title: A G Protein-Coupled Receptor with a Lipid Kinase Domain Is Involved in Cell-Density Sensing.

status: Public on May 16 2007

city: Cologne

first: Ludwig

last: Eichinger

address: Joseph-Stelzmann-Strasse 52

institute: Institute for Biochemistry 1

summary:
One mechanism multicellular structures use for controlling cell number [1, 2] involves the secretion and sensing of a factor, such as leptin [3] or myostatin [4], in mammals. Dictyostelium cells secrete autocrine factors for sensing cell density prior to aggregation and multicellular development [5, 6] such as CMF (conditioned-medium factor), which enables starving cells to respond to cAMP pulses [7, 8, 9]. Its actions are mediated by two receptors. CMFR1 activates a G protein-independent signaling pathway regulating gene expression [10]. An unknown Gα1-dependent receptor activates phospholipase C (PLC), which regulates the lifetime of Gα2-GTP [11, 12, 13]. Here, we describe RpkA, an unusual seven-transmembrane receptor that is fused to a C-terminal PIP5 kinase domain and that localizes in membranes of a late endosomal compartment. Loss of RpkA resulted in formation of persistent loose aggregates and altered expression of cAMP-regulated genes. The developmental defect can be rescued by full-length RpkA and the transmembrane domain only. The PIP5 kinase domain is dispensable for the developmental role of RpkA. rpkA− cells secrete and bind CMF but are unable to induce downstream responses. Inactivation of Gα1, a negative regulator of CMF signaling, rescued the developmental defect of the rpkA− cells, suggesting that RpkA actions are mediated by Gα1.
Keywords: Comparison of wild type AX2 cells with rpkA minus cells

overall_design:
Microarray analysis for the rpkA- mutant.

Developmental
stage      Microarray	Wild type	     Mutant
		             Total RNA     dye	  Total RNA     dye
T0	12874460	Culture A      Cy3	  Culture A        Cy5
	12945001	Culture B      Cy3	  Culture B        Cy5
	12874461	Culture A      Cy5	  Culture A        Cy3
	12945002	Culture B      Cy5	  Culture B        Cy3
T8	12882232	Culture A      Cy3	  Culture A        Cy5
	12953218	Culture B      Cy3	  Culture B        Cy5
	12882233	Culture A      Cy5	  Culture A        Cy3
	12953220	Culture B      Cy5	  Culture B        Cy3

AX2 cells and rpkA- cells were starved (5 × 10E6 cells/cm2) on phosphate agar plates and total RNA was prepared at 0 and 8 hrs of development. cDNA was prepared from total RNA (10 ug) using the Fairplay Kit (Stratagene Europe, Amsterdam, The Netherlands) as described in the manufacturers protocol. For each microarray experiment, an experimental (mutant, rpkA-) sample was labeled with Cy5 or Cy3 and the reference (wild type, AX2) sample with Cy3 or Cy5, respectively. Fluorescently labeled cDNA of the rpkA- mutant was generated and compared to labeled cDNA from AX2. Microarray production, expression profiling and data analysis were carried out as described in [Farbrother et al., Cell. Microbiology 2006, 3:438-456].

first: Deenadayalan

last: Bakthavatsalam


title: Aerobic and anaerobic transcriptional responses of wild type, hmp and norR to strains to NO in a chemostat

status: Public on Sep 01 2006

city: Sheffield

first: Robert

last: Poole

state: South Yorkshire

address: Firth Court, Western Bank

department: Molecular Biology & Biotechnology

country: United Kingdom

institute: University of Sheffield

summary: This SuperSeries is composed of the SubSeries listed below.

overall_design: Refer to individual Series


title: Comparison of NCTC 11168 isolate vs. genome-sequenced variant, microaerobic and anaerobic conditions

status: Public on Dec 31 2005

city: Stanford

last: Stanford Microarray Database

state: CA

address: 300 Pasteur Drive

department: Stanford University, School of Medicine

country: USA

institute: Stanford Microarray Database (SMD)

summary:
Transcriptional analysis (RNA hybridization) for 11168-G (genome sequenced) vs. 11168-O (original), cultured microaerobically or anaerobically (published as "severely O2-limited).  Published in Gaynor et al, J. Bacteriology 186:503-517
The genome sequence of the enteric bacterial pathogen Campylobacter jejuni NCTC 11168 (11168-GS) was published in 2000, providing a valuable resource for the identification of C. jejuni-specific colonization and virulence factors. Surprisingly, the 11168-GS clone was subsequently found to colonize 1-day-old chicks following oral challenge very poorly compared to other strains. In contrast, we have found that the original clinical isolate from which 11168-GS was derived, 11168-O, is an excellent colonizer of chicks. Other marked phenotypic differences were also identified: 11168-O invaded and translocated through tissue culture cells far more efficiently and rapidly than 11168-GS, was significantly more motile, and displayed a different morphology. Serotyping, multiple high-resolution molecular genotyping procedures, and subtractive hybridization did not yield observable genetic differences between the variants, suggesting that they are clonal. However, microarray transcriptional profiling of these strains under microaerobic and severely oxygen-limited conditions revealed dramatic expression differences for several gene families. Many of the differences were in respiration and metabolism genes and operons, suggesting that adaptation to different oxygen tensions may influence colonization potential. This correlates biologically with our observation that anaerobically priming 11168-GS or aerobically passaging 11168-O caused an increase or decrease, respectively, in colonization compared to the parent strain. Expression differences were also observed for several flagellar genes and other less well-characterized genes that may participate in motility. Targeted sequencing of the sigma factors revealed specific DNA differences undetected by the other genomic methods
An all pairs experiment design type is where all labeled extracts are compared to every other labeled extract.
Keywords: all_pairs

overall_design: Computed

first: Erin

last: Gaynor


title: Genomotyping of 11168-GS vs. 11168-O

summary:
DNA hybridizations to compare genomic content of 11168-gs (genome-sequenced) to 11168-o (original)
The genome sequence of the enteric bacterial pathogen Campylobacter jejuni NCTC 11168 (11168-GS) was published in 2000, providing a valuable resource for the identification of C. jejuni-specific colonization and virulence factors. Surprisingly, the 11168-GS clone was subsequently found to colonize 1-day-old chicks following oral challenge very poorly compared to other strains. In contrast, we have found that the original clinical isolate from which 11168-GS was derived, 11168-O, is an excellent colonizer of chicks. Other marked phenotypic differences were also identified: 11168-O invaded and translocated through tissue culture cells far more efficiently and rapidly than 11168-GS, was significantly more motile, and displayed a different morphology. Serotyping, multiple high-resolution molecular genotyping procedures, and subtractive hybridization did not yield observable genetic differences between the variants, suggesting that they are clonal. However, microarray transcriptional profiling of these strains under microaerobic and severely oxygen-limited conditions revealed dramatic expression differences for several gene families. Many of the differences were in respiration and metabolism genes and operons, suggesting that adaptation to different oxygen tensions may influence colonization potential. This correlates biologically with our observation that anaerobically priming 11168-GS or aerobically passaging 11168-O caused an increase or decrease, respectively, in colonization compared to the parent strain. Expression differences were also observed for several flagellar genes and other less well-characterized genes that may participate in motility. Targeted sequencing of the sigma factors revealed specific DNA differences undetected by the other genomic methods
An all pairs experiment design type is where all labeled extracts are compared to every other labeled extract.
Keywords: all_pairs


title: Comparison of the genome-sequenced variant of Campylobacter jejuni NCTC 11168 and the original clonal clinical isolate

type:
Expression profiling by array
Genome variation profiling by array

summary:
Abstract: The genome sequence of the enteric bacterial pathogen Campylobacter jejuni NCTC 11168 (11168-GS) was published in 2000, providing a valuable resource for the identification of C. jejuni-specific colonization and virulence factors. Surprisingly, the 11168-GS clone was subsequently found to colonize 1-day-old chicks following oral challenge very poorly compared to other strains. In contrast, we have found that the original clinical isolate from which 11168-GS was derived, 11168-O, is an excellent colonizer of chicks. Other marked phenotypic differences were also identified: 11168-O invaded and translocated through tissue culture cells far more efficiently and rapidly than 11168-GS, was significantly more motile, and displayed a different morphology. Serotyping, multiple high-resolution molecular genotyping procedures, and subtractive hybridization did not yield observable genetic differences between the variants, suggesting that they are clonal. However, microarray transcriptional profiling of these strains under microaerobic and severely oxygen-limited conditions revealed dramatic expression differences for several gene families. Many of the differences were in respiration and metabolism genes and operons, suggesting that adaptation to different oxygen tensions may influence colonization potential. This correlates biologically with our observation that anaerobically priming 11168-GS or aerobically passaging 11168-O caused an increase or decrease, respectively, in colonization compared to the parent strain. Expression differences were also observed for several flagellar genes and other less well-characterized genes that may participate in motility. Targeted sequencing of the sigma factors revealed specific DNA differences undetected by the other genomic methods

This SuperSeries is composed of the SubSeries listed below.


title: Microarray analysis of human spermatogenic dysfunction

status: Public on May 12 2006

city: 20246 Hamburg

first: Andrej-Nikolai

last: Spiess

address: Martinistr. 52

department: Andrology

country: Germany

institute: University Hospital Hamburg-Eppendorf

summary:
We investigated testicular biopsies from 28 men which presented with full spermatogenesis (Johnsen Score 10, 12 samples), arrest at the spermatid stage (Johnsen Score 8, 6 samples), arrest at spermatocyte stage (Johnsen Score 5, 5 samples) and Sertoli-cell-only syndrom (Johnsen Score 2, 5 samples). Testicular biopsies were obtained by the "sandwich" method and conserved in RNALater (Ambion) during surgery.

Keywords:
spermatogenesis
germ cell differentiation
Keywords: disease state analysis

overall_design: Total RNA was extracted from the biopsies by desintegration in RNAPure (Biozym) and post-cleanup with RNEasy columns (Qiagen). 2 ug of total RNA were labeled with the Amersham Codelink Expression Kit according to the manufacturers protocol, obtaining 40-80 ug of Biotin-labeled cRNA. 10 ug of fragmented cRNA were hybridized for 16h to Human 20K Codelink microarrays (Amersham), washed and stained with Cy5-Streptavidin according to manufacturers protocols and scanned with an Affymetrix 428 Array Scanner at gain 70. Spots were quantified by Codelink Expression software v4.0.

first: Caroline

last: Feig

first: Christiane

last: Kirchhoff

first: Richard

last: Ivell

first: Olaf

last: Naether

first: Wolfgang

last: Schulze


title: Yeast linker histone HHO1p effect on transcription before and after heat shock

status: Public on Jul 01 2008

city: Ramat Gan

first: Don

middle: Jay

last: Katcoff

address: Ramat Gan

department: Life Sciences

country: Israel

institute: Bar Ilan University

summary:
DNA array expression analysis comparing RNA transcripts in wild type and hho1 strains before and after a heat-shock from 25 to 37 degrees.
Keywords: expression, heat shock

overall_design: We used a reference design to hybridize three microarrays with the following cRNA samples labeled with Cy3 and Cy5, respectively: 1) wt25 and wt37, 2) mu37 and wt25, 3) mu25 and wt25. wt25, wt37, mu37 and mu25 represent cRNA made on RNA templates isolated from wild type cells grown at 25 degrees C, wild type cells following heat shock at 37 degrees C, hho1 delta cells following heat shock at 37 degrees C and hho1 delta cells grown at 25 degrees C, respectively.

middle: J

first: Mali

last: Salmon-Divon

first: Miri

last: Eyal