Reverse Northern hybridization (“cDNA Southern”)

Leaf blight caused by Cylindrocladium quinqueseptatum Boedijn & Reitsma is a major disease occurring in Eucalyptus plantlets and saplings worldwide. Current studies on the resistance to this disease in eucalypti have focused primarily on analyzing the relationship between the disease and the metabolic products from the anatomic structure of Eucalyptus leaves and their physiological responses to pathogen invasion. There have been no reports on the mechanisms of molecular responses. Previous research suggests that Eucalyptus grandis × Eucalyptus urophylla 9224 is the highest resistant cultivar among the 11 ones planted in Fujian Province. Therefore, in this study, the molecular responses of E. grandis × E. urophylla 9224 to C. quinqueseptatum were investigated by means of suppression subtractive hybridization (SSH). In total, 928 clones were obtained from the forward SSH cDNA library of E. grandis × E. urophylla 9224, and 25 up-regulated genes were identified by screening the library using the method of reverse northern hybridization. The results show that among these, 23 up-regulated genes with known functions were involved in signal transduction, plant resistance response, photosynthesis, energy transfer, protein metabolism, and plant growth.

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Mutations in nuclear gene cyt-4 of Neurospora crassa result in pleiotropic defects in processing and splicing of mitochondrial RNAs.

Genetics ◽

10.1093/genetics/123.1.97 ◽

1989 ◽

Vol 123 (1) ◽

pp. 97-108 ◽

Cited By ~ 1

Author(s):

K F Dobinson ◽

M Henderson ◽

R L Kelley ◽

R A Collins ◽

A M Lambowitz

Keyword(s):

Neurospora Crassa ◽

Mitochondrial Protein ◽

Nuclear Gene ◽

Northern Hybridization ◽

Rrna Gene ◽

Mitochondrial Rna ◽

Group I Introns ◽

Group I ◽

Processing Pathways ◽

Aberrant Rna

Abstract The nuclear cyt-4 mutants of Neurospora crassa have been shown previously to be defective in splicing the group I intron in the mitochondrial large rRNA gene and in 3' end synthesis of the mitochondrial large rRNA. Here, Northern hybridization experiments show that the cyt-4-1 mutant has alterations in a number of mitochondrial RNA processing pathways, including those for cob, coI, coII and ATPase 6 mRNAs, as well as mitochondrial tRNAs. Defects in these pathways include inhibition of 5' and 3' end processing, accumulation of aberrant RNA species, and inhibition of splicing of both group I introns in the cob gene. The various defects in mitochondrial RNA synthesis in the cyt-4-1 mutant cannot be accounted for by deficiency of mitochondrial protein synthesis or energy metabolism, and they suggest that the cyt-4-1 mutant is defective in a component or components required for processing and/or turnover of a number of different mitochondrial RNAs. Defective splicing of the mitochondrial large rRNA intron in the cyt-4-1 mutant may be a secondary effect of failure to synthesize pre-rRNAs having the correct 3' end. However, a similar explanation cannot be invoked to account for defective splicing of the cob pre-mRNA introns, and the cyt-4-1 mutation may directly affect splicing of these introns.

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Riboflavin Production in Lactococcus lactis: Potential for In Situ Production of Vitamin-Enriched Foods

Applied and Environmental Microbiology ◽

10.1128/aem.70.10.5769-5777.2004 ◽

2004 ◽

Vol 70 (10) ◽

pp. 5769-5777 ◽

Cited By ~ 125

Author(s):

Catherine Burgess ◽

Mary O'Connell-Motherway ◽

Wilbert Sybesma ◽

Jeroen Hugenholtz ◽

Douwe van Sinderen

Keyword(s):

Functional Analysis ◽

Lactic Acid ◽

Lactococcus Lactis ◽

Regulatory Region ◽

Northern Hybridization ◽

Vitamin B ◽

Fermented Foods ◽

Riboflavin Production ◽

In Situ Production

ABSTRACT This study describes the genetic analysis of the riboflavin (vitamin B2) biosynthetic (rib) operon in the lactic acid bacterium Lactococcus lactis subsp. cremoris strain NZ9000. Functional analysis of the genes of the L. lactis rib operon was performed by using complementation studies, as well as by deletion analysis. In addition, gene-specific genetic engineering was used to examine which genes of the rib operon need to be overexpressed in order to effect riboflavin overproduction. Transcriptional regulation of the L. lactis riboflavin biosynthetic process was investigated by using Northern hybridization and primer extension, as well as the analysis of roseoflavin-induced riboflavin-overproducing L. lactis isolates. The latter analysis revealed the presence of both nucleotide replacements and deletions in the regulatory region of the rib operon. The results presented here are an important step toward the development of fermented foods containing increased levels of riboflavin, produced in situ, thus negating the need for vitamin fortification.

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DNA damage and heat shock dually regulate genes in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.6.1.90 ◽

1986 ◽

Vol 6 (1) ◽

pp. 90-96 ◽

Cited By ~ 39

Author(s):

T McClanahan ◽

K McEntee

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Damage ◽

Heat Shock ◽

Shock Treatment ◽

Northern Hybridization ◽

Heat Shock Treatment ◽

Base Pairs ◽

S1 Nuclease ◽

Hsp70 Family ◽

Transcriptional Start Sites

Two Saccharomyces cerevisiae genes isolated in a differential hybridization screening for DNA damage regulation (DDR genes) were also transcriptionally regulated by heat shock treatment. A 0.45-kilobase transcript homologous to the DDRA2 gene and a 1.25-kilobase transcript homologous to the DDR48 gene accumulated after exposure of cells to 4-nitroquinoline-1-oxide (NQO; 1 to 1.5 microgram/ml) or brief heat shock (20 min at 37 degrees C). The DDRA2 transcript, which was undetectable in untreated cells, was induced to high levels by these treatments, and the DDR48 transcript increased more than 10-fold as demonstrated by Northern hybridization analysis. Two findings argue that dual regulation of stress-responsive genes is not common in S. cerevisiae. First, two members of the heat shock-inducible hsp70 family of S. cerevisiae, YG100 and YG102, were not induced by exposure to NQO. Second, at least one other DNA-damage-inducible gene, DIN1, was not regulated by heat shock treatment. We examined the structure of the induced RNA homologous to DDRA2 after heat shock and NQO treatments by S1 nuclease protection experiments. Our results demonstrated that the DDRA2 transcript initiates equally frequently at two sites separated by 5 base pairs. Both transcriptional start sites were utilized when cells were exposed to either NQO or heat shock treatment. These results indicate that DDRA2 and DDR48 are members of a unique dually regulated stress-responsive family of genes in S. cerevisiae.

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Activation of the ATP-ubiquitin-proteasome pathway in skeletal muscle of cachectic rats bearing a hepatoma

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1995.268.5.e996 ◽

1995 ◽

Vol 268 (5) ◽

pp. E996-E1006 ◽

Cited By ~ 41

Author(s):

V. E. Baracos ◽

C. DeVivo ◽

D. H. Hoyle ◽

A. L. Goldberg

Keyword(s):

Muscle Protein ◽

Northern Hybridization ◽

Proteolytic System ◽

Atp Production ◽

Tumor Bearing ◽

No Weight Loss ◽

Ubiquitin Proteasome ◽

Proteasome Pathway ◽

And Control ◽

Dependent Pathway

Rats implanted with Yoshida ascites hepatoma (YAH) show a rapid and selective loss of muscle protein due mainly to a marked increase (63-95%) in the rate of protein degradation (compared with rates in muscles of pair-fed controls). To define which proteolytic pathways contribute to this increase, epitrochlearis muscles from YAH-bearing and control rats were incubated under conditions that modify different proteolytic systems. Overall proteolysis in either group of rats was not affected by removal of Ca2+ or by blocking the Ca(2+)-dependent proteolytic system. Inhibition of lysosomal function with methylamine reduced proteolysis (-12%) in muscles from YAH-bearing rats, but not in muscles of pair-fed rats. When ATP production was also inhibited, the remaining accelerated proteolysis in muscles of tumor-bearing rats fell to control levels. Muscles of YAH-bearing rats showed increased levels of ubiquitin-conjugated proteins and a 27-kDa proteasome subunit in Western blot analysis. Levels of mRNA encoding components of proteolytic systems were quantitated using Northern hybridization analysis. Although their total RNA content decreased 20-38%, pale muscles of YAH-bearing rats showed increased levels of ubiquitin mRNA (590-880%) and mRNA for multiple subunits of the proteasome (100-215%). Liver, kidney, heart, and brain showed no weight loss and no change in these mRNA species. Muscles of YAH-bearing rats also showed small increases (30-40%) in mRNA for cathepsins B and D, but not for calpain I or heat shock protein 70. Our findings suggest that accelerated muscle proteolysis and muscle wasting in tumor-bearing rats result primarily from activation of the ATP-dependent pathway involving ubiquitin and the proteasome.

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IL-8 secretion and neutrophil activation by HT-29 colonic epithelial cells

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1994.267.6.g991 ◽

1994 ◽

Vol 267 (6) ◽

pp. G991-G997 ◽

Cited By ~ 6

Author(s):

C. P. Kelly ◽

S. Keates ◽

D. Siegenberg ◽

J. K. Linevsky ◽

C. Pothoulakis ◽

...

Keyword(s):

Epithelial Cells ◽

Intestinal Inflammation ◽

Neutrophil Migration ◽

Intercellular Adhesion Molecule ◽

Northern Hybridization ◽

Intercellular Adhesion Molecule 1 ◽

Colonic Epithelial Cells ◽

Neutrophil Extravasation ◽

Cytokine Stimulation ◽

Ht 29

This study examines the ability of HT-29 human colonic epithelial cells to stimulate neutrophil migration and adhesion. Interleukin-8 (IL-8), a potent neutrophil chemoattractant, was detected in conditioned media from both unstimulated (1.1 ng/ml) and IL-1 beta-stimulated (16.1 ng/ml) HT-29 cultures. Conditioned medium from IL-1 beta-exposed HT-29 cells stimulated neutrophil migration (395% of control, P < 0.01), and this effect was completely inhibited by anti-IL-8 antibody. HT-29 medium also induced shedding of neutrophil L-selectin and increased expression of neutrophil CD11/CD18 adhesion receptors. Coculture of HT-29 cells with human endothelial cell monolayers resulted in increased neutrophil transendothelial migration (169% of control, P < 0.01), which was blocked by both anti-IL-8 and anti-CD18 antibody. Northern hybridization analysis demonstrated increased levels of mRNA for IL-8 and intercellular adhesion molecule-1 (ICAM-1) in cytokine-treated HT-29 cells. Cytokine stimulation of HT-29 monolayers was also associated with increased neutrophil adhesion to these cells. Neutrophil-HT-29 cell adhesion was blocked by monoclonal antibodies to neutrophil CD18 or to ICAM-1 on the HT-29 cells (86% and 56% inhibition, respectively, P < 0.01 for both). These data suggest that IL-8 secretion by activated colonic epithelial cells may contribute to neutrophil extravasation and tissue infiltration in intestinal inflammation.

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Novel Endogenous Retrovirus in Rabbits Previously Reported as Human Retrovirus 5

Journal of Virology ◽

10.1128/jvi.76.14.7094-7102.2002 ◽

2002 ◽

Vol 76 (14) ◽

pp. 7094-7102 ◽

Cited By ~ 28

Author(s):

David J. Griffiths ◽

Cécile Voisset ◽

Patrick J. W. Venables ◽

Robin A. Weiss

Keyword(s):

Sequence Analysis ◽

Dna Sequences ◽

Inflammatory Diseases ◽

Endogenous Retrovirus ◽

European Rabbit ◽

Pcr Analysis ◽

Northern Hybridization ◽

Human Dna ◽

Integration Sites ◽

Human Retrovirus

ABSTRACT Human retrovirus 5 (HRV-5) represented a fragment of a novel retrovirus sequence identified in human RNA and DNA preparations. In this study, the genome of HRV-5 was cloned and sequenced and integration sites were analyzed. Using PCR and Southern hybridization, we showed that HRV-5 is not integrated into human DNA. A survey of other species revealed that HRV-5 is present in the genomic DNA of the European rabbit (Oryctolagus cuniculus) and belongs to an endogenous retrovirus family found in rabbits. The presence of rabbit sequences flanking HRV-5 proviruses in human DNA extracts suggested that rabbit DNA was present in our human extracts, and this was confirmed by PCR analysis that revealed the presence of rabbit mitochondrial DNA sequences in four of five human DNA preparations tested. The origin of the rabbit DNA and HRV-5 in human DNA preparations remains unclear, but laboratory contamination cannot explain the preferential detection of HRV-5 in inflammatory diseases and lymphomas reported previously. This is the first description of a retrovirus genome in rabbits, and sequence analysis shows that it is related to but distinct from A-type retroelements of mice and other rodents. The species distribution of HRV-5 is restricted to rabbits; other species, including other members of the order Lagomorpha, do not contain this sequence. Analysis of HRV-5 expression by Northern hybridization and reverse transcriptase PCR indicates that the virus is transcribed at a low level in many rabbit tissues. In light of these findings we propose that the sequence previously designated HRV-5 should now be denoted RERV-H (for rabbit endogenous retrovirus H).

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Two Internal Origins of Replication in Streptomyces Linear Plasmid pFRL1

Applied and Environmental Microbiology ◽

10.1128/aem.02905-09 ◽

2010 ◽

Vol 76 (17) ◽

pp. 5676-5683 ◽

Cited By ~ 6

Author(s):

Ran Zhang ◽

Shiyuan Peng ◽

Zhongjun Qin

Keyword(s):

Reverse Transcription ◽

Northern Hybridization ◽

Linear Plasmids ◽

Time Points ◽

Linear Mode ◽

Quantitative Reverse Transcription Pcr ◽

Reverse Transcription Pcr ◽

Origins Of Replication ◽

Additional Locus

ABSTRACT Previous reports showed that Streptomyces linear plasmids usually contain one internal replication locus. Here, we identified two new replication loci on pFRL1, one (rep1A-ncs1) next to a telomere and another (rep2A-ncs2) ∼10 kb from it. The rep1A-ncs1 locus was able to direct replication independently in both linear and circular modes, whereas rep2A-ncs2 required an additional locus, rlrA-rorA, in order to direct propagation in linear mode. Rep1A protein bound to ncs1 in vitro. By quantitative reverse transcription-PCR and Northern hybridization, we showed that transcription of rep1A and rep2A varied during development and that each dominated at different time points. pFRL1-derived linear plasmids were inherited through spores more stably than circular plasmids and were more stable with pSLA2 telomeres than with pFRL1 telomeres in Streptomyces lividans.

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RT-PCR and Northern blot analysis in search for a putative Paramecium beta-adrenergic receptor.

Acta Biochimica Polonica ◽

10.18388/abp.1999_4153 ◽

1999 ◽

Vol 46 (3) ◽

pp. 813-821

Author(s):

A Płatek ◽

J Wiejak ◽

E Wyroba

Keyword(s):

Northern Blot ◽

Blot Analysis ◽

Adrenergic Receptor ◽

Northern Blot Analysis ◽

Molecular Probes ◽

Northern Hybridization ◽

Rt Pcr ◽

Beta Adrenergic Receptor ◽

Beta Adrenergic ◽

Sequence Tagged Sites

RT-PCR and Northern blot analysis were performed in order to search for a putative beta-adrenergic receptor (beta-AR) in Paramecium using several beta2-adrenergic-specific molecular probes. Under strictly defined RT-PCR conditions DNA species of expected molecular size about 360 bp were generated with the primers corresponding to the universal mammalian beta2-AR sequence tagged sites (located within the 4th and the 6th transmembrane regions of the receptor). This RT-PCR product hybridized in Southern blot analysis with the oligonucleotide probe designed to the highly conservative beta2-AR region involved in G-proteins interaction and located within the amplified region. Northern hybridization was performed on Paramecium total RNA and mRNA with human beta2-AR cDNA and two oligonucleotide probes: the first included Phe 290 involved in agonist binding (Strader et al., 1995) and the second was the backward RT-PCR primer. All these probes revealed the presence of about 2 kb mRNA which is consistent with the size of beta2-AR transcripts found in higher eukaryotes.

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Increased levels of clusterin (SGP-2) mRNA and protein accompany rat ventral prostate involution following finasteride treatment

Journal of Endocrinology ◽

10.1677/joe.0.1670197 ◽

2000 ◽

Vol 167 (2) ◽

pp. 197-204 ◽

Cited By ~ 17

Author(s):

S Astancolle ◽

G Guidetti ◽

C Pinna ◽

A Corti ◽

S Bettuzzi

Keyword(s):

Ventral Prostate ◽

Northern Hybridization ◽

Androgen Ablation ◽

Rat Ventral Prostate ◽

Hybridization In Situ ◽

Cell Atrophy ◽

High Level ◽

Epithelial Cell Population

Finasteride is a well-known inhibitor of the prostatic enzyme 5 alpha-reductase type 2 which prevents conversion of testosterone into 5 alpha-dihydrotestosterone, the active intraprostatic androgen, which causes prostate involution through a combination of cell atrophy and cell death. The drug is widely used to improve symptoms of benign prostatic hyperplasia in man. Clusterin, a glycoprotein which is generally up-regulated under conditions inducing cell atrophy or organ involution, is produced at a high level in the regressing rat ventral prostate following androgen ablation. According to several authors, clusterin does not respond to finasteride treatment, suggesting a different action of testosterone and 5 alpha-dihydrotestosterone. We show here that, under our conditions, finasteride was capable of inducing production of both clusterin mRNA and protein in the rat ventral prostate. In fact, by using different and converging techniques, such as Northern hybridization, in situ hybridization histochemistry and immunohistochemistry, we were able to show a strong induction of the clusterin gene in the epithelial cell population of the gland. The response to finasteride, which was similar to that seen with castration, occurred with a delay of a few days. In situ and immunohistochemistry experiments indicated that both orchidectomy and finasteride administration resulted in increased transition of the epithelial cells from the columnar to the cuboidal (atrophic) shape, and this was accompanied by an increased intensity of the signal for clusterin. Thus, it appears that induction of clusterin is part of the molecular process leading to prostate involution caused by either orchidectomy or finasteride administration.

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