Genome maps 9. Arabidopsis thaliana. Wall chart.
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Arabidopsis↗
Biomedical subjects
C Dean
Publications and source records attributed to C Dean.
Two erbB-4 transcripts are expressed in normal breast and in most breast cancers.
ErbB-4 is a recently described member of the epidermal growth factor receptor (EGFR) family which together with erbB-3 acts as a receptor for a group of ligands known as the neuregulins (NRGs) or heregulins (HRGs). Unlike the EGFR and erbB-2 relatively little is known about the expression of erbB-4 in human tumours. Using RT-PCR and Southern blotting analysis we have investigated the expression of erbB-4 mRNA in a range of human tumour cell lines and in normal and malignant breast tissue. Using primers which amplified a 658 base pair (bp) region corresponding to part of the cytoplasmic domain of c-erbB-4 we found the receptor was expressed in some but not all breast and ovarian tumour cell lines and also in a glioma cell line. The highest level of erbB-4 expression was found in the ovarian carcinoma OVCAR-3 and the breast carcinoma T-47D. In all cell lines where the 'full-length' erbB-4 was detected, a second previously undescribed c-erbB-4 sequence was also found as a 610 bp PCR product. The alternative PCR product was identical in sequence to c-erbB-4 except for a deletion of 48 bp which encodes a consensus phosphatidylinositol 3-kinase (PI3K) binding site. This suggested that the two forms of erbB-4 might interact with different intracellular signalling pathways and therefore influence a wider variety of cellular responses to heregulin than previously thought. Expression of both erbB-4 variants was found in 7/7 normal breast tissues but only in 9/12 breast tumours analysed. In line with the terminology of Elenius et al. (1997b) we have designated the two isoforms of the C-terminal transcripts as CT-a (full-length) and CT-b which lacks the P13K binding motif. These results identify suitable cell lines for the further investigation of erbB-4 expression and function and suggest that the role of erbB-4 in breast cancer warrants further investigation with larger numbers of normal and malignant breast tissues.
Alternative Splicing↗
Analysis of 1.9 Mb of contiguous sequence from chromosome 4 of Arabidopsis thaliana.
The plant Arabidopsis thaliana (Arabidopsis) has become an important model species for the study of many aspects of plant biology. The relatively small size of the nuclear genome and the availability of extensive physical maps of the five chromosomes provide a feasible basis for initiating sequencing of the five chromosomes. The YAC (yeast artificial chromosome)-based physical map of chromosome 4 was used to construct a sequence-ready map of cosmid and BAC (bacterial artificial chromosome) clones covering a 1.9-megabase (Mb) contiguous region, and the sequence of this region is reported here. Analysis of the sequence revealed an average gene density of one gene every 4.8 kilobases (kb), and 54% of the predicted genes had significant similarity to known genes. Other interesting features were found, such as the sequence of a disease-resistance gene locus, the distribution of retroelements, the frequent occurrence of clustered gene families, and the sequence of several classes of genes not previously encountered in plants.
Arabidopsis↗
Anti-EGFR monoclonal antibodies which act as EGF, TGF alpha, HB-EGF and BTC antagonists block the binding of epiregulin to EGFR-expressing tumours.
Epiregulin is the newest member of the epidermal growth factor (EGF) family of ligands that was isolated from conditioned medium of the murine fibroblast-derived tumour cell line NIH3T3/T7. Here, using a panel of anti-EGFR receptor (EGFR) monoclonal antibodies (MAbs) directed against 4 distinct epitopes on the external domain of the receptor, we have investigated the importance of the EGFR in transmitting the biological action of epiregulin. We found that MAb ICR9, which enhances the binding of EGF, TGF alpha, HB-EGF and betacellulin to the EGFR, also increases the binding of 125I-epiregulin to a number of EGFR-expressing tumour cell lines, including EJ, SKBR3, SKOV3, MDA-MB468 and HN5. In addition, anti-EGFR MAbs ICR15, ICR16, ICR61, ICR62 and ICR80, which block the binding of 125I-EGF to the EGFR, inhibit the binding of 125I-epiregulin to these tumour cell lines. Like EGF, we found that both the epiregulin-induced growth inhibition of HN5 and MDA-MB468 cells and tyrosine phosphorylation of the 170 kDa EGFR on HN5 cells are reversed in the presence of anti-EGFR MAbs ICR62 and ICR80. Surprisingly and unlike 125I-EGF, radiolabelled epiregulin bound very poorly to human bladder carcinoma EJ cells and its binding to SKOV3 cells was not inhibited efficiently in the presence of blocking antibodies. We conclude that the EGFR plays an important role in transmitting the biological action of epiregulin and that these effects could be blocked in the presence of anti-EGFR MAbs. The low level of binding of epiregulin compared with EGF to EJ cells suggests that the EGFR may not be the primary receptor for epiregulin.
3T3 Cells↗
Control of flowering time.
The multiple promotive and repressive pathways controlling flowering have been further defined by analysis of genetic interactions and the activation of floral meristem identity genes. Cloning of additional genes in these pathways has uncovered some of the molecular processes that control the timing of the transition to reproductive development.
Arabidopsis↗
Effects of real and imagined training on voluntary muscle activation during maximal isometric contractions.
In this study we directly tested the hypothesis that isometric strength training increases voluntary drive to muscles. In addition, it was attempted to replicate the findings of an earlier study that showed imagined training increases voluntary strength as much as actual training, as this finding provides key support for the hypothesis that training increases voluntary drive (Yue & Cole 1992). Fifty-four subjects were randomly allocated to groups that performed 8 weeks of isometric training of the elbow flexor muscles, imagined isometric training, or a control task involving the lower limbs. Voluntary isometric strength and activation of the elbow flexor muscles were measured before and after training. Voluntary activation was measured with a sensitive form of twitch interpolation. Training, imagined training and control groups increased voluntary isometric elbow flexor strength by means of 17.8% (+/- 3.1 SEM), 6.8% (+/- 2.6) and 6.5% (+/- 3.0), respectively. The training group increased in strength significantly more than imagined training and control groups (P = 0.01 for both comparisons), but the small difference between imagined training and control groups was not significant (P = 0.31). Prior to training, voluntary activation of all subjects was high (96.2 +/- 0.5%). This did not change significantly with training and there were no significant differences between groups. These data challenge the hypothesis that training of the elbow flexor muscles increases isometric strength by inducing adaptations of the central nervous system, because they show that training does not increase voluntary activation and imagined training does not increase strength.
Adult↗
Development of an AFLP based linkage map of Ler, Col and Cvi Arabidopsis thaliana ecotypes and construction of a Ler/Cvi recombinant inbred line population.
An amplified fragment polymorphism (AFLP) based linkage map has been generated for a new Landsberg erecta/ Cape Verde Islands (Ler/Cvi) recombinant inbred line (RIL) population. A total of 321 molecular PCR based markers and the erecta mutation were mapped. AFLP markers were also analysed in the Landsberg erecta/Columbia (Ler/Col) RIL population (Lister and Dean, 1993) and 395 AFLP markers have been integrated into the previous Arabidopsis molecular map of 122 RFLPs, CAPSs and SSLPs. This enabled the evaluation of the efficiency and robustness of AFLP technology for linkage analyses in Arabidopsis. AFLP markers were found throughout the linkage map. The two RIL maps could be integrated through 49 common markers which all mapped at similar positions. Comparison of both maps led to the conclusion that segregating bands from a common parent can be compared between different populations, and that AFLP bands of similar molecular size, amplified with the same primer combination in two different ecotypes, are likely to correspond to the same locus. AFLPs were found clustering around the centromeric regions, and the authors have established the map position of the centromere of chromosome 3 by a quantitative analysis of AFLP bands using trisomic plants. AFLP markers were also used to estimate the polymorphism rate among the three ecotypes. The larger polymorphism rate found between Ler and Cvi compared to Ler and Col will mean that the new RIL population will provide a useful material to map DNA polymorphisms and quantitative trait loci.
Arabidopsis↗
The transition to flowering
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Journal Article↗
The transposition frequency of Tag1 elements is increased in transgenic Arabidopsis lines.
Tag1 was identified as a highly active endogenous transposable element in transgenic Arabidopsis thaliana Landsberg erecta plants carrying the maize transposable element Activator (Ac). Here, we describe experiments designed to determine the basis for the high activity of Tag1. The frequency of transposition of Tag1 elements was compared in lines containing or lacking Ac transposase to assess the effect of Ac transposase on Tag1 activity. Three populations of nontransgenic plants, including nontransformed regenerants, were also analyzed. The high level of activity of Tag1 did not correlate with the presence or absence of Ac transposase but was significantly higher in transgenic lines. This result was maintained through at least six generations after transformation. These data suggest that Tag1 transposition is stimulated by processes that occur during the Agrobacterium transformation and that thereafter remain active. Two Tag1 elements are tightly linked in the Landsberg erecta genome and map to the lower arm of chromosome 1. Tag1 elements were found in only a few A. thaliana ecotypes but were present in four other Arabidopsis species.
Arabidopsis↗
p21waf1/cip1mda-6 expression in non-small-cell lung cancer: relationship to survival.
In order to clarify critical events during bronchial carcinogenesis, and to evaluate a possible prognostic role for p21 immunohistochemical detection, we assessed the immunohistochemical expression of p21 protein in 60 surgically resected non-small-cell lung cancers (NSCLCs) that had been investigated previously for their p53 protein status. We found that p21 protein was expressed in both normal and neoplastic tissue. In normal tissue, p21 immunoreactivity was detectable in a low percentage of well-differentiated cells. We found immunostaining for p21 in 80% of the investigated neoplasms. In 73.3% of the neoplasms, p21 was considered to be overexpressed. No relationship was found between p21 overexpression and tumor stage or tumor-nodal-metastatic (TNM) status. The histologic grading was slightly correlated with the p21 status (P = -0.51), with no significant differences noted between squamous carcinomas and adenocarcinomas. Survival percentage curves for our lung-cancer patients, based on a comparison of different p21 expression levels and constructed through a Kaplan-Meier analysis, showed significant differences in mean (P < 0.001) and overall (P < 0.001) survival time between patients of different p21 status, suggesting a favorable prognostic value of p21 immunostaining for NSCLC patients.
Adenocarcinoma↗
EGFR blockade by tyrosine kinase inhibitor or monoclonal antibody inhibits growth, directs terminal differentiation and induces apoptosis in the human squamous cell carcinoma HN5.
Human squamous cell carcinomas frequently overexpress the epidermal growth factor receptor (EGFR) and this is often associated with poor prognosis in patients with these cancers. The high level of expression of the EGFR provides an important target for therapy and we and others have shown that monoclonal antibodies (mAbs) which block the activation of the receptor by the EGF family of ligands inhibit the growth of EGFR overexpressing tumours in vitro and induce the regression of established tumours grown as xenografts in athymic mice. Inhibitors of the tyrosine kinase associated with the EGFR have also been shown to block receptor activation and prevent tumour cell proliferation. Using the EGFR-overexpressing head and neck carcinoma cell line HN5, we have compared the biological consequences of treatment with an inhibitor of EGFR tyrosine kinase (PD153035) with anti-EGFR monoclonal antibodies (mAbs) ICR63 or ICR80. We found that both the anti-EGFR mAbs and the TK inhibitor produce similar biological changes namely, they inhibit the EGF and TGFá-induced tyrosine phosphorylation of the receptor and the growth in culture of HN5 cells. At concentrations above 100 nM, the TK inhibitor prevented the growth in culture of HN5 cells completely with an IC50 of 40 nM. With the anti-EGFR mAbs, growth of HN5 cells was inhibited completely at concentrations above 4 nM with an IC50 of 1 nM. More importantly we found that, like the anti-EGFR mAbs, treatment with the TK inhibitor directs HN5 cells to undergo terminal differentiation as monitored by the expression of cytokeratin 10. In addition, our results indicate that the growth inhibitory effects of the anti-EGFR agents also lead to induction of apoptosis as determined by 7-amino actinomycin D staining (7-AAD). We conclude that EGFR blockade by anti-EGFR mAbs or TK inhibitor influences the growth in culture of EGFR overexpressing tumours by directing terminal differentiation and inducing apoptosis.
Antibodies, Monoclonal↗
Collinearity between a 30-centimorgan segment of Arabidopsis thaliana chromosome 4 and duplicated regions within the Brassica napus genome.
Arabidopsis thaliana (the model dicotyledonous plant) is closely related to Brassica crop species. Genome collinearity, or conservation of marker order, between Brassica napus (oilseed rape) and A. thaliana was assessed over a 7.5-Mbp region of the long arm of A. thaliana chromosome 4, equivalent to 30 cM. Estimates of copy number indicated that sequences present in a single copy in the haploid genome of A. thaliana (n = 5) were present in 2-8 copies in the haploid genome of B. napus (n = 19), while sequences present in multiple copies in A. thaliana were present in over 10 copies in B. napus. Genetic mapping in B. napus of DNA markers derived from a segment of A. thaliana chromosome 4 revealed duplicated homologous segments in the B. napus genome. Physical mapping in A. thaliana of homologues of Brassica clones derived from these regions confirmed the identity of six duplicated segments with substantial homology to the 7.5-Mbp region of chromosome 4 in A. thaliana. These six duplicated Brassica regions (on average 22 cM in length) are collinear, except that two of the six copies contain the same large internal inversion. These results have encouraging implications for the feasibility of shuttling between the physical map of A. thaliana and genetic maps of Brassica species, for identifying candidate genes and for map based gene cloning in Brassica crops.
Arabidopsis↗
Recurrent vocal fold papilloma: resection using cold instruments.
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Adult↗
AAC-11, a novel cDNA that inhibits apoptosis after growth factor withdrawal.
Many growth factors and cytokines act as cellular survival factors by preventing programmed cell death (apoptosis). However, the specific genes and corresponding proteins that mediate survival are poorly defined. To identify potential survival genes, a cDNA library was prepared from murine fibroblasts and screened by a functional expression cloning approach. A 1023-bp cDNA, AAC-11, was identified that encodes a protein of approximately 25 kDa. The AAC-11 gene shows strong species conservation and is ubiquitously expressed in embryonic and adult tissues with multiple transcripts, as well as in various human tumor cell lines. The predicted protein contains a leucine zipper domain but lacks a DNA-binding domain. BALB/c3T3 fibroblasts that were stably transfected with AAC-11 cDNA were viable in serum-free medium for up to 12 weeks. The protective action of AAC-11 was abolished by mutation of leucines to arginines within the leucine zipper domain. We also isolated a longer AAC-11 cDNA that codes for up to an additional 290 amino-terminal amino acids but did not protect against apoptosis. The cDNA for human AAC-11 was identified and exhibits strong homology with the murine species and retains the leucine zipper domain. Western immunoblots of BALB/c3T3 cells using rabbit anti-AAC-11 polyclonal serum revealed a major native 55-kDa AAC-11 protein and a minor 25-kDa protein corresponding to the long and short forms of AAC-11 cDNA, respectively. In summary, we report a cDNA whose expression supports cell viability after withdrawal of growth factors. The corresponding native protein may function as a novel inhibitor of apoptosis.
Alternative Splicing↗
A unique domain of pRb2/p130 acts as an inhibitor of Cdk2 kinase activity.
The Cdk2 kinase has long been known to be involved in the progression of mammalian cells past the G1 phase restriction point and through DNA replication in the cell cycle. The Rb family of proteins, consisting of pRb, p107, and pRb2/p130, has also been shown to monitor progression of G1 phase, mostly through their interaction with E2F family members. p107 is able to inhibit Cdk2 kinase activity through this interaction via a p21-related domain present in the C terminus of the protein. We show here that pRb2/p130 also possesses this activity, but through a separate domain. Moreover, we correlate the increased expression of pRb2/p130 during various cellular processes with the decreased kinase activity of Cdk2. We hypothesize that pRb2/p130 may act not only to bind and modify E2F activity, but also to inhibit Cdk2 kinase activity in concert with p21 in a manner different from p107.
Animals↗
Mapping of carotid baroreceptor subtype projections to the nucleus tractus solitarius using c-fos immunohistochemistry.
This study has combined physiological pressure stimulation of carotid baroreceptors via a vascularly isolated carotid sinus and anodal block of baroreceptor afferent fibers in the carotid sinus nerve to examine the medullary projections of type I vs. type II (large A- vs. small A- and C-fiber afferent) baroreceptors. The control distribution of cells in the nucleus tractus solitarius expressing c-fos in response to physiological activation of carotid baroreceptors in the isolated sinus was compared to that during anodal block of large A-fibers in the carotid sinus nerve. Carotid baroreceptor stimulation primarily activated neurons in the ipsilateral commissural and medial subnuclei of the caudal nucleus tractus solitarius and the dorsolateral, dorsomedial and medial subnuclei in the intermediate and rostral levels of the nucleus tractus solitarius. Elimination of large A-fiber carotid baroreceptor afferents, during similar carotid baroreceptor stimulation resulted in a decrease in the number of cells expressing c-fos in the dorsomedial subnucleus of the rostral nucleus tractus solitarius. These data indicate that projections of larger A-fiber (type I) carotid baroreceptors are localized primarily to the rostral dorsomedial subnucleus, while those of smaller A- and C-fiber baroreceptors are more widely distributed to the commissural, medial and dorsal subnuclei of the nucleus tractus solitarius.
Afferent Pathways↗
FCA, a gene controlling flowering time in Arabidopsis, encodes a protein containing RNA-binding domains.
A strong promoter of the transition to flowering in Arabidopsis is encoded by FCA. FCA has been cloned and shown to encode a protein containing two RNA-binding domains and a WW protein interaction domain. This suggests that FCA functions in the posttranscriptional regulation of transcripts involved in the flowering process. The FCA transcript is alternatively spliced with only one form encoding the entire FCA protein. Plants carrying the FCA gene fused to the strong constitutive 35S promoter flowered earlier, and the ratio and abundance of the different FCA transcripts were altered. Thus, FCA appears to be a component of a posttranscriptional cascade involved in the control of flowering time.
Amino Acid Sequence↗
Methodology for selection of human antibodies to membrane proteins from a phage-display library.
We describe a simple antigen capture technique for the selection of a specific human antibody to p185erbB-2, a transmembrane glycoprotein, from a library of human Fab genes expressed on the surface of bacteriophage. Magnetic beads coated with the rat antibody ICR55 have been used to capture erbB-2 antigen from Triton X-100 extracts of SKOV3 cells. The antigen-coated beads have then been used to select bacteriophage displaying human Fab with affinity for p185erbB-2. After 4 rounds of selection, 65 phage clones were isolated which bound specifically to p185erbB-2 in a capture assay. Nine of the clones which gave the strongest reaction in an ELISA were selected for further development and the Fab genes were subcloned into the expression vector pUC119his6mycXba and electroporated into E. coli TG1. Colonies were grown, induced and the supernatants tested for the presence of secreted human Fab. Supernatants from two of the 9 clones contained human Fab and one of these bound specifically to erbB-2 in a capture assay, stained the membranes of the erbB-2 overexpressing cell lines BT474 and SKBR3 and immunoprecipitated a protein of molecular weight 185 000 kDa from SKOV3 cells. We conclude that a membrane antigen captured by specific monoclonal antibody can be used successfully to select phage displaying human antibodies specific for the antigen.
Animals↗