Differential regulation of keratin 8 and 18 messenger RNAs in differentiating F9 cells.

F9 embryonal carcinoma cells (F9EC) can be induced to differentiate in vitro into epithelial cells expressing keratin 8 (K8) and keratin 18 (K18). cDNAs corresponding to K8 and K18 mRNAs were cloned and used to study the change in the abundance of these mRNAs during differentiation of F9 cells into parietal endoderm-like cells by treatment with retinoic acid (RA) or with RA and dibutyryl cAMP (Bt2cAMP). Using an RNase protection assay, it was determined that K8 mRNA was induced slightly before K18 mRNA and that it accumulated to a greater extent than K18 mRNA. Furthermore, differentiation in presence of Bt2cAMP plus RA resulted in an earlier induction of the two mRNAs and a higher level of expression of K8 mRNA. These results indicate that K8 and K18 mRNAs are regulated differently in F9 cells.

Complete sequence of the mouse type-II keratin EndoA: its amino-terminal region resembles mitochondrial signal peptides.

We have isolated a clone, pKA56, from a cDNA library prepared from poly(A)/RNA of F9ACc19 cells. Northern-blot analysis showed that this clone recognizes a 1.9-kb mRNA which is expressed strongly in F9 differentiated cells but only faintly detected in F9 stem cells. Sequence determination revealed that this mRNA codes for EndoA, the murine homologue of the human type-II keratin No. 8. This is the first report of the complete coding sequence of a mammalian keratin No. 8. Comparison of mouse EndoA with keratin No. 8 of humans, cows and frogs indicated a strong evolutionary conservation. The first 16 amino acid residues of the N-terminal domain of EndoA are also homologous to other type-II keratins and, to a lesser extent, to other intermediate filament (IF) proteins. Furthermore, this region is predicted to adopt an amphiphilic alpha-helical conformation similar to that of mitochondrial signal peptides. Conservation of that sequence and of other segments of the end domains of EndoA supports the idea that those regions are implicated in the specific organization of the IF network in the cell and in the interactions of IF with other cell constituents.

Mouse keratin 19: complete amino acid sequence and gene expression during development.

The complete amino acid sequence of the mouse keratin 19 (K19) was determined from a partial sequence of cDNA isolated from a mouse (day 10.5) embryo library and an amplified genomic fragment. Analysis of the sequence reveals strong evolutionary conservation with other K19s. Examination of the expression of the gene encoding K19 (K19) during development using an RNase protection assay reveals it is expressed in extra-embryonic tissues by day 8.5 and in the embryo proper by at least day 9.5. Furthermore, the K19 gene is induced in differentiating F9 embryonal carcinoma cells. These results indicate that K19 is another keratin, in addition to the K8-K18 pair, which is synthesized early during mouse development. Finally, Southern analysis of the K19 gene reveals that it is found as a unique copy in the mouse genome, in contrast to what is found in humans, which have at least one processed pseudogene.

Interaction of Fusarium graminearum and F. moniliforme in Maize Ears: Disease Progress, Fungal Biomass, and Mycotoxin Accumulation.

ABSTRACT To investigate the interaction between two major ear-rotting pathogens, maize ears were inoculated with either Fusarium graminearum, F. moniliforme, or an equal mixture of the two. Silk and kernel tissues were periodically harvested throughout the growing season so that a time course of the experimental variables (disease severity, ergosterol content, fungal DNA content, and mycotoxin concentration) could be recorded. Over the 3 years tested (1992 to 1994), the highest levels of disease and ergosterol were found in the F. graminearum treatment, followed by the mixture treatment (F. graminearum plus F. moniliforme) and, finally, the F. moniliforme treatment. Kernel ergosterol content and disease rating were correlated for both pathogens, but the highest correlation coefficients were obtained in the F. graminearum treatment. The DNA analysis revealed that, in the mixed inoculum, F. moniliforme had a greater growth rate than did F. graminearum. In 1994, appreciable F. moniliforme from natural inoculum was found in the F. graminearum treatment. Fumonisin B(1) levels did not differ between the F. moniliforme treatment and the mixed inoculum treatment. The effect of temperature on the growth rate of the two species explained some of the field results, with temperatures in the silks being more favorable to F. moniliforme. Data on the growth rate on silks obtained by the incorporation of radiolabeled precursor to ergosterol demonstrated that F. graminearum was able to grow well at 26 to 28 degrees C, whereas F. moniliforme grew well over a broader range, including at higher temperatures.

Low temperature induced defence gene expression in winter wheat in relation to resistance to snow moulds and other wheat diseases.

Cold hardening of winter wheat at 2 °C for 1-6 wks increased resistance to the snow mould pathogens LTB, Typhula incarnata, and Microdochium nivale as well as to powdery mildew (Blumaria graminis f. sp. graminis) and stripe rust (Puccinia striiformis). Using microarrays and hardening of winter wheat for 0.25, 0.5, 1, 7, 21 and 49 d, an upregulation of a wide range of stress-response genes that include defence-related and abiotic stress-related genes, transcription factors including several lipoxygenases and ethylene responsive factors, and WRKY genes was observed. For the majority of these genes, the upregulation occurred later in the 21-49 d hardening treatments and coincided with the highest expression levels of snow mould resistance. Defence-related sequences were upregulated to a greater extent and were more numerous in the snow mould resistant line CI14106 compared to cold hardy DH+268. Transcript profiling of candidate defence and other stress-related genes under prolonged conditions at -3 °C with or without snow mould infection showed that there was a decline in transcripts of the defence-related genes PR1.1b and NPR3 during the 12wks incubation. Additionally, 14 d hardening was insufficient to permit full expression of the jasmonic acid synthesis gene, allene oxide synthase (AOS) and the fructan degrading enzyme ß-fructofuranosidase compared the 42 d hardening treatment. The snow mould resistant line CI14106 was able to maintain higher transcript levels of AOS for longer conditions compared to the susceptible line Norstar under artificial snow mould conditions. These results explain the nature of cold-induced resistance to snow moulds and provide direction on establishing selection criteria for improving resistance and cold tolerance in winter wheat.

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.

Characterization of a novel glycine-rich protein from the cell wall of maize silk tissues.

The isolation, characterization and regulation of expression of a maize silk-specific gene is described. zmgrp5 (Zea mays glycine-rich protein 5) encodes a 187 amino acid glycine-rich protein that displays developmentally regulated silk-specific expression. Northern, Western, in situ mRNA hybridization and transient gene expression analyses indicate that zmgrp5 is expressed in silk hair and in cells of the vascular bundle and pollen tube transmitting tissue elements. The protein is secreted into the extracellular matrix and is localized in the cell wall fraction mainly through interactions mediated by covalent disulphide bridges. Taken together, these results suggest that the protein may play a role in maintaining silk structure during development. This is the first documented isolation of a stigma-specific gene from maize, an important agronomic member of the Poaceae family.

Metal ions modulate gene expression and accumulation of the mycotoxins aflatoxin and zearalenone.

AIMS: To determine the modulating action of some metal ions (Zn+2, Fe+2, Cu+2) on gene expression of enzymes related to fungal growth and accumulation of the mycotoxins aflatoxin and zearalenone. METHODS AND RESULTS: The effect of the metal ions, as single or mixed treatments, was observed in submerged cultures of toxigenic Aspergillus flavus or Fusarium graminearum, which produce the mycotoxins aflatoxin or zearalenone, respectively. The enzyme-linked immunosorbent assay results showed that the single metals Zn+2 or Cu+2 stimulated aflatoxin accumulation while Cu+2 or Fe+2 stimulated zearalenone in fungal cultures. Single Zn+2 treatment also affected conidial differentiation and pigmentation. A cDNA suppression subtractive library was also produced and followed by sequencing of potential metal treatment-specific clones, thus determining induced genes. The genes uncovered included enzymes and regulators of cell growth and division, including many genes with unknown functions were uncovered. A Northern blot analysis was used to verify the expression pattern of the corresponding genes under metal treatment. The metal ions enhanced the expression of alcohol dehydrogenase Adh1 homologue by up to 33-fold in A. flavus and ca fourfold in F. graminearum. Encoding homologues of a neutral amino acid permease, were also used in the Northern analysis. However, the expression of the permease was not significantly affected by metal ion treatments. CONCLUSIONS: The results showed a significant effect of metal ions on expression of gene related to fungal growth, development, conidiation and production of both aflatoxin and zearalenone. SIGNIFICANT AND IMPACT OF THE STUDY: At the molecular and cellular level, the significant effects of metal ions on fungal growth and development, conidiation, and production of both aflatoxin and zearalenone were demonstrated.

Mapping of genes expressed in Fusarium graminearum-infected heads of wheat cultivar 'Frontana'.

The isolation, physical, and genetic mapping of a group of wheat genes expressed in infected heads of Triticum aestivum 'Frontana' resistant to Fusarium head blight is reported. A cDNA library was built from heads of 'Frontana' through suppressive subtractive hybridization, to enrich for sequences induced by the pathogen Fusarium graminearum during infection. A group of 1794 clones was screened by dot blot hybridization for differential gene expression following infection. Twenty of these clones showed a strong difference in intensity of hybridization between infected and mock-inoculated wheat head samples, suggesting that they corresponded to genes induced during infection. The 20 clones were sequenced and used for mapping analysis. We determined a precise chromosomal location for 14 selected clones by using series of chromosome deletion stocks. It was shown that the 14 clones detected 90 fragments with the use of the restriction enzyme EcoRI; 52 bands were assigned to chromosome bins, whereas 38 fragments could not be assigned. The selected clones were also screened for polymorphisms on a 'Wuhan' x 'Maringa' wheat doubled haploid mapping population. One clone, Ta01_02b03, was related to a quantitative trait locus for type II resistance located on chromosome 2AL, as determined with simple sequence repeat markers on another mapping population, but did not map in the same location on our population. Another clone, Ta01_06f04, was identified by BLAST (basic local alignment search tool) search in public databases to code for a novel beta-1,3-glucanase, homologous to a major pathogenesis-related protein. This clone mapped to chromosomal regions on chromosome 3, including 3BL and 3DL, where B glucanase gene clusters are known to exist. Seven other clones, including 1 coding for an ethylene-response element binding protein and 3 for ribosomal proteins, and 4 clones corresponding to proteins with unknown function, were also mapped.

The maize An2 gene is induced by Fusarium attack and encodes an ent-copalyl diphosphate synthase.

Using the technique of differential display, a maize transcript was identified whose silk tissue expression is induced in the presence of the ear rot pathogen Fusarium graminearum. The 3445 nt transcript includes a 727 nt 5' untranslated leader with the potential for extensive secondary structure and represents the maize gene An2. An2 encodes a copalyl diphosphate synthase (CPS)-like protein with 60% amino acid sequence identity with the maize An1 gene product involved in gibberellin (GA) biosynthesis. Recombinant expression and functional analysis demonstrated that both AN1 and AN2 are ent-copalyl diphosphate (ent-CPP) synthases (ent-CPS). Notably, the presence of an additional ent-CPS gene is consistent with previous reports that maize GA biosynthesis can proceed in the absence of An1. In addition, northern blot analysis showed that An2 transcript levels were strongly up-regulated by Fusarium attack, with an increase in silk, husk and ear tip tissues as early as 6 h after inoculation of silk channels with spore suspensions of various Fusarium sp. Gene expression of a third maize CPS-like gene, Cpsl1, is not affected by Fusarium infection. The Fusarium-inducible nature of An2 is also consistent with a previous report that cell-free extracts from maize seedlings produce ent-CPP derived diterpenes in response to Fusarium infection. However, it is not known whether An2 is involved in defense-related secondary metabolism in addition to GA synthesis.

Members of the acetohydroxyacid synthase multigene family of Brassica napus have divergent patterns of expression.

Brassica species possess the most complex acetohydroxyacid synthase (AHAS) multigene family reported for plants. The AHAS genes code for an essential enzyme in branched-chain amino acid biosynthesis. In the allotetraploid species, B. napus, four (AHAS1-4) of the five AHAS genes have been cloned and sequenced. The transcripts were examined by RNase protection assays using gene-specific, antisense RNA probes. Only AHAS1, AHAS2 and AHAS3 were shown to be expressed in B. napus and one of the diploid progenitor species B. campestris or B. oleracea. AHAS1 and AHAS3 are highly conserved genes that presumably code for the essential AHAS housekeeping functions. They were expressed as low abundance mRNA in all somatic and reproductive tissues examined. AHAS2, which is structurally distinct from all other plant AHAS genes, was only expressed in mature ovules and extraembryonic tissues of immature seeds. This study provides direct evidence for multiple AHAS isoforms in plants and for an AHAS gene which is developmentally regulated in a tissue-specific manner. The discovery raises questions concerning the functional significance of AHAS in seed development.

Rapid genomic changes in interspecific and intergeneric hybrids and allopolyploids of Triticeae.

Allopolyploidy is preponderant in plants, which often leads to speciation. Some recent studies indicate that the process of wide hybridization and (or) genome doubling may induce rapid and extensive genetic and epigenetic changes in some plant species and genomic stasis in others. To further study this phenomenon, we analyzed three sets of synthetic allopolyploids in the Triticeae by restriction fragment length polymorphism (RFLP) using a set of expressed sequence tags (ESTs) and retrotransposons as probes. It was found that 40-64.7% of the ESTs detected genomic changes in the three sets of allopolyploids. Changes included disappearance of parental hybridization fragment(s), simultaneous appearance of novel fragment(s) and loss of parental fragment(s), and appearance of novel fragment(s). Some of the changes occurred as early as in the F1 hybrid, whereas others occurred only after allopolyploid formation. Probing with retrotransposons revealed numerous examples of disappearance of sequences. No gross chromosome structural changes or physical elimination of sequences were found. It is suggested that DNA methylation and localized recombination at the DNA level were probably the main causes for the genomic changes. Possible implications of the genomic changes for allopolyploid genome evolution are discussed.

Metal ion enhancement of fungal growth, gene expression and aflatoxin synthesis in Aspergillus flavus: RT-PCR characterization.

AIMS: To determine the effect of mineral ions (e.g., Zn2+, Cu2+, and Fe2+) on the enhancement of fungal growth, total RNA, aflatoxin pathway gene expression, and production of aflatoxin and its precursor O-methylsterigmatocystin (OMST). METHODS AND RESULTS: The influence of the metal ions, as a single or mixed treatments, was observed in submerged cultures of toxigenic Aspergillus flavus through changes in the fungal RNA or aflatoxin pathway gene (omtA) by reverse transcription-polymerase chain reaction (RT-PCR) levels, and also in fungal dry-weight accumulation, aflatoxin and OMST production. CONCLUSIONS: The ion treatments induced changes of fungal total RNA, mRNA levels, associate fungal growth, biosynthesis of aflatoxin and OMST, and enhanced expression of RT-PCR. SIGNIFICANCE AND IMPACT OF STUDY: Demonstrates at the cellular and molecular level, the significant effects of metal ions on both fungal growth and production of aflatoxin.

Differential expression of keratin genes during mouse development.

Suprabasal layers of the newborn mouse epidermis contain two mRNAs of 2.0 and 2.4 kb which are translated into keratins of 59 and 67 kDa, respectively. To study their expression during development, cDNA sequences corresponding to the 2.0- and the 2.4-kb mRNAs were cloned, characterized by hybridization selection assay, and used as probes to detect keratin sequences in polyadenylated RNA from Day 11, 13, 15, and 17 embryos. In RNA from Day 11 of gestation, two RNAs of 2.8 and 1.8 kb were identified. They were found to have homologies with both epidermal RNAs, suggesting that they are coding for proteins of the keratin family. These two sequences were not detected in sample of later stages. RNAs comigrating with the two epidermal keratin RNAs were identified only in Day 15 and 17 embryos indicating that their expression was induced between Day 13 and 15. Finally, the localization of the 59-kDa keratin mRNA was examined by in situ hybridization. The spinous and granulous cell layers were found to be heavily covered with grains while other regions of the tissue sections were unlabeled. All these results support the hypothesis of a sequential expression of keratins during differentiation of epidermal cells and suggest that proteins related to the keratins expressed specifically in keratinizing cells are expressed earlier during development.

Characterization of six wheat x Thinopyrum intermedium derivatives by GISH, RFLP, and multicolor GISH.

Restriction fragment length polymorphism (RFLP) analysis and multicolor genomic in situ hybridization (GISH) are useful tools to precisely characterize genetic stocks derived from crosses of wheat (Triticum aestivum) with Thinopyrum intermedium and Thinopyrum elongatum. The wheat x Th. intermedium derived stocks designated Z1, Z2, Z3, Z4, Z5, and Z6 were initially screened by multicolor GISH using Aegilops speltoides genomic DNA for blocking and various combinations of genomic DNA from Th. intermedium, Triticum urartu, and Aegilops tauschii for probes. The probing (GISH) results indicated that lines Z1 and Z3 were alien disomic addition lines with chromosome numbers of 2n = 44. Z2 was a substitution line in which chromosome 2D was substituted by a pair of Th. intermedium chromosomes; this was confirmed by RFLP and muticolour GISH. Z4 (2n = 44) contained two pairs of wheat--Th. intermedium translocated chromosomes; one pair involved A-genome chromosomes, the other involved D- and A- genome chromosomes. Z5 (2n = 44) contained one pair of wheat--Th. intermedium translocated chromosomes involving the D- and A-genome chromosomes of wheat. Z6 (2n = 44) contained one pair of chromosomes derived from Th. intermedium plus another pair of translocated chromosomes involving B-genome chromosomes of wheat Line Z2 was of special interest because it has some resistance to infection by Fusarium graminearum.

Differential expression of the epidermal K1 and K10 keratin genes during mouse embryo development.

Induction of genes coding for the K1 and K10 keratins during mouse development was studied by measuring the accumulation of their respective mRNAs in day 10 to 17 embryos using an RNase protection assay. Although these two keratins are coexpressed in the suprabasal layers of the epidermis, it was found that while K1 mRNA was detectable as soon as day 10, K10 mRNA was not detectable before day 12. The expression of these genes at this stage of development was not expected since they are specifically associated with keratinization, a process that does not begin before day 17 of gestation. Histological examination of the epidermis of day 10 to 17 embryos suggests that both genes are induced in cells committed to epidermal differentiation, after stratification has started but before the onset of keratinization. It was also found that the two mRNAs increased in abundance steadily and significantly until day 16 and that, in spite of the expectation that filaments should contain equivalent amounts of each subunit, K1 mRNA remained more abundant than K10 mRNA at all times including in adult epidermis. These observations indicate that the two genes are regulated independently during development.

Tri1 in Fusarium graminearum encodes a P450 oxygenase.

Gibberella zeae (asexual state Fusarium graminearum) is a major causal agent of wheat head blight and maize ear rot in North America and is responsible for contamination of grain with deoxynivalenol and related trichothecene mycotoxins. To identify additional trichothecene biosynthetic genes, cDNA libraries were prepared from fungal cultures under trichothecene-inducing conditions in culture and in planta. A gene designated LH1 that was highly expressed under these conditions exhibited only moderate (59%) similarity to known trichothecene biosynthetic cytochrome P450s. To determine the function of LH1, gene disruptants were produced and assessed for trichothecene production. Gene disruptants no longer produced 15-acetyldeoxynivalenol, which is oxygenated at carbon 7 (C-7) and C-8, but rather accumulated calonectrin and 3-deacetylcalonectrin, which are not oxygenated at either C-7 or C-8. These results indicate that gene LH1 encodes a cytochrome P450 responsible for oxygenation at one or both of these positions. Despite the relatively low level of DNA and amino acid sequence similarity between the two genes, LH1 from G. zeae is the probable homologue of Tri1, which encodes a cytochrome P450 required for C-8 oxygenation in F. sporotrichioides.

Molecular verification and characterization of BYDV-resistant germ plasms derived from hybrids of wheat with Thinopyrum ponticum and Th. intermedium.

Twenty-five partial amphiploids (2n=8x=56), which were derived from hybrids of wheat (Triticum aestivum L.) with either Thinopyrum ponticum (Podpera) Liu & Wang, Th. intermedium (Host) Barkworth & D. Dewey, or Th. junceum (L.) A. Löve, were assayed for resistance to BYDV serotype PAV by slot-blot hybridization with viral cDNA of a partial coat protein gene. Three immune lines were found among seven partial amphiploids involving Th. ponticum. Seven highly resistant lines were found in ten partial amphiploids involving Th. intermedium. None of eight partial amphiploids or 13 addition lines of Chinese Spring - Th. junceum were resistant to BYDV. Genomic in situ hybridization demonstrated that all of the resistant partial amphiploids, except TAF46, carried an alien genome most closely related to St, whether it was derived from Th. ponticum or Th. intermedium. The two partial amphiploids carrying an intact E genome of Th. ponticum are very susceptible to BYDV-PAV. In TAF46, which contains three pairs of St- and four pairs of E-genome chromo somes, the gene for BYDV resistance has been located to a modified 7 St chromosome in the addition line L1. This indicates that BYDV resistance in perennial polyploid parents, i.e., Th. ponticum and Th. intermedium, of these partial amphiploids is probably controlled by a gene(s) located on the St-genome chromosome(s).

An acetohydroxy acid synthase mutant reveals a single site involved in multiple herbicide resistance.

Acetohydroxy acid synthase (AHAS) is an essential enzyme for many organisms as it catalyzes the first step in the biosynthesis of the branched-chain amino acids valine, isoleucine, and leucine. The enzyme is under allosteric control by these amino acids. It is also inhibited by several classes of herbicides, such as the sulfonylureas, imidazolinones and triazolopyrimidines, that are believed to bind to a relic quinone-binding site. In this study, a mutant allele of AHAS3 responsible for sulfonylurea resistance in a Brassica napus cell line was isolated. Sequence analyses predicted a single amino acid change (557 Trp-->Leu) within a conserved region of AHAS. Expression in transgenic plants conferred strong resistance to the three classes of herbicides, revealing a single site essential for the binding of all the herbicide classes. The mutation did not appear to affect feedback inhibition by the branched-chain amino acids in plants.

Transformation of Brassica napus canola cultivars with Arabidopsis thaliana acetohydroxyacid synthase genes and analysis of herbicide resistance.

A survey of selected crop species and weeds was conducted to evaluate the inhibition of the enzyme acetohydroxyacid synthase (AHAS) and seedling growth in vitro by the sulfonylurea herbicides chlorsulfuron, DPX A7881, DPX L5300, DPX M6316 and the imidazolinone herbicides AC243,997, AC263,499, AC252,214. Particular attention was given to the Brassica species including canola cultivars and cruciferous weeds such as B. kaber (wild mustard) and Thlaspi arvense (stinkweed). Transgenic lines of B. napus cultivars Westar and Profit, which express the Arabidopsis thaliana wild-type AHAS gene or the mutant gene csr1-1 at levels similar to the resident AHAS genes, were generated and compared. The mutant gene was essential for resistance to the sulfonylurea chlorsulfuron but not to DPX A7881, which appeared to be tolerated by certain Brassica species. Cross-resistance to the imidazolinones did not occur. The level of resistance to chlorsulfuron in transgenic canola greatly exceeded the levels that were toxic to the Brassica species or cruciferous weeds. Direct selection of transgenic lines with chlorsulfuron sprayed at field levels under greenhouse conditions was achieved.