Expression and potential role of Fsrg1, a murine bromodomain-containing homologue of the Drosophila gene female sterile homeotic

1998 ◽  
Vol 111 (23) ◽  
pp. 3541-3550 ◽  
Author(s):  
K. Rhee ◽  
M. Brunori ◽  
V. Besset ◽  
R. Trousdale ◽  
D.J. Wolgemuth
Keyword(s):  
Cultured Cells ◽  
Polyclonal Antibodies ◽  
Blot Hybridization ◽  
Immunoblot Analysis ◽  
Hybridization Analysis ◽  
Relative Molecular Mass ◽  
Northern Blot Hybridization ◽  
Specific Substrate ◽  
Drosophila Gene

We have isolated a cDNA which is a murine homologue of the Drosophila gene female sterile homeotic (fsh). This homologue, which we have designated Fsrg1*, contains two bromodomains and an ET motif characteristic of the Fsh sub-class of bromodomain-containing proteins. Northern blot hybridization analysis of adult tissues revealed that Fsrg1 was expressed at low levels rather ubiquitously, but most abundantly in the testis and ovary. Polyclonal antibodies raised against an Fsrg1 fusion protein were used to characterize the Fsrg1 gene product in tissues. Constructs were also generated in which the Fsrg1 cDNA was tagged with epitopes for hemaglutinin and used in transfection experiments. Immunoblot analysis revealed that the Fsrg1 protein migrates with a relative molecular mass of approximately 110 kDa, although the cDNA sequence would predict a protein of approximately 88 kDa. The migration at approximately 110 kDa was observed for both in vivo protein and protein produced in cultured cells. The Fsrg1 protein was localized to the nucleus when expressed in cultured cells, consistent with the presence of a nuclear localization signal motif in the Fsrg1 sequence. No kinase activity was detected for this nuclear protein as assessed in either autokinase or specific substrate assays. In situ hybridization analysis revealed strikingly high expression of Fsrg1 in granulosa cells of growing follicles in the adult ovary and suggested its possible involvement in folliculogenesis. Additional clues to its potential function were provided by the demonstration of its high level of expression in epithelia of tissues which undergo hormonally-modulated remodeling.

scholarly journals Deletion of the anion exchanger Slc26a4 (pendrin) decreases apical Cl−/HCO3− exchanger activity and impairs bicarbonate secretion in kidney collecting duct

2010 ◽  
Vol 299 (1) ◽  
pp. C33-C41 ◽  
Author(s):  
Hassane Amlal ◽  
Snezana Petrovic ◽  
Jie Xu ◽  
Zhaohui Wang ◽  
Xuming Sun ◽  
...  
Keyword(s):  
Anion Exchanger ◽  
Ph Monitoring ◽  
Collecting Duct ◽  
Blot Hybridization ◽  
Elevated Serum ◽  
Northern Blot Hybridization ◽  
Ammonium Excretion ◽  
Bicarbonate Secretion ◽  
Immunofluorescence Labeling

The anion exchanger Pendrin, which is encoded by SLC26A4 (human)/Slc26a4 (mouse) gene, is localized on the apical membrane of non-acid-secreting intercalated (IC) cells in the kidney cortical collecting duct (CCD). To examine its role in the mediation of bicarbonate secretion in vivo and the apical Cl−/HCO3− exchanger in the kidney CCD, mice with genetic deletion of pendrin were generated. The mutant mice show the complete absence of pendrin expression in their kidneys as assessed by Northern blot hybridization, Western blot, and immunofluorescence labeling. Pendrin knockout (KO) mice display significantly acidic urine at baseline [pH 5.20 in KO vs. 6.01 in wild type (WT); P < 0.0001] along with elevated serum HCO3− concentration (27.4 vs. 24 meq/l in KO vs. WT, respectively; P < 0.02), consistent with decreased bicarbonate secretion in vivo. The urine chloride excretion was comparable in WT and KO mice. For functional studies, CCDs were microperfused and IC cells were identified by their ability to trap the pH fluorescent dye BCECF. The apical Cl−/HCO3− exchanger activity in B-IC and non-A, non-B-IC cells, as assessed by intracellular pH monitoring, was significantly reduced in pendrin-null mice. The basolateral Cl−/HCO3− exchanger activity in A-IC cells and in non-A, non-B-IC cells, was not different in pendrin KO mice relative to WT animals. Urine NH4+ (ammonium) excretion increased significantly, consistent with increased trapping of NH3 in the collecting duct in pendrin KO mice. We conclude that Slc26a4 (pendrin) deletion impairs the secretion of bicarbonate in vivo and reduces apical Cl−/HCO3− exchanger activity in B-IC and non-A, non-B-IC cells in CCD. Additional apical Cl−/HCO3− exchanger(s) is (are) present in the CCD.

Comparison of human and porcine group C rotaviruses by Northern blot hybridization analysis

1991 ◽  
Vol 118 (3-4) ◽  
pp. 269-277 ◽  
Author(s):  
Y. Qian ◽  
L. J. Saif ◽  
A. Z. Kapikian ◽  
S. Y. Kang ◽  
B. Jiang ◽  
...  
Keyword(s):  
Northern Blot ◽  
Blot Hybridization ◽  
Hybridization Analysis ◽  
Northern Blot Hybridization

Localization of Z-RNA in Normal Lens Epithelium: Middle Fibers

2000 ◽  
Vol 6 (S2) ◽  
pp. 900-901
Author(s):  
C.E. Gagna ◽  
H.R. Kuo ◽  
R. Schulz ◽  
R. Cordova ◽  
G. Crippen ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Cellular Localization ◽  
Cultured Cells ◽  
Polyclonal Antibodies ◽  
Image Analyzer ◽  
Left Handed ◽  
Z Dna ◽  
Normal Lens

The goal of this project was to analyze the cellular localization of Z-RNA, within middle fibers (MF) of the adult dog ocular lens (1.5 yr) (Fig. 1), using anti-Z-RNA IgG polyclonal antibodies. B-DNA can adopt the left-handed Z-DNA conformation in vitro (1). Right-handed A-RNA can be transformed into left-handed Z-RNA (2). Z-RNA has been studied in cultured cells (3). Evidence supports the presence of Z-DNA in vivo (1). Removal of DNA binding proteins by fixatives can initiate supercoiling which stabilizes Z-DNA sequences (1).Anti-Z-RNA polyclonal antibody probes were developed in rabbits immunized with multiple injections of Z-RNA: Br-poly[ribosomal(G-C)]. Regarding immunohistochemistry, lens tissues were fixed in Carnoy's, embedded in paraffin and sectioned (2.5 μm) (Fig. 2). Computerized image analysis was performed using a Leitz DM-RB microscope and Leica Quantiment 500 + image analyzer.

Fgf-4 expression during gastrulation, myogenesis, limb and tooth development in the mouse

Development ◽  
1992 ◽  
Vol 114 (3) ◽  
pp. 755-768 ◽  
Author(s):  
L. Niswander ◽  
G.R. Martin
Keyword(s):  
Tooth Development ◽  
Inner Cell Mass ◽  
Expression Patterns ◽  
Blot Hybridization ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Hybridization Analysis ◽  
Limb Bud ◽  
Northern Blot Hybridization ◽  
Tail Bud

Fgf-4, initially isolated as a transforming gene from human tumors, is a member of the Fibroblast Growth Factor (FGF) family. It has previously been shown by northern blot hybridization analysis to be expressed in teratocarcinoma and embryonic stem cells, suggesting that it plays a role in embryonic development. We have carried out an RNA in situ hybridization analysis of Fgf-4 expression in the developing mouse embryo, from fertilization through the 14th day of gestation (E14.5). Our results show that Fgf-4 RNA is first detected at the late blastocyst stage in cells that give rise to all of the embryonic lineages (inner cell mass cells). During the early stages of gastrulation, expression becomes restricted to the primitive streak where mesoderm and definitive endoderm are formed. Expression continues in the distal (rostral) two-thirds of the streak through approx. E10, and then is detected in the tail bud, which replaces the streak as the primary source of mesoderm. Additional sites of expression are found after the three primary germ layers are established and organogenesis begins. Fgf-4 RNA is detected transiently in the branchial arch units, the somitic myotome, the apical ectodermal ridge of the developing limb bud and the tooth bud, suggesting that the gene has multiple roles during embryogenesis. These results are compared with the expression patterns of other FGF genes. Taken together, the data suggest that individual members of the gene family are expressed sequentially in developmental pathways such as mesoderm formation and myogenesis, and play a role in specific epithelial-mesenchymal interactions.

Identification of mRNAs encoding low molecular mass heat-shock proteins in maize (Zea mays L.)

1986 ◽  
Vol 28 (6) ◽  
pp. 1106-1114 ◽  
Author(s):  
C. A. B. Rees ◽  
N. C. Hogan ◽  
D. B. Walden ◽  
B. G. Atkinson
Keyword(s):  
Heat Shock ◽  
Heat Shock Proteins ◽  
Molecular Mass ◽  
Polyclonal Antibodies ◽  
Relative Molecular Mass ◽  
Temperature Heat ◽  
Free Translation ◽  
Shock Proteins

Subjecting 5-day-old maize seedlings to a rapid elevation in growth temperature (heat shock; 25–42 °C) results in a shift in the pattern of protein synthesis in maize plumules from the production of a broad spectrum of proteins to the new and (or) enhanced synthesis of a small number of heat-shock proteins (HSPs). The low relative molecular mass (Mr) HSPs, and more specifically an 18-kDa HSP with four major isoelectric variants, represent the majority of HSP synthesis following cell-free translation of total cellular poly (A)+ RNAs and polyribosomal RNAs extracted from heat-shocked plumules. Immunochemical studies, using polyclonal antibodies raised against the 18-kDa HSPs, show that the 18-kDa HSPs synthesized in vitro share immunochemical properties with HSPs of the same Mr synthesized in vivo by heat-shocked plumules. Furthermore, size fractionation and translation analyses of total cellular poly(A)+ RNAs extracted from heat-shocked plumules demonstrate that poly(A)+ RNAs encoding an 18-kDa HSP(s) have an estimated size of 0.6–0.95 kilobases. The observation that 18-kDa HSPs are absent among the translation products and immunoprecipitates of proteins synthesized in vitro by RNAs extracted from control plumules (25 °C) suggests that the mRNAs encoding 18-kDa HSPs are heat-shock induced.Key words: mRNA, maize, heat shock.

Demonstration of Z-RNA in the Dog Eye Lens Epithelium (Germinative Zone)

1998 ◽  
Vol 4 (S2) ◽  
pp. 1108-1109
Author(s):  
C.E. Gagna ◽  
J.H. Chen ◽  
H.R. Kuo ◽  
W.C. Lambert
Keyword(s):  
Cellular Localization ◽  
Cultured Cells ◽  
Polyclonal Antibodies ◽  
Phosphate Buffer Solution ◽  
Buffer Solution ◽  
Solution Ph ◽  
Immunogold Staining ◽  
Z Dna

The purpose of this scientific investigation was to determine the presence and specific cellular localization of left-handed Z-RNA, within germinative zone (GZ) epithelium of the lens (Fig. 1), using anti-Z-RNA IgG polyclonal antibodies. Right-handed B-DNA has the ability to adopt the Z-DNA conformation in vitro (Sinden, 1994). Right-handed A-RNA can be transformed into Z-RNA under specific conditions (Hall et al., 1984), and Z-RNA has been identified in cultured cells (Zarling et al., 1990). Strong evidence supports the idea of Z-DNA in vivo (Sinden, 1994). Removal of proteins by fixatives can induce supercoiling which stabilizes Z-DNA (Sinden, 1994).Anti-Z-RNA antibodies were produced in rabbits immunized with injections of Z-RNA: brominated-poly[ribosomal(G-C)]. For light microscopy, immunohistochemical studies (ABC method), normal dog lens tissues (1 yr old) were fixed in Carnoy's, embedded in paraffin and sectioned 2 μm thick. For electron microscopy (immunogold staining), pieces of epithelium from the GZ of normal dog lens (1 yr old) were fixed with 5% glutaraldehyde in 0.05 M phosphate buffer solution, pH 7.3.

scholarly journals Characterization of Bacteroides fragilis Hemolysins and Regulation and Synergistic Interactions of HlyA and HlyB

2006 ◽  
Vol 74 (4) ◽  
pp. 2304-2316 ◽  
Author(s):  
Kirstin P. Robertson ◽  
C. Jeffrey Smith ◽  
Andrea M. Gough ◽  
Edson R. Rocha
Keyword(s):  
Hemolytic Activity ◽  
Blot Hybridization ◽  
Bacteroides Fragilis ◽  
Blood Agar ◽  
Northern Blot Hybridization ◽  
Rich Medium ◽  
Independent Manner ◽  
Hemolytic Assay ◽  
Growth Deficiency

ABSTRACT This study describes the presence of 10 hemolysin orthologs in the genome of the opportunistic human anaerobic pathogen Bacteroides fragilis, which is currently classified as a nonhemolytic bacterium. The hemolysins were designated HlyA through HlyI plus HlyIII. All cloned hemolysin genes were able to confer hemolytic activity to a nonhemolytic Escherichia coli strain on blood agar plates. Interestingly, HlyH was found to be present in the genome of the B. fragilis NCTC9343 strain but absent in strains 638R, YCH46, and Bacteroides thetaiotaomicron VPI-5482. The hemolysins HlyA, HlyB, and HlyIII were selected for further characterization. HlyA, HlyB, and HlyIII were cytolytic to erythrocytes on liquid hemolytic assay. When hlyA and hlyB were expressed together in a nonhemolytic E. coli strain, the strain showed enhanced hemolytic activity on blood agar plates. Further analysis revealed that HlyA and HlyB have synergistic hemolytic activity as detected by the liquid hemolytic assay. In addition, the two-component hemolysins HlyA and HlyB form a protein-protein complex in vivo as determined by bacterial two-hybrid system assay. The hlyB and hlyA genes are organized in an operon that is coordinately regulated by iron and oxygen. Northern blot hybridization analysis revealed that hlyBA were expressed as a bicistronic mRNA induced approximately 2.5-fold under low-iron conditions and repressed in iron-rich medium. The normal iron-regulated expression of hlyBA mRNA was lost in the furA mutant strain. In contrast, the hlyA gene was also expressed as a single mRNA in iron-rich medium, but its expression was reduced approximately threefold under low-iron conditions in a Fur-independent manner. This suggests that hlyA alone is regulated by an unidentified iron-dependent regulator. Moreover, the expression levels of hlyBA and hlyA were reduced about threefold following oxygen exposure and treatment with hydrogen peroxide. Taken together, these results suggest that iron and oxidative stress have an effect on the control of hlyBA and hlyA transcriptional levels. A hlyBA mutant was constructed, and its hemolytic activity was greatly diminished compared to those of the hlyIII mutant and parent strains. In addition, the hlyBA mutant had a significant modification in colony morphology and growth deficiency compared to the parent strain. The implications of these findings for the pathophysiology of B. fragilis in extraintestinal infections and competition in ecological systems for this organism are discussed.

scholarly journals Improving Crustacean Aquaculture Production Efficiencies through Development of Monosex Populations Using Endocrine and Molecular Manipulations

2010 ◽  
Author(s):  
Abigail Elizur ◽  
Amir Sagi ◽  
Gideon Hulata ◽  
Clive Jones ◽  
Wayne Knibb
Keyword(s):  
Cdna Library ◽  
Developmental Stages ◽  
Blot Hybridization ◽  
Repeated Administration ◽  
Sex Reversal ◽  
Northern Blot Hybridization ◽  
Female Population ◽  
Original Proposal ◽  
Aquaculture Production

Background Most of Australian prawn aquaculture production is based on P. monodon. However, the Australian industry is under intense competition from lower priced overseas imports. The availability of all-female monosex populations, by virtue of their large size and associated premium prize, will offer competitive advantage to the industry which desperately needs to counteract competitors within this market. As for the redclaw production in Israel, although it is at its infancy, the growers realized that the production of males is extremely advantageous and that such management strategy will change the economic assumptions and performances of this aquaculture to attract many more growers. Original objectives (as in original proposal) Investigating the sex inheritance mechanism in the tiger prawn. Identification of genes expressed uniquely in the androgenic gland (AG) of prawns and crayfish. The above genes and/or their products will be used to localize the AG in the prawn and manipulate the AG activity in both species. Production of monosex populations through AG manipulation. In the prawn, production of all-female populations and in the crayfish, all-male populations. Achievements In the crayfish, the AG cDNA library was further screened and a third AG specific transcript, designated Cq-AG3, had been identified. Simultaneously the two AG specific genes, which were previously identified, were further characterized. Tissue specificity of one of those genes, termed Cq-AG2, was demonstrated by northern blot hybridization and RNA in-situ hybridization. Bioinformatics prediction, which suggested a 42 amino acid long signal anchor at the N-terminus of the deduced Cq-AG2, was confirmed by immunolocalization of a recombinant protein. Cq-IAG's functionality was demonstrated by dsRNA in-vivo injections to intersex crayfish. Cq-IAGsilencing induced dramatic sex-related alterations, including male feature feminization, reduced sperm production, extensive testicular apoptosis, induction of the vitellogeningene expression and accumulation of yolk proteins in the ovaries. In the prawn, the AG was identified and a cDNA library was created. The putative P. monodonAG hormone encoding gene (Pm-IAG) was identified, isolated and characterized for time of expression and histological localization. Implantation of the AG into prawn post larvae (PL) and juveniles resulted in phenotypic transformation which included the appearance of appendix masculina and enlarged petasma. The transformation however did not result in sex change or the creation of neo males thus the population genetics stage to be executed with Prof. Hulata did not materialized. Repeated AG implantation is currently being trialed. Major conclusions and Implications, both scientific and agricultural Cq-IAG's involvement in male sexual differentiation had been demonstrated and it is strongly suggested that this gene encodes an AG hormone in this crayfish. A thorough screening of the AG cDNA library shows Cq-IAG is the prominent transcript within the library. However, the identification of two additional transcripts hints that Cq-IAG is not the only gene mediating the AG effects. The successful gene silencing of Cq-IAG, if performed at earlier developmental stages, might accomplish full and functional sex reversal which will enable the production of all-male crayfish populations. Pm-IAG is likely to play a similar role in prawns. It is possible that repeated administration of the AG into prawn will lead to the desired full sex reversal, so that WZ neo males, crossed with WZ females can result in WW females, which will form the basis for monosex all-female population.  

Repeat-induced point mutations of HSP80 gene of Neurospora crassa: methylation of duplicated DNA sequences in the vegetative state

1996 ◽  
Vol 74 (1) ◽  
pp. 41-50 ◽  
Author(s):  
Y. Vijayaraghavan ◽  
M. Kapoor
Keyword(s):  
Neurospora Crassa ◽  
Dna Sequences ◽  
Gene Sequence ◽  
Vegetative State ◽  
Blot Hybridization ◽  
Point Mutations ◽  
Immunoblot Analysis ◽  
Gene Copy ◽  
Northern Blot Hybridization ◽  
Inducible Protein

The process of repeat-induced point mutations (RIP) was used to disrupt the gene encoding the 80-kDa heat-inducible protein of Neurospora crassa. Germinated conidia of the wild-type recipient strain were electrotransformed with a plasmid containing a 7-kb fragment harbouring the complete hsp80 gene sequence. Some of the transformants with a duplication of hsp80 gene sequence showed extensive methylation of these sequences even in vegetatively growing cells. The presence of an extra gene copy in transformants of this type resulted in a marked reduction in the expression of this gene. Progeny of a cross of one such transformant, showing methylation of hsp80, was analyzed by Southern blot and Northern blot hybridization to examine the relationship between methylation and the accumulation of hsp80 mRNA under hyperthermia. In addition, HSP80 polypeptide levels were monitored in stressed and unstressed cells by immunoblot analysis using polyclonal anti-HSP80 IgG preparations. A correlation between the extent of RIP and expression of this gene was observed in the progeny isolates.Key words: 80-kDa heat shock protein, RIP, DNA methylation, expression, HSP.

DNA Polymerase-Associated Lectin (DPAL) and Its Binding to the Galactose-Containing Glycoconjugate of the Replication Complex

1999 ◽  
Vol 19 (5) ◽  
pp. 433-447
Author(s):  
Thomas J. Kelley ◽  
Tara St. Amand ◽  
Jeremy M. Groll ◽  
Satyajit Ray ◽  
Subhash Basu
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibody ◽  
Dna Polymerase ◽  
Recombinant Dna ◽  
Cultured Cells ◽  
Polyclonal Antibodies ◽  
Immunoblot Analysis ◽  
Post Translational Modification ◽  
Human Neuroblastoma ◽  
Dna Polymerase Α

The highly purified DNA Pol-α from rat prostate tumor (PA-3) and human neuroblastoma (IMR-32) cells appeared to be inhibited by Ricin (RCA-II), and Con-A. Loss of activity (40 to 60%) of a specific form of DNA polymerase from IMR-32 was observed when the cells were treated with tunicamycin [Bhattacharya, P. and Basu, S. (1982) Proc. Natl. Acad. Sci., USA79:1488–1492]. Binding of ConA and RCA to human recombinant DNA polymerase-α showed a specific labile site in the N-terminus [Hsi et al.. (1990) Nucleic Acid Res.18:6231–6237]. The catalytic polypeptide, DNA polymerase-α of eukaryotic origin, was isolated from developing tissues or cultured cells as a family of 180 to 120 kDa polypeptides, perhaps derived from a single primary structure. Immunoblot analysis with a monoclonal antibody (SJK-237-71) indicated that the lower molecular weight polypeptides resulted from either proteolytic cleavage of post-translational modification after specific cleavages. Present results suggest DNA polymerase-α from embryonic chicken brain (ECB) contains an α-galactose-binding subunit which may be involved in developmental regulation of the enzyme. It was shown before that the catalytic subunit of DNA polymerase-α reduces from 186 kDa in 11-day-old ECB to 120 kDa in 19-day-old ECB [Ray, S. et al. Cell Growth and Differentiation2:567–573] by the treatment with methyl-α-galactose. The low molecular weight DNA polymerase activity (120 kDa) can be reconstituted to high molecular weight (Mr = 186 kDa) with an α-galactose binding, 56 kDa lectin-like protein. Polyclonal antibodies raised against the purified lectin were able to precipitate DNA. Pol-α as determined by immunostaining with the polymerase-α-specific monoclonal antibody SJK 132-20, suggesting this is a DNA polymerase associated-lectin (DPAL). RCA-II and GS-I-Sepharose 4B chromatographies resulted in significant purification of DNA-α and a complete separation of polymerase complex and primase.

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