[Tinea corporis within recent tattoos]. / Dermatophytose sur des tatouages récents.

INTRODUCTION: Tattoos are occasionally associated with cutaneous infections. Diagnosis can be challenging as the clinical presentation of such infections may differ from those on plain skin. Herein we report an atypical form of tinea corporis restricted to two recent tattoos during healing, caused by environmental contamination. We reviewed the literature for all cases of fungal infection after tattooing. PATIENTS AND METHODS: A 27-year-old female patient was seen for ring-shaped, erosive, oozing, pruritic and rapidly extensive skin lesions as well as infiltrated papular lesions occurring on tattoos done 6 and 12 days earlier. Fungal analysis revealed Microsporum canis. History-taking indicated that the patient's cat had ringworm and that the patient's sister also had skin lesions consistent with tinea corporis. DISCUSSION: Tinea on tattoos is rarely reported. We found ten additional cases in the literature, as well as five cases of less common fungal infections. These could be explained by the skin break created by the needle during tattooing resulting in an impaired skin barrier, or by accidental self-inoculation (e.g. foot-tattoos). The hypothesis of local immune deficiency induced by tattoo inks strikes us as rather improbable. Unlike usual cases of infections (pyogenic bacteria, mycobacteria, viral hepatitis), fungal infections are not related to a lack of hygiene on the part of the tattooist, but rather to contamination during the healing phase. Their clinical presentation may be atypical, resulting in diagnostic difficulties.

Think global, act local--how to regulate S phase from individual replication origins.

All eukaryotes use similar proteins to licence replication origins but, paradoxically, origin DNA is much less conserved. Specific binding sites for these proteins have now been identified on fission yeast and Drosophila chromosomes, suggesting that the DNA-binding activity of the origin recognition complex has diverged to recruit conserved initiation factors on polymorphic replication origins. Once formed, competent origins are activated by cyclin- and Dbf4-dependent kinases. The latter have been shown to control S phase in several organisms but, in contrast to cyclin-dependent kinases, seem regulated at the level of individual origins. Global and local regulations generate specific patterns of DNA replication that help establish epigenetic chromosome states.

Hierarchy of S-phase-promoting factors: yeast Dbf4-Cdc7 kinase requires prior S-phase cyclin-dependent kinase activation.

In all eukaryotes, the initiation of DNA synthesis requires the formation of prereplicative complexes (pre-RCs) on replication origins, followed by their activation by two S-T protein kinases, an S-phase cyclin-dependent kinase (S-CDK) and a homologue of yeast Dbf4-Cdc7 kinase (Dbf4p-dependent kinase [DDK]). Here, we show that yeast DDK activity is cell cycle regulated, though less tightly than that of the S-CDK Clb5-Cdk1, and peaks during S phase in correlation with Dbf4p levels. Dbf4p is short-lived throughout the cell cycle, but its instability is accentuated during G(1) by the anaphase-promoting complex. Downregulating DDK activity is physiologically important, as joint Cdc7p and Dbf4p overexpression is lethal. Because pre-RC formation is a highly ordered process, we asked whether S-CDK and DDK need also to function in a specific order for the firing of origins. We found that both kinases are activated independently, but we show that DDK can perform its function for DNA replication only after S-CDKs have been activated. Cdc45p, a protein needed for initiation, binds tightly to chromatin only after S-CDK activation (L. Zou and B. Stillman, Science 280:593-596, 1998). We show that Cdc45p is phosphorylated by DDK in vitro, suggesting that it might be one of DDK's critical substrates after S-CDK activation. Linking the origin-bound DDK to the tightly regulated S-CDK in a dependent sequence of events may ensure that DNA replication initiates only at the right time and place.

Monitoring S phase progression globally and locally using BrdU incorporation in TK(+) yeast strains.

Eukaryotic chromosome replication is initiated from numerous origins and its activation is temporally controlled by cell cycle and checkpoint mechanisms. Yeast has been very useful in defining the genetic elements required for initiation of DNA replication, but simple and precise tools to monitor S phase progression are lacking in this model organism. Here we describe a TK(+) yeast strain and conditions that allow incorporation of exogenous BrdU into genomic DNA, along with protocols to detect the sites of DNA synthesis in yeast nuclei or on combed DNA molecules. S phase progression is monitored by quantification of BrdU in total yeast DNA or on individual chromosomes. Using these tools we show that yeast chromosomes replicate synchronously and that DNA synthesis occurs at discrete subnuclear foci. Analysis of BrdU signals along single DNA molecules from hydroxyurea-arrested cells reveals that replication forks stall 8-9 kb from origins that are placed 46 kb apart on average. Quantification of total BrdU incorporation suggests that 190 'early' origins have fired in these cells and that late replicating territories might represent up to 40% of the yeast genome. More generally, the methods outlined here will help understand the kinetics of DNA replication in wild-type yeast and refine the phenotypes of several mutants.

[Malakoplakia of the submandibular gland in a renal transplant patient]. / Malakoplakie de la glande sous-mandibulaire chez un patient transplanté rénal.

INTRODUCTION: Malakoplakia (MP) is a rare granulomatous disease, usually occurring in immunocompromised patients, linked to Escherichia coli infection. The lesions are usually located in the genitourinary tract, but there is a great variability in the topography and the clinical presentation. CASE REPORT: A 70-year-old diabetic kidney transplant patient under immunosuppressive treatment presented with a voluminous submandibular chronic lesion, involving the skin, associated with a burgeoning lesion of the oral mucosa. Histological examination of biopsies concluded to MP and bacteriological samples were positive for E. coli. Antibiotic treatment allowed for the regression of the lesion before surgical removal. Histological examination of resected material confirmed the diagnosis of invasive MP of the submandibular gland. DISCUSSION: The diagnosis of MP relies on histological examination, showing the presence of von Hansemann's cells and Michaelis- Gutmann bodies. The treatment is based on active antibiotics targeted against intracellular bacteria, possibly associated with surgery. We report the first case of MP involving the submandibular gland.

Selectivity and specificity in the recognition of tRNA by E coli glutaminyl-tRNA synthetase.

The specific recognition by Escherichia coli glutaminyl-tRNA synthetase (GlnRS) of tRNA(Gln) is mediated by extensive protein:RNA contacts and changes in the conformation of tRNA(Gln) when complexed with GlnRS. In vivo accuracy of aminoacylation depends on two factors: competition between synthetases, and the context and recognition of identity elements in the tRNA. The structure of the tRNA(Gln):GlnRS complex supports studies from amber and opal suppressor tRNAs, complemented by in vitro aminoacylation of the mutated tRNA transcripts, that the glutamine identity elements are located in the anticodon and acceptor stem of tRNA(Gln). Recognition of individual functional groups in tRNA, for example the 2-amino group of guanosine, is also evident from the result with inosine-substituted tRNAs. Communication between anticodon and acceptor stem recognition is indicated by mutants in GlnRS isolated by genetic selection with opal suppressor tRNAs which are altered in interactions with the inside of the L-shaped tRNA. We have also used genetic selection to obtain mutants of GlnRS altered in acceptor stem recognition with relaxed specificity for amber suppressor tRNAs, and a more extensive mutational analysis shows the importance of the acceptor binding domain to accurate recognition of tRNA.

Nucleotide sequence of the mitochondrial 5S rRNA gene from lupine (Lupinus luteus).

A lupine mitochondrial clone containing 5S rRNA gene is characterized. The gene is located on the same strand as 18S rRNA and separated from it by 190 nucleotides. The intergenic region in different plants shows high degree of homology. In the case of lupine and soybean 43 nucleotides upstream of 5S rRNA gene exhibits 100% of homology. Comparisons of lupine 5S rRNA gene sequence with other plant mitochondrial 5S rRNA genes displays high degree of homology (from 89.8% to 95.8%).

Elongated unique DNA strand deposition on microstructured substrate by receding meniscus assembly and capillary force.

Ordered deposition of elongated DNA molecules was achieved by the forced dewetting of a DNA solution droplet over a microstructured substrate. This technique allows trapping, uncoiling, and deposition of DNA fragments without the need of a physicochemical anchoring of the molecule and results in the combing of double stranded DNA from the edge of microwells on a polydimethylsiloxane (PDMS) substrate. The technique involves scanning a droplet of DNA solution caught between a movable blade and a PDMS substrate containing an array of microwells. The deposition and elongation appears when the receding meniscus dewets microwells, the latter acting here as a perturbation in the dewetting line forcing the water film to break locally. Thus, DNA molecules can be deposited in an ordered manner and elongated conformation based solely on a physical phenomenon, allowing uncoiled DNA molecules to be observed in all their length. However, the exact mechanism that governs the deposition of DNA strands is not well understood. This paper is an analysis of the physical phenomenon occurring in the deposition process and is based on observations made with the use of high frame/second rate video microscopy.

Selection of a 'minimal' glutaminyl-tRNA synthetase and the evolution of class I synthetases.

The evolution of the aminoacyl-tRNA synthetases is intriguing in light of their elaborate relationship with tRNAs and their significance in the decoding process. Based on sequence motifs and structure determination, these enzymes have been assigned to two classes. The crystal structure of Escherichia coli glutaminyl-tRNA synthetase (GlnRS), a class I enzyme, complexed to tRNA(Gln) and ATP has been described. It is shown here that a 'minimal' GlnRS, i.e. a GlnRS from which domains interacting with the acceptor-end and the anticodon of the tRNA have been deleted, has enzymatic activity and can charge a tRNA(Tyr)-derived amber suppressor (supF) with glutamine. The catalytic core of GlnRS, which is structurally conserved in other class I synthetases, is therefore sufficient for the aminoacylation of tRNA substrates. Some of these truncated enzymes have lost their ability to discriminate against non-cognate tRNAs, implying a more specific role of the acceptor-end-binding domain in the recognition of tRNAs. Our results indicate that the catalytic and substrate recognition properties are carried by distinct domains of GlnRS, and support the notion that class I aminoacyl-tRNA synthetases evolved from a common ancestor, jointly with tRNAs and the genetic code, by the addition of non-catalytic domains conferring new recognition specificities.

CLB5 and CLB6, a new pair of B cyclins involved in DNA replication in Saccharomyces cerevisiae.

The functions of the Cdc28 protein kinase in DNA replication and mitosis in Saccharomyces cerevisiae are thought to be determined by the type of cyclin subunit with which it is associated. G1-specific cyclins encoded by CLN1, CLN2, and CLN3 are required for entry into the cell cycle (Start) and thereby for S phase, whereas G2-specific B-type cyclins encoded by CLB1, CLB2, CLB3, and CLB4 are required for mitosis. We describe a new family of B-type cyclin genes, CLB5 and CLB6, whose transcripts appear in late G1 along with those of CLN1, CLN2, and many genes required for DNA replication. Deletion of CLB6 has little or no effect, but deletion of CLB5 greatly extends S phase, and deleting both genes prevents the timely initiation of DNA replication. Transcription of CLB5 and CLB6 is normally dependent on Cln activity, but ectopic CLB5 expression allows cells to proliferate in the absence of Cln cyclins. Thus, the kinase activity associated with Clb5/6 and not with Cln cyclins may be responsible for S-phase entry. Clb5 also has a function, along with Clb3 and Clb4, in the formation of mitotic spindles. Our observation that CLB5 is involved in the initiation of both S phase and mitosis suggests that a single primordial B-type cyclin might have been sufficient for regulating the cell cycle of the common ancestor of many, if not all, eukaryotes.

New yeast actin-like gene required late in the cell cycle.

Actin, a major cytoskeletal component of all eukaryotic cells, is one of the most highly conserved proteins. It is involved in various cellular processes such as motility, cytoplasmic streaming, chromosome segregation and cytokinesis. The actin from the yeast Saccharomyces cerevisiae, encoded by the essential ACT1 gene, is 89% identical to mouse cytoplasmic actin and is involved in the organization and polarized growth of the cell surface. We report here the characterization of ACT2, a previously undescribed yeast split gene encoding a putative protein (391 amino acids, relative molecular mass (Mr) 44,073) that is 47% identical to yeast actin. The requirement of the ACT2 gene for vegetative growth of yeast cells and the existence of related genes in other eukaryotes indicate an important and conserved role for these actin-like proteins. Superimposition of the Act2 polypeptide onto the three-dimensional structure of known actins reveals that most of the divergence occurred in loops involved in actin polymerization, DNase I and myosin binding, leaving the core domain mainly unaffected. To our knowledge, the Act2 protein from S. cerevisiae is the first highly divergent actin molecule described. Structural and physiological data suggest that the Act2 protein might have an important role in cytoskeletal reorganization during the cell cycle.

Purification of the yeast mitochondrial methionyl-tRNA synthetase. Common and distinctive features of the cytoplasmic and mitochondrial isoenzymes.

Yeast-mitochondrial methionyl-tRNA synthetase was purified 1060-fold from mitochondrial matrix proteins of Saccharomyces cerevisiae using a four-step procedure based on affinity chromatography (heparin-Ultrogel, tRNA(Met)-Sepharose, Agarose-hexyl-AMP) to yield to a single polypeptide of high specific activity (1800 U/mg). Like the cytoplasmic methionyl-tRNA synthetase (Mr 85,000), the mitochondrial isoenzyme is a monomer, but of significantly smaller polypeptide size (Mr 65,000). In contrast, the corresponding enzyme of Escherichia coli is a dimer (Mr 152,000) made up of identical subunits. The measured affinity constants of the purified mitochondrial enzyme for methionine and tRNA(Met) are similar to those of the cytoplasmic isoenzyme. However, the two yeast enzymes exhibit clearly different patterns of aminoacylation of heterologous yeast and E. coli tRNA(Met). Furthermore, polyclonal antibodies raised against the two proteins did not show any cross-reactivity by inhibition of enzymatic activity and by the highly sensitive immunoblotting technique, indicating that the two enzymes share little, if any, common antigenic determinants. Taken together, our results further support the belief that the yeast mitochondrial and cytoplasmic methionyl-tRNA synthetases are different proteins coded for by two distinct nuclear genes. Like the yeast cytoplasmic aminoacyl-tRNA synthetases, the mitochondrial enzymes displayed affinity for immobilized heparin. This distinguishes them from the corresponding enzymes of E. coli. Such an unexpected property of the mitochondrial enzymes suggests that they have acquired during evolution a domain for binding to negatively charged cellular components.

Acceptor end binding domain interactions ensure correct aminoacylation of transfer RNA.

The recognition of the acceptor stem of tRNA(Gln) is an important element ensuring the accuracy of aminoacylation by Escherichia coli glutaminyl-tRNA synthetase (GlnRS; EC 6.1.1.18). On the basis of known mutations and the crystal structure of the tRNA(Gln).GlnRS complex, we mutagenized at saturation two motifs in the acceptor end binding domain of GlnRS. Mutants with lowered tRNA specificity were then selected in vivo by suppression of a glutamine-specific amber mutation (lacZ1000) with an amber suppressor tRNA derived from tRNA(1Ser). The mischarging GlnRS mutants obtained in this way retain the ability to charge tRNA(Gln), but in addition, they misacylate a number of noncognate amber suppressor tRNAs. The critical residues responsible for specificity are Arg-130 and Glu-131, located in a part of GlnRS that binds the acceptor stem of tRNA(Gln). On the basis of the spectrum of tRNAs capable of being misacylated by such mutants we propose that, in addition to taking part in productive interactions, the acceptor end binding domain contributes to recognition specificity by rejecting noncognate tRNAs through negative interactions. Analysis of the catalytic properties of one of the mischarging enzymes, GlnRS100 (Arg-130-->Pro, Glu-131-->Asp), indicates that, while the kinetic parameters of the mutant enzyme are not dramatically changed, it binds noncognate tRNA(Glu) more stably than the wild-type enzyme does (Kd is 1/8 that of the wild type). Thus, the stability of the noncognate complex may be the basis for mischarging in vivo.

The B-type cyclin kinase inhibitor p40SIC1 controls the G1 to S transition in S. cerevisiae.

When yeast cells reach a critical size, they initiate bud formation, spindle pole body duplication, and DNA replication almost simultaneously. All three events depend on activation of Cdc28 protein kinase by the G1 cyclins Cln1, -2, and -3. We show that DNA replication also requires activation of Cdc28 by B-type (Clb) cyclins. A sextuple clb1-6 mutant arrests as multibudded G1 cells that resemble cells lacking the Cdc34 ubiquitin-conjugating enzyme. cdc34 mutants cannot enter S phase because they fail to destroy p40SIC1, which is a potent inhibitor of Clb but not Cln forms of the Cdc28 kinase. In wild-type cells, p40SIC1 protein appears at the end of mitosis and disappears shortly before S phase. Proteolysis of a cyclin-specific inhibitor of Cdc28 is therefore an essential aspect of the G1 to S phase transition.

Cloning of the two essential yeast genes, PRP6 and PRP9, and their rapid mapping, disruption and partial sequencing using a linker insertion strategy.

In the yeast Saccharomyces cerevisiae, some thermosensitive (ts) mutants have been shown to be impaired in pre-mRNA splicing (prp mutants). From a yeast genomic library, we have isolated plasmids that complement prp6 or prp9 ts mutations. These plasmids also complement the ts growth defect of additional independent mutants identified as new prp6 and prp9 ts alleles, indicating that the cloned DNAs encode PRP6 and PRP9 genes, respectively. Here, we describe the restriction maps of these loci which are localized on chromosome II and IV, respectively. The limits of open reading frames (ORFs) within the cloned inserts have been determined using a linker insertion strategy combined with the ts complementation assay. Double-strand DNA sequencing was also performed directly on the yeast expression vector from the inserted linkers. Gene disruption experiments demonstrate that both genes are essential for viability.

The 'SUN' family: yeast SUN4/SCW3 is involved in cell septation.

SUN4 is the fourth member of the SUN gene family from S. cerevisiae, whose products display high homology in their 258 amino acid C-terminal domain. SIM1, UTH1, NCA3 (the founding members) are involved in different cellular processes (DNA replication, ageing, mitochondrial biogenesis) and it is shown herein that SUN4 plays a role in the cell septation process. sun4 delta cells are larger than wild-type and begin a new cell cycle before they have separated from their mother cell. This phenotype is more pronounced in sun4Delta cells also deleted for UTH1. FACS analysis shows apparent polyploidy which disappears when the cell cycle is arrested by mating factor or nocodazole, indicating that cell septation is delayed without modification of the doubling time. Elutriated sun4 delta uth1 delta daughter cells are born larger, and therefore enter S phase sooner than their wild-type counterpart. S phase duration, as well as timing of Clb2 degradation, is normal, but cell septation is delayed. Sun4p/Scw3p was recently described as a cell wall protein (Cappellaro et al., 1998) and, consistent with this notion, electron micrographs of sun4 delta cells show defects in the final steps of cell wall septation. Our data suggest that Sun4p and Uth1p might contribute to the regulated process of cell wall morphogenesis and septation.

RPK1, an essential yeast protein kinase involved in the regulation of the onset of mitosis, shows homology to mammalian dual-specificity kinases.

We report here the sequence of RPK1 (for Regulatory cell Proliferation Kinase), a new Saccharomyces cerevisiae gene coding for a protein with sequence similarities to serine/threonine protein kinases. The protein sequence of 764 amino acids includes an amino-terminal domain (residues 1-410), which may be involved in regulation of the kinase domain (residues 411-764). The catalytic domain of Rpk1 is not closely related to other known yeast protein kinases but exhibits strong homology to a newly discovered group of mammalian kinases (PYT, TTK, esk) with serine/threonine/tyrosine kinase activity. Null alleles of RPK1 are lethal and thus this gene belongs to the small group of yeast protein kinase genes that are essential for cell growth. In addition, eliminating the expression of RPK1 gives rise to the accumulation of non-viable cells with less than a 1 N DNA content suggesting that cells proceed into mitosis without completion of DNA synthesis. Therefore, the Rpk1 kinase may function in a checkpoint control which couples DNA replication to mitosis. The level of the RPK1 transcript is extremely low and constant throughout the mitotic cycle. However it is regulated during cellular differentiation, being decreased in alpha-factor-treated a cells and increased late in meiosis in a/alpha diploids. Taken together, our results suggest that Rpk1 is involved in a pathway that coordinates cell proliferation and differentiation.

A role for the Cdc7 kinase regulatory subunit Dbf4p in the formation of initiation-competent origins of replication.

Using a reconstituted DNA replication assay from yeast, we demonstrate that two kinase complexes are essential for the promotion of replication in vitro. An active Clb/Cdc28 kinase complex, or its vertebrate equivalent, is required in trans to stimulate initiation in G(1)-phase nuclei, whereas the Dbf4/Cdc7 kinase complex must be provided by the template nuclei themselves. The regulatory subunit of Cdc7p, Dbf4p, accumulates during late G(1) phase, becomes chromatin associated prior to Clb/Cdc28 activation, and assumes a punctate pattern of localization that is similar to, and dependent on, the origin recognition complex (ORC). The association of Dbf4p with a detergent-insoluble chromatin fraction in G(1)-phase nuclei requires ORC but not Cdc6p or Clb/Cdc28 kinase activity, and correlates with competence for initiation. We propose a model in which Dbf4p targets Cdc7p to the prereplication complex prior to the G(1)/S transition, by a pathway parallel to, but independent of, the Cdc6p-dependent recruitment of MCMs.