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PubMed1982

Biosynthesis of enzymes of peroxisomal beta-oxidation.

Male Wistar rats were fed a diet with or without di(2-ethylhexyl)phthalate (DEHP), a peroxisome proliferator, for 2 weeks. The increases in the individual enzymes of the hepatic peroxisomal beta-oxidation system after administration of DEHP were 31- to 33-fold. It was found by in vivo experiments using L-[4,5-3H]leucine and the immunoprecipitation technique that the rates of synthesis of the enzymes were 16- to 20-fold higher and those of degradation were 1.7- to 1.9-fold lower in the DEHP group. The translation rates of these enzymes in vitro with liver RNA in the reticulocyte-lysate system were 12- to 14-fold higher in the DEHP group. Short-term kinetic labeling experiments on acyl-CoA oxidase consisting of three subunits were conducted in vivo to explore the biogenesis of peroxisomes. The label was found in the biggest subunit of the enzyme in the supernatant fraction shortly after the label injection, but was distributed to the smaller subunits later. The labeling in the smaller subunits in the peroxisomal fraction was greater than that of the supernatant. The distribution of the label among the subunits in these subcellular fractions was the same as that of the protein amounts 1 day after the label injection. This paper reports that the increase in the quantities of peroxisomal enzymes upon administration of DEHP is mainly due to the increase in their synthesis rates caused by the increase in amounts of mRNA coding for these enzymes.

Acyl-CoA Oxidase↗
PubMed1982

The heat shock response is self-regulated at both the transcriptional and posttranscriptional levels.

When Drosophila cells are shifted from 25 degrees C to 37 degrees C, the synthesis of a small group of proteins (the heat shock proteins or HSPs) is rapidly induced, while most preexisting synthesis is repressed. On return to normal growing temperatures, synthesis of HSPs is gradually repressed and normal synthesis is restored. We show that production of HSP 70 (the major heat-induced protein in these cells) is quantitatively correlated with the degree of stress. The level of synthesis is controlled both transcriptionally and posttranscriptionally through repression of HSP 70 mRNA synthesis and destabilization of HSP 70 transcripts. These regulatory mechanisms depend upon the accumulation of the HSPs themselves; when the production of functional HSPs is blocked, HS transcription continues and HS mRNAs are stable, accumulating in vast quantities; if the block is released, a specific quantity of functional HSP must accumulate before HS transcription is repressed and preexisting HS mRNAs are destabilized. Evidence is also presented that indicates that the same quantity of HSP 70 is required to release the block in normal protein synthesis.

Animals↗
PubMed1989

Effect of age on cutaneous interleukin 1 expression.

Age-related changes in epidermal immune function include morphologic and functional changes in Langerhans cells and decreased epidermal cytokine activity. In this study, expression of interleukin 1 (IL-1) mRNA was quantitated in 6-102-week old mice of different strains. A significant decline in cutaneous IL-1 expression occurred in mice of all strains examined; the rate of decline was most prominent in MRL/MpJ+ mice who also had highest initial levels of IL-1. This observation is in keeping with previous observations that the quantity of several gene products in youth is directly proportional to the rate of change of the quantity of that gene product. The term "economic correction" is used to describe this phenomenon.

Aging↗
PubMed2006

Caffeine biosynthesis and adenine metabolism in transgenic Coffea canephora plants with reduced expression of N-methyltransferase genes.

In anti-sense and RNA interference transgenic plants of Coffea canephora in which the expression of CaMXMT1 was suppressed, caffeine biosynthesis from [8-(14)C]adenine was investigated, together with the overall metabolism of [8-(14)C]adenine. Compared with wild type control plants, total purine alkaloid biosynthesis from adenine and conversion of theobromine to caffeine were both reduced in the transgenic plants. As found previously, [8-(14)C]adenine was metabolised to salvage products (nucleotides and RNA), to degradation products (ureides and CO(2)) and to purine alkaloids (theobromine and caffeine). In the transgenic plants, metabolism of [8-(14)C]adenine shifted from purine alkaloid synthesis to purine catabolism or salvage for nucleotides. HPLC analysis revealed a significantly reduced caffeine content in the transgenic plants. A small quantity (less than 20 nmol g(-1) fresh weight) of xanthosine had accumulated in at least one of the transgenic plants.

Adenine↗
PubMed2025

GENOME TARGETED ENRICHMENT AND SEQUENCING OF HUMAN-INFECTING CRYPTOSPORIDIUM spp.

Cryptosporidium spp. are parasites that cause severe illness in vulnerable human populations. Obtaining pure and sufficient Cryptosporidium DNA from clinical and environmental samples is a challenging task. Oocysts shed in available fecal samples can be limited in quantity, require purification (biased towards dominant strains), and yield limited DNA (<40 fg/oocyst). Here, we use updated genomic sequences from a broad diversity of human-infecting Cryptosporidium species ( C. cuniculus , C. hominis , C. meleagridis , C. parvum , C. tyzzeri , and C. viatorum ) to develop and validate a set of 100,000 RNA baits (CryptoCap_100k) with the aim of enriching Cryptosporidium spp. DNA from varied samples. Compared to unenriched libraries, CryptoCap_100k increases the percentage of reads mapping to target genome sequences, increases the depth and breadth of genome coverage and the reliability of detecting species and mixed infections within a sample, and allows assessment of genetic variation via SNP calling, while decreasing costs.

Journal Article↗
PubMed1994

Ty1 in vitro integration: effects of mutations in cis and in trans.

Mutations within the TYB gene of Ty1 encoding integrase (IN) as well as alterations in its substrate, a linear DNA molecule, were examined for their effects on in vitro IN activity, using a recently developed physical assay. Five different codon-insertion mutations, two frameshift mutations, and one missense mutation, previously identified as transposition-deficient mutations, were tested. Virus-like particles, the source of IN, from two different protease mutants and a reverse transcriptase mutant exhibited near-normal to normal IN activity. Two frameshift mutations mapping within the phylogenetically variable C-terminal domain of IN resulted in significant in vitro IN activity. In contrast, three mutations within the amino-terminal conserved domain of IN completely abolished IN activity. When the substrate termini were mutated, we found that substrates with as few as 4 bp of Ty1 termini were capable of efficiently generating integration products. Surprisingly, certain substrates that lacked obvious similarity to Ty1 termini were also readily integrated into both linear and circular targets, whereas others were not used as substrates at all. Termini rich in adenosine residues were among the more active substrates; however, certain substrates lacking terminal adenosine residues can form small quantities of integration products, including complete integration reactions.

Amino Acid Sequence↗
PubMed2005

Normalization of soil DNA extraction for accurate quantification of target genes by real-time PCR and DGGE.

The analysis of microbial communities in environmental samples requires accurate and reproducible methods for extraction of DNA from sample matrices that have different physical and chemical characteristics. Even with the same sample type, variations in laboratory methods can result in different DNA yields. To circumvent this problem, we have developed an easy and inexpensive way to normalize the quantities of DNA that involves the addition of an internal standard prepared from plasmid DNA. The method was evaluated by comparing DNA yields using different DNA extraction procedures, after which the DNA was used for microbial community analysis by PCR-denaturing gradient gel electrophoresis (PCR-DGGE) of 16S ribosomal RNA (rRNA) and for quantification of 16S rRNA gene copy numbers in environmental samples by real-time PCR. Our results show that use of the internal standard allows normalization of the resulting data and more accurate quantification of gene copy numbers in soil samples. These methods should also have broad application for various other types of environmental samples.

DNA Primers↗
PubMed1994

In situ detection of cytokine messenger RNAs in the eccrine sweat gland of normal human skin.

Human sweat and eccrine sweat glands contained the multifunctional polypeptide cytokines, interleukin-1 alpha and beta (IL-1 alpha and beta). To determine whether the sweat gland itself is an actual site of transcription for cytokines we performed in situ hybridization on histologic sections of normal human skin. In this report we show that the mRNAs encoding the cytokines IL-1 alpha, IL-1 beta, and tumor necrosis factor-alpha (TNF-alpha) are present in normal human skin eccrine sweat gland duct and secretory coil epithelium. These results suggest that in vivo the sweat gland is a production site for cytokine polypeptides and may contribute to the exceedingly large quantities of these cytokine proteins found in the epidermis.

Cytokines↗
PubMed2006

Analysis of human telomerase activity and function by two color single molecule coincidence fluorescence spectroscopy.

Telomerase is a nonclassical DNA polymerase that uses its integral RNA as a template to synthesize telomeric repeats onto chromosome ends. The molecular mechanism of telomerase is unique and involves a translocation step after the synthesis of each telomeric repeat. To directly measure the enzymatic turnover of substrate and the efficiency of the translocation step we have extended our two-color single molecule fluorescence coincidence method (Anal.Chem. 2003, 75, 1664-1670). The method employs Cy5-dATP incorporation into a DNA primer that has been prelabeled with a reference fluorophore. Measurements are performed in the single molecule regime and products, which necessarily have both fluorophores, are excited by two independent lasers, and give rise to coincident events. By counting the number of coincident events and using the coincidence detection efficiency, it is possible to determine the number of the extended products generated by attomole quantities of telomerase, without separation or the use of PCR or radioactivity. Histograms of the logarithms of the ratios of the Cy5 to the reference fluorophore fluorescence can be used to determine the length distribution of the products and hence the enzyme processivity. The mean processivity obtained from the single molecule fluorescence coincidence assay is 0.32 +/- 0.04, in good agreement with the value of 0.37 +/- 0.05 derived from the direct radioactive assay approach. The function of the alignment domain of human telomerase RNA in sustaining catalytic activity in vitro has been reevaluated using this method. Together with our previous results (Nucleic Acids Res. 2002, 30, 4470-4480) these experiments identify the essential residues in the alignment domain of human telomerase RNA that contribute to the activity and processivity of telomerase.

Catalysis↗
PubMed1987

Myelin basic protein-specific RNA levels in interleukin-2-stimulated oligodendrocytes.

In the immune system, T- and B-cell proliferation, as well as B-cell immunoglobulin secretion, is induced by interleukin-2 (IL-2), a T-cell-derived lymphokine. IL-2 also influences the growth of glial cells, specifically, the proliferation and maturation of oligodendrocytes. Studies were conducted to investigate further IL-2-induced maturation of oligodendrocytes through its effect on the regulation of the myelin basic protein (MBP) gene. A cDNA probe specific for rat MBP and a double-antibody radioimmunoassay for MBP were used to quantitate MBP mRNA and protein levels in oligodendrocytes under different experimental conditions. We demonstrate that both MBP mRNA and protein levels are increased in IL-2-stimulated oligodendrocytes. MBP mRNA levels increase within 8 h after IL-2 stimulation, peak between 24 and 48 h, and then decline slightly. MBP protein levels increase 24 h after stimulation and peak at 72 h. MBP mRNA transcripts in the range of 2.0-2.4 kilobases are present in cultured rat oligodendrocytes, which are similar to the MBP mRNA transcripts detected in whole rat and mouse brain. These mRNA transcripts are specifically increased in quantity after oligodendrocyte stimulation with IL-2. These results suggest that one component of oligodendrocyte differentiation/maturation--MBP mRNA and protein expression--can be regulated in part by IL-2.

Animals↗
PubMed1974

Isolation and in vitro translation of delta-crystallin mRNA from embryonic chick lens fibers.

Of the protein synthesized and accumulated during differentiation of embryonic chick lens fibers, 70-80% is the tissue specific protein delta-crystallin. We have isolated and partially characterized the total cytoplasmic mRNA from purified lens fibers of 15-day-old embryos as an initial step toward understanding the regulated expression of delta-crystallin during development. Each lens fiber mass contained an average of 10 mug of cytoplasmic RNA; approximately 0.1 mug per fiber mass was recovered in the mRNA fraction by oligo(dT)-cellulose chromatography. The mRNA electrophoresed primarily as a single peak on a polyacrylamide-agarose gel with an apparent molecular weight of about 9 x 10(5) estimated by comparison with 28S and 18S rRNA markers. Of the protein synthesized in response to the mRNA in cell-free systems derived from Krebs II ascites tumor cells or rabbit reticulocytes, 70-80% comigrated with delta-crystallin on sodium dodecyl sulfatepolyacrylamide-agarose gels. Comparison of the tryptic peptides of delta-crystallin with those of the in vitro products from both heterologous systems established that the lens fiber mRNA contained delta-crystallin mRNA, and that no other functional mRNAs were present in detectable quantities. Thus, the specialization of protein synthesis in embryonic chick lens fibers apparently results from an accumulation of delta-crystallin mRNA in the cytoplasm.

Acrylamides↗
PubMed1983

A stable transcription complex directs mouse ribosomal RNA synthesis by RNA polymerase I.

Ribosomal RNA is synthesized from template molecules that are activated by a stable association with essential transcription factors. This activated template assembles prior to the onset of transcription as a preinitiation complex and factors remain firmly attached during active elongation as well. Sequential addition of differently marked rRNA genes to an S-100 mouse cell extract shows that the DNA binding factors of the stable complex are present in limiting quantities. They associate rapidly with template molecules and the resultant transcription complex remains intact over prolonged periods of incubation in the presence of competitor DNA. The resistance of the stable complex to the usual inhibitory effect of high DNA concentration suggests that more than one DNA binding factor recognizes the rDNA promoter region and is needed to direct faithful transcription. Finally, although the stable complex is specific for the rRNA initiation region, added vector sequences can neutralize nonspecific DNA binding components that are also present in the cell extract. This lowers the requirement for rDNA template and demonstrates that each activated rRNA gene can direct at least 10 rounds of elongation and reinitiation.

Animals↗
PubMed2006

A SAGE approach to identifying novel trans-acting factors involved in the X inactivation process.

X chromosome inactivation ensures the dosage compensation of X-linked genes in XX females compared to their XY male counterpart. It is characterised by the specific recruitment of an inhibitory ribonucleoprotein complex involving the non-coding Xist RNA to the presumptive inactive X chromosome and associated chromatin modifications, which result in the transcriptional silencing of the X chromosome. As an approach to the identification of some of the potential molecular players in this process we have performed comparative transcriptional profiling of mouse 6.5-dpc (days post-coitum) female and male embryos using a modified SAGE (Serial analysis of gene expression) technique which allows the analysis of small quantities of biological material. At 6.5 dpc, a moment when random X inactivation of embryonic tissues has just been achieved, some two hundred transcripts that were significantly enriched in the female gastrula compared to its male counterpart could be identified. The validation of an association with the X inactivation process of a subset of these transcripts has been studied, ex vivo, in differentiating female and male ES cells and in female ES cells in which the establishment of X inactivation is interrupted through the post-transcriptional inhibition of Xist synthesis.

Animals↗
PubMed1991

Mutations affecting hepadnavirus plus-strand DNA synthesis dissociate primer cleavage from translocation and reveal the origin of linear viral DNA.

Hepadnaviruses replicate their circular DNA genomes via reverse transcription of an RNA intermediate. The initial product of reverse transcription, minus-strand DNA, contains two copies of a short direct repeat (DR) sequence, termed DR1 and DR2. Plus-strand DNA synthesis initiates at DR2 on minus-strand DNA, using as a primer a short, DR1-containing oligoribonucleotide derived by cleavage and translocation from the 5' end of pregenomic RNA. To clarify the sequence requirements for plus-strand primer cleavage and translocation, we have constructed mutants of the duck hepatitis B virus bearing base changes in or around the DR1 sequence in the primer. A point mutation at the terminal nucleotide of DR1 has a striking phenotype: normal levels of duplex viral DNA are produced, but nearly all of the DNA is linear rather than circular. Mapping of the 5' end of plus-strand DNA reveals that primer cleavage occurs with normal efficiency and accuracy, but the primer is not translocated to DR2; rather, it is extended in situ to generate duplex linear DNA. Other mutations just 3' to DR1 similarly affect primer translocation, although with differing efficiencies. Linear DNA found in wild-type virus preparations has the same fine structure as the mutant linears described above. These results indicate that (i) plus-strand primer cleavage and translocation are distinct steps that can be dissociated by mutation, (ii) lesions in sequences not included in the primer can severely inhibit primer translocation, and (iii) elongation of such untranslocated primers is responsible for the variable quantities of linear DNA that are found in all hepadnaviral stocks.

Animals↗
PubMed1993

Structure and expression of the gene encoding rat nonspecific form delta-aminolevulinate synthase.

To understand the regulatory mechanisms controlling the heme biosynthetic pathway in animal liver, RNA blot hybridization analysis was used to examine the developmental stage-specific transcription of the gene encoding nonspecific form delta-aminolevulinate synthase (ALAS-N). The expression of the erythroid-specific delta-aminolevulinate synthase (ALAS-E) mRNA was also studied. The results demonstrated that, while ALAS-E is the key enzyme which supplies large quantities of heme for hemoglobin synthesis in fetal rat liver, ALAS-N functions to supply heme for the cytochrome P-450 system in fetal, newborn, and adult rat liver. ALAS-N was also suggested to work as a housekeeper gene to supply heme for respiratory cytochromes and other hemoproteins in various tissues. The structure and organization of the rat ALAS-N gene were next analyzed to study the molecular mechanisms regulating ALAS-N gene transcription. The ALAS-N gene was found to span more than 14 kb in the rat genome, encompassing eleven exons. The promoter region of the gene was found to contain several potential cis-acting regulatory elements, including motifs matching the TATA box sequence and the nuclear respiratory factor 1 binding sequence. The organization of the rat ALAS-N gene was determined to be quite similar to that of the ALAS-E gene in mouse; the mouse ALAS-E gene consists of eleven exons. This observation suggested that the ancestral gene for ALA synthase in animals was probably composed of eleven exons, and both the ALAS-N and ALAS-E genes were derived from this ancestral gene.

5-Aminolevulinate Synthetase↗
PubMed1994

Type-I insulin-like growth factor receptor gene expression in the chick. Developmental changes and the effect of selection for increased growth on the amount of receptor mRNA.

An RNase protection assay is described that allowed the quantitative analysis of chicken type-I IGF receptor mRNA transcripts. The transcripts were measured in extracts of total nucleic acid (TNA) and, under the hybridization conditions described, protected probes of the expected size were obtained. The RNA-RNA hybrids could be quantified in the presence of at least a 1000-fold molar excess of DNA containing sequences which were complimentary to the RNA probe. The amount of protected probe was linearly related to the amount of TNA in the hybridization reaction medium, and this allowed the results to be expressed in the form of mRNA molecules/cell. Type-I IGF receptor mRNA transcripts were detected in all the tissues examined from a 20-day-old chick embryo. Their amount ranged from 5 to 24 molecules/cell, in the order liver < breast muscle < leg muscle < heart < brain. The amount of receptor mRNA was 65- to 300-fold less than that of beta-actin mRNA. The quantity of type-I IGF receptor mRNA varied significantly throughout embryonic and post-hatch development. Maximum amounts were measured in 21-day-old embryos (a two- to fourfold increase relative to 16-day-old embryos). Thereafter the amount of receptor mRNA decreased, during the 4-week period after hatching, to levels which were significantly lower than that observed in 16-day-old embryos. Throughout the period of embryonic and post-hatch development described here the amount of beta-actin mRNA remained constant, indicating that the changes in the quantity of receptor mRNA were due to specific mechanisms acting directly on the steady-state levels of type-I IGF receptor mRNA. Selection for increased growth had no effect on the amount of type-I IGF receptor mRNA. The result was the same when expressed either as molecules/cell or as a percentage of beta-actin mRNA.

Actins↗
PubMed1996

Trypanosomatidae: a spliced-leader-derived probe specific for the genus Phytomonas.

We probed DNA from all trypanosomatid genera by slot blot hybridization with an oligonucleotide (SL3') complementary to a sequence of the Phytomonas spliced-leader or mini-exon RNA. The 19-nucleotide probe target site was previously shown to be highly conserved among a limited number of Phytomonas isolates, but diverges in other kinetoplastid genera. Our examination of 84 isolates of various genera of trypanosomatids showed hybridization of this probe exclusively with isolates from plants or insects which could, by morphological, biochemical, and molecular criteria, be considered to belong to the genus Phytomonas. In contrast, no hybridization was observed with flagellates of the genera Blastocrithidia, Crithidia, Endotrypanum, Herpetomonas, Leptomonas, Leishmania, and Trypanosoma. The method detected DNA quantities as low as 50 ng using either radioactive or nonradioactive probes, and was effective with as few as 10(4) intact flagellates. Together, these results suggest that this probe will serve as a convenient marker for taxonomic and epidemiological studies requiring reliable identification of Phytomonas spp. in plants or in putative insect vectors.

Animals↗