[Functions of phosphate-responsible genes in plants].
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Acid Phosphatase↗
Biomedical subjects
D Shibata
Publications and source records attributed to D Shibata.
Establishment of framework P1 clones for map-based cloning and genome sequencing: direct RFLP mapping of large clones.
Large insert capacity, clone stability and convenient propagation in Escherichia coli have made bacterial artificial chromosome and phage P1 vector-based libraries the first choice for large-scale sequencing projects, and these libraries have also proven useful for chromosome walking. The application of these libraries for either purpose is greatly facilitated by the establishment of a set of framework clones distributed across the genome. Using a P1-based library of Arabidopsis thaliana with genomic inserts of 70-90kb (Liu, Y.-G., Mitsukawa, N., Vazquez-Tello, A., Whittier, R.F., 1995. Generation of a high-quality P1 library of Arabidopsis suitable for chromosome walking. Plant J. 7, 351-358), we have now established such a set of framework clones. To date, such clones have usually been identified by hybridization to smaller, previously mapped clones that detect restriction fragment length polymorphisms (RFLPs). In order to establish framework clones more efficiently, we refined protocols for P1 clone DNA isolation and RFLP detection in order to employ whole P1 clones directly as probes. This strategy enabled a very high rate of RFLP detection, and obviated the need to screen the P1 library with smaller RFLP probes. Altogether 95 clones were mapped providing a framework into which further clones can be integrated by physical overlap.
Arabidopsis↗
Phosphate transporter gene family of Arabidopsis thaliana.
Using a high-affinity phosphate transporter gene of Arabidopsis thaliana, PHT1, as a probe, three Arabidopsis homologs were isolated, named PHT4, PHT5 and PHT6, in addition to the previously isolated PHT2 and PHT3. The amino acid sequences deduced from the three nucleotides were 32-42% homologous with microbial phosphate transporters of Saccharomyces cerevisiae (PHO84), Neurospora crassa (PHO-5) and Glomus versiforme (GvPT). PHT1, PHT2, PHT3 and PHT6 were clustered in a 25-kbp region of chromosome V. PHT1 and PHT4 transcripts were detected in roots. Interestingly, suspension-cultured cells expressed only PHT4. PHT4 and PHT5 located within a genetic distance of 6.4 cM on chromosome II, and were close to a phosphate accumulation mutant pho2. Genomic sequencing revealed no difference in the sequences of the two genes in both pho2 and wild-type. The PHT4 transcript was expressed at similar levels in the mutant and wild-type. These results demonstrate that neither PHT4 nor PHT5 is allelic to PHO2.
Amino Acid Sequence↗
A physical map of Arabidopsis thaliana chromosome 3 represented by two contigs of CIC YAC, P1, TAC and BAC clones.
We have constructed a physical map of Arabidopsis thaliana chromosome 3 by ordering the clones from CIC YAC, P1, TAC and BAC libraries using the sequences of a variety of genetic and EST markers and terminal sequences of clones. The markers used were 112 DNA markers, 145 YAC end sequences, and 156 end sequences of P1, TAC and BAC clones. The entire genome of chromosome 3, except for the centromeric and telomeric regions, was covered by two large contigs, 13.6 Mb and 9.2 Mb long. This physical map will facilitate map-based cloning experiments as well as genome sequencing of chromosome 3. The map and end sequence information are available on the KAOS (Kazusa Arabidopsis data Opening Site) web site at http://www.kazusa.or.jp/arabi/.
Arabidopsis↗
Enhanced intestinal adenomatous polyp formation in Pms2-/-;Min mice.
Analysis of two human familial cancer syndromes, hereditary nonpolyposis colorectal cancer and familial adenomatous polyposis, indicates that mutations in either one of four DNA mismatch repair gene homologues or the adenomatous polyposis coli (APC) gene, respectively, are important for the development of colorectal cancer. To further investigate the role of DNA mismatch repair in intestinal tumorigenesis, we generated mice with mutations in both Apc and the DNA mismatch repair gene, Pms2. Whereas Pms2-deficient mice do not develop intestinal tumors, mice deficient in Pms2 and heterozygous for Min, an allele of Apc, develop approximately three times the number of small intestinal adenomas and four times the number of colon adenomas relative to Min and Pms2+/-;Min mice. Although Pms2 deficiency clearly increases adenoma formation in the Min background, histological analysis indicated no clear evidence for progression to carcinoma.
Adenoma↗
Tracing cell fates in human colorectal tumors from somatic microsatellite mutations: evidence of adenomas with stem cell architecture.
Occult aspects of tumor proliferation are likely recorded genetically as their microsatellite (MS) loci become polymorphic. However, MS mutations generated by division may also be eliminated with death as noncoding MS loci lack selective value. Therefore, highly polymorphic MS loci cannot exist unless mutation rates are high, or unless mutation losses are inherently minimized. Mutations accumulate differently when cell fates are determined intrinsically before or extrinsically after division. Stem cell (asymmetrical division as in intestinal crypts) and random (asymmetrical and symmetrical division) proliferation, respectively, represent simulated cell fates determined before or after division. Whereas mutations regardless of selection systematically persist once inherited with stem cell proliferation, mutations are eliminated by the symmetrical losses of both daughter cells with random proliferation. Therefore, greater genetic diversity or MS variance accumulate with stem cell compared with random proliferation. MS loci in normal murine intestinal mucosa and xenografts of cancer cell lines accumulated mutations, respectively, consistent with stem cell and random proliferation. Tumors from patients with hereditary nonpolyposis colorectal cancer (HNPCC) demonstrated polymorphic MS loci. Overall, three of five adenomas and one of six cancers exhibited high MS variances. Assuming mutation rates are not significantly greater in adenomas than in cancers, these studies suggest the stem cell proliferation and hierarchy of normal intestines persists in many HNPCC adenomas and some cancers. An adenoma stem cell architecture can explain the complex polymorphic MS loci observed in HNPCC adenomas and account for many adenoma features. In contrast, cancers may lose intrinsic control of cell fate. These studies illustrate a feasible phylogenetic approach to unravel and describe occult aspects of human tumor proliferation. The switch from predominantly stem cell to random proliferation may be a critical and defining characteristic of malignancy.
Adenoma↗
Overexpression of pp60c-src elicits invasive behavior in rat colon epithelial cells.
BACKGROUND & AIMS: Src activation is reported as an early event found in preneoplastic colonic adenomas and in 70% of colon carcinomas. The aim of this study was to identify the biological consequences of c-src overexpression in rat colon epithelial cells. METHODS: Introduction and overexpression of c-src in an immortalized rat colon epithelial cell line was achieved using lipofection. Transfectants were tested for changes in growth and cell behavior using different in vitro assay systems. RESULTS: Colon epithelial cells overexpressing c-src showed the ability to form microcolonies in soft agar without acquiring tumorigenic potential. In in vitro assays, c-src transfectants displayed a gain of invasive potential through Matrigel without an accompanying change in migrational ability. No discernible qualitative changes were observed in the phosphotyrosyl protein profile between c-src and v-src transfectants. Assessment of the cadherin/catenin status in these cells revealed an intact, functional complex with no detectable tyrosine phosphorylation of different components of the complex. CONCLUSIONS: Overexpression of c-src in an immortalized rat colon epithelial cell line does not elicit full neoplastic transformation but enhances anchorage-independent growth and confers invasion capability. Increased invasion through Matrigel was not linked to inactivation of the cadherin complex in c-src transfectants.
Animals↗
Tumour susceptibility and spontaneous mutation in mice deficient in Mlh1, Pms1 and Pms2 DNA mismatch repair.
Germline mutations in the human MSH2, MLH1, PMS2 and PMS1 DNA mismatch repair (MMR) gene homologues appear to be responsible for most cases of hereditary non-polyposis colorectal cancer (HNPCC; refs 1-5). An important role for DNA replication errors in colorectal tumorigenesis has been suggested by the finding of frequent alterations in the length of specific mononucleotide tracts within genes controlling cell growth, including TGF-beta receptor type II (ref. 6), BAX (ref. 7) and APC (ref. 8). A broader role for MMR deficiency in human tumorigenesis is implicated by microsatellite instability in a fraction of sporadic tumours, including gastric, endometrial and colorectal malignancies. To better define the role of individual MMR genes in cancer susceptibility and MMR functions, we have generated mice deficient for the murine homologues of the human genes MLH1, PMS1 and PMS2. Surprisingly, we find that these mice show different tumour susceptibilities, most notably, to intestinal adenomas and adenocarcinomas, and different mutational spectra. Our results suggest that a general increase in replication errors may not be sufficient for intestinal tumour formation and that these genes share overlapping, but not identical functions.
Adaptor Proteins, Signal Transducing↗
Increase of foreign gene expression in monocot and dicot cells by an intron in the 5' untranslated region of a soybean phosphoenolpyruvate carboxylase gene.
A genomic clone containing part of the coding region and upstream sequences of a phosphoenolpyruvate carboxylase (PEPC) gene was isolated from a soybean genomic library. The first intron of this gene is located in the 5' untranslated region. This intron-spanning fragment is capable of increasing GUS activity in plant cells.
Amino Acid Sequence↗
The SURF technique. Selective genetic analysis of microscopic tissue heterogeneity.
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Cell Separation↗
The dynamics of early intestinal tumour proliferation: to be or not to be.
New data and approaches bring novel perspectives and possibilities to old problems. The speculation of this chapter attempts to merge the puzzling MS data observed in human tumours (Figs. 3 and 4B) within a multistep tumour progression model. Clearly the current models are gross simplifications and other, more sophisticated models may better account for the distribution of MS alleles found in human tumours. The findings and the data are also limited to MMR deficient tumours, and studies in non-mutator phenotype tumours may be more difficult since fewer polymorphisms will arise during progression. The current model, however, precisely defines proliferation and clearly delineates two very distinct patterns. Further studies using MS loci in MMR deficient tumours will allow fairer tests of alternative pathways (Fig. 4C) to cancer. Evolution proceeding in occult progenitor populations allows mutations to accumulate throughout life and not just in the last decades after polyps appear.
Animals↗
Epstein-Barr virus-associated nuclear antigen-1 carboxy-terminal gene sequences in Japanese and American patients with gastric carcinoma.
We examined Epstein-Barr virus (EBV)-associated nuclear antigen-1 (EBNA-1) gene carboxy (C)-terminal deletions occurring in gastric carcinoma and noncarcinoma tissues from two different ethnic populations. Previously reported EBNA-1 C-terminal region amino acid sequence variations include five subtypes based on the amino acid at codon 487: Prototype (P)-ala, which is found in the B95.8-derived prototype virus; P-thr; Variant (V)-pro; V-leu; and V-val. Using polymerase chain reaction to amplify fragments of the EBNA-1 gene, followed by gene sequencing, we identified a single EBNA-1 gene sequence variant (V-val') in all 25 cases of known EBV-positive gastric carcinoma and all 8 cases of known EBV-positive reactive follicular hyperplasia from Japan. All 17 cases of known EBV-positive American gastric carcinomas had a single EBNA-1 gene sequence, including 3 P-ala or P-ala variants, 9 P-thr, 4 V-leu or V-leu variants, and 1 V-val'. All 11 cases of American reactive lymphoid tissue also had a single EBNA-1 gene sequence, including 2 P-ala variants, 5 P-thr or P-thr variants, and 4 V-leu. Variant EBNA-1 sequences were more common in both carcinoma and non-neoplastic tissues than the prototype sequence, whether from Japan or America. The EBNA-1 gene sequences found in the two ethnic populations differed, suggesting that variation in EBNA-1 gene sequence is due to polymorphisms present within pre-existing viral strains prevalent within various ethnic populations. The EBNA-1 gene sequences in reactive tissues were similar to those in malignant tissues from the same ethnic population, suggesting that the differences in EBNA-1 gene sequences do not result from mutation occurring during neoplastic transformation and that there may not be a selective cell tropism for different EBNA-1 proteins, as previously suggested.
Amino Acid Substitution↗
A fine physical map of Arabidopsis thaliana chromosome 5: construction of a sequence-ready contig map.
A fine physical map of Arabidopsis thaliana chromosome 5 was constructed by ordering the clones from YAC, P1, TAC and BAC libraries of the genome using the sequences of a variety of genetic and EST markers and terminal sequences of clones. The markers used were 88 genetic markers, 13 EST markers, 87 YAC end probes, 100 YAC subclone end probes, and 390 end probes of P1, TAC and BAC clones. The entire genome of chromosome 5, except for the centromeric and telomeric regions, was covered by two large contigs 11.6 Mb and 14.2 Mb long separated by the centromeric region. The minimum tiling path of the chromosome was constituted by a total of 430 P1, TAC and BAC clones. The map information is available at the Web site http://www.kazusa.or.jp/arabi/.
Arabidopsis↗
Overexpression of an Arabidopsis thaliana high-affinity phosphate transporter gene in tobacco cultured cells enhances cell growth under phosphate-limited conditions.
A higher plant homologue to the high-affinity phosphate transporter gene of yeast (Saccharomyces cerevisiae) PHO84 was isolated from Arabidopsis thaliana. Expression of the Arabidopsis gene PHT1 at high levels in tobacco-cultured cells increased the rate of phosphate uptake. The uptake activity attributable to the transgene was inhibited by protonophores, suggesting an H+ cotransport mechanism of phosphate uptake, and had a Km of 3.1 microM which is within limits characteristic of high-affinity transport mechanisms. These results indicate that PHT1 encodes a high-affinity phosphate transporter. The transgenic cells exhibited increased biomass production when the supply of phosphate was limited, establishing gene engineering of phosphate transport as one approach toward enhancing plant cell growth.
Arabidopsis↗
Microsatellite instability is rare in B-cell non-Hodgkin's lymphomas.
Microsatellite instability (MSI), a symptom of defect in DNA mismatch repair function, represents a type of genomic instability frequently detected in many types of cancers. However, the involvement of MSI in non-Hodgkin's lymphomas (NHL) has not been conclusively investigated. In this study, we have tested the presence of MSI in 69 cases of B-cell NHL (B-NHL) representative of the various histologic categories of the disease and including 17 cases of acquired immunodeficiency syndrome (AIDS)-related B-NHL (AIDS-NHL). In addition, for selected B-NHL cases, consecutive samples obtained before and after clinical progression (with and without concomitant histologic transformation) were also investigated. Five distinct microsatellite repeats (2 dinucleotide, 2 trinucleotide, and 1 tetranucleotide repeats) were analyzed by polymerase chain reaction in all cases. MSI, defined by the presence of microsatellite alterations in two or more of the five microsatellite loci tested, was not found in NHL. In contrast to a previous study reporting the frequent association between MSI and AIDS-NHL, we found this abnormality in only 1 of 17 cases of AIDS-NHL representative of the major subtypes. Overall, these data indicate that defects in DNA mismatch repair do not contribute significantly to the molecular pathogenesis of B-NHL.
Acquired Immunodeficiency Syndrome↗
DNA mismatch repair in mammals: role in disease and meiosis.
The understanding of mammalian mismatch repair (MMR) gene function has been accelerated as a result of progress on several fronts. First, the biochemical analysis of MMR has been advanced by the production of purified human MMR proteins which will eventually allow reconstitution of MMR activity in vitro. Second, a wealth of clinical studies on colon cancer patients have begun to allow correlations to be made among MMR mutations, tumor types, therapeutic approaches and clinical outcomes. Finally, new unexpected meiotic phenotypes have been associated with mutations in certain mouse MMR genes.
Animals↗
AtPLC2, a gene encoding phosphoinositide-specific phospholipase C, is constitutively expressed in vegetative and floral tissues in Arabidopsis thaliana.
A cDNA encoding a phosphoinositide-specific phospholipase C (PI-PLC) from the higher plant Arabidopsis thaliana was cloned and characterized. The gene corresponding to this cDNA is designated AtPLC2. The overall structure of the predicted AtPLC2 protein is similar to those of plant PI-PLCs and mammalian delta-type PI-PLCs. Northern blot analysis revealed that AtPLC2 is expressed constitutively whereas AtPLC1S, another gene for PI-PLC of Arabidopsis, is induced by environmental stresses such as dehydration and salinity, indicating that the function of AtPLC2 is distinct from that of AtPLC1S. The AtPLC2 mRNA was detected in vegetative and floral tissues. We determined the positions of these two PI-PLCs genes on Arabidopsis chromosomes by RFLP mapping using P1 genomic clones.
Amino Acid Sequence↗
Localization of a susceptibility locus for Peutz-Jeghers syndrome to 19p using comparative genomic hybridization and targeted linkage analysis.
Many human cancer susceptibility genes have been successfully mapped by genetic linkage studies. One that has so far eluded researchers is that for Peutz-Jeghers (P-J) syndrome, a condition characterized by intestinal hamartomatous polyposis and melanin spots of the lips, buccal mucosa and digits. A dramatically elevated risk of malignancy has also been documented. Gastrointestinal tumours as well as cancers of the breast, ovary, testis and uterine cervix appear to be overrepresented in families with this syndrome. The nature of hamartomatous polyps is equivicol. Hamartomas are usually considered histologically benign, but in the case of Peutz-Jeghers patients, there are reports of adenomatous and malignant changes in the polyps, and the possibility of a hamartoma-carcinoma sequence has been discussed. A search for a putative tumour suppressor locus was made using comparative genomic hybridization (CGH) of Peutz-Jeghers polyps, combined with loss of heterozygosity (LOH) study. Genetic linkage analysis in 12 families using markers from a deletion site demonstrated the presence of a high-penetrance locus in distal 19p with a multipoint lod score of 7.00 at marker D19S886 without evidence of genetic heterogeneity. The study demonstrates the power of CGH combined with LOH analysis in identifying putative tumour suppressor loci, and provides molecular evidence of malignant potential in hamartomas.
Adenocarcinoma↗