The effect of cycloheximide on the expression of cell cycle dependent genes

We have investigated the inducibility of several cell cycle-dependent genes (plus control sequences, not expressed in a cell cycle-dependent manner) in the presence of cycloheximide, an inhibitor of protein synthesis. The genes studied include: 1) five cDNA clones that are preferentially expressed in the G1 phase of the cell cycle: KC-1, JE-3, 2F1, 4F1 and 2A9; 2) one gene preferentially expressed in late G1/S phase: histone H3; and 3) the cell cycle-dependent oncogene p53. All the genes studied are induced by serum even in the presence of cycloheximide. Previous results in the literature have shown that 2 other oncogenes, c-myc and c-fos, can be induced by growth factors in the presence of cycloheximide. Together with our results, these findings indicate that protein synthesis is not required for the induction of at least nine cell cycle genes by growth factors.     

Adenovirus type 2 activates cell cycle-dependent genes that are a subset of those activated by serum.

We have studied a panel of 10 genes and cDNA sequences that are expressed in a cell cycle-dependent manner in different types of cells from different species and that are inducible by different mitogens. These include five sequences (c-myc, 4F1, 2F1, 2A9, and KC-1) that are preferentially expressed in the early part of the G1 phase, three genes (ornithine decarboxylase, p53, and c-rasHa) preferentially expressed in middle or late G1, and two genes (thymidine kinase and histone H3) preferentially expressed in the S phase of the cell cycle. We have studied the expression of these genes in nonpermissive (tsAF8) and semipermissive (Swiss 3T3) cells infected with adenovirus type 2. Under the conditions of these experiments, adenovirus type 2 infection stimulates cellular DNA synthesis in both tsAF8 and 3T3 cells. However, four of the five early G1 genes (c-myc, 4F1, KC-1, and 2A9) and one of the late G1 genes (c-ras) are not induced by adenovirus infection, although they are strongly induced by serum. The other sequences (2F1, ornithine decarboxylase, p53, thymidine kinase, and histone H3) are activated by both adenovirus and serum. We conclude that the cell cycle-dependent genes activated by adenovirus 2 are a subset of the cell cycle-dependent genes activated by serum. The data suggest that the mechanisms by which serum and adenovirus induce cellular DNA synthesis are not identical.     

Analysis of expressed sequence tags (EST) obtained from common carp,Cyprinus carpio L., head kidney cells after stimulation by two mitogens,lipopolysaccharide and concanavalin-A

A representative cDNA library from mRNA obtained from lipopolysaccharide and concanavalin-A-induced head kidney cells of carp, Cyprinus carpio, was constructed. Two hundred single pass and partially sequenced clones (AU183343 to AU183542) were generated from expressed sequence tags (ESTs) and these were searched for homology in the DDBJ/GENBANK with blastN and blastX programs. Clones matching known genes were classified according to their function and distribution. One hundred and twenty-nine genes showed homology with known genes in databases, whereas 71 (35.5%) clones did not show any significant homology to sequences in the public database. Known genes also showed homology to fish genes deposited in the database. Twenty-two clones (11%), encoding 16 different sequences, were identified as putative biodefense and oncogenes, associated with an immune response. High expression of lysozyme (3%) was detected. Putatively identified biodefense-related sequences such as Lectin type 2, MHC class II invariant chain, mcl-1a and lysozyme were aligned with known homologues from the database and the percentage identity determined. A time course evaluation of gene expression due to mitogen stimulation by RT-PCR revealed the above mentioned gene homologues were switched on early during the cell proliferation.     

Two amidophosphoribosyltransferase genes of Arabidopsis thaliana expressed in different organs

Amidophosphoribosyltransferase (ATase: EC 2.4.2.14) is a key enzyme in the pathway of purine nucleotide biosynthesis. We have identified several cDNA clones whose amino acid sequences exhibit similarity with the known ATases in a cDNA library of young floral buds of Arabidopsis thaliana. The cDNA clones are derived from two genes homologous with each other. These cDNAs represent the first plant representatives of ATase gene. Structural comparison with ATases of other organisms has revealed that the two genes encode [4Fe-4S] cluster-dependent ATases. Northern blot analysis showed that expression level of the genes is different in three organs; one gene is expressed in flowers and roots, while the other gene is mainly expressed in leaves.     

Expressed sequence tags from persimmon at different developmental stages

Persimmon (Diospyros kaki Thunb.) is an important fruit in Asian countries, where it is eaten as a fresh fruit and is also used for many other purposes. To understand the molecular mechanism of fruit development and ripening in persimmon, we generated a total of 9,952 expressed sequence tags (ESTs) from randomly selected clones of two different cDNA libraries. One cDNA library was derived from fruit of “Saijo” persimmon at an early stage of development, and the other from ripening fruit. These ESTs were clustered into 6,700 non-redundant sequences. Of the 6,700 non-redundant sequences evaluated, the deduced amino acid sequences of 4,356 (65%) showed significant homology to known proteins, and 2,344 (35%) showed no significant similarity to any known proteins in Arabidopsis databases. We report comparison of genes identified in the two cDNA libraries and describe some putative genes involved in proanthocyanidin and carotenoid synthesis. This study provides the first global overview of a set of genes that are expressed during fruit development and ripening in persimmon.     

Mass isolation of cuticle protein cDNAs from wing discs of Bombyx mori and their characterizations

Multiple cloning of cuticle protein genes was performed by sequencing of cDNAs randomly selected from a cDNA library of wing discs just before pupation, and nine different cuticular protein genes were identified. Thirty-one clones of a cuticle protein gene were identified from the 1050 randomly sequenced clones; about 3% were cuticle protein genes in the W3-stage wing disc cDNA library. The sequence diversity of the deduced amino acid sequences of isolated Bombyx cuticle genes was examined along with the expression profiles. The deduced amino acid sequences of the nine cuticle protein genes contained a putative signal peptide at the N-terminal region and a very conserved hydrophilic region known as the R and R motif. The developmental expression of cuticle genes was classified into two types: pupation (five clones were expressed only around pupation) and pupation and mid-pupal (four clones were expressed around this stage). All the isolated genes were expressed in the head, thoracic, and abdominal regions of the epidermis at different levels around pupation, but no expression was observed in the epidermis at the fourth molting stage.     

Prediction of the Coding Sequences of Unidentified Human Genes. IV. The Coding Sequences of 40 New Genes (KIAA0121-KIAA0160) Deduced by Analysis of cDNA Clones from Human Cell Line KG-1

In this series of projects regarding the accumulation of sequenceinformation of unidentified human genes, we newly deduced thesequences of 40 full-length cDNA clones of human cell line KG-1,and predicted the coding sequences of the corresponding genes,named KIAA0121 to 0160. The results of a computer search ofpublic databases indicated that the sequences of 13 genes wereunrelated to any reported genes, while the remaining 27 genescarried sequences which showed some similarities to known genes.Obvious unique sequences noted were as follows. A stretch oftriplet repeats was contained in each of three genes: Thesewere GAG(Glu) in KIAA0122 and KIAA0147, and TCC(Ser) in KIAA0150.A stretch of 10 amino acidresidues was repeated 21 times inKIAA0139, and a homologous sequence of 76–78 nucleotideswas found repeated 6 times in the untranslated region of KIAA0125.northern hybridization analysis demonstrated that 13 genes wereexpressed in a cell- or tissue-specific manner. Although a vastnumber of expressed sequence tags (ESTs) have been registeredfor comprehensive analysis of cDNA clones, our sequence dataindicated that their distribution is very unbalanced: e.g. whileno EST hit 7 genes, 85 ESTs fell in a single gene.     

cDNA Libraries from Single Human Preimplantation Embryos

In this paper, the construction, evaluation, and application of cDNA libraries from eight unfertilized oocytes and single four-cell-, seven-cell-, and blastocyst-stage embryos are described. Rapid, reproducible, and efficient procedures for the construction of PCR-based cDNA libraries from fewer than 10 cells were first developed in small populations of fibroblast cells. The human embryo libraries display complexities sufficient (between 10⁵and 10⁶clones) to represent the entire active gene population at these early stages of human development. The ubiquitous cytoskeletal elements, β-actin, keratin-18, and α-tubulin, were detected at the expected frequency. Sequencing of consecutively picked random clones, without selection, showed the presence of a variety of sequences, such as the human transposable element, LINE-1 andAlurepeat sequences, housekeeping genes, and tissue-specific genes, such as α-globin and FMR-1. In addition to cDNAs corresponding to known ESTs (expressed sequence tags) in the GenBank and dbEST databases, a high proportion of novel sequences were detected. Applications of the libraries to several areas of interest, such as expression of CpG-island-containing “tissue-specific” genes, developmental genes expressed in a stage-specific manner, and a search for monoallelic expression of imprinted genes, are described. The libraries are a valuable resource for the study of gene expression during human preimplantation development and obviate the need for research on the human embryos themselves.     

Screening and analysis of differentially expressed genes from an alien addition line of wheat Thinopyrum intermedium induced by barley yellow dwarf virus infection.

The alien addition line TAI-27 contains a pair of chromosomes of Thinopyrum intermedium that carry resistance against barley yellow dwarf virus (BYDV). A subtractive library was constructed using the leaves of TAI-27, which were infected by Schizaphis graminum carrying the GAV strain of BYDV, and the control at the three-leaf stage. Nine differentially expressed genes were identified from 100 randomly picked clones and sequenced. Two of the nine clones were highly homologous with known genes. Of the remaining seven cDNA clones, five clones matched with known expressed sequence tag (EST) sequences from wheat and (or) barley whereas the other two clones were unknown. Five of the nine differentially expressed sequences (WTJ9, WTJ11, WTJ15, WTJ19, and WTJ32) were highly homologous (identities >94%) with ESTs from wheat or barley challenged with pathogens. These five sequences and another one (WTJ18) were also highly homologous (identities >86%) with abiotic stress induced ESTs in wheat or barley. Reverse Northern hybridization showed that seven of the nine differentially expressed cDNA sequences hybridized with cDNA of T. intermedium infected by BYDV. Three of these also hybridized with cDNA of line 3B-2 (a parent of TAI-27) infected by BYDV. The alien chromosome in TAI-27 was microdissected. The second round linker adaptor mediated PCR products of the alien chromosomal DNA were labeled with digoxygenin and used as the probe to hybridize with the nine differentially expressed genes. The analysis showed that seven differentially expressed genes were homologous with the alien chromosome of TAI-27. These seven differentially expressed sequences could be used as ESTs of the alien chromosome of TAI-27. This research laid the foundation for screening and cloning of new specific functional genes conferring resistance to BYDV and probably other pathogens.     

Nad+-dependent isocitrate dehydrogenase from Arabidopsis thaliana. Characterization of two closely related subunits

Two cDNA clones which appear to encode different subunits of NAD⁺-dependent isocitrate dehydrogenase (IDH; EC 1.1.1.41) were identified by homology searches from the Arabidopsis EST database. These cDNA clones were obtained and sequenced; both encoded full-length messages and displayed 82.7% nucleotide sequence identity over the coding region. The deduced amino acid sequences revealed preprotein lengths of 367 residues, with an amino acid identity of 86.1%. Genomic Southern blot analysis showed distinct single-copy genes for both IDH subunits. Both IDH subunits were expressed as recombinant proteins in Escherichia coli, and polyclonal antibodies were raised to each subunit. The Arabidopsis cDNA clones were expressed in Saccharomyces cerevisiae mutants which were deficient in either one or both of the yeast NAD⁺-dependent IDH subunits. The Arabidopsis cDNA clones failed to complement the yeast mutations; although both IDH-I and IDH-II were expressed at detectable levels, neither protein was imported into the mitochondria.     

Combining SSH and cDNA microarrays for rapid identification of differentially expressed genes

Comparing patterns of gene expression in cell lines and tissues has important applications in a variety of biological systems. In this study we have examined whether the emerging technology of cDNA microarrays will allow a high throughput analysis of expression of cDNA clones generated by suppression subtractive hybridization (SSH). A set of cDNA clones including 332 SSH inserts amplified by PCR was arrayed using robotic printing. The cDNA arrays were hybridized with fluorescent labeled probes prepared from RNA from ER-positive (MCF7 and T47D) and ER-negative (MDA-MB-231 and HBL-100) breast cancer cell lines. Ten clones were identified that were over-expressed by at least a factor of five in the ER-positive cell lines. Northern blot analysis confirmed over-expression of these 10 cDNAs. Sequence analysis identified four of these clones as cytokeratin 19, GATA-3, CD24 and glutathione-S-transferase mu-3. Of the remaining six cDNA clones, four clones matched EST sequences from two different genes and two clones were novel sequences. Flow cytometry and immunofluorescence confirmed that CD24 protein was over-expressed in the ER-positive cell lines. We conclude that SSH and microarray technology can be successfully applied to identify differentially expressed genes. This approach allowed the identification of differentially expressed genes without the need to obtain previously cloned cDNAs.     

Two distinct classes of prestalk-enriched mRNA sequences in Dictyostelium discoideum

We have isolated cDNA clones derived from three mRNA sequences which are inducible by DIF, the putative stalk-specific morphogen of Dictyostelium. The three mRNA sequences are selectively expressed in cells on the stalk cell pathway of differentiation and we have compared them with previously characterized prestalk-enriched mRNA sequences. We find these latter sequences are expressed without a dependence on DIF, are much less highly enriched in prestalk over prespore cells and are expressed earlier during development than the DIF-inducible mRNA sequences. We propose two distinct mechanisms whereby a mRNA may become enriched in prestalk cells. An apparently small number of genes, represented by those we have isolated, is inducible by DIF and accumulates only in prestalk cells. We suggest that a second class of prestalk-enriched mRNA sequences are induced by cAMP to accumulate in all cells during aggregation and then become enriched in prestalk cells by selective loss from prespore cells.     

Identification of differentially expressed genes in isogenic highly metastatic and poorly metastatic cell lines of R3230AC rat mammary adenocarcinoma

Tumour metastasis occurs as a result of a cascade of events including alterations in the expression of various genes. The identification of such genes is essential to understanding formation of metastasis. In a previous study, highly metastatic (LN4.D6) and poorly metastatic (CAb.D5) cell lines were obtained from the rat mammary adenocarcinoma cell line R3230AC. Subtractive hybridization was used to identify differentially expressed genes between these two cell lines. We identified eight cDNA clones in CAb.D5 and six cDNA clones in LN4.D6 that were differentially expressed. One of the cDNA clones in each cell line had no homology with known sequences. Expression patterns of these differentially expressed genes were examined in a pair of rat mammary and prostate adenocarcinoma cell lines. Compared with cell lines examined, cDNA FF-10 was only expressed in CAb.D5; however, cDNA RB-8, RE-1, RF-5 were only expressed in the highly metastatic LN4.D6. No correlation was observed between expression patterns of the differentially expressed genes and metastatic potential of these cells. However, differential expression of genes, especially cytokeratins (CK8 and CK5) and collagens (III and IV) between highly metastatic and low metastatic rat mammary adenocarcinoma cell lines might initiate further investigation of these genes in metastatic process.     

cDNA cloning,expression levels and gene mapping of photosynthetic and non-photosynthetic ferredoxin genes in sunflower (<Emphasis Type="Italic">Helianthus annuus</Emphasis> L.)

Fatty acid desaturation in plastids and chloroplasts depends on the electron-donor activity of ferredoxins. Using degenerate oligonucleotides designed from known photosynthetic and heterotrophic plant ferredoxin sequences, two full-length ferredoxin cDNAs were cloned from sunflower (Helianthus annuus L.) leaves and developing seeds, HaFd1 and HaFd2, homologous to photosynthetic and non-photosynthetic ferredoxins, respectively. Based on these cDNAs, the respective genomic sequences were obtained and the presence of DNA polymorphisms was investigated. Complete sequencing of the HaFd1 and HaFd2 genes in different lines indicated the presence of two haplotypes for HaFd2 and their alignment showed that sequence polymorphisms are restricted to the 5′-NTR intron. In addition, specific DNA markers for the HaFd1 and HaFd2 genes were developed that enabled the genes to be mapped. Accordingly, the HaFd1 locus maps to linkage group 10 of the public sunflower map, while the HaFd2 locus maps to linkage group 11. Both ferredoxins display different spatial-temporal patterns of expression. While HaFd2 is expressed at similar levels in all tissues tested (leaves, stem, roots, cotyledons and developing seeds), HaFd1 is more strongly expressed in green tissues than in all the other tissues tested. Both photosynthetic- and heterotrophic-ferredoxins are present in sunflower seeds and may contribute to fatty acid desaturation during oil accumulation. Nevertheless, the levels of HaFd2 expression during seed formation are distinct in lines that only varied in the HaFd2 haplotypes they expressed.     

人参植物与皂苷生物合成相关的差减cDNA文库构建及基因差异表达分析

应用抑制差减杂交技术,分别以源于4年和1年生人参根组织cDNA群体作为检测子(tester)与驱赶子(driver),成功构建了与人参植物皂苷生物合成相关的差减cDNA文库,并时从中筛选的阳性cDNA克隆进行DNA测序及其序列分析、PCR及Northern印迹杂交鉴定．结果显示,获得的13个克隆为新基因序列．其中6个差减克隆系人参植物根生长发育阶段差异表达基因．目前,6个差异表达新基因的结构与功能仍在进一步研究中．     

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.