Simultaneous Fluorescent Identification of Odontoblasts and Ameloblasts.

The tooth is mainly composed of dentin and enamel. Identification of dentin-producing odontoblasts and enamel-producing ameloblasts using reporter techniques is useful to study tooth development and regeneration with tissue engineering. Ameloblasts express Amelogenin, Ameloblastin, Enamelin, and Amelotin, whereas odontoblasts express Dentin sialophosphoprotein (Dspp) and Dentin matrix protein1 (Dmp1). Although there are several transgenic lines using promoter elements or bacterial artificial chromosomes (BACs) to label odontoblasts and ameloblasts, there is a possibility that the expression patterns vary from the endogenous genes. Here, we established 2 lines of mice where tdTomato was knocked into the second exon of X-chromosomal Amelogenin (Amelx), and green fluorescent protein (GFP) was knocked into the second exon of Dspp. tdTomato and GFP were highly expressed on secretory ameloblasts and secretory and fully differentiated odontoblasts, respectively. In addition, DSPP and AMELX were not produced in the dentin matrix and enamel matrix of DsppGFP/GFP and AmelxtdTomato male mice (as representative of AmelxtdTomato/Y hemizygous male mice), respectively. Moreover, micro-computed tomography analysis of AmelxtdTomato male mice revealed a notable reduction in enamel volume but increased dentin mineral density. DsppGFP/GFP mice had reduced dentin mineral density. To identify odontoblasts and ameloblasts from developing tooth, we examined the expression of mesenchymal cell surface molecules CD90, CD166 and epithelial cell surface molecules CD49f, Epcam1 with fluorescence on odontoblasts and ameloblasts in these mice. We found that GFP+ odontoblasts and tdTomato+ ameloblasts in tooth germ from 0.5-d-old DsppGFP/+ mice and AmelxtdTomato male mice were enriched in CD45-/Ter119-/Epcam1-/CD90+/Integrin α4+cell fractions and CD45-/Ter119-/Epcam1+/CD49f+/CD147+ cell fractions, respectively. By using antibodies against mesenchymal and epithelial cell surface molecules and fluorescence, we can easily distinguish odontoblasts from ameloblasts and isolate each cell for further studies. These mice would serve as useful models for tooth development and regeneration as well as provide concurrent observation for the differentiation processes of odontoblasts and ameloblasts in vivo and in vitro.

Cell-cell interaction in graft rejection responses: induction of anti-allo-class I H-2 tolerance is prevented by immune responses against allo-class II H-2 antigens coexpressed on tolerogen.

The intravenous sensitization of C57BL/6 (B6) mice with class I H-2-disparate B6-C-H-2bm1 (bm1) spleen cells results in almost complete abrogation of anti-bm1 CD8+ helper (proliferative and interleukin 2-producing) T cell (Th) activities. Although an appreciable portion of CD8+ cytotoxic T lymphocyte (CTL) precursors themselves remained after this regimen, such a residual CTL activity was eliminated after the engrafting of bm1 grafts, and these grafts exhibited prolonged survival. In contrast, the intravenous sensitization with (bm1 x B6-C-H-2bm12 [bm12])F1 cells instead of bm1 cells failed to induce the prolongation of bm1 graft survival as well as bm12 and (bm1 x bm12)F1 graft survival. In the (bm1 x bm12)F1-presensitized B6 mice before as well as after the engrafting of bm1 grafts, anti-bm1 CTL responses that were comparable to or slightly stronger than those observed in unpresensitized mice were induced in the absence of anti-bm1 Th activities. bm1 graft survival was also prolonged by intravenous presensitization with a mixture of bm1 and bm12 cells but not with a mixture of bm1 and (bm1 x bm12)F1 cells. The capacity of CD4+ T cells to reject bm12 grafts was eliminated by intravenous presensitization with antigen-presenting cell (APC)-depleted bm12 spleen cells. However, intravenous presensitization with APC-depleted (bm1 x bm12)F1 cells failed to induce the prolongation of bm1 graft survival under conditions in which appreciably prolonged bm12 graft survival was induced. More surprisingly, bm1 graft survival was not prolonged even when the (bm1 x bm12)F1 cell presensitization was performed in CD4+ T cell-depleted B6 mice. This contrasted with the fact that conventional class I-disparate grafts capable of activating self Ia-restricted CD4+ as well as allo-class I-reactive CD8+ Th exhibited prolonged survival in CD4+ T cell-depleted, class I-disparate cell-presensitized mice. These results indicate that: (a) intravenous presensitization with class I- and II-disparate cells fails to reduce anti-allo-class I rejection responses that would otherwise be eliminated using only class I-disparate cells; (b) such failure is generated according to the coexpression of both classes of alloantigens on a single cell as tolerogen; and (c) allo-class II antigens coexpressed on tolerogen function to activate CD4+ as well as non-CD4+ Th leading to the generation of anti-class I effector T cell responses.

Uncoupled cell cycle without mitosis induced by a protein kinase inhibitor, K-252a.

The staurosporine analogues, K-252a and RK-286C, were found to cause DNA re-replication in rat diploid fibroblasts (3Y1) without an intervening mitosis, producing tetraploid cells. Analysis of cells synchronized in early S phase in the presence of K-252a revealed that initiation of the second S phase required a lag period of 8 h after completion of the previous S phase. Reinitiation of DNA synthesis was inhibited by cycloheximide, actinomycin D, and serum deprivation, but not by Colcemid, suggesting that a functional G1 phase dependent on de novo synthesis of protein and RNA is essential for entry into the next S phase. In a src-transformed 3Y1 cell line, as well as other cell lines, giant cells containing polyploid nuclei with DNA contents of 16C to 32C were produced by continuous treatment with K-252a, indicating that the agent induced several rounds of the incomplete cell cycle without mitosis. Although the effective concentration of K-252a did not cause significant inhibition of affinity-purified p34cdc2 protein kinase activity in vitro, in vivo the full activation of p34cdc2 kinase during the G2/M was blocked by K-252a. On the other hand, the cyclic fluctuation of partially activated p34cdc2 kinase activity peaking in S phase still continued. These results suggest that a putative protein kinase(s) sensitive to K-252a plays an important role in the mechanism for preventing over-replication after completion of previous DNA synthesis. They also suggest that a periodic activation of p34cdc2 is required for S phases in the cell cycle without mitosis.

Phosrestin I undergoes the earliest light-induced phosphorylation by a calcium/calmodulin-dependent protein kinase in Drosophila photoreceptors.

Activation of PI-PLC initiates two independent branches of protein phosphorylation cascades catalyzed by either PKC or Ca2+/calmodulin-dependent protein kinase (CaMK). We find that phosrestin I (PRI), a Drosophila homolog of vertebrate photoreceptor arrestin, undergoes light-induced phosphorylation on a subsecond time scale which is faster than that of any other protein in vivo. We determine that a CaMK activity is responsible for in vitro PRI phosphorylation at Ser366 in the C-terminal tryptic segment, MetLysSer(P)IleGluGlnHisArg, in which Ser(P) represents phosphoserine366. We also demonstrate that Ser366 is the phosphorylation site of PRI in vivo by identifying the molecular species resulting from in-gel tryptic digestion of purified phospho-PRI using HPLC-electrospray ionization tandem quadrupole mass spectroscopy. From these data, we conclude that the CaMK pathway, not the PKC pathway, is responsible for the earliest protein phosphorylation event following activation of PI-PLC in living Drosophila photoreceptors.

Role of epidermal growth factor receptor in maintaining airway goblet cell hyperplasia in rats sensitized to allergen.

BACKGROUND: Stimulation of epidermal growth factor receptor (EGFR) induces airway goblet cell hyperplasia, but the role of this molecule in the maintenance of this pathologic change remains uncertain. OBJECTIVE: To determine the mechanisms by which goblet cell hyperplasia is maintained in airway epithelium, we investigated EGFR-induced signalling pathways that lead to both mucin production and antiapoptosis in vitro. We also tested whether the inhibition of EGFR tyrosine kinase speeds reversal of established goblet cell hyperplasia to normal epithelial phenotype in vivo. METHODS: MUC5AC production was measured by immunoassay, and antiapoptotic responses were determined by Bcl-2 expression and terminal deoxynucleotidyl transferase-mediated dUTP-biotin Nick End Labelling staining using NCI-H292 cells. The effect of an inhibitor of EGFR tyrosine kinase (AG1478) on goblet cell hyperplasia was also determined in rats sensitized with ovalbumin (OVA). RESULTS: MUC5AC was constitutively expressed and few apoptotic cells were observed in NCI-H292 cells under non-stimulated condition. TGF-alpha increased MUC5AC and Bcl-2 expression, an effect that was prevented by inhibitors of EGFR tyrosine kinase (AG1478), MEK (PD98059), and NF-kappaB (CAPE). After the addition of TGF-alpha, AG1478 and an inhibitor of phosphatidylinositol 3 kinase/Akt (LY294002), but not PD98059, induced a marked apoptotic response, which was prevented by the caspase inhibitor Z-VAD fmk. Goblet cell hyperplasia and EGFR expression in airway epithelium were noted in the OVA-sensitized rats. Intratracheal instillation of AG1478 induced apoptosis of goblet cells, reverting the airway epithelium to normal epithelial phenotype. CONCLUSION: These findings indicate that EGFR plays an important role in the maintenance of goblet cell hyperplasia. We speculate that inhibitors of the EGFR cascade might be an effective therapy of airway remodelling.

Trehalose sensitivity in Drosophila correlates with mutations in and expression of the gustatory receptor gene Gr5a.

Drosophila taste gene Tre is located on the distal X chromosome and controls gustatory sensitivity to a subset of sugars [1, 2]. Two adjacent, seven-transmembrane domain genes near the Tre locus are candidate genes for Tre. One (CG3171) encodes a rhodopsin family G protein receptor [3, 4], and the other (Gr5a) is a member of a chemosensory gene family encoding a putative gustatory receptor [5-7]. We carried out molecular analyses of mutations in Tre to elucidate their involvement in the gustatory phenotype. Here, we show that Tre mutations induced by P element-mediated genomic deletions disrupt Gr5a gene organization and the expression of Gr5a mRNA, while disruption of the CG3171 gene or its expression was not always associated with mutations in Tre. In flies with the spontaneous mutation Tre(01), both CG3171 and Gr5a mRNAs are transcribed. Coding sequences of these two candidate genes were compared among various strains. A total of three polymorphic sites leading to amino acid changes in CG3171 were not correlated with the gustatory phenotype. Among four nonsynonymous sites in Gr5a, a single nucleotide polymorphism leading to an Ala218Thr substitution in the predicted second intracellular loop cosegregated with Tre(01). Taken together, the mutation analyses support that Gr5a is allelic to Tre.

Living Donor Liver Transplantation for Progressive Familial Intrahepatic Cholestasis Type 1: Two Reported Cases.

BACKGROUND: Progressive familial intrahepatic cholestasis type 1 (PFIC1) is an inherited disease characterized by cholestatic features. We report two patients with PFIC1 who underwent liver retransplantation. CASE REPORT: One patient was a 3-year-old female who underwent liver transplantation for PFIC1. She presented with severe diarrhea and fatty liver, and went into liver failure. She therefore underwent liver retransplantation and external biliary diversion 8 years after the initial liver transplantation. The explanted liver was histologically diagnosed with chronic rejection. Her intractable diarrhea stopped after the retransplantation. She was diagnosed with a fatty liver 8 months after the retransplantation and died 4 years after retransplantation due to bleeding from an ileostomy. The other patient was a 3-year-old male. This patient underwent liver retransplantation due to liver cirrhosis caused by steatohepatitis 9 years after the initial liver transplantation. The biliary tract was not diverted. He also experienced severe diarrhea after the retransplantation and requires home parenteral nutrition due to an eating disorder. CONCLUSIONS: Liver transplantation is the only treatment to resolve life-threatening issues due to PFIC1, but requires further improvement as a therapeutic modality.

Crosstalk between NRF2 and HIPK2 shapes cytoprotective responses.

Homeodomain interacting protein kinase-2 (HIPK2) is a member of the HIPK family of stress-responsive kinases that modulates cell growth, apoptosis, proliferation and development. HIPK2 has several well-characterised tumour suppressor roles, but recent studies suggest it can also contribute to tumour progression, although the underlying mechanisms are unknown. Herein, we have identified novel crosstalk between HIPK2 and the cytoprotective transcription factor NRF2. We show that HIPK2 is a direct transcriptional target of NRF2, identifying a functional NRF2 binding site in the HIPK2 gene locus and demonstrating for the first time a transcriptional mode of regulation for this kinase. In addition, HIPK2 is required for robust NRF2 responsiveness in cells and in vivo. By using both gain-of-function and loss-of-function approaches, we demonstrate that HIPK2 can elicit a cytoprotective response in cancer cells via NRF2. Our results have uncovered a new downstream effector of HIPK2, NRF2, which is frequently activated in human tumours correlating with chemoresistance and poor prognosis. Furthermore, our results suggest that modulation of either HIPK2 levels or activity could be exploited to impair NRF2-mediated signalling in cancer cells, and thus sensitise them to chemotherapeutic drugs.

An Arabidopsis cDNA encoding a DNA-binding protein that is highly similar to the DEAH family of RNA/DNA helicase genes.

A cDNA encoding a putative RNA and/or DNA helicase has been isolated from Arabidopsis thaliana cDNA libraries. The cloned cDNA is 5166 bases long, and its largest open reading frame encodes 1538 amino acids. The central region of the predicted protein is homologous to a group of nucleic acid helicases from the DEAD/H family. However, the N- and C-terminal regions of the Arabidopsis cDNA product are distinct from these animal DEIH proteins. We have found that the C-terminal region contains three characteristic sequences: (i) two DNA-binding segments that form a probe helix (PH) involved in DNA recognition; (ii) an SV40-type nuclear localization signal; and (iii) 11 novel tandem-repeat sequences each consisting of about 28 amino acids. We have designated this cDNA as NIH (nuclear DEIH-boxhelicase). Functional character-ization of a recombinant fusion product containing the repeated region indicates that NIH may form homodimers, and that this is the active form in solution. Based on this information and the observation that the sequence homology is limited to the DEAH regions, we conclude that the biological roles of the plant helicase NIH differ from those of the animal DEIH family.

Elevated expression of the ornithine decarboxylase gene in human esophageal cancer.

The mechanisms involved in sustaining the high levels of ornithine decarboxylase (ODC) activity in human cancers are not well defined. We examined the level of expression of ODC mRNA together with ODC activity in surgically excised human cancers, including esophagus, stomach, colon, and liver tumors, the objective being to determine whether the ODC mRNA level correlates with enhancement of ODC activity in these cancers. Among these tumors, the esophageal cancers had the highest ODC activity (120 +/- 43.9 pmol of CO2/h/mg of protein), compared with the stomach (37.6 +/- 13.7), colon (22.8 +/- 5.9), and liver (10.2 +/- 5.6) cancers. A remarkable increase in ODC mRNA was seen in all of the esophageal cancers. The ratio of ODC mRNA in the tumors, relative to the paired normal tissues, was 14.6 +/- 3.7. Some increase was noted in some of the stomach (2.9 +/- 0.9) and colon (2.1 +/- 0.9) cancers, but there was no increase in the liver tumors (0.9 +/- 0.2). A significant correlation was noted between ODC activity and mRNA expression in cancerous and noncancerous tissues of the esophagus, stomach, and colon, thereby suggesting that increased steady-state mRNA may be responsible for the high ODC activity in these tumors. Southern blot analysis of the DNA from the esophageal cancers revealed no amplification or significant rearrangement of the gene. Mechanisms sustaining high ODC mRNA levels in esophageal cancers may be an enhancement of the promoter activity of this gene or stabilization of the mRNA.

Randomly controlled study of chemotherapy versus chemoimmunotherapy in postoperative gastric cancer patients.

From September 1979 through March 1981, a total of 302 patients with gastric cancer and undergoing gastrectomy at the Department of Surgery at Chiba University Hospital and its 14 affiliated hospitals was studied for clinical effectiveness of immunotherapy with Nocardia rubra cell wall skeleton. The patients were stratified by gross stage of cancer and degree of operative curability. They were then assigned randomly to either chemotherapy group or chemotherapy plus immunotherapy group. Immunotherapy used was intradermal injection of 400 micrograms of N. rubra cell wall skeleton which was given weekly for the first month and monthly thereafter. After the specimen was examined microscopically, the patients were classified by histological stage of cancer and radicality of surgical intervention into curative or noncurative groups. The patients were surveyed for survival period in December 1981. The postoperative survival rate was compared in patients of histologically curative or noncurative resection cases between the two treatment groups. No statistical difference was detected between the groups in age, sex, or operative procedures that might influence the patient's survival. As a result, statistical intergroup difference in survival rates was not seen in patients of the curative group, probably due to a short observation period. However, the intergroup difference in survival rates was statistically significant in patients of the noncurative group (p less than 0.01). These results indicate the adjunctive effect of N. rubra cell wall skeleton as an immunotherapeutic agent in patients undergoing gastrectomy for gastric cancer.

Molecular cloning of a cDNA encoding a novel Ca(2+)-dependent nuclease of Arabidopsis that is similar to staphylococcal nuclease.

We have isolated a cDNA from Arabidopsis thaliana for a protein consisting of 323 amino acids with similarity to an extracellular nuclease from Staphylococcus. Nuclease assay using toluidine blue-DNA plates has demonstrated that the gene product has nuclease activity dependent on Ca(2+) and inhibited by Zn(2+), designated CAN (Ca(2+)-dependent nuclease). Differing from the staphylococcal nuclease, CAN has neither a signal peptide nor any long hydrophobic regions, suggesting that it is not a secreted protein.

Cloning and characterization of two transcripts generated from the mel-13 gene positioned adjacent to the mammalian Polycomb group-related gene mel-18.

We previously isolated the mel-18 gene, a mammalian Polycomb group (PcG)-related gene with homology to bmi-1 oncogene. We show in this paper the existence of a new gene, mel-13, which overlapped with the mel-18 anti-oncogene. We discuss the relationships between mel-13 and the mel-18, bup, and Su(z)2 genes.

Dependency on light and vitamin A derivatives of the biogenesis of 3-hydroxyretinal and visual pigment in the compound eyes of Drosophila melanogaster.

When the fruitfly, Drosophila melanogaster, was reared on media deficient in carotenoids and retinoids, the level of 3-hydroxyretinal (the chromophore of fly rhodopsin) in the retina decreased to less than 1% compared with normal flies. The level of 3-hydroxyretinal increased markedly in flies that were given a diet supplemented with retinoids or carotenoids. The retinas of flies fed on all-trans retinoids and maintained in the dark predominantly contained the all-trans form of 3-hydroxyretinal, and showed no increase in the level of either the 11-cis isomer or the visual pigment. Subsequent illumination of the flies converted substantial amounts of all-trans 3-hydroxyretinal to its 11-cis isomer. The action spectrum of the conversion by illumination showed the optimum wavelength to be approximately 420 nm, which is significantly greater than the absorption maximum of free, all-trans 3-hydroxyretinal. Flies that were fed on carotenoids showed a rapid increase of the levels of 11-cis 3-hydroxyretinal and of visual pigment in the absence of light.

Current progress on nucleoside antibiotics.

Structure and biological activity of thirty-six new nucleoside antibiotics which appeared after the 1988 review are described. New synthetic analogs of neplanocin and oxetanocin are also described with special emphasis on their antiviral activities. New biosynthetic findings on nikkomycins, blasticidin S, and griseolic acid are also reviewed.

Taste sensitivity to trehalose and its alteration by gene dosage in Drosophila melanogaster.

The taste sensitivity to the disaccharide trehalose of Drosophila melanogaster is under the genetic control by the Tre gene on the X chromosome. The gene is genetically dimorphic for high and low sensitivity and is likely to be functioning in the primary step of chemoreception. We have determined the cytological localization of the Tre gene to be between 5A10 and 5B1-3 by analyzing the sensitivity to trehalose in flies which are segmentally aneuploid bearing either deficiencies or duplicated fragments of T(X;Y) translocations. We also constructed flies which are aneuploidy and thus carry different dosage of Tre and/or Tre(+) alleles in order to examine the gene dosage effect on trehalose sensitivity and to deduce the nature of the gene's action. Trehalose sensitivity decreased in females carrying half the normal dosage of a given Tre allele, but a proportional increase in sensitivity was not observed in flies bearing a duplication of the Tre alleles. The changes in sensitivity in various aneuploid flies suggest that there is an upper limit to the number of molecules that can be incorporated into the receptor membrane. Genetic evidence strongly suggests that Tre is the structural gene for the trehalose receptor. We present a model to account for the mechanism of genetical control on the sensitivity to trehalose.

The role of Bcl6 in mature cardiac myocytes.

OBJECTIVE: The Bcl6 gene encodes a sequence-specific transcriptional repressor and is ubiquitously expressed in adult murine tissues including heart muscle. The objective of this study was to examine the role of Bcl6 in cardiac myocytes. METHOD: We developed Bcl6-deficient (Bcl6-/-) mice and histologically examined hearts from these mice. RESULTS: Massive myocarditis with eosinophilic infiltration occurred in Bcl6-/- mice after 4-6 weeks of age. Since expression of the Bcl6 gene was induced in normal cardiac myocytes after 2 weeks of age and thereafter detected through adulthood, loss of Bcl6 in mature cardiac myocytes may be related to the induction of eosinophilic myocarditis. To examine the effects of eosinophils from Bcl6-/- mice on normal hearts, bone marrow cells from Bcl6-/- mice were adoptively transferred into sublethally irradiated RAG1-deficient mice. Although massive eosinophilic infiltration was detected in conjunctivas and spleens from the chimeric mice, myocarditis was never observed. Electron microscopic analysis of cardiac myocytes from Bcl6-/- mice revealed a spectrum of degenerative changes prior to eosinophilic infiltration. CONCLUSION: Bcl6 maynot be essential for the maturation of cardiac myocytes but may play a role in protecting mature cardiac myocytes from eosinophilic inflammation.

Identification of likely genes on chromosome VI of Saccharomyces cerevisiae by correlating transcripts and nucleotide sequence data.

Most of the 97 transcripts of the genes on chromosome VI of Saccharomyces cerevisiae that were identified by a series of Northern hybridization experiments (Yoshikawa and Isono, Nucl. Acids Res., 19, 1189-1195, 1991) have been correlated with the open reading frames (ORFs) deduced from the nucleotide sequence data of this chromosome (Murakami et al., Nature Genet., 10, 261-268, 1995). This was performed by comparing the experimentally constructed physical map and the one produced from the nucleotide sequence data, as well as the sizes and positions of observed transcripts and those of sequenced ORFs. Thus, 75 ORFs of chromosome VI were correlated uniquely with the corresponding transcripts and 3 ORFs with two transcripts of different sizes. Comparing the relative abundance levels of individual transcripts with that of the RPO41 transcript, highly expressed genes of chromosome VI were found to be located almost exclusively on the Crick strand. Based on the correlation between the abundance level of the experimentally identified transcripts and the codon adaptation indices of the corresponding gene, the genes on chromosome VI of S. cerevisiae were classified into three groups. The data thus provides information concerning their chromosomal locations as well as their likely levels of expression in vegetatively growing cells.

Cloning and characterization of the ribosomal protein genes in the spc operon of a prokaryotic endosymbiont of the pea aphid, Acyrthosiphon kondoi.

To correlate a prokaryotic endosymbiont in the pea aphid, Acyrthosiphon kondoi, with the endosymbionts in related aphid species as well as with free-living bacteria and subcellular organelles, and to study the mode of its gene expression within aphid cells, we have cloned and characterized the genes encoding ribosomal proteins S3, L16, L29, S17, L14, L24, L5, S14, S8, L6, L18, S5, L30, L15 and secretion protein Y (Sec Y) from the S10 and spc ribosomal protein gene operons of this endosymbiont. The organization of these genes is identical to that in Escherichia coli, and their nucleotide sequences are highly similar (87% identity) to the corresponding E. coli genes. They are much less similar to the corresponding chloroplast and mitochondrial genes. The guanine plus cytosine G+C content of the genes of the A. kondoi endosymbiont is much higher than those of the endosymbionts in related aphid species reported so far. It appears either that the A. kondoi endosymbiont is derived from an ancestral bacterium different from those in other aphids or that its G+C content increased in a relatively short time after the evolutionary divergence of its host.

Characteristic features of the nucleotide sequences of yeast mitochondrial ribosomal protein genes as analyzed by computer program GeneMark.

The nucleotide sequence data for yeast mitochondrial ribosomal protein (MRP) genes were analyzed by the computer program GeneMark which predicts the presence of likely genes in sequence data by calculating statistical biases in the appearance of consecutive nucleotides. The program uses a set of standard sequence data for this calculation. We used this program for the analysis of yeast nucleotide sequence data containing MRP genes, hoping to obtain information as to whether they share features in common that are different from other yeast genes. Sequence data sets for ordinary yeast genes and for 27 known MRP genes were used. The MRP genes were nicely predicted as likely genes regardless of the data sets used, whereas other yeast genes were predicted to be likely genes only when the data set for ordinary yeast genes was used. The assembled sequence data for chromosomes II, III, VIII and XI as well as the segmented data for chromosome V were analyzed in a similar manner. In addition to the known MRP genes, eleven ORF's were predicted to be likely MRP genes. Thus, the method seems very powerful in analyzing genes of heterologous origins.