Transcript levels for a mung bean cysteine protease during early seedling growth

1997 ◽  
Vol 7 (4) ◽  
pp. 359-372 ◽  
Author(s):  
Kangmoon Lee ◽  
Zhouwen Liu ◽  
Anna Tan-Wilson ◽  
Karl Wilson
Keyword(s):  
Mung Bean ◽  
Protease Activity ◽  
Blot Hybridization ◽  
Northern Blot Hybridization ◽  
Specific Primers ◽  
System A ◽  
Early Seedling ◽  
Polymerase Chain ◽  
Normal Increase ◽  
Putative Translation Product

AbstractcDNAs for the major cysteine endopeptidase (CEpase) of mung bean (Vigna radiata [L.] Wilczek) seedling cotyledons have been cloned using gene-specific primers with the polymerase chain reaction (PCR) in a 3'-RACE system. A cDNA clone for CEpase, pKL042, that is 1221 bp long excluding the poly A tail was isolated. It appears to contain the entire coding sequence for a 362-residue-long polypeptide. The N-terminal sequence for the mature CEpase begins at position 128 of the putative translation product, suggesting removal of an N-terminal hydrophobic signal peptide and additional sequences to produce the mature protease. Northern blot hybridization with CEpase cDNA pKL042 as probe indicates that CEpase transcript is not detectable in the cotyledons or the embryonic axis of dry seeds, but is first detectable in the day 1 cotyledons and in the day 3 axis. The level of CEpase mRNA in both cotyledons and axis increases as growth proceeds. A decline of protease activity, however, is observed after day 3 in the cotyledons, even though the level of protease transcripts continues to increase until day 8. Detachment of the axis from the cotyledons before day 3 results in the prevention of the normal increase in both protease activity and CEpase mRNA.

scholarly journals Detection and Differentiation of Bovine Group A Rotavirus Serotypes using Polymerase Chain Reaction-Generated Probes to the VP7 Gene

1992 ◽  
Vol 4 (2) ◽  
pp. 148-158 ◽  
Author(s):  
Anil V. Parwani ◽  
Blair I. Rosen ◽  
Jorge Flores ◽  
Malcolm A. McCrae ◽  
Mario Gorziglia ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Blot Hybridization ◽  
Northern Blot Hybridization ◽  
Chain Reaction ◽  
Diarrhea Virus ◽  
Bovine Rotavirus ◽  
Group A ◽  
Polymerase Chain ◽  
Vp7 Gene ◽  
Hybridization Assays

Dot and Northern blot hybridization assays were developed to detect and differentiate group A bovine rotavirus serotypes using radiolabeled serotype 6 (Nebraska calf diarrhea virus [NCDV] and United Kingdom [UK] strains) or serotype 10 (Cracker [Cr] strain) VP7 gene probes. Partial length VP7-specific cDNA encompassing areas of major sequence diversity were generated by the polymerase chain reaction (PCR) using either cloned VP7 genes (NCDV and UK strains) or reverse transcribed mRNA (Cr strain) as templates. Radiolabeled probes prepared from the PCR-generated cDNA were tested at various stringency conditions to optimize the hybridization assays. At high stringency conditions (52 C, 50% formamide, 5 x standard saline citrate), the NCDV, UK, and Cr probes serotypically differentiated bovine rotavirus isolates in RNA samples prepared from cell culture-propagated viruses or in fecal specimens from infected gnotobiotic calves. The sensitivity and specificity of NCDV and Cr VP7 probes were characterized in dot blot hybridization assays, and the probes were estimated to detect at least 1 ng of viral RNA. The serotyping results obtained using VP7 probes were similar to those obtained using serologic assays. Further development of these assays may provide a useful means for the rapid detection and differentiation of bovine rotavirus serotypes in fecal samples from calves in the field.

scholarly journals Citrus viroid V: Occurrence, Host Range, Diagnosis, and Identification of New Variants

2008 ◽  
Vol 98 (11) ◽  
pp. 1199-1204 ◽  
Author(s):  
P. Serra ◽  
M. Eiras ◽  
S. M. Bani-Hashemian ◽  
N. Murcia ◽  
E. W. Kitajima ◽  
...  
Keyword(s):  
Sweet Orange ◽  
Blot Hybridization ◽  
The United States ◽  
Northern Blot Hybridization ◽  
Sultanate Of Oman ◽  
Sweet Lime ◽  
The Family ◽  
Commercial Species ◽  
Polymerase Chain ◽  
Amplification Host

The recently described Citrus viroid V (CVd-V) has been proposed as a new species of the genus Apscaviroid within the family Pospiviroidae. Analysis of 64 samples from different citrus-growing areas has shown that CVd-V is present in the United States, Spain, Nepal, and the Sultanate of Oman. CVd-V found in six sweet orange sources from the Sultanate of Oman was identical to the reference CVd-V variant, whereas three new variants with sequence identities of 98.6% (CVd-VCA), 97.3% (CVd-VST), and 94.9% (CVd-VNE) were identified in sources from California, Spain, and Nepal, respectively. These results suggest that this viroid has not emerged recently and that it is relatively widespread. Transmission assays to sweet orange, mandarin, and mandarin hybrids, clementine, satsuma, lemon, sour orange, Tahiti lime, Palestine sweet lime, calamondin, bergamot, and kumquat have shown that all these citrus species and citrus relatives are hosts for CVd-V. Several indexing approaches, including slot blot, northern blot hybridization, and reverse transcription-polymerase chain reaction, have been evaluated for detecting CVd-V, either using Etrog citron as an amplification host or directly from commercial species and cultivars.

Total RNA Extraction from Saccharomyces cerevisiae Using Hot Acid Phenol

2021 ◽  
Vol 2021 (12) ◽  
pp. pdb.prot101691
Author(s):  
Michael R. Green ◽  
Joseph Sambrook
Keyword(s):  
Blot Hybridization ◽  
Rna Extraction ◽  
Sodium Dodecyl ◽  
Northern Blot Hybridization ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Low Salt ◽  
Rigid Cell Wall ◽  
Polymerase Chain

Isolation of RNA from yeast is complicated by the need to first break the thick, rigid cell wall. The protocol provided here uses a cycle of heating and freezing of cells in the presence of phenol and the detergent sodium dodecyl sulfate (SDS). The extraction is performed in the presence of low salt so that, following separation of the aqueous and phenol phases by centrifugation, DNA can be collected from the interface while RNA remains in the aqueous phase. This protocol should yield ∼50–250 µg of RNA from 10 mL of culture. The RNA isolated using this approach is suitable for most follow-up applications such as northern blot hybridization, reverse transcriptase-polymerase chain reaction (RT-PCR), and cDNA construction.

scholarly journals Location of the initial cleavage sites in mouse pre-rRNA.

1983 ◽  
Vol 3 (8) ◽  
pp. 1501-1510 ◽  
Author(s):  
L H Bowman ◽  
W E Goldman ◽  
G I Goldberg ◽  
M B Hebert ◽  
D Schlessinger
Keyword(s):  
Blot Hybridization ◽  
Northern Blot Hybridization ◽  
28S Rrna ◽  
Cleavage Sites ◽  
S1 Nuclease ◽  
5.8S Rrna ◽  
Initial Cleavage ◽  
S1 Nuclease Mapping ◽  
Mapping Techniques ◽  
Nuclease Mapping

The locations of three cleavages that can occur in mouse 45S pre-rRNA were determined by Northern blot hybridization and S1 nuclease mapping techniques. These experiments indicate that an initial cleavage of 45S pre-rRNA can directly generate the mature 5' terminus of 18S rRNA. Initial cleavage of 45S pre-rRNA can also generate the mature 5' terminus of 5.8S rRNA, but in this case cleavage can occur at two different locations, one at the known 5' terminus of 5.8S rRNA and another 6 or 7 nucleotides upstream. This pattern of cleavage results in the formation of cytoplasmic 5.8S rRNA with heterogeneous 5' termini. Further, our results indicate that one pathway for the formation of the mature 5' terminus of 28S rRNA involves initial cleavages within spacer sequences followed by cleavages which generate the mature 5' terminus of 28S rRNA. Comparison of these different patterns of cleavage for mouse pre-rRNA with that for Escherichia coli pre-rRNA implies that there are fundamental differences in the two processing mechanisms. Further, several possible cleavage signals have been identified by comparing the cleavage sites with the primary and secondary structure of mouse rRNA (see W. E. Goldman, G. Goldberg, L. H. Bowman, D. Steinmetz, and D. Schlessinger, Mol. Cell. Biol. 3:1488-1500, 1983).

scholarly journals Tissue-specific regulation of dipeptidyl peptidase IV expression during development

1991 ◽  
Vol 277 (2) ◽  
pp. 331-334 ◽  
Author(s):  
M Hildebrandt ◽  
W Reutter ◽  
J D Gitlin
Keyword(s):  
Lung Development ◽  
Kidney Development ◽  
Blot Hybridization ◽  
Dipeptidyl Peptidase ◽  
Transcriptional Level ◽  
Northern Blot Hybridization ◽  
Dpp Iv ◽  
Organ Specific ◽  
Specific Regulation ◽  
Lung And Kidney

The patterns of dipeptidyl peptidase (DPP) IV activity and protein amount in different rat organs during development were compared. In order to elucidate the molecular basis for these patterns, total RNA was isolated from lung and kidney at different stages of development and analysed by Northern-blot hybridization using an oligonucleotide derived from the DPP IV cDNA sequence. This oligonucleotide hybridized to two distinct mRNAs of approx. 3.2 and 4.8 kb respectively. During kidney development, the pattern for DPP IV mRNA paralleled that of DPP IV activity and protein amount, suggesting that, in kidney, the expression of DPP IV is primarily controlled at the transcriptional level. In contrast, the magnitude of DPP IV activity during lung development compared with that of DPP IV mRNA in lung suggests that post-transcriptional mechanisms are involved in regulating the expression of DPP IV in lung. Organ-specific regulation of DPP IV expression may provide a useful model for further comparative studies of transcriptional and post-transcriptional mechanisms of DPP IV expression within the same organism.

Location of the initial cleavage sites in mouse pre-rRNA

1983 ◽  
Vol 3 (8) ◽  
pp. 1501-1510
Author(s):  
L H Bowman ◽  
W E Goldman ◽  
G I Goldberg ◽  
M B Hebert ◽  
D Schlessinger
Keyword(s):  
Blot Hybridization ◽  
Northern Blot Hybridization ◽  
28S Rrna ◽  
Cleavage Sites ◽  
S1 Nuclease ◽  
5.8S Rrna ◽  
Initial Cleavage ◽  
S1 Nuclease Mapping ◽  
Mapping Techniques ◽  
Nuclease Mapping

The locations of three cleavages that can occur in mouse 45S pre-rRNA were determined by Northern blot hybridization and S1 nuclease mapping techniques. These experiments indicate that an initial cleavage of 45S pre-rRNA can directly generate the mature 5' terminus of 18S rRNA. Initial cleavage of 45S pre-rRNA can also generate the mature 5' terminus of 5.8S rRNA, but in this case cleavage can occur at two different locations, one at the known 5' terminus of 5.8S rRNA and another 6 or 7 nucleotides upstream. This pattern of cleavage results in the formation of cytoplasmic 5.8S rRNA with heterogeneous 5' termini. Further, our results indicate that one pathway for the formation of the mature 5' terminus of 28S rRNA involves initial cleavages within spacer sequences followed by cleavages which generate the mature 5' terminus of 28S rRNA. Comparison of these different patterns of cleavage for mouse pre-rRNA with that for Escherichia coli pre-rRNA implies that there are fundamental differences in the two processing mechanisms. Further, several possible cleavage signals have been identified by comparing the cleavage sites with the primary and secondary structure of mouse rRNA (see W. E. Goldman, G. Goldberg, L. H. Bowman, D. Steinmetz, and D. Schlessinger, Mol. Cell. Biol. 3:1488-1500, 1983).

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