Transcriptional control of delta-crystallin gene expression in the chicken embryo lens: demonstration by a new method for measuring mRNA metabolism

1993 ◽  
Vol 13 (6) ◽  
pp. 3282-3290
Author(s):  
X Li ◽  
D C Beebe
Keyword(s):  
Chicken Embryo ◽  
Transcriptional Control ◽  
Cultured Cells ◽  
Pcr Primers ◽  
High Refractive Index ◽  
In Vitro Transcription ◽  
Equilibrium Density ◽  
Specific Pcr ◽  
High Concentrations

Crystallins are proteins that accumulate to very high concentrations in the fiber cells of the lens of the eye. Crystallins are responsible for the transparency and high refractive index that are essential for lens function. In the chicken embryo, delta-crystallin accounts for more than 70% of the newly synthesized lens proteins. We used density labeling and gene-specific polymerase chain reaction (PCR) to determine the mechanism regulating the expression of the two very similar delta-crystallin genes. Newly synthesized RNA was separated from preexisting RNA by incubating the lenses with 15N- and 13C-labeled ribonucleosides and then separating newly synthesized, density-labeled RNA from the bulk of light RNA by equilibrium density centrifugation in NaI-KI gradients. The relative abundances of the two crystallin mRNAs in the separated fractions were then determined by PCR. This method permitted the quantitation of newly synthesized processed and unprocessed delta-crystallin mRNAs. Additional studies used intron- and gene-specific PCR primers to determine the relative expression of the two delta-crystallin genes in processed RNA and unprocessed RNA extracted from different regions of the embryonic lens. Results of these tests indicated that the differential expression of the delta-crystallin genes was regulated primarily at the level of transcription. This outcome was not expected on the basis of the results of previous studies, which used in vitro transcription and transfection methods to evaluate the relative strengths of delta-crystallin promoter and enhancer sequences. Our data suggest that the cultured cells used in these earlier studies may not have provided an accurate view of delta-crystallin regulation in the intact lens.

scholarly journals Transcriptional control of delta-crystallin gene expression in the chicken embryo lens: demonstration by a new method for measuring mRNA metabolism.

1993 ◽  
Vol 13 (6) ◽  
pp. 3282-3290 ◽  
Author(s):  
X Li ◽  
D C Beebe
Keyword(s):  
Chicken Embryo ◽  
Transcriptional Control ◽  
Cultured Cells ◽  
Pcr Primers ◽  
High Refractive Index ◽  
In Vitro Transcription ◽  
Equilibrium Density ◽  
Specific Pcr ◽  
High Concentrations

Crystallins are proteins that accumulate to very high concentrations in the fiber cells of the lens of the eye. Crystallins are responsible for the transparency and high refractive index that are essential for lens function. In the chicken embryo, delta-crystallin accounts for more than 70% of the newly synthesized lens proteins. We used density labeling and gene-specific polymerase chain reaction (PCR) to determine the mechanism regulating the expression of the two very similar delta-crystallin genes. Newly synthesized RNA was separated from preexisting RNA by incubating the lenses with 15N- and 13C-labeled ribonucleosides and then separating newly synthesized, density-labeled RNA from the bulk of light RNA by equilibrium density centrifugation in NaI-KI gradients. The relative abundances of the two crystallin mRNAs in the separated fractions were then determined by PCR. This method permitted the quantitation of newly synthesized processed and unprocessed delta-crystallin mRNAs. Additional studies used intron- and gene-specific PCR primers to determine the relative expression of the two delta-crystallin genes in processed RNA and unprocessed RNA extracted from different regions of the embryonic lens. Results of these tests indicated that the differential expression of the delta-crystallin genes was regulated primarily at the level of transcription. This outcome was not expected on the basis of the results of previous studies, which used in vitro transcription and transfection methods to evaluate the relative strengths of delta-crystallin promoter and enhancer sequences. Our data suggest that the cultured cells used in these earlier studies may not have provided an accurate view of delta-crystallin regulation in the intact lens.

scholarly journals Evaluation of a Novel Boron-Containing α-d-Mannopyranoside for BNCT

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1277 ◽  
Author(s):  
Takao Tsurubuchi ◽  
Makoto Shirakawa ◽  
Wataru Kurosawa ◽  
Kayo Matsumoto ◽  
Risa Ubagai ◽  
...  
Keyword(s):  
Normal Tissue ◽  
Cultured Cells ◽  
Tumor Model ◽  
Intracellular Distribution ◽  
Inductively Coupled ◽  
Inductively Coupled Mass Spectrometry ◽  
Icp Ms ◽  
High Concentrations

Boron neutron capture therapy (BNCT) is a unique anticancer technology that has demonstrated its efficacy in numerous phase I/II clinical trials with boronophenylalanine (BPA) and sodium borocaptate (BSH) used as 10B delivery agents. However, continuous drug administration at high concentrations is needed to maintain sufficient 10B concentration within tumors. To address the issue of 10B accumulation and retention in tumor tissue, we developed MMT1242, a novel boron-containing α-d-mannopyranoside. We evaluated the uptake, intracellular distribution, and retention of MMT1242 in cultured cells and analyzed biodistribution, tumor-to-normal tissue ratio and toxicity in vivo. Fluorescence imaging using nitrobenzoxadiazole (NBD)-labeled MMT1242 and inductively coupled mass spectrometry (ICP-MS) were performed. The effectiveness of BNCT using MMT1242 was assessed in animal irradiation studies at the Kyoto University Research Reactor. MMT1242 showed a high uptake and broad intracellular distribution in vitro, longer tumor retention compared to BSH and BPA, and adequate tumor-to-normal tissue accumulation ratio and low toxicity in vivo. A neutron irradiation study with MMT1242 in a subcutaneous murine tumor model revealed a significant tumor inhibiting effect if injected 24 h before irradiation. We therefore report that 10B-MMT1242 is a candidate for further clinical BNCT studies.

scholarly journals In VitroCulture of Previously Uncultured Oral Bacterial Phylotypes

2015 ◽  
Vol 81 (24) ◽  
pp. 8307-8314 ◽  
Author(s):  
Hayley Thompson ◽  
Alexandra Rybalka ◽  
Rebecca Moazzez ◽  
Floyd E. Dewhirst ◽  
William G. Wade
Keyword(s):  
Culture Media ◽  
Pcr Primers ◽  
Oral Bacteria ◽  
Subgingival Plaque ◽  
Community Studies ◽  
Shotgun Metagenomics ◽  
Content Type ◽  
Specific Pcr ◽  
Proteose Peptone

ABSTRACTAround a third of oral bacteria cannot be grown using conventional bacteriological culture media. Community profiling targeting 16S rRNA and shotgun metagenomics methods have proved valuable in revealing the complexity of the oral bacterial community. Studies investigating the role of oral bacteria in health and disease require phenotypic characterizations that are possible only with live cultures. The aim of this study was to develop novel culture media and use anin vitrobiofilm model to culture previously uncultured oral bacteria. Subgingival plaque samples collected from subjects with periodontitis were cultured on complex mucin-containing agar plates supplemented with proteose peptone (PPA), beef extract (BEA), or Gelysate (GA) as well as on fastidious anaerobe agar plus 5% horse blood (FAA).In vitrobiofilms inoculated with the subgingival plaque samples and proteose peptone broth (PPB) as the growth medium were established using the Calgary biofilm device. Specific PCR primers were designed and validated for the previously uncultivated oral taxaBacteroidetesbacteria HOT 365 and HOT 281,Lachnospiraceaebacteria HOT 100 and HOT 500, andClostridialesbacterium HOT 093. All agar media were able to support the growth of 10 reference strains of oral bacteria. One previously uncultivated phylotype,Actinomycessp. HOT 525, was cultivated on FAA. Of 93 previously uncultivated phylotypes found in the inocula, 26 were detected inin vitro-cultivated biofilms.Lachnospiraceaebacterium HOT 500 was successfully cultured from biofilm material harvested from PPA plates in coculture withParvimonas micraorVeillonella dispar/parvulaafter colony hybridization-directed enrichment. The establishment ofin vitrobiofilms from oral inocula enables the cultivation of previously uncultured oral bacteria and provides source material for isolation in coculture.

scholarly journals Identification by PCR of Fusarium culmorum Strains Producing Large and Small Amounts of Deoxynivalenol

2002 ◽  
Vol 68 (11) ◽  
pp. 5472-5479 ◽  
Author(s):  
B. Bakan ◽  
C. Giraud-Delville ◽  
L. Pinson ◽  
D. Richard-Molard ◽  
E. Fournier ◽  
...  
Keyword(s):  
Intergenic Region ◽  
Sequence Data ◽  
Fusarium Culmorum ◽  
Pcr Primers ◽  
Wheat Grains ◽  
Specific Pcr ◽  
Trichodiene Synthase

ABSTRACT Thirty deoxynivalenol-producing F. culmorum strains, isolated from wheat grains, were incubated in vitro and analyzed for trichothecene production. Seventeen strains produced more than 1 ppm of deoxynivalenol and acetyldeoxynivalenol and were considered high-deoxynivalenol-producing strains, whereas 13 F. culmorum strains produced less than 0.07 ppm of trichothecenes and were considered low-deoxynivalenol-producing strains. For all strains, a 550-base portion of the trichodiene synthase gene (tri5) was amplified and sequenced. According to the tri5 data, the F. culmorum strains tested clustered into two groups that correlated with in vitro deoxynivalenol production. For three high-producing and three low-producing F. culmorum strains, the tri5-tri6 intergenic region was then sequenced, which confirmed the two separate clusters within the F. culmorum strains. According to the tri5-tri6 sequence data, specific PCR primers were designed to allow differentiation of high-producing from low-producing F. culmorum strains.

Alterations in hepatic chromatin template availability during infection

1975 ◽  
Vol 228 (4) ◽  
pp. 1183-1187 ◽  
Author(s):  
HS Earp
Keyword(s):  
Transcriptional Control ◽  
Metabolic Response ◽  
Systemic Infection ◽  
In Vitro Transcription ◽  
Stimulatory Action ◽  
Chromatin Template ◽  
Severity Of The Disease ◽  
Dna Template

Hepatic chromatin was isolated from rats at various times after inoculation with either live or heat-killed bacteria. The chromatin was assayed under conditions that allow determination of the DNA template available to support in vitro transcription. Both a fulminant Diplococcus pneumoniae and a milder Salmonella typhimurium infection produced time-related increases in hepatic chromatin template availability when compared to chromatin isolated from rats inoculated with heat-killed bacteria. Both timing and magnitude of increased template availability correlated with the severity of the infection. The earliest change observed was a 50 percent rise in availability noted 4 h after inoculation with D. pneumoniae. This preceded the onset of fever, as well as other known heaptic consequences of systemic infection. After 24 h of infection the maximum rise of 90 percent occurred. Similar changes developed during S. typhimurium infection, but were slower in onset and smaller in magnitude. Adrenalectomy prior to infection enhanced the severity of the disease but markedly blunted the increase in template availability. The data are consistent with the hypothesis that systemic infection regulates the hepatic metabolic response to infection through transcriptional control and that a permissive or stimulatory action of glucocorticoids is involved in the increases in template availability effected.

In vitro transcription systems from cultured cells and fat-body tissue of the silkworm,Bombyx mori

1997 ◽  
Vol 8 (1) ◽  
pp. 79-88 ◽  
Author(s):  
Eriko Mine ◽  
Hiroshi Sakurai ◽  
Susumu Izumi ◽  
Shiro Tomino
Keyword(s):  
Bombyx Mori ◽  
Cultured Cells ◽  
Fat Body ◽  
In Vitro Transcription ◽  
Body Tissue ◽  
Silkworm Bombyx Mori

scholarly journals Reversal of in vitro p53 squelching by both TFIIB and TFIID.

1995 ◽  
Vol 15 (11) ◽  
pp. 6474-6478 ◽  
Author(s):  
X Liu ◽  
A J Berk
Keyword(s):  
Transcriptional Activation ◽  
Human Tumor ◽  
Activation Function ◽  
In Vitro Transcription ◽  
Activation Domains ◽  
Double Stranded Dna ◽  
P53 Activation ◽  
High Concentrations ◽  
Tata Box Binding Protein

p53, the protein encoded by one of the most significant human tumor suppressor genes, is a sequence-specific transcriptional activator. When activated by a double-stranded DNA break, p53 function arrests cells in G1 and can induce apoptosis. Transcriptional activation function is critical for p53 tumor suppression, although transcriptional repressing and nontranscriptional functions of p53 may contribute. p53 activation requires that it bind to TFIID through interactions with TATA box-binding protein (TBP)-associated factors and potentially with TBP. Here, we studied the mechanism of p53 activation using in vitro transcription and a sufficiently high p53 concentration to squelch activated transcription. Squelching is thought to result when target molecules that interact with activation domains are titrated by binding to excess activator. Addition of either excess TFIIB or TFIID but not other proteins required for p53-activated transcription reversed squelching by high p53 concentrations, whereas neither stimulated transcription in reactions without excess p53. These results reveal that both TFIIB and TFIID are inhibited by high concentrations of p53 and suggest that p53 activation may work through direct or indirect interactions with both TFIIB and TFIID.

scholarly journals Fur: Find unique genomic regions for diagnostic PCR

2021 ◽  
Author(s):  
Bernhard Haubold ◽  
Fabian Klötzl ◽  
Lars Hellberg ◽  
Daniel Thompson ◽  
Markus Cavalar
Keyword(s):  
Similarity Search ◽  
Pcr Primers ◽  
The Other ◽  
Supplementary Information ◽  
Lactobacillus Species ◽  
Diagnostic Pcr ◽  
Specific Pcr ◽  
The Many ◽  
Genomic Regions

Abstract Motivation Unique marker sequences are highly sought after in molecular diagnostics. Nevertheless, there are only few programs available to search for marker sequences, compared to the many programs for similarity search. We therefore wrote the program Fur for Finding Unique genomic Regions. Results Fur takes as input a sample of target sequences and a sample of closely related neighbors. It returns the regions present in all targets and absent from all neighbors. The recently published program genmap can also be used for this purpose and we compared it to fur. When analyzing a sample of 33 genomes representing the major phylogroups of E.coli, fur was 40 times faster than genmap but used three times more memory. On the other hand, genmap yielded three times more markers, but they were less accurate when tested in silico on a sample of 237 E.coli genomes. We also designed phylogroup-specific PCR primers based on the markers proposed by genmap and fur, and tested them by analyzing their virtual amplicons in GenBank. Finally, we used fur to design primers specific to a Lactobacillus species, and found excellent sensitivity and specificity in vitro. Availability and implementation Fur sources and documentation are available from https://github.com/evolbioinf/fur. The compiled software is posted as a docker container at https://hub.docker.com/r/haubold/fox. Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals Multiple Roles of the τ131 Subunit of Yeast Transcription Factor IIIC (TFIIIC) in TFIIIB Assembly

2002 ◽  
Vol 22 (1) ◽  
pp. 298-308 ◽  
Author(s):  
Hélène Dumay-Odelot ◽  
Joël Acker ◽  
Rosalia Arrebola ◽  
André Sentenac ◽  
Christian Marck
Keyword(s):  
Transcription Factor ◽  
Transcription Initiation ◽  
In Vitro Transcription ◽  
Initiation Factor ◽  
Physical Interaction ◽  
Restrictive Temperature ◽  
Class Iii ◽  
High Concentrations

ABSTRACT Yeast transcription factor IIIC (TFIIIC) plays a key role in assembling the transcription initiation factor TFIIIB on class III genes after TFIIIC-DNA binding. The second largest subunit of TFIIIC, τ131, is thought to initiate TFIIIB assembly by interacting with Brf1/TFIIIB70. In this work, we have analyzed a TFIIIC mutant (τ131-ΔTPR2) harboring a deletion in τ131 removing the second of its 11 tetratricopeptide repeats. Remarkably, this thermosensitive mutation was selectively suppressed in vivo by overexpression of B”/TFIIIB90, but not Brf1 or TATA-binding protein. In vitro, the mutant factor preincubated at restrictive temperature bound DNA efficiently but lost transcription factor activity. The in vitro transcription defect was abolished at high concentrations of B” but not Brf1. Copurification experiments of baculovirus-expressed proteins confirmed a direct physical interaction between τ131 and B”. τ131, therefore, appears to be involved in the recruitment of both Brf1 and B”.

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