbers of minutes before freezing the infected cells and storing them in liquid nitrogen. The longer development had been allowed to proceed at 37°C. before freezing, the slower the inactivation of the frozen infective centers by P³² decay. Samples which were frozen after incubation for 9 minutes were completely stable. Experiment II. Radioactive B/r bacteria in radioactive growth medium were infected with non-radioactive (i.e. stable) T2 and incubated for various lengths of time before being frozen and stored in liquid nitrogen, like those of Experiment I. In this case, the infective centers were stable to P³² decay as long as they were frozen before the end of the eclipse period. The T2 progeny phages issuing from the infected bacteria were P³²-unstable. Experiment III. Radioactive B/r bacteria in radioactive medium were infected with radioactive (i.e. P³²-unstable) T2 and otherwise incubated and frozen like those of the first two experiments. In this case, the same progressive stabilization, of the infective centers towards inactivation by P³² decay was observed as that found in Experiment I. The ability to yield infective progeny of infected bacteria incubated for 10 minutes at 37°C. before freezing could no longer be destroyed by P³² decay. The progeny issuing from the infected cells were as unstable as the parental phage. These results could be explained by one of three general hypotheses. As vegetative phage begins to multiply, it is possible that: (a) there is a high probability that any part of the vegetative phage already duplicated can be saved after its destruction by P³² decay through a process analogous to multiplicity reactivation or, (b) there occurs a change in state of the deoxyribonucleic acid (DNA) preliminary to or in the course of its replication that renders it refractory to destruction by P³² decay, or, finally (c) there occurs a transfer of the genetic factors from the DNA of the infecting phage to another substance not sensitive to destruction by P³² decay.     

Isolation and Partial Characterization of Two Aeromonas hydrophila Bacteriophages

Two Aeromonas hydrophila bacteriophages, Aeh1 and Aeh2, were isolated from sewage. Both phages showed binal symmetry. The dimensions of A. hydrophila phages Aeh1 and Aeh2 differed from those of the other Aeromonas phages. Also, phage Aeh2 was the largest Aeromonas phage studied to date. Phage Aeh1 formed small, clear plaques, and phage Aeh2 formed turbid plaques with clear centers. Both phages were sensitive to chloroform treatment, being totally inactivated after treatment for 1 h at 60°C at pH 3 and 11. However, the infectivity of Aeh1 phage stocks increased by approximately fivefold after they were treated at pH 10 for 1 h at 22°C. Phages Aeh1 and Aeh2 were serologically unrelated and had latent periods of 39 and 52 min, respectively. The average burst sizes of phages Aeh1 and Aeh2 were 17 and 92 PFU per cell, respectively. Phage Aeh1 infected 13 of 22 A. hydrophila strains tested, whereas phage Aeh2 infected only its original host. Phage Aeh1 infected some A. hydrophila strains only at or below 37°C. Neither phage infected the two A. (Plesiomonas) shigelloides strains used in this study.     

Effect of Incubation Temperature on the Detection of Thermophilic Campylobacter Species from Freshwater Beaches,Nearby Wastewater Effluents,and Bird Fecal Droppings

This large-scale study compared incubation temperatures (37°C versus 42°C) to study the detection of thermophilic Campylobacter species, including Campylobacter jejuni, C. coli, and C. lari, in various surface water samples and bird fecal droppings around Hamilton Harbor, Lake Ontario. The putative culture isolates obtained from incubation temperatures of 37 and 42°C were confirmed by Campylobacter genus- and species-specific triplex PCR assays targeting the 16S rRNA gene and the 16S-23S rRNA gene internal transcribed spacer (ITS) region. A total of 759 water, wastewater, and bird fecal dropping samples were tested. Positive amplification reactions for the genus Campylobacter were found for 454 (60%) samples incubated at 37°C, compared to 258 (34%) samples incubated at 42°C. C. jejuni (16%) and C. lari (12%) were detected significantly more frequently at the 42°C incubation temperature than at 37°C (8% and 5%, respectively). In contrast, significantly higher rates of C. coli (14%) and other Campylobacter spp. (36%) were detected at the 37°C incubation temperature than at 42°C (8% and 7%, respectively). These results were consistent across surface water, wastewater, and bird fecal dropping samples. At times, Campylobacter spp. were recovered and detected at 37°C (3% for C. jejuni, 10% for C. coli, and 3% for C. lari) when the same samples incubated at 42°C were negative. A significantly higher rate of other Campylobacter spp. was detected only at 37°C (32%) than only at 42°C (3%). These results indicate that incubation temperature can significantly influence the culturability and detection of thermophilic and other fastidious Campylobacter spp. and that a comprehensive characterization of the Campylobacter spp. in surface water, wastewaters, or bird fecal droppings will require incubation at both 37 and 42°C.     

Storage Temperature Alters the Expression of Differentiation-Related Genes in Cultured Oral Keratinocytes

Purpose

Storage of cultured human oral keratinocytes (HOK) allows for transportation of cultured transplants to eye clinics worldwide. In a previous study, one-week storage of cultured HOK was found to be superior with regard to viability and morphology at 12°C compared to 4°C and 37°C. To understand more of how storage temperature affects cell phenotype, gene expression of HOK before and after storage at 4°C, 12°C, and 37°C was assessed.

Materials and Methods

Cultured HOK were stored in HEPES- and sodium bicarbonate-buffered Minimum Essential Medium at 4°C, 12°C, and 37°C for one week. Total RNA was isolated and the gene expression profile was determined using DNA microarrays and analyzed with Partek Genomics Suite software and Ingenuity Pathway Analysis. Differentially expressed genes (fold change > 1.5 and P < 0.05) were identified by one-way ANOVA. Key genes were validated using qPCR.

Results

Gene expression of cultures stored at 4°C and 12°C clustered close to the unstored control cultures. Cultures stored at 37°C displayed substantial change in gene expression compared to the other groups. In comparison with 12°C, 2,981 genes were differentially expressed at 37°C. In contrast, only 67 genes were differentially expressed between the unstored control and the cells stored at 12°C. The 12°C and 37°C culture groups differed most significantly with regard to the expression of differentiation markers. The Hedgehog signaling pathway was significantly downregulated at 37°C compared to 12°C.

Conclusion

HOK cultures stored at 37°C showed considerably larger changes in gene expression compared to unstored cells than cultured HOK stored at 4°C and 12°C. The changes observed at 37°C consisted of differentiation of the cells towards a squamous epithelium-specific phenotype. Storing cultured ocular surface transplants at 37°C is therefore not recommended. This is particularly interesting as 37°C is the standard incubation temperature used for cell culture.     

Stability of Staphylococcus aureus Phage ISP after Freeze-Drying (Lyophilization)

Staphylococcus aureus phage ISP was lyophilized, using an Amsco-Finn Aqua GT4 freeze dryer, in the presence of six different stabilizers at different concentrations. Stability of the lyophilized phage at 4°C was monitored up to 37 months and compared to stability in Luria Bertani broth and physiological saline at 4°C. Sucrose and trehalose were shown to be the best stabilizing additives, causing a decrease of only 1 log immediately after the lyophilization procedure and showing high stability during a 27 month storage period.     

FURTHER OBSERVATIONS ON THE MECHANISM OF PHAGE ACTION

1. The reaction between an antistaphlycoccal phage and the homologous bacterium has been studied, applying the following experimental technics not used in earlier work reported from this laboratory: (a) Both the activity assay and the plaque count were utilized for determining [phage]. (b) Sampling was done at short intervals; i.e., every 0.1 hour. (c) Extracellular phage was separated from the cell-bound fraction by a filtration procedure permitting passage of < 95 per cent of free phage. 2. Using these technics, the reaction was followed: (a) with pH maintained at 6.10 and temperature at 28°C. to slow the process; (b) with pH maintained at 7.2 and temperature at 36°C. 3. In addition separate experiments were performed on the sorption of phage by bacteria at 30°, 23°, and 0°C. 4. At pH 6.10 and 28°C. the phage-bacterium reaction proceeds in the following sequence: (a) There is an initial phase of rapid logarithmic sorption of phage to susceptible cells, during which the total phage activity and the plaque numbers in the mixtures remain constant. (b) When 90 per cent of the phage has been bound, there is a sudden very rapid increase in phage activity not paralleled by an increase in plaques; i.e., phage is formed intracellularly, but is retained within cellular confines. (c) After a further drop in the extracellular phage fraction there occurs a pronounced increase in the total phage plaque count not accompanied by any increase in total activity. This indicates a redistribution of phage formed intracellularly. At the same time there is a rise in the extracellular phage curves (both activity and plaque). (d) With the concentrations of phage and bacteria used in the experiment carried out at pH 6.1 and 28°C. there are two further increments in [phage]_act. before massive lysis begins. (e) During terminal lysis there are sharp rises in the curves for [total phage]_plaq., [extracellular phage]_act., and [extracellular phage]_plaq.. (f) Immediately after the completion of lysis there is a considerable disparity between measurements of total phage and extracellular phage, probably occasioned by the association of phage molecules with cellular debris, the latter being of sufficient size to be removed by the super-cel filters. 5. At pH 7.2 and 36°C. the steps in the phage production curve as determined by activity assay and plaque count are much less prominent than those observed at pH 6.1 and 28°C. However, the plateaus described by Ellis and Delbrück (10) for B. coli and coli phage can be detected also in the present case if frequent samples are taken. 6. The sorption experiments show a significant rise in the rate of phage uptake with increase in temperature, again supporting the view that the reaction involves more than a purely physical adsorption. 7. Delbrück''s objections to: (a) the use of the activity assay for determining [total phage] in mixtures of phage and susceptible cells, and (b), to the demonstration of phage precursor in "activated" bacteria have been analyzed. 8. The activity assay has been demonstrated to be an accurate procedure for determining either phage free in solution or phage bound to living susceptible cells, under the conditions of the experiments reported here and in earlier work. 9. The titration values obtained in the experiments designed to exhibit intracellular phage precursor are not the result of artifacts as Delbrück has inferred. The data can be interpreted in terms of the precursor theory, although other explanations are not ruled out.     

Temperature-Regulated Formation of Mycelial Mat-Like Biofilms by Legionella pneumophila

Fifty strains representing 38 species of the genus Legionella were examined for biofilm formation on glass, polystyrene, and polypropylene surfaces in static cultures at 25°C, 37°C, and 42°C. Strains of Legionella pneumophila, the most common causative agent of Legionnaires' disease, were found to have the highest ability to form biofilms among the test strains. The quantity, rate of formation, and adherence stability of L. pneumophila biofilms showed considerable dependence on both temperature and surface material. Glass and polystyrene surfaces gave between two- to sevenfold-higher yields of biofilms at 37°C or 42°C than at 25°C; conversely, polypropylene surface had between 2 to 16 times higher yields at 25°C than at 37°C or 42°C. On glass surfaces, the biofilms were formed faster but attached less stably at 37°C or 42°C than at 25°C. Both scanning electron microscopy and confocal laser scanning microscopy revealed that biofilms formed at 37°C or 42°C were mycelial mat like and were composed of filamentous cells, while at 25°C, cells were rod shaped. Planktonic cells outside of biofilms or in shaken liquid cultures were rod shaped. Notably, the filamentous cells were found to be multinucleate and lacking septa, but a recA null mutant of L. pneumophila was unaffected in its temperature-regulated filamentation within biofilms. Our data also showed that filamentous cells were able to rapidly give rise to a large number of short rods in a fresh liquid culture at 37°C. The possibility of this biofilm to represent a novel strategy by L. pneumophila to compete for proliferation among the environmental microbiota is discussed.     

H-NS Regulates DNA Repair in Shigella

We report a new role for H-NS in Shigella spp.: suppression of repair of DNA damage after UV irradiation. H-NS-mediated suppression of virulence gene expression is thermoregulated in Shigella, being functional at 30°C and nonfunctional at 37 to 40°C. We find that H-NS-mediated suppression of DNA repair after UV irradiation is also thermoregulated. Thus, Shigella flexneri M90T, incubated at 37 or 40°C postirradiation, shows up to 30-fold higher survival than when incubated at 30°C postirradiation. The hns mutants BS189 and BS208, both of which lack functional H-NS, show a high rate of survival (no repression) whether incubated at 30 or 40°C postirradiation. Suppression of DNA repair by H-NS is not mediated through genes on the invasion plasmid of S. flexneri M90T, since BS176, cured of plasmid, behaves identically to the parental M90T. Thus, in Shigella the nonfunctionality of H-NS permits enhanced DNA repair at temperatures encountered in the human host. However, pathogenic Escherichia coli strains (enteroinvasive and enterohemorrhagic E. coli) show low survival whether incubated at 30 or 40°C postirradiation. E. coli K-12 shows markedly different behavior; high survival postirradiation at both 30 and 40°C. These K-12 strains were originally selected from E. coli organisms subjected to both UV and X irradiation. Therefore, our data suggest that repair processes, extensively described for laboratory strains of E. coli, require experimental verification in pathogenic strains which were not adapted to irradiation.     

Survival of F-Specific RNA Coliphage, Feline Calicivirus, and Escherichia coli in Water: a Comparative Study

The relationship between the survival of enteric viral pathogens and their indicators (coliform bacteria and coliphages) is not well understood. We compared the survival rates of feline calicivirus (FCV), Escherichia coli, and a male-specific RNA coliphage MS2 at 4, 25, and 37°C for up to 28 days in dechlorinated water. The survival rates of E. coli and FCV, a surrogate of noroviruses (NV), had a high degree of correlation at 4 and 25°C, while MS2 phage survived significantly longer (P < 0.05) at these two temperatures. At 37°C, the survival rates for all three organisms were highly correlated. Decimal reduction values indicating the number of days needed for 90% reduction in titer (D values) decreased for all three organisms as storage temperatures increased. FCV had the shortest D value among all three organisms at all temperatures investigated. These findings indicate that F-specific RNA phages may be useful indicators of NV in the environment.     

Control of Lignin Peroxidase Production by Phanerochaete chrysosporium INA-12 by Temperature Shifting

There are two temperature optima connected with lignin peroxidase synthesis by Phanerochaete chrysosporium INA-12. One, at 37°C, is for the mycelium-growing phase; the other, at 30°C, is for the lignin peroxidase-producing phase. One of six extracellular proteins with ligninase activity increased when cultures were grown at 30°C for the entire fermentation period or when cultures were grown at 37°C for the first 2 days of incubation and then shifted to 30°C, compared with the activity of control cultures grown at 37°C for the entire fermentation period. The unsaturation of fatty acid (Δ/mole) of P. chrysosporium INA-12 mycelium decreased from 1.25 to 1.03 when the growth temperature was shifted from 20 to 40°C.     

Cold-sensitive mutants of Taq DNA polymerase provide a hot start for PCR

Although the thermophilic bacterium Thermus aquaticus grows optimally at 70°C and cannot grow at moderate temperatures, its DNA polymerase I has significant activity at 20–37°C. This activity is a bane to some PCRs, since it catalyzes non-specific priming. We report mutations of Klentaq (an N-terminal deletion variant) DNA polymerase that have markedly reduced activity at 37°C yet retain apparently normal activity at 68°C and resistance at 95°C. The first four of these mutations are clustered on the outside surface of the enzyme, nowhere near the active site, but at the hinge point of a domain that has been proposed to move at each cycle of nucleotide incorporation. We show that the novel cold-sensitive mutants can provide a hot start for PCR and exhibit slightly improved fidelity.     

THE RATE OF BACTERIOPHAGE INACTIVATION BY FILTRATES OF ESCHERICHIA COLI CULTURES

1. The rate of inactivation of an anti-coli phage by filtrates of cultures of the homologous bacteria has been studied. 2. The inactivation rate at 37°C. is proportional to phage concentration and filtrate concentration. 3. At 0°C. the rate of phage inactivation becomes proportional to the square root of the filtrate concentration. 4. A reaction scheme to account for these observations is suggested and discussed. 5. This coli-phage is also inactivated by relatively large concentrations of soluble starch, inulin, gum arabic, and acetylated gum arabic. 6. The inactivation is markedly influenced by salt concentration, being rapid at moderate salt concentrations and slow at high or extremely low salt concentrations. 7. The inactivated phage cannot be regenerated by high salt concentrations, or by soaps.     

Resistance against Industrial Bacteriophages Conferred on Lactococci by Plasmid pAJ1106 and Related Plasmids

Plasmid pAJ1106 and its deletion derivative, plasmid pAJ2074, conferred lactose-fermenting ability (Lac) and bacteriophage resistance (Hsp) at 30°C to Lac⁻ proteinase (Prt)-negative Lactococcus lactis subsp. lactis and L. lactis subsp. lactis var. diacetylactis recipient strains. An additional plasmid, pAJ331, isolated from the original source strain of pAJ1106, retained Hsp and conjugative ability without Lac. pAJ331 was conjugally transferred to two L. lactis subsp. lactis and one L. lactis subsp. cremoris starter strains. The transconjugants from such crosses acquired resistance to the phages which propagated on the parent recipient strains. Of 10 transconjugant strains carrying pAJ1106 or one of the related plasmids, 8 remained insensitive to phages through five activity test cycles in which cultures were exposed to a large number of industrial phages at incubation temperatures used in lactic casein manufacture. Three of ten strains remained phage insensitive through five cycles of a cheesemaking activity test in which cultures were exposed to approximately 80 different phages through cheesemaking temperatures. Three phages which propagated on transconjugant strains during cheesemaking activity tests were studied in detail. Two were similar (prolate) in morphology and by DNA homology to phages which were shown to be sensitive to the plasmid-encoded phage resistance mechanism. The third phage was a long-tailed, small isometric phage of a type rarely found in New Zealand cheese wheys. The phage resistance mechanism was partially inactivated in most strains at 37°C.     

A role for MHR1, a gene required for mitochondrial genetic recombination, in the repair of damage spontaneously introduced in yeast mtDNA

A nuclear recessive mutant in Saccharomyces cerevisiae, mhr1-1, is defective in mitochondrial genetic recombination at 30°C and shows extensive vegetative petite induction by UV irradiation at 30°C or when cultivated at a higher temperature (37°C). It has been postulated that mitochondrial DNA (mtDNA) is oxidatively damaged by by-products of oxidative respiration. Since genetic recombination plays a critical role in DNA repair in various organisms, we tested the possibility that MHR1 plays a role in the repair of oxidatively damaged mtDNA using an enzyme assay. mtDNA isolated from cells grown under standard (aerobic) conditions contained a much higher level of DNA lesions compared with mtDNA isolated from anaerobically grown cells. Soon after a temperature shift from 30 to 37°C the number of mtDNA lesions increased 2-fold in mhr1-1 mutant cells but not in MHR1 cells. Malonic acid, which decreased the oxidative stress in mitochondria, partially suppressed both petite induction and the temperature-induced increase in the amount of mtDNA damage in mhr1-1 cells at 37°C. Thus, functional mitochondria require active MHR1, which keeps the extent of spontaneous oxidative damage in mtDNA within a tolerable level. These observations are consistent with MHR1 having a possible role in mtDNA repair.     

Gene Expression and Stability of mRNA Affected by DNA-Arrested Synthesis in Gene 59, 46, and 47 Mutants of Bacteriophage T4

The effect of bacteriophage T4 gene 59 mutations (DNA-arrested synthesis) on kinetics of DNA synthesis, gene expression, and stability of mRNA has been studied. When Escherichia coli B was infected by a T4 gene 59 mutant, DNA synthesis proceeded to increase linearly after initiation, but started to decrease at 8 min and was completely arrested at 12 min at 37°C. At various incubation temperatures (20 to 42°C), the initial rates and times of arrest of DNA synthesis were different, but the total amount of DNA synthesized was constant. This result supports the hypothesis that function of gene 59 is required for the conversion of 63S DNA molecules to other replicative intermediates (39). The abnormality in protein synthesis caused by gene 59 mutation is manifested by (i) a delayed shutoff in the expression of early proteins (gene 43, 46, 39, 52, 63, 42-45, and some unidentified proteins), (ii) a reduced rate of late gene expression (gene 34, 37, 18, 20, 23, wac, 24, 22, 38, and 19), and (iii) an absence of cleavage of certain late proteins (23, 24, IPIII and 22 to 23^*, 24^*, IPIII^*, and small fragments). It appears that there was no effect on the expression of gene 33, 55, and 32 by a mutation in gene 59. Results obtained from an addition of rifampin at the prereplicative cycle after infection indicated that mRNA from genes 43, rIIA, 46, 39, 52, and 63 are more stable in T4amC5 (gene 59) than in wild-type-infected cells. mRNA remained functional longer in mutant-infected cells, and this may explain the prolonged synthesis of certain early proteins. The gene expression of other DNA arrested mutants—those in genes 46 and 47—showed a pattern of abnormal protein synthesis similar to that found in gene 59 mutant-infected cells, except more late proteins are synthesized. The gene expression in terms of phage DNA structure is discussed.