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Following mating or conjugation, the hypotrichous ciliate Euplotes crassus undergoes a massive genome reorganization process. While the nature of the rearrangement events has been well studied, little is known concerning proteins that carry out such processes. As a means of identifying such proteins, differential screening of a developmental cDNA library, as well as construction of a cDNA subtraction library, was used to isolate genes expressed only during sexual reproduction. Five different conjugation-specific genes have been identified that are maximally expressed early in conjugation, during the period of micronuclear meiosis, which is just prior to macronuclear development and the DNA rearrangement process. All five genes are retained in the mature macronucleus. Micronuclear, macronuclear, and cDNA clones of one gene (conZA7) have been sequenced, and the results indicate that the gene encodes a putative DNA binding protein. In addition, the presence of an internal eliminated sequence in the micronuclear copy of the conZA7 gene indicates that this conjugation-specific gene is transcribed from the old macronucleus.

Genetic linkage map is being constructed for watermelon based on a testcross population and an F2 population. The testcross map comprises 262 markers (RAPD, ISSR, AFLP, SSR and ASRP markers) and covers 1,350 cM. The map comprises 11 large linkage groups (50.7–155.2 cM), 5 medium-size linkage groups (37.5–46.2 cM), and 16 small linkage groups (4.2–31.4 cM). Most AFLP markers are clustered on two linkage regions, while all other marker types are randomly dispersed on the genome. Many of the markers in this study are skewed from the classical (Mendelian) segregation ratio of1:1 in the testcross or the 3:1 ratio in the F2 population. Although the skewed segregation, marker order appeared to be consistent in linkage groups of the testcross and F2 population. A cDNA library was constructed using RNA isolated from watermelon flesh 1 week (rapid cell division stage), 2 weeks (cell growth and storage deposition stage, 4 weeks (maturation stage), and 5 weeks (postmaturation stage) post pollination. Over 1,020 cDNA clones were sequenced, and were analyzed using the Basic Local Alignment Search Tool (BLAST). The sequenced cDNA clones were designated as expressed sequenced tag (EST) markers and will be used in mapping analysis of watermelon genome.

A new and highly effective method, termed suppression subtractive hybridization (SSH), has been developed for the generation of subtracted cDNA libraries. It is based primarily on a recently described technique called suppression PCR and combines normalization and subtraction in a single procedure. The normalization step equalizes the abundance of cDNAs within the target population and the subtraction step excludes the common sequences between the target and driver populations. In a model system, the SSH technique enriched for rare sequences over 1,000-fold in one round of subtractive hybridization. We demonstrate its usefulness by generating a testis-specific cDNA library and by using the subtracted cDNA mixture as a hybridization probe to identify homologous sequences in a human Y chromosome cosmid library. The human DNA inserts in the isolated cosmids were further confirmed to be expressed in a testis-specific manner. These results suggest that the SSH technique is applicable to many molecular genetic and positional cloning studies for the identification of disease, developmental, tissue-specific, or other differentially expressed genes.

We used a PCR-based subtraction cloning procedure with concanavalin A-treated and -untreated animal caps from stage 9 Xenopus embryos to search for genes up-regulated during early neural development. One such gene was found to encode a protein homologous to several known glutamine synthetases, and we named it xGS. Molecular hybridization studies revealed that xGS mRNA is maternally transmitted and abundantly expressed in neuroectoderm-derived tissues during the gastrula and neurula stages. The expression of xGS mRNA in the nervous system continues until the larval stages, but declines thereafter and becomes undetectable in adult brain. Considering its metabolic activity and potential neuroprotective effect against the neurotoxic substances such as glutamate and ammonia, the glutamine synthetase may play an important role in the early stages of vertebrate neural development.

Cell-suspension cultures of Lithospermum erythrorhizon Sieb. et Zucc. (Boraginaceae) grown in the dark produce a large amount of red naphthoquinone pigments that are shikonin derivatives when transferred to the production medium M9 (Fujita et al., 1981) from the Linsmaier Skoog liquid medium (Linsmaier and Skoog, 1965). It is known that shikonin biosynthesis is completely inhibited by irradiation with either white or blue light (Heide et al., 1989). In most plant cell cultures, secondary metabolism, in particular the production of phenolic compounds, is stimulated by light, and genes related to light-inducible biosynthesis of secondary metabolites have recently been investigated to identify cis-acting elements in their promoter regions and to elucidate the function of trans-acting factors (Feldbriigge et al., 1994). However, the molecular mechanism of the induction of secondary metabolism in the dark is an open question. The Lithospermum cell culture seems to offer an ideal system to study dark-inducible genes, since the shikonin production is specifically inhibited by light, whereas it can be induced rapidly when the cultures are placed in darkness. In the present study, an attempt was made to clone cDNAs expressed dominantly in the dark-grown Lithospermum cell cultures. Two different cDNA samples, one corresponding to mRNA from Lithospermum cells cultured for 2 d after inoculation in M9 medium in the dark, and the other corresponding to that from the cells cultured under continuous illumination, were prepared. cDNAs expressed preferably in the dark were enriched by the cDNA subtraction method of Wang and Brown (1991). A mixture of the subtracted cDNA was used as a probe for plaque hybridization, to detect about 360 positive signals among 6000 plaques of the A-ZAPII cDNA library prepared from Lithospermum cells cultured in M9 medium in the dark. Of the positive clones 160 single plaques were randomly picked, and a dot blot filter containing each A clone was prepared. Since about half of the A clones were found to be identical in a crosshybridization experiment with the dot blot filter, one of these plaques was subjected to further analysis.

Expression of major histocompatibility complex (MHC) class II molecules and ability to present anti gen to T lymphocytes is acquired upon activation of the macrophage by intcrfcron-γ (IFN-γ). Little information is available concerning immune regulation of protease gene expression in mouse macrophages. We have isolated a eDNA clone for cathepsin H, a lysosomal cysteine pro teinase from a cDNA subtraction library of mouse macro phage genes induced by IFN-γ, and have characterized its expression. The level of cathepsin H mRNA increased in mouse peritoneal macrophages following addition of IFN-γ. Cathepsin H mRNA levels began to increase 8 h after the addition of IFN-γ and was maximal at 24–48 h. This increase was concordant in time with appearance of MHC class II Eβ mRNA and Ia invariant chain mRNA. The increase in cathepsin H mRNA levels by IFN-γ was dose dependent. Cycloheximide treatment of peritoneal macrophages inhibited the increase in cathepsin H mRNA levels induced by IFN-γ, suggesting that the in crease in cathepsin mRNA levels requires de novo protein synthesis. Lipopolysaccharide and cytokines interleukin-2 (IL-2), IL-4, IL-10, and tumor necrosis factor α were found to have no effect on cathepsin H mRNA levels in mouse peritoneal macrophages. J. Leukoc. Biol. 57: 663–669; 1995.

In screening a rat mucosa cDNA subtraction library, a clone that exhibited a remarkable degree of homology with a previously described cDNA from the green garden pea, designated the 26g pea turgor protein, was found. A partial cDNA sequence from rat and a complete cDNA sequence from human were obtained. The deduced human protein had a molecular weight of 55,285 and was designated antiquitin because of its remarkable level of conservation through evolution. Human antiquitin was 60% homologous to the green pea 26g with only a single amino acid gap in each sequence. The 66 amino acids at the carboxyl ends of the human antiquitin and pea 26g proteins were 86% identical, and one segment of 52 amino acids was 92% identical. A similar partial sequence encoding 164 amino acids has been detected in Caenorhabditis elegans. Yeast DNA was found to have sequences that hybridize with a human antiquitin probe on Southern blotting. Analysis of the amount of mRNA in various rat and human tissues indicated that the largest amounts were found in rat kidney and liver and in cultured human hepatoma cells. Only minimal amounts were detected in human peripheral blood leukocytes, rat lung, or cultured human fibroblasts. Attempts to induce the mRNA by heat-shock, dehydration, ionizing irradiation, or treatment with iron, t-butylhydroperoxide, or glucocorticoids were unsuccessful. The function of the protein remains unknown.

The subtractive-hybridization procedure (see e.g. (1)) is a powerful procedure for obtaining cDNA populations which are enriched for upand down-regulated gene products. The method is based on the elimination of the common fragments within the cDNA populations being compared and is straight forward (1). Unfortunately, the analysis of the outcome is based on a screening hybridization assay and, hence, is indirect and time-consuming since individual molecules have to be cloned and analyzed. Another useful procedure for the isolation of specific gene products is the differential display method as developed by Liang and Pardee (2), recently optimized (3) and automated (4). In this procedure differential gene expression, in cells or tissues, is visualized by differences in the intensities of cDNA fragments after electrophoretic separation on a sequencing gel. After conversion into a cDNA using reverse transcriptase all gene products can be amplified as distinct, primer-dependent groups of cDNA by choosing a distinct oligo-dT-NN primer and a set of (short) random primers which facilitates identification (2). Although this procedure allows in principle for the identification of all mRNAs it can not be used for the isolation of underrepresented gene products. Here we report that the combination of both procedures, i.e. enriching a cDNA population by the subtractive-hybridization method (1) and identifying the enriched fragments by the differential display method (2) on a polyacrylamide gel yields a very powerful procedure that allows the identification of cDNAs that are enriched as little as 3-fold in the test fraction. In our laboratory we are studying the altered gene expression in a rat liver after a 70% hepatectomy. cDNA populations have been prepared from a normal and a regenerating (partially hepatectomized) rat liver (5). The subtractive-hybridization method as described by Wang and Brown (1), with minor modifications, was used to obtain the upand down-regulated gene products. Both cDNA populations were tagged with their own unique set of primers/adapters, CTCTTGCTTGAATTCGGACTA-3' and AATTCAGGCCAAGTCGGCCGG-3' for the normal and regenerating liver cDNA, respectively. In this way the necessity of linker removal, as described in the original procedure (1), at any point during the method was eliminated. The cDNA needed for each round of subtraction was amplified with Taq DNA polymerase (94°C for 1 min, 58°C for 90 s, and 72°C for 2 min with 30 cycles, followed by a final 5-min elongation-step at 72°C). The respective primers could not be interchanged since no product was detectable after a complete amplification cycle with a mismatched primer (not shown). In order to identify the upand down-regulated gene products after a completed subtractive-hybridization procedure, we adapted the display method from Liang and Pardee (2) with modifications. The two cDNA populations obtained after each round of subtractive-hybridization were amplified using Taq polymerase and the distinct primers in the presence of 0.5 jtM [a-S]dATP (1200 Ci/mmol) (final volume: 25 /xl). The experimental conditions were exactly the same as those used to generate the

Enchytraeus japonensis is a small oligochaete species that proliferates asexually via fragmentation and regeneration. As sexual reproduction can also be induced, it is a good model system for the study of both regenerative and germline stem cells. It has been shown by histological study that putative mesodermal stem cells called neoblasts, and dedifferentiated epidermal and endodermal cells are involved in blastema formation. To clarify the molecular biological mechanism operating at an initial stage of regeneration in E. japonensis , we isolated five genes whose expression levels changed in the regeneration process occurring between growing and early regenerating worms by cDNA subtraction method. One of the isolated genes (a novel gene named grimp ) was expressed transiently from 3 to 12 h post-amputation only in neoblasts and a population of mesodermal cells (the non-neoblast grimp-expressing cells) incorporating BrdU simultaneously showed mitotic activity. We carried out functional analysis by using RNA interference (RNAi). In knock-down worms, the number of BrdU-positive neoblasts and the non-neoblast grimp-expressing cells in the coelom drastically decreased. Also the elongation and segmentation of blastema were inhibited, while no statistically significant inhibitory effect was observed in epidermal and intestinal cells. These results suggest that grimp is required for initial proliferation of neoblasts and some mesodermal cells for regeneration. We also investigate the function of grimp at the protein level. cDNA sequence of grimp contains a putative open reading frame and poly (A). The deduced polypeptide contains a triplicate repeat of a very similar sequence containing integrin-binding RGDS sequences and protein kinase C phosphorylation sites. However, it is hard to predict its function from the sequence. Thus we took several approaches to clarify the localization and the function of grimp protein.

Background Dengue virus, a mosquito-borne flavivirus, is the etiological agent of dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. It generally induces apoptosis in mammalian cells, but frequently results in persistent infection in mosquito cells. That mechanism remains to be explored. In turn, a genomic survey through subtractive hybridization (PCR-select cDNA subtraction) was conducted in order to find gene(s) that may play a role in interactions between the virus and its host cells. Results Through this technique, we identified a novel eukaryotic translation initiation factor 5A (eIF5A) which is upregulated in Aedes albopictus-derived C6/36 cells infected by the type 2 dengue (Den-2) virus. The full-length of the identified eIF5A gene consisted of 1498 bp of nucleotides with a 41.39% G+C content, and it possessed a higher similarity and shorter evolutionary distance with insects than with other organisms. Upregulation of eIF5A in response to Den-2 virus infection was validated at both the RNA and protein levels. This phenomenon was also observed by confocal microscopy. In addition, cell death obviously occurred when eIF5A activity was inhibited in C6/36 cells even when they were infected by the virus. However, viral multiplication was not obviously affected in infected C6/36 cells when eIF5A activity was reduced. Conclusions Taken together, we postulated that eIF5A plays a role in preventing mosquito cells from death in response to Den-2 viral infection, thus facilitating continued viral growth and potential persistent infection in mosquito cells. It would be worthwhile to further investigate how its downstream factors or cofactors contribute to this phenomenon of dengue infection.

CN09-04 Although infection contributes to over 20% of human cancers worldwide, the list of confirmed carcinogenic infectious agents is surprisingly short[1]. Epidemiologic studies strongly suggest that novel infectious agents remain to be discovered and contribute to a broad range of diseases, including cancers, autoimmune disorders, and degenerative diseases. Although current methods have been successful in identifying human viruses, most pathogen discovery approaches suffer from several shortcomings. None are quantitative, so if no candidate sequence is found, it is not possible to estimate how likely it is that an agent is present but missed in the search. There are no simple ways to scale up pathogen discovery with these methods short of analyzing additional samples and it is unlikely that any technique can universally identify all human tumor viruses. But with the near completion of the human genome project, high-throughput sequencing can be exploited to overcome many of these problems. We developed digital transcript subtraction (DTS) to subtract in silico known human sequences from expression library data sets, leaving candidate nonhuman sequences for further analysis [2]. This approach requires precise discrimination between human and nonhuman cDNA sequences. Database comparisons show high likelihood that small viral cDNA sequences can be successfully distinguished from human sequences. We pilot tested DTS on 9,026 20-bp cDNA tags from an expression library of BCBL-1 cells infected with Kaposi's sarcoma-associated herpesvirus (KSHV). In this initial test, we succeeded in complete in silico cDNA subtraction. Only three candidate sequences were identified as being of nonhuman origin: two of these sequences belonged to highly-expressed KSHV transcripts, and the third belonged to an unannotated human expression sequence tag. Overall, 0.24% of transcripts from this cell line were of viral origin. DTS was then applied to 241,122 expression tags from three squamous cell conjunctival carcinomas (SCCC), a cancer strongly associated with immunosuppression. Only 21 of these sequences did not align to human databases and all 21 candidates were ruled out as viral sequences by experimental isolation. This analysis shows that it is unlikely that distinguishable viral transcripts are present in conjunctival carcinomas at 20 transcripts per million or higher, which is the equivalent of approximately 4 transcripts per cell. Merkel cell carcinoma (MCC) is a malignant skin tumor arising from mechanoreceptor Merkel cells. MCC also occurs more frequently than expected among immunosuppressed transplant and AIDS patients. To search for viral sequences in MCC, we performed DTS on cDNA from 4 MCC cases, analyzing 395,734 cDNA sequences [3]. One transcript was similar to but distinct from African green monkey (AGM) lymphotropic polyomavirus (LPV) and human BK polyomavirus T antigen sequences, defining a new human polyomavirus, Merkel cell polyomavirus (MCV or McPyV). We sequenced the complete close circular genome of MCV (5387 bp), which encodes a T antigen locus, VP1, VP2/3, and replication origin sequences. MCV has highest homology to LPV belonging to MuPyV subgroup, the first human member of this subgroup, The MCV transcript was present at 10 transcript per million or abut 5 transcripts per cell. MCV sequences were detected in 8 of 10 (80%) MCC tumors but only 5 of 59 (8%) control tissues from various body sites and 4 of 25 (16%) control skin tissues. Rapid amplification of cDNA ends (3’-RACE) revealed a T antigen fusion transcript with intron 1 of the human receptor tyrosine phosphatase type G gene (PTPRG), suggesting viral integration in the tumor. In six of eight MCV-positive MCCs, viral DNA was also found integrated within the tumor genome in a clonal pattern, suggesting that MCV infection and integration preceded clonal expansion of the tumor cells. 5’-RACE and northern analysis defined large (LT), small (ST) and variably spliced MCV T transcripts that retain polyomaviral CR1 (LXXLL), DnaJ (HPDKGG), PP2A binding (CXCXXC), Rb binding (LXCXE) motifs as well as origin-binding and helicase/ATPase domains. We further sequenced nine MCC tumor-derived LT genomic region and all were found to harbor mutations prematurely truncating the MCV LT helicase [4]. In contrast, four presumed episomal viruses from nontumor sources did not process this T antigen signature mutation. MCV-positive MCC tumor cells undergo selection for LT mutations to prevent autoactivation of integrated virus replication detrimental to cell survival. Because these mutations render the virus replication-incompetent, MCV cannot be a “passenger virus” that secondarily infects MCC tumors. Overall, DTS is a simple screening method to discover novel viral nucleic acids. It provides, for the first time, quantitative evidence against some classes of viral etiology when no viral transcripts are found, thereby reducing the uncertainty involved in new pathogen discovery. Citation Information: Cancer Prev Res 2008;1(7 Suppl):CN09-04.

It is reasonable to propose that gene expression profiles of purified stem cells could give clues for the molecular mechanisms of stem cell behavior. We took advantage of cDNA subtraction to identify a set of genes selectively expressed in mouse adult hematopoietic stem cells (HSC) as opposed to bone marrow (BM). Analysis of HSC-enriched genes revealed several key regulatory gene candidates, including two novel seven transmembrane (7TM) receptors. Furthermore, by using cDNA microarray techniques we found a large set of HSC-enriched genes that are expressed in mouse neurospheres (a population greatly enriched for neural progenitor cells), but not present in terminally differentiated neural cells.In situhybridization demonstrated that many of them, including one HSC-enriched 7TM receptor, were selectively expressed in the germinal zones of fetal and adult brain, the regions harboring mouse neural stem cells. We propose that at least some of the transcripts that are selectively and commonly expressed in two or more types of stem cells define a functionally conserved group of genes evolved to participate in basic stem cell functions, including stem cell self-renewal.

Background Fluctuations in external salinity force eukaryotic cells to respond by changes in the gene expression of proteins acting in protective biochemical processes, thus counteracting the changing osmotic pressure. The high-osmolarity glycerol (HOG) signaling pathway is essential for the efficient up-regulation of the osmoresponsive genes. In this study, the differential gene expression of the extremely halotolerant black yeast Hortaea werneckii was explored. Furthermore, the interaction of mitogen-activated protein kinase HwHog1 and RNA polymerase II with the chromatin in cells adapted to an extremely hypersaline environment was analyzed. Results A cDNA subtraction library was constructed for H. werneckii, adapted to moderate salinity or an extremely hypersaline environment of 4.5 M NaCl. An uncommon osmoresponsive set of 95 differentially expressed genes was identified. The majority of these had not previously been connected with the adaptation of salt-sensitive S. cerevisiae to hypersaline conditions. The transcriptional response in hypersaline-adapted and hypersaline-stressed cells showed that only a subset of the identified genes responded to acute salt-stress, whereas all were differentially expressed in adapted cells. Interaction with HwHog1 was shown for 36 of the 95 differentially expressed genes. The majority of the identified osmoresponsive and HwHog1-dependent genes in H. werneckii have not been previously reported as Hog1-dependent genes in the salt-sensitive S. cerevisiae. The study further demonstrated the co-occupancy of HwHog1 and RNA polymerase II on the chromatin of 17 up-regulated and 2 down-regulated genes in 4.5 M NaCl-adapted H. werneckii cells. Conclusion Extremely halotolerant H. werneckii represents a suitable and highly relevant organism to study cellular responses to environmental salinity. In comparison with the salt-sensitive S. cerevisiae, this yeast shows a different set of genes being expressed at high salt concentrations and interacting with HwHog1 MAP kinase, suggesting atypical processes deserving of further study.

PURPOSE. To identify and characterize genes differentially expressed in anterior segment tissues of eyes with pseudoexfoliation (PEX) syndrome and glaucoma. METHODS. Anterior segment tissues (iris, ciliary processes, lens epithelium) were obtained from eight surgically enucleated eyes with PEX-associated open-angle or closed-angle glaucomas and eight age-matched glaucomatous control eyes without PEX. cDNA libraries were generated from three PEX and three control specimens, and their gene expression patterns were compared by means of cDNA subtraction. Differentially expressed clones from the subtracted cDNA libraries were sequenced, and their differential expression was verified by means of RT-PCR, virtual Northern blot analysis, and in situ hybridization with specific RNA probes. RESULTS. Subtraction of cDNA libraries identified 27 candidate genes for differential expression in PEX tissues, of which 23 genes were confirmed by virtual Northern blot, RT-PCR, and in situ hybridization. One set of genes consistently upregulated in anterior segment tissues from different patients with PEX comprised latent transforming growth factor binding proteins (LTBP-1 and -2), which are structural components of elastic microfibrils, the cross-linking enzyme transglutaminase-2 (TGase-2), tissue inhibitor of matrix metalloproteinase-2 (TIMP-2), A-kinase anchor protein-2 (AKAP-2), apolipoprotein D, and the adenosine receptor-A3 (AdoR-A3). Genes reproducibly downregulated in PEX tissues included TIMP-1, clusterin, microsomal glutathione-S-transferase-1 (mGST-1), and serum amyloid A1. Further transcripts, such as elastase, GST-T1, integrin 4, and dehydrocholesterol reductase, did not show a consistent differential expression pattern in tissues obtained from different patients. Although fibrillin-1 was not isolated from subtracted cDNA libraries, upregulated expression of this elastic microfibrillar component was also demonstrated by RT-PCR and in situ hybridization. CONCLUSIONS. Differentially expressed genes with a high level of reproducibility in different tissues and different patients with PEX syndrome are mainly related to extracellular matrix metabolism and cellular stress. The underlying pathophysiology of PEX syndrome appears to be associated with an excessive production of elastic microfibril components, enzymatic crosslinking processes, a proteolytic imbalance between matrix metalloproteinases and their inhibitors, and increased cellular and oxidative stress supporting the notion of PEX syndrome as a stress-induced elastic microfibrillopathy. (Invest Ophthalmol Vis Sci. 2005;46:3742‐3752) DOI:10.1167/iovs.05-0249

Expression of the common leukocyte antigen, CD45 is quite variable in human myeloma cells and cell lines, such as U266, and CD45+ U266 proliferates in response to a growth factor, interleukin-6 (IL-6). IL-6 as a mitogenic stimulus, activated a src family kinase, Lyn and phospholipase C (PLC)-g2 followed by Ca2+ influxes and protein kinase C (PKC) activation in CD45+ U266. Here we show that compared with CD45− U266, CD45+ U266 was more sensitive to various apoptotic stimuli, such as oxidative stress and endoplasmic reticulum (ER)-stress. Both release of apoptosis-inducing factor or cytochrome c and the activation of caspase-9 and caspase-3 were enhanced in CD45+ U266 more than in CD45− U266. Increased susceptibility to the stress-induced apoptosis was also observed in other CD45-expressing myeloma cell lines and the CD45-transfected U266. Reactive oxygen species (ROS) and calcium ion (Ca2+) seem to be involved in the susceptibility to apoptosis of CD45+ U266 because either an anti-oxidant, DTT or a Ca2+ chelating agent, BAPTA blocked the apoptosis. As the oxidative stress-induced Ca2+ influxes and apoptosis of CD45+ U266 were blocked by a src family kinase inhibitor, PP2, CD45 phosphatase regulating the src family kinase activity plays an important role in the augmented apoptosis in CD45+ U266 by oxidative stress. The intracellular accumulation of ROS may activate src associated with CD45, and the activated src kinases accelerate the Ca2+ influxes followed by calicineurin activation, mitochondrial translocation of Bad, and inhibition of Bcl-2 function. These results indicate that the CD45-expression renders myeloma cells competent with not only mitogenic but also apoptotic stimuli, resulting in either proliferation or apoptosis of CD45+ myeloma cells dependently upon the stimuli. Furthermore, voltage-dependent anion channel (VDAC)-1 was identified as a gene highly expressed in CD45+ U266 by cDNA subtraction. The increased expression of VDAC-1 seemed to augment the sensitivity to the ER-stress because the VDAC-1-transfected CD45− U266 was confirmed to be susceptible to the thapsigargin-induced apoptosis. Thus, CD45 may define the signaling thresholds that are critical for the IL-6-induced proliferation and the oxidative stress-induced apoptosis of myeloma cells, and the CD45 expression accompanied by the increased VDAC-1 expression sensitizes myeloma cells to the various extracellular stimuli triggering apoptosis via the mitochondrial pathways.

The main objective of this project was to isolate and identify important genes specifically expressed in oocytes. These genes are characterized and may uncover the molecular mechanisms related to the unique functions found in the oocyte. Total RNA (1μg) from denuded germinal vesicle-stage (GV) oocytes and somatic tissues were used for cDNA production. The mRNAs were reverse-transcribed and the cDNAs were amplified using the Smart cDNA amplification kit (Clontech, BD Biosciences, San Jose, CA, USA). SSH was performed with the PCR-Select cDNA Subtraction Kit (Clontech). Briefly, pools consisted of bovine oocytes for the tester and bovine somatic tissues for the driver. The same procedure was repeated with mouse and xenopus tissues. All of the 3500 subtracted PCR products generated by SSH were cloned, PCR amplified and sequenced. The resulting sequences were compared against the GenBank database using online computer BLASTn program. For microarray analysis, purified PCR products were spotted on GAPS II glass slides (Corning, Corning, NY, USA) using a VersArray Chip WriterPro robot (BioRad, Hercules, CA, USA). Forward- and reverse-subtracted PCR products were used as probes labeled with Cy-3 and Cy-5 dyes (Amersham, Piscataway, NJ, USA) using the Amino Allyle cDNA Labeling Kit (Ambion, Austin, TX, USA). Slides were scanned and analyzed using the ChipReader and ArrayPro Analyser software (Media Cybernetics, Carlsbad, CA, USA). Detection of the oocyte-specific zona pellucida (ZP), growth differentiation factor-9 (GDF-9), bone morphogenetic protein 15 (BMP15), H1 histone family, member O, oocyte-specific (H1oo), and cyclin B1 transcript in the oocyte subtracted library increased confidence in the validity of the subtraction procedure. All of these transcripts account for 12 % of the clones for the mouse subtracted oocyte library. Microarray analysis performed with the mouse arrays revealed that 33% (382) of the clones were differentially expressed (ratio>10) in the oocyte for the mouse library. Of the 139 clones (12%) that seemed to be present only in the oocyte, 65 were found to be expressed in both the bovine and the xenopus subtracted oocyte library. The most interesting clones to date are #1518, a gene associated with pluripotency;; #1776, a cDNA associated with stem cells in mouse, and #1906, a protein that interacts with chromatin. Since they are conserved across species, the chances that their function is important are quite high. A validation step with RT-PCR analysis will need to be performed on genes identified as oocyte-specific, and the functionality of these genes will be determined with the RNAi technique. WEB page http://www.begc.crbr.ulaval.ca/ (Supported by NSERC).

During the first month after birth, synchronous follicular growth occurs in the ovary of immature mice (first wave). Previously, we showed that mouse oocytes during the first wave were more competent developmentally in older females (Suzuki O et al., 2002 Theriogenology 57, 628 abst), although the numbers of mature oocytes did not differ with female age (17, 18, and 24 days old). In this study, we examined the genetic factors that affect the developmental competence of mouse oocytes during the first wave using suppression subtractive hybridization (SSH). Oocytes collected from 17- and 24-day-old B6D2F1 females (D17 and D24, respectively) without hormonal treatment were matured in Waymouth medium supplemented with pyruvate (0.23mM), antibiotics, bovine fetuin (1mgmL−1), and polyvinylpyrrolidone (3mgmL−1). After 17-h culture at 37°C in an atmosphere of 5% CO2, 5% O2, and 90% N2, total RNA was isolated from oocytes whose germinal vesicles had broken down (mature oocytes), separately, in three independent culture groups per age (each group contained oocytes from four animals) using Cell-to-cDNA Cell Lysis Buffer (Ambion, Austin, TX, USA). Some of the total RNA from each independent group was pooled by age (total of RNA from approximately 100 oocytes per age) and used for SSH. A SMART cDNA Synthesis Kit (Clontech, Palo Alto, CA, USA) was used to reverse-transcribe total RNA to cDNA. SSH was performed with a PCR-Select cDNA Subtraction Kit (Clontech). The subtracted PCR products were cloned into pGEM-T vector (Promega, Madison, WI, USA). Clones from the subtracted library (D24−D17) were sequenced and their identities were examined using the NCBI BLAST search. The differential expression of candidate genes preferentially expressed in mature D24 oocytes suggested by SSH was confirmed with cDNA transcribed separately in the three independent culture groups per age using real-time quantitative PCR with an ABI Prism 7900HT with TaqMan technology (Applied Biosystems, Foster City, CA, USA). Of 513 clones sequenced, the top six preferentially-expressed candidate genes in more developmentally-competent D24 oocytes were spindlin (20 clones), bmi-1 (4 clones), cyclin B1 (4 clones), E330034G19Rik (4 clones), Jagged1 (4 clones), and Ndfip2 (4 clones). The expression of spindlin in mature D24 oocytes (relative threshold cycle: −3.8±0.7, mean±SD) was confirmed to be approximately 11-fold higher than in D17 oocytes (−0.3±1.5) when GAPDH was used as an internal control (P

Self-incompatibility (SI) in Brassica is controlled by a single locus, termed the S locus. There is evidence that two of the S locus genes, SLG , which encodes a secreted glycoprotein, and SRK , which encodes a putative receptor kinase, are required for SI on the stigma side. The current model postulates that a pollen ligand recognizing the SLG/SRK receptors is encoded in the genomic region defined by the SLG and SRK genes. A fosmid contig of ∼65 kb spanning the SLG-910 and SRK-910 genes was isolated from the Brassica napus W1 line. A new gene, SLL3 , was identified using a novel approach combining cDNA subtraction and direct selection. This gene encodes a putative secreted small peptide and exists as multiple copies in the Brassica genome. Sequencing analysis of the 65-kb contig revealed seven additional genes and a transposon. None of these seven genes exhibited features expected of S genes on the pollen side. An ∼88-kb contig of the A14 S region also was isolated from the B. napus T2 line and sequenced. Comparison of the two S regions revealed that (1) the gene organization downstream of SLG in both S haplotypes is highly colinear; (2) the distance between SLG-A14 and SRK-A14 genes is much larger than that between SLG-910 and SRK-910 , with the intervening region filled with retroelements and haplotype-specific genes; and (3) the gene organization downstream of SRK in the two haplotypes is divergent. These observations lead us to propose that the SLG downstream region might be one border of the S locus and that the accumulation of heteromorphic sequences, such as retroelements as well as haplotype-unique genes, may act as a mechanism to suppress recombination between SLG and SRK.

Recently, we reported that acetylcholine-induced hypoxia-inducible factor-1α protects cardiomyocytes from hypoxia; however, the downstream factors reducing hypoxic stress are unknown. We identified apoptosis inhibitor (AI) gene as being differentially expressed between von Hippel Lindau (VHL) protein-positive cells with high levels of GRP78 expression and VHL-negative cells with lower GRP levels, using cDNA subtraction. AI decreased GRP78 level, suppressed mitochondrial function, reduced oxygen consumption and, ultimately, suppressed hypoxia-induced apoptosis. By contrast, knockdown of the AI gene increased mitochondrial function. Hypoxic cardiomyocytes and ischemic myocardium showed increased AI mRNA expression. These findings suggest that AI is involved in suppressing mitochondrial function, thereby leading to cellular stress eradication and consequently to protection during hypoxia.

Although the gross embryology of inner ear development has been documented for several different vertebrate species at a descriptive level, our understanding of the molecular mechanisms involved remains rudimentary. Therefore, we have used cDNA subtraction and normalization procedures to define genes upregulated in the 13.5dpc mouse inner ear, a developmental stage where inner ear morphogenesis and tissue remodeling is active and differentiation of future hair cells is being initiated. We recovered 33 different genes from this subtraction and using gene-specific primers have confirmed the transcriptional upregulation of 26 of these in the 13.5dpc inner ear. Northern analyses were used to investigate splicing differences between the inner ear and the whole embryo at 13.5dpc. Spatial localization of expression was determined through whole-ear in situ hybridization analysis, and selected genes were analyzed in more detail through in situ hybridization of tissue sections. These data illustrate that the genes isolated in this study are expressed in the developing otic capsule and/or neuroepithelium. Furthermore, the expression patterns also reveal molecular heterogeneity in the developing capsule and indicate that for some genes, the chondrogenic otic capsule is composed of distinct domains of gene expression.

We report the isolation of genomic and cDNA clones encoding the human T-lymphocyte cell-surface differentiation antigen, Leu-2 (T8), by use of a combination of transfection, fluorescence-activated cell-sorting, and subtractive cDNA hybridization. We constructed a cDNA library with mRNA from a mouse L-cell transfectant in which the human Leu-2 gene is expressed and amplified. We identified Leu-2 cDNA clones by screening with a selected cDNA probe from a second amplified Leu-2 transfectant. This probe contained cDNA species not removed by hybridization with L-cell mRNA. A Leu-2 cDNA clone was used to isolate a genomic clone. Transfection with DNA from this clone resulted in a high number of Leu-2 transfectants. This approach can be used to clone genes coding for other cell-surface molecules.

application/pdf, From previous cDNA subtraction studies analyzing gene expression in equine endometrium, high lipocalin 2 (LCN2) mRNA expression was found in the gravid endometrium. In the uterus, LCN2 may transport hydrophobic molecules and siderophores with iron, or may form a complex with another protein, however, the expression of uterine LCN2 beyond day 20 of equine pregnancy and its receptor has not been characterized. To study the expression and potential roles of uterine LCN2 from pre-implantation to mid-gestation period, stage-specific endometrial samples were obtained from day 13 (day 0 = ovulation) cyclic and days 13, 19, 25, and 60 to 131 pregnant mares. Expression of LCN2 mRNA increased in day 19 gravid endometrium and was abundant from day 60 onward. The expression of LCN2 mRNA was localized to the glandular epithelium. LCN2 protein was detected in day 25 gravid endometrium and luminal fluid, and the protein was localized to the glandular epithelium and luminal cavity, whereas LCN2 receptor expression was found in luminal and glandular epithelium and trophectoderm throughout the experimental period. The presence of matrix metalloproteinase-9 (MMP9) was also examined because MMP9 is known to form a complex with LCN2. Although MMP9 and LCN2 were both found in luminal fluid from day 25 pregnant uterus, the complex of these proteins was not detected. Localization of the receptor in the trophectoderm suggests that endometrial LCN2 could play a role in carrying small substances from the mother to fetus in the equine species.

Hepatitis B x antigen (HB x Ag) is a trans-activating protein that may be involved in hepatocarcinogenesis, although few natural effectors of HB x Ag that participate in this process have been identified. To identify additional effectors, whole cell RNA isolated from HB x Ag-positive and HB x Ag-negative HepG2 cells were compared by polymerase chain reaction select cDNA subtraction, and one clone, upregulated gene, clone 11 (URG11), was chosen for further characterization. Elevated levels of URG11 mRNA and protein were observed in HB x Ag-positive compared to HB x Ag-negative HepG2 cells. Costaining was observed in infected liver (P < 0.01). URG11 stimulated cell growth in culture (P < 0.01), anchorage-independent growth in soft agar (P < 0.001), and accelerated tumor formation (P < 0.01), and yielded larger tumors (P < 0.02) in SCID mice injected subcutaneously with HepG2 cells. These data suggest that URG11 is a natural effector of HB x Ag that may promote the development of hepatocellular carcinoma.