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The soil-borne oomycete Phytophthora cinnamomi is a highly destructive Phytophthora species associated with the decline of forest. This pathogen secretes a novel class of necrosis-inducing proteins known as Nep1-like proteins (NLPs). In this work, we report the sequencing and molecular characterization of one of these proteins, more specifically the necrosis-inducing Phytophthora protein 1 (NPP1). The ORF of the npp1 gene (EMBL database AM403130) has 768 bp encoding a putative peptide of 256 amino acids with a molecular weight of approximately 25 kD. In order to understand its function, in vitro gene expression was studied during growth in different carbon sources (glucose, cellulose, and sawdust), and at different times of infection, in vivo by RT-qPCR. The highest expression of the npp1 gene occurred in glucose medium followed by sawdust. In vivo infection of Castanea sativa roots with P. cinnamomi revealed a decrease in npp1 expression from 12 to 24 h; at 36 h its expression increased suggesting the existence of a complex mechanism of defense/attack interaction between the pathogen and the host. Expression of recombinant npp1 gene was achieved in Pichia pastoris and assessed by SDS-PAGE analysis of the protein secreted into the culture supernatant, revealing the presence of the NPP1 protein., Project COMBATINTA/SP2.P11/02 Interreg IIIA—Cross-Border Cooperation Spain-Portugal, financed by The European Regional Development Fund and by national funding from the Portuguese Ministério da Ciência e do Ensino Superior (MCES) (PTDC/AGR-AAM/67628/2006)., info:eu-repo/semantics/publishedVersion

[EN] Background: Phlomis plants are a source of biological active substances with potential applications in the control of phytopathogens. Phlomis purpurea (Lamiaceae) is autochthonous of southern Iberian Peninsula and Morocco and was found to be resistant to Phytophthora cinnamomi. Phlomis purpurea has revealed antagonistic effect in the rhizosphere of Quercus suber and Q. ilex against P. cinnamomi. Phlomis purpurea roots produce bioactive compounds exhibiting antitumor and anti-Phytophthora activities with potential to protect susceptible plants. Although these important capacities of P. purpurea have been demonstrated, there is no transcriptomic or genomic information available in public databases that could bring insights on the genes underlying this anti-oomycete activity. Results: Using Illumina technology we obtained a de novo assembly of P. purpurea transcriptome and differential transcript abundance to identify putative defence related genes in challenged versus non-challenged plants. A total of 1,272,600,000 reads from 18 cDNA libraries were merged and assembled into 215,739 transcript contigs. BLASTX alignment to Nr NCBI database identified 124,386 unique annotated transcripts (57.7%) with significant hits. Functional annotation identified 83,550 out of 124,386 unique transcripts, which were mapped to 141 pathways. 39% of unigenes were assigned GO terms. Their functions cover biological processes, cellular component and molecular functions. Genes associated with response to stimuli, cellular and primary metabolic processes, catalytic and transporter functions were among those identified. Differential transcript abundance analysis using DESeq revealed significant differences among libraries depending on post-challenge times. Comparative cyto-histological studies of P. purpurea roots challenged with P. cinnamomi zoospores and controls revealed specific morphological features (exodermal strips and epi-cuticular layer), that may provide a constitutive efficient barrier against pathogen penetration. Genes involved in cutin biosynthesis and in exodermal Casparian strips formation were up-regulated. Conclusions: The de novo assembly of transcriptome using short reads for a non-model plant, P. purpurea, revealed many unique transcripts useful for further gene expression, biological function, genomics and functional genomics studies. The data presented suggest a combination of a constitutive resistance and an increased transcriptional response from P. purpurea when challenged with the pathogen. This knowledge opens new perspectives for the understanding of defence responses underlying pathogenic oomycete/plant interaction upon challenge with P. cinnamomi., This work was supported by the project PTDC/AGR-CFL/100217/2008 and the grant SFRH/BD/66016/2009, both funded by Fundacao para a Ciencia e Tecnologia (FCT). The APC charge was supported by the project UID/BIA/04325/2013 - MEDTBIO (FCT). The COST Office and the European Council provided financial support to the Short Term Scientific Mission (COST-STSM-FP0801-10084).

The scope of this study was to identify sequence polymorphisms in the β -1,4-galactosyltransferase-I gene (B4GALT1), the gene which encodes the catalytic part of lactose synthase enzyme. Exons of the gene were scanned for the presence of allelic variation among a sample of 400 Iranian Holsteins, using the single strand conformation polymorphism technique followed by sequencing. Nine polymorphic nucleotide sites and 16 different genotypes of B4GALT1 were identified. We concluded therefore that the β-1,4-galactosyltransferase-I gene was polymorphic in Holsteins. Considering the biological roles of the B4GALT1 gene, we suggest that further studies are critical to investigate the biological effects of the SNPs reported in this study.

The differential expression of four Phytophthora cinnamomi elicitin genes was analysed by Real Time RT- PCR. In in vitro cultures, the a-cinnamomin gene showed the highest level of expression, the b-cinna- momin gene (b-cin) was the most inducible, and the HAE transcripts were in low abundance. Tran- scription of all the elicitins was active during the active growth of the pathogen when infecting cork oak (Quercus suber) roots, and as host colonization progressed, the level of b-cin expression fell, while that of a-cin rose. In an antisense transgenic strain, the silencing of b-cin also negatively affected the expression of other elicitin genes in the cluster. The reduction in planta growth of the b-cin knock-out is related to the altered pattern of elicitin gene expression, supporting the idea that one of the functions of elicitins is related, directly or indirectly, with pathogenesis. 2009 Published by Elsevier Ltd.

cDNA-AFLP methodology was used to gain insight into gene fragments differentially present in the mRNA profiles of Quercus suber roots infected with zoospores of Phytophthora cinnamomi at different post challenge time points. Fifty-three transcript-derived fragments (TDFs) were identified and sequenced. Six candidate genes were selected based on their expression patterns and homology to genes known to play a role in defence. They encode a cinnamyl alcohol dehydrogenase2 (QsCAD2), a protein disulphide isomerase (QsPDI), a CC-NBS-LRR resistance protein (QsRPc), a thaumatin-like protein (QsTLP), a chitinase (QsCHI) and a 1,3-β-glucanase (QsGlu). Evaluation of the expression of these genes by quantitative polymerase chain reaction (qPCR) revealed that transcript levels of QsRPc, QsCHI, QsCAD2 and QsPDI increased during the first 24 h post-inoculation, while those of thaumatin-like protein decreased. No differential expression was observed for 1,3-β-glucanase (QsGlu). Four candidate reference genes, polymerase II (QsRPII), eukaryotic translation initiation factor 5A (QsEIF-5A), β-tubulin (QsTUB) and a medium subunit family protein of clathrin adaptor complexes (QsCACs) were assessed to determine the most stable internal references for qRT-PCR normalization in the Phytophthora-Q. suber pathosystem in root tissues. Those found to be more stable, QsRPII and QsCACs, were used as internal reference in the present work. Knowledge on the Quercus defence mechanisms against biotic stress is scarce. This study provides an insight into the gene profiling of a few important genes of Q. suber in response to P. cinnamomi infection contributing to the knowledge of the molecular interactions involving Quercus and root pathogens that can be useful in the future to understand the mechanisms underlying oak resistance to soil-borne oomycetes. Electronic supplementary material The online version of this article (doi:10.1186/2193-1801-3-613) contains supplementary material, which is available to authorized users.

Background: Cork oak (Quercus suber) is one of the rare trees with the ability to produce cork, a material widely used to make wine bottle stoppers, flooring and insulation materials, among many other uses. The molecular mechanisms of cork formation are still poorly understood, in great part due to the difficulty in studying a species with a long life-cycle and for which there is scarce molecular/genomic information. Cork oak forests are of great ecological importance and represent a major economic and social resource in Southern Europe and Northern Africa. However, global warming is threatening the cork oak forests by imposing thermal, hydric and many types of novel biotic stresses. Despite the economic and social value of the Q. suber species, few genomic resources have been developed, useful for biotechnological applications and improved forest management. Results: We generated in excess of 7 million sequence reads, by pyrosequencing 21 normalized cDNA libraries derived from multiple Q. suber tissues and organs, developmental stages and physiological conditions. We deployed a stringent sequence processing and assembly pipeline that resulted in the identification of ~159,000 unigenes. These were annotated according to their similarity to known plant genes, to known Interpro domains, GO classes and E.C. numbers. The phylogenetic extent of this ESTs set was investigated, and we found that cork oak revealed a significant new gene space that is not covered by other model species or EST sequencing projects. The raw data, as well as the full annotated assembly, are now available to the community in a dedicated web portal at www.corkoakdb.org. Conclusions: This genomic resource represents the first trancriptome study in a cork producing species. It can be explored to develop new tools and approaches to understand stress responses and developmental processes in forest trees, as well as the molecular cascades underlying cork differentiation and disease response., This project was funded by "Fundacao para a Ciencia e a Tecnologia" (FCT) within a National Consortium (COEC - Cork Oak ESTs Consortium) that supported 12 sub-projects (SOBREIRO/033, 035, 014, 034, 015, 017, 038, 019, 029, 039, 030, 036/2009). The authors further wish to acknowledge FCT for ten doctoral (BD) and post doctoral (BPD) fellowships (Tania Almeida: SFRH/BD/44410/2008, Tiago Capote: SFRH/BD/69785/2010, Ines Chaves: SFRH/BPD/20833/2004, Ana S. Fortunato: SFRH/BPD/47563/2008, Marilia Horta: SFRH/BPD/63213/2009, Liliana Marum: "SFRH/BPD/47679/2008, Andreia Miguel: SFRH/BD/44474/2008, Margarida Rocheta: SFRH/BPD/64905/2009, Tatiana E. Santo: SFRH/BD/47450/2008, Monica Sebastiana: SFRH/BPD/25661/2005). Andreas Bohn, Nelson J.M. Saibo, Rita Teixeira were supported by the Programa Ciencia 2007, financed by POPH (QREN) and Isabel A. Abreu, Susana Araujo, Dora Batista, A. Margarida Fortes, Jorge A.P. Paiva, Sonia Goncalves by Programa Ciencia 2008, also funded by POPH (QREN). A Margarida Santos was funded through iBET (PEst-OE/EQB/LA0004/2011). Maintenance of the CorkOakDB is supported by the Instituto Gulbenkian de Ciencia.

Oomycetes from the genus Phytophthora are fungus-like plant pathogens that are devastating for agriculture and natural ecosystems. They are able to secrete a glucanase inhibitor protein (GIP) that inhibits the activity of endoglucanases (EGases) involved in defense responses against infection. One of the most widely distributed and aggressive Phytophthora species, with more than 1,000 host plants is P. cinnamomi. In this work we report the sequencing and characterization of a class of GIPs secreted by Phytophthora cinnamomi. The gip gene from P. cinnamomi has a 937 bp ORF encoding a putative peptide of 312 deduced amino acids. The expression of this gene was studied during growth in different carbon sources (glucose, cellulose and sawdust), by RT-qPCR and its level of expression was evaluated at five time points. The highest expression of gip gene occurred in sawdust at 8 h of induction. In vivo infection of C. sativa revealed an increase in gip expression from 12 to 24 h. At 36 h its expression decreased suggesting that a compensatory mechanism must occur in plant.

The present preliminary study attempts to establish associations between milk production traits and genetic polymorphisms at the GH gene in the Algarvia goat. The DNA of 108 goats of the indigenous Portuguese Algarvia breed was evaluated. Single-strand conformation polymorphisms (SSCP) were identified at the five exons of the goat growth hormone (gGH) gene. Two conformational patterns were found in each of exons 1 and 2, four in exon 3, six in exon 4 and five in exon 5. An association between these SSCP patterns with milk, fat and protein production, and fat and protein content was examined. Patterns F/F of exon 4 and A/A of exon 5 were positively associated with milk production (P

Elicitins are a group of highly conserved proteins secreted by species of Phytophthora and a species of the related genus Pythium, Pythium vexans. Some of these proteins act as inducers of the necrotic hypersensitive-like response and the associated systemic acquired resistance phenomenon, in some species. We cloned and characterised the cinnamomin-beta and -alpha genes and two related elicitin genes from Phytophthora cinnamomi. These four open reading frames (ORFs) are clustered in tandem pairs. Two out of these four genes present homologies with the basic and acidic elicitin groups; but the two others encode, if expressed, elicitin isoforms exhibiting homologies with the class II of highly acidic elicitins.

In response to the need for a faster, more reliable method for identifying Phytophthora cinnamomi in cork oak soils in Portugal, a simple, fast, sensitive molecular identification method is described. It is based on a colorimetric assay which involves an oligonucleotide capture probe covalently immobilised on microtitration wells, a multi-biotinylated oligonucleotide detection probe and the PCR-amplified target DNA. The target DNA is a 349 bp DNA fragment partially covering the 3′-translated and 3′-untranslated regions of the cinnamomin gene. When the specificity of the PCR reaction was evaluated in vitro using isolates of P. cinnamomi and eight other Phytophthora species, including the related P. cambivora , it was specific to P. cinnamomi . When 30 isolates of P. cinnamomi from oak roots in southern Portugal were assayed, 26 gave a strong positive response. The assay has a sensitivity of about 2–5 genome equivalents of P. cinnamomi . The reason for the negative response of four isolates remains unclear.

The β(1,4)-galactosyltransferase-I gene (β4galt1) encodes the catalytic part of the enzyme lactose synthase, responsible of lactose synthesis in the mammary gland. The complete coding region of the gene was screened for the presence of allelic variation among a sample of 1,200 Iranian Holstein cows, using PCR-SSCP technique followed by sequencing. Nine polymorphic nucleotide sites were identified-one in exons I and VI, two in exons II and III, and three in exon V. Altogether 18 different genotypes were assigned. Statistical analysis showed that the genotypes of Β4GALT1 significantly affect milk, lactose, protein and total solid productions in both the first and second lactation (P < 0.001). Variance component analysis considering restricted maximum likelihood showed that the major factor making differences in milk, lactose, protein and total solid productions among the studied cow is the β4galt1 genotype. We concluded that the β4galt1 gene is potentially associated with milk production traits in dairy cows and should be considered for further studies on genetics of the milk production traits.

Expression of POU1F1 gene, a member of the POU homeodomain family of transcription factors, is necessary for normal differentiation, development and survival of three anterior pituitary cell types (thyrotrophs, somatotrophs and lactotrophs) and for the proper expression of growth hormone (GH), prolactin (PRL), thyroid-stimulating hormone (TSH) genes and POU1F1 gene itself. Alternative splicing forms of this gene have been reported in different species, with few functional studies. Apart from the POU1F1-Wild-type with the expected length, in this work we isolated three additional splicing variants: POU1F1-beta, with a 78 bp insert in the trans-activation domain; POU1F1-gamma that lacks exon 3 and POU1F1-delta that lacks exons 3, 4 and 5. Four different protein isoforms were also detected by Western blot in the sheep pituitary tissue. Functional assays were performed to study the trans-activation of GH and PRL promoters by the splicing variants. Regarding the PRL promoter, the beta variant presented only 12% of the Wild-type trans-activation capacity. Variants gamma and delta showed no capacity to trans-activate PRL promoter. Both gamma and delta variants acted as repressors of Wt, reducing significantly the trans-activation made by Wt alone (p

POU1F1 (PIT-1/GHF-1) is a transcription factor with critical role in the transcriptional regulation of multiple genes in the pituitary and also important for the survival, differentiation and proliferation of three pituitary cell types. To understand the regulation of POU1F1 gene in Ovis aries we report its cloning, sequencing and characterization. The sequenced 5787 bp included six exons and two complete introns. Ovine POU1F1 gene has a high level of conservation with its bovine, human and rat counterparts showing 98.2%, 91.2% and 86.2% of similarity at the coding level, respectively. All six exons were analyzed for polymorphism detection in 100 animals of the Portuguese indigenous ovine breed 'Churra da Terra Quente'. One polymorphism was found at codon 58 in exon 2, in one allele of 4 animals leading to a change from cysteine to tyrosine (2% allelic frequency). In exon 3 two polymorphisms were detected: a G to A transition altering a glycine to an asparagine at codon 89 in one allele of one animal (0.5% allelic frequency) and another G to A transition at codon 105 converting an alanine into a threonine in one allele of 3 animals (1.5% allelic frequency). These polymorphisms might change the structure of the POU1F1 protein and modify gene-expression. In intron 4, an A to G transition was detected in one allele of six animals (3% allelic frequency). Exons 1, 4 and 6 showed no polymorphisms.

A gene encoding a potential NADPH-dependent cinnamyl alcohol dehydrogenase (QsCAD1) (GenBank accession no: AY362455) was identified in Quercus suber (cork oak). Its complete cDNA sequence was obtained by RACE-PCR, starting from total RNA extracted from roots of seedlings of Q. suber, infected with Phytophthora cinnamomi, the causal agent of the decline and sudden death of Q. suber and Quercus ilex subsp. rotundifolia in the Iberian Peninsula. Sequence information to perform the RACE-PCR was acquired from a polymorphic fragment (C9), specifically identified by cDNA-AFLP, in leaves of epicormic shoots of a cork oak tree that suffered sudden death. RT-PCR and hybridization analysis showed that the QsCAD1 gene is up-regulated in root seedlings of Q. suber infected with P. cinnamomi. QsCAD1 has a high structural homology with VR-ERE (Vigna radiata), an enzyme that detoxifies eutypine (produced by Eutypa lata, the causal agent of eutypa dieback of grapevines), to eutypinol, and with QrCAD1 (Q. ilex subsp. rotundifolia), EgCAD1 (Eucalyptus gunnii), MdCAD1 (Malus x domestica). Taken together, these results suggest that these enzymes, and namely QsCAD1 belong to a new group of CAD potentially involved in deactivation of toxins produced by phytopathogens. r 2007 Elsevier Ltd. All rights reserved.

The genetic variability of cork oak (Quercus suber, L.) in Portugal was evaluated by AFLP using five primer combinations. Three hundred and thirteen trees from three geographically contrasting regions exhibited a high level of genetic variation. The genetic profile of each individual is composed of 291 loci, randomly positioned in the genome and consists of monomorphic and polymorphic fragments. Similarities and dissimilarities among the individuals were quantitatively evaluated by numerical taxonomy. The overall sample shows a proportion of AFLP polymorphic markers of 71%, denoting a high level of variability. Ninety percent of the polymorphic markers identified in cork oak genotypes are uniformly distributed throughout the cork oak populations of Algarve, Alentejo and Trás-os-Montes regions. The coefficients of genetic similarity vary from 0.61 to 0.88 implying that 60% of fragments found are common. A sample of 52 holm oak [Quercus ilex subsp. rotundifolia (Lam.)] trees from overlapping areas was also analysed by AFLP with the same five primer combinations. However the codification of markers together with those selected on cork oak profiles was feasible with only one primer combination due to an apparent much higher polymorphism. AFLP and numerical taxonomy analysis enabled to differentiate the taxa and showed that the level of similarity observed between the profiles of the individuals from holm oak species was lower than that observed in cork oak, implying that apparently the degree of polymorphism is higher in Q. ilex subsp. rotundifolia than that quantified in Q. suber. A Bayesian approach was used to assess Q. suber total genetic diversity (Ht = 0.2534, P < 0.001) of which 1.7% (Fst = 0.0172, P < 0.001) was assigned to differences among populations. Analysis of molecular variance (AMOVA) showed that most genetic variation is comprised within populations (96%) while 3.6% is among populations (Φst = 0.036, P < 0.001). Differences among populations within geographic regions account for 2.6% (Φsc = 0.026, P < 0.001) of the total variation and only 1.3% (Φct = 0.013, P = 0.007) is attributed to variation among regions denoting little differentiation of populations over a range of 700 km.

The present study suggests the existence of an association between milk production traits and genetic polymorphisms at the growth hormone (GH) gene in the Portuguese indigenous Serrana goat. TheDNAfrom 229 animals of two ecotypes (Jarmelista and Ribatejano)was analysed by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique revealing a high degree of genetic polymorphism at the goat GH (gGH) gene. Two conformation patterns were detected in exons 1 and 2, 6 in exon 3, 10 in exon 4 and 5 in exon 5. The evaluation of an association effect between these SSCP patterns with milk, fat and protein yields and fat and protein percentages suggests a positive effect of pattern A/B of exon 4 for Ribatejano ecotype and of pattern A/B of exon 2 for Jarmelista ecotype with milk yield (P < 0.05) and of pattern A/B of exon 1 and pattern B/B of exon 2 with protein percentage (P < 0.05) for Ribatejano ecotype. The results support the hints suggested by previous studies of the importance of the gGH gene as a candidate gene for marker-assisted selection in goat breeds and suggest that exon 4 is a preferential target for further investigation on mutations that influence milk yield variation.

Phosphoramidites of thymidine or 2′-deoxyinosine, modified in 5′ by the addition of an aminoalkylcarbamate function, were prepared. The derivatized nucleotides can be used in automatic DNA synthesis to tag any oligodeoxynucleotide chain and provide a convenient reactive group for labelling with non radioactive reporters. As an example of application, we show the specific detection of Human Papilloma Virus DNA using a biotin-labelled 29-mer oligodeoxynucleotide entirely prepared on solid support.

A cDNA library derived from human carcinoma cells was used to isolate a clone, pULB1000, coding for the preproenzyme form of human urokinase. This clone carries the full-length sequence coding for the signal peptide and for the A chain (157 amino acids) and B chain (253 amino acids) of urokinase in tandem. The sequence of the cDNA predicts the presence of a single lysine residue between the last amino acid of the mature A polypeptide (Phe-157) and the first amino acid of the mature B polypeptide (Ile-1). The amino acid sequence deduced from the cDNA sequence fits the published amino acid sequence data with three exceptions, the reported cysteine residue at position 131 in the A chain is a tryptophan, and glycine 366 and alanine 410 in the B chain are, respectively, a cysteine and a valine in our clone. A large Bgl I fragment (1482 bp), derived from the clone pULB1000 coding for most of the signal peptide and for the A and B chains, has been subcloned into the expression vector pCQV2. Heat induction of E. coli cells carrying the recombinant plasmid leads to the production of urokinase-like polypeptides having the expected molecular weights and being specifically recognized by antibodies raised against natural human urokinase.

A cDNA library prepared from human liver was screened for alpha 1-antitrypsin, a major constituent of plasma which functions as inhibitor of proteolytic enzymes. The library was screened using a 12-base-long synthetic oligodeoxyribonucleotide corresponding to a known DNA fragment of human alpha 1-antitrypsin and by hybrid-selection of alpha 1-antitrypsin mRNA. A plasmid, pULB1523, was identified carrying a cDNA insert of about 1400 bp coding for human alpha 1-antitrypsin. Restriction mapping and DNA sequence analysis indicated that the 1400 bp code for the signal peptide and for the complete mature alpha 1-antitrypsin molecule. In addition, a solid-phase enzyme-linked immunoassay showed that pULB1523 expresses human alpha 1-antitrypsin in bacteria. Fusion of the alpha 1-antitrypsin sequence to the leader sequence of the beta-lactamase gene (plasmid pKT287) resulted also in the expression of the protein in bacteria.

Oligodeoxynucleotide probes derived from the published amino acid (aa) sequence for d -amino acid oxidase (DAO) [Ronchi et al. J. Biol. Chem. 259 (1982) 8824–8834] were used to screen cDNA libraries made from porcine kidney cortex and liver. Whereas no clones were obtained from kidney mRNAs, 20 independent ones were isolated from the liver library. Surprisingly, all of them carried only partial cDNAs for DAO starting around aa 100 in the coding sequence and extending for up to 250 bp in the 3′-noncoding sequence. One of these clones, pULB9103, was used to screen a porcine genomic library and allowed the isolation of DAO gene clone phULB001. Four exons encoding aa 1–151 were identified and sequenced, as well as the relevant exon-intron junctions. The mRNA sequence coding for DAO has been reconstituted from the genomic and cDNA sequences; its analysis by computer did not reveal any significant secondary structure, or particular feature, which could explain the failure to obtain full-length cDNAs.

A human liver cDNA library was used to isolate a clone coding for apolipoprotein A-I (Apo A-I). The clone carries the sequence for the prepeptide (18 amino acids), the propeptide (6 amino acids), and the mature protein (243 amino acids). A coding cassette for the proapo A-I molecule was reconstructed by fusing synthetic sequences, chosen to optimize expression and specifying the amino-terminal methionine and amino acids -6 to +14, to a large fragment of the cDNA coding for amino acids 15-243. The module was expressed in pOTS-Nco, an Escherichia coli expression vector carrying the regulatable lambda PL promoter, leading to the production of proapolipoprotein A-I at up to 10% of total soluble proteins. The recombinant polypeptide was purified and characterized in terms of apparent molecular mass, isoelectric point, and by both chemical and enzymatic peptide mapping. In addition, it was assayed in vitro for the stimulation of the enzyme lecithin: cholesterol acyltransferase. The data show for the first time that proapo A-I can be produced efficiently in E. coli as a stable and undegraded protein having physical and functional properties indistinguishable from those of the natural product.