Macular corneal dystrophy type I and type II are caused by distinct mutations in a new sulphotransferase gene.

Macular corneal dystrophy (MCD; MIM 217800) is an autosomal recessive hereditary disease in which progressive punctate opacities in the cornea result in bilateral loss of vision, eventually necessitating corneal transplantation. MCD is classified into two subtypes, type I and type II, defined by the respective absence and presence of sulphated keratan sulphate in the patient serum, although both types have clinically indistinguishable phenotypes. The gene responsible for MCD type I has been mapped to chromosome 16q22, and that responsible for MCD type II may involve the same locus. Here we identify a new carbohydrate sulphotransferase gene (CHST6), encoding an enzyme designated corneal N-acetylglucosamine-6-sulphotransferase (C-GlcNAc6ST), within the critical region of MCD type I. In MCD type I, we identified several mutations that may lead to inactivation of C-GlcNAc6ST within the coding region of CHST6. In MCD type II, we found large deletions and/or replacements caused by homologous recombination in the upstream region of CHST6. In situ hybridization analysis did not detect CHST6 transcripts in corneal epithelium in an MCD type II patient, suggesting that the mutations found in type II lead to loss of cornea-specific expression of CHST6.

A radiation hybrid map of the rat genome containing 5,255 markers.

A whole-genome radiation hybrid (RH) panel was used to construct a high-resolution map of the rat genome based on microsatellite and gene markers. These include 3,019 new microsatellite markers described here for the first time and 1,714 microsatellite markers with known genetic locations, allowing comparison and integration of maps from different sources. A robust RH framework map containing 1,030 positions ordered with odds of at least 1,000:1 has been defined as a tool for mapping these markers, and for future RH mapping in the rat. More than 500 genes which have been mapped in mouse and/or human were localized with respect to the rat RH framework, allowing the construction of detailed rat-mouse and rat-human comparative maps and illustrating the power of the RH approach for comparative mapping.

CREB-H: a novel mammalian transcription factor belonging to the CREB/ATF family and functioning via the box-B element with a liver-specific expression.

The expression of liver-specific genes is regulated by unequivocally allocated transcription factors via proper responsible elements within their promoters. We identified a novel transcription factor, CREB-H, and found that its expression was restricted in the liver among 16 human tissues tested. A region of CREB-H exhibited significant homology to the basic leucine zipper (b-Zip) domain of members of the CREB/ATF family: mammalian LZIP and Drosophila BBF-2 that binds to box-B, a Drosophila enhancer modulating the fat-body-specific gene expression. CREB-H contained a hydrophobic region representing a putative transmembrane domain, like LZIP. Constructing a variety of CREB-H fusion proteins with the GAL4 DNA-binding domain disclosed that CREB-H functioned as a transcriptional activator and its N-terminal 149 amino acids accounted for the activation ability. Gel mobility sift assays revealed that CREB-H did not bind to the C/EBP, AP-1 and NF-kappaB elements but specifically bound to CRE and the box-B element. Luciferase reporter assays demonstrated that like BBF-2, CREB-H activated transcription via the box-B element and that a deletion of the putative transmembrane domain increased the activation of reporter expression significantly. Furthermore, a fusion protein of GFP and full-length CREB-H was localized in reticular structures surrounding the nucleus, whereas a fusion protein of GFP and a deletion mutant lacking the putative transmembrane domain was mainly in the nucleus. These findings suggest that CREB-H plays an important role in transcriptional regulation of genes specifically expressed in the liver, and that the putative transmembrane domain may be associated with modulation of its function as the transcriptional activator.

Allelotype of breast cancer: cumulative allele losses promote tumor progression in primary breast cancer.

Allele loss on a specific chromosome has implied the existence of a tumor suppressor gene such as the p53 gene and the RB gene. In order to determine which chromosome(s) carries a tumor suppressor gene(s) that contributes to tumor progression in primary breast cancer, we analyzed the loss of heterozygosity for each autosomal chromosome arm by using 39 restriction fragment length polymorphism markers including 25 variable numbers of tandem repeat probes. In 79 primary breast cancers, we found the frequent loss on the long arm of chromosome 13 (21%), the long arm of chromosome 16 (45%), and the short arm of chromosome 17 (56%). Interestingly, breast cancers in which loss of both chromosomes 13q and 17p was detected showed more malignant histopathological features, and a group of the tumors in which chromosome 16q loss was detected presented with frequent lymph node metastasis. Furthermore, the result of the deletion mapping on chromosome 17p implied the existence of a tumor suppressor gene distal to the p53 gene as well as the p53 gene itself for primary breast cancer. These results suggest that at least 4 tumor suppressor genes exist on chromosomes 13q, 16q, and 17p for primary breast cancer.

Deletion within the D17S34 locus in a primitive neuroectodermal tumor.

Loss of heterozygosity on chromosome 17p13.3 is frequently observed in solid tumors, and the presence of a tumor suppressor gene has been predicted in this region of chromosome 17. We have analyzed a primitive neuroectodermal tumor sample exhibiting loss of heterozygosity at the D17S34 locus, a commonly used telomeric marker on the short arm of chromosome 17. The remaining allele showed a rearrangement. Cosmids spanning the D17S34 locus and probes from that region were used to demonstrate a 9-kb deletion within the D17S34 locus and were found to contain evolutionary, conserved sequences. Genetic alterations in this region may also affect expression of immediately adjacent genes, such as ABR, and could be a common mechanism in the causation of primitive neuroectodermal tumors.

Isolation and mapping of a human lung-specific gene, TSA1902, encoding a novel chitinase family member.

Using differential display technique, we have isolated a novel human gene expressed specifically in the lung. Two forms of the gene, designated TSA1902, were transcribed by alternate mRNA splicing. The transcribed mRNAs, termed TSA1902-L and TSA1902-S, putatively encode proteins of 368 and 315 amino acids, respectively, which show high similarity to human chitotriosidase protein. The N-terminal region of TSA1902-L protein contains the conserved active site residues (DXXDXDXE) of the catalytic center of various chitinases which are essential for chitinase activity. The deduced protein sequence of TSA1902-S, however, does not possess this active site, with the N-terminal 54 amino acids present in TSA1902-L protein having been deleted. Both proteins lacked the secretory sequence of N-termini and, judging from the hydropathy profile, may be soluble proteins in the cytoplasm. Chromosomal mapping by radiation hybrid analysis localized this gene to the chromosome 1p13.1-p21.3.

Expression of TMEFF1 mRNA in the mouse central nervous system: precise examination and comparative studies of TMEFF1 and TMEFF2.

TMEFF1 and TMEFF2 are putative transmembrane proteins comprised of one epidermal growth factor (EGF)-like domain and two follistatin-like domains. Both TMEFF1 and TMEFF2 are predominantly expressed in the brain. We previously demonstrated that recombinant TMEFF2 protein can promote survival of neurons in primary culture and determined expression sites of TMEFF2 mRNA in the mouse central nervous system. To extend our understanding of TMEFF protein functions, we compared precise sites of expression of TMEFF1 and TMEFF2 mRNA using in situ hybridization analysis. Although both TMEFF genes are widely expressed in the brain, they exhibit different patterns of expression. TMEFF1 showed comparatively higher signals in the pyramidal cells of fifth layer of the cerebral neocortex, CA3, CA1 and subiculum regions of the hippocampus, locus coeruleus, and dentate cerebellar nucleus. In contrast, TMEFF2 is highly expressed in the medial habenular, CA2, CA3 and dentate gyrus region of the hippocampus, corpus callosum, cerebellar cortex and cranial nerve nuclei (III, IV, VII, X, XII). The results presented here indicate that expression of TMEFF1 and TMEFF2 are regulated differently and that they play region-specific roles in the central nervous system.

Molecular characterization of a recurring complex chromosomal translocation in two human extragonadal germ cell tumors.

The molecular characterization of a recurring complex chromosomal translocation involving 6p21, 6p22, 6q23, and 11q13 in two independent but similar extragonadal human germ cell tumors was initiated using fluorescence in situ hybridization (FISH) and pulse field gel electrophoresis (PFGE) techniques. By using a series of specific probes from the 11q13 region, the translocation breakpoint in this chromosomal band could be located within a long-range restriction enzyme map in between the markers D11S457 and D11S546. In addition, aberrantly hybridizing restriction fragments were revealed by PFGE in both tumors, indicating that the breakpoint region must be located within a distance of at maximum 200 kilobase pairs (kbp) from the nearest DNA marker (D11S546).

HPV33 DNA in premalignant and malignant breast lesions in Chinese and Japanese populations.

The relationship between human papillomavirus (HPV) infection and breast cancer is controversial. In this study, paraffin-embedded tissue blocks prepared from 72 patients with benign, premalignant or malignant mammary lesions were randomly collected from the Shanghai region of China and Tokushima in Japan. DNA specimens extracted from all tissues were amplified by the polymerase chain reaction (PCR) using HPV16, 18 and 33 primers. Southern blot hybridization showed 19 cases to be positive for HPV33 DNA: The positive rate for HPV33 DNA in Chinese (41.7%) was significantly higher than in Japanese (11.1%) (P < 0.01): The positive rate for HPV33 DNA in invasive ductal carcinoma (34.1%) was higher than in benign or borderline mammary lesions (5%) (P < 0.02). There were no statistically significant difference among the relationship of the nuclear grade of breast cancers with HPV33 DNA-positivity. This is the first report of a positive correlation between HPV33 DNA and breast lesions in Chinese and Japanese populations. These results suggest that the infection by HPV33, but not HPV 16 or HPV 18, may be involved in breast hyperplastic lesions, especially breast cancer, in humans.

Human papillomavirus type 33 DNA in breast cancer in Chinese.

BACKGROUND: The association between human papillomavirus (HPV) and anogenital tumors, especially cervical cancer, is well documented. However, it remains unclear whether there is also a correlation between HPV infection and human breast cancer. METHODS: We used PCR and Southern blot hybridization to analyze HPV-related DNA specimens from 32 cases of invasive ductal carcinoma operated upon in the Shanghai region of China. RESULTS: DNA derived from HPV33 was detected in 14 cases (43.8%). No HPV16 or HPV18 DNA was detected in any of the cases in this study. This is the first report demonstrating a correlation between HPV33 infection and breast cancer. CONCLUSIONS: Our results suggest that HPV33 infection may be involved in the pathogenesis of breast cancer in Chinese.

[Self-evaluation on the ability to retrieve Kanji words: a comparison between an aphasiac and normal subjects].

The present study investigated the accuracy of an aphasiac's self-evaluation on his ability to retrieve words. The aphasiac and six normal subjects were shown a list of words and that of drawings. The words were written in Kanji and the drawings could be translated into the Kanji words. For each word, the subjects rated the likelihood that they could write it, and for each drawing, they rated the likelihood that they could translate it into the Kanji word. A week later, they were asked to translate drawings into Kanji words, and the performance and self-ratings were compared. Although the aphasiac could write far less Kanji words than the normals, his self-rating was as high as the normals. It was suggested that metamnemonic judgments are based on the indirect inferential processes, not on the direct access to the memory-traces. In other words, the aphasiac might raise his rating scores when Kanji words or drawings seemed easy to understand.

Comparative PCR: a simple and sensitive method for quantifying low-abundance mRNA species.

To measure low-abundance messenger RNA species comparatively, we developed a simple and highly sensitive quantification method designated comparative PCR. Messenger RNAs from two samples were converted into cDNAs with modified oligo(dT) primers (designated RT primers) containing a sample-specific sequence and a common sequence. After equal amounts of the cDNAs were mixed together, a target gene was amplified by competitive PCR with additional primers: a gene-specific primer and a primer consisting of the common sequence of the RT primers. The amplified products were visualized by the final PCR, designated fluorescence PCR, with an additional three primers: two different fluorescence-labeled primers consisting of the sample-specific sequence within the RT primers and a nested gene-specific primer. Expression levels of the target gene in the two samples were measured by calculating ratios of two different fluorescence intensities. We could quantify 0.1-0.3 copies of the target mRNA per cell from only 0.5 ng of poly(A)(+) RNA for a single detection. This system should be useful for sensitive measurement of scarce transcripts from small samples with a limited amount of RNA such as biopsy specimens.

Automated extraction of information on protein-protein interactions from the biological literature.

MOTIVATION: To understand biological process, we must clarify how proteins interact with each other. However, since information about protein-protein interactions still exists primarily in the scientific literature, it is not accessible in a computer-readable format. Efficient processing of large amounts of interactions therefore needs an intelligent information extraction method. Our aim is to develop an efficient method for extracting information on protein-protein interaction from scientific literature. RESULTS: We present a method for extracting information on protein-protein interactions from the scientific literature. This method, which employs only a protein name dictionary, surface clues on word patterns and simple part-of-speech rules, achieved high recall and precision rates for yeast (recall = 86.8% and precision = 94.3%) and Escherichia coli (recall = 82.5% and precision = 93.5%). The result of extraction suggests that our method should be applicable to any species for which a protein name dictionary is constructed. AVAILABILITY: The program is available on request from the authors.

Cloning, expression, and chromosomal mapping of the human 14-3-3gamma gene (YWHAG) to 7q11.23.

The 14-3-3 family of proteins exerts diverse influences on the signal transduction pathways of cells. We have newly identified a human cDNA encoding the gamma subtype of the 14-3-3 family of genes. The deduced amino acid sequence of human 14-3-3gamma was identical to that of rat 14-3-3gamma. The human 14-3-3gamma gene (HGMW-approved symbol YWHAG) is highly expressed in brain, skeletal muscle, and heart. By fluorescence in situ hybridization analysis, the human 14-3-3gamma gene was mapped to chromosome 7q11.23. Radiation hybrid mapping has shown that this gene is localized 2.33 cR telomeric to D7S1870, a polymorphic marker located at the most telomeric end of the common deletion region of Williams-Beuren syndrome (WBS). This suggests that haploinsufficiency of 14-3-3gamma may not contribute to the WBS phenotype. However, information regarding the precise chromosomal location of a member of the 14-3-3 family of genes will aid in examining the relationship between this family of proteins and human disorders.

Physical mapping of a 950-kb region surrounding a locus (D10S102) tightly linked to the MEN2A gene.

We have constructed a long-range contig of cosmid and YAC clones around D10S102, a locus that is tightly linked to the gene responsible for multiple endocrine neoplasia type 2A (MEN2A). With D10S102 as a starting point, a 360-kb cosmid contig was constructed by bidirectional genomic walking, and at least six fragments from these cosmids showed high sequence homology to other species. Five YAC clones were also isolated at the D10S102 locus, and they formed a contig covering 950 kb of genomic DNA. Furthermore, we obtained six RFLP systems from the contig, which will serve as new resources for fine-scale genetic linkage mapping of the MEN2A locus.

Thirty-one new RFLP systems detected by twenty-four DNA markers on human chromosome 10.

Thirty-one new RFLP systems corresponding to 24 loci have been identified from a chromosome 10-specific cosmid library. Twelve of the markers on the proximal long arm (cen-q11.2) of this chromosome, including four RFLP systems for the RET locus, will be especially useful in efforts to identify the gene responsible for multiple endocrine neoplasia type 2A (MEN2A). The new panel of markers also may contribute to fine-scale mapping of tumor suppressor genes associated with glioblastoma multiforme or renal cell carcinoma, because allelic deletions in these tumors have implied the presence of a tumor suppressor gene(s) on chromosome 10.

A high-resolution cytogenetic map of 168 cosmid DNA markers for human chromosome 11.

We have constructed a high-resolution cytogenetic map with 168 DNA markers, including 90 RFLP markers for human chromosome 11. The cosmid clones were mapped by fluorescence in situ suppression hybridization, in which discrete fluorescent signals can be detected directly on prometaphase R-banded chromosomes. Although these cosmid clones were distributed throughout the chromosome, they had some tendency to localize in the regions of R-positive band, such as 11p15, 11p11.2, 11q13, 11q23, and 11q25. Since these regions of chromosome 11 are considered to contain genes responsible for certain genetic diseases, cancer breakpoints involved in chromosome rearrangements, and tumor-suppressor genes, this high-resolution cytogenetic map will contribute to the molecular characterization of such genes. This map will also provide many landmarks essential for construction of the complete physical map with contigs of cosmid and YAC clones.

A 14-Mb physical map of the region at chromosome 11q13 harboring the MEN1 locus and the tumor amplicon region.

We have constructed a physical map of chromosome 11q13, using 54 DNA markers that had been localized to 11q13.1----q13.5 by means of somatic hybrid cell panels. Although the map has some gaps, it spans nearly 14 Mb and includes the region containing the gene responsible for multiple endocrine neoplasia type 1 (MEN1) and also the region that is amplified in several types of malignant tumors. As the estimated average distance between each locus is roughly 300 kb, the markers reported here will be valuable resources for construction of contig maps with yeast artificial chromosomes and/or cosmid clones. Furthermore, these clones will be useful in efforts to identify the MEN1 gene and in analyses of the amplification units present at 11q13 in certain tumors.

Identification of 57 conventional RFLP and 6 VNTR systems with 32 DNA clones on chromosome 11p15.

Fifty-four clones containing human inserts were selected from a cosmid library constructed from a somatic cell hybrid containing chromosome 11p15.3-p15.5 as its only human complement. In 32 of these clones, 63 polymorphic systems were identified with a panel of restriction enzymes: 57 conventional RFLP systems and 6 highly polymorphic VNTR systems. Although we examined the cosmid with only seven enzymes, 18 clones (including 6 VNTRs) were polymorphic with three or more enzymes. The results suggested that DNA sequences on the peritelomeric region of chromosome 11p tend to be highly variable. Because these markers are highly informative, they will be excellent resources for investigations of hereditary diseases and tumor suppressor genes in this region of chromosome 11.