Electron microscope heteroduplex study of sequence relations of T2, T4, and T6 bacteriophage DNAs

The regions of sequence homology and nonhomology between the DNA molecules of bacteriophages T2, T4, and T6 have been mapped by the electron microscope heteroduplex method. The heteroduplexes show characteristic reproducible patterns of substitution and deletion loops. The heteroduplex maps have been oriented with respect to the T4 genetic map by observing the positions of several T4 rII deletion loops and a lysozyme deletion loop relative to the heteroduplex patterns. All heteroduplexes show more than 85% homology. Some of the loop patterns in $\text{T}\text{2}\text{T}\text{4}$ heteroduplexes are similar to those in $\text{T}\text{4}\text{T}\text{6}$.We find that the rII, the lysozyme and ac genes, the D region, and gene 52 are homologous in T2, T4, and T6. Genes 43 and 47 are probably homologous between T2 and T4. The region of greatest homology is that bearing the late genes. The host range region, which comprises a part of gene 37 and all of gene 38, is heterologous in T2, T4, and T6. The remainder of gene 37 is partially homologous in the $\text{T}\text{2}\text{T}\text{4}$ heteroduplex (Beckendorf et al. 1972), but it is heterologous in $\text{T}\text{4}\text{T}\text{6}$ and in $\text{T}\text{2}\text{T}\text{6}$. Some of the tRNA genes are homologous, and some are not. The internal protein genes in general seem to be nonhomologous.Most of the regions of homology and nonhomology are gene size or larger. There is no evidence for many partially homologous sequences. This is the expected situation for phages which undergo genetic recombination.The molecular lengths of the T-even DNA's are the same within experimental error; the ratio of this molecular length to that of λ DNA is 3.63 ± 0.06, corresponding to a molecular length of 170 kilobase pairs (kb). This suggests that the molecular weight of the nonglucosylated T-even DNAs, carrying hydroxymethylcytosine, is 112 ± 4 × 10⁶ daltons.Circular duplexes with single-stranded tails are observed by denaturation and renaturation of any one of the T-even DNAs because of its circular permutation and terminal repetition. The observed lengths of the circular duplexes indicate that the genome sizes are 166 ± 2 kb (T4), 164 ± 2 kb (T6), and 160 ± 2 kb (T2). Thus, T2 has a smaller genome than T4 and T6. The mean lengths of the terminal repetitions are 3.3 ± 1 kb (T4 and T6) and 9 ± 2 kb (T2). These differences in length of the terminal repetitions are consistent with the observed differences in genome size. The variability in length of the terminal repetition in any one phage is consistent with the interpretation that there is a variability of about 1% of the full molecular length in the amount of DNA packaged by the head-full mechanism.     

Protein synthesis in virus-infected plants. 3. Effects of tobacco mosaic virus mutants on protein synthesis in Nicotiana tabacum

Nicotiana tabacum, infected with three widely differing chemically produced and one natural mutant of tobacco mosaic virus (TMV), was pulse labeled with [³H]leucine. Uninfected plants were similarly pulse labeled with [¹⁴C]leucine. The ³H- and ¹⁴C-labeled leaf tissues were combined, fractionated into subcellular fractions, denatured, dialyzed, and electrophoresed on polyacrylamide gels in SDS. Increases or decreases in the ${}^3\text{H}{}^{14}\text{C}$ ratio in gel slices indicated changes in the proteins synthesized in the infected fractions as compared to the uninfected control fractions.In contrast to wild-type TMV, where only coat protein was detected (Singer, 1971), protein synthesis differed markedly among the mutants and their subcellular fractions in both the number and molecular weights of the protein peaks. No protein was found to be common to all strains in the same subcellular fractions except the coat protein. In addition, decreases in the ${}^3\text{H}{}^{14}\text{C}$ ratio were found, indicative of suppression of specific host proteins. The total molecular weights of proteins appearing upon infection with some mutants exceeded the coding capacity of the viral nucleic acid, and there was no indication that any of the very large proteins found were precursors. It appears probable that these multiple responses specifically produced by mutants are the result of changes in host protein synthesis caused by the infection.     

Control of breathing during acute change in cardiac preload in a patient with partial cardiopulmonary bypass

We recently had the opportunity to investigate the ventilatory effects of changing the rate of venous return to the heart (and thus pulmonary gas exchange) in a patient equipped with a venous-arterial oxygenated shunt (extracorporeal membrane oxygenation (ECMO) support). The presence of the ECMO support provided a condition wherein venous return to the right heart could be increased or decreased while maintaining total aortic blood flow and arterial blood pressure (ABP) constant. The patient, who had received a heart transplant 12 years ago, was admitted for acute cardiac failure related to graft rejection. The clinical symptomatology was that of right heart failure. We studied the patient on the 4th day of ECMO support, while she was breathing spontaneously. The blood flow diverted through the ECMO system represented 2/3 of the total aortic flow (4 l min⁻¹). With these ECMO settings, the baseline level of ventilation was low (3.89 ± 0.99 l min⁻¹), but PET_CO2$\text{PE}{\text{T}}_{\text{C}{\text{O}}_2}$ was not elevated (37 ± 2 mmHg). When Pa_CO2$\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$ in the blood coming from the ECMO was increased, no stimulatory effect on ventilation was observed. However, when the diversion of the venous return to the ECMO was stopped then restored, minute ventilation respectively increased then decreased by more than twofold with opposite changes in PET_CO2$\text{PE}{\text{T}}_{\text{C}{\text{O}}_2}$. These maneuvers were associated with large changes in the size of the right atrium and ventricle and of the left atrium. This observation suggests that the change in venous return affects breathing by encoding some of the consequences of the changes in cardiac preload. The possible sites of mediation are discussed.     

Emergence of a new human adenovirus type 4 (Ad4) genotype: identification of a novel inverted terminal repeated (ITR) sequence from majority of Ad4 isolates from US military recruits.

BACKGROUND: Ad4 is the principal etiological agent of acute respiratory disease (ARD) in the US military. Discovery of the novel 208bp inverted terminal repeated (ITR) sequence from a recent Ad4 Jax78 field isolate was totally distinct from the analogous 116bp ITR of Ad4 prototype. OBJECTIVES: To investigate the origin and distribution of the novel Ad4 ITR sequence from ARD infections. STUDY DESIGN: Direct sequencing of ligated Ad ITR termini. RESULTS: The new Ad4 ITR was highly homologous with the ITRs of human Ad subgroup B. The left post-ITR region of Ad4 Jax78 was found to be highly homologous to the corresponding region of subgroup B Ads: 81% for Ad11 and 98% for Ad3 and Ad7. The right post-ITR region of Ad4 Jax78 contained a truncated classic ITR of the Ad4 prototype. CONCLUSIONS: The Ad4 Jax78 ITR most likely evolved from Ad4 prototype by substituting the Ad4 prototype ITR with the subgroup B Ads ITR. The ITR-based PCR assays developed from this study can be used to distinguish the new Ad4 genotype from the classical Ad4 prototype. The new Ad4 genotype was first detected in 1976 from Georgia, USA, and is the main causative agent of ARD infections in US military population.     

Thirty-one human adenovirus serotypes (Ad1-Ad31) form five groups (A-E) based upon DNA genome homologies.

The DNA homology relationships among 31 human adenovirus serotypes (Ad1–Ad31) were investigated by liquid-phase molecular hybridization, using in vitro labeled viral DNA as probe. Hybridizations were carried to 40 times the $\text{C}{}_0\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ and were assayed by batchwise chromatography on hydroxylapatite (HAP), and in some experiments by use of the more stringent S1 nuclease procedure. Five distinct DNA homology groups, A to E, were identified. DNAs of group A Ads (Ad12, 18, and 31) hybridized 48 to 69% with each other and 8 to 20% with DNAs of other serotypes (HAP). DNAs of group B Ads (Ad3, 7, 11, 14, 16, and 21) hybridized 89 to 94% (HAP; 81 to 89% by S1 nuclease) with each other and 9 to 20% (HAP; 8 to 15% by S1 nuclease) with DNAs of other types. DNAs of group C Ads (Ad1, 2, 5, and 6) hybridized 99 to 100% with each other and 10 to 16% with DNAs of other types (HAP). DNAs of group D Ads (Ad8–10, 13, 15, 17, 19, 20, and 22–30) hybridized 95 to 99% (HAP; 88 to 98% by S1 nuclease) with each other and 4 to 17% with DNAs of other types. Ad4 DNA hybridized to 4 to 23% (HAP; 3 to 22% by S1 nuclease) with DNAs of other types, and thus Ad4 is the only member of group E. Members within all groups except group A were closely related. Members within group A showed considerable heterology, and six isolates, classified as Ad12 by neutralization tests, were much more related to Ad31 than to Ad12 prototype Huie strain. These DNA homology groupings are consistent in the main with the properties of other “groupings” of human Ads, e.g., oncogenic groups (tumorigenicity in newborn hamsters), T-antigen groups, G + C content of viral DNA, hemagglutination groups, molecular characteristics of subviral particles and virion proteins (e.g., length of fiber), and human epidemiology and pathogenicity.     

Functions of baseplate components in bacteriophage T4 infection. III. The functional organization of the baseplate.

Thermolabile (hs) mutants of bacteriophage T4 have been used to investigate the functions of tail proteins in phage adsorption. The thermolabilities of the mutants before and after conversion to urea-treated particles, which bypass the first two stages of adsorption, were examined. The conformational integrity of gene products P5, P12, P18, P25, and P48 is essential for the formation of irreversibly adsorbed, contracted phage particles. P27 is apparently irrelevant to these processes, but structurally necessary for the final stages of infection when the phage DNA enters the host. On the basis of these results, and those reported earlier (Dawes and Goldberg, Virology55, 391–396, 1973), functional regions have been delineated in the T4 baseplate.     

Analysis of human cancers,normal tissues,and verruce plantares for DNA sequences of human papillomavirus types 1 and 2

Comparatively little is known about human papillomaviruses (HPV) because they cannot be grown in tissue culture. We have in vitro labeled DNAs from two HPVs, HPV-1 which was isolated from plantar warts, and HPV-2 which was isolated from common hand warts, and used these DNAs to examine the homology between HPV-1 and HPV-2, to examine the state of the HPV genome in papillomavirus lesions, and to assay human cancer DNAs for HPV. The specific activities of the DNAs were 5.0 × 10⁷ to 1.1 × 10⁸ cpm/μg. The $\text{C}{}_0\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of the HPV-1 and HPV-2 DNAs were 5 and 7 × 10^?4, respectively, consistent with a genome molecular weight of about 5.2 × 10⁶. Cross-hybridization of HPV-1 and HPV-2 DNAs revealed only 5–7% homology, confirming that these are distinct viruses. HPV-1 DNA was detected by Southern blot analysis in 9 of 10 plantar warts examined. No clear evidence was found for integrated viral sequences in DNAs from eight of the nine warts analyzed. Using these HPV-1 and HPV-2 probes, we have performed the first extensive and definitive molecular hybridization analysis of human cancer DNAs for HPV sequences. Human tumor DNAs were analyzed for HPV sequences by saturation hybridization using nick-translated HPV-1 and HPV-2 DNA probes. Reconstruction experiments with added HPV-1 or HPV-2 DNAs indicated that the probes could detect 0.1 copy of the viral genome per diploid equivalent of cellular DNA. No HPV-1 sequences were detected in DNAs from 156 human cancers (14 melanoma, 3 Ca skin, 5 Ca pharynx, 1 Ca esophagus, 4 Ca stomach, 5 Ca small intestine, 22 Ca colon, 14 Ca rectum, 25 squamous cell Ca lung, 3 adenocarcinoma lung, 4 oat cell Ca lung, 21 Ca kidney, 7 Ca bladder, 3 Ca ovary, 3 Ca cervix, 4 Ca prostate, 10 non-Hodgkin lymphoma, 2 reticulum cell sarcoma [spleen]), or 27 normal human tissues (1 skin, 10 tonsil, 8 colon, 8 kidney). No HPV-2 sequences were detected in DNAs from 145 human cancers (13 melanoma, 4 Ca skin, 2 Ca pharynx, 3 Ca mouth, 7 Ca esophagus, 4 Ca stomach, 3 Ca small intestine, 29 Ca colon, 15 Ca rectum, 25 Ca kidney, 15 Ca bladder, 2 Ca ovary, 6 Ca cervix, 4 Ca prostate, 2 Ca seminoma testes, 11 non-Hodgkin lymphoma) or 1 normal human ovary. These data are strong evidence that none of the cancer specimens assayed were induced by HPV-1 or HPV-2. However, additional work is required to fully evaluate whether HPVs are possible agents of human cancers, because the cancer types assayed in this study represent only about 50% of the cancer incidence in the United States, and because our probes would not detect sequences of other recognized HPV types (HPV-3, HPV-4, and HPV-5).