The essential role of the cro gene in lytic development by bacteriophage λ

In an effort to understand the physiological role of the Cro repressor protein of bacteriophage λ we have studied the influence of cro^? mutation on lytic and lysogenic development. Effective lytic growth is blocked by cro^? mutation under conditions in which the cI repressor protein is fully active or in which active cI protein is not made; however, a partially active cI protein can substitute for Cro. The blocked lytic development under cro^?cI^? conditions is characterized by a severe inhibition of λ DNA synthesis, and the newly replicated DNA has an aberrant structure. Under cro^?cI⁺ conditions, we have studied the frequency of lysogenization to determine whether the failure of lytic development in this situation results from a complete channeling into the lysogenic pathway. However, the major alteration in the lysogenization pattern for cro^? phage is the appearance of a large fraction of surviving, nonlysogenic cells; we interpret this as a failure of normal lytic growth without a concomitant switch to stable lysogeny. From these results and from previous work on Cro function, we conclude that the repression exerted by Cro is essential for normal lytic development, that Cro probably functions as a “weak” repressor compared to cI, and that at least one of the essential regulatory activities of Cro concerns viral DNA replication.     

Coordinated regulation of the cI and int genes in coliphage λ and specificity of the cII/cIII activators

Expression of the cI (repressor) and int (integrase) genes in the λ lysogenic pathway has been studied by following protein synthesis in uv-irradiated cells. Gene expression following infection by λ⁺, a phage carrying an intact repressor gene, and by λvir, a phage carrying operator mutations with reduced affinity for the repressor, substantiated previous findings on the coordinated regulation of these two genes, and showed that their expression can be dissociated from that of other early genes. Experiments with λ hybrid phages showed that the cII gene products of λimm21 and of λimm22, which are of origin of phages 21 and P22, can activate Int synthesis from P_I of λ origin. Possible homology between the various cII genes is discussed.     

An early regulatory gene of Salmonella phage P22 analogous to gene N of coliphage λ

Mutants of the Salmonella phage P22 that are pleiotropically deficient in early phage functions, e.g., lysogeny and DNA replication, have been isolated. These mutants define a new early regulatory cistron called gene 24. Gene 24 has been located on the genetic map and is adjacent to immC between genes c₃ and c₂ (which encodes the immC repressor). P22 24^? mutants are grossly analogous in phenotype as well as in map position to N^? mutants of coliphage λ.     

Mutants of bacteriophage λ which do not require the cIII gene for efficient lysogenization

The λ cIII gene is ordinarily required for the high rate, establishment mode of repressor synthesis and efficient lysogenization by phage λ We have isolated phage mutants (called can) which no longer require cIII for efficient lysogenization by selecting turbid plaque-forming pseudorevertants of phages carrying a deletion of cIII. Three different kinds of can mutations have been characterized: can10, which is located in the cro gene; can15, which appears to be identical to cin1 and which is located in the left part of the y region; and can1, which is located in the right part of they region, among the clear cy mutations. Can10 and the previously identified cro^? mutation, cro27, both improve lysogenization frequency in the absence of cIII function, possibly because of overproduction of cII protein. In addition, lysogenization by λcIII⁺ phages carrying these mutations is more efficient than by λ⁺ at low multiplicity of infection. The cro27 mutation also improves lysogenization by cII^? phages, suggesting that Cro may prevent premature synthesis of repressor in the maintenance mode. Can1 is dominant to can⁺ and partially relieves the lysogenization defect of λcIII^? in a cap^?cya^? host. The ability of λcIII^?cI^?can1 to stimulate lysogenization by λcIII^?cI⁺ indicates that can1 does not require an adjacent functional cI gene for its action and that can1 affects the activity of a diffusible product, presumably cII protein. This proposal is supported by a cis-trans test showing that can1 requires an adjacent functional cII gene. Although can1 does not increase the frequency of lysogenization in the absence of cII function, it does increase lysogenization frequency when cII activity is present but limiting. These observations suggest that can1 increases the level or activity of cII protein. Based on the ability of can1 and can10, to bypass the need for cIII, we propose that the role of cIII protein in stimulating repressor synthesis is indirect; it acts by increasing activity of the cII protein.     

Physical mapping of coliphage λatt2

The transducing phage lines of the type λatt², which arise by abnormal excision of an integrated prophage, carry bacterial DNA from both the left and right of the prophage including the two prophage attachment sites, attL and attR. The excision sites, X_L and X_R, have been mapped with respect to the attachment sites by DNA heteroduplex analysis of a particular λatt². The X_L-attL segment, containing no known E. coli genes, is about 1300 base-pairs long (2.78 ± 0.13 %λ units). The att_R-X_R segment, including the bio operons and the uvrB locus, is about 7800 base-pairs long (16.8 ± 0.5 %λ units). X_L and X_R appear to be favored sites for the generation of λatt².     

Transfer of the lambda dv plasmid to new bacterial hosts

λdv, which was derived from bacteriophage λ, replicates autonomously as a plasmid in Escherichia coli and consists of only the immunity region (imm^λ) and DNA replication genes (O, P) of the ancestral phage. Addition phages (λmm²¹-λdv) carrying the λdv fragment inserted as a tandem duplication in their genome (sequence Aimm²¹OPimm^λOPR) are formed as recombinants after λimm²¹ infection of strains carrying λdv. Addition phages were used to transfer λdv. to new bacterial hosts. λdv. transfer by excision of the λdv. segment from the addition phage genome requires a bacterial Rec or a phage Red recombination system. Successful transfer is stimulated by UV irradiation of the addition phage before infection. Some properties of the newly transferred λdv. plasmids are described.     

Deletions and insertions in the immunity region of coliphage lambda: revised measurement of the promoter-startpoint distance

Plaque-forming λcI^? mutants which carry internal deletions in the immunity region, including genes cI and rex, were isolated. Among seven independent isolates, only three demonstrably different deletions were obtained. The largest deletion (KH54 or KH115), which eliminates almost all of the DNA between promoters p_L and p_R, spans the distance from 74.1 to 78.4 %λ, as measured in relation to the left (0 %λ) and right (100 %λ) termini of the mature λ DNA molecule. There were four representatives of a second (74.7–77.7%λ) and one of a third (75.2–78.1%λ) deletion. In addition, a mutant carrying a 1400-nucleotide pair-long insertion in gene cI was isolated.The cI-rex deletions served as markers for more accurate measurements of the physical positions of the endpoints of several bio insertions, a dy plasmid, and the endonucleolytic cleavage sites for the restriction nucleases by electron micrography of DNA heteroduplexes. This led to a reevaluation of our earlier estimate of the interval length between the p_L promoter mutation sex1 and the s_L startpoint for the major leftward operon of λ. In agreement with the independent results of others, we find that the p_L to s_L distance is within the limits of 24–62 nucleotide pairs (s_L-sex1) or 11 to 45 nucleotide pairs (s_L-HinII cut in o_L).     

Hypoxia induced changes in lung fluid balance in humans is associated with beta-2 adrenergic receptor density on lymphocytes

Background

Previous studies have demonstrated an important role for beta-2 adrenergic receptors (β₂AR) in lung fluid clearance. The purpose of this investigation was to examine the relationship between β₂AR density on lymphocytes and indices of lung water in healthy humans exposed to ∼17 h of hypoxia (F_IO2=12.5%$F_{{\text{IO}}_2}=12.5\%$ in a hypoxia tent).

Methods

Thirteen adults (mean ± SEM; age = 31 ± 3 years, BMI = 24 ± 1 kg/m², V_O2 Peak=40±2 ml/kg/min$V_{{\text{O}}_{2\text{Peak}}}=40\pm 2\text{ml}/\text{kg}/\text{min}$) participated. Pulmonary function, CT derived lung tissue volume (V_tis-tissue, blood and water), lung diffusing capacity for carbon monoxide (D_CO) and nitric oxide (D_NO), alveolar-capillary conductance (D_M), pulmonary capillary blood volume (V_c) and lung water (CT V_tis − V_c) were assessed before and after ∼17 h normobaric hypoxia (F_IO2=12.5%$F_{{\text{IO}}_2}=12.5\%$). β₂AR density on lymphocytes was measured via radioligand binding. Arterial oxygen saturation (Sa_O2${\text{Sa}}_{{\text{O}}_2}$), cardiac output (Q), right ventricular systolic pressure (RVSP) and blood pressure (BP) were also assessed.

Results

After 17 h hypoxia, Sa_O2${\text{Sa}}_{{\text{O}}_2}$ decreased from 97 ± 1 (normoxia) to 82 ± 4% and RVSP increased from 14 ± 3 (normoxia) to 29 ± 2 mmHg (p < 0.05) with little change in Q or BP. V_c and D_M both increased with hypoxia with a small increase in D_M/V_c ratio (p > 0.05). CT V_tis decreased and lung water was estimated to decline 7 ± 13%, respectively. β₂AR density averaged 1497 ± 187 receptors/lymphocyte and increased 21 ± 34% with hypoxia (range −31 to +86%). The post-hypoxia increase in β₂AR density was significantly related to the reduction in lung water (r = −0.64, p < 0.05), with the subjects with the greatest increase in density demonstrating the largest decline in lung water.

Conclusions

Lung water decreases with 17 h normobaric hypoxia are associated with changes in beta adrenergic receptor density on lymphocytes in healthy adults.     

The genomic RNA of mengovirus: I. Location of the poly(C) tract

Strains of λ phage containing a mutated cII, cIII, or cy gene in addition to a cro^? mutation were constructed. Each single mutant and the double mutants were analyzed for growth behavior and for expression of specific early and late λ proteins. λc1857cro 16 is unable to grow well at 30° and at 43°. This strain is incapable of turning off early genes (of the b region, of the P_L and P_R operons, and the cU and int genes) and is defective in the expression of late functions. The clear mutations permit phage growth at 30°. They relieve the inhibitory effect conferred by the cro^? mutation on phage growth at 43° to different extents. Our results also indicate that the cII and cIII proteins do not necessarily act at y in the regulation of late functions.     

Isolation and characterization of mutants of lambda recA which synthesize a hyperactive recA protein.

λrecA cI⁺ makes turbid plaques on spr^? and lexA^? mutants of Escherichia coli, in which much and very little recA protein synthesis can occur, respectively. On the other hand, λtif-1 cI⁺ makes clear plaques on a spr^? host mutant and turbid plaques on a lexA^? host mutant. Clear plaque development in the spr^? host appears to be due to proteolytic cleavage of phage repressor due to high levels of hyperactive tif-1 mutant protein. This plaque type difference between λrecA⁺cI⁺ and λtif-1 cI⁺ provides a method for detection of mutants of λrecA⁺cI⁺ which synthesize a hyperactive, tif-like recA protein.     

The identification of a VH subgroup allotypic specificity,y30, which differentiates the y33 allele into two variants. Implications for rabbit VH gene organization and evolution

A new anti-allotype Ab,^∥ designated anti-y30, was produced by immunizing rabbits of the VH genotype a²x³²y³³/a²x³²y³³ with purified IgG from $\text{a}{}^1\text{x}{}^{\mathrm{?}}\text{y}{}^{33}\text{a}{}^1\text{x}{}^{\mathrm{?}}\text{y}{}^{33}$ rabbits suppressed for al Ig synthesis. Genetic analysis of 200 rabbits representing 12 heavy chain (VH-CH) haplotypes indicated that y30 was controlled by the heavy chain chromosomal region and was expressed in rabbits of the VH haplotype a¹x^?y³³ but not of theVH haplotypes a¹x^?y^?, a²x³²y³³ or a³x³²y^?. Thus, some rabbits express y33 with y30 whereas others express y33 without y30. By immunodiffusion, radioimmunoassay and sequential precipitation techniques, y30 and y33 were found to be co-expressed on most of the IgG molecules from $\text{a}{}^1\text{x}{}^{\mathrm{?}}\text{y}{}^{33}\text{a}{}^1\text{x}{}^{\mathrm{?}}\text{y}{}^{33}$ rabbits suppressed for the al allotype. Also, when newborn $\text{a}{}^1\text{x}{}^{\mathrm{?}}\text{y}{}^{\mathrm{?}}\text{a}{}^1\text{x}{}^{\mathrm{?}}\text{y}{}^{33}$ rabbits were injected at birth with either anti-y33 or anti-y30 Ab, both allotypic specificities were initially suppressed and subsequently escaped suppression in unison. From this, we suggest that two variants of the y³³ allele exist, y^33,30 and y³³,?; an inhibition of radioprecipitation assay indicated that the y33 specificity was qualitatively similar in both variants. These allelic variants, presumably representing differences in amino acid sequence, may hve resulted from an ancestral recombinational event within a VH Cistron prior to gene duplication and expansion into present day VH subgroups. The highly restricted association observed between genetic markers of the a,x, and y VH subgroups argues against recombination between the VH subgroups.     

Bronchoconstriction during alveolar hypocapnia and systemic hypercapnia in dogs with a cardiopulmonary bypass

The roles of the alveolar and systemic CO₂ on the lung mechanics were investigated in dogs subjected to cardiopulmonary bypass. Low-frequency pulmonary impedance data (Z_L) were collected in open-chest dogs with an alveolar CO₂ level (FA_CO2)$(\text{F}{\text{A}}_{\text{C}{\text{O}}_2})$ of 0.2–7% and during systemic hypercapnia before and after elimination of the vagal tone. Airway resistance (R_aw), inertance (I_aw), parenchymal damping (G) and elastance (H) were estimated from the Z_L. The highest R_aw observed at 0.2% FA_CO2$\text{F}{\text{A}}_{\text{C}{\text{O}}_2}$, which decreased markedly up to a FA_CO2$\text{F}{\text{A}}_{\text{C}{\text{O}}_2}$ of 2% (212 ± 24%), and remained unchanged under normo- and hypercapnia (FA_CO2$\text{F}{\text{A}}_{\text{C}{\text{O}}_2}$ 2–7%). These changes were associated with smaller decreases in I_aw (−16.6 ± 3.7%), mild elevations in G (25.7 ± 4.7%), and no change in H. Significant increases in all mechanical parameters were observed following systemic hypercapnia; atropine counteracted the R_aw rises. We conclude that severe alveolar hypocapnia may contribute to minimization of the ventilation–perfusion mismatch by constricting the airways in poorly perfused lung regions. The constrictor potential of systemic hypercapnia is mediated by vagal reflexes.     

Isolation and characterization of mutations in the cIII gene of bacteriophage lambda which increase the efficiency of lysogenization of Escherichia coli K-12.

Mutations of phage λ are described which permit lysogenization of singly infected cells under conditions where λ⁺ lysogenizes only by multiple infection. Deletion mapping places them between the end points of bio252 and bio10, most probably within the cIII gene. In addition to their effects on lysogenization, these mutations (cIIIs1 and cIIIs2) cause a lengthening of the latent period in one-step growth experiments with lytic infections. This growth defect is relieved by a mutation in the cII gene, but not by a mutation in the Y region. A one-step growth experiment in which cells were mixedly infected by cIIIs cI^? and cIII+ cI^? showed a latent period intermediate between that of infections by each strain alone. A similar result was obtained in a mixed infection with cIIIs cI^? and cIII^?cI^?. I conclude that the intermediate latent period obtained in mixed infections depends upon the ratio of cIIIs cI^? to cIII⁺cI^? or cIII^?cI^? chromosomes. cIIIs mutants accumulate more repressor than cIII⁺ by 30 min after infection, as assayed by the DNA filter binding technique. These properties are consistent with the idea of a “super cIII” protein, which may be more stable or more active than the wild-type protein.     

Cerebral blood flow and oxygenation at maximal exercise: the effect of clamping carbon dioxide

During exercise, as end-tidal carbon dioxide (P_ETCO2$P_{\text{E}{\text{T}}_{\text{C}{\text{O}}_2}}$) drops after the respiratory compensation point (RCP), so does cerebral blood flow velocity (CBFv) and cerebral oxygenation. This low-flow, low-oxygenation state may limit work capacity. We hypothesized that by preventing the fall in P_ETCO2$P_{\text{E}{\text{T}}_{\text{C}{\text{O}}_2}}$ at peak work capacity (W_max) with a newly designed high-flow, low-resistance rebreathing circuit, we would improve CBFv, cerebral oxygenation, and W_max. Ten cyclists performed two incremental exercise tests, one as control and one with P_ETCO2$P_{\text{E}{\text{T}}_{\text{C}{\text{O}}_2}}$ constant (clamped) after the RCP. We analyzed , middle cerebral artery CBFv, cerebral oxygenation, and cardiopulmonary measures. At W_max, when we clamped P_ETCO2$P_{\text{E}{\text{T}}_{\text{C}{\text{O}}_2}}$ (39.7 ± 5.2 mmHg vs. 29.6 ± 4.7 mmHg, P < 0.001), CBFv increased (92.6 ± 15.9 cm/s vs. 73.6 ± 12.5 cm/s, P < 0.001). However, cerebral oxygenation was unchanged (ΔTSI −21.3 ± 13.1% vs. −24.3 ± 8.1%, P = 0.33), and W_max decreased (380.9 ± 20.4 W vs. 405.7 ± 26.8 W, P < 0.001). At W_max, clamping P_ETCO2$P_{\text{E}{\text{T}}_{\text{C}{\text{O}}_2}}$ increases CBFv, but this does not appear to improve W_max.