Cloning of PCP1, a member of a family of pollen coat protein (PCP) genes from Brassica oleracea encoding novel cysteine-rich proteins involved in pollen-stigma interactions

The pollen coatings of both Brassica oleracea and Brassica napus contain a small family of basic 6–8 kDa proteins which are released on to the stigmatic surface on pollination. Following partial amino-acid sequencing of one of these pollen coat proteins (PCPs), PCR primers were constructed to isolate the PCP sequence from anther mRNA using RT-PCR. A cDNA was obtained which, in Northern hybridization experiments, revealed a characteristic pattern of expression during late stages of anther development. Interestingly, in situ hybridization revealed expression of this sequence to be confined to the cytoplasm of the trinucleate pollen grains: no signal was detected in the tapetum. Southern hybridization experiments have shown the gene ( PCP1 ) to be a member of a large family of between 30 and 40 PCP genes in the genome of Brassica oleracea , Surprisingly, RFLP experiments showed reduced copy number (one to two copies) in some of the F₂ segregants, perhaps resulting from the clustering of PCP sequences. PCP1 contains a single intron and encodes a small, basic peptide 83 amino acids in length featuring a hydrophobic signal peptide sequence separated from the more hydrophilic, cysteine-rich mature protein. The central part and C-terminal region of the peptide contain a characteristic and invariant pattern of eight cysteines which show clear homology with a number of other anther-specific genes; the remainder of the sequence shows little similarity to other sequences on the data bases. The product of PCP1 is a member of a large family of similar proteins, some of which have been demonstrated to bind specifically to S-locus glycoproteins, but does not appear to be genetically linked to the S-locus .     

Spatial expression dynamics of Men-9 delineate the third floral whorl in male and female flowers of dioecious Silene latifolia

Sex determination in Silene latifolia is controlled by heteromorphic sex chromosomes. Female flowers have five fused carpels and ten arrested stamen primordia. The male-determining Y chromosome overrides female development to suppress carpel formation and promote stamen development. The isolation and characterization of two S. latifoliaM ale en hanced cDNAs, Men-9a and Men-9b, which probably represent different alleles of a novel gene are reported here. Men-9a and Men-9b share 91.8% coding sequence nucleotide identity, yet only 85.4% amino acid identity. The Men-9 cDNAs are related to the previously reported MROS3 cDNA from S. latifolia. However, MROS3 is not present in the S. latifolia population used in these studies and the expression dynamics of Men-9a and Men-9b contrast dramatically with those reported for MROS3. Men-9 cDNAs are expressed primarily in anthers of young male flowers, with highest expression in 1–2 mm buds. Men-9 expression is also observed at a low level in female flowers. In situ hybridization analysis reveals two phases of Men-9 expression. The first phase is during a common stage of early stamen development in male and female flowers prior to stamen arrest in female flowers. The second phase of Men-9 expression is maximal in the epidermis and endothecium of Y chromosome- and Ustilago violacea-induced stamens; expression in male and female flowers extends to the epidermis of the staminal nectaries with strict boundaries at the second and fourth whorls. Men-9 gene expression therefore delineates the boundaries of the third floral whorl in S. latifolia flowers.     

Stimulation of CFU-f formation by prostaglandin E2 is mediated in part by its degradation product, prostaglandin A2

Prostaglandins (PG) of the E series are known to rapidly undergo non-enzymatic dehydration in culture medium containing serum albumin to produce the cyclopentenone PGs of the A series. We investigated the actions of PGA1 and A2 in the in vitro calcifying fibroblastic-colony forming unit assay which can partially mimic the in vivo anabolic effects of PGE2. It was found that PGA1 and A2 both stimulated colony formation in a dose-dependent manner with a maximum at 10(-6) M and to a similar degree to PGE2. In contrast to PGE2, PGA1 and PGA2 both caused an inhibition of cAMP accumulation. Furthermore, the addition of protein kinase A inhibitors, H8 and H89, had no significant effect on the stimulation of colony number by PGE2. These data suggest that (a) the bone anabolic effects of PGE1 and E2 are, in part at least, mediated by their dehydration products PGA1 and A2 and (b) that they are mediated via pathways not necessarily involving the cAMP/protein kinase A cascade.     

Intrachromosomal recombination between attP regions as a tool to remove selectable marker genes from tobacco transgenes

Recombinant genes conferring resistance to antibiotics or herbicides are widely used as selectable markers in plant transformation. Once transgenic material has been selected, the marker gene is dispensable. We report a novel strategy to remove undesirable parts of a transgene after integration into the tobacco genome. This approach is based on the transfer of a vector containing a NPTII gene flanked by two 352 bp attachment P (attP) regions of bacteriophage lambda, and the identification of somatic tissue with deletion events following intrachromosomal recombination between the attP regions. This system was used to delete a 5.9 kb region from a recombinant vector that had been inserted into two different genomic regions. As the attP system does not require the expression of helper proteins to induce deletion events, or a genetic segregation step to remove recombinase genes, it should provide a useful tool to remove undesirable transgene regions, especially in vegetatively propagated species.     

The Men-10 cDNA encodes a novel form of proline-rich protein expressed in the tapetum of dioecious Silene latifolia

 The Men-10 gene is expressed specifically in the tapetum tissue that surrounds and nourishes the developing microspores in the dioecious plant species, Silene latifolia. Men-10 encodes a proline-rich protein that contains a predicted signal region, indicating that it may be secreted from the tapetal cells and function in the extracellular domain of the tapetum or be translocated to the developing microspores. Here we report the sequence and precise expression pattern of the Men-10 cDNA and demonstrate a high level of restriction fragment length polymorphism associated with the Men-10 locus. The possible classification of Men-10 amongst known groups of proline- and hydroxyproline-rich glycoproteins, such as the arabinogalactan proteins, is discussed. Received: 20 December 1997 / Revision accepted: 2 July 1998     

The evolutionary-developmental analysis of plant microRNAs

MicroRNAs (miRNAs) control many important aspects of plant development, suggesting these molecules may also have played key roles in the evolution of developmental processes in plants. However, evolutionary-developmental (evo-devo) studies of miRNAs have been held back by technical difficulties in gene identification. To help solve this problem, we have developed a two-step procedure for the efficient identification of miRNA genes in any plant species. As a test case, we have studied the evolution of the MIR164 family in the angiosperms. We have identified novel MIR164 genes in three species occupying key phylogenetic positions and used these, together with published sequence data, to partially reconstruct the evolution of the MIR164 family since the last common ancestor of the extant flowering plants. We use our evolutionary reconstruction to discuss potential roles for MIR164 genes in the evolution of leaf shape and carpel closure in the angiosperms. The techniques we describe may be applied to any miRNA family and should thus enable plant evo-devo to begin to investigate the contributions miRNAs have made to the evolution of plant development.     

Current trends and future directions in flower development research

Flowers, the reproductive structures of the approximately 400 000 extant species of flowering plants, exist in a tremendous range of forms and sizes, mainly due to developmental differences involving the number, arrangement, size and form of the floral organs of which they consist. However, this tremendous diversity is underpinned by a surprisingly robust basic floral structure in which a central group of carpels forms on an axis of determinate growth, almost invariably surrounded by two successive zones containing stamens and perianth organs, respectively. Over the last 25 years, remarkable progress has been achieved in describing the molecular mechanisms that control almost all aspects of flower development, from the phase change that initiates flowering to the final production of fruits and seeds. However, this work has been performed almost exclusively in a small number of eudicot model species, chief among which is Arabidopsis thaliana. Studies of flower development must now be extended to a much wider phylogenetic range of flowering plants and, indeed, to their closest living relatives, the gymnosperms. Studies of further, more wide-ranging models should provide insights that, for various reasons, cannot be obtained by studying the major existing models alone. The use of further models should also help to explain how the first flowering plants evolved from an unknown, although presumably gymnosperm-like ancestor, and rapidly diversified to become the largest major plant group and to dominate the terrestrial flora. The benefits for society of a thorough understanding of flower development are self-evident, as human life depends to a large extent on flowering plants and on the fruits and seeds they produce. In this preface to the Special Issue, we introduce eleven articles on flower development, representing work in both established and further models, including gymnosperms. We also present some of our own views on current trends and future directions of the flower development field.     

An Integrated Mass-Spectrometry Pipeline Identifies Novel Protein Coding-Regions in the Human Genome

Background

Most protein mass spectrometry (MS) experiments rely on searches against a database of known or predicted proteins, limiting their ability as a gene discovery tool.

Results

Using a search against an in silico translation of the entire human genome, combined with a series of annotation filters, we identified 346 putative novel peptides [False Discovery Rate (FDR)<5%] in a MS dataset derived from two human breast epithelial cell lines. A subset of these were then successfully validated by a different MS technique. Two of these correspond to novel isoforms of Heterogeneous Ribonuclear Proteins, while the rest correspond to novel loci.

Conclusions

MS technology can be used for ab initio gene discovery in human data, which, since it is based on different underlying assumptions, identifies protein-coding genes not found by other techniques. As MS technology continues to evolve, such approaches will become increasingly powerful.     

Development and Validation of a Risk Model for Predicting Adverse Drug Reactions in Older People during Hospital Stay: Brighton Adverse Drug Reactions Risk (BADRI) Model

Background

Older patients are at an increased risk of developing adverse drug reactions (ADR). Of particular concern are the oldest old, which constitute an increasingly growing population. Having a validated clinical tool to identify those older patients at risk of developing an ADR during hospital stay would enable healthcare staff to put measures in place to reduce the risk of such an event developing. The current study aimed to (1) develop and (2) validate an ADR risk prediction model.

Methods

We used a combination of univariate analysis and multivariate binary logistic regression to identify clinical risk factors for developing an ADR in a population of older people from a UK teaching hospital. The final ADR risk model was then validated in a European population (European dataset).

Results

Six-hundred-ninety patients (median age 85 years) were enrolled in the development stage of the study. Ninety-five reports of ADR were confirmed by independent review in these patients. Five clinical variables were identified through multivariate analysis and included in our final model; each variable was attributed a score of 1. Internal validation produced an AUROC of 0.74, a sensitivity of 80%, and specificity of 55%. During the external validation stage the AUROC was 0.73, with sensitivity and specificity values of 84% and 43% respectively.

Conclusions

We have developed and successfully validated a simple model to use ADR risk score in a population of patients with a median age of 85, i.e. the oldest old. The model is based on 5 clinical variables (≥8 drugs, hyperlipidaemia, raised white cell count, use of anti-diabetic agents, length of stay ≥12 days), some of which have not been previously reported.