Cellular localization of the embryo-specific hybrid PRP from Zea mays, and characterization of promoter regulatory elements of its gene.

The expression, regulation and cellular localization of ZmHyPRP, a gene marker of embryo differentiation whose expression declines after ABA induction, was studied. ZmHyPRP is a proline-rich protein with a C-terminal domain having eight cysteines in a CM8 pattern. Transient expression in onion epidermal cells, transformed with a 2x35S::ZmHyPRP-GFP construction, indicated the protein is present in vesicles lining the membrane of the cell. The ZmHyPRP gene expression is under the control of classic promoter seed-specific regulatory elements such as Sph/RY and G-boxes, suggesting regulation by B3 and b-ZIP transcription factors. Promoter deletion analysis, by particle-bombardment transient transformation of maize immature embryos with serial deletions of the promoter fused to GUS, showed the presence of two negative regulatory elements, NE1 (-2070 to -1280) and NE2 (-232 to -178), in the ZmHyPRP promoter. By selective deletion or mutation of ZmHyPRP regulatory promoter elements we conclude that the promoter expression is attenuated by the NE2 element as well as by the G-box2 and the Sph1-2 box together with the G-box2.

A maize defective-kernel mutant (longcell) characterized by tubular cells, severe morphological alterations and induction of cell death.

Seeds of the longcell mutant in maize (Zea mays L) have a defective-kernel phenotype: the embryo aborts at the early coleoptilar stage and the endosperm is reduced in size. Mutant embryos have severe alterations in morphogenesis. They have a suspensor-, an embryo axis- and a scutellum-like structure, but the shoot apical meristem (SAM) is not formed. Scanning electron microscopy showed that most of the cells in longcell embryos are tubular and abnormally enlarged. The level of expression of several genes involved in basic metabolism is not severely affected during early and mid embryogenesis, but storage molecule accumulation is reduced. Genes which in normal conditions are only expressed after germination, are expressed during kernel development in the longcell seeds. Mutant embryos undergo cell death in late embryogenesis. Nuclei in dying embryos are TUNEL positive, and different genes coding for hydrolytic enzymes are up-regulated. The expression of genes related to oxidative stress is also altered in longcell embryos. These results lead us to suggest that the longcell mutant may be cytokinesis-defective.

Adsorption of recombinant human bone morphogenetic protein rhBMP-2m onto hydroxyapatite.

Recombinant human mature bone morphogenetic protein 2 (rhBMP-2m) has been expressed to study its adsorption onto precipitated hydroxyapatite (HA). The influence on the adsorption process of different parameters such as pH and concentrations of calcium, phosphate or NaCl has been investigated. Although the adsorption proceeds rapidly at the initial stages, the maximum adsorbed amount is reached after four hours. The process is notably influenced by adding calcium or phosphate to the system but, while calcium ions increase the adsorption of rhBMP-2m onto HA, phosphate ions inhibit it. The influence of pH and NaCl concentration are notable but less important than those of calcium and phosphate. The adsorption data fit well to a Langmuir adsorption isotherm. The values of the isotherm parameters have been calculated and discussed, and an adsorption mechanism has been proposed.

Analysis of regulatory elements of the promoter and the 3' untranslated region of the maize Hrgp gene coding for a cell wall protein.

Hydroxyproline-rich glycoproteins (HRGP) are structural components of the plant cell wall. Hrgp genes from maize and related species have a conserved 500 bp sequence in the 5'-flanking region, and all Hrgp genes from monocots have an intron located in the 3' untranslated region. To study the role of these conserved regions, several deletions of the Hrgp gene were fused to the beta-glucuronidase ( GUS) gene and used to transform maize tissues by particle bombardment. The overall pattern of GUS activity directed by sequential deletions of the Hrgp promoter was different in embryos and young shoots. In embryos, the activity of the full-length Hrgp promoter was in the same range as that of the p35SI promoter construct, based on the strong 35S promoter, whereas in the fast-growing young shoots it was 20 times higher. A putative silencer element specific for young shoots was found in the -1,076/-700 promoter region. Other major cis elements for Hrgp expression are probably located in the regions spanning -699/-510 and -297/-160. Sequences close to the initial ATG and mRNA leader were also important since deletion of the region -52/+16 caused a 75% reduction in promoter activity. The presence of the Hrgp intron in the 3' untranslated region changed the levels of GUS activity directed by the Hrgp and the 35S promoters. This pattern of activity was complex, and was dependent on the promoter and cell type analysed.

Three Tnt1 subfamilies show different stress-associated patterns of expression in tobacco. Consequences for retrotransposon control and evolution in plants.

The genomes of most Nicotiana species contain three different subfamilies of the Tnt1 retrotransposon, which differ completely in their U3 sequence, whereas the rest of the sequence is relatively constant. The results presented here show that all three Tnt1 subfamilies are expressed in tobacco (Nicotiana tabacum) and that the U3 sequence variability correlates with differences in the pattern of expression of the Tnt1 elements. Each of the three Tnt1 subfamilies is induced by stress, but their promoters have a different response to different stress-associated signaling molecules. The Tnt1A subfamily is particularly strongly induced by elicitors and methyl jasmonate, whereas expression of the Tnt1C subfamily is more sensitive to salicylic acid and auxins. The direct relationship between U3 sequence variability and differences in the stress-associated expression of the Tnt1 elements present in a single host species gives support to our model that postulates that retrotransposons have adapted to their host genomes through the evolution of highly regulated promoters that mimic those of the stress-induced plant genes. Moreover, here we show that the analysis of the transcriptional control of a retrotransposon population such as Tnt1 provides new insights into the study of the complex and still poorly understood network of defense- and stress-induced plant signal transduction pathways.

Early and multiple Ac transpositions in rice suitable for efficient insertional mutagenesis.

A GFP excision assay was developed to monitor the excision of Ac introduced into rice by Agrobacterium-mediated transformation. The presence of a strong double enhancer element of the CaMV 35S promoter adjacent to the Ac promoter induced very early excision, directly after transformation into the plant cell, exemplified by the absence of Ac in the T-DNA loci. Excision fingerprint analysis and characterization of transposition events from related regenerants revealed an inverse correlation between the number of excision events and transposed Ac copies, with single early excisions after transformation generating Ac amplification. New transpositions were generated at a frequency of 15-50% in different lines, yielding genotypes bearing multiple insertions, many of which were inherited in the progeny. The sequence of DNA flanking Ac in three representative lines provided a database of insertion tagged sites suitable for the identification of mutants of sequenced genes that can be examined for phenotypes in a reverse genetics strategy to elucidate gene function. Remarkably, two-thirds of Ac tagged sites showing homology to sequences in public databases were in predicted genes. A clear preference of transposon insertions in genes that are either predicted by protein coding capacity or by similarity to ESTs suggests that the efficiency of recovering knockout mutants of genes could be about three times higher than random. Linked Ac transposition, suitable for targeted tagging, was documented by segregation analysis of a crippled Ac element and by recovery of a set of six insertions in a contiguous sequence of 70 kb from chromosome 6 of rice.

Differential distribution of cAMP-specific phosphodiesterase 7A mRNA in rat brain and peripheral organs.

We investigated the regional distribution and cellular localization of mRNA coding for the cAMP-specific phosphodiesterase 7A (PDE7A) in rat brain and several peripheral organs by in situ hybridization histochemistry. The regional expression of two splice variants, PDE7A1 and PDE7A2, was examined by RT-PCR using RNA extracted from several brain regions. PDE7A mRNA was found to be widely distributed in rat brain in both neuronal and nonneuronal cell populations. The highest levels of hybridization were observed in the olfactory bulb, olfactory tubercle, hippocampus, cerebellum, medial habenula nucleus, pineal gland, area postrema, and choroid plexus. Positive hybridization signals were also detected in other areas, such as raphe nuclei, temporal and entorhinal cortex, pontine nuclei, and some cranial nerve motor nuclei. Both mRNA splice forms were differentially distributed in several areas of the brain with the striatum expressing only PDE7A1 and the olfactory bulb and spinal cord expressing PDE7A2 exclusively. In peripheral organs the highest levels of PDE7A hybridization were seen in kidney medulla, although testis, liver, adrenal glands, thymus, and spleen also presented high hybridization signal. These results are consistent with PDE7A being involved in the regulation of cAMP signaling in many brain functions. The consistent colocalization with PDE4 mRNAs suggests that PDE7A could have an effect on memory, depression, and emesis. The results offer clear anatomical and functional systems in which to investigate future specific PDE7 inhibitors.

Sequence and analysis of chromosome 3 of the plant Arabidopsis thaliana.

Arabidopsis thaliana is an important model system for plant biologists. In 1996 an international collaboration (the Arabidopsis Genome Initiative) was formed to sequence the whole genome of Arabidopsis and in 1999 the sequence of the first two chromosomes was reported. The sequence of the last three chromosomes and an analysis of the whole genome are reported in this issue. Here we present the sequence of chromosome 3, organized into four sequence segments (contigs). The two largest (13.5 and 9.2 Mb) correspond to the top (long) and the bottom (short) arms of chromosome 3, and the two small contigs are located in the genetically defined centromere. This chromosome encodes 5,220 of the roughly 25,500 predicted protein-coding genes in the genome. About 20% of the predicted proteins have significant homology to proteins in eukaryotic genomes for which the complete sequence is available, pointing to important conserved cellular functions among eukaryotes.

Distribution of microsatellites in relation to coding sequences within the Arabidopsis thaliana genome.

The distribution of repetitive sequences, or microsatellites, formed by either one or two base pairs and longer than eight units, has been studied in almost 1 Mb of the sequenced Arabidopsis thaliana genome. Except for those formed by only G and C residues, the repetitions are more abundant in the Arabidopsis genome than can be calculated from its nucleotide composition. They are distributed in proportions higher than expected in introns, and in the intergenic regions both proximal and distal to the coding sequences. In exons, only the TC/GA microsatellite seems to be particularly abundant. The AT/TA microsatellites produce more length variation between Arabidopsis ecotypes than the A/T repeated sequences. These two classes are more abundant per kilobase than coding sequences in the Arabidopsis genome. The results indicate not only that the presence of microsatellites is not an effect of random distribution of nucleotides, but that their resolution as molecular markers may be equivalent to the number of genes and also that they do not seem to be systematically linked to specific regulatory sequences proximal to genes.

Phosphodiesterases 4D and 7A splice variants in the response of HUVEC cells to TNF-alpha(1).

The mRNA accumulation of phosphodiesterases PDE4D and PDE7A was studied by RNA blot analysis in human umbilical vein endothelial cells (HUVEC) incubated with TNFalpha for different periods. A contrasting behaviour was observed in the mRNA accumulation of the two genes. Further analysis by RT-PCR of the PDE4D and PDE7A splice variants gave different accumulation patterns which may indicate that differential splicing has a role in the regulation of these enzymes. Three previously undescribed PDE4D isoforms, with different accumulation patterns, were also detected. They code for truncated PDE4D isoforms, which could participate in the regulation of PDE4D activity.

Progress in Arabidopsis genome sequencing and functional genomics.

Arabidopsis thaliana has a relatively small genome of approximately 130 Mb containing about 10% repetitive DNA. Genome sequencing studies reveal a gene-rich genome, predicted to contain approximately 25000 genes spaced on average every 4.5 kb. Between 10 to 20% of the predicted genes occur as clusters of related genes, indicating that local sequence duplication and subsequent divergence generates a significant proportion of gene families. In addition to gene families, repetitive sequences comprise individual and small clusters of two to three retroelements and other classes of smaller repeats. The clustering of highly repetitive elements is a striking feature of the A. thaliana genome emerging from sequence and other analyses.

Benzyl derivatives of 2,1,3-benzo- and benzothieno[3,2-a]thiadiazine 2,2-dioxides: first phosphodiesterase 7 inhibitors.

The synthesis of a new family of benzyl derivatives of 2,1,3-benzo- and benzothieno[3,2-a]thiadiazine 2,2-dioxides was achieved. The biological data revealed the first heterocyclic family of compounds with PDE 7 inhibitory properties appearing to be a new objective for the treatment of T-cell-dependent disorders. The IC(50) values or percent inhibition values of the compounds against PDE 7 were calculated by testing them against human recombinant PDE 7 expressed in S. cerevisiae. In this expression system the only cyclic nucleotide hydrolyzing activity present in cell extracts corresponded to human PDE 7. Isoenzyme selectivity PDE 7 versus PDE 4 and PDE 3 was also measured. Considering simultaneously inhibition of the three different isoenzymes, monobenzyl derivatives 15 and 23 showed interesting PDE 7 potency (around 10 microM); although not statistically significant, a trend toward selectivity with respect to PDE 3 and PDE 4 was obtained. Benzothiadiazine 16, although less potent at PDE 7 (IC(50) = 25 microM), also showed a trend of selectivity toward PDE 3 and PDE 4. These compounds are considered the best leads for further optimization.

Phosphodiesterase type 4 isozymes expression in human brain examined by in situ hybridization histochemistry and[3H]rolipram binding autoradiography. Comparison with monkey and rat brain.

We have examined the distribution of four different cyclic AMP-specific phosphodiesterase isozyme (PDE4A, PDE4B, PDE4C and PDE4D) mRNAs in the brain of different species by in situ hybridization histochemistry and by autoradiography with [3H]rolipram. We have compared the localization of each isozyme in human brain with that in rat and monkey brain. We have found that the four PDE4 isoforms display a differential expression pattern at both regional and cellular level in the three species. PDE4A, PDE4B and PDE4D are widely distributed in human brain, with the two latter appearing more abundant. In contrast, PDE4C in human brain, presents a more restricted distribution, limited to cortex, some thalamic nuclei and cerebellum. This is at variance with the distribution of PDE4C in rat brain, where it is found exclusively in olfactory bulb. In monkey brain, the highest expression for this isoform is found in the claustrum, and at lower levels in cortical areas and cerebellum. PDE4B presented a broad distribution, being expressed in both neuronal and non neuronal cell populations. In general, the distribution of binding sites visualized with [3H]rolipram correlated well with the expression of each PDE4 isozyme.

Structure, organization and expression of the eukaryotic translation initiation factor 5, eIF-5, gene in Zea mays.

The maize genomic DNA sequence encoding the eukaryotic translation initiation factor 5 (eIF-5) has been isolated from genomic library of maize seedlings and the exon-intron structure determined (accession number AJ132240). The length of genomic DNA sequenced was about 7kb and contained two exons with the translation start site in exon 2. The only intron is located in the non-coding 5' region and it is 1298bp long with the splice acceptor and donor sites conforming to the AG/GT rules. Repetitive sequence fragments are located in the 5' and 3' intergenic region. The accumulation of eIF-5 mRNA was studied by RNA blot and in situ hybridization. The observed distribution of mRNA may correlate with the function of the protein, as it appears to be highly abundant in tissues where the proportion of cells actively dividing is very high, such as meristematic regions.

TM20, a gene coding for a new class of transmembrane proteins expressed in the meristematic tissues of maize.

In the course of the analysis of lachrima, a recessive, defective kernel, embryo-lethal mutation in Zea mays that alters embryo and endosperm development, a gene coding for a new class of transmembrane proteins was isolated. The mutant was produced by Ac transposon tagging, and a gene located in the insertion region of the transposon was isolated as well as the corresponding cDNA. The predicted protein contains twenty hydrophobic segments that can be grouped in five repeats formed by four segments that fulfill the criteria for membrane spanning domains, and for this reason the gene has been named TM20. The sequences of the domains in each position of each group can be aligned, indicating that TM20 is formed by a four-domain structure duplicated five times. During embryogenesis in wild-type embryos and in the growing plant, TM20 gene expression is associated with meristems.

Involvement of a maize proline-rich protein in secondary cell wall formation as deduced from its specific mRNA localization.

A clone encoding a proline-rich protein (ZmPRP) has been obtained from maize root by differential screening of a maturing elongation root cDNA library. The amino acid sequence deduced from the full-length cDNA contains a putative signal peptide and a highly repetitive sequence containing the PEPK motif, indicating that the ZmPRP mRNA may code for a cell wall protein. The PEPK repeat is also found in a previously reported wheat sequence but differs from the repeated sequences found in hydroxyproline-rich glycoproteins (HRGP) and in dicot proline-rich proteins (PRP). In the maize genome, the ZmPRP protein is encoded by a single gene that is expressed in maturing regions of the root, in the hypocotyl and in the pericarp. In these organs, the ZmPRP mRNA accumulates in the xylem and surrounding cells, and in the epidermis. No ZmPRP mRNA was found in the phloem. The pattern of mRNA accumulation is very similar to the one observed for genes coding for proteins involved in lignin biosynthesis and, like most cell wall proteins, ZmPRP synthesis is also induced by wounding. These data support the hypothesis that ZmPRP is a member of a new class of fibrous proteins involved in the secondary cell wall formation in monocot species.

Sequence and analysis of chromosome 4 of the plant Arabidopsis thaliana.

The higher plant Arabidopsis thaliana (Arabidopsis) is an important model for identifying plant genes and determining their function. To assist biological investigations and to define chromosome structure, a coordinated effort to sequence the Arabidopsis genome was initiated in late 1996. Here we report one of the first milestones of this project, the sequence of chromosome 4. Analysis of 17.38 megabases of unique sequence, representing about 17% of the genome, reveals 3,744 protein coding genes, 81 transfer RNAs and numerous repeat elements. Heterochromatic regions surrounding the putative centromere, which has not yet been completely sequenced, are characterized by an increased frequency of a variety of repeats, new repeats, reduced recombination, lowered gene density and lowered gene expression. Roughly 60% of the predicted protein-coding genes have been functionally characterized on the basis of their homology to known genes. Many genes encode predicted proteins that are homologous to human and Caenorhabditis elegans proteins.

Presence of miniature inverted-repeat transposable elements (MITEs) in the genome of Arabidopsis thaliana: characterisation of the Emigrant family of elements.

Although the genome of Arabidopsis thaliana has a small amount of repetitive DNA, it contains representatives of most classes of mobile elements. However, to date, no miniature inverted-repeat transposable element (MITE) has been described in this plant. Here, we describe a new family of repeated sequences that we have named Emigrant, which are dispersed in the genome of Arabidopsis and fulfil all the requirements of MITEs. These sequences are short, AT-rich, have terminal inverted repeats (TIRs), and do not seem to have any coding capacity. Evidence for the mobility of Emigrant elements has been obtained from the absence of one of these elements in a specific Arabidopsis ecotype. Emigrant is also present in the genome of different Brassicae and its TIRs are 74% identical to those of Wujin elements, a recently described family of MITEs from the yellow fever mosquito Aedes aegypti.