Construction of an equalized cDNA library fromColletotrichum lagenariumand its application to the isolation of differentially expressed genes

2000 ◽  
Vol 46 (2) ◽  
pp. 150-158 ◽  
Author(s):  
Atsuko Inagaki ◽  
Yoshitaka Takano ◽  
Yasuyuki Kubo ◽  
Kazuyuki Mise ◽  
Iwao Furusawa
Keyword(s):  
Amino Acid ◽  
Cdna Library ◽  
Differentially Expressed Genes ◽  
Kinetic Method ◽  
Differential Screening ◽  
Differentially Expressed ◽  
Phytopathogenic Fungus ◽  
Cdna Clones ◽  
Appressorium Formation ◽  
Colletotrichum Lagenarium

To establish an efficient screening system for differentially expressed genes of a phytopathogenic fungus Colletotrichum lagenarium, we constructed an equalized (normalized) cDNA library from C. lagenarium and used this library for differential screening. For the isolation of genes involved in infection-related developments of conidia, conidia undergoing appressorium differentiation were selected as the source of materials for construction of the cDNA library. The equalization of cDNA was performed twice using a kinetic method, and the products were cloned into a plasmid vector. Colony hybridization with nine probes of different abundance showed a reduction in abundance variation from at least 276-fold in the original library to 10-fold in the equalized cDNA library, which demonstrated that the cDNA was successfully equalized. By differential hybridization of 1900 cDNA clones in the equalized cDNA library and RNA blot analysis of candidate clones, we identified 11 independent cDNA clones, designated CAD1 through CAD11, that were expressed in appressorium-differentiating conidia, but not in vegetative mycelia. The transcripts of CAD1 and CAD2 hardly accumulated in preincubated conidia, whereas those of CAD3 and CAD4 accumulated highly and slightly, respectively. The amount of the four CAD transcripts increased at the early stage of the appressorium formation process. Sequence analysis of CAD1 revealed that CAD1 would encode for 101 amino acid polypeptides, which showed homology to metallothioneins. Deduced amino acid sequence of CAD2 would encode 278 amino acid polypeptides, and showed high homology to genes in aflatoxin, and sterigmatocystin gene clusters of Aspergillus parasiticus and A. nidulans, respectively. Key words: equalized cDNA library, differential screening, Colletotrichum lagenarium, appressorium formation, CAD genes.

Differential screening of a PCR-generated mouse embryo cDNA library: glucose transporters are differentially expressed in early postimplantation mouse embryos

Development ◽  
1992 ◽  
Vol 116 (3) ◽  
pp. 555-561 ◽  
Author(s):  
D.E. Smith ◽  
T. Gridley
Keyword(s):  
Cdna Library ◽  
Pcr Amplification ◽  
Glucose Transporters ◽  
Distal Portion ◽  
Mouse Embryos ◽  
Differential Screening ◽  
Differentially Expressed ◽  
Nucleotide Sequence Analysis ◽  
Cdna Clones ◽  
Glut 1

Differential screening of a cDNA library constructed using PCR amplification techniques from RNA isolated from the distal portion (embryonic ectoderm, mesoderm and visceral endoderm) of 7.5 days post coitum (dpc) mouse embryos led to the isolation of two cDNA clones expressed at higher levels in 7.5 dpc embryos than 12.5 dpc embryos. Nucleotide sequence analysis revealed that each of these clones was a different member of the family of facilitative glucose transporters (Glut genes). The differentially expressed cDNA clones represent mouse Glut-1 and Glut-3. Levels of the Glut-3 mRNA declined 14-fold between days 7.5 and 12.5 of gestation, and were under our limits of detection by 14.5 dpc. The levels of the Glut-1 mRNA declined about 3-fold between days 7.5 and 12.5 of gestation. Analysis of the expression of these genes by in situ hybridization revealed striking differences in transcript localization in early postimplantation mouse embryos. At 7.5 dpc, both transporters were expressed more strongly in extraembryonic tissues than in the embryo proper. While both transporters were expressed in the amnion and chorion, only Glut-1 was expressed in the ectoplacental cone. In the yolk sac, Glut-3 appeared to be expressed only in the endoderm while Glut-1, although expressed in both layers, was expressed more strongly in the mesoderm layer. Thus, the two transporters have relatively reciprocal sites of expression in the developing extraembryonic membranes. Expression of Glut-1 was fairly widespread in the embryo at 8.5 dpc, but by 10.5 dpc expression was down-regulated and was observed in the eye and the spinal cord.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Cloning and sequencing of protochlorophyllide reductase

1990 ◽  
Vol 265 (3) ◽  
pp. 789-798 ◽  
Author(s):  
P M Darrah ◽  
S A Kay ◽  
G R Teakle ◽  
W T Griffiths
Keyword(s):  
Amino Acid ◽  
Chemical Analysis ◽  
Amino Acid Sequence ◽  
Cdna Library ◽  
Avena Sativa ◽  
Cdna Clones ◽  
Lambda Phage ◽  
Cloning And Sequencing ◽  
Protochlorophyllide Reductase ◽  
Cnbr Cleavage

Putative protochlorophyllide reductase cDNA clones (252 and 113) were isolated from an etiolated-oat (Avena sativa) cDNA library. These were used to indirectly characterize a further clone, p127, isolated from a lambda-phage gt11 cDNA library. The latter (1.15 kb in length) was sequenced, and the derived amino acid sequence was shown to be remarkably similar to that derived from chemical analysis of a CNBr-cleavage fragment of the purified reductase, p127 codes for more than 95% of the reductase protein.

Isolation and characterization of related cDNA clones encoding skeletal muscle beta-tropomyosin and a low-molecular-weight nonmuscle tropomyosin isoform

1989 ◽  
Vol 9 (1) ◽  
pp. 185-192
Author(s):  
J A Bradac ◽  
C E Gruber ◽  
S Forry-Schaudies ◽  
S H Hughes
Keyword(s):  
Skeletal Muscle ◽  
Molecular Weight ◽  
Amino Acid ◽  
Cdna Library ◽  
Low Molecular Weight ◽  
Cdna Clones ◽  
Protein Coding ◽  
Coding Sequence ◽  
Untranslated Sequence ◽  
Complete Protein

We have isolated and characterized cDNA clones from chicken cDNA libraries derived from skeletal muscle, body wall, and cultured fibroblasts. A clone isolated from a skeletal muscle cDNA library contains the complete protein-coding sequence of the 284-amino-acid skeletal muscle beta-tropomyosin together with 72 bases of 5' untranslated sequence and nearly the entire 3' untranslated region (about 660 bases), lacking only the last 4 bases and the poly(A) tail. A second clone, isolated from the fibroblast cDNA library, contains the complete protein-coding sequence of a 248-amino-acid fibroblast tropomyosin together with 77 bases of 5' untranslated sequence and 235 bases of 3' untranslated sequence through the poly(A) tract. The derived amino acid sequence from this clone exhibits only 82% homology with rat fibroblast tropomyosin 4 and 80% homology with human fibroblast tropomyosin TM30nm, indicating that this clone encodes a third 248-amino-acid tropomyosin isoform class. The protein product of this mRNA is fibroblast tropomyosin 3b, one of two low-molecular-weight isoforms expressed in chicken fibroblast cultures. Comparing the sequences of the skeletal muscle and fibroblast cDNAs with a previously characterized clone which encodes the smooth muscle alpha-tropomyosin reveals two regions of absolute homology, suggesting that these three clones were derived from the same gene by alternative RNA splicing.

Screening of Pathogenicity-Related Genes in Bone Marrow CD4+ T Cells of Aplastic Anemia Patients.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3760-3760
Author(s):  
Miao Zheng ◽  
Wenli Liu ◽  
Hanying Sun ◽  
Jianfeng Zhou ◽  
Yicheng Zhang
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Aplastic Anemia ◽  
Differentially Expressed Genes ◽  
Cd4 T Cells ◽  
Translation Initiation Factor ◽  
Differentially Expressed ◽  
Cdna Clones ◽  
Sorting Technique ◽  
Ssh Library

Abstract Recent clinical and experimental data have proved that CD4+ T cells play a crucial role in the pathogenesis of aplastic anemia (AA). However, the mechanisms how CD4+ T cells in AA over-proliferate, activate, infiltrate bone marrow and damage hematopoietic cells have largely remained unknown. Therefore we adopted the suppressive subtractive hybridization (SSH) method so as to screen differentially expressed genes of bone marrow CD4+ T cells between AA patients and normal donors, which may hopefully help clear up the mystery of the pathogenesis of AA. Firstly, we selected a pair of subjects: a typical first-visit AA patient and a healthy donor of the same age and sex. Their bone marrow (BM) mononuclear cells were isolated using lymphocyte separating medium by density gradient centrifugation. After a 24-hour expansion culture, CD4+ T cells were separated with magnetic bead sorting technique. Secondly, with CD4+ T cells of the patient as “tester” and those of the normal donor as “driver”, a cDNA library was established by the SSH method. 110 clones were detected by means of PCR in the established subtractive library, which contained an inserted fragment with 200∼700bp respectively, and the positive detected rate was up to 88%. Then 10 of the resulting subtracted cDNA clones were randomly selected for a respective DNA sequencing and honologic analysis. Among 10 sequenced clones, there were 8 known genes including 2 repeated genes. Lastly, we collected BM samples of 20 AA patients and 20 healthy donors so as to validate the expression levels of differentially expressed genes from SSH library with semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR). We found, compared with normal donors, there were 6 genes among these 8 genes from SSH library up-expressed in BM CD4+ T cells of patients with AA. These genes were transducin (beta) -like 1X-linked receptor 1 (TBL1XR1) (NM024665); ATP-binding cassette, sub-family B (MDR/TAP), member 6 (NM005689); ARP2 actin-related protein 2 homolog (ACTR 2) (NM005722); zinc finger protein 561 (ZNF561) (NM152289); NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 6 (NM002493); eukaryotic translation initiation factor 3, subunit 5(NM003754). These results indicate that some genes are found able to regulate functions of BM CD4+ T cells in AA, concerned with protein synthesis, biology oxidation, signal transduction and cell migration, etc. Therefore, screening and cloning these genes are helpful to elucidate the possible mechanisms of AA.

scholarly journals The identification of a novel synaptosomal-associated protein, SNAP-25, differentially expressed by neuronal subpopulations.

1989 ◽  
Vol 109 (6) ◽  
pp. 3039-3052 ◽  
Author(s):  
G A Oyler ◽  
G A Higgins ◽  
R A Hart ◽  
E Battenberg ◽  
M Billingsley ◽  
...  
Keyword(s):  
Amino Acid ◽  
Metal Binding ◽  
Granule Cells ◽  
Molecular Layer ◽  
Synaptic Function ◽  
Differentially Expressed ◽  
Predicted Amino Acid Sequence ◽  
Cdna Clones ◽  
Thalamic Nuclei ◽  
Electron Microscopic

cDNA clones of a neuronal-specific mRNA encoding a novel 25-kD synaptosomal protein, SNAP-25, that is widely, but differentially expressed by diverse neuronal subpopulations of the mammalian nervous system have been isolated and characterized. The sequence of the SNAP-25 cDNA revealed a single open reading frame that encodes a primary translation product of 206 amino acids. Antisera elicited against a 12-amino acid peptide, corresponding to the carboxy-terminal residues of the predicted polypeptide sequence, recognized a single 25-kD protein that is associated with synaptosomal fractions of hippocampal preparations. The SNAP-25 polypeptide remains associated with synaptosomal membrane components after hypoosmotic lysis and is released by nonionic detergent but not high salt extraction. Although the SNAP-25 polypeptide lacks a hydrophobic stretch of residues compatible with a transmembrane region, the amino terminus may form an amphiphilic helix that may facilitate alignment with membranes. The predicted amino acid sequence also includes a cluster of four closely spaced cysteine residues, similar to the metal binding domains of some metalloproteins, suggesting that the SNAP-25 polypeptide may have the potential to coordinately bind metal ions. Consistent with the protein fractionation, light and electron microscopic immunocytochemistry indicated that SNAP-25 is located within the presynaptic terminals of hippocampal mossy fibers and the inner molecular layer of the dentate gyrus. The mRNA was found to be enriched within neurons of the neocortex, hippocampus, piriform cortex, anterior thalamic nuclei, pontine nuclei, and granule cells of the cerebellum. The distribution of the SNAP-25 mRNA and the association of the protein with presynaptic elements suggest that SNAP-25 may play an important role in the synaptic function of specific neuronal systems.

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