Isolation of carp cDNA clones, representing developmentally-regulated genes, using a subtractive-hybridization strategy

1996 ◽  
Vol 205 (7-8) ◽  
pp. 460-467 ◽  
Author(s):  
C. J. M. Stevens ◽  
G. Kronnie ◽  
J. Samallo ◽  
H. Schipper ◽  
H. W. J. Stroband
Keyword(s):  
Subtractive Hybridization ◽  
Cdna Clones ◽  
Developmentally Regulated

Characterization of colorectal-cancer-related cDNA clones obtained by subtractive hybridization screening

1997 ◽  
Vol 123 (8) ◽  
pp. 447-451 ◽  
Author(s):  
Jiang Cao ◽  
Xinhan Cai ◽  
Lei Zheng ◽  
Liyi Geng ◽  
Zhengzheng Shi ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Subtractive Hybridization ◽  
Cdna Clones

Isolation of transcripts overexpressed in the human pathogenTrichophyton rubrumgrown in lipid as carbon source

2011 ◽  
Vol 57 (4) ◽  
pp. 333-338 ◽  
Author(s):  
Fernanda C.A. Maranhão ◽  
Henrique C.S. Silveira ◽  
Antonio Rossi ◽  
Nilce M. Martinez-Rossi
Keyword(s):  
Carbon Source ◽  
Trichophyton Rubrum ◽  
Cdna Array ◽  
Subtractive Hybridization ◽  
Cellular Responses ◽  
Cdna Clones ◽  
Medical Importance ◽  
Insight Into ◽  
Dot Blotting ◽  
Host Invasion

Trichophyton rubrum is the most common etiological agent of human dermatophytosis. Despite the incidence and medical importance of this dermatophyte, little is known about the mechanisms of host invasion and pathogenicity. Host invasion depends on the adaptive cellular responses of the pathogen that allow it to penetrate the skin layers, which are mainly composed of proteins and lipids. In this study, we used suppression subtractive hybridization to identify transcripts overexpressed in T. rubrum cultured in lipid as carbon source. Among the subtractive cDNA clones isolated, 85 clones were positively screened by cDNA array dot blotting and were sequenced. The putative proteins encoded by the isolated transcripts showed similarities to fungal proteins involved in metabolism, signaling, defense, and virulence, such as the MDR/ABC transporter, glucan 1,3-β-glucosidase, chitin synthase B, copper-sulfate-regulated protein, and serine/threonine phosphatase (calcineurin A). These results provide the first molecular insight into the genes differentially expressed during the adaptation of T. rubrum to a lipidic carbon source.

scholarly journals Microarray screening of suppression subtractive hybridization-PCR cDNA libraries identifies novel RNAs regulated by dehydration in the rat supraoptic nucleus

2006 ◽  
Vol 24 (2) ◽  
pp. 163-172 ◽  
Author(s):  
Mohamed T. Ghorbel ◽  
Greig Sharman ◽  
Charles Hindmarch ◽  
Kevin G. Becker ◽  
Tanya Barrett ◽  
...  
Keyword(s):  
General Circulation ◽  
Supraoptic Nucleus ◽  
Subtractive Hybridization ◽  
Biologically Active ◽  
Differentially Expressed ◽  
Cdna Libraries ◽  
Suppressive Subtractive Hybridization ◽  
Cdna Clones ◽  
Microarray Screening ◽  
Mechanistic Basis

The magnocellular neurons (MCNs) of the supraoptic nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus are the principal site of biosynthesis of prepropeptide precursor of the antidiuretic hormone vasopressin (VP). This precursor is processed during anterograde axonal transportation to terminals in the posterior pituitary gland, where biologically active VP is stored until release into the general circulation in response to physiological activation of the SON by osmotic cues. By binding to V2-type receptors located in the kidney, VP decreases the amount of water lost in urine. Osmotic activation of the SON is accompanied by a dramatic morphological and functional remodeling. We have sought to understand the mechanistic basis of this plasticity in terms of the differential expression of genes. To identify such genes, we adopted an unbiased global approach based on suppressive subtractive hybridization-polymerase chain reaction (SSH-PCR) Using this method, we generated libraries of clones putatively differentially expressed in control vs. dehydrated SON. To rapidly screen these libraries, 1,152 clones were subjected to microarray analysis, resulting in the identification of 459 differentially expressed transcripts. cDNA clones corresponding to 56 of these RNAs were sequenced, revealing many of them to be novel expressed sequence tags (ESTs). Four transcripts were shown by in situ hybridization (ISH) to be significantly up- or downregulated in the SON after dehydration. These genes may represent novel effectors or mediators of SON physiological remodeling.

Characterization of colorectal-cancer-related cDNA clones obtained by subtractive hybridization screening

1997 ◽  
Vol 123 (8) ◽  
pp. 447-451 ◽  
Author(s):  
Jiang Cao ◽  
Xinhan Cai ◽  
Lei Zheng ◽  
Liyi Geng ◽  
Zhengzheng Shi ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Subtractive Hybridization ◽  
Cdna Clones

scholarly journals Two muscle-specific LIM proteins in Drosophila.

1996 ◽  
Vol 134 (5) ◽  
pp. 1179-1195 ◽  
Author(s):  
B E Stronach ◽  
S E Siegrist ◽  
M C Beckerle
Keyword(s):  
Gene Expression ◽  
Muscle Fibers ◽  
Binding Motif ◽  
Cdna Clones ◽  
Lim Domain ◽  
Developmentally Regulated ◽  
Pharyngeal Muscles ◽  
Regulate Cell Growth ◽  
Lim Proteins ◽  
Muscle Lim Proteins

The LIM domain defines a zinc-binding motif found in a growing number of eukaryotic proteins that regulate cell growth and differentiation during development. Members of the cysteine-rich protein (CRP) family of LIM proteins have been implicated in muscle differentiation in vertebrates. Here we report the identification and characterization of cDNA clones encoding two members of the CRP family in Drosophila, referred to as muscle LIM proteins (Mlp). Mlp60A encodes a protein with a single LIM domain linked to a glycine-rich region. Mlp84B encodes a protein with five tandem LIM-glycine modules. In the embryo, Mlp gene expression is spatially restricted to somatic, visceral, and pharyngeal muscles. Within the somatic musculature, Mlp84B transcripts are enriched at the terminal ends of muscle fibers, whereas Mlp60A transcripts are found throughout the muscle fibers. The distributions of the Mlp60A and Mlp84B proteins mirror their respective mRNA localizations, with Mlp84B enrichment occurring at sites of muscle attachment. Northern blot analysis revealed that Mlp gene expression is developmentally regulated, showing a biphasic pattern over the course of the Drosophila life cycle. Peaks of expression occur late in embryogenesis and during metamorphosis, when the musculature is differentiating. Drosophila Mlp60A and Mlp84B, like vertebrate members of the CRP family, have the ability to associate with the actin cytoskeleton when expressed in rat fibroblast cells. The temporal expression and spatial distribution of muscle LIM proteins in Drosophila are consistent with a role for Mlps in myogenesis, late in the differentiation pathway.

scholarly journals Structure of transcripts from the homeotic Antennapedia gene of Drosophila melanogaster: two promoters control the major protein-coding region.

1986 ◽  
Vol 6 (12) ◽  
pp. 4676-4689 ◽  
Author(s):  
A Laughon ◽  
A M Boulet ◽  
J R Bermingham ◽  
R A Laymon ◽  
M P Scott
Keyword(s):  
Drosophila Melanogaster ◽  
Alternative Polyadenylation ◽  
Transcription Unit ◽  
Cdna Clones ◽  
Major Protein ◽  
Coding Region ◽  
Developmentally Regulated ◽  
Protein Coding ◽  
Reading Frame ◽  
C Terminus

The Antennapedia (Antp) homeotic gene of Drosophila melanogaster regulates segmental identity in the thorax. Loss of Antp function results in altered development of the embryonic thoracic segments or can cause legs to be transformed into antennae. Certain combinations of Antp recessive lethal alleles complement to permit normal development. The structure of the Antp gene, analyzed by sequencing cDNA clones and exons and by transcript mapping, revealed some of the basis for its genetic complexity. It has two promoters governing two nested transcription units, one unit 36 and one 103 kilobase pairs (kb) long. Both units incorporated the same protein-coding exons, all of which are located in the 3'-most 13 kb of the gene. The two promoters resulted in the attachment of either of two long noncoding leader sequences (1.5 and 1.7 kb) to a 1.1-kb open reading frame. Both transcription units used the same pair of alternative polyadenylation sites 1.4 kb apart; the choice of sites was developmentally regulated. Some of the mutations that disrupt the larger transcription unit complemented a mutation affecting the smaller one. Dominant mutations that transform antennae into legs split the gene but left the coding exons intact. The encoded protein has unusually long runs of glutamine and a homeodomain near the C terminus.

scholarly journals Identification of developmentally regulated mesodermal-specific transcript in mouse embryonic metanephros

2002 ◽  
Vol 282 (5) ◽  
pp. F953-F965 ◽  
Author(s):  
Yashpal S. Kanwar ◽  
Anil Kumar ◽  
Kosuke Ota ◽  
Sun Lin ◽  
Jun Wada ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Mesenchymal Cell ◽  
Subtractive Hybridization ◽  
Antisense Oligodeoxynucleotide ◽  
Postnatal Life ◽  
Metanephric Mesenchyme ◽  
Developmentally Regulated ◽  
Newborn Mice ◽  
Gene Treatment ◽  
Dose Dependent Decrease

Mesodermal-specific cDNA or transcript (MEST) was identified by suppression subtractive hybridization-PCR of cDNA isolated from embryonic day 13vs. newborn mice kidneys. At day 13 of mouse gestation, a high expression of MEST, with a single ∼2.7-kb transcript that was exclusively localized to the metanephric mesenchyme was observed. The MEST mRNA expression gradually decreased during the later stages and then abruptly decreased in the newborn kidneys and subsequent postnatal life, after which a very mild expression persisted in the glomerular mesangium. Regression in mRNA expression during embryonic renal development appears to be related to methylation of the MEST gene. Treatment of metanephroi, harvested at day 13 of gestation with MEST-specific antisense oligodeoxynucleotide resulted in a dose-dependent decrease in the size of the explants and the nephron population. This was associated with a selective decrease in MEST mRNA expression and accelerated apoptosis of the mesenchyme. These findings suggest that MEST, a gene with a putative mesenchymal cell-derived protein, conceivably plays a role in mammalian metanephric development.

Role of protein synthesis in decay and accumulation of mRNA during spore germination in the cellular slime mold Dictyostelium discoideum

1987 ◽  
Vol 7 (2) ◽  
pp. 799-805
Author(s):  
R Kelly ◽  
D R Shaw ◽  
H L Ennis
Keyword(s):  
Protein Synthesis ◽  
Dictyostelium Discoideum ◽  
Spore Germination ◽  
Mrna Decay ◽  
Protein Synthesis Inhibitor ◽  
Cdna Clones ◽  
Cellular Slime Mold ◽  
Suitable Model ◽  
Mrna Synthesis ◽  
Developmentally Regulated

Spore germination in Dictyostelium discoideum is a particularly suitable model for studying the regulation of gene expression, since developmentally regulated changes in both protein and mRNA synthesis occur during the transition from dormant spore to amoeba. The previous isolation of three cDNA clones specific for mRNA developmentally regulated during spore germination allowed for the quantitation of the specific mRNAs during this process. The three mRNAs specific to clones pLK109, pLK229, and pRK270 have half-lives much shorter (minutes) than those of constitutive mRNAs (hours). Using spore germination as a model, we studied the roles of ribosome-mRNA interactions and protein synthesis in mRNA degradation by using antibiotics that inhibit specific reactions in protein biosynthesis. Cycloheximide inhibits the elongation step of protein synthesis. Polysomes accumulate in inhibited cells because ribosomes do not terminate normally and new ribosomes enter the polysome, eventually saturating the mRNA. Pactamycin inhibits initiation, and consequently polysomes break down in the presence of this drug. Under this condition, the mRNA is essentially free of ribosomes. pLK109, pLK229, and pRK270 mRNAs were stabilized in the presence of cycloheximide, but pactamycin had no effect on their normal decay. Since it seems likely that stability of mRNA reflects the availability of sites for inactivation by nucleases, it follows that in the presence of cycloheximide, these sites are protected, presumably by occupancy by ribosomes. No ribosomes are bound to mRNA in the presence of pactamycin, and therefore mRNA degrades at about the normal rate. The data further indicate that a labile protein is probably not involved in mRNA decay or stabilization, since protein synthesis is inhibited equally by both antibiotics. We conclude that it may be important to use more than one type of protein synthesis inhibitor to evaluate whether protein synthesis is required for mRNA decay. The effect of protein synthesis inhibition on mRNA synthesis and accumulation was also studied. mRNA synthesis continues in the presence of inhibitors, albeit at a diminished rate relative to that of the uninhibited control.

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