[Platypnea-orthodeoxia syndrome secondary to pneumonia: Two cases]. / Syndrome platypnée-orthodéoxie secondaire à une pneumopathie : à propos de deux cas.

INTRODUCTION: The platypnea-orthodeoxia syndrome is a rare situation characterized by the appearance of dyspnea and/or hypoxemia during the transition to orthostatism. OBSERVATIONS: We report the case of two patients, who presented with a platypnea-orthodeoxia syndrome following pneumocystis pneumonia and COVID-19, revealing an intracardiac communication with a right-left shunt on contrast ultrasound. CONCLUSION: This syndrome can be detected easily at the bedside with positional maneuvers and the shunt demonstrated by a hyperoxia test. Non-reversible situations may require correction of the anatomical anomaly by transcatheter intervention or surgery.

Hemoglobin biosynthesis in individual erythropoietic bursts in culture. Studies of adult peripheral blood.

By using a methylcellulose clonal assay, we cultured peripheral blood erythropoietic precursors from a patient with sickle cell anemia, a patient with sickle cell hemoglobin C disease, and a normal volunteer. We then analyzed the synthetic rates of adult and fetal hemoglobins (Hb) in individual erythropoietic bursts. Bb were labeled with 14C-amino acids in culture, separated by slab gel isoelectric focusing techniques, and quantitated by fluorographic methods. All bursts exhibited both fetal and adult Hb in varying ratios. Frequency distributions of the individual burst differing in percentage of BbF biosynthesis approached normal distributions. Further stimulation of HbF synthesis by higher erythropoietin in culture was associated with increased HbF biosynthesis in individual bursts. Augmentation of human HbF synthesis in culture appears to be controlled by qualitative intracellular changes rather than by changes in cellular population.

Nucleotide-sequence-specific and non-specific interactions of T4 DNA polymerase with its own mRNA.

The DNA-binding DNA polymerase (gp43) of phage T4 is also an RNA-binding protein that represses translation of its own mRNA. Previous studies implicated two segments of the untranslated 5'-leader of the mRNA in repressor binding, an RNA hairpin structure and the adjacent RNA to the 3' side, which contains the Shine-Dalgarno sequence. Here, we show by in vitro gp43-RNA binding assays that both translated and untranslated segments of the mRNA contribute to the high affinity of gp43 to its mRNA target (translational operator), but that a Shine-Dalgarno sequence is not required for specificity. Nucleotide sequence specificity appears to reside solely in the operator's hairpin structure, which lies outside the putative ribosome-binding site of the mRNA. In the operator region external to the hairpin, RNA length rather than sequence is the important determinant of the high binding affinity to the protein. Two aspects of the RNA hairpin determine specificity, restricted arrangement of purine relative to pyrimidine residues and an invariant 5'-AC-3' in the unpaired (loop) segment of the RNA structure. We propose a generalized structure for the hairpin that encompasses these features and discuss possible relationships between RNA binding determinants of gp43 and DNA binding by this replication enzyme.

DNA polymerase of the T4-related bacteriophages.

The DNA polymerase of bacteriophage T4, product of phage gene 43 (gp43), has served as a model replicative DNA polymerase in nucleic acids research for nearly 40 years. The base-selection (polymerase, or Pol) and editing (3'-exonuclease, or Exo) functions of this multifunctional protein, which have counterparts in the replicative polymerases of other organisms, are primary determinants of the high fidelity of DNA synthesis in phage DNA replication. T4 gp43 is considered to be a member of the "B family" of DNA-dependent DNA polymerases (those resembling eukaryotic Pol alpha) because it exhibits striking similarities in primary structure to these enzymes. It has been extensively analyzed at the genetic, physiological, and biochemical levels; however, relationships between the in vivo properties of this enzyme and its physical structure have not always been easy to explain due to a paucity of structural data on the intact molecule. However, gp43 from phage RB69, a phylogenetic relative of T4, was crystallized and its structure solved in a complex with single-stranded DNA occupying the Exo site, as well as in the unliganded form. Analyses with these crystals, and crystals of a T4 gp43 proteolytic fragment harboring the Exo function, are opening new avenues to interpret existing biological and biochemical data on the intact T4 enzyme and are revealing new aspects of the microanatomy of gp43 that can now be explored further for functional significance. We summarize our current understanding of gp43 structure and review the physiological roles of this protein as an essential DNA-binding component of the multiprotein T4 DNA replication complex and as a nucleotide-sequence-specific RNA-binding translational repressor that controls its own biosynthesis and activity in vivo. We also contrast the properties of the T4 DNA replication complex to the functionally analogous complexes of other organisms, particularly Escherichia coli, and point out some of the unanswered questions about gp43 and T4 DNA replication.

Mutational analysis of the mRNA operator for T4 DNA polymerase.

Biosynthesis of bacteriophage T4 DNA polymerase is autogenously regulated at the translational level. The enzyme, product of gene 43, represses its own translation by binding to its mRNA 5' to the initiator AUG at a 36-40 nucleotide segment that includes the Shine-Dalgarno sequence and a putative RNA hairpin structure consisting of a 5-base-pair stem and an 8-base loop. We constructed mutations that either disrupted the stem or altered specific loop residues of the hairpin and found that many of these mutations, including single-base changes in the loop sequence, diminished binding of purified T4 DNA polymerase to its RNA in vitro (as measured by a gel retardation assay) and derepressed synthesis of the enzyme in vivo (as measured in T4 infections and by recombinant-plasmid-mediated expression). In vitro effects, however, were not always congruent with in vivo effects. For example, stem pairing with a sequence other than wild-type resulted in normal protein binding in vitro but derepression of protein synthesis in vivo. Similarly, a C----A change in the loop had a small effect in vitro and a strong effect in vivo. In contrast, an A----U change near the base of the hairpin that was predicted to increase the length of the base-paired stem had small effects both in vitro and in vivo. The results suggest that interaction of T4 DNA polymerase with its structured RNA operator depends on the spatial arrangement of specific nucleotide residues and is subject to modulation in vivo.

Expression of a DNA replication gene cluster in bacteriophage T4: genetic linkage and the control of gene product interactions.

The results of this study bear on the relationship between genetic linkage and control of interactions between the protein products of different cistrons. In T4 bacteriophage, genes 45 and 44 encode essential components of the phage DNA replication multiprotein complex. T4 gene 45 maps directly upstream of gene 44 relative to the overall direction of reading of this region of the phage chromosome, but it is not known whether these two genes are cotranscribed. It has been shown that a nonsense lesion of T4 gene 45 exerts a cis-dominant inhibitory effect on growth of a missense mutant of gene 44 but not on growth of phage carrying the wild-type gene 44 allele. In previous work, we confirmed these observations on polarity of the gene 45 mutation but detected no polar effects by this lesion on synthesis of either mutant or wild-type gene 44 protein. In the present study, we demonstrate that mRNA for gene 44 protein is separable by gel electrophoresis from gene 45-protein-encoding mRNA. That is, the two proteins are not synthesized from one polycistronic message, and the cis-dominant inhibitory effect of the gene 45 mutation on gene 44 function is probably expressed at a posttranslational stage. We propose that close genetic linkage, whether or not it provides shared transcriptional and translational regulatory signals for certain clusters of functionally related cistrons, may determine the intracellular compartmentalization for synthesis of proteins encoded by these clusters. In prokaryotes, such linkage-dependent compartmentation may minimize the diffusion distances between gene products that are synthesized at low levels and are destined to interact.

Functional interactions beween the DNA ligase of Escherichia coli and components of the DNA metabolic apparatus of T4 bacteriophage.

T4 phage completely defective in both gene 30 (DNA ligase) and the rII gene (function unknown) require at least normal levels of host-derived DNA ligase (E. coli lig gene) for growth. Viable E. coli mutant strains that harbor less than 5% of the wild-type level of bacterial ligase do not support growth of T4 doubly defective in genes 30 and rII (T4 30- rII- mutants). We describe here two classes of secondary phage mutations that permit the growth of T4 30- rII- phage on ligase-defective hosts. One class mapped in T4 gene su30 (Krylov 1972) and improved T4 30- rII- phage growth on all E. coli strains, but to varying degrees that depended on levels of residual host ligase. Another class mapped in T4 gene 32 (helix-destabilizing protein) and improved growth specifically on a host carrying the lig2 mutation, but not on a host carrying another lig- lesion (lig4). Two conclusions are drawn from the work: (1) the role of DNA ligase in essential DNA metabolic processes in T4-infected E. coli is catalytic rather than stoichiometric, and (2) the E. coli DNA ligase is capable of specific functional interactions with components of the T4 DNA replication and/or repair apparatus.

Transcriptional mapping of a DNA replication gene cluster in bacteriophage T4. Sites for initiation, termination, and mRNA processing.

A phage T4 genetic cluster that encodes DNA polymerase, several other DNA replication proteins, transcriptional factors, and the translation repressor RegA has been shown to be controlled by overlapping modes of transcription which initiate at several promoters. The promoters were mapped by using a combination of assays including Northern blotting, S1-mapping, RNA sequencing, and analysis of products of radioactive labeling of 5' ends on T4-induced RNA in vitro via the reaction catalyzed by eukaryotic guanylyl transferase (RNA capping assay). The most proximal in the cluster are two promoters that do not require any phage-induced factors for activation, i.e. they are T4 early promoters. Initiation at these promoters yields several RNA species having overlapping 5'-terminal sequences, the largest of which is estimated to be about 15,000 nucleotides long and to include all the cistrons of the cluster. A third early promoter maps inside the protein encoding segment of one of the cistrons (T4 gene 47), while at least five additional promoters map in intercistronic regions and are T4 middle promoters, i.e. they require the T4-induced DNA-binding transcription factor MotA. Transcriptional readthrough at a termination site within the T4 gene 45-44 intercistronic region is required for synthesis of gp44 and gp62, two essential T4 DNA-polymerase (gp43) accessory proteins. In contrast, transcription of T4 gene 43 is serviced by readthrough across a termination site in the regA-43 intercistronic region as well as by a MotA-dependent promoter that maps downstream of the termination site, and the region contains a site for processing by a T4-induced enzyme that also cleaves elsewhere in the polycistronic mRNA from the cluster (i.e. in the Shine-Dalgarno sequence of the gene 45.2 mRNA). The termination events in the gene 45-44 and regA-43 intercistronic regions both occur downstream of RNA stem-loop structures containing the sequence 5'CUUCGG3' in the loop segments. Transcription termination in the 78-base-pair regA-43 intercistronic region occurs about 60 nucleotides away from the gp43 initiator AUG, transcription initiation occurs at 38-40 nucleotides upstream from the AUG, and T4-dependent RNA processing occurs at several sites (including a GGAG sequence) between the transcription termination and initiation sites. Thus, all gp43-encoding mRNAs contain the translational operator (residues -40 to -1 relative to the AUG) for autogenous repression by this DNA polymerase (Andrake et al., 1988).(ABSTRACT TRUNCATED AT 400 WORDS)

Mutation to overproduction of bacteriophage T4 gene products.

R9 was isolated as one of several mutations that enhanced the growth of a leaky amber (am) mutant of bacteriophage T4 gene 62 (product required for phage DNA synthesis) under conditions of partial suppression by ribosomal ambiguity. R9 also enhanced the growth of leaky am mutants of some, but not all, other T4 "early" gene functions. R9 mapped between mutations in genes 43 and 62. By using assays involving polyacrylamide slab gel electrophoresis in the presence of sodium dodecyl sulfate, we observed the following. (i) R9 resulted in an overproduction of many T4 "early" proteins in infected cells. The most pronounced effects of R9 were observed when phage DNA synthesis and/or the functions of maturation genes 55 and 33 were not expressed. (ii) In rifampintreated infected cells, the capacity to synthesize T4 "early" proteins decayed more slowly in the presence of the R9 mutation than in the presence of the wild-type counterpart of R9. R9 appeared to have no effect on the rates of RNA synthesis either during early or late times after infection. The results suggest that the R9 mutation leads to increased functional stability of T4 "early" messengers.

Diversity of structure and function of DNA polymerase (gp43) of T4-related bacteriophages.

The replication DNA polymerase (gp43) of the bacteriophage T4 is a member of the pol B family of DNA polymerases, which are found in all divisions of life in the biosphere. The enzyme is a modularly organized protein that has several activities in one polypeptide chain (approximately 900 amino acid residues). These include two catalytic functions, POL (polymerase) and EXO (3 -exonuclease), and specific binding activities to DNA, the mRNA for gp43, deoxyribonucleotides (dNTPs), and other T4 replication proteins. The gene for this multifunctional enzyme (gene 43) has been preserved in evolution of the diverse group of T4-like phages in nature, but has diverged in sequence, organization, and specificity of the binding functions of the gene product. We describe here examples of T4-like phages where DNA rearrangements have created split forms of gene 43 consisting of two cistrons instead of one. These gene 43 variants specify separate gp43A (N-terminal) and gp43B (C-terminal) subunits of a split form of gp43. Compared to the monocistronic form, the interruption in contiguity of the gene 43 reading frame maps in a highly diverged sequence separating the code for essential components of two major modules of this pol B enzyme, the FINGERS and PALM domains, which contain the dNTP binding pocket and POL catalytic residues of the enzyme. We discuss the biological implications of these gp43 splits and compare them to other types of pol B splits in nature. Our studies suggest that DNA mobile elements may allow genetic information for pol B modules to be exchanged between organisms.

Binding specificity of T4 DNA polymerase to RNA.

Bacteriophage T4 DNA polymerase, product of phage gene 43 (gp43), is a multifunctional DNA-binding protein and a key component of the phage DNA replicase. It is also an RNA-binding protein that selectively recognizes a site on its mRNA (the translational operator) and represses its own translation. We examined the ability of the protein to discriminate between DNA and RNA by using a gel mobility shift assay with defined RNA and DNA substrates. A higher affinity to RNA as compared with DNA (about 100-fold) was observed in assays that utilized synthetic DNA and in vitro transcribed RNA substrates bearing the T4 gene 43 translational operator sequence. The replacement of thymine with uracil in the synthetic DNA did not improve binding. The results suggest that the protein's selectivity for RNA is based in structure (intramolecular interactions) specific to the ribonucleotide sequence of the operator. Competition studies suggest that the protein determinants for RNA and DNA recognition are only partially overlapping.

Analysis of expression of the rII gene function of bacteriophage T4.

Temperature-sensitive (ts) mutants of the T4 phage rII gene were islated and used in temperature shift experiments that revelaed two different expressions for the normal rII (rII+) gene function in vivo: (i) an early expression (0 to 12 min postinfection at 30 C) that prevents restriction of T4 growth in Escherichia coli hosts lysogenic for gamma phage, and (ii) a later expression (12 to 18 min postinfection at 30 C) that results in restriction of T4 growth when the phage DNA ligase (gene 30) is missing. The earlier expression appeared to coincide with the period of synthesis of the protein product of the T4 rIIA cistron, whereas the later expression occurred after rIIA protein synthesis had stopped. The synthesis of the protein product of the rIIB cistron continues for several minutes after rIIA protein synthesis ceases (O'Farrell and Gold, 1973). The two rII+ gene expressions might require different molar ratios of the rIIA and rIIB proteins. It is possible that the separate expressions of rII+ gene function are manifestations of different associations between the two rII proteins and other T4-induced proteins that are synthesized or activated at different times after phage infection.

Properties of bacteriophage T4 mutants defective in gene 30 (deoxyribonucleic acid ligase) and the rII gene.

In Escherichia coli K-12 strains infected with phage T4 which is defective in gene 30 [deoxyribonucleic acid (DNA) ligase] and in the rII gene (product unknown), near normal levels of DNA and viable phage were produced. Growth of such T4 ligase-rII double mutants was less efficient in E. coli B strains which show the "rapidlysis" phenotype of rII mutations. In pulse-chase experiments coupled with temperature shifts and with inhibition of DNA synthesis, it was observed that DNA synthesized by gene 30-defective phage is more susceptible to breakdown in vivo when the phage is carrying a wild-type rII gene. Breakdown was delayed or inhibited by continued DNA synthesis. Mutations of the rII gene decreased but did not completely abolish the breakdown. T4 ligase-rII double mutants had normal sensitivity to ultraviolet irradiation.

Suppression of amber mutations of bacteriophage T4 gene 43 (DNA polymerase) by translational ambiguity.

The growth properties of twelve different amber (am) mutants of bacteriophage T4 gene 43 (DNA polymerase) were examined by using nonpermissive (su(-)) as well as permissive (su(+)) Escherichia coli hosts. It was found that most of these mutants were measurably suppressed in su(-) hosts by translational ambiguity (misreading of codons during protein synthesis). The ability of these mutants to grow in response to this form of weak suppression probably means that the T4 gene 43 DNA polymerase can be effective in supporting productive DNA replication when it is supplied in small amounts. By similar criteria, studies with other phage mutants suggested that the products of T4 genes 62 (uncharacterized), 44 (uncharacterized), 42 (dCMP-hydroxymethylase), and 56 (dCTPase) are also effective in small amounts. Some T4 gene products, such as the product of gene 41 (uncharacterized), seem to be partially dispensable for phage growth since am mutants of such genes do propagate, although weakly, in streptomycin-resistant su(-) hosts which appear to have lost the capacity to suppress am mutations by ambiguity.

Evolution of RNA-binding specificity in T4 DNA polymerase.

DNA polymerase of phage T4 (T4 gp43), an essential component of the T4 DNA replicase, is a multifunctional single-chained (898-amino acid) protein that catalyzes the highly accurate synthesis of DNA in phage replication. The enzyme functions both as a DNA-binding replication protein and as a sequence-specific RNA-binding autogenous translational repressor. We have utilized a phylogenetic approach to study the relationships between the two nucleic acid-binding functions of the protein. We found that autogenous translational control of gp43 biosynthesis has been conserved in phage RB69, a distant relative of T4, although we also found that the RB69 system differs from its T4 counterpart in two regards: (a) nucleotide sequence and predicted secondary structure of the RNA target (translational operator), and (b) RNA specificity of the protein. T4 gp43 is specific to the RNA operator sequence of the T4 genome whereas RB69 gp43 can bind and repress operator RNA from both phages equally well. In studies with T4-RB69 gp43 chimeras, we mapped T4 gp43 RNA-binding specificity to a protein segment that also harbors important determinants for DNA binding and the polymerase catalytic function. Our results suggest that RNA functions as a regulator of both the dosage and activity of this DNA replication enzyme.

Divergence of a DNA replication gene cluster in the T4-related bacteriophage RB69.

The genomes of bacteriophages T4 and RB69 are phylogenetically related but diverge in nucleotide sequence at many loci and are incompatible with each other in vivo. We describe here the biological implications of divergence in a genomic segment that encodes four essential DNA replication proteins: gp45 (sliding clamp), gp44/62 complex (clamp loader), and gp46 (a recombination protein). We have cloned, sequenced, and expressed several overlapping segments of the RB69 gene 46-45.2-(rpbA)-45-44-62 cluster and compared its features to those of the homologous gene cluster from T4. The deduced primary structures of all four RB69 replication proteins and gp45.2 from this cluster are very similar (80 to 95% similarity) to those of their respective T4 homologs. In contrast, the rpbA region (which encodes a nonessential protein in T4) is highly diverged (approximately 49% similarity) between the two phage genomes and does not encode protein in RB69. Expression studies and patterns of high divergence of intercistronic nucleotide sequences of this cluster suggest that T4 and RB69 evolved similar transcriptional and translational control strategies for the cistrons contained therein, but with different specificities. In plasmid-phage complementation assays, we show that posttranslationally, RB69 and T4 homologs of gp45 and the gp44/62 complex can be effectively exchanged between the two phage replicase assemblies; however, we also show results which suggest that mixed clamp loader complexes consisting of T4 gp62 and RB69 gp44 subunits are not active for phage DNA replication. Thus, specificity of the gp44-gp62 interaction in the clamp loader marks a point of departure between the T4 and RB69 replication systems.

Modular organization of T4 DNA polymerase. Evidence from phylogenetics.

We describe the use of a phylogenetic approach to analyze the modular organization of the single-chained (898 amino acids) and multifunctional DNA polymerase of phage T4. We have identified, cloned in expression vectors, and sequenced the DNA polymerase gene (gene 43) of phage RB69, a distant relative of T4. The deduced primary structure of the RB69 protein (RB69 gp43) differs from that of T4 gp43 in discrete clusters of short sequence that are interspersed with clusters of high similarity between the two proteins. Despite these differences, the two enzymes can substitute for each other in phage DNA replication, although T4 gp43 does exhibit preference to its own genome. A 55-amino acid internal gp43 segment of high sequence divergence between T4 and RB69 could be replaced in RB69 gp43 with the corresponding segment from T4 without loss of replication function. The reciprocal chimera and a deletion mutant of the T4 gp43 segment were both inactive for replication and specifically inhibitory ("dominant lethal") to the T4 wild-type allele. The results show that phylogenetic markers can be used to construct chimeric and truncated froms of gp43 that, although inactive for replication, can still exhibit biological specificity.