Updated model of group A Streptococcus M proteins based on a comprehensive worldwide study.

Group A Streptococcus (GAS) M protein is an important virulence factor and potential vaccine antigen, and constitutes the basis for strain typing (emm-typing). Although >200 emm-types are characterized, structural data were obtained from only a limited number of emm-types. We aim to evaluate the sequence diversity of near-full-length M proteins from worldwide sources and analyse their structure, sequence conservation and classification. GAS isolates recovered from throughout the world during the last two decades underwent emm-typing and complete emm gene sequencing. Predicted amino acid sequence analyses, secondary structure predictions and vaccine epitope mapping were performed using MUSCLE and Geneious software. A total of 1086 isolates from 31 countries were analysed, representing 175 emm-types. emm-type is predictive of the whole protein structure, independent of geographical origin or clinical association. Findings of an emm-type paired with multiple, highly divergent central regions were not observed. M protein sequence length, the presence or absence of sequence repeats and predicted secondary structure were assessed in the context of the latest vaccine developments. Based on these global data, the M6 protein model is updated to a three representative M protein (M5, M80 and M77) model, to aid in epidemiological analysis, vaccine development and M protein-related pathogenesis studies.

Regulation of the alpha-fetoprotein promoter: Ku binding and DNA spatial conformation.

This work shows that the proximal promoter of the mouse Afp gene contains a Ku binding site and that Ku binding is associated with down-regulation of the transcriptional activity of the Afp promoter. The Ku binding site is located in a segment able to adopt a peculiar structured form, probably a hairpin structure. Interestingly, the structured form eliminates the binding sites of the positive transcription factor HNF1. Furthermore, a DNAse hypersensitive site is detected in footprinting experiments done with extracts of AFP non-expressing hepatoma cells. These observations suggest that the structured form is stabilised by Ku and is associated with extinction of the gene in AFP non-expressing hepatic cells.

Identification of KLF13 and KLF14 (SP6), novel members of the SP/XKLF transcription factor family.

Using the sequence of the SP1 zinc-finger DNA-binding domain as a probe to screen a mouse EST database, we identified two novel members of the SP/XKLF transcription factor family, KLF13 and KLF14. The mouse Klf13 cDNA (1310 bp in length) contains a single open reading frame of 288 amino acids with a DNA-binding domain closely related to that of the human RFLAT-1 protein and a putative transactivator N-terminal domain rich in proline and alanine residues. The mouse Klf13 gene seems to be the homologue of the human RFLAT1 gene. The mouse Klf14 sequence is homologous to a human genomic sequence from chromosome 17 that is believed to code for a protein with three zinc fingers at the end of its C-terminal domain. Using reverse transcription-polymerase chain reaction, we showed ubiquitous expression of Klf13 and Klf14 in adult mice. A third member of this family was also identified in a human EST database; this sequence was found to be identical to KLF11 (TIEG2), recently identified by Cook et al. (1998, J. Biol. Chem. 273: 25929-25936). The corresponding mouse cDNA was isolated and sequenced. The three genes were localized in the human and the rat: chromosomes 15 (human KLF13), 17q21.3-q22 (human KLF14; HGMW-approved symbol SP6), and 2p25 (human KLF11) and chromosomes 1q31-q32 (rat Klf13), 10q31-q32.1 (rat Klf14) (SP6), and 6q16-q21 (rat Klf11).

New positive selection system based on the parD (kis/kid) system of the R1 plasmid.

The use of vectors that are designed to allow positive selection of recombinants facilitates cloning experiments in E. coli. Using kid, a lethal gene of the R1 plasmid parD locus, we generated pKID vectors leading to high selective efficiency of recombinants (greater than 90%). The E. coli bacterial host used to propagate these vectors produces the Kis protein, the natural antagonist of Kid. This new positive-selection system exhibits the same efficiency as the original ccdB-based selection vectors, pKIL (4). We also show that the ccdB and kid systems are independent. This property increases the potential of plasmidic poison-antidote systems for genetic applications and opens the door to a generation of new vectors containing the two selection systems.

Positive selection vectors to generate fused genes for the expression of his-tagged proteins.

Epitope tagging simplifies detection, characterization and purification of proteins. Gene fusion to combine the coding region of a well-characterized epitope with the coding region for a protein of interest generally requires several subcloning steps. Alternatively, a PCR strategy can be used to generate such a chimeric gene. In addition to its simplicity, this approach allows one to limit the size of the multiple cloning sites present in conventional expression vectors, thus reducing the introduction of artifactual amino-acid sequences into the fused protein. In this communication, we describe new vectors that allow PCR cloning and selection of chimeric genes coding for N- or C-terminal His-tagged proteins. These vectors are based on the control of cell death CcdB direct selection technology and are well adapted to the cloning of blunt-ended PCR products that were generated by using thermostable polymerases that provide proofreading activity.

Negative regulation of the alpha-foetoprotein gene in fibroblasts: identification and characterization of cis and trans elements.

The alpha-foetoprotein (AFP) gene is extinguished in hybrids formed between hepatoma cells (expressing cells) and fibroblasts (non-expressing cells). Transfection experiments with constructions containing segments from the promoter region of the AFP-gene, placed upstream of an ubiquitously expressed promoter (the Herpes virus thymidine kinase gene promoter), showed that the AFP gene-derived sequence contains at least one negative element active in fibroblasts (while this sequence behaves as an enhancer in hepatoma cells). We identified such a fibroblast negative region, localized between nucleotide positions -80 to -38 (FNE1). Gel retardation experiments showed that FNE1 specifically binds fibroblast nuclear proteins, generating three complexes. The sequence from -57 to -43 was shown to be responsible for both the formation of these complexes and the negative activity of FNE1. These results suggest that the binding of nuclear factors to the AFP promoter region contributes to silencing the AFP gene in non-expressing cells, such as fibroblasts, and thus to establishing lineage-specific expression of the AFP gene.

Bifunctional lacZ alpha-ccdB genes for selective cloning of PCR products.

The use of PCR-amplified DNA-fragments is a classical approach to generate recombinant DNA. To facilitate the cloning of PCR products, we have constructed two new pKIL vectors that allow selection of recombinants. The multiple cloning sites (MCS) of these plasmids contain two adjacent Aspel sites and a unique HindII site. Cleavage of these vectors with Aspel produce linearized molecules with a single thymidine nucleotide at the 3' ends allowing TA cloning of Taq-amplified fragments. On the other hand, cleavage with HindII can be used for the cloning of blunt-ended PCR products generated by other DNA polymerases. The LacZ alpha-CcdB fusion protein produced by these plasmids has retained both the CcdB killer activity and the ability to alpha-complement the truncated LacZ delta M15. This bifunctionality allowed us to show that small PCR products (< 1000 bp) that do not disrupt lacZ alpha efficiently do inactivate CcdB, which demonstrates that the CcdB-based selection is well adapted for cloning of PCR products, especially for small size fragments.

The thyroid hormone down-regulates the mouse alpha-foetoprotein promoter.

The thyroid hormone (T3) was shown to down regulate the level of alpha-foetoprotein (AFP) mRNA in hepatoma cells HepG2. Recombinant plasmids containing segments from the mouse AFP gene promoter were transfected in HepG2 cells and transient expression assays showed that the T3 inhibitory effect depends on the sequence limited by positions -80 and -38, upstream from the TATA box. This sequence is able to confer T3 sensitivity to a heterologous promoter and contains a putative T3-responsive element, as well as likely CEBP- and HNF1-responsive elements. These observations suggest that T3 is a good candidate for hormonal control of the AFP gene expression and especially for the neonatal shut off of the gene.

Replicon typing of virulence plasmids of enterotoxigenic Escherichia coli isolates from cattle.

Plasmid DNA hybridization with probes for virulence factors used for basic replicons of plasmids was used to identify the virulence plasmids of a collection of enterotoxigenic Escherichia coli isolates from cattle. The virulence probes were derived from the genes coding for the heat-stable enterotoxin STaP and for the F5 (K99) and F41 fimbrial adhesins. The replicon probes were derived from 16 different basic replicons of plasmids (probes repFIA, repFIB, repFIC, repFIIA, repI1, repHI1, repHI2, repL/M, repN, repP, repQ, repT, repU, repW, repX, and repY). The virulence genes coding for the STaP enterotoxin and for the F5 adhesin were located on a single plasmid band in each isolate. The sizes of most of these virulence plasmids were from 65 to 95 MDa. The F41 probe failed to hybridize with any plasmid band. The virulence plasmids had multireplicon types typical of plasmids of the IncF groups. The most common basic replicon association was the triple RepFIA-RepFIB-RepFIC family association.

Dexamethasone bovine pharmacokinetics.

Dexamethasone phosphate (DXM-PHO) is an ester which is quickly hydrolysed by the bovine and the dexamethasone (DXM) plasma half-life was 5.16 h. It has been demonstrated that 54 h after DXM-PHO injection, DXM concentrations were lower than 0.1 mg/ml. Tritiated dexamethasone was also administered twice to an another young bull for metabolite investigation. The elapsed time required to recover, in plasma, half of the radioactivity injected was 8.8 h. Radioactivity recovery in the urine reached 36.4% and 22.6% for the first and the second injections respectively.

Assignment of 12 loci to rat chromosome 5: evidence that this chromosome is homologous to mouse chromosome 4 and to human chromosomes 9 and 1 (1p arm).

Twelve loci have been assigned to rat chromosome 5: aldolase B (ALDOB), atrial natriuretic factor (ANF = pronatriodilatin, PND), D4RP1, DSI1, galactosyltransferase (GGTB2), glucose transporter (GLUT1), interferon alpha 1 and related interferon alpha (INFA), interferon beta (INFB), lymphocyte-specific protein-tyrosine kinase (LCK), oncogene MOS, alpha 2U-globulin (major urinary protein, MUP), and orosomucoid (ORM, also called alpha 1-acid glycoprotein, AGP). Among these, the interferon alpha and beta genes map in the q22-23 region, which also contains a transformation suppressor gene (SAI1). The other loci reside outside this region. This study also indicated that the rat genome contains 2 LCK genes, unlike the human and murine genomes. These new assignments on rat chromosome 5 demonstrate that this chromosome is highly homologous to mouse chromosome 4 and carries synteny groups conserved on human chromosome 9 (interferon alpha and beta, galactosyltransferase, orosomucoid, and aldolase B genes) and on the short arm of human chromosome 1 (MYCL, glucose transporter, protein kinase LCK, and atrial natriuretic factor genes).

Validation of a quantitative procedure for the extraction of sterols from edible oils using radiolabelled compounds.

The work described in this paper is integrated in an analytical programme organised by the Community Bureau of Reference with the aim of developing reference materials certified for sterol content. Preliminary inter-comparison of methods showed that the level of agreement of the results was insufficient for certification purposes. Errors could occur in the different steps before the final determination by gas-liquid chromatography. It was, therefore, decided to validate a quantitative procedure for the isolation of sterols. A well defined saponification-extraction method was tested using labelled sterols ([3H]cholesterol and [3H]cholesteryl oleate) and radiochemical measurements. The study has shown that total cholesterol recovery reached 100.5 +/- 1.4%, that cholesteryl ester was saponified quantitatively and that there were no appreciable amounts of degradation products. The procedure has been used as the basis for the certification of three reference materials and it has been shown that the saponification and extraction procedure leads to the quantitative recovery of sterols regardless of the nature of the fatty material tested.

[Phenotypic and genotypic properties of 4 virulence plasmids of Salmonella typhimurium]. / Propriétés phénotypiques et génotypiques de quatre plasmides de virulence de Salmonella typhimurium.

Virulence-associated plasmids pLT2, pIP1350, pIP1360 and pIP1370 coded for the fi+ character. These plasmids did not code for the synthesis of classical adhesins, aerobactin or colicins. Nor did they mediate resistance to the metals Hg, Te, As, Cd or flavomycin. Plasmids pLT2, pIP1350 and pIP1370 contained DNA sequences homologous to the incFIIA and incFIB replicons. Plasmid pIP1360 contained a single sequence homologous to incFIIA replicon.

Mini-F E protein: the carboxy-terminal end is essential for E gene repression and mini-F copy number control.

Mini-F is a segment of the conjugative plasmid F consisting of two origins of replication flanked by regulatory regions, which ensure a normal control of replication and partitioning. Adjacent to the ori-2 origin is a complex coding region that consists of the E gene overlapped by three open reading frames with the coding potential for 9000 Mr polypeptides here designated 9 kd-1, 9 kd-2 and 9 kd-3. In this paper, we show that open reading frame 9 kd-3 is preceded by active promoter and Shine-Dalgarno sequences. The E coding region specifies: an initiator of replication, which acts at the ori-2 site; a function that negatively regulates the expression of the E gene; and a function involved in mini-F copy number control. To assign one of these functions to one of the overlapping coding sequence, we have isolated, characterized and sequenced mutations mapping in the E coding region. In this paper, we analyse two mutations (cop5 and pla25) that abolish the repression of the E gene. As these mutations affect the primary structure of protein E itself but not the 9 kd polypeptides, we conclude that protein E takes part in the negative regulation of its own synthesis. In addition, the localization of the cop5 and pla25 mutations indicates that the carboxy-terminal end of the E protein is involved in the autorepression function. The cop5 mutation causes an eightfold increase of the mini-F copy number. The pla25 mutation leads to the inability of the derived mini-F plasmid to give rise to plasmid-harbouring bacteria. The ways in which the cop5 and pla25 mutations may lead to such phenotypes are discussed in relation to the different functions mapping in the E coding sequence.

Mini-F encoded proteins: identification of a new 10.5 kilodalton species.

The elements which ensure the maintenance of the F plasmid are located in its f5 EcoRI restriction fragment. This f5 fragment constitutes a mini-F plasmid showing the same stability and copy number control as the entire F plasmid. The proteins expressed in minicells by wild-type or mutated f5 fragments were analysed by pH gradient two-dimensional electrophoresis. We identified seven f5-encoded polypeptides and located their genes on the F map. Among them, H1, an acidic polypeptide of mol. wt. 10.5 K, had not been detected before. It is in fact the most abundant f5-encoded polypeptide identified so far. In addition, we showed that both 10.5-K and 12-K protein bands detected by SDS-polyacrylamide gel electrophoresis are, respectively, composed of two polypeptides, H1 and H2, G1 and G2, of different isoelectric points. Polypeptides H2 and G2, respectively, share common coding sequences with polypeptides H1 and G1. Their possible biological significance is discussed. The sequences coding for polypeptides H1/H2 and G1/G2 are clustered in a 800-bp long region located between the two mini-F origin sites and are proposed to be organized as an operon. The results reported in the accompanying paper point out the importance of polypeptides G1/G2 and H1/H2 in the relationship between the F plasmid and its host.

Ham22, a mini-F mutation which is lethal to host cell and promotes recA-dependent induction of lambdoid prophage.

A mini-F region 800 bp long, located between the two F origin sites, plays an essential role in the relationship between the F plasmid and its host. This region comprises two sets of overlapping coding sequences: the first set codes for the newly identified H1 and H2 polypeptides; the second set codes for polypeptides G1 and G2. A mini-F amber mutation (Ham22) causes the virtual disappearance of polypeptides H1 and H2 but only slightly reduces synthesis of polypeptides G1 and G2. This mutation: (i) renders mini-F hybrids lethal to the host cells (conditional Hos- phenotype for host survival) and (ii) causes the induction of a resident prophage in recA+ strains (conditional Map- phenotype for maintenance of the prophage). When an additional mutation prevents the synthesis of polypeptides G1 and G2, both the lethal character and the induction of the prophage are abolished. We conclude: (i) that polypeptides G1 and/or G2 are specific mini-F polypeptides involved in the plasmid-mediated killing effect and in the recA-dependent induction of the resident prophage and (ii) that, in normal conditions, polypeptides H1 and/or H2 negatively control (directly or indirectly) the action of polypeptides G1 and/or G2. In relation to the analysis of indirect induction mediated by u.v.-irradiated lambda mini-F hybrids, we propose that polypeptides G1 and/or G2 are specific mini-F products involved in the activation of the bacterial SOS pathway. The H1/H2 and G1/G2 polypeptides could constitute the controlled mini-F signal enabling the coordination between cell division and F plasmid replication.