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We present data suggesting that corticosteroid-binding globulin (CBG) may be the causal gene of a previously identified quantitative trait locus (QTL) associated with cortisol levels, fat, and muscle content in a pig intercross. Because Cbg in human and mouse maps in the region orthologous to the pig region containing this QTL, we considered Cbg as an interesting positional candidate gene because CBG plays a major role in cortisol bioavailability. Firstly, we cloned pig Cbg from a bacterial artificial chromosome library and showed by fluorescent in situ hybridization and radiation hybrid mapping that it maps on 7q26 at the peak of the QTL interval. Secondly, we detected in a subset of the pig intercross progeny a highly significant genetic linkage between CBG plasma binding capacity values and the chromosome 7 markers flanking the cortisol-associated QTL. In this population, CBG capacity is correlated positively to fat and negatively to muscle content. Thirdly, CBG capacity was three times higher in Meishan compared with Large White parental breeds and a 7-fold difference was found in Cbg mRNA expression between the two breeds. Overall, the data accumulated in this study point to Cbg gene as a key regulator of cortisol levels and obesity susceptibility.

The contribution of in vitro methods and molecular tools to pear genetic studies and breeding has advanced considerably during the last decade. Haploidization via in situ parthenogenesis induced by irradiated pollen and in vitro rescue of the haploid plantlets has been developed for pear. Techniques of adventitious bud regeneration from in vitro leaves have been developed for several genotypes of European and Asian pear and for quince. So far, applications of these techniques for the induction of somaclonal variation have been very limited. Increased tolerance to fire blight has been obtained in somaclonal variants of the cultivar Durondeau, and selection of quince rootstocks tolerant to iron deficiency has been achieved with adventious regeneration followed by in vitro selection. A major evolution of pear biotechnology is the recent development of Agrobacterium-mediated transformation. Since the first report in 1996, several teams have succeeded to transform about ten different genotypes of varieties and rootstocks. Projects have started in several countries to express various trangenes in pear (lytic peptides, rolC and SAMase genes). Molecular markers developed on pear now include isozymes, RAPDs and RFLPs. They have been used mostly for varietal identification of european as well as asian pears. Markers have been developed recently for a few genes of interest, such as a black-spot resistance gene and an ACC synthase gene. But no genetic map of pear is yet available. Finally, about 20 genes have already been cloned in pear, most of them from P. pyrifolia. They are mostly involved in fruit ripening or quality and self-incompatibility.

Expression of the adrenocorticotropin receptor (MC2-R) is restricted to adrenocortical cells and is up-regulated by both adrenocorticotropin and angiotensin II through the activation of protein kinase A and protein kinase C pathways, respectively. After cloning of the promoter region of the human MC2-R gene (hMC2-R), we have shown that cyclic AMP-induced regulation of transcriptional activity of the gene is achieved through two SF1 binding elements located in the proximal promoter. On the other hand, regulation by angiotensin II partly involved two AP1 binding sites. Using different primary cell cultures, we have also been able to delineate a region inside the promoter which is responsible for part of the tissue-specific expression of the gene.

The disruption of eight novel genes was realized in two genetic backgrounds. Among these open reading frames, NO333, NO348 and NO364 presented homologies with other proteins of yeast or other organisms, whereas NO320, NO325, NO339, NO384 and NO388 showed no similarity with any protein. Tetrad analysis of heterozygous deletant strains revealed that NO348, NO364 and NO388 are essential genes for vegetative growth, whereas NO320, NO325, NO333, NO339 and NO384 are non-essential. Basic phenotypic analyses of the non-lethal deletant strains as suggested in the six-pack B0 programme did not reveal any significant differences between parental and mutant strains. © 1998 John Wiley & Sons, Ltd.

The genomic region encoding the type IIS restriction-modification (R-M) system HphI (enzymes recognizing the asymmetric sequence 5'-GGTGA-3'/5'-TCACC-3') from Haemophilus parahaemolyticus were cloned into Escherichia coli and sequenced. Sequence analysis of the R-M HphI system revealed three adjacent genes aligned in the same orientation: a cytosine 5 methyltransferase (gene hphIMC), an adenine N6 methyltransferase (hphIMA) and the HphI restriction endonuclease (gene hphIR). Either methyltransferase is capable of protecting plasmid DNA in vivo against the action of the cognate restriction endonuclease. hphIMA methylation renders plasmid DNA resistant to R.Hindill at overlapping sites, suggesting that the adenine methyltransferase modifies the 3'-terminal A residue on the GGTGA strand. Strong homology was found between the N-terminal part of the m6A methyltransferasease and an unidentified reading frame interrupted by an incomplete gaIE gene of Neisseria meningitidis. The HphI R-M genes are flanked by a copy of a 56 bp direct nucleotide repeat on each side. Similar sequences have also been identified in the non-coding regions of H.influenzae Rd DNA. Possible involvement of the repeat sequences in the mobility of the HphI R-M system is discussed.

The cDNAs of hypodermins, enzymes secreted by the larvae of the parasitic fly Hypoderma lineatum , were sequenced. Four cDNA clones were isolated, one encoding hypodermin A (HA), one encoding hypodermin C (HC), and the two others encoding proteins related to hypodermin B (HB). The amino acid sequences deduced from the nucleotide sequences confirmed that these enzymes are serine proteases. HA and one of the HB proteins had potential N -linked glycosylation sites. Analysis of hypodermin protein, RNA and DNA at different larval stages indicated that protein overexpression is regulated transcriptionally for HA and HB, and by transcriptional and DNA amplification for HC.

The F17b fimbriae encoded by the transmissible virulence plasmid Vir, also coding for cytotoxic necrotizing factor type 2, were characterized. A 5.7-kb region of Vir mediates in vitro N-acetylglucosamine-sensitive adhesion to calf intestinal villi. Sequence analysis revealed that this region codes for a structural subunit and an adhesin closely related to the F17-A and F17-G proteins encoded by the F17 fimbrial gene cluster. The F17b-A gene presents an open reading frame of 540 bp encoding a polypeptide of 180 amino acids with a putative signal peptide of 21 residues. The mature protein shows an identity of 74% with the F17-A structural subunit. This 20-kDa protein is recognized by antiserum directed against F17 fimbriae. The F17b-G gene shows an open reading frame of 1,029 bp encoding a polypeptide of 343 amino acids with a putative signal peptide of 22 residues. The F17b-G polypeptide exhibits 95% identity with the F17-G adhesin. The functional homology of the gene products was further confirmed by demonstrating that mutants in the F17-A gene can be complemented by the F17b-A gene and vice versa. These results prove that fimbriae belonging to the F17 family are also found on pathogenic Escherichia coli strains other than enterotoxigenic isolates producing heat-labile or heat-stable enterotoxin.

We previously described the gene structure of murine cathepsin B. OUr results suggested that the 5′-untranslated region (leader) is interrupted by a large intron. The second exon (exon 2) contains the translation initiation site. To characterize the leader region, a rapid amplification of cDNA ends (RACE) procedure was developed. The PCR products were directly cloned and sequenced. Nucleotide sequence analyses revealed three different 5′-cDNA ends, suggesting the existence of three different leader regions. In addition to the leader (LA) previously characterized, we now describe two other 5′-untranslated regions, LB and LC. Leader LB is located 2.3 kb upstream exon-2, and leader LC corresponds to the 3′-end of the first intron and is thus contiguous to exon-2. Our results suggest for murine cathepsin B gene the presence of multiple promoters, and possibly the expression of multiple mRNAs differing in their leader region.

Resistance to coffee leaf rust is conferred by S(H)3, a major dominant gene that has been introgressed from a wild coffee species Coffea liberica (genome L) into the allotetraploid cultivated species, Coffea arabica (genome (CEa)-E-a). As the first step toward the map-based cloning of the S(H)3 gene, using a bacterial artificial chromosome (BAC) library, we describe the construction of a physical map in C. arabica spanning the resistance locus. This physical map consists in two homeologous BAC-contigs of 1,170 and 1,208 kb corresponding to the subgenomes C-a and E-a, respectively. Genetic analysis was performed using a single nucleotide polymorphism detection assay based on Sanger sequencing of amplicons. The C. liberica-derived chromosome segment that carries the S(H)3 resistance gene appeared to be introgressed on the sub-genome C-a. The position of the S(H)3 locus was delimited within an interval of 550 kb on the physical map. In addition, our results indicated a sixfold reduction in recombination frequency in the introgressed S(H)3 region compared to the orthologous region in Coffea canephora.

The genes of Lactococcus lactis subsp. lactis involved in histidine biosynthesis were cloned and characterized by complementation of Escherichia coli and Bacillus subtilis mutants and DNA sequencing. Complementation of E. coli hisA, hisB, hisC, hisD, hisF, hisG, and hisIE genes and the B. subtilis hisH gene (the E. coli hisC equivalent) allowed localization of the corresponding lactococcal genes. Nucleotide sequence analysis of the 11.5-kb lactococcal region revealed 14 open reading frames (ORFs), 12 of which might form an operon. The putative operon includes eight ORFs which encode proteins homologous to enzymes involved in histidine biosynthesis. The operon also contains (i) an ORF encoding a protein homologous to the histidyl-tRNA synthetases but lacking a motif implicated in synthetase activity, which suggests that it has a role different from tRNA aminoacylation, and (ii) an ORF encoding a protein that is homologous to the 3'-aminoglycoside phosphotransferases but does not confer antibiotic resistance. The remaining ORFs specify products which have no homology with proteins in the EMBL and GenBank data bases.

CS31A is a plasmid-encoded K88-related fimbrial antigen. A Sau3AI library was constructed from p31A, a 180 kb CS31A encoding plasmid, in the pSUP202 vector. Bacterial recombinant clones expressing CS31A were isolated. A 8.5 kb EcoRI-HindIII DNA fragment from one of them was subcloned in pBR322 and pHSG575 vectors, leading to pAG315 and pEH524 recombinant plasmids respectively. Escherichia coli harboring pAG315 or pEH524 expressed CS31A fimbrial antigens on their cell surface. Analysis of these plasmids in minicells showed that at least seven mature polypeptides were encoded by the EcoRI-HindIII DNA fragment, with apparent molecular masses of 76000, 54000, 30000, 29000, 28000, 15500 and 13500 daltons respectively. The genetic organization of the CS31A gene cluster was determined and showed to be similar to that of the K88 operon. The nucleotide sequence homology between CS31A and K88 determinants was investigated by Southern blot hybridization at high stringency. This indicated that extensive nucleotide sequence homology exists throughout both gene clusters except for the subunit structural genes.

We have cloned an infectious genome of the Junonia coenia densonucleosis virus (JcDNV) into the bacterial plasmid pBR322. The viral genome could be rescued from the recombinant plasmid pBRJ by transfection of pBRJ DNA to sensitive Spodoptera littoralis larvae. pBRJ DNA produced a typical viral infection and a comparable percentage of larvae became infected following inoculation of equivalent amounts of purified virion DNA or cloned viral DNA. Virions extracted from transfected larvae were indistinguishable from wild-type (wt) virions with regard to their biophysical and biological properties. In particular, rescued virions were as infectious as wt virions and showed identical restriction profiles of their genome. In contrast, subcloning of JcDNV DNA deleted at both extremities of a sequence of ca 250 or ca 100 bp resulted in the inability of the recombinant plasmids to initiate a viral infection. These data suggest that, as for vertebrate parvoviruses, the inverted terminal repeats display essential functions in the rescue process and replicative cycle of densoviruses. This is the first report of the molecular cloning of the infectious genome from an insect parvovirus, and more generally from an invertebrate virus. pBRJ should provide an efficient tool to further define the organization of the JcDNV genome and compare it to other parvoviruses.

The mRNA transcribed from the N gene of viral haemorrhagic septicaemia virus (VHSV) of salmonids has been cloned in Escherichia coli and expressed. Fusion proteins were recognized by monoclonal antibody directed against the N protein from the viral particle. A 1212 bp long open reading frame (ORF) coding for 404 amino acids with a calculated M r of 44590 was deduced from the nucleotide sequence. The ORF was preceded by a 93 bp segment including in position 42 the AACAC pentanucleotide which is presumed to be the start signal for transcription by analogy with other rhabdoviral mRNAs. The upstream 41 bp region could correspond to the covalently linked positive polarity leader RNA as also found on the N mRNA from infectious haematopoietic necrosis virus (IHNV). This may be a characteristic of fish lyssaviruses. The AAACC sequence, which is part of the leader, was not found. Amino acids 44 to 359 from IHNV and 45 to 360 from VHSV are 45.3% homologous. A strong homology which could reflect functional importance was also found for potential phosphorylation sites and hydrophobic peaks despite the fact that the two viruses evolved on different continents.

Viral haemorrhagic septicaemia virus (VHSV), a rhabdovirus, is a major threat for continental European trout fish farming. The development of a recombinant subunit vaccine could solve that problem. The neutralizing epitopes are located on the glycoprotein or G protein, the surface antigen. The G protein has a molecular weight of 65 kDa, reduced to 55 kDa by deglycosylation. cDNA was synthetized from mRNA of VHS virus infected cells, and cloned in E. coli. The viral cDNA was recognized by positive hybridization with a labelled probe made from infected cell RNA, and negative hybridization with labelled cDNA made from cellular RNA. The Northern blot hybridization with different clones on VHS infected cell RNA revealed two VHS mRNA whose lengths, 2.0 and 1.5 kb, were compatible with the mRNA length for G and N proteins respectively. This mRNA must contain about 400 bp of untranslated sequence.

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A cathepsin L protease full-length cDNA (named Mi-cpl-1 ) was characterised from infective second-stage juveniles of the root-knot nematode Meloidogyne incognita by SL1-primed PCR and 3′ rapid amplification of cDNA ends (3′-RACE). A single open reading frame was identified, encoding a putative 383-amino acid protein predicted to contain a putative N-terminal short secretion signal peptide, which suggests that Mi-CPL-1 should function as an extracellular protein. The putative mature enzyme included several conserved amino acid residues/motives, among which the typical catalytic triad of the active site. In situ mRNA hybridization analyses showed that transcripts of Mi-cpl-1 accumulated specifically in the intestinal cells of specimens, which suggests that the protein could be involved in the digestive processes of the nematode. However, expression of Mi-cpl-1 was shown to occur exclusively in the developmental stages which are in close interaction with the root tissues (i.e. second-stage juveniles and females). This may indicate that some function of the cathepsin L cysteine protease in M. incognita is more directly related to the parasitic aspects of the plant-nematode relationship, e.g. pathogenicity and/or evasion of primary host plant defence systems.

We have previously characterized an 18-19 kDa cationic protein, SOB3, that was detected in the epididymis and localized within the acrosome and on the neck region of human spermatozoa. We suggested that it is involved in secondary sperm binding to the zona pellucida. The present study describes its purification to homogeneity by preparative electrophoresis and non-equilibrium pH gradient electrophoresis. Degenerate primers deduced from microsequencing were used to amplify a specific fragment from human epididymal RNA by reverse transcription-polymerase chain reaction (RT-PCR). This 164 bp fragment was extended by 5' and 3'-RACE to obtain the 548 bp full length cDNA. The open reading frame encodes a 170 amino acid protein. SOB3 is a single copy gene. It is 98% identical to prepro-FALL39 and 100% identical to CAP18, two human genes which were initially identified by screening a human bone marrow (lambda)gt11 library, and which encode an antimicrobial protein. Northern blots of human tissues revealed a 1 kb transcript in corpus and cauda epididymis only, while RT-PCR showed presence of the mRNA in the three epididymal regions and also in round spermatids. The above results suggest that SOB3 has two roles in sperm protection and fertilization, depending on its dual origin and final sperm localization.

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A cDNA clone encoding for the glycoprotein of the viral haemorrhagic scepticaemia virus, a fish rhabdovirus, has been sequenced. The cDNA was 2035 bp long and contained two open reading frames (ORF). A 1523 bp ORF corresponded to the glycoprotein and was adjacent, on its 5' side, to an incomplete 372 bp ORF. Although the protein encoded by this ORF displayed no similarity with other rhabdovirus proteins, it was supposed that the cDNA had been reverse-transcribed from a readthrough mRNA encoding successively for the M2 and the G proteins.

International audience; The ACO3 gene, which encodes one of the acyl-CoA oxidase isoenzymes, was isolated from the alkane-utilizing yeast Yarrowia lipolytica as a 10 kb genomic fragment. It was sequenced and found to encode a 701-amino acid protein very similar to other ACOs, 67.5% identical to Y. lipolytica Aco1p and about 40% identical to S. cerevisiae Pox1p. Haploid strains with a disrupted allele were able to grow on fatty acids. The levels of acyl-CoA oxidase activity in the ACO3 deleted strain, in an ACO1 deleted strain and in the wild-type strain, suggested that ACO3 encodes a short chain acyl-CoA oxidase isoenzyme. This narrow substrate spectrum was confirmed by expression of Aco3p in E. coli.

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A Clostridium difficile isolate was found to produce an actin-specific ADP-ribosyltransferase (CDT) homologous to the enzymatic components of Clostridium perfringens iota toxin and Clostridium spiroforme toxin (M. R. Popoff, E. J. Rubin, D. M. Gill, and P. Boquet, Infect. Immun. 56:2299-2306, 1988). The CDT locus from C. difficile CD196 was cloned and sequenced. It contained two genes (cdtA and cdtB) which display organizations and sequences similar to those of the iota toxin gene. The deduced enzymatic (CDTa) and binding (CDTb) components have 81 and 84% identity, respectively, with the corresponding components of iota toxin. CDTa and CDTb induced actin cytoskeleton alterations similar to those caused by other clostridial binary toxins. The lower level of production of binary toxin by CD196 than of iota toxin by C. perfringens was related to a lower transcript level, possibly due to a promoter region different from that of iota toxin genes. The cdtA and cdtB genes have been detected in 3 of 24 clinical isolates examined, and cdtB alone was found in 2 additional strains. One strain (in addition to CD196) was shown by Western blotting to produce CDTa and CDTb. These results indicate that some C. difficile strains synthesize a binary toxin that could be an additional virulence factor.

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The 6.8-kb rDNA intergenic spacer region of F. excelsior was isolated from a CsCl/actinomycin-D gradient and cloned into pUC18 for further characterization. We observed the presence of subrepeats delimited by HaeIII enzyme sites. These subrepeats were sub-cloned and 11 clones were sequenced. These corresponded to subrepeated elements of either 32 bp or 41 bp that shared a 23-bp common sequence in the 5' end. Within each family of subrepeats, the percentage of common nucleotides was 84.4% for the 5 32-bp subrepeats and 67.4% for the 640-bp subrepeats. Non-repeated HaeIII fragments of 450 bp and 650 bp were also sub-cloned. To compare homology at the IGS region between the rDNA spacers of F. excelsior and the three related species (F. oxyphylla, F. americana, F. ornus), we conducted Southern hybridization analyses using each member of the 32-bp and 40-bp subrepeat families and the unique 450-bp and 650-bp fragments as probes. These analyses indicated that (1) the American ash is more genetically distant from the other three species that the latter are from each other and (2) F. oxyphylla and F. excelsior are more closely related to each other than to F. ornus.

The gene coding for a new class of proteins rich in glycine and proline (GPRP) was cloned in Arabidopsis thaliana. In the protein sequence, five amino acids-glycine, proline, alanine, tyrosine and histidine-account for 79.4% of the total composition. The protein has two different glycine-rich domains interrupted by a hydrophobic segment having a high probability of helix formation. The protein synthesized in vitro interacts with microsomes possibly through the hydrophobic domain. The gene in Arabidopsis has two introns, one in the coding region and the other one in the 5′ non-coding region. The later one is 778 bp long. Homologous sequences are found in carrot, tomato and tobacco. GPRP mRNA is found in the different organs of the plant analyzed except in mature seeds and anthers, and mostly in epidermal and vascular tissues. Possible hypotheses about the function of GPRP are discussed., I.M. was supported by a fellowship from European Community (BRIDGE Programme). The work was supported by grant 094-648 to P.P. from Plant Nacional de Investigacion Cientifica y Tecnica. The present work has been carried out within the framework of the Laboratoire Europen Associ CNRS-CSIC de Biologie Molrculaire Vegetale.

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A highly significant genetic association has been found between some alleles of the swine Major Histocompatibility Complex SLA (Swine Leukocyte Antigen genetic complex) and the cytosolic malic enzymatic activity level in muscles. The aim of this study was to find out whether this genetic association was due to a close linkage of the SLA region and the gene coding for the enzyme. Since no swine cytosolic malic enzyme sequence (ME1) was available, we isolated several overlapping fragments that spanned the almost entire malic enzyme transcript both by screening of a swine cDNA library and by RT-PCR. The results indicated the existence of two transcripts of 2. 0 and 3.1 kb, which probably correspond to two alternative forms of one gene. The sequence of the transcript was highly similar to the other published mammalian cytosolic NADP+-dependent malic enzyme cDNA, especially within the four functional domains. Two major bands at 3.7 and 2.4 kb were detected on Northern blots containing the RNA from 25 tissues from fetuses and adult pigs. A high expression level was found in the adrenal gland, muscle, liver, and peripheral nerves. The analysis of malic enzyme RFLPs in five SLA informative families revealed an independent segregation of the ME1 gene from the SLA region. In situ hybridization results localized the cytosolic malic enzyme on the swine Chromosome (Chr) 1p1.2, except that the association between SLA and the malic enzyme activity level was due to a physical genetic linkage. Thus, the mechanisms underlying this association remain to be elucidated.

Diacetyl is an important food flavor compound produced by certain strains of citrate-metabolizing lactic acid bacteria. Citrate is converted to pyruvate, from which diacetyl is produced via intermediate alpha-acetolactate. This paper reports the cloning and analysis of the gene (aldB) encoding alpha-acetolactate decarboxylase from Lactococcus lactis MG1363. Deletion of the MG1363 chromosomal aldB gene was achieved by double crossover homologous recombination. The mutant strain was found to produce diacetyl; alpha-acetolactate decarboxylase activity was eliminated. Overexpression of the cloned ilvBN genes (encoding an alpha-acetolactate synthase) in the aldB deletion strain produced even higher levels of alpha-acetolactate, acetoin, and diacetyl.

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Most organisms appear to have a molybdenum cofactor consisting of a complex of molybdenum and a pterin derivative. Very little is known about molybdenum cofactor biosynthesis in plants or other eukaryotes, because the instability of the cofactor and its precursors makes it difficult to analyze this pathway. We have isolated two cDNA clones from the higher plant Arabidopsis thaliana encoding genes involved in early steps of molybdenum cofactor biosynthesis. The cDNAs were obtained by functional complementation of two Escherichia coli mutants deficient in single steps of molybdenum cofactor biosynthesis. The two cDNAs, designated Cnx2 and Cnx3, encode proteins of 43 and 30 kDa, respectively. They have significant identity to the E. coli genes, moaA and moaC, involved in molybdenum cofactor biosynthesis. Both genes have N-terminal extensions that resemble targeting signals for the chloroplasts or the mitochondria. Import studies with the translated proteins and purified mitochondria and chloroplasts did not show import of these proteins to either of these organelles. Northern analysis show that Cnx2 is expressed in all organs and strongest in roots. Cnx3 is not expressed in abundant levels in any tissue but roots. For both genes there is no detectable difference in the expression level from plants grown with nitrate or with ammonium. The Cnx2 gene has been mapped to chromosome II. Southern analysis suggests that both genes exist as single copies in the genome.

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This chapter describes two vectors used to introduce and express foreign genes in Spiroplasma citri. One is the replicative form of spiroplasmavirus SpV1 of S. citri, and the other is a plasmid whose replication is mediated by the chromosomal replication origin (oriC) of S. citri. This chapter also describes a protocol for efficient electrotransformation of S. citri. The use of recombinant DNA technology to study expression of mollicute genes by cloning in Escherichia coli has been limited because many mollicutes use the codon UGA to encode for tryptophan. Cloning in a spiroplasma host overcome these difficulties and help to investigate gene expression and the function of the polypeptides they encode. Transfer of genetic material into mollicutes also provides a way to complement mutations. Electroporation refers to the process of subjecting living cells to a rapidly changing, high-strength electric field, thereby producing transient pores in their outer membranes. This process has been used to introduce DNA into eukaryotic and prokaryotic cells.

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cDNA clones encoding the 65-kDa (PR65) and 55-kDa (PR55) regulatory subunits of protein phosphatase 2A from Xenopus laevis were isolated by homology screening with the corresponding human cDNAs, and used to analyze the developmental expression patterns of these genes. The PR65 subunit was found to be encoded by two genes, termed XPR65 alpha and XPR65 beta. The open reading frames of the alpha and beta cDNAs both span 1767 bp, and predict proteins of 64.4 kDa and 65.3 kDa, respectively, that are 87% identical. The predicted amino acid sequence of XPR65 alpha showed 95% and 84% identity with human PR65 alpha and PR65 beta proteins, respectively, whereas the identity of XPR65 beta with the same proteins was 87% and 86.5%, respectively. Only one type of Xenopus PR55 (XPR55) was isolated that showed 93% and 84% similarity to human PR55 alpha and PR55 beta, respectively. Analysis of the N-terminal region of XPR55 with the same regions of human PR55 alpha and PR55 beta, indicates that the XPR55 is the Xenopus homolog of the human PR55 alpha isoform. Despite the overall similarity with PR55 from other species, XPR55 has an N-terminal extention of at least 24 amino acids. In the ovary, a transcript of 2.8 kb, encoding the XPR65 beta, was predominantly expressed and these XPR65 beta mRNAs are present at a constant level during oogenesis until late embryogenesis. Expression of the 2.4-kb XPR65 alpha was low until the larval stage, then dramatically increased. In all adult tissues except ovary, the 2.4-kb alpha-specific mRNA was more abundant than the 2.8-kb beta transcript. Two transcripts of 2.4 kb and 2.5 kb, encoding the XPR55 subunit, were detected at a constant level throughout Xenopus oogenesis and during embryogenesis. Both transcripts were also expressed at similar levels in all adult tissues, but in a tissue-specific manner. Analysis of the XPR55 and XPR65 proteins using antibodies to recombinant proteins revealed that the overall levels of the two proteins were constant, in good agreement with mRNA data.

To obtain large amounts of the X-prolyl dipeptidyl aminopeptidase from Lactococcus lactis subsp lactis (PepX, E.C. 3.4.14.5), PepX was purified from a commercial L. lactis cell extract. The enzyme was purified in only three steps and the last one was performed by HPLC on a C4 reverse-phase column using acetonitrile as an eluent. Despite its high molecular mass (175 kDa), the enzyme was recovered with a good activity yield (75%). Advantages and drawbacks of this technique compared to the classical ones are discussed. The stability of the enzyme in aqueous solutions and in the presence of 10 water-miscible solvents was also investigated. PepX was found to be stabilized by dimethyl sulfoxide, triglyme, and glycerol.

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