Long-term effects of neonatal exposure to MK-801 on recognition memory and excitatory-inhibitory balance in rat hippocampus.

Blockade of the N-methyl-d-aspartate receptors (NMDARs) during the neonatal period has been reported to induce long-term behavioral and neurochemical alterations that are relevant to schizophrenia. In this study, we examined the effects of such treatment on recognition memory and hippocampal excitatory and inhibitory (E/I) balance in both adolescence and adulthood. After exposure to the NMDAR antagonist, MK-801, at postnatal days (PND) 5-14, male Sprague-Dawley rats were tested for object and object-in-context recognition memory during adolescence (PND 35) and adulthood (PND 63). The parvalbumin-positive (PV+) γ-aminobutyric acid (GABA)-ergic interneurons and presynaptic markers for excitatory and inhibitory neurons, vesicular glutamate transporter-1 (VGLUT1) and vesicular GABA transporter (VGAT) were examined in the hippocampus to reflect the E/I balance. We found that rats receiving MK-801 treatment showed deficits of recognition memory, reduction in PV+ cell counts and upregulation of the VGLUT1/VGAT ratio in both adolescence and adulthood. Notably, the changes of the VGLUT1/VGAT ratio at the two time points exhibited distinct mechanisms. These results parallel findings of hippocampal abnormalities in schizophrenia and lend support to the usefulness of neonatal NMDAR blockade as a potential neurodevelopmental model for the disease.

Mapping quantitative trait loci for nitrogen uptake and utilization efficiency in rice (Oryza sativa L.) at different nitrogen fertilizer levels.

Genetic improvement is the fundamental basis for improving nitrogen-use efficiency. A better understanding of genetic factors controlling nitrogen uptake and utilization is required for crop genetic improvement. In this study, we identified the quantitative trait loci (QTLs) associated with traits of nitrogen uptake and utilization by using the single-sequence repeat marker method and a recombinant inbred line (RIL) population derived from a super hybrid Xieyou9308. All the traits investigated were inherited quantitatively by continuous variation and showed normal distribution in phenotype with transgressive segregation in the RIL population. Most of the traits were significantly correlated with each other except for nitrogen absorption ability (NAA) with nitrogen harvest index (NHI) and NHI with agricultural nitrogen-absorption efficiency (ANAE). At logarithmic odds value of 2.3, total 13 candidate QTLs, including 4 for NAA, 2 for NHI, 2 for physiological nitrogen-use efficiency, 1 for agricultural nitrogen-use efficiency (ANUE), and 4 for ANAE, were detected and mapped on chromosomes 2, 3, 4, 5, 8, 9, 10, and 12. Significant pleiotropic effect or neighboring expression of QTLs was observed among traits. At position 64.8 cM on chromosome 4 near the marker RM5757, there was a QTL cluster of NAA, ANUE, and ANAE, and at chromosome 5 near the marker RM5968, there was a QTL cluster of NAA and ANUE. The QTL clusters might provide partial explanation and genetic mechanism for the observed correlations between nitrogen uptake and utilization efficiency traits and might form a basis for future breeding programs.

Development of a human mitochondria-focused cDNA microarray (hMitChip) and validation in skeletal muscle cells: implications for pharmaco- and mitogenomics.

Mitochondrial research has influenced our understanding of human evolution, physiology and pathophysiology. Mitochondria, intracellular organelles widely known as 'energy factories' of the cell, also play fundamental roles in intermediary metabolism, steroid hormone and heme biosyntheses, calcium signaling, generation of radical oxygen species, and apoptosis. Mitochondria possess a distinct DNA (mitochondrial DNA); yet, the vast majority of mitochondrial proteins are encoded by the nuclear DNA. Mitochondria-related genetic defects have been described in a variety of mostly rare, often fatal, primary mitochondrial disorders; furthermore, they are increasingly reported in association with many common morbid conditions, such as cancer, obesity, diabetes and neurodegenerative disorders, although their role remains unclear. This study describes the creation of a human mitochondria-focused cDNA microarray (hMitChip) and its validation in human skeletal muscle cells treated with glucocorticoids. We suggest that hMitChip is a reliable and novel tool that will prove useful for systematically studying the contribution of mitochondrial genomics to human health and disease.

Isolation of tumor suppressor genes in melanoma by cDNA microarray.

The multistep genetic alterations thought to involve both oncogenes and tumor suppressor genes that are causally related to melanocytic transformation remain largely undetermined (1). Mapping of alterations to chromosome 6 indicates that multiple genetic loci on 6q contribute causally to the development and progression of malignant melanoma (1). This notion is also supported by the introduction of chromosome 6 in malignant melanoma cell lines suppressing either their tumorigenicity (2) or metastasis (3,4). However, the suppressor genes involved have yet to be identified.

Identification of tumor-suppressor genes using human melanoma cell lines UACC903, UACC903(+6), and SRS3 by comparison of expression profiles.

The development and progression of cancer are believed to be due to multiple genetic alterations resulting in complex changes in expression of many genes. The parental malignant melanoma cell line UACC903 displays anchorage-independent growth, and the chromosome 6-suppressed subline UACC903(+6) displays anchorage-dependent growth. The anchorage-independent revertant cell line SRS3 derived from UACC903(+6) by retroviral transduction resembles the phenotype of UACC903. In this study, we first compared the expression profiles of 3317 genes between these three cell lines in pairs by cDNA microarrays, resulting in identification of genes with known suppressor activities. We then demonstrated connexin 43 (Cx43)-suppressing anchorage-independent growth of UACC903 on overexpression. Of 3317 genes with informative expression detected by cDNA microarray, 321 (9.68%) showed expression changes between at least one pair of the three cell lines. Notably, 12 genes displayed higher levels of expression in UACC903(+6) than in both UACC903 and SRS3, providing candidates for further identification of melanoma-suppressor genes. Genes encoding Cx43 (suppressor activity), monocyte chemotactic protein 1 (suppressor activity), and cysteine proteinase P32alpha (apoptotic activity) were all upregulated in UACC903(+6), in contrast to both UACC903 and SRS3. Transfection of Cx43, encoded on human chromosome 6q21-q23, a region frequently altered in malignant melanoma, resulted in its overexpression and the suppression of anchorage-independent growth of UACC903. Thus, our result proves the principle that the combination of the ability to alter cellular phenotype by successive genetic alterations and the ability to examine the global expression profiles facilitates the identification of tumor suppressor genes. Mol. Carcinog. 28:119-127, 2000.

Characterization of a highly conserved gene (OS4) amplified with CDK4 in human sarcomas.

Amplification and overexpression of genes involved in cellular growth control occur frequently in human cancers. Here, we report characterization of the full length OS4 cDNA derived from 12q13-q15 (Su et al., Proc. Natl. Acad. Sci. USA, 91: 9121-9125, 1994), a region frequently amplified in sarcomas and brain tumors. This cDNA consists of 4833 base pairs (bp) encoding an open reading frame (ORF) of 283 amino acids. The ORF predicts a water-soluble acidic (pI 5.50) polypeptide with a molecular weight of 31759. Database searches revealed highly significant similarity between OS4 and eight proteins predicted from genomic sequences of Caenorhabditis elegans, Schizosaccaharomyces pombe, and Saccharomyces cerevisiae. Thus, OS4 defines a novel evolutionarily conserved gene superfamily. Northern and database analyses revealed OS4 transcripts in numerous human tissues demonstrating its ubiquitous expression. We also observed overexpression of OS4 in three cancer cell lines with amplification of this gene. Furthermore, we detected OS4 amplification in 5/5 primary sarcomas with known amplification of the closely linked marker CDK4. These results demonstrate that the highly conserved OS4 gene is frequently included in the 12q13-q15 amplicon and may contribute to the development of a subset of sarcomas.

Cloning a novel member of the human interferon-inducible gene family associated with control of tumorigenicity in a model of human melanoma.

Chromosome 6-mediated suppression of tumorigenicity in malignant melanoma cell lines provides a model system to identify genes associated with the reversion of the tumorigenic phenotype. Using subtractive cDNA selection, we recently identified a series of novel genes which are differentially expressed in association with chromosome 6-mediated suppression. We now report the molecular characterization of a novel gene termed AIM2 for (Absent In Melanoma), which represents a 1485 bp cDNA. An open reading frame of 1032 base pairs, corresponding to 344 amino acid residues, is predicted. The predicted protein shares a conserved sequence domain of approximately 200 amino acids with known interferon-inducible genes of both human and mouse. We demonstrate that the AIM2 gene encodes a transcript of approximately 2 kb which is expressed in spleen, small intestine, and peripheral blood leukocytes. In addition, we have localized AIM2 to the long arm of human chromosome 1 (band q22) in a highly conserved region which also contains the known interferon-inducible genes IFI16 and MNDA. We have also demonstrated that, like IFI16 and MNDA, AIM2 is induced in HL60 cells by interferon gamma. Our findings support the existence of a family of genes in this region similar to the well-characterized mouse Ifi200 gene family.

AIM1, a novel non-lens member of the betagamma-crystallin superfamily, is associated with the control of tumorigenicity in human malignant melanoma.

AIM1 is a novel gene whose expression is associated with the experimental reversal of tumorigenicity of human malignant melanoma. The predicted protein product of the major 4.1-kb transcript shows striking similarity to the betagamma-crystallin superfamily. All known members of this superfamily contain two or four characteristic motifs arranged as one or two symmetrical domains. AIM1, in contrast, contains 12 betagamma motifs, suggesting a 6-domain structure resembling a trimer of beta- or gamma-crystallin subunits. The structure of the AIM1 gene shows remarkable similarity to beta-crystallin genes, with homologous introns delineating equivalent protein structural units. AIM1 is the first mammalian member of the betagamma superfamily with a primarily non-lens role. Other parts of the predicted AIM1 protein sequence have weak similarity with filament or actin-binding proteins. AIM1 is a good candidate for the putative suppressor of malignant melanoma on chromosome 6, possibly exerting its effects through interactions with the cytoskeleton.

Identification of region specific genes by chromosome microdissection.

Chromosome microdissection is an extremely useful molecular cytogenetic tool for the characterization of chromosomal abnormalities in tumor cells. Although it has been used primarily in conjunction with fluorescence in situ hybridization (FISH), microdissection has also been useful for molecular analysis via microclone library construction. Recently, microdissection has been applied to the isolation of region specific cDNAs with sufficient success that gene discovery can now be added to the list of applications of this versatile molecular cytogenetic technique.

Use of a cDNA microarray to analyse gene expression patterns in human cancer.

The development and progression of cancer and the experimental reversal of tumorigenicity are accompanied by complex changes in patterns of gene expression. Microarrays of cDNA provide a powerful tool for studying these complex phenomena. The tumorigenic properties of a human melanoma cell line, UACC-903, can be suppressed by introduction of a normal human chromosome 6, resulting in a reduction of growth rate, restoration of contact inhibition, and suppression of both soft agar clonogenicity and tumorigenicity in nude mice. We used a high density microarray of 1,161 DNA elements to search for differences in gene expression associated with tumour suppression in this system. Fluorescent probes for hybridization were derived from two sources of cellular mRNA [UACC-903 and UACC-903(+6)] which were labelled with different fluors to provide a direct and internally controlled comparison of the mRNA levels corresponding to each arrayed gene. The fluorescence signals representing hybridization to each arrayed gene were analysed to determine the relative abundance in the two samples of mRNAs corresponding to each gene. Previously unrecognized alterations in the expression of specific genes provide leads for further investigation of the genetic basis of the tumorigenic phenotype of these cells.

Reversion of monochromosome-mediated suppression of tumorigenicity in malignant melanoma by retroviral transduction.

We have developed a general strategy to reverse monochromosome suppression of the malignant phenotypes by retroviral transduction. Our approach involved the introduction of a retroviral expression vector-carried cDNA library into a chromosome 6-suppressed melanoma subline UACC-903(+6) [J. M. Trent et al., Science (Washington DC), 247: 568-571, 1990]. The cDNA library was constructed from polyadenylated RNA isolated from the suppressed UACC-903(+6) cells, packaged into high-titer amphotropic retrovirus particles, and transduced into UACC-903(+6) cells. Revertant his(R) transductants were selected by isolating colony-forming cells in soft agar. A total of 121 large (> 150 microm) colonies was picked from soft agar culture with 18 of 121 (15%) established as permanent sublines. The revertant sublines demonstrated 7-58% cloning efficiency upon plating in agar, in contrast to <0.05% for the UACC-903(+6) subline. All 18 revertant sublines, termed SRS1-SRS18 (for "selection of revertants for suppression"), displayed a reduced population-doubling time, with 9 of 18 showing focus formation in monolayer similar to the parental (nonsuppressed) cell line. Preliminary evidence for reversion of the suppressed phenotype by injection of cells into athymic nude mice has been completed for one revertant subline. Southern analysis has demonstrated integration of the retroviral vector sequence in all 18 sublines. This approach should facilitate the identification of genes involved in the tumorigenic phenotype of malignant melanoma, and is readily adaptable to other model systems.

Isolation and characterization of genes associated with chromosome-6 mediated tumor suppression in human malignant melanoma.

Melanocytic transformation is thought to occur by the sequential accumulation of genetic alterations. Evidence implicating human chromosomes as a site for a gene(s) involved in melanoma suppression comes from studies of LOH [loss of heterozygosity], cytogenetics and biologic reversion of tumorigenicity following the introduction of a normal chromosome 6 by microcell-mediated chromosome transfer (Trent et al., 1990). Using a tumorigenic melanoma cell line (UACC 903) and a chromosome-6 suppressed melanoma subline [UACC 903 (+6)], we have isolated a series of genes uniquely expressed in the suppressed subline. A modified PCR-based cDNA subtraction technique was used to generate subtracted cDNA sublibraries for both the parental and (+6) suppressed cells. A total of 32 randomly selected clones from the suppressed sublibrary were isolated and examined, with 24 detecting a transcript by Northern analysis. Of these 24 clones, 21 (88%) demonstrated elevated expressed by Northern analysis in the suppressed subline relative to the tumorigenic parental cell line. In 6/21 differentially expressed clones (29%), expression was exclusive to the suppressed subline. Partial sequence analysis and database searching of these clones indicated that 5/6 were novel with one representing a previously characterized gene. Chromosomal localization of the five novel clones was performed following PCR amplification of a human/rodent somatic cell hybrid mapping panel or fluorescent in situ hybridization. One cDNA (termed AIM1) was localized to a band-region of chromosome 6 frequently deleted in melanomas (6q21). This novel approach should facilitate the identification of genes whose expression is causally related to the suppressed phenotype.

Complete sequence analysis of a gene (OS-9) ubiquitously expressed in human tissues and amplified in sarcomas.

Amplification and overexpression of genes involved in cellular growth control occur frequently in human tumors. Using a chromosome microdissection-based hybrid-selection strategy, we recently identified two novel genes (OS-9 and OS-4) within 12q13-15, a region frequently amplified in human cancers. We now report further characterization of the full-length OS-9 cDNA sequence consists of 2785 bp from which an open reading frame (ORF) with 667 amino-acid residues was deduced, The predicted polypeptide was water soluble and acidic. We also demonstrate that the OS-9 gene encoded a 2.8-kb mRNA transcribed in all 16 human tissues examined, suggesting that OS-9 is ubiquitously expressed in human tissues. OS-9 was co-amplified with CDK4 in three of five sarcoma tissues. Homology analysis of the amino-acid sequence reveals significant similarities between OS-9 and two ORFs deduced from genomic sequences in Caenorhabditis elegans and Saccharomyces cerevisiae. The region of similarity extended over 200 residues (approximately one-third of each ORF), and eight cysteines were conserved in all three ORFs. These observations suggest that this region comprises a functional domain present in a novel evolutionarily conserved gene family defined by OS-9.

Nucleotide sequence of the 18-kb conjugative transposon Tn916 from Enterococcus faecalis.

Conjugative transposon Tn916 from Enterococcus faecalis DS16 encodes tetracycline resistance (Tet M) as well as determinants necessary for its own movement. Determination of the nucleotide sequence of Tn916 has been completed. The element is 18,032 bp in length and has an overall G+C content of 38.8%. Twenty-four potential open reading frames (ORFs) were identified based on sequence analysis. Similarities of the ORFs to other known determinants, which were revealed by database searches, are discussed.

Direct isolation of genes encoded within a homogeneously staining region by chromosome microdissection.

Identification of genes involved in recurring chromosome rearrangements has provided significant insight into the molecular basis of malignancy. We describe here a strategy combining chromosome microdissection and hybrid selection for the direct isolation of chromosome region-specific genes. We modeled this strategy by using sequences recovered from the microdissection of a homogeneously staining region to allow isolation of genes that were overexpressed and present at high copy number within the homogeneously staining region, including the direct isolation of two genes encoded within a 12q homogeneously staining region found in the osteosarcoma cell line OsA-CL. Although first applied to amplified genes, this strategy should be applicable to the isolation of cDNAs from any chromosomal region.

Characterization of the left 4 kb of conjugative transposon Tn916: determinants involved in excision.

The rate-limiting step in movement of the conjugative transposon Tn916, originally identified in Enterococcus faecalis, is believed to be an excision event that generates a non-replicative circular intermediate. When present on a plasmid vector in Escherichia coli, Tn916 generally excises at a high frequency. It was reported previously that insertion of Tn5 in a region near the left end of Tn916 eliminated the ability to excise; and the mutation could be complemented in trans. In this communication the nucleotide sequence of 4 kb of Tn916 DNA connecting the recently sequenced tet(M) determinant (Su et al., 1992; Burdett, 1990) with the left end of the transposon. Ten open reading frames (ORFs) were deduced, two of which (ORF3 and ORF4) were encoded in-frame within a third (ORF2). Mutants with Tn5 insertions in the ORF1 or ORF2 (ORF3 and ORF4) were defective in excision, but could be complemented in vivo by a co-resident plasmid containing the ORF1 or ORF2 determinant, respectively. The data support the view that both ORF1 and ORF2 are essential for excision. ORF1 and ORF2 are essentially identical to determinants designated xis-Tn and int-Tn, respectively, in the closely related Tn1545. A Tn5 insertion in ORF5 eliminated conjugative transfer between E. faecalis strains. Functions for the remaining ORFs (ORF6 through ORF10) remain unknown; however, nucleotide sequences within ORF6 and ORF9 had significant homology with sequence downstream of other tet(M) determinants.

Characterization of the tet(M) determinant of Tn916: evidence for regulation by transcription attenuation.

The nucleotide sequence of the tetracycline resistance determinant tet(M), located on conjugative transposon Tn916 of Enterococcus faecalis, was determined and found to encode a 72,486-dalton protein exhibiting a high degree of homology with other tet(M) determinants. A short open reading frame corresponding to a 28-amino-acid peptide and containing a number of inverted repeat sequences was noted immediately upstream of tet(M), suggesting that regulation might occur by a mechanism involving transcriptional attenuation. Transcription analyses found this to indeed be the case, showing that the expression of tet(M) resulted from an extension of a small transcript representing the upstream leader region into the resistance determinant. Exposure of cells to tetracycline resulted in a significant increase in the amount of tet(M) transcription; this increase could be explained on the basis of increased transcriptional read-through from the upstream transcript. A model suggesting how transcriptional attenuation might operate in this system is presented.

Nucleotide sequence of the gelatinase gene (gelE) from Enterococcus faecalis subsp. liquefaciens.

The gene coding for gelatinase (also called metalloendopeptidase II; microbial proteinase, EC 3.4.24.4) of Enterococcus faecalis subsp. liquefaciens strain OG1-10 was cloned in an Escherichia coli-Enterococcus shuttle vector, and its nucleotide sequence was determined. The DNA sequence encodes one large open reading frame (ORF) with 509 amino acid residues. The ORF contains a signal sequence in its N-terminal region, whereas the N-terminal amino acid sequence determined from the purified extracellular proteinase starts at residue 192 deduced from the ORF. This implies that the gelatinase is synthesized as a prepropolypeptide or prezymogen. The mature gelatinase contains 318 amino acid residues (molecular weight, 34,582) and has significant homology with neutral proteinases from Bacillus species and elastase from Pseudomonas aeruginosa.

[Evolution of human esophageal cancer cell in the process of cell line establishment].

An esophageal cancer cell line EC8501 was established by tissue culture technique in vitro. Biologic appraisements demonstrated that this cell line was certainly a malignant one. The authors counted chromosome number of 1,284 cells from 10 to 30 passages and discovered that the modal chromosome number was 46 in 10 and 13 passages, 47 in 14 passage and 65 or so after 25 passage. It was a cell population with subtriploid G-banded chromosome analysis of 73 cells from 7 passages (13, 15, 25-27, 29 and 30) showed that structural chromosome aberrations were manifold, complicated and changeable. Sixteen marker chromosomes were present at appearance rate of 11-97%, 6 of which appeared in every passage. Many derivative chromosomes in the 13-30 passages were derived from marker chromosomes. Thirteen markers were first discovered in the 13 passage and 16 of rearrangement points on the markers were close to 7 oncogenes and 7 cancer breakpoints. Two of thirteen markers (del 1p13 and der 2) were similar or same to markers found in epithelium adjacent to esophageal cancer of two patients. The authors suggested that the two markers may reflect the chromosome changes in early carcinogenesis of esophageal epithelium. According to this research, the authors consider that the tumor cell lines cultured in vitro for many years can not reflect the characteristics of tumor cells in vivo.