[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.

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.

Interacting fidelity defects in the replicative DNA polymerase of bacteriophage RB69.

The DNA polymerases (gp43s) of the related bacteriophages T4 and RB69 are B family (polymerase alpha class) enzymes that determine the fidelity of phage DNA replication. A T4 whose gene 43 has been mutationally inactivated can be replicated by a cognate RB69 gp43 encoded by a recombinant plasmid in T4-infected Escherichia coli. We used this phage-plasmid complementation assay to obtain rapid and sensitive measurements of the mutational specificities of mutator derivatives of the RB69 enzyme. RB69 gp43s lacking proofreading function (Exo(-) enzymes) and/or substituted with alanine, serine, or threonine at the conserved polymerase function residue Tyr(567) (Pol(Y567(A/S/T)) enzymes) were examined for their effects on the reversion of specific mutations in the T4 rII gene and on forward mutation in the T4 rI gene. The results reveal that Tyr(567) is a key determinant of the fidelity of base selection and that the Pol and Exo functions are strongly coupled in this B family enzyme. In vitro assays show that the Pol(Y567A) Exo(-) enzyme generates mispairs more frequently but extends them less efficiently than does a Pol(+) Exo(-) enzyme. Other replicative DNA polymerases may control fidelity by strategies similar to those used by RB69 gp43.

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.

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.

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.

Retention of replication fidelity by a DNA polymerase functioning in a distantly related environment.

The primary structures of the replicative DNA polymerases (gp43s) of bacteriophage T4 and its distant phylogenetic relative RB69 are diverged, retaining only 61% identity and 74% similarity. Nevertheless, RB69 gp43 substitutes effectively for T4 gp43 in T4 DNA replication in vivo. We show here that RB69 gp43 replicates T4 genomes in vivo with a fidelity similar to that achieved by T4 gp43. Furthermore, replication by RB69 gp43 in the distantly related environment does not enhance the mutator activities of mutations in T4 genes that encode other components of the multienzyme DNA replicase. We also show that the fidelities of RB69 gp43 and T4 gp43 are both high in vitro and that they are similarly and sharply reduced in vivo by mutations that eliminate the 3'-exonucleolytic proofreading function. We conclude that gp43 interactions with the other replication proteins are probably nonessential for polymerase fidelity.

Crystal structure of a pol alpha family replication DNA polymerase from bacteriophage RB69.

The 2.8 A resolution crystal structure of the bacteriophage RB69 gp43, a member of the eukaryotic pol alpha family of replicative DNA polymerases, shares some similarities with other polymerases but shows many differences. Although its palm domain has the same topology as other polymerases, except rat DNA polymerase beta, one of the three carboxylates required for nucleotidyl transfer is located on a different beta strand. The structures of the fingers and thumb domains are unrelated to all other known polymerase structures. The editing 3'-5' exonuclease domain of gp43 is homologous to that of E. coli DNA polymerase I but lies on the opposite side of the polymerase active site. An extended structure-based alignment of eukaryotic DNA polymerase sequences provides structural insights that should be applicable to most eukaryotic DNA polymerases.

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.

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.

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.

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)

Primary structure of T4 DNA polymerase. Evolutionary relatedness to eucaryotic and other procaryotic DNA polymerases.

Bacteriophage T4 gene 43 codes for the viral DNA polymerase. We report here the sequence of gene 43 and about 70 nucleotides of 5'- and 3'-flanking sequences, determined by both DNA and RNA sequencing. We have also purified T4 DNA polymerase from T4 infected Escherichia coli and from E. coli containing a gene 43 overexpression vector. A major portion of the deduced amino acid sequence has been verified by peptide mapping and sequencing of the purified DNA polymerase. All these results are consistent with T4 DNA polymerase having 898 amino acids with a calculated Mr = 103,572. Comparison of the primary structure of T4 DNA polymerase with the sequence of other procaryotic and eucaryotic DNA polymerases indicates that T4 DNA polymerase has regions of striking similarity with animal virus DNA polymerases and human DNA polymerase alpha. Surprisingly, T4 DNA polymerase shares only limited similarity with E. coli polymerase I and no detectable similarity with T7 DNA polymerase. Based on the location of specific mutations in T4 DNA polymerase and the conservation of particular sequences in T4 and eucaryotic DNA polymerases, we propose that the NH2-terminal half of T4 DNA polymerase forms a domain that carries out the 3'----5' exonuclease activity whereas the COOH-terminal half of the polypeptide contains the dNTP-binding site and is necessary for DNA synthesis.

Identification of two new bacteriophage T4 genes that may have roles in transcription and DNA replication.

We have identified two bacteriophage T4 genes, 45.1 and 45.2, that map in the intergenic space between phage replication genes 46 (which encodes a recombination initiation protein) and 45 (which encodes a bifunctional protein required in replication and transcription). The existence of genes 45.1 and 45.2 had not been previously recognized by mutation analysis of the T4 genome. We cloned the T4 gene 45.1/45.2 segment, determined its nucleotide sequence, and expressed its two reading frames at high levels in bacterial plasmids. The results predicted molecular weights of 11,400 (100 amino acids) for gp45.1 and 7,500 (62 amino acids) for gp45.2. We also determined that in T4-infected Escherichia coli, genes 45.1 and 45.2 are cotranscribed with their distal neighbor, gene 45, by at least one mode of transcription. In an accompanying report (K. P. Williams, G. A. Kassavetis, F. S. Esch, and E. P. Geiduschek, J. Virol. 61:600-603, 1987), it is shown that the product of gene 45.1 is the so-called T4-induced 15K protein, an RNA polymerase-binding protein of unknown role in phage development. Possibly, T4 genes 45.2, 45.1, and 45 constitute an operon for host RNA polymerase-binding phage proteins. Jointly with Williams et al., we propose the term rpb (RNA polymerase-binding) to refer to T4 genes whose products bind to the host RNA polymerase and have adopted the name rpbA for T4 gene 45.1.

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.

G gamma and A gamma globin-chain biosynthesis by adult and umbilical cord blood erythropoietic bursts and reticulocytes.

We examined gamma-globin-chain biosynthesis by adult and umbilical cord blood or erythropoietic bursts in methylcellulose clonal culture and gamma-chain synthesis by cord blood reticulocytes. Globin chains were labeled with 14C-amino acids and guantitated by using autoradiography or fluorography. Alpha, beta, and G gamma and A gamma chains were separated by isoelectric focusing in polyacrylamide gels containing 8 M urea and 3% Nonidet P-40 (a nonionic detergent). Time course examinations of the gamma-chains synthesized by the bursts revealed no changes in the G gamma:A gamma ratio between days 10 and 18 of culture. The ratio of G gamma/(G gamma + A gamma) in cultures of adult circulating erythroid precursors was 0.38 +/- 0.09, which corresponds to the known ratio in adult peripheral blood erythrocytes. The relative G gamma-chain biosyntheses in the cord blood bursts and reticulocytes were 0.56 +/- 0.02 and 0.66 +/- .008, respectively. Both are intermediate between the accepted newborn and adult ratios. Natal erythropoietic precursors appear to be in the transitional stage with respect to the switching of G gamma-A gamma ratios.

Biosynthesis of hemoglobin F Malta-I in culture by adult circulating erythropoietic precursors.

By using a methylcellulose clonal assay, we cultured peripheral blood erythropoietic precursors (BFU-E) from an adult couple whose child had HbF Malta-I(gamma 117 His leads to Arg), a G gamma variant, and measured the synthetic rates of HbA, HbF, and HbF Malta-I. Hemoglobin was labeled with 14C-amino acid in culture, separated by slab gel isoelectric focusing technique, and quantitated by autoradiographic or fluorographic method. Culture of BFU-E from both parents revealed significant HbF biosynthesis. HbF Malta-I was present in culture of the father's cells and comprised about 24% of total HbF. When we analyzed Hb biosynthesis in individual bursts, all bursts contained HbA and HbF in varying ratios. The frequency distribution of the individual bursts differing in percentages of HbF biosynthesis approached normal distribution. While the relative ratio of HbF Malta-I to total HbF biosynthesis in individual bursts also revealed significant variation, its frequency distribution did not show a normal distribution. There was a positive correlation between the ratios of HbF/Hb and HbF Malta-I/HbF in individual bursts.