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Lantibiotics are antimicrobial peptides from the bacteriocin family, secreted by Gram-positive bacteria. These peptides differ from other bacteriocins by the presence of (methyl)lanthionine residues, which result from enzymatic modification of precursor peptides encoded by structural genes. Several groups of lantibiotics have been distinguished, the largest of which is the lacticin 481 group. This group consists of at least 16 members, including lacticin 481, streptococcin A-FF22, mutacin II, nukacin ISK-1, and salivaricins. We present the first review devoted to this lantibiotic group, knowledge of which has increased significantly within the last few years. After updating the group composition and defining the common properties of these lantibiotics, we highlight the most recent developments. The latter concern: transcriptional regulation of the lantibiotic genes; understanding the biosynthetic machinery, in particular the ability to perform in vitro prepeptide maturation; characterization of a novel type of immunity protein; and broad application possibilities. This group differs in many aspects from the best known lantibiotic group (nisin group), but shares properties with less-studied groups such as the mersacidin, cytolysin and lactocin S groups.

We previously characterized three Lactococcus lactis promoters, P170, P1 and P3, which are induced by low pH. Here, we identified a novel 14 bp regulatory DNA region centred at around -41.5 and composed of three tetranucleotide sequences, boxes A, C and D. Boxes A and C contribute to P1 activity, whereas box D and the position of boxes ACD (renamed ACiD-box) are essential to P1 activity and acid response. We also identified a trans -acting protein, RcfB, which is involved in P170 and P1 basal activity and is essential for their pH induction. The regulator belongs to the Crp-Fnr family of transcription regulators. Overexpression of rcfB resulted in increased beta-galactosidase activities and lantibiotic lacticin 481 production from P170- and P1-controlled genes, respectively, in acid condition. RcfB is thus probably activated when cells encounter an acid environment. rcfB is co-transcribed with genes encoding an universal stress-like protein and a multidrug transporter. RcfB plays a role in acid adaptation, as the survival rate of an rcfB mutant after a lethal acid challenge was 130-fold lower than that of the wild-type strain, when the bacteria were first grown in acidic medium. The groESL promoter includes a sequence resembling an ACiD-box and the chaperone GroEL production is partly RcfB dependent in acid condition. Our results suggest that the ACiD-box could be the DNA target site of RcfB.

In lantibiotic lacticin 481 biosynthesis, LctT cleaves the precursor peptide and exports mature lantibiotic. Matrix-assisted laser desorption ionization-time of flight mass spectrometry revealed that a truncated form of lacticin 481 is produced in the absence of LctT or after cleavage site inactivation. Production of truncated lacticin 481 is 4-fold less efficient, and its specific activity is about 10-fold lower.

The lantibiotic lacticin 481 operon (lctAMTFEG) is mainly transcribed from P1 and P3, two promoters lying upstream of lctA. A weak additional promoter allows independent expression of the immunity genes (lctFEG). Lacticin 481 production by Lactococcus lactis is stimulated by the acidification due to lactic acid production, and by artificially lowering the pH of the medium. This regulation occurs at the transcriptional level, since P1 and P3 are both acid-induced. P1 is weaker but more tightly regulated than P3. As no specific regulator is encoded by the lacticin 481 operon, P1 and P3 are likely controlled by a general regulator.

Class I bacteriocins (lantibiotics) and class II bacteriocins are antimicrobial peptides secreted by gram-positive bacteria. Using two lantibiotics, lacticin 481 and nisin, and the class II bacteriocin coagulin, we showed that bacteriocins can be detected without any purification from whole producer bacteria grown on plates by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS). When we compared the results of MALDI-TOF-MS performed with samples of whole cells and with samples of crude supernatants of liquid cultures, the former samples led to more efficient bacteriocin detection and required less handling. Nisin and lacticin 481 were both detected from a mixture of their producer strains, but such a mixture can yield additional signals. We used this method to determine the masses of two lacticin 481 variants, which confirmed at the peptide level the effect of mutations in the corresponding structural gene.

Galectins are animal lectins that can specifically bind beta-galactosides. Thirteen galectins have already been described. This review focuses on a specific member of this family: galectin-8. This galectin was discovered in prostate cancer cells eight years ago and has been studied extensively in the last few years. The galectin-8 gene ( LGALS8) encodes numerous mRNAs by alternate splicing and the presence of three unusual polyadenylation signals. These mRNAs encode six different isoforms of galectin-8: three belong to the tandem-repeat galectin group (with two CRDs linked by a hinge peptide) and three to the prototype group (with one CRD). Various studies showed that galectin-8 is widely expressed in tumor tissues as well as in normal tissues. The level of galectin-8 expression may correlate with the malignancy of human colon cancers and the degree of differentiation of lung squamous cell carcinomas and neuro-endocrine tumors. Recently, the differences in galectin-8 expression levels between normal and tumor tissues have been used as a guide for the selection of strategies for the prevention and treatment of lung squamous cell carcinoma. These experiments are still under investigation, but demonstrate the potential of galectin-8 research to enhance our understanding of, and possibly prevent, the process of neoplastic transformation.

Galectins are animal proteins which specifically bind beta-D-galactoside residues and their specific cellular function is not yet clearly established. However, these proteins seem to play a role in neoplastic transformations. Po66 is a murine monoclonal antibody directed against a protein from human lung carcinoma, Po66 Carbohydrate-Binding-Protein (Po66-CBP), which belongs to the galectin-8 family. Our results show that the Po66-CBP gene generates five transcripts by alternative splicing, which could give rise to five proteins: two proteins belong to the tandemly repeated galectin family and three belong to the single carbohydrate recognition domain galectins. All these proteins are encoded by a unique gene located in 1q42. Experiments carried out by reverse transcriptase-polymerase chain reaction show that the levels of expression of these five galectin-8 isoforms are variable during the culture time in SK-MES-1, a human lung squamous carcinoma cell line. Cancer Genome Anatomy Project database analysis confirms the presence of Po66-CBP in lung cancer and its absence in healthy lung.

Two copies of IS1675, a novel lactococcal insertion element from the IS4 family, are present on a 70-kb plasmid, where they frame the lantibiotic lacticin 481 operon. The whole structure could be a composite transposon designated Tn5721. This study shows that the lacticin 481 operon does not include any regulatory gene and provides a new example of a transposon-associated bacteriocin determinant. We identified five other IS1675 copies not associated with the lacticin 481 operon. The conservation of IS1675 flanking sequences suggested a 24-bp target site.

Class AII and AIII lantibiotics and mersacidin are antibacterial peptides containing unusual residues obtained by posttranslational modifications of prepeptides, presumably catalyzed by LanM. LctM, the LanM for lacticin 481, is essential for the production of this class AII lantibiotic. Using the yeast two-hybrid system, we showed direct contact between the prelacticin 481 and LctM, supporting the proposed LctM function. Sixteen domains are conserved between the 10 known LanM proteins, whereas three additional domains were found only in class AII LanM proteins and in MrsM, the LanM for mersacidin. All the truncated LctM proteins that we tested presented impaired LctA-binding activity.

Vitellogenins (Vtg) are the major yolk proteins in most oviparous organisms. They are encoded by a small number of genes--between one and four depending on the species. Characterization of the Vtg region in the genome of the rainbow trout reveals unusual features, however, in that this locus contains twenty complete genes and ten pseudogenes per haploid genome. The Vtg genes differ from each other by insertion, deletion and rearrangement events, although, at the sequence level, they show a high degree of similarity. Fluorescent in situ hybridization (FISH), pulsed-field gel electrophoresis (PFGE) and Southern analysis indicate that all gene copies are contained in a single 1,500-kb region, and that most of the genes form tandem arrays separated by a conserved 4.5-kb intergenic region. The presence of large reiterated fragments indicates that this region has been subjected to several amplification events. The presence of a retroposon element (called 19) in Vtg intron 9 appears to be responsible for the silencing of at least nine of the ten pseudogenes. Two other incomplete retrotransposons (one LTR- and one LINE-type) and sequences derived from a HIV-like retrovirus are inserted into the conserved intergenic region, very close to the transcription start site. Their presence in all Vtg 5'-flanking regions suggests a possible role in gene amplification at this locus.

Growth inhibition of Lactococcus lactis provoked by increasing osmolarity is reversed when glycine betaine (GB) or its analogs are added to a defined medium. Lacticin 481 production increased sharply with growth medium osmolarity in the absence of osmoprotectant but remained unaffected when GB was supplied in media of increasing osmolarity.

The lantibiotic lacticin 481 is a bacteriocin produced by Lactococcus lactis strains. The genetic determinants of lacticin 481 production are organized as an operon encoded by a 70-kb plasmid. We previously reported the first three genes of this operon, lctA, lctM, and lctT, which are involved in the bacteriocin biosynthesis and export (A. Rincé, A. Dufour, S. Le Pogam, D. Thuault, C. M. Bourgeois, and J.-P. Le Pennec, Appl. Environ. Microbiol. 60:1652-1657, 1994). The operon contains three additional open reading frames: lctF, lctE, and lctG. The hydrophobicity profiles and sequence similarities strongly suggest that the three gene products associate to form an ABC transporter. When the three genes were coexpressed into a lacticin 481-sensitive L. lactis strain, the strain became resistant to the bacteriocin. This protection could not be obtained when any of the three genes was deleted, confirming that lctF, lctE, and lctG are all necessary to provide immunity to lacticin 481. The quantification of the levels of immunity showed that lctF, lctE, and lctG could account for at least 6% and up to 100% of the immunity of the wild-type lacticin 481 producer strain, depending on the gene expression regulation. The lacticin 481 biosynthesis and immunity systems are discussed and compared to other lantibiotic systems.

In this paper we describe the first complete structure of a fish vitellogenin gene. A 22 kb genomic region from rainbow trout (Oncorhynchus mykiss) was cloned and analysed. This region was shown to contain two tandemly arranged vitellogenin genes. Both genes are 98.7% similar, indicating that they result from a recent local duplication. The complete sequence encoding one of the two genes was determined and the gene organization was established. The gene is 10.3 kb long and has 34 exons, it lacks one exon compared to amphibian and avian vitellogenin genes. Exons 22 and 23 of the Xenopus and chicken genes were shown to be merged into a single exon in the trout genome. Other splicing sites appeared highly conserved between the three vertebrate genes. In contrast, little similarity between invertebrate and vertebrate vitellogenin genes was observed with respect to the number and organization of introns. The comparison of 17 independent invertebrate splicing sites with the 34 vertebrate sites indicated that a few sites are probably ancient. However, most of the splicing junctions compared appeared unrelated. Results suggest that vitellogenin genes have been reshaped through multiple insertions and deletions of intervening sequences during evolution.

The nucleotide sequence of the vitellogenin cDNA from the rainbow trout Oncorhynchus mykiss was determined. Analysis of the deduced amino acid sequence (1659 residues) places the lipovitellin I, phosvitin and lipovitellin II domains between amino acids 16 to 1088, 1089 to 1145 and 1146 to 1659, respectively. The general structure is similar to other vertebrate vitellogenins except for the serine rich phosvitin domain which is the shortest identified so far in vertebrates (57 amino acids), being 2 to 4 times smaller than in other species. Sequence comparisons between vertebrate and invertebrate vitellogenins as well as with distantly related proteins allowed to identify two short amino acid motifs particularly well conserved, RGILN and TCGLCG in lipovitellin I and II domains, respectively, and strongly suggest that the lipovitellin II domain is involved in protein interactions via disulfide bridge formation.

CDK2 (cyclin-dependent kinase 2) is a serine/threonine kinase which is involved in regulating S-phase entry in higher eukaryotes. To investigate the transcriptional control of this gene, a 13-kb Xenopus laevis genomic clone containing the 5' flanking sequences was isolated. A 2.7-kb fragment containing the promoter region was sequenced and the transcription start point (tsp) was determined by primer extension. Several putative regulatory elements, such as the E2F-binding site, Y box and octamer-binding site, were localized in this region, but no TATA box was found. When fused to cat, a reporter gene encoding chloramphenicol acetyltransferase, the 5' flanking sequences were shown to function in oocytes and an enhancer activity was found in this region. During early embryogenesis, cdk2 promoter activity was tested and de novo transcription was detected at the mid-blastula transition.

The partial nucleotide sequence of a Lactococcus lactis subsp. lactis ADRIA 85LO30 bacteriocin-producing operon was determined. The first two open reading frames of the operon are necessary to get bacteriocin expression in L. lactis IL1403R.

Twenty-four bacteriophages of Lactococcus lactis subsp. lactis and L. lactis subsp. cremoris were classified. Two groups of bacteriophages morphologically defined as prolate or isometric types by electron microscopy were examined for their genome sizes, protein patterns and DNA homologies. These criteria showed that prolate phages are quite homogeneous. In contrast, isometric phages exhibit more differences, particularly in particle sizes and protein compositions. Analysis of DNA hybridizations confirmed that prolate phages can be grouped together as can be isometric phages but for one exception, phage I52. These two families were clearly defined. The unique phage which does not fit in either group probably belongs to a third one which is much less represented. No obvious relationships between these criteria and the lytic spectra were detected. Evidence of the presence of cohesive ends in phage genomes is also presented in this study. A more detailed analysis performed on one member of the prolate group revealed 3' protruding ends made up of around 13 nucleotides on complementary single strands.

A cDNA encoding a lectin secreted by the oviduct of Pleurodeles waltl has been isolated and sequenced. The cDNA was identified by comparing the N-terminal amino-acid sequence of the purified P. waltl lectin polypeptides with the amino-acid sequence deduced from the cDNA. The two chains of the mature protein can be encoded within a unique mRNA. Two mRNA were also found in the oviduct extracts. However, they probably result from differential polyadenylation events. The mRNA are strictly localized in the anterior part of the oviduct and increase after estradiol stimulation, two characteristics which have been previously demonstrated for the protein. P. waltl is known to possess a very high DNA content (approximately 2 x 10(10) bp) but the aforementioned results and Southern-blot experiments suggest a unique or at least a very low gene-copy number for this protein. The amino-acid sequence of the P. waltl lectin deduced from the cDNA sequence shows similarities with the C-type carbohydrate-recognition domains of animal lectins as defined by Drickamer [Drickamer, K. (1988) J. Biol. Chem. 263, 9557-9560]. Although it is regulated by estradiol, the P. waltl lectin amino-acid sequence shows a higher similarity with animal lectins involved in the defence of the organism than with those involved in reproduction and development.

The complete coding region of the estrogen receptor gene was isolated from a rainbow trout genomic library. This gene is divided into ten exons spanning at least 30 kb of genomic DNA. With two exceptions, intron positions are identical to those of the human estrogen receptor gene. The 5' end of the gene, including 1.7 kb of the promoter region, was sequenced. This region exhibits several putative regulatory elements. Localization of a potential estrogen responsive element to the first exon suggests that this gene is autoregulated. This 5' end region was also shown to be able to drive the expression of a CAT reporter gene in Xenopus laevis oocytes.

1H-NMR techniques have been used to study the metal binding properties of a synthetic peptide of 15 amino acids corresponding to a highly conserved domain of Pleurodeles lectin. The addition of lanthanum chloride or praseodymium chloride in a peptide solution induces some conformational changes as displayed by several concerted variations of peptide resonances. The Ln3+ concentration dependence of the chemical shifts was used to calculate the Ln3+ binding constants. The dissociation constants of 95 microM and 280 microM were found for La3+ and Pr3+, respectively.

Provirus ev21 was found in both K- and k(+)-feathering Rhode Island Red commercial layers. Probe EV21-int revealed the presence of two distinct but similar regions, US (unoccupied site) and OS (occupied site). Restriction analysis showed that these regions had at least 19 kb structural homology but were distinguishable by ev21 proviral sequences, OS, and possibly three polymorphics US. The loci OS and US were both located on Chromosome Z. The k(+)-feathering birds were found to have only one site (either OS or US) per individual Z chromosome, whereas K-feathering birds had at least one Z chromosome with both regions in cis configuration. It has been possible to show that the reversion to the k(+)-feathering phenotype is accompanied by the loss of either a US or OS region that disrupts the cis configuration in K-feathering birds.

Lactococcin, a bacteriocin produced by Lactococcus lactis subsp. lactis ADRIA 85LO30, was purified as a 2.3-2.4 kDa peptide. Six non-bacteriocin-producing (Bac-) and non-immune (Imm-) strains were isolated after curing experiments. These strains had in common the loss or modification of two plasmids: pOS4 (32 kb) and pOS5 (70 kb). By comparing pOS5 and several modified plasmids, a DNA region from pOS5 of about 10 kb, which was necessary for wild-type bacteriocin production and immunity, was identified.

Four component lines of a commercial broiler stock were analyzed by restriction fragment length polymorphism (RFLP) analysis, for the presence of endogenous avian leukosis virus loci. The resulting RFLP patterns of endogenous virus (ev) loci for the four strains were further characterized in terms of number of loci per animal and frequency. Loci were also analyzed within limits for major structural alterations and deletions to discern whether certain loci were similar to previously identified loci in White Leghorn layer birds. The ev RFLP patterns for these broiler lines were found to be highly complex and contained many loci unreported in White Leghorn layer birds.

An electrophoretic method for separating well-defined large DNA fragments from higher and lower molecular weight molecules is described. It combines in a first dimension either a contour-clamped homogeneous electric field or an orthogonal field alternation gel electrophoresis technique followed by a perpendicular field inversion gel electrophoresis (FIGE) in a second dimension. A complex migration curve after the FIGE run is obtained depending on the applied pulse time, the forward/reverse ratio being kept constant at 3. However, a part of the curve appears as a straight line where the migration is inversely proportional to the molecular weight of the DNA fragments. In this zone, DNA molecules are particularly well separated from other fragments. When the forward pulse time increases, this part is displaced toward the higher molecular weights. Moreover a simple relationship between the middle part of the straight line and the forward pulse time has been established.

The Eco RI fragment "b" of chicken DNA (Breathnach, Mandel and Chambon, 1977), which contains the sequences coding for the 5' quarter of ovalbumin mRNA (ov mRNA), has been isolated by molecular cloning using a "shotgun" approach. Electron microscopy and restriction enzyme analysis have revealed that the sequences coding for the 5' quarter (approximately 500 nucleotides) of ov mRNA are split into four regions separated by three intervening sequences. The cloning procedure seems to be reliable, since the restriction enzyme pattern of the cloned Econ RI fragment "b" is similar to that of the corresponding chromosomal DNA fragment. There is no evidence supporting the existence of a 150-200 nucleotide long sequence at the 5' end of the ov mRNA similar to the "leader" sequences found at the 5' end of some adenovirus and SV40 mRNAs.