Genetic bases for variation in structure and biological activity of trichothecene toxins produced by diverse fungi.

Trichothecenes are sesquiterpene toxins produced by diverse but relatively few fungal species in at least three classes of Ascomycetes: Dothideomycetes, Eurotiomycetes, and Sordariomycetes. Approximately 200 structurally distinct trichothecene analogs have been described, but a given fungal species typically produces only a small subset of analogs. All trichothecenes share a core structure consisting of a four-ring nucleus known as 12,13-epoxytrichothec-9-ene. This structure can be substituted at various positions with hydroxyl, acyl, or keto groups to give rise to the diversity of trichothecene structures that has been described. Over the last 30 years, the genetic and biochemical pathways required for trichothecene biosynthesis in several species of the fungi Fusarium and Trichoderma have been elucidated. In addition, phylogenetic and functional analyses of trichothecene biosynthetic (TRI) genes from fungi in multiple genera have provided insights into how acquisition, loss, and changes in functions of TRI genes have given rise to the diversity of trichothecene structures. These analyses also suggest both divergence and convergence of TRI gene function during the evolutionary history of trichothecene biosynthesis. What has driven trichothecene structural diversification remains an unanswered question. However, insight into the role of trichothecenes in plant pathogenesis of Fusarium species and into plant glucosyltransferases that detoxify the toxins by glycosylating them point to a possible driver. Because the glucosyltransferases can have substrate specificity, changes in trichothecene structures produced by a fungus could allow it to evade detoxification by the plant enzymes. Thus, it is possible that advantages conferred by evading detoxification have contributed to trichothecene structural diversification. KEY POINTS : • TRI genes have evolved by diverse processes: loss, acquisition and changes in function. • Some TRI genes have acquired the same function by convergent evolution. • Some other TRI genes have evolved divergently to have different functions. • Some TRI genes were acquired or resulted from diversification in function of other genes. • Substrate specificity of plant glucosyltransferases could drive trichothecene diversity.

Trichodiene Production in a Trichoderma harzianum erg1-Silenced Strain Provides Evidence of the Importance of the Sterol Biosynthetic Pathway in Inducing Plant Defense-Related Gene Expression.

Trichoderma species are often used as biocontrol agents against plant-pathogenic fungi. A complex molecular interaction occurs among the biocontrol agent, the antagonistic fungus, and the plant. Terpenes and sterols produced by the biocontrol fungus have been found to affect gene expression in both the antagonistic fungus and the plant. The terpene trichodiene (TD) elicits the expression of genes related to tomato defense and to Botrytis virulence. We show here that TD itself is able to induce the expression of Botrytis genes involved in the synthesis of botrydial (BOT) and also induces terpene gene expression in Trichoderma spp. The terpene ergosterol, in addition to its role as a structural component of the fungal cell membranes, acts as an elicitor of defense response in plants. In the present work, using a transformant of T. harzianum, which is silenced in the erg1 gene and accumulates high levels of squalene, we show that this ergosterol precursor also acts as an important elicitor molecule of tomato defense-related genes and induces Botrytis genes involved in BOT biosynthesis, in both cases, in a concentration-dependent manner. Our data emphasize the importance of a balance of squalene and ergosterol in fungal interactions as well as in the biocontrol activity of Trichoderma spp.

Effects of Trichothecene Production on the Plant Defense Response and Fungal Physiology: Overexpression of the Trichoderma arundinaceum tri4 Gene in T. harzianum.

Trichothecenes are fungal sesquiterpenoid compounds, the majority of which have phytotoxic activity. They contaminate food and feed stocks, resulting in potential harm to animals and human beings. Trichoderma brevicompactum and T. arundinaceum produce trichodermin and harzianum A (HA), respectively, two trichothecenes that show different bioactive properties. Both compounds have remarkable antibiotic and cytotoxic activities, but in addition, trichodermin is highly phytotoxic, while HA lacks this activity when analyzed in vivo. Analysis of Fusarium trichothecene intermediates led to the conclusion that most of them, with the exception of the hydrocarbon precursor trichodiene (TD), have a detectable phytotoxic activity which is not directly related to the structural complexity of the intermediate. In the present work, the HA intermediate 12,13-epoxytrichothec-9-ene (EPT) was produced by expression of the T. arundinaceum tri4 gene in a transgenic T. harzianum strain that already produces TD after transformation with the T. arundinaceum tri5 gene. Purified EPT did not show antifungal or phytotoxic activity, while purified HA showed both antifungal and phytotoxic activities. However, the use of the transgenic T. harzianum tri4 strain induced a downregulation of defense-related genes in tomato plants and also downregulated plant genes involved in fungal root colonization. The production of EPT by the transgenic tri4 strain raised levels of erg1 expression and reduced squalene accumulation while not affecting levels of ergosterol. Together, these results indicate the complex interactions among trichothecene intermediates, fungal antagonists, and host plants.

Temporal dynamics and population genetic structure of Fusarium graminearum in the upper Midwestern United States.

Fusarium graminearum sensu stricto causes Fusarium head blight (FHB) in wheat and barley, and contaminates grains with several trichothecene mycotoxins, causing destructive yield losses and economic impact in the United States. Recently, a F. graminearum strain collected from Minnesota (MN) was determined to produce a novel trichothecene toxin, called NX-2. In order to determine the spatial and temporal dynamics of NX-2 producing strains in MN, North Dakota (ND) and South Dakota (SD), a total of 463 F. graminearum strains were collected from three sampling periods, 1999-2000, 2006-2007 and 2011-2013. A PCR-RFLP based diagnostic test was developed and validated for NX-2 producing strains based on polymorphisms in the Tri1 gene. Trichothecene biosynthesis gene (Tri gene)-based polymerase chain reaction (PCR) assays and ten PCR-restriction fragment length polymorphism (RFLP) markers were used to genotype all strains. NX-2 strains were detected in each sampling period but with a very low overall frequency (2.8%) and were mainly collected near the borders of MN, ND and SD. Strains with the 3ADON chemotype were relatively infrequent in 1999-2000 (4.5%) but increased to 29.4% in 2006-2007 and 17.2% in 2011-2013. The distribution of 3ADON producing strains also expanded from a few border counties between ND and MN in 1999-2000, southward toward the border between SD and MN in 2006-2007 and westward in 2011-2013. Genetic differentiation between 2006-2007 and 2011-2013 populations (3%) was much lower than that between 1999-2000 and 2006-2007 (22%) or 1999-2000 and 2011-2013 (20%) suggesting that most change to population genetic structure of F. graminearum occurred between 1999-2000 and 2006-2007. This change was associated with the emergence of a new population consisting largely of individuals with a 3ADON chemotype. A Bayesian clustering analysis suggested that NX-2 chemotype strains are part of a previously described Upper Midwestern population. However, these analyses also suggest that the NX-2 isolates could represent a distinct population, but that interpretations of population assignment are influenced by the small number of NX-2 strains available for analysis.

Involvement of Trichoderma trichothecenes in the biocontrol activity and induction of plant defense-related genes.

Trichoderma species produce trichothecenes, most notably trichodermin and harzianum A (HA), by a biosynthetic pathway in which several of the involved proteins have significant differences in functionality compared to their Fusarium orthologues. In addition, the genes encoding these proteins show a genomic organization differing from that of the Fusarium tri clusters. Here we describe the isolation of Trichoderma arundinaceum IBT 40837 transformants which have a disrupted or silenced tri4 gene, a gene encoding a cytochrome P450 monooxygenase that oxygenates trichodiene to give rise to isotrichodiol, and the effect of tri4 gene disruption and silencing on the expression of other tri genes. Our results indicate that the tri4 gene disruption resulted in a reduced antifungal activity against Botrytis cinerea and Rhizoctonia solani and also in a reduced ability to induce the expression of tomato plant defense-related genes belonging to the salicylic acid (SA) and jasmonate (JA) pathways against B. cinerea, in comparison to the wild-type strain, indicating that HA plays an important function in the sensitization of Trichoderma-pretreated plants against this fungal pathogen. Additionally, the effect of the interaction of T. arundinaceum with B. cinerea or R. solani and with tomato seedlings on the expressions of the tri genes was studied.

Greenhouse Studies Reveal Increased Aggressiveness of Emergent Canadian Fusarium graminearum Chemotypes in Wheat.

The role of Fusarium graminearum trichothecene-chemotypes in disease outcomes was evaluated by point inoculation in a series of wheat lines with different levels of resistance to Fusarium head blight (FHB). Four inocula, each consisting of a composite of four strains with either 15-acetyldeoxynivalenol (ADON) chemotypes from "traditional" or emergent populations, a 3-ADON chemotype, or a nivalenol (NIV) chemotype, were compared. The evaluated wheat included Canadian lines with different levels of FHB resistance/susceptibility and double haploid lines developed from crosses of these lines. Highly resistant lines were resistant to infection by all of the F. graminearum chemotypes evaluated. In the moderately susceptible/resistant wheat lines, the 3-ADON producers and the emergent 15-ADON population were, in some instances, more aggressive and resulted in higher Fusarium damaged kernel scores and levels of trichothecene accumulation. The data presented in this study demonstrate the importance of growing highly resistant wheat cultivars in the current climate of an evolving F. graminearum population, and suggest that moderate levels of FHB resistance may not be sufficient to minimize trichothecene contamination of grain from F. graminearum-infected wheat.

Identification of loci and functional characterization of trichothecene biosynthesis genes in filamentous fungi of the genus Trichoderma.

Trichothecenes are mycotoxins produced by Trichoderma, Fusarium, and at least four other genera in the fungal order Hypocreales. Fusarium has a trichothecene biosynthetic gene (TRI) cluster that encodes transport and regulatory proteins as well as most enzymes required for the formation of the mycotoxins. However, little is known about trichothecene biosynthesis in the other genera. Here, we identify and characterize TRI gene orthologues (tri) in Trichoderma arundinaceum and Trichoderma brevicompactum. Our results indicate that both Trichoderma species have a tri cluster that consists of orthologues of seven genes present in the Fusarium TRI cluster. Organization of genes in the cluster is the same in the two Trichoderma species but differs from the organization in Fusarium. Sequence and functional analysis revealed that the gene (tri5) responsible for the first committed step in trichothecene biosynthesis is located outside the cluster in both Trichoderma species rather than inside the cluster as it is in Fusarium. Heterologous expression analysis revealed that two T. arundinaceum cluster genes (tri4 and tri11) differ in function from their Fusarium orthologues. The Tatri4-encoded enzyme catalyzes only three of the four oxygenation reactions catalyzed by the orthologous enzyme in Fusarium. The Tatri11-encoded enzyme catalyzes a completely different reaction (trichothecene C-4 hydroxylation) than the Fusarium orthologue (trichothecene C-15 hydroxylation). The results of this study indicate that although some characteristics of the tri/TRI cluster have been conserved during evolution of Trichoderma and Fusarium, the cluster has undergone marked changes, including gene loss and/or gain, gene rearrangement, and divergence of gene function.

Species diversity and mycotoxin production by members of the Fusarium tricinctum species complex associated with Fusarium head blight of wheat and barley in Italy.

Fusarium head blight (FHB) is a global cereal disease caused by a complex of Fusarium species. In Europe, the main species responsible for FHB are F. graminearum, F. culmorum and F. poae. However, members of the F. tricinctum species complex (FTSC) have become increasingly important. FTSC fusaria can synthesize mycotoxins such as moniliformin (MON), enniatins (ENNs) and several other biologically active secondary metabolites that could compromise food quality. In this study, FTSC isolates primarily from Italian durum wheat and barley, together with individual strains from four non-graminaceous hosts, were collected to assess their genetic diversity and determine their potential to produce mycotoxins in vitro on rice cultures. A multilocus DNA sequence dataset (TEF1, RPB1 and RPB2) was constructed for 117 isolates from Italy and 6 from Iran to evaluate FTSC species diversity and their evolutionary relationships. Phylogenetic analyses revealed wide genetic diversity among Italian FTSC isolates. Among previously described FTSC species, F. avenaceum (FTSC 4) was the most common species in Italy (56/117 = 47.9%) while F. tricinctum (FTSC 3), and F. acuminatum (FTSC 2) accounted for 11.1% (13/117) and the 8.5% (10/117), respectively. The second most detected species was a new and unnamed Fusarium sp. (FTSC 12; 32/117 = 19%) resolved as the sister group of F. tricinctum. Collectively, these four phylospecies accounted for 111/117 = 94.9% of the Italian FTSC collection. However, we identified five other FTSC species at low frequencies, including F. iranicum (FTSC 6) and three newly discovered species (Fusarium spp. FTSC 13, 14, 15). Of the 59 FTSC isolates tested for mycotoxin production on rice cultures, 54 and 55 strains, respectively, were able to produce detectable levels of ENNs and MON. In addition, we confirmed that the ability to produce bioactive secondary metabolites such as chlamydosporol, acuminatopyrone, longiborneol, fungerin and butanolide is widespread across the FTSC.

CLM1 of Fusarium graminearum encodes a longiborneol synthase required for culmorin production.

Fusarium graminearum is a fungal pathogen of cereal crops (e.g., wheat, barley, maize) and produces a number of mycotoxins, including 15-acetyldeoxynivalenol, butenolide, zearalenone, and culmorin. To identify a biosynthetic gene for the culmorin pathway, an expressed-sequence-tag database was examined for terpene cyclase genes. A gene designated CLM1 was expressed under trichothecene-inducing conditions. Expression of CLM1 in yeast (Saccharomyces cerevisiae) resulted in the production of a sesquiterpene alcohol, longiborneol, which has the same ring structure as culmorin. Gene disruption and add-back experiments in F. graminearum showed that CLM1 was required for culmorin biosynthesis. CLM1 gene disruptants were able to convert exogenously added longiborneol to culmorin. Longiborneol accumulated transiently in culmorin-producing strains. The results indicate that CLM1 encodes a longiborneol synthase and is required for culmorin biosynthesis in F. graminearum.

Novel rare mutations and promoter haplotypes in ABCA1 contribute to low-HDL-C levels.

The ATP-binding cassette A1 (ABCA1) protein regulates plasma high-density lipoprotein (HDL) levels. Mutations in ABCA1 can cause HDL deficiency and increase the risk of premature coronary artery disease. Single nucleotide polymorphisms (SNPs) in ABCA1 are associated with variation in plasma HDL levels. We investigated the prevalence of mutations and common SNPs in ABCA1 in 154 low-HDL individuals and 102 high-HDL individuals. Mutations were identified in five of the low-HDL subjects, three having novel variants (I659V, R2004K, and A2028V) and two with a previously identified variant (R1068H). Analysis of four SNPs in the ABCA1 gene promoter (C-564T, G-407C, G-278C, and C-14T) identified the C-14T SNP and the TCCT haplotype to be over-represented in low-HDL individuals. The R1587K SNP was over-represented in low-HDL individuals, and the V825I and I883M SNPs over-represented in high-HDL individuals. We conclude that sequence variation in ABCA1 contributes significantly to variation in HDL levels.

Apolipoprotein B-32: a new truncated mutant of human apolipoprotein B capable of forming particles in the low density lipoprotein range.

We have identified a new species of apolipoprotein (apo) B in an individual with heterozygous hypobetalipoproteinemia. The new apo B (apo B-32) is the result of a single point mutation (1450 Gln----Stop) in the apo B gene that prevents full length translation. Apo B-32 is predicted to contain the 1449 amino-terminal amino acids of apo B-100 and is associated with a markedly decreased low density lipoprotein (LDL) cholesterol level. The density distribution of apo B-32 in the plasma lipoproteins makes it unique amongst other truncated apo B species. Normally, apo B-100 is found in both very low density lipoprotein (VLDL) and LDL particles. However, the majority of the apo B-32 protein was found in the high density lipoprotein (HDL) and lipoprotein-deplete (d greater than 1.21 g/ml) fractions, suggesting that it was mainly assembled into abnormally dense lipoprotein particles. A small amount of apo B-32 was also found in the LDL, making it the shortest known apo B variant capable of forming particles in this density range. Apo B-32 was undetected in VLDL. The apo B-32 mutation further defines the minimum length of the apo B protein that is required for the assembly of LDL.

Characterization of lipoproteins containing a truncated form of apolipoprotein B, apolipoprotein B32.

We have characterized the lipoproteins isolated from the plasma of a human subject who was heterozygous for a mutation yielding a truncated apolipoprotein B (apo-B32). The apo-B32 lipoproteins were isolated from the plasma high density lipoprotein (HDL) fraction by heparin-Sepharose chromatography. Although the chemical composition of the apo-B32-containing lipoproteins was similar to that of normal HDL, the mean diameter of the apo-B32 lipoproteins was larger than typical apo-AI-containing HDL particles. On agarose gels, the apo-B32 lipoproteins had pre-beta mobility similar to that of normal very low density lipoproteins. Analysis of the purified apo-B32 lipoproteins by SDS-polyacrylamide gel electrophoresis revealed the presence of both apo-AI and apo-E. In order to further analyze the properties of apo-B32, we developed an apo-B32 expression vector and generated stable rat hepatoma cell lines expressing apo-B32. In these cell lines, the apo-B32 protein was secreted in a d > 1.21 g/ml lipoprotein. Oleic acid supplementation of the cell-culture media had no measurable affect on the density distribution of the apo-B32 lipoproteins that were secreted by the cells.

Generation of transgenic mice from yeast artificial chromosome DNA that has been modified by gene targeting.

Yeast artificial chromosome (YAC) vectors permit the cloning of up to megabase fragments of human genomic DNA in yeast (Saccharomyces cerevisiae). Efficient recombination between homologous segments of DNA is one of the hallmark genetic features of yeast. This characteristic facilitates the introduction of specific mutations into YACs by gene targeting. Gene targeting has been used recently to introduce specific mutations into YACs spanning the human ß-globin locus and the human apolipoprotein (apo)-B gene, and the mutated YAC DNA has been used to generate transgenic mice. This approach has been useful for the study of the regulatory elements controlling ß-globin gene expression and for the study of apo-B structure and function. This article reviews the techniques for introducing mutations into YACs, for the purpose of expression in transgenic mice.

Reduced virulence of Gibberella zeae caused by disruption of a trichothecene toxin biosynthetic gene.

The production of trichothecene mycotoxins by some plant pathogenic species of Fusarium is thought to contribute to their virulence. Gibberella zeae (F. graminearum) is an important cereal pathogen that produces the trichothecene deoxynivalenol. To determine if trichothecene production contributes to the virulence of G. zeae, we generated trichothecene-deficient mutants of the fungus by gene disruption. The disrupted gene, Tri5, encodes the enzyme trichodiene synthase, which catalyzes the first step in trichothecene biosynthesis. To disrupt Tri5, G. zeae was transformed with a plasmid carrying a doubly truncated copy of the Tri5 coding region interrupted by a hygromycin B resistance gene. Tri5- transformants were selected by screening for the inability to produce trichothecenes and by Southern blot analysis. Tri5- strains exhibited reduced virulence on seedlings of Wheaton wheat and common winter rye, but wild-type virulence on seedlings of Golden Bantam maize. On Caldwell and Marshall wheat and Porter oat seedlings, Tri5- strains were inconsistent in causing less disease than their wild-type progenitor strain. Head blight developed more slowly on Wheaton when inoculated with Tri5- mutants than when inoculated with wild-type strains. These results suggest that trichothecene production contributes to the virulence of G. zeae on some hosts.

Aflatoxin production in cultures of Aspergillus flavus incubated in atmospheres containing selected cotton leaf-derived volatiles.

Aspergillus flavus, in solid culture, was exposed to individual selected, commercially obtained volatile compounds that were similar to volatile cotton leaf-derived components. The radial growth pattern of A. flavus and the production of aflatoxin were determined on these volatile exposed cultures. The most bioactive compounds C6-C9 alkenals, completely inhibited the growth of the fungus. Unexpected results demonstrated that 3-methyl-1-butanol and 3-methyl-2-butanol inhibited A. flavus growth by 20% but increased production of aflatoxin B1 by 1.5 to 2-fold. Other relationships of growth and aflatoxin production resulting from exposure to the bioactive volatiles are discussed.

Murine lymphocyte proliferation impaired by substituted neosolaniols and calonectrins--Fusarium metabolites associated with trichothecene biosynthesis.

The capacity of Fusarium secondary metabolites associated with trichothecene biosynthesis to inhibit murine spleen lymphocyte proliferation was evaluated and compared to that for well known trichothecenes. Activity of these compounds was not specific for B and T lymphocytes since they inhibited [3H]thymidine (TdR) incorporation in unstimulated, Con A- and LPS-stimulated lymphocytes to the same extent. Concentrations of 8-propionyl neosolaniol and 8-butyrylneosolaniol which inhibited [3H]Tdr uptake by 50% (ID50s) were 0.95 and 0.34 ng/ml, respectively. The ID50 for T-2 toxin was 0.26 ng/ml, indicating that there are minor alterations in 12,13-epoxytrichothecene toxicity resulting from the replacement of the isovaleryl moiety on C8 of the trichothecene skeleton with other bulky acyl groups. ID50 values for 4,15-diacetylnivalenol, fusarenon X, deoxynivalenol and 3-acetyldeoxynivalenol were 25, 38, 120 and 1800 ng/ml, respectively. Comparatively, ID50 values for 3,15-dideacetylcalonectrin, 15-deacetylcalonectrin, and 7,8-dihydroxycalonectrin were 390, 2700 and 2400 ng/ml, respectively, indicating that the modified calonectrins had equivalent or less toxicity. Lymphotoxicity of trichothecenes thus decreased upon substitution of acyl groups at the C8 with keto or hydroxy moieties and was also dependent on the nature of substitutions at the C3, C4 and C15 positions. Sambucinol and the trichothecene precursor trichodiene, metabolites which do not contain a 12,13-epoxide, did not inhibit lymphocyte proliferation. The results suggest the need for further assessment of occurrence and in vivo toxicity of Fusarium metabolites, particularly the substituted neosolaniols and calonectrins.

Occurrence of Fusarium species and mycotoxins in nepalese maize and wheat and the effect of traditional processing methods on mycotoxin levels.

Maize (Zea mays) and wheat (Triticum aestivum) collected in the foothills of the Nepal Himalaya Mountains were analyzed for Fusarium species and mycotoxins: fumonisins, nivalenol (NIV), and deoxynivalenol (DON). Predominant species were Gibberella fujikuroi mating population A (F. moniliforme) in maize and F. graminearum in maize and wheat; G. fujikuroi mating population D (F. proliferatum), F. acuminatum, F. avenaceum, F. chlamydosporum, F. equiseti, F. oxysporum, F. semitectum, and F. torulosum were also present. Strains of G. fujikuroi mating population A produced fumonisins, and strains of F. graminearum produced NIV or DON. By immunoassay or high-performance liquid chromatography, fumonisins were >1000 ng/g in 22% of 74 maize samples. By immunoassay or fluorometry, NIV and DON were >1000 ng/g in 16% of maize samples but were not detected in wheat. Fumonisins and DON were not eliminated by traditional fermentation for producing maize beer, but Nepalese rural and urban women were able to detoxify contaminated maize by hand-sorting visibly diseased kernels.

Apolipoprotein E variation in patients with hyperlipidaemia: DNA and protein phenotyping.

Two patients with marked elevation of plasma and very low density lipoprotein (VLDL) lipids were investigated to establish the molecular basis of their hyperlipidaemia. In one the demonstration of the apolipoprotein E 2/2 phenotype substantiated the diagnosis of type III hyperlipidaemia. In the other the E4/3 phenotype excluded this diagnosis. In both cases oligonucleotide probing of amplified DNA and isoelectric focusing (IEF) of apo VLDL identified the correct apolipoprotein E phenotype as defined by peptide mapping and IEF of purified apolipoprotein E after modification with iodoacetic acid. Probing of amplified DNA clearly distinguishes the three common variants of apolipoprotein E (E2, E3, E4) and facilitates the diagnosis of type III hyperlipidaemia.

A new approach for studying gene regulation by distant DNA elements in transgenic mice.

Apolipoprotein B (apo-B) plays a crucial role in the assembly of lipoproteins in the liver and the intestine. Here, we review how transgenic mouse expression studies with large genomic clones have been used to define distant cis-acting regulatory DNA sequences that control the expression of the apo-B gene. In early studies, apo-B transgenic mice were generated with approximately 80-kb P1 bacteriophage clones spanning either the human or the mouse apo-B genes. Both the human and mouse clones directed high levels of transgene expression in the liver, but transgene expression was absent in the intestine. The absence of transgene expression in the intestine was surprising because both P1 clones contained more than 11 kb of flanking sequences both 5' and 3' to the gene. Subsequently, we isolated and characterized 145-kb and 207-kb bacterial artificial chromosome (BAC) clones that spanned the human apo-B gene. Each of these BAC's contained extensive 5 and 3' flanking sequences and each directed spatially and physiologically appropriate apo-B gene expression in the intestines of transgenic mice. To define the location of the sequences that control intestinal expression of the apo-B gene, we generated transgenic mice by co-microinjecting the approximately 80-kb P1 bacteriophage clone (which did not confer intestinal expression of apo-B) with either the 5' sequences or the 3' sequences from the 145-kb BAC. Analysis of the apo-B expression pattern in those mice revealed that the DNA sequences controlling intestinal expression were located 5' to the apo-B gene. Next, we used recA-assisted restriction endonuclease (RARE) cleavage to truncate specific segments of the 5' and 3' flanking sequences from the 145-kb BAC. A series of the truncated BAC's containing different lengths of 5' and 3' sequences was used to generate more than 40 additional lines of human apo-B transgenic mice. Analysis of human apo-B gene expression in those mice demonstrated that the sequences controlling the expression of the apo-B gene in the intestine are located more than 50 kb 5' to the apo-B gene. Our studies demonstrate that the RARE cleavage/transgenic expression strategy is a powerful approach for examining gene regulation by distant gene-regulatory elements.

A novel gene cluster in Fusarium graminearum contains a gene that contributes to butenolide synthesis.

The development of expressed sequence tag (EST) databases, directed transformation and a sequenced genome has facilitated the functional analysis of Fusarium graminearum genes. Extensive analysis of 10,397 ESTs, derived from thirteen cDNA libraries of F. graminearum grown under diverse conditions, identified a novel cluster of eight genes (gene loci fg08077-fg08084) located within a 17kb region of genomic sequence contig 1.324. The expression of these genes is concomitantly up-regulated under growth conditions that promote mycotoxin production. Gene disruption and add-back experiments followed by metabolite analysis of the transformants indicated that one of the genes, fg08079, is involved in butenolide synthesis. The mycotoxin butenolide is produced by several Fusarium species and has been suggested, but not proven, to be associated with tall fescue toxicoses in grazing cattle. This is the first report of the identification of a gene involved in the biosynthetic pathway of butenolide.