RNA Blots (Northern Blots)

2002 ◽  
Author(s):  
J. M. Gershoni
Keyword(s):  
Northern Blots ◽  
Rna Blots

scholarly journals Cloning and characterization of four SIR genes of Saccharomyces cerevisiae.

1986 ◽  
Vol 6 (2) ◽  
pp. 688-702 ◽  
Author(s):  
J M Ivy ◽  
A J Klar ◽  
J B Hicks
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mating Type ◽  
Blot Analysis ◽  
Transcriptional Control ◽  
Genomic Library ◽  
Yeast Saccharomyces Cerevisiae ◽  
Mating Type Genes ◽  
Rna Blots

Mating type in the yeast Saccharomyces cerevisiae is determined by the MAT (a or alpha) locus. HML and HMR, which usually contain copies of alpha and a mating type information, respectively, serve as donors in mating type interconversion and are under negative transcriptional control. Four trans-acting SIR (silent information regulator) loci are required for repression of transcription. A defect in any SIR gene results in expression of both HML and HMR. The four SIR genes were isolated from a genomic library by complementation of sir mutations in vivo. DNA blot analysis suggests that the four SIR genes share no sequence homology. RNA blots indicate that SIR2, SIR3, and SIR4 each encode one transcript and that SIR1 encodes two transcripts. Null mutations, made by replacement of the normal genomic allele with deletion-insertion mutations created in the cloned SIR genes, have a Sir- phenotype and are viable. Using the cloned genes, we showed that SIR3 at a high copy number is able to suppress mutations of SIR4. RNA blot analysis suggests that this suppression is not due to transcriptional regulation of SIR3 by SIR4; nor does any SIR4 gene transcriptionally regulate another SIR gene. Interestingly, a truncated SIR4 gene disrupts regulation of the silent mating type loci. We propose that interaction of at least the SIR3 and SIR4 gene products is involved in regulation of the silent mating type genes.

scholarly journals The genomic organization of the region containing the Drosophila melanogaster rpL7a (Surf-3) gene differs from those of the mammalian and avian Surfeit loci.

1995 ◽  
Vol 15 (5) ◽  
pp. 2367-2373 ◽  
Author(s):  
N Armes ◽  
M Fried
Keyword(s):  
Drosophila Melanogaster ◽  
Gene Cluster ◽  
Ribosomal Protein Gene ◽  
Single Copy ◽  
Gene Organization ◽  
Genomic Region ◽  
Divergent Evolution ◽  
Close Proximity ◽  
Rapid Amplification ◽  
Rna Blots

The Surf-3 gene of the unusually tight mouse Surfeit locus gene cluster has been identified as the highly conserved ribosomal protein gene L7a (rpL7a). The topography and juxtaposition of the Surfeit locus genes are conserved for the 600 million years of divergent evolution between mammals and birds. This suggests cis interaction and/or coregulation of the genes and suggests that, within this locus, gene organization plays an important role in gene expression. The further evolutionary conservation of the organization of the Surfeit locus was investigated. A cDNA encoding the Drosophila melanogaster homolog of the Surf-3/rpL7a gene was cloned, was shown to be present as a single copy, and was expressed constitutively at high levels throughout development. Genomic cosmid clones encompassing the gene and its surrounding DNA were isolated. The gene was determined to have five introns, of which two were located in the 5' untranslated region of the gene. The remaining three introns had splice sites at positions equivalent to those found in the Surf-3/rpL7a mammalian homologs. S1 analysis and 5' rapid amplification of cDNA ends both confirmed the start of transcription to occur in a polypyrimidine tract in the absence of a TATA box in the promoter. The genomic region around the Surf-3/rpL7a gene was analyzed by low-stringency hybridization with murine Surfeit gene probes, by partial sequence analysis, and by hybridization of fragments to Northern (RNA) blots. No homologs of other members of the Surfeit gene cluster were detected in close proximity to the D. melanogaster Surf-3/rpL7a gene. However, a gene which was detected directly 3' to the Surf-3/rpL7a gene was shown to encode a homolog of a mammalian serine-pyruvate aminotransferase.

scholarly journals Differential mucin gene expression in human pancreatic and colon cancer cells

1991 ◽  
Vol 276 (3) ◽  
pp. 599-605 ◽  
Author(s):  
S Yonezawa ◽  
J C Byrd ◽  
R Dahiya ◽  
J J L Ho ◽  
J R Gum ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Colon Cancer ◽  
Cancer Cells ◽  
Cell Lines ◽  
Cancer Cell ◽  
Cancer Cell Lines ◽  
Human Pancreatic Cancer ◽  
Northern Blots ◽  
Western Blots ◽  
Pancreatic Cancer Cells

The purpose of this study was to determine the quantity and nature of the mucins synthesized and secreted by four different pancreatic cancer cell lines. Well- to moderately-differentiated SW1990 and CAPAN-2 human pancreatic cancer cells were found to produce more high-Mr glycoprotein (HMG) than less-differentiated MIA PaCa-2 and PANC-1 cells. Most of the labelled HMG was secreted within 24 h. The results of chemical and enzymic degradation, ion-exchange chromatography and density-gradient centrifugation indicated that the HMG in SW1990 and CAPAN-2 cells has the properties expected for mucins, whereas much of the HMG in MIA PaCa-2 and PANC-1 cells may not be mucin, but proteoglycan. These results are consistent with immunoblots and Northern blots showing the presence of apomucin and apomucin mRNA in SW1990 and CAPAN-2 cells, but not in MIA PaCa-2 and PANC-1 cells. The Western blots and Northern blots also show that SW1990 and CAPAN-2 cells, like breast cancer cells, have the mammary-type apomucin and mRNA coded by the MUC1 gene, but lack the intestinal type apomucin and mRNA coded by the MUC2 gene. In contrast, the colon cancer cell lines tested in culture express apomucin and mRNA coded by MUC2 but not by MUC1.

scholarly journals Coat protein and RNAs of Cole latent virus are not present within chloroplasts of Chenopodium quinoa-infected cells

2003 ◽  
Vol 28 (1) ◽  
pp. 84-88 ◽  
Author(s):  
Priscila Belintani ◽  
José O. Gaspar
Keyword(s):  
Electron Microscopy ◽  
Coat Protein ◽  
Chenopodium Quinoa ◽  
Latent Virus ◽  
Percoll Gradient ◽  
Northern Blots ◽  
Virus Particles ◽  
Ultrastructural Studies ◽  
Particle Aggregates ◽  
Infected Cells

Cole latent virus (CoLV), genus Carlavirus, was studied by electron microscopy and biochemical approaches with respect both to the ultrastructure of the Chenopodium quinoa infected cells and to its association with chloroplasts. The CoLV was observed to be present as scattered particles interspersed with membranous vesicles and ribosomes or as dense masses of virus particles. These virus particles reacted by immunolabelling with a polyclonal antibody to CoLV. Morphologically, chloroplasts, mitochondria and nuclei appeared to be unaltered by virus infection and virus particles were not detected in these organelles. However, virus particle aggregates were frequently associated with the outer membrane of chloroplasts and occasionally with peroxisomes. Chloroplasts were purified by Percoll gradient, and the coat protein and virus-associated RNAs were extracted and analyzed by Western and Northern blots respectively. Coat protein and CoLV-associated RNAs were not detected within this organelle. The results presented in this work indicate that the association CoLV/chloroplasts, observed in the ultrastructural studies, might be a casual event in the host cell, and that the virus does not replicate inside the organelle.

Selective expression of intracisternal A-particle genes in established mouse plasmacytomas

1993 ◽  
Vol 13 (12) ◽  
pp. 7439-7446
Author(s):  
K K Lueders ◽  
J W Fewell ◽  
V E Morozov ◽  
E L Kuff
Keyword(s):  
B Cells ◽  
Regulatory Sequence ◽  
Cdna Clones ◽  
Sequence Variants ◽  
Long Terminal Repeats ◽  
Specific Sequence ◽  
Rna Transcripts ◽  
Sequence Selection ◽  
Intracisternal A Particle ◽  
Rna Blots

Mouse plasmacytomas generally express higher levels of RNA transcripts from endogenous intracisternal A-particle (IAP) proviral elements than do lipopolysaccharide-stimulated normal lymphocytes. Lymphocytes express a limited and highly characteristic set of IAP elements (lymphocyte-specific [LS] elements). In this study, we examined whether LS elements are expressed at higher levels after transformation of the cells and/or whether new IAP elements are activated. The IAP elements expressed in plasmacytoma MPC11 were characterized by sequence analysis of 22 cDNA clones. The long terminal repeats (LTRs) of the tumor cDNAs proved to be highly related in sequence. None of the clones was of the LS cDNA type. The MPC11 LTRs were five- to sixfold more active than an LS cDNA LTR when tested for promoter activity by transfection into plasmacytoma cells. The LTRs of the tumor-derived cDNAs contained a canonical ATF core sequence (ATF-PC), while the LS cDNAs contained an altered sequence (ATF-LS). An ATF-PC oligonucleotide probe detected multiple IAP transcripts on Northern (RNA) blots of RNA from several plasmacytoma but gave no reaction with RNA from stimulated B lymphocytes. In contrast, an ATF-LS probe detected higher levels of RNA in lymphocyte than in tumor RNAs. Thus, expression of IAP elements in transformed B cells is selective for a different set of regulatory sequence variants than those expressed in normal B cells. Other oligonucleotide probes representing LS- and PC-specific sequence variants detected multiple common hypomethylated IAP proviral loci in three independently derived plasmacytomas. Overall, the results show that established plasmacytomas exhibit a characteristic pattern of IAP proviral hypomethylation and regulatory sequence selection.

Molecular biology approaches to comparative study of Na(+)-glucose cotransport

1992 ◽  
Vol 263 (3) ◽  
pp. R489-R495 ◽  
Author(s):  
A. M. Pajor ◽  
B. A. Hirayama ◽  
E. M. Wright
Keyword(s):  
Amino Acid ◽  
Species Distribution ◽  
Evolutionary Conservation ◽  
Structure And Function ◽  
Rabbit Kidney ◽  
Predicted Amino Acid Sequence ◽  
Northern Blots ◽  
Intestinal Brush Border ◽  
Marine Mussels ◽  
And Function

The rabbit intestinal Na(+)-glucose cotransporter has been cloned and sequenced. The cDNA encoding the cotransporter has been used in two general lines of research related to comparative aspects of Na(+)-glucose cotransport that are reviewed here. First, defined regions of the predicted amino acid sequence were used to raise antibodies, and the species distribution of epitopes recognized by those antibodies was investigated. Intestinal brush-border membranes from mammals, birds, an amphibian, and a reptile were all found to contain protein that were recognized by the antibodies in Western analysis. The cDNA encoding the rabbit intestinal Na(+)-glucose cotransporter was also used directly to examine the species distribution of related mRNA in Northern studies and to isolate new cDNAs encoding other Na(+)-glucose cotransporters. Northern blots revealed the presence of related mRNAs in intestines of mammals and a fish, as well as rabbit kidney and gills of marine mussels. The cDNAs encoding mammalian Na(+)-glucose cotransporters and bacterial Na(+)-dependent cotransporters for proline and pantothenate share sequence homology. There has been evolutionary conservation of the structure and function of the Na(+)-glucose cotransporter, and there appears to be a gene family that codes for the Na(+)-coupled cotransporters.

scholarly journals Downregulation of the Cl-/HCO3-Exchanger Pendrin in Kidneys of Mice with Cystic Fibrosis: Role in the Pathogenesis of Metabolic Alkalosis

2018 ◽  
Vol 45 (4) ◽  
pp. 1551-1565 ◽  
Author(s):  
Mujan Varasteh Kia ◽  
Sharon Barone ◽  
Alicia A. McDonough ◽  
Kamyar Zahedi ◽  
Jie Xu ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Metabolic Alkalosis ◽  
Collecting Duct ◽  
Cortical Collecting Duct ◽  
Acid Base ◽  
Volume Depletion ◽  
Northern Blots ◽  
Western Blots ◽  
Salt Restriction ◽  
Immunofluorescence Labeling

Background/Aims: Patients with cystic fibrosis (CF) are prone to the development of metabolic alkalosis; however, the pathogenesis of this life threatening derangement remains unknown. We hypothesized that altered acid base transport machinery in the kidney collecting duct underlies the mechanism of impaired bicarbonate elimination in the CF kidney. Methods: Balance studies in metabolic cages were performed in WT and CFTR knockout (CF) mice with the intestinal rescue in response to bicarbonate loading or salt restriction, and the expression levels and cellular distribution of acid base and electrolyte transporters in the proximal tubule, collecting duct and small intestine were examined by western blots, northern blots and/or immunofluorescence labeling. Results: Baseline parameters, including acid-base and systemic vascular volume status were comparable in WT and CF mice, as determined by blood gas, kidney renin expression and urine chloride excretion. Compared with WT animals, CF mice demonstrated a significantly higher serum HCO3- concentration (22.63 in WT vs. 26.83 mEq/l in CF mice; n=4, p=0.013) and serum pH (7.33 in WT vs. 7.42 in CF mice; n=4, p=0.00792) and exhibited impaired kidney HCO3- excretion (urine pH 8.10 in WT vs. 7.35 in CF mice; n=7, p=0.00990) following a 3-day oral bicarbonate load. When subjected to salt restriction, CF mice developed a significantly higher serum HCO3- concentration vs. WT animals (29.26 mEq/L in CF mice vs. 26.72 in WT; n=5, p=0.0291). Immunofluorescence labeling demonstrated a profound reduction in the apical expression of the Cl-/HCO3- exchanger pendrin in cortical collecting duct cells and western and northern blots indicated diminished plasma membrane abundance and mRNA expression of pendrin in CF kidneys. Conclusions: We propose that patients with cystic fibrosis are prone to the development of metabolic alkalosis secondary to the inactivation of the bicarbonate secreting transporter pendrin, specifically during volume depletion, which is a common occurrence in CF patients.

scholarly journals Persistence of chronic myelocytic leukemia despite deletion of rearranged bcr/c-abl sequences in blast crisis.

1986 ◽  
Vol 164 (5) ◽  
pp. 1389-1396 ◽  
Author(s):  
C R Bartram ◽  
J W Janssen ◽  
R Becher ◽  
A de Klein ◽  
G Grosveld
Keyword(s):  
In Situ Hybridization ◽  
T Cell ◽  
Southern Blot ◽  
Blast Crisis ◽  
Cell Lineage ◽  
Chronic Myelocytic Leukemia ◽  
Northern Blots ◽  
Myelocytic Leukemia ◽  
Ph Chromosome

We report on a Ph+ chronic myelocytic leukemia (CML) case, cytogenetically characterized by the occurrence of a second Philadelphia (Ph) chromosome in lymphoid blast crisis of T cell lineage. In situ hybridization analyses showed a deletion of translocated c-abl sequences, present on the Ph during chronic state, from both Ph in acute state. Moreover, Southern blot analyses of blastic cells exhibited a rearrangement within bcr, but a deletion of 5' bcr sequences, and Northern blots failed to detect the hybrid 8.5 kb bcr/c-abl transcript usually observed in Ph+ CML.

Expression of heat shock genes of Neurospora crassa: effect of hyperthermia and other stresses on mRNA levels

1988 ◽  
Vol 66 (2) ◽  
pp. 81-92 ◽  
Author(s):  
Carol A. Curle ◽  
M. Kapoor
Keyword(s):  
Heat Shock ◽  
Neurospora Crassa ◽  
Sodium Arsenite ◽  
Gene Probe ◽  
Mrna Levels ◽  
Hsp 70 ◽  
Northern Blots ◽  
Stressed Cells ◽  
Shock Proteins

Neurospora crassa mycelium was heat shocked for intervals varying from 15–180 min. Heat shock mRNA was monitored by hybridization of Northern blots with the Drosophila hsp-70 gene probe and an inducible member of the yeast hsp-70 gene family, YG100. A 2.7 kilobase (kb) transcript, with homology to these two probes, was detected in cultures shocked for 15 min; its levels increased up to 60–90 min and declined thereafter. Sodium arsenite, too, induced the synthesis of this transcript. An additional, constitutively synthesized 2.4-kb transcript was revealed by hybridization with the yeast probe. The synthesis of this message was terminated during heat shock. Hybridization of Northern blots with the Drosophila actin gene probe demonstrated two size classes, 1.85 and 1.63 kb; the former decreased dramatically following heat shock. Recovery, as assessed by the disappearance of the 2.7-kb hsp-70-mRNA and restoration of the 1.85-kb actin message to the prestress levels, was essentially complete within 60 min of transfer to 28 °C. In vitro translations of RNA from stressed cells showed the heat shock messages to be stable and readily translatable. RNA of cells subjected to heat shock plus CdCl2 showed a higher content of messages for heat shock proteins of 70, 80, and 90 kilodaltons.

Sign in / Sign up

Export Citation Format

Share Document