Robert Ivarie - Academia.edu (original) (raw)
Papers by Robert Ivarie
Journal of Cellular Physiology, 1975
Anucleate HTC cells have been used to determine the importance of the nucleus in the regulation o... more Anucleate HTC cells have been used to determine the importance of the nucleus in the regulation of the intracellular levels of tyrosine aminotransferase (TAT) in hepatoma tissue culture (HTC) cells. In the absence of the nucleus, neither the induction of the enzyme by dexamethasone nor its deinduction upon removal of the hormone occurs. Degradation of the enzyme takes place when protein synthesis is inhibited in anucleates by cycloheximide. Therefore, the maintenance of induced levels of enzyme activity after dexamethasone withdrawal from pre-induced anucleates suggest that the nucleus is required for the inactivation of the TAT mRNA.
Journal of Animal Science
Nat Biotechnol, 2002
labeled by random priming using 3 nmol of Cy3-dCTP or Cy5-dCTP (NEN, Boston, MA) and 0.6 nmol dCT... more labeled by random priming using 3 nmol of Cy3-dCTP or Cy5-dCTP (NEN, Boston, MA) and 0.6 nmol dCTP. For array hybridization, 0.5 µg of labeled tumor DNA was co-precipitated with 0.5 µg of normal control, 5 µg Cot-I DNA, and 20 µg sheared salmon testis DNA. The probe mixture was dissolved in 5 µl H 2 O, and SSPE and SDS were added to a final concentration of 6× SSPE and 2% SDS in a final volume of 15 µl. The probe solution was denatured at 98°C for 2 min, cooled to room temperature, and spread onto the array and covered with a 22 × 22 mm coverslip. The slide was enclosed in a Corning hybridization chamber and incubated at 62°C for 16-22 h. After hybridization, the array was rinsed with H 2 O twice at room temperature and then washed in 2× SSC, 0.5% SDS at 65°C with shaking for 45 min. The arrays were rinsed with H 2 O and air-dried right before scanning. Array image acquisition and data processing. The fluorescent signals were scanned into image files using the Scan Array 3000 (General Scanning, Watertown, MA). Images were quantified using Imagen software (Biodiscovery, San Diego, CA). The relative fluorescent level or fluorescent ratio, representing the relative amounts of target sequences in the probe mix, was analyzed by comparing the fluorescent intensity of corresponding individual spots after background subtraction. When equal amounts of genomic probes labeled with Cy5 and Cy3 were comparatively hybridized, the variation of fluorescence ratio of the two colors was within 10% for different BACs on the array. The average local background and standard deviation over all the array spots was calculated. Local background value for a spot was replaced with the average background if it was 2 s.d. units above the average value. The mean ratio over all the unique clones was calculated, as well as the s.d. Those ratios beyond 1.5 s.d. from the mean were discarded from the calculation, and the mean and s.d. were recalculated. The final average ratio value was used as the normalization factor to normalize all the ratios. The ratios of clones were plotted along individual chromosomes for easy inspection. For each sample two experiments were done with reversal of the dye labels. The Cy5:Cy3 ratios were plotted together along individual chromosomes. The two ratio curves should be reciprocal; thus any ratio artifact can be easily identified. A BAC clone was judged to be significant only if two or all three replicate spots passed the criteria in both experiments.
Exp Cell Res, 1998
these factors to elicit the myogenic lineage. In verte-Myogenic regulatory factors (MRFs) are hie... more these factors to elicit the myogenic lineage. In verte-Myogenic regulatory factors (MRFs) are hierarchi-brates, MRFs are variously expressed in muscle precurcal regulators of skeletal myogenesis. Many MRF prosor tissues, in differentiating muscle cells, and in fetal moters have been well characterized with respect to and adult muscle. All four MRF family members are flanking sequences that control their expression. Yet found in vertebrates, and homologs of the MRF family the promoter elements that regulate Myf-5, the first have been found in Drosophila (6) and Caenorhabditis MRF expressed during mammalian embryogenesis, elegans [7]. Among the MRFs, both Myf-5 and MyoD are still largely unknown. Comparison of Myf-5 5 act to convert mesodermal stem cells to proliferating flanking regions from bovine, mouse, and chicken myoblasts [8, 9], yet each factor exhibits distinct regugenes revealed three evolutionarily conserved elelatory and functional properties. In proliferating culments proximal to the transcription start site: the tures of established muscle cell lines, either MyoD or TATA box, an octamer motif, termed OLS, and a 6-Myf-5 is expressed [reviewed in Ref. 3], suggesting that bp C-rich element. Mobility shift assays and DNase I they negatively regulate each other's expression and footprinting analysis demonstrated that a nuclear faccontrol independent myogenic lineages. Most importor(s) present in both bovine and avian muscle and tantly, recent studies in transgenic knockout mice have nonmuscle tissues specifically recognized OLS. Furshown that Myf-5 and the paired-type homeobox gene thermore, this binding activity reacted with a poly-Pax-3 genetically lie upstream of MyoD and indepenclonal Oct-1 antibody. In avian primary myoblast and fibroblast cultures, CAT reporter constructs under dently control two separate myogenic pathways that regulation of the proximal Myf-5 5 flanking sequence lead to the body wall and limb musculature [10]. were expressed preferentially in myoblasts with CAT Promoter sequences controlling transcription have levels approximately 12-fold higher than in fibrobeen well characterized for several MRFs [7-17]. Howblasts. The TATA box and octamer motif were imever, little is known about how the Myf-5 gene is reguportant for expression in both myoblasts and fibrolated. In transgenic mice, the 5 flanking region from blasts: loss of the TATA box abolished activity, and mouse Myf-5 can direct the correct spatial and tempodisruption of the OLS resulted in 50-75% loss of proral expression of a lacZ reporter in visceral arches moter activity. ᭧ 1998 Academic Press while expression of the reporter in somites is delayed [18]. Thus, the region contains at least a subset of the muscle-specific regulatory elements that control Myf-5
Trends in Biotechnology, Jan 31, 2003
Biotechniques, Sep 1, 1994
We have tested the ability of polyvinyl alcohol (PVA) to enhance the indoxyl-nitro blue tetrazoli... more We have tested the ability of polyvinyl alcohol (PVA) to enhance the indoxyl-nitro blue tetrazolium alkaline phosphatase color reaction in whole mount in situ hybridizations of early stage quail embryos. Using detection of the myogenic factor qmyoD in hybridizations of 13-14 somite-pair quail embryos as a test system, 10% solutions of 13-23 kDa and 31-50 kDa PVA optimally enhanced the alkaline phosphatase color reaction. With the enhanced detection conditions, we have been able to measure qmyoD expression as early as the 3 somite-pair stage, whereas previously we were unable to detect qmyoD prior to the 10 somite-pair stage. PVA-enhanced detection of somitic qmyoD expression was specific as no corresponding signal was found in control hybridizations with a sense qmyoD probe.
Journal of Cellular Physiology, 1975
Anucleate HTC cells have been used to determine the importance of the nucleus in the regulation o... more Anucleate HTC cells have been used to determine the importance of the nucleus in the regulation of the intracellular levels of tyrosine aminotransferase (TAT) in hepatoma tissue culture (HTC) cells. In the absence of the nucleus, neither the induction of the enzyme by dexamethasone nor its deinduction upon removal of the hormone occurs. Degradation of the enzyme takes place when protein synthesis is inhibited in anucleates by cycloheximide. Therefore, the maintenance of induced levels of enzyme activity after dexamethasone withdrawal from pre-induced anucleates suggest that the nucleus is required for the inactivation of the TAT mRNA.
Journal of Animal Science
Nat Biotechnol, 2002
labeled by random priming using 3 nmol of Cy3-dCTP or Cy5-dCTP (NEN, Boston, MA) and 0.6 nmol dCT... more labeled by random priming using 3 nmol of Cy3-dCTP or Cy5-dCTP (NEN, Boston, MA) and 0.6 nmol dCTP. For array hybridization, 0.5 µg of labeled tumor DNA was co-precipitated with 0.5 µg of normal control, 5 µg Cot-I DNA, and 20 µg sheared salmon testis DNA. The probe mixture was dissolved in 5 µl H 2 O, and SSPE and SDS were added to a final concentration of 6× SSPE and 2% SDS in a final volume of 15 µl. The probe solution was denatured at 98°C for 2 min, cooled to room temperature, and spread onto the array and covered with a 22 × 22 mm coverslip. The slide was enclosed in a Corning hybridization chamber and incubated at 62°C for 16-22 h. After hybridization, the array was rinsed with H 2 O twice at room temperature and then washed in 2× SSC, 0.5% SDS at 65°C with shaking for 45 min. The arrays were rinsed with H 2 O and air-dried right before scanning. Array image acquisition and data processing. The fluorescent signals were scanned into image files using the Scan Array 3000 (General Scanning, Watertown, MA). Images were quantified using Imagen software (Biodiscovery, San Diego, CA). The relative fluorescent level or fluorescent ratio, representing the relative amounts of target sequences in the probe mix, was analyzed by comparing the fluorescent intensity of corresponding individual spots after background subtraction. When equal amounts of genomic probes labeled with Cy5 and Cy3 were comparatively hybridized, the variation of fluorescence ratio of the two colors was within 10% for different BACs on the array. The average local background and standard deviation over all the array spots was calculated. Local background value for a spot was replaced with the average background if it was 2 s.d. units above the average value. The mean ratio over all the unique clones was calculated, as well as the s.d. Those ratios beyond 1.5 s.d. from the mean were discarded from the calculation, and the mean and s.d. were recalculated. The final average ratio value was used as the normalization factor to normalize all the ratios. The ratios of clones were plotted along individual chromosomes for easy inspection. For each sample two experiments were done with reversal of the dye labels. The Cy5:Cy3 ratios were plotted together along individual chromosomes. The two ratio curves should be reciprocal; thus any ratio artifact can be easily identified. A BAC clone was judged to be significant only if two or all three replicate spots passed the criteria in both experiments.
Exp Cell Res, 1998
these factors to elicit the myogenic lineage. In verte-Myogenic regulatory factors (MRFs) are hie... more these factors to elicit the myogenic lineage. In verte-Myogenic regulatory factors (MRFs) are hierarchi-brates, MRFs are variously expressed in muscle precurcal regulators of skeletal myogenesis. Many MRF prosor tissues, in differentiating muscle cells, and in fetal moters have been well characterized with respect to and adult muscle. All four MRF family members are flanking sequences that control their expression. Yet found in vertebrates, and homologs of the MRF family the promoter elements that regulate Myf-5, the first have been found in Drosophila (6) and Caenorhabditis MRF expressed during mammalian embryogenesis, elegans [7]. Among the MRFs, both Myf-5 and MyoD are still largely unknown. Comparison of Myf-5 5 act to convert mesodermal stem cells to proliferating flanking regions from bovine, mouse, and chicken myoblasts [8, 9], yet each factor exhibits distinct regugenes revealed three evolutionarily conserved elelatory and functional properties. In proliferating culments proximal to the transcription start site: the tures of established muscle cell lines, either MyoD or TATA box, an octamer motif, termed OLS, and a 6-Myf-5 is expressed [reviewed in Ref. 3], suggesting that bp C-rich element. Mobility shift assays and DNase I they negatively regulate each other's expression and footprinting analysis demonstrated that a nuclear faccontrol independent myogenic lineages. Most importor(s) present in both bovine and avian muscle and tantly, recent studies in transgenic knockout mice have nonmuscle tissues specifically recognized OLS. Furshown that Myf-5 and the paired-type homeobox gene thermore, this binding activity reacted with a poly-Pax-3 genetically lie upstream of MyoD and indepenclonal Oct-1 antibody. In avian primary myoblast and fibroblast cultures, CAT reporter constructs under dently control two separate myogenic pathways that regulation of the proximal Myf-5 5 flanking sequence lead to the body wall and limb musculature [10]. were expressed preferentially in myoblasts with CAT Promoter sequences controlling transcription have levels approximately 12-fold higher than in fibrobeen well characterized for several MRFs [7-17]. Howblasts. The TATA box and octamer motif were imever, little is known about how the Myf-5 gene is reguportant for expression in both myoblasts and fibrolated. In transgenic mice, the 5 flanking region from blasts: loss of the TATA box abolished activity, and mouse Myf-5 can direct the correct spatial and tempodisruption of the OLS resulted in 50-75% loss of proral expression of a lacZ reporter in visceral arches moter activity. ᭧ 1998 Academic Press while expression of the reporter in somites is delayed [18]. Thus, the region contains at least a subset of the muscle-specific regulatory elements that control Myf-5
Trends in Biotechnology, Jan 31, 2003
Biotechniques, Sep 1, 1994
We have tested the ability of polyvinyl alcohol (PVA) to enhance the indoxyl-nitro blue tetrazoli... more We have tested the ability of polyvinyl alcohol (PVA) to enhance the indoxyl-nitro blue tetrazolium alkaline phosphatase color reaction in whole mount in situ hybridizations of early stage quail embryos. Using detection of the myogenic factor qmyoD in hybridizations of 13-14 somite-pair quail embryos as a test system, 10% solutions of 13-23 kDa and 31-50 kDa PVA optimally enhanced the alkaline phosphatase color reaction. With the enhanced detection conditions, we have been able to measure qmyoD expression as early as the 3 somite-pair stage, whereas previously we were unable to detect qmyoD prior to the 10 somite-pair stage. PVA-enhanced detection of somitic qmyoD expression was specific as no corresponding signal was found in control hybridizations with a sense qmyoD probe.