Hybridization and detection of digoxigenin probes on RNA blots

1996 ◽  
Vol 6 (1) ◽  
pp. 75-77
Author(s):  
Elizabeth Davies ◽  
Rachel Hodge ◽  
Peter G. Isaac
Keyword(s):  
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.

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.

scholarly journals Design and Evaluation of a Lactobacillus manihotivorans Species-Specific rRNA-Targeted Hybridization Probe and Its Application to the Study of Sour Cassava Fermentation

2000 ◽  
Vol 66 (5) ◽  
pp. 2224-2226 ◽  
Author(s):  
Frédéric Ampe
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Sequence Comparison ◽  
Cassava Starch ◽  
Rrna Sequence ◽  
16S Rrna Sequence ◽  
Hybridization Probe ◽  
Culture Collections ◽  
Species Specific ◽  
Rna Blots

ABSTRACT Based on 16S rRNA sequence comparison, we have designed a 20-mer oligonucleotide that targets a region specific to the speciesLactobacillus manihotivorans recently isolated from sour cassava fermentation. The probe recognized the rRNA obtained from all the L. manihotivorans strains tested but did not recognize 56 strains of microorganisms from culture collections or directly isolated from sour cassava, including 29 species of lactic acid bacteria. This probe was then successfully used in quantitative RNA blots and demonstrated the importance of L. manihotivoransin the fermentation of sour cassava starch, which could represent up to 20% of total lactic acid bacteria.

scholarly journals Control of hsp70 RNA levels in human lymphocytes.

1987 ◽  
Vol 104 (2) ◽  
pp. 183-187 ◽  
Author(s):  
L Kaczmarek ◽  
B Calabretta ◽  
H T Kao ◽  
N Heintz ◽  
J Nevins ◽  
...  
Keyword(s):  
Culture Medium ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Human Lymphocytes ◽  
Peripheral Blood Mononuclear ◽  
Growth State ◽  
Steady State Levels ◽  
Rna Blots

The expression of a hsp70 gene in human cells has previously been shown to be related to the growth state of the cells. As an alternative to in vitro synchronization procedures, we have measured steady-state levels of the RNA for a heat-shock protein 70 (hsp70) in human peripheral blood mononuclear cells (PBMC) that are naturally quiescent in a G0 state. The probe used recognized, on RNA blots, one single band. The levels of this hsp70 RNA are elevated in circulating PBMC and decrease when the cells are incubated with serum, or phytohemagglutinin, or simply when they are incubated in culture medium. The levels of hsp70 RNA decrease within 30 min after in vitro culture, and are accompanied by an increase in the levels of c-fos RNA. These findings, together with other recent reports in the literature, suggest a possible role of the hsp70 proteins in the regulation of cell growth.

scholarly journals A study of the role of metallothionein in the inherited copper toxicosis of dogs

1986 ◽  
Vol 236 (2) ◽  
pp. 409-415 ◽  
Author(s):  
D M Hunt ◽  
S A Wake ◽  
J F B Mercer ◽  
D M Danks
Keyword(s):  
Ion Exchange ◽  
Liver Tissue ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Copper Accumulation ◽  
Total Liver ◽  
Liver Copper ◽  
Rna Blots ◽  
Dog Liver

The role of metallothionein (MT) was assessed in the copper-loading disease prevalent in Bedlington terriers. Fractionation of tissue supernatants over Sephadex G-75 showed that most of the additional cytosolic copper present in liver tissue of these dogs was bound to MT, and that substantially more MT-bound copper could be solubilized by detergent plus mercaptoethanol. Zinc contents were only slightly raised, although most of the extra zinc was associated with a 4000-Mr ligand. Ion-exchange chromatography revealed two isoproteins, MT1 and MT2, in all the dog liver samples examined. In Bedlington terrier liver, copper associated with both isoproteins was increased, although the increase for MT2 was greater than for MT1. The content of MT protein was also raised, although cell-free translations and RNA blots of total liver RNA showed that this increase was not associated with a rise in MT mRNA. The significance of these results to the mechanism of copper accumulation in the Bedlington terrier disorder is discussed.

scholarly journals Molecular basis for the CAT-2 null phenotype in maize.

Genetics ◽  
1988 ◽  
Vol 118 (1) ◽  
pp. 149-153
Author(s):  
L A Bethards ◽  
J G Scandalios
Keyword(s):  
Gene Expression ◽  
Molecular Basis ◽  
Protein Identification ◽  
Null Line ◽  
Maize Line ◽  
S1 Nuclease ◽  
Developmental Patterns ◽  
Catalase Gene ◽  
Null Phenotype ◽  
Rna Blots

Abstract Previous reports have described several maize lines whose developmental patterns of catalase gene expression vary from the "typical" maize line, W64A. Among these variants are the lines A16 and A338, both found to be null for the CAT-2 protein. Identification of a third CAT-2 null line, designated A340, is described. RNA blots and S1 nuclease protection analysis indicate that all three CAT-2 null lines produce a similarly shortened Cat2 transcript. The molecular basis for this aberrant Cat2 transcript is discussed.

scholarly journals Cloning and Expression of Dehydrin Genes in Blueberry

HortScience ◽  
1996 ◽  
Vol 31 (4) ◽  
pp. 585a-585 ◽  
Author(s):  
Lisa J. Rowland ◽  
Mubarack Muthalif ◽  
Amnon Levi ◽  
Rajeev Arora
Keyword(s):  
Low Temperature ◽  
Cdna Clone ◽  
Cloning And Expression ◽  
Sequence Information ◽  
Floral Buds ◽  
Growth Characterization ◽  
Dna Primers ◽  
Rna Blots ◽  
Dehydrin Genes

Previous studies identified three major chilling-responsive proteins of 65, 60, and 14 kDa whose levels increase in floral buds of blueberry during cold acclimation and decrease during deacclimation and resumption of growth. Characterization of these proteins found them to be members of a family of proteins responsive to drought and low temperature stress called dehydrins. The 65- and 60-kDa proteins were purified, digested into peptides, and several peptides from each were sequenced. The sequence information was used to synthesize degenerate DNA primers for amplification of a part of the gene(s) encoding these proteins. One pair of primers amplified a 200-bp fragment, which now has been cloned and sequenced. Within the 200-bp sequence is a motif conserved amongst dehydrins. Hybridization of the 200-bp fragment to RNA blots revealed homology to two chilling-responsive messages of 3.7 and 1.6 kb. The 200-bp fragment currently is being used to screen a cDNA library (prepared from RNA from cold acclimated blueberry floral buds) to isolate the full length cDNA clone.

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