Cloning and characterization of preferentially expressed genes in an aluminum-tolerant mutant derived from Penicillium chrysogenum IFO4626

cDNAs expressed preferentially in an Al-tolerant microorganism were isolated by subtraction hybridization with cDNAs of Al-sensitive Penicillium chrysogenum IFO4626 as driver cDNA and cDNAs of the Al-tolerant mutant derived from the wild cells by UV irradiation as tester cDNA. Northern blot analysis revealed that mRNA levels of six genes were increased significantly in the Al-tolerant mutant after exposure to Al stress when compared with the wild cells. Two genes accumulated in both the presence and absence of Al stress and four genes were induced by Al stress in the Al-tolerant mutant. cDNA fragments were amplified by rapid amplification of cDNA ends and sequenced to obtain full-length cDNAs of the six genes. Two genes were novel or predicted ones and the others showed significant homology to known genes, ADP/ATP translocase, enolase, cysteine synthase, and glucoamylase, which are induced by environmental stresses in prokaryotic and eukaryotic cells. These enzyme activities increased in the Al-tolerant mutant when compared to those in the wild cells, showing that not only the levels of gene expression but also the levels of enzyme activities increased in the Al-tolerant mutant.     

Combination of PCR subtraction and cDNA microarray for differential gene expression profiling.

PCR subtraction hybridization has been used effectively to enrich and single out differentially expressed genes. However identification of these genes by means of cloning and sequencing individual cDNAs is a tedious and lengthy process. In this report, an attempt has been made to combine the use of PCR select cDNA subtraction hybridization and cDNA microarrays to identify differentially expressed genes using a nonradioactive chemiluminescent detection method. mRNA from human prolactin (hPRL) or human prolactin antagonist (hPRL-G129R) treated and non-treated breast cancer cells was isolated, and cDNAs were synthesized and used for the PCR subtraction to enrich the differentially expressed genes in the treated cells. The PCR-amplified and subtracted cDNA pools were purified and labeled using the digoxigenin method. Labeled cDNAs were hybridized to a human apoptosis cDNA microarray membrane and identified by chemiluminescence. The results suggest that the strategy of combining all three methods will allow for a more efficient, nonradioactive way of identifying differentially expressed genes in target cells.     

Identification of Up-Regulated Genes After Complete Spinal Cord Transection in Adult Rats

Spinal cord injury (SCI) initiates a cascade of events and these responses to injury are likely to be mediated and reflected by changes in mRNA concentrations. As a step towards understanding the complex mechanisms underlying repair and regeneration after SCI, the gene expression pattern was examined 4.5 days after complete transection at T8-9 level of rat spinal cord. Improved subtractive hybridization was used to establish a subtracted cDNA library using cDNAs from normal rat spinal cord as driver and cDNAs from injured spinal cord as tester. By expressed sequence tag (EST) sequencing, we obtained 73 EST fragments from this library, representing 40 differentially expressed genes. Among them, 32 were known genes and 8 were novel genes. Functions of all annotated genes were scattered in almost every important field of cell life such as DNA repair, detoxification, mRNA quality control, cell cycle control, and signaling, which reflected the complexity of SCI and regeneration. Then we verified subtraction results with semiquantitative RT-PCR for eight genes. These analyses confirmed, to a large extent, that the subtraction results accurately reflected the molecular changes occurring at 4.5 days post-SCI. The current study identified a number of genes that may shed new light on SCI-related inflammation, neuroprotection, neurite-outgrowth, synaptogenesis, and astrogliosis. In conclusion, the identification of molecular changes using improved subtractive hybridization may lead to a better understanding of molecular mechanisms responsible for repair and regeneration after SCI.     

Isolation and characterization of the major form of human MUC18 cDNA gene and correlation of MUC18 over-expression in prostate cancer cell lines and tissues with malignant progression.

Ectopical expression of huMUC18, a cell adhesion molecule in the immunoglobulin gene superfamily, causes a non-metastatic human melanoma cell line to become metastatic in a nude mouse system. To determine if MUC18 expression correlates with the development and malignant progression of prostate cancer, we investigated differential expression of human MUC18 (huMUC18) in normal prostate epithelial cells, prostate cancer cell lines, and prostatic normal and cancer tissues. We cloned and characterized the human MUC18 (huMUC18) cDNA gene from three human prostate cancer cell lines and three human melanoma cell lines. The cDNA sequences from the six human cancer cell lines were identical except differences in one to five nucleotides. The deduced amino acid sequences of the longest ORF were 646 amino acids that were identical in these cDNAs except for one to three amino acid residues. The amino acid sequences of all our huMUC18 cDNA genes are similar to that cloned by other group (GenBank access #M28882) except differences in the same seven amino acids. We conclude that huMUC18 cDNA gene reported here represents the gene product from a major allele. The MUC18 mRNA and protein was expressed in three metastatic prostate cancer cell lines (TSU-PR1, DU145, and PC-3), but not in one non-metastatic prostate cancer cell line (LNCaP.FGC). The expression of huMUC18 in these four cell lines is positively related to their extent of in vitro motility and invasiveness and in vivo metastasis in nude mice. HuMUC18 protein was also expressed at high levels in extracts prepared from tissue sample sections containing high grade prostatic intraepithelial neoplasia (PIN), but weakly expressed in extracts prepared from cultured primary normal prostatic epithelial cells and the normal prostate gland. Immunohistochemical analysis showed that huMUC18 was expressed at higher levels in the epithelial cells of high-grade PIN and prostatic carcinomas, and in cells of a perineural invasion, a lymph node, and a lung metastases compared to that in normal or benign hyperplastic epithelium (BPH). We therefore conclude that MUC18 expression is increased during prostate cancer initiation (high grade PIN) and progression to carcinoma, and in metastatic cell lines and metastatic carcinoma. Increased expression of MUC18 is implicated to play an important role in developing and malignant progression of human prostate cancer. Furthermore, the lacking of predominant cytoplasmic membrane expression of MUC18 appeared to correlate with malignant progression of prostate cancer.     

Serological cloning of PARIS-1: a new TBC domain-containing, immunogenic tumor antigen from a prostate cancer cell line.

Identifying immunogenic tumor antigens plays a critical role in developing efficient diagnostic and therapeutic strategies for treatment of cancer. Using a recently developed technology, serological identification of antigens by recombinant expression cloning (SEREX), we identified a total of 8 genes whose expression elicited antibody responses in prostate cancer patients. Of the 8 genes, 5 represented known genes in the GenBank database, 2 were previously uncharacterized genes, and 1 showed sequence homology to a mouse gene. The sequence feature and the expression of one of the novel genes, prostate antigen recognized and identified by SEREX (PARIS-1), are determined in this study. The PARIS-1 cDNA is 3257 bp in length and contains a complete open reading frame of 2751 bp encoding for a primary translation product of 917 amino acids. Using Northern blot hybridization assay, we detected a single species of approximately 3.3 kb PARIS-1 mRNA that is differentially expressed in prostate normal and cancer cells. Western blot analysis confirmed the expression of the PARIS-1 protein in these cells. Structure analysis revealed that PARIS-1 protein contains a TBC domain that is conserved in the family of cell cycle-regulatory and Rab GTPase-activating proteins (Rab-GAP). Thus, the PARIS-1 protein may play a role in regulation of cell differentiation and growth or represent a new member of the Rab-GAP family.     

Identification of iron responsive genes by screening cDNA libraries from suppression subtractive hybridization with antisense probes from three iron conditions

The goal of the present study is to identify genes that respond to iron availability. Suppression subtraction hybridization (SSH) was used to generate cDNA libraries from iron loaded and control human astrocytoma cells (SW1088). The cDNA libraries were screened with antisense cDNA probes obtained from mRNA isolated from astrocytoma cells exposed to three conditions: (i) normal media (control), (ii) deferoxamine treated (iron deficient) or (iii) iron loaded. The screening of the cDNA libraries with antisense probes from the three conditions enhanced the screening efficiency and decreased the number of false positives. Positive clones were identified and sequenced. The genes of interest were further analyzed by determining changes in hybridization signal on northern blots from astrocytoma cells exposed to iron or deferoxamine over different time intervals. Our analysis identified cDNAs corresponding to known iron responsive genes such as L-chain ferritin, but also revealed a number of mRNAs with novel sequences and mRNAs previously not known to be responsive to iron such as one of the ABC transporters and Thy-1 glycoprotein. Thus our results suggest that the expression of a number of genes may be influenced by changes in iron availability.     

Characterization of a novel human breast cancer associated gene (BCA3) encoding an alternatively spliced proline-rich protein

As part of an integrated study of breast cancer gene expression, partial cDNAs were cloned from normal and tumor breast cells by subtractive-hybridization and differential display cloning. The DNA sequence for one of these breast cancer associated genes was used to construct the larger 1319 bp BCA3 cDNA sequence using ESTs without assigned names or functions. High-level BCA3 mRNA expression was found in breast and prostate tumor cell lines whereas normal breast and prostate tissues have low-level expression. Further analysis revealed possible functional domains and alternative splicing of BCA3 that we confirmed by RT-PCR analysis. Immunohistochemistry revealed that the protein is expressed in breast tumor cells in vivo, and not in surrounding stromal tissue.     

Suppression subtractive hybridization identifies differentially expressed genes in Brassica napus chlorophyll-reduced mutant

Suppressive subtraction hybridization (SSH) was used to identify differentially expressed genes caused by a chlorophyll-reduced mutation in B. napus. The cDNA fragments, derived from SSH positive subtractive library (tester: normal wild type, driver: mutant) were cloned into pMD18-T vector. Two hundred SSH cDNA clones were screened by dot blot array, and 151 clones were identified as differentially expressed cDNA fragments in Cr3529 line. Thirty-six positive clones which showed marked expression differences were selected and sequenced. After redundant cDNAs were removed, 33 differentially expressed unique cDNA section clones were obtained. Among the 33 clones, two clones possess different parts of the cDNA sequence of the same gene coding geranylgeranyl reductase, four clones belong to unknown proteins, and the rest share homology to genes of diverse class. Sequence analysis showed that at least 12 genes were discovered to be related to the photosynthesis, seven of them coded the proteins which belong to the subunit of photosystem 2. RNA gel blot analysis showed that compared with 3529, the gene expression of the chlorophyll a/b-binding protein Lhcb2 in photosystem 2 declined markedly in the cotyledons and seedling leaves of Cr3529, indicating that the reduced light-harvesting complex 2 accumulation in thylakoid membrane of Cr3529 was due to the decrease of the related gene mRNA level for translation.     

Cloning and Characterization of Two Novel Human cDNAs (NELL1 and NELL2) Encoding Proteins with Six EGF-like Repeats

From a human fetal-brain cDNA library we isolated two novel genes encoding peptides containing six EGF-like repeats. Both showed significant homologies with nel, a gene strongly expressed in neural tissues of chicken. The cDNAs, designated NELL1 (nel-like, type 1) and NELL2 (nel-like, type 2), contained open reading frames encoding 810 and 816 amino acids, respectively. NELL2 is strongly expressed in brain of adult and fetus but only weakly in fetal kidney. NELL1 and NELL2 were mapped by FISH to chromosomal bands 11p15.1–p15.2 and 12q13.11–q13.12, respectively.     

Biochemical characterization and expression analysis of neural thrombospondin-1-like proteins NELL1 and NELL2.

Two closely related genes coding for NELL proteins (NELL1 and NELL2) have been cloned by the yeast two-hybrid screening of a rat brain cDNA library with the regulatory domain of protein kinase C betaI (PKCbetaI) as bait. The rat NELL proteins show about 55% identity with each other and contain several protein motifs assigned to a secretion signal peptide, an NH(2)-terminal thrombospondin-1 (TSP-1)-like module, five von Willebrand factor C domains, and six epidermal growth factor-like domains; the NELL proteins share many protein motifs with TSP-1. The NELL proteins expressed in COS-7 cells are homotrimeric glycoproteins and possess heparin-binding activity. Furthermore, while NELL1 and NELL2 show distinct subcellular localization in cytoplasm, they both are partially secreted into the culture medium of COS-7 cells. Although the NELL1 mRNA is faintly expressed in adult neural cells, the NELL2 mRNA is expressed abundantly, particularly in the pyramidal cells of rat hippocampus, showing neuronal high plasticity. During mouse embryogenesis, expression of the NELL2 mRNA is initiated 7-11 days postcoitum, simultaneously with neural plate formation. These results strongly suggest that the NELL2 protein, similar to but not identical with TSP-1, is involved in the growth and differentiation of neural cells. Additionally, the NELL1 and NELL2 mRNAs were found to be expressed abundantly in Burkitt's lymphoma Raji cells and colorectal adenocarcinoma SW480 cells, respectively. Thus, it is likely that the NELL proteins also participate in the growth, differentiation, and oncogenesis of cancer cell lines.     

Analysis of Medicago truncatula nodule expressed sequence tags

Systematic sequencing of expressed sequence tags (ESTs) can give a global picture of the assembly of genes involved in the development and function of organs. Indeterminate nodules representing different stages of the developmental program are especially suited to the study of organogenesis. With the vector lambdaHybriZAP, a cDNA library was constructed from emerging nodules of Medicago truncatula induced by Sinorhizobium meliloti. The 5' ends of 389 cDNA clones were sequenced, then these ESTs were analyzed both by sequence homology search and by studying their expression in roots and nodules. Two hundred fifty-six ESTs exhibited significant similarities to characterized data base entries and 40 of them represented 26 nodulin genes, while 133 had no similarity to sequences with known function. Only 60 out of the 389 cDNA clones corresponded to previously submitted M. truncatula EST sequences. For 117 cDNAs, reverse Northern (RNA) hybridization with root and nodule RNA probes revealed enhanced expression in the nodule, 48 clones are likely to code for novel nodulins, 33 cDNAs are clones of already known nodulin genes, and 36 clones exhibit similarity to other characterized genes. Thus, systematic analysis of the EST sequences and their expression patterns is a powerful way to identify nodule-specific and nodulation-related genes.     

Use of SMART-generated cDNA for gene expression studies in multiple human tumors

We demonstrate here that SMART PCR-amplified cDNAs arrayed on a nylon membrane are suitable for high-throughput tissue expression profiling when starting biological materials are limited. We show that SMART cDNA accurately reflects gene expression patterns found in total RNA by comparing the expression level of several target genes in SMART PCR-amplified cDNAs and their corresponding total RNAs. We also arrayed cDNAs from 68 matched tumor and normal samples on a nylon membrane to determine whether SMART PCR-amplified cDNA could be used for detecting differentially expressed genes in these tissues. These arrays containing normalized tumor and normal cDNAs were hybridized with probes for glutathione peroxidase and gelsolin. The hybridization results revealed cancer-related and patient-specific gene expression differences between tumor and normal tissues for these genes. These studies show that SMART PCR-amplified cDNAs maintain the complexity of the original mRNA population and are thus suitable for high-throughput studies to compare the relative abundance of target genes and to detect differentially expressed genes in a wide variety of tissues simultaneously.     

Identification of receptor genes in renal cell carcinoma associated with angiogenesis by differential hybridization technique

To screen the receptor genes in renal cell carcinoma (RCC) associated with angiogenesis, we performed differential hybridization of the cDNA library of membrane-type protein tyrosine kinases (mPTKs). Three thousand plaques of a mPTKs-enriched cDNA library were screened with mPTKs mixture probes produced from hypervascular RCC tissues and RCC cell lines. Six different cDNA fragments of the PTK genes were isolated, and the sequence analysis showed that these represented cDNAs for TIE1, KDR, FMS, FGFR-4, JAK1 and HCK. Of these genes, the expression of TIE1, KDR, and FGFR-4 was studied in RCC tissue and cell lines by Northern blot analysis. We also investigated the expression of vascular endothelial growth factor (VEGF), placenta growth factor (PlGF) and their receptor FLT-1. In all the hypervascular RCC tissues, the amounts of mRNAs for KDR and FLT-1 were increased compared to adjacent normal tissues. The TIE1 and FGFR-4 genes were also overexpressed in most of the hypervascular RCC tissues, while no mRNA of KDR, FLT-1, or TIE1 could be detected in any of the four human RCC cell lines. The amounts of the VEGF and PlGF mRNAs were increased in hypervascular RCC tissues, while VEGF mRNA was detected in the four cell lines but PlGF mRNA was not. FGFR-4 mRNA was expressed in three of the four cell lines. These results suggest that KDR, FLT-1, PlGF and TIE1 mRNAs are present in the mesenchymal cells of RCC, while VEGF and FGFR-4 genes are expressed in RCC cells themselves in vivo.     

Differential mRNA display analysis of two related but functionally distinct rat insulinoma (RIN) cell lines: identification of CD24 and its expression in the developing pancreas

To identify genes that might play a role in growth and differentiation of pancreatic beta-cells, we have applied the technique of differential mRNA display to the lineage-related, but functionally distinct rat insulinoma (RIN) cell lines RIN-5AH and RIN-A12. Direct comparison of PCR-generated RIN-5AH and RIN-A12 cDNAs on DNA sequencing gels revealed 31 differentially expressed bands. By Northern blot hybridization, authentic differential expression was confirmed for three cDNAs derived from RIN-5AH cells and four cDNAs from RIN-A12 cells. Nucleotide sequences were determined for these cDNAs and database searches identified one known gene that encoded heat stable antigen CD24. Of the remaining six genes, three matched with established sequence tags from fetal tissue, and three were potentially novel. By RT-PCR analysis, five of the seven genes were expressed in normal fetal and/or adult pancreas. In a detailed survey of CD24 protein expression in the pancreas using the CD24-specific monoclonal antibody J11d, CD24 was predominantly expressed in ductal epithelial cells (E13.5-15.5), developing endocrine (alpha, beta and delta) and exocrine cells (E15.5-20.5) and mature exocrine and peripheral islet delta-cells post E20.5. The retention of CD24 expression in a large proportion of delta-cells but only in a minority of alpha- and beta-cells leads us to hypothesize that CD24 may mark a pool of precursor endocrine cells within adult islets.     

Regulation of ipsilateral and contralateral bovine oviduct epithelial cell function in the postovulation period: a transcriptomics approach

We studied differential gene expression in ipsilateral and contralateral bovine oviduct epithelial cells using a combination of subtracted cDNA libraries and cDNA array hybridization. Four Simmental heifers were synchronized and slaughtered 3.5 days after they entered standing heat. Epithelial cells were isolated from ipsilateral and contralateral oviducts. To identify genes that are differentially regulated in ipsilateral and contralateral epithelium, subtracted cDNA libraries were produced by suppression subtractive hybridization and analyzed by cDNA array hybridization. Sequencing of cDNAs showing differential expression levels in ipsilateral and contralateral epithelium revealed 35 different cDNAs, 30 of which matched genes with known functions and 5 of which matched genes without a known function. The majority of genes (n = 27) were expressed at a higher level in the ipsilateral oviduct, but for some genes (n = 8), mRNA abundance was higher in the contralateral oviduct. The regulated genes or their products include a variety of functional classes such as cell-surface proteins, cell-cell interaction proteins, members of signal transduction pathways, immune-related proteins, and enzymes. Identification of genes differentially regulated in ipsilateral and contralateral oviduct epithelial cells is the first step toward a systematic analysis of local mechanisms that regulate the function of the bovine oviduct epithelium in the postovulation period.